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Illness in the intensive care unit is defined not by pathologic changes in a particular tissue or by structural changes in a specific organ, but by a constellation of quantifiable changes in physiological and biochemical measures . To round in an intensive care unit is to be exposed to a cacophony of numbers the ph, the glasgow coma scale, the fibrinogen level . To be an intensivist means to take this chaotic melange of digits and to transform them into a clinical profile that will support a therapeutic decision . An uninitiated visitor to a contemporary intensive care unit could be forgiven for concluding that the intensivity of intensive care referred to the zeal with which its practitioners measure things: the continuous recording of the pulse, the blood pressure and the transcutaneous oxygen saturation, and the frequent assay of circulating factors whose function is familiar (e.g. Potassium or hemoglobin) as well as those factors whose biologic significance is less so . High on the list of those less significant factors is procalcitonin . In the present issue of critical care, level and colleagues report the results of a carefully conducted cohort study of 15 patients undergoing continuous venovenous hemodialysis . They show that procalcitonin (pct) is cleared by continuous venovenous hemodialysis at conventional filtration rates and that the protein adsorbs to the filter, so as much as 20% of pct is removed through the membrane . The consequences of this removal are modest, however, and are probably not clinically significant . What message should the beleaguered intensivist, struggling to maintain a focus in the face of an onslaught of new measures and new sources of uncertainty, take from this report? I believe there are two: one message regarding the utility of pct as a diagnostic marker, and the second message addressing the more fundamental question of how to interpret the masses of numeric information generated within the intensive care unit . A report by assicot and colleagues a decade ago, evaluating 79 children suspected of being infected, suggested that elevated levels of pct could reliably discriminate patients who were truly infected from those patients in whom clinical signs of acute inflammation were initiated by noninfectious stimuli . Since that report, and driven in no small part by the development of a reliable assay for pct, a medline search using the keyword' procalcitonin' currently identifies 483 publications these studies suggest that, although pct levels can be elevated in noninfectious conditions such as the treatment of transplant rejection with antibodies to cd3, elevated levels of pct are a reliable and specific marker of invasive infection [4 - 6], and that adequate treatment of such infection results in a reduction in the levels of circulating pct . The utility of pct as a diagnostic marker appears to be less in its sensitivity to detect infection than in its specificity to rule it out . In particular, a low level of pct permits the clinician to be confident that infection is not present with greater than 90% certainty . But if pct is a promising marker that permits us to conclude that a critically ill patient is not infected (and so to avoid noninformative diagnostic investigations or exposure to unnecessary antibiotics), how confident can we be that the information it provides can be applied in all critically ill patients? Clinicians must make categorical, yes / no decisions based on data that are continuous in character . A culture of a venous catheter tip is considered positive if more than 15 colonies of bacteria are present following a standardized method of culture, or transfusion is administered if the hemoglobin level is less than 70 the validity of each of these thresholds has been established empirically, but their successful application depends on the reliability of the measure that is used . Can that reliability be significantly jeopardized by an artifact resulting from the confounding effects of the underlying disease or its treatment? This is the question that level and colleagues sought to address, and a question of practical importance to the interpretation of diagnostic tests in the intensive care unit . The circulating level of a given molecule depends on three factors: the rate of production and release of the molecule, the rate of its removal, and the volume within which it is diluted . When rates of production and removal are equal, a steady - state constant level results . However, the actual measured level of that steady state will depend on the volume of distribution . Although the kinetics of the synthesis and release of pct are not well understood, its synthesis and release appear to be triggered by invasive infection, with the result that levels in the circulation increase . The magnitude of this increase is clearly large enough to offset the reduction in concentration that might result from the presence of a larger volume of redistribution in the resuscitated, septic patient . Is it, therefore, either artefactually increased in renal failure or reduced by hemodialysis? Herget - rosenthal and colleagues studied pct levels in 68 patients with acute or chronic renal failure treated by intermittent hemodialysis . They found that elevated pct levels had an 84% positive predictive value and an 87% negative predictive value for the diagnosis of infection . Low flux membranes did not alter these figures after the start of dialysis, while high flux membranes did result in a significant reduction in the negative predictive value to 54% . In contrast, in a study of 26 patients undergoing continuous venovenous hemofiltration, meisner showed that although pct was adsorbed to the membrane, and removed in the ultrafiltrate, plasma levels remained constant . It thus appears that continuous venovenous hemodialysis at conventional flow rates does not jeopardize the diagnostic utility of pct; whether high - volume hemofiltration has an effect remains to be determined . From a broader perspective, the evolving literature on pct underlines the conceptual quandaries that confront the contemporary intensivist . Why is the pro form of a calcium - regulating hormone released during bacterial infection? Is its release a marker of an appropriate host response to that infection, or is it a marker of a maladaptive response that might contribute to the morbidity of sepsis? Is pct simply a convenient diagnostic marker, or is it an appropriate target for therapy? Are the diagnostic criteria of infection used to evaluate pct performance indicative of a disease process (infection) whose timely and appropriate treatment might improve outcome? Or, rather, are the criteria surrogate markers of an alternate disease (hyperprocalcitonemia) that merits therapy in its own right? The emergence of the intensive care unit as a locus for providing supportive care for critically ill patients has confronted us with challenges that are unprecedented in medical history . Intensive care units care for a population of patients who, if nature were permitted to take her course, would die a rapid death . In the absence of fluid replacement and circulatory support with exogenous catecholamines and vasoactive agents, the end result of shock is a quiet death from circulatory insufficiency; without the mechanical ventilator, hypoxemia similarly leads inevitably to a rapid demise . But if survival under these circumstances is unprecedented, how should we interpret the biochemical and physiologic events that occur in patients who remain alive only because of the intervention of the intensivist? There is no compelling evolutionary argument to support the advantages of one physiologic state over another: those who in an earlier era would have died do not contribute to the gene pool, and even in our own brave new age reproduction while on the ventilator is distinctly uncommon . Under these circumstances, what is normal may not be what is optimal, and what is abnormal may not be reliable.
Aloe vera gel is a colorless substance obtained from the parenchymatous cells in fresh leaves of aloe vera (l) burm . Native to north africa, aloe has been introduced and is being cultivated in the warmer areas of the world . Aloe vera gel, rich in polysaccharides (pectins, hemicelluloses, glucomannan, acemannan, and other mannose derivatives) should not be confused with the drug aloe dried juice of aloe vera leaves, bitter yellow exudate containing anthracene glycosides, mainly of the aloe - emodin anthrone 10-c - glucoside type [1, 2]. Consumed as a beverage it was not toxic in vivo for mice . On the contrary, aloe latex and its hydroxyanthrone derivatives (aloin, aloe - emodin etc .) Have strong laxative properties and their longer use requires medical supervision . Traditionally, aloe gel was widely used for the treatment of minor wounds, inflammatory skin disorders, and thermal and radiation burns . In vitro, aloe gel suppressed bacteria - induced pro - inflammatory [tumor necrosis factor (tnf-) and interleukin 1 (il-1)] cytokines and matrix metalloproteinase 9 (mmp-9) production in human mononuclear leukocytes [4, 5]. In vivo, polysaccharides derived from aloe vera gel, injected into mice, potently stimulated migration of macrophages to the peritoneal cavity . In human, oral aloe vera gel was used by patients with inflammatory bowel disease, osteoarthritis, and other inflammatory conditions . Oral and topical administration of aloe vera gel diminished inflammation and eased joint immobility and pain [811]. In ophthalmology, aloe vera extracts may be used in eye drops to treat inflammations and other cornea ailments . Besides its anti - inflammatory activity, aloe vera gel has antimicrobial properties and in vivo exerts a protective effect on polymicrobial sepsis in mice [1317]. Anthraquinones, compounds present in the outer part of aloe leaves and in their succus or extract, have been shown to have direct anti - cancer activity in different kinds of human cancer cell lines . Moreover, aloe - emodin, a hydroxyanthraquinone from aloe vera, can act as an anti - angiogenic agent . Some data suggest that the inner part of aloe vera leaves, aloe vera gel and their polysaccharide components also have tumor growth modulatory properties, probably connected with their immunomodulatory activity [20, 21]. In our previous paper we reported the inhibitory effect of aloe barbadensis fresh leaves aqueous extract (herbal drug biostymina) on tumor - induced cutaneous angiogenesis in mice . The aim of the present study was to evaluate in balb / c mice the in vivo influence of commercial aloe vera gel product (aloe vera drinking gel) on the syngeneic l-1 sarcoma tumor growth and its vascularization: a) early cutaneous neovascular response, tumor - induced angiogenesis (tia test), and b) tumor hemoglobin content measured 14 days after l-1 sarcoma cell grafting . Drug . Tru - alo 99% aloe vera drinking gel (aloe barbadensis miller folium succus), aloin content <40 ppm; produced by hi tech aloe vera pty ltd, bundaberg, australia the study was performed on 59 female inbred balb / c mice 6 - 8 weeks old, weighing about 20 g, delivered from the polish academy of sciences breeding colony . For all performed experiments animals were handled according to the polish law on the protection of animals and nih (national institutes of health) standards . Mice were housed 4 - 5 per cage and maintained under conventional conditions (room temperature 22.5 - 23.0c, relative humidity 50 - 70%, 12 h day / night cycle) with free access to standard rodent diet and water . Ketamina 10%, biowet, pulawy, poland); xylazine 10 mg / kg (prep . Sedazin, biowet, pulawy, poland); 3.6% chloral hydrate 0.1 ml per 10 g of body mass (sigma aldrich, usa); morbital (biowet pulawy, poland). Evaluation of sarcoma l-1 growth and angiogenic activity was performed as previously described [23, 24]. L-1 sarcoma cells were delivered from warsaw oncology center collection, passaged twice in vivo and grafted subcutaneously (for evaluation of tumor growth and its hemoglobin (hb) content) or intradermally (for evaluation of their angiogenic activity) to syngeneic balb / c mice . Briefly, sarcoma l-1 cells from in vitro stock were grafted (106/0.1 ml) subcutaneously into the subscapular region of balb / c mice . After 14 days the tumors were excised, cut to smaller pieces, rubbed through the sieve and suspended in 5 ml of phosphate buffered saline (pbs). After sedimentation, the supernatant was collected and centrifuged for 10 min at 1500 rpm . Obtained sarcoma cells were washed once with pbs for 10 min, then centrifuged at 1500 rpm, and resuspended in parker medium in concentration of 4 10/ml or 10/ml . Multiple 0.05 ml samples of 200 thousand sarcoma cells were injected intradermally into partly shaved, narcotized balb / c mice (at least 2 - 4 mice per group). In order to facilitate the localization of cell injection sites, the suspension mice obtained aloe vera gel (150 l for one mouse daily) in drinking water for 3 days . After 72 hours mice were sacrificed with a lethal dose of morbital . All newly formed blood vessels were identified and counted in the dissection microscope, on the inner skin surface, at magnification of 6, in 1/3 of the central area of the microscopic field . Identification was based on the fact that new blood vessels are thin, directed to the point of cell injection, with ramifications, and some of them are tortuous (fig . Neovascular reaction 3 days after the intradermal injection of tumor cells 0.1 ml samples of 1 million sarcoma cells were grafted subcutaneously into the sub - scapular region of balb / c mice . On the day of cell grafting and on the following 13 days mice obtained 150 l of aloe vera gel in drinking water, or water as a control . Tumors were removed, weighed and measured with an electronic caliper (the fowler ultra - cal mark iii caliper). Briefly, tumors were homogenized in pbs using an ultrasonic sonificator (virsonic, usa), then centrifuged for 20 min at 4000 g. 20 l of the supernatant was added to 5 ml of drabkin reagent . The absorbance was read in a spectrophotometric reader elx800 (biotek instruments, usa) at 570 nm . The reader for the hb measurement was calibrated with hemoglobin standard solutions (sigma). Multiple 0.05 ml samples of 200 thousand sarcoma cells were injected intradermally into partly shaved, narcotized balb / c mice (at least 2 - 4 mice per group). In order to facilitate the localization of cell injection sites, the suspension mice obtained aloe vera gel (150 l for one mouse daily) in drinking water for 3 days . After 72 hours mice were sacrificed with a lethal dose of morbital . All newly formed blood vessels were identified and counted in the dissection microscope, on the inner skin surface, at magnification of 6, in 1/3 of the central area of the microscopic field . Identification was based on the fact that new blood vessels are thin, directed to the point of cell injection, with ramifications, and some of them are tortuous (fig . 0.1 ml samples of 1 million sarcoma cells were grafted subcutaneously into the sub - scapular region of balb / c mice . On the day of cell grafting and on the following 13 days mice obtained 150 l of aloe vera gel in drinking water, or water as a control . Tumors were removed, weighed and measured with an electronic caliper (the fowler ultra - cal mark iii caliper). Briefly, tumors were homogenized in pbs using an ultrasonic sonificator (virsonic, usa), then centrifuged for 20 min at 4000 g. 20 l of the supernatant was added to 5 ml of drabkin reagent . The absorbance was read in a spectrophotometric reader elx800 (biotek instruments, usa) at 570 nm . The reader for the hb measurement was calibrated with hemoglobin standard solutions (sigma). Two - tailed p value was lower than 0.0001 (t = = 4.467; df = 70). Hence, the mean number of newly formed blood vessels in the experimental, aloe - fed group of mice was highly significantly lower than in the corresponding controls (fig . 2) mean number of newly - formed blood vessels counted 3 days after the intradermal injection of sarcoma l-1 cells the difference between the groups of control and aloefed mice (150 l daily dose for 14 days) with respect to the 14 days tumor volume was on the border of statistical significance (fig . No difference was observed in hemoglobin content between control and experimental tumors (21.3 3.1 vs. 24.1 3.6 g / mg, respectively). The effect of feeding mice with aloe vera drinking gel for 14 days after l-1 sarcoma subcutaneous grafting on the tumor volume (difference on the border of statistical significance) it was shown by other authors that some aloe vera active components slow down the experimental tumor growth . Three anthraquinones (aloesin, aloe - emodin and barbaloin) extracted from aloe vera leaves may exert their chemo - preventive effect through modulating antioxidant and detoxification enzyme activity levels . Aloe - emodin induces cell death through s - phase arrest and apoptosis in the dose- and time - dependent manner . Other researchers describe the anti - tumour effect of specific derivatives of the aloe vera plant . Di(2-ethylhexyl)phthalate isolated from aloe vera linne may have anti - leukemic and anti - mutagenic properties . The anti - tumor effect was also documented for the aloe vera leaf pulp extract and the main lectin (aloctin i) present in it, in the ehrlich ascites tumor model . Acemannan, the compound of the extract from the parenchyma of aloe vera / aloe barbadensis, stimulates the synthesis of monokines and recruitment of immune cells and, by this mechanism, necrosis and regression of murine sarcoma . The results of these studies suggest that this effect could be due to its immunomodulatory activity . Acemannan has been approved by the fda - us as a potent immunomodulating and anti - viral agent . It was approved as an aid in the treatment of canine and feline fibrosarcoma . However, the critical condition for the tumor to effectively metastasize is formation of the new vessels prompted by a group of cancer cells derived from the primary, transported by the blood circulation and grafted in permissible tissue environment . This permissiveness is conditioned by the agents released by the tumor cells that drive recipient tissue to facilitate new vessel growth in it . We have been able to show that aloe vera drinking gel slows down an early phase of new vessel formation and their in - growth in hosting tissue . However, aloe vera drinking gel has not caused the necrotical effect on the tumor volume and it has not influenced the vascularity of the mature tumor (as indicated by the lack of differences in hemoglobin content of tumors between groups). It may suggest that its effect is exerted only on the newly forming vessels during micrometastasis implantation . Few cytotoxic and targeted drugs have been proven effective in adjuvant systemic therapies after most of the tumor was removed by surgery or radiotherapy . The presence of micrometastases at the time of primary therapy is emphasized as the cause of failure of loco regional therapies . The effective chemoprevention should be directed at the micrometastasis priming mechanism, that is among most important angiogenesis . Aloe vera drinking gel, having a low profile of side effects, may be a good candidate for supplemental therapy . However, aloe vera is also known for its beneficial wound healing impact that might be partly attributed to its compound, -sitosterol, pro - angiogenic properties . It was shown that in the presence of heparin, beta - sitosterol stimulated neovascularization in the mouse matrigen plug assay, and the motility of human umbilical vein endothelial cells in an in vitro wound migration assay . Therefore, further detailed studies on the specific compounds contribution of the antiangiogenic effect and its mechanism are warranted . Performed a randomized study of chemotherapy versus chemotherapy plus aloe arborescens, in 240 patients with lung, colorectal, gastric and pancreatic metastatic cancers . Aloe arborescens was given orally at a dose of 10 ml thrice daily of a mixture consisting of 300 g of aloe fresh leaves in 500 g of honey plus 40 ml of 40% alcohol, every day without interruption, either during or after chemotherapy, until the progression of disease, starting 6 days prior to the onset of chemotherapy . The results of this study suggest that aloe may be successfully associated with chemotherapy to increase the tumor regression rate and survival time.
The evolution of implant dentistry has continued with significant advances in biomaterials and clinical techniques through ongoing research and development . To a large extent this evolution has reflected the change in the thinking pattern of individuals coming to dental clinics for treatment now - a - days . Implants can be placed at different timings after tooth extraction; accordingly descriptive terminologies used are immediate placement (in post extraction sockets), early placement with soft tissue healing (after 4 to 8 weeks of extraction), early placement with partial bone healing (after 12 to 16 weeks of extraction), and late placement (after 6 months or greater than 6 months after extraction). It has been proposed that implant placement in the fresh extraction socket has many advantages as this procedure can counteract the tissue modeling that occurs after the extraction of the tooth, hence preserve the dimensions of the alveolar ridge giving an esthetic benefit, along with additional benefits like fewer surgical sessions and shorter treatment periods . However, animal studies done on dogs proved that implant placement in a fresh extraction socket might result in an early hard tissue fill of marginal defect, but in the later phase results of tissue remodeling and newly formed bone may in part be lost . The buccal bone wall was proved to be more susceptible to resorption than lingual due to thinness and the presence of a large amount of bundle bone . Surgical trauma inflicted along with flap elevation, root extraction and implant installation were also proposed to influence the bone remodeling . Thus, immediate implant installation apparently failed to interfere with the process of hard tissue resorption and consideration of this factor while placing an implant in fresh extraction socket was suggested . However, a human study to evaluate the dimensional alterations of hard tissues that occur after immediate placement of implant following tooth extraction noted that the marginal gaps in buccal and palatal / lingual locations were resolved through new bone formation from inside of the defects and substantial bone resorption from the outside of the ridge, but at no site was the surface of the implant devoid of bone coverage . Esthetic success of these implants was also studied and proposed to depend on ideal three dimensional implant position, adequate buccal bone over the implant surface, and tissue biotype . A retrospective review on the esthetic outcomes of immediate implant placement was reported with a high degree of predictability to integrate, thus suggesting the requirement of careful case selection and high surgical skills for achieving a good esthetic outcome . Accordingly, recommendations were made to place implant shoulder 1 to 2 mm lingual to the emergence of the adjacent tooth to ensure maintenance of an adequate width of buccal bone as well as stable mucosa over the buccal implant surface, proper apico - coronal position or depth of implant placement and selecting thick tissue biotype to minimize resorption and maximize esthetics . It has been postulated that the location of implant placement, thickness of buccal bone crest and the size of the horizontal buccal gap significantly influence the ridge alterations following immediate implant placement and only if situation demands augmentation procedures are needed to achieve adequate bony contours around the implant . There is also development toward new and innovative implant designs with surface treatments and improved abutment - implant connections for decreasing the crestal bone loss . Keeping in mind these factors, the clinician must make very important decisions, as to recommend the best time to place an implant and also the appropriate biomaterials to be used . The need for functional and esthetic outcomes as well as desire for reduced treatment times between tooth extraction and implant placement must be weighed carefully against the pretreatment conditions of the site, dimensional changes that will occur following extraction, the predictability of the planned treatment approach and the related risk of complications . Another concept reported in literature that is proved to give an esthetic benefit is platform switching, which is designated to the usage of smaller diameter abutments to larger diameter implants . A study pointed out that platform switching concept might compromise the emergence profile especially in anterior cases . However, subsequent biomechanical and histological studies, case reports and randomized clinical trials reported on this concept have proved that the marginal bone loss around platform switched implants was significantly less than around platform matched implants . The esthetic advantage is ascribed for two reasons: first one being the increased surface area to which the soft tissue could attach and the decrease in crestal resorption needed to establish a biologic width; second one being the increased distance between the implant abutment junction and the adjacent bone, which in turn limits the resorptive effect of the implant - abutment junction associated inflammatory cell infiltrate . The following are some cases where in immediate placement of implant after tooth extraction was done with applied platform switching concept . A 30-year - old male patient reported to the clinic with a traumatic injury to the upper jaw . He was diagnosed with complicated crown - root fracture of maxillary right lateral incisor [figure 1a] and subluxation of maxillary right and left central incisors . The lateral incisor had worst prognosis and there was no provision to plan for a post core because of the difficulty to get a ferrule, hence extraction and prosthetic replacement were the treatment of choice . He was informed about the possibilities of removable denture, fixed prosthesis, and implant therapy . As patient was interested in implant therapy, and as we felt him to be an ideal patient, we planned for immediate placement of implant after extraction of the tooth . Patient received 1 g of amoxicillin / clavulanate 1 h before surgery and continued with 2 g per day for 5 days . Under local anesthesia, a platform switched (0.6 mm), sand blasted, large grit, acid etched surface treated implant of length 11 mm and diameter 3.5 mm (ankylos implant design, friadent gmbh, mannheim, germany) was placed in the post extraction socket with the platform of the implant positioned 2 mm below the alveolar crest level . [figures 1b and 2a] after a 2 month healing period, definitive prosthesis for the lateral incisor was planned with a zirconium ceramic abutment and a zirconium crown . [figures 2b and 3a] in the recall visits as there was discoloration of right and left central incisors and as both the teeth did not respond for vitality tests, they were planned for endodontic therapy . Radiographs taken immediately after placement of implant, in 3 months, 1 year, and 2 year follow up are represented in figure 4 and clinical picture in the 2 year follow up in figure 3b . (a) pre - operative photograph showing fractured maxillary right lateral incisor (b) photograph showing immediate implant placement in the extraction socket (a) gingival former in place (b) zirconium abutment in place (a) permanent crown in place (b) photograph showing soft tissue level 2 years after loading (a) radiograph immediately after implant placement (b) radiograph taken 3 months after loading (c) radiograph taken 1 year after loading (d) radiograph taken 2 years after loading a 23-year - old female patient reported to the clinic with a chief complaint of over retained primary canine in the right mandibular region . The primary tooth was extracted and a platform switched implant of length 11 mm and 3.5 mm diameter (ankylos implant design) was placed in the post extraction socket . Radiographs taken pre - operatively, immediately, and 3 months after placement of implant and 1 year after loading are represented in figure 5 . (a) pre - operative orthopantomograph (b) radiograph immediately after implant placement in extraction socket (c) radiograph taken 3 months after placement (d) radiograph taken 1 year after loading a 35-year - old female patient reported to the clinic with a chief complaint of fractured root canal treated maxillary left lateral incisor which was extracted and followed with the placement of a platform switched implant of length 11 mm and 3.5 mm diameter (ankylos implant design) in the post extraction socket . Radiographs taken immediately and 3 months after placement of implant; and 6 months and 1 year after loading are represented in figure 6 . (a) radiograph immediately after implant placement in extraction socket (b) orthopantomograph 3 months after implant placement (c) radiograph taken 6 months after loading (d) radiograph taken 1 year after loading a 28-year - old male patient reported to the clinic with a chief complaint of fractured root canal treated maxillary right central incisor which was extracted and followed with the placement of a platform switched implant of length 15 mm and 3.5 mm diameter (ankylos implant design) in the post extraction socket . Radiographs taken pre - operatively and immediately after placement of implant; and 1 year after loading are represented in figure 7 . (a) pre - operative radiograph (b) orthopantomograph immediately after implant placement in extraction socket (c) radiograph taken 1 year after loading a 30-year - old male patient reported to the clinic with a traumatic injury to the upper jaw . He was diagnosed with complicated crown - root fracture of maxillary right lateral incisor [figure 1a] and subluxation of maxillary right and left central incisors . The lateral incisor had worst prognosis and there was no provision to plan for a post core because of the difficulty to get a ferrule, hence extraction and prosthetic replacement were the treatment of choice . He was informed about the possibilities of removable denture, fixed prosthesis, and implant therapy . As patient was interested in implant therapy, and as we felt him to be an ideal patient, we planned for immediate placement of implant after extraction of the tooth . Patient received 1 g of amoxicillin / clavulanate 1 h before surgery and continued with 2 g per day for 5 days . Under local anesthesia, a platform switched (0.6 mm), sand blasted, large grit, acid etched surface treated implant of length 11 mm and diameter 3.5 mm (ankylos implant design, friadent gmbh, mannheim, germany) was placed in the post extraction socket with the platform of the implant positioned 2 mm below the alveolar crest level . [figures 1b and 2a] after a 2 month healing period, definitive prosthesis for the lateral incisor was planned with a zirconium ceramic abutment and a zirconium crown . [figures 2b and 3a] in the recall visits as there was discoloration of right and left central incisors and as both the teeth did not respond for vitality tests, they were planned for endodontic therapy . Radiographs taken immediately after placement of implant, in 3 months, 1 year, and 2 year follow up are represented in figure 4 and clinical picture in the 2 year follow up in figure 3b . (a) pre - operative photograph showing fractured maxillary right lateral incisor (b) photograph showing immediate implant placement in the extraction socket (a) gingival former in place (b) zirconium abutment in place (a) permanent crown in place (b) photograph showing soft tissue level 2 years after loading (a) radiograph immediately after implant placement (b) radiograph taken 3 months after loading (c) radiograph taken 1 year after loading (d) radiograph taken 2 years after loading a 23-year - old female patient reported to the clinic with a chief complaint of over retained primary canine in the right mandibular region . The primary tooth was extracted and a platform switched implant of length 11 mm and 3.5 mm diameter (ankylos implant design) was placed in the post extraction socket . Radiographs taken pre - operatively, immediately, and 3 months after placement of implant and 1 year after loading are represented in figure 5 . (a) pre - operative orthopantomograph (b) radiograph immediately after implant placement in extraction socket (c) radiograph taken 3 months after placement (d) radiograph taken 1 year after loading a 35-year - old female patient reported to the clinic with a chief complaint of fractured root canal treated maxillary left lateral incisor which was extracted and followed with the placement of a platform switched implant of length 11 mm and 3.5 mm diameter (ankylos implant design) in the post extraction socket . Radiographs taken immediately and 3 months after placement of implant; and 6 months and 1 year after loading are represented in figure 6 . (a) radiograph immediately after implant placement in extraction socket (b) orthopantomograph 3 months after implant placement (c) radiograph taken 6 months after loading (d) radiograph taken 1 year after loading a 28-year - old male patient reported to the clinic with a chief complaint of fractured root canal treated maxillary right central incisor which was extracted and followed with the placement of a platform switched implant of length 15 mm and 3.5 mm diameter (ankylos implant design) in the post extraction socket . Radiographs taken pre - operatively and immediately after placement of implant; and 1 year after loading are represented in figure 7 . (a) pre - operative radiograph (b) orthopantomograph immediately after implant placement in extraction socket (c) radiograph taken 1 year after loading the success rate of the single implant therapy depends not only on restoring the function, but also on the level of integration that we provide for the restoration so that it harmoniously sinks into the patient's overall appearance . In clinician's view, success of implant restoration depends on the change in the peri - implant bone level and the surrounding soft tissue; and for the patient it is the esthetics and his / her level of satisfaction . The patient satisfaction has more importance when we have the restoration located in the esthetic zone . However, the esthetics associated with the final implant restoration is greatly affected by both the soft and hard tissue changes . Hence, if clinician's criteria are met, indirectly they will fulfill the patient's criteria . Thus, if we can take care of the shrinkage of the adjacent interdental papillae and the loss of the scalloped tissue contour around the implant restoration and minimize the crestal bone loss; the anterior implant restoration will have good esthetics . Crestal bone remodeling and resorption during the first year following immediate implant placement leads to compromise in the treatment outcome that are reported in literature, and ways to overcome this also described . In the cases presented we could appreciate a good esthetic outcome, the success of which might be ascribed to many reasons [table 1]. Neither periapical infection nor periodontal is responsible for extracting the tooth, which might be the first and foremost reason . The surgical trauma was also minimal as there was careful extraction of the tooth and no flap elevation procedure . Sand blasted, large grit, acid etched implant with a diameter 2 mm less than the diameter of extraction socket and with a conical abutment - implant connection (morse taper) was selected . The implant placement factors that might have led to success were palatal / lingual placement of shoulder, almost 2 mm gap between the labial plate and the implant, 2 mm below the crest of the socket and the implant was not immediately loaded . The implant abutment factors were conical connection that lacks a microgap between abutment and implant, and usage of zirconium ceramic abutment which is proved to be biocompatible, both not favoring the accumulation of bacteria when compared to external / internal hex and titanium abutments, respectively . Incorporation of platform switching concept with a circumferential horizontal mismatch of 0.6 mm added to the clinical success as it was proved to have minimal ill effects on the biomechanical environment of implants . The factors to be considered while placing an implant in immediate extraction socket thus, by integrating immediate placement with platform switching concept and careful case selection, we could achieve a very good esthetic overcome . Platform switching is not the only way to control circumferential bone loss around dental implants, but it is one of the ways to get a better results . Diagnosing and treatment planning are very critical when the timing of the implant placement is concerned . When we integrate everything we can get a very good esthetic outcome as presented, thus increasing the quality of life of patients.
Anterior cruciate ligament (acl) has a primary role in the limitation of anterior translation of the tibia as regards to the femur . They are also important for the compensation of stability in the acl in the knee lesions and patients with acl . The hamstring muscle weakness and hamstrings weak relationship with the quadriceps are risk factors for acl injuries . One of the most commonly used treatments is the method in which knee flexor tendons are used as autograft . As shown in previous studies, after using this method reconstructed knee recovered up to the 90% of flexion muscle strength of counter knee (harter et al ., 1990; lipscomb et al ., 1982; simonian et al ., 1997). It is also shown in previous studies that semitendinosus which is also used as acl graft has regeneration potential from more proximal and with similar morphology (eriksson et al ., 2001; ferretti et al . Although there is a development of regeneration close to normal morphology; knee flexors abnormalities in neurological function can be a barrier to motor units function by preventing their healing . However, morphological factors such as muscle atrophy in these patients is not the only factor that determines the maximum power; in previous studies, it is shown that despite the absence of morphological abnormalities the afferent feedback deficiency from acl may interfere with the activation of gamma motor unit of the muscles around the knee (konishi et al . Even if the knee flexor tendon regeneration is morphologically normal and that motor unit complex may prevent the function of the knee flexors by causing neurologic abnormalities . Therefore, while comparing knee flexor muscle strength of patients who underwent acl reconstruction with normal uninjured knee, muscle weakness due to the possibility of neurologic abnormalities cannot be ignored . After acl reconstruction, which muscle groups are more affected from frequently developing thigh muscle atrophy is a matter of debate . Muscle strength tests are used to evaluate recovery of acl after treatment and the effectiveness of treatment . According to the study carried out, after acl reconstruction using hamstring tendon grafts, weakness in the knee flexor muscle strength can be observed up to 24 months . In addition, previous studies showed that in patients who underwent acl reconstruction quadriceps muscle torque per unit volume is significantly lower compared the intact knee (konishi et al ., 2007b). In our study, we aimed to evaluate the effect of thigh circumference difference between patients knees who were administered the acl reconstruction with hamstring tendon autograft and intact knees, on torque between the hamstring and quadriceps muscles . As is known, development of muscle weakness in quadriceps after acl lesions caused by neurological dysfunction was shown to be a natural consequence . In our study, we planned to evaluate the effect of thigh circumference difference between operated extremity and intact extremity on knee flexorsmuscle strength . Fifty - five patients (54 males, 1 female, mean age 28.156.47) who underwent acl reconstruction in our clinic and with at least 6 months follow - up period available were included in our study . While choosing the patients, not having any disease nor interventions previously on intact knees has taken into account . Patients who have symptoms and signs such as discharge, inflammation, instability, locking, limitation of motion, anxiety while exercising were removed from the study . All operations were applied in teaching and research hospital by the same surgeon . The condition that autogenous hamstring tendon graft usage in acl reconstruction was searched for the patients . In all patients, same postoperative rehabilitation program which is indicated below for the first week walking with a full load, 090 of passive knee extension, bringing the active flexion, quadriceps and hamstring muscle training, heel shift, straight leg rise; between 12 weeks, hamstring training in the prone position, asideleg lift, walkingin the water if a swimming pool exist; between 23 weeks, terminal extension and hamstring stretch studies with weight; 34 weeks, if knee flexion has reached to 90 the pedals ergonometric work, walking back in the pool (if possible), doing full daily activities; 612 weeks, rising at finger tips and starting to closed kinetic chain exercises, preparations for the transition to the sport aiming to increase strength, durability and propsiosepsiyo; in the 3rd month, cycling, running and scissoring in water; in the 4th month, in addition to weight training activities beginning to proprioceptive and on the stairs activities, doing straight running; in 68 months beginning to sport - specific movements, ensuring to return to contact sports were taken into account . Each patient was laid in the supine position, knees at full extension and relaxed position prior to measurement of the circumference of the thigh muscles . Both thigh circumference were measured and recorded from 15 cm proximal to the upper limit of the patella for measuring . The determined length difference between there constructed knee and the intact knee power measurements of quadriceps and hamstring muscle groups in patients extremities who underwent operation and who did not in were done by using cybex ii dynamometer (humac). During the measurements the maximum torque (peak torque) values at 60/sec, 240/sec infrequency, the application of standard equipment, data collection and heating procedures were performed before the measurement . Patients were told to continue their usual daily activities not to do tiring activities in the day before the test . On the test day before the beginning of measurements patients patients pelvis were stabilized with the help of a belt, thighs were supported with pillows, ankle cuff was placed directly on top of malleolar . Patients forearms were positioned so that the rotational axis of the forearms were aligned with rotational axis of knees . During the test the range of motion was adjusted to be 0 extension and 90 flexion . In order to get support patients were allowed to hold seats on the sidebar during the test . Before starting the recording of the data patients did three times sample repetition at both angle rates . Concentric exercise were carried out for 5 times in maximal flexion and extension to patients after one minute rest . First, 60/sec speed after a minute rest the test was continued with 240/sec speed . Several patients developed thigh pain during measurements and measurements were repeated after the test was terminated . Number cruncher statistical system (ncss) 2007 and power analysis and sample size (pass) 2008 statistical software (utah, usa) were used for statistical analysis . Besides descriptive statistical methods (mean, standard deviation, median, frequency, rate, minimum, maximum) for qualitative comparison of data pearson s chi - square test, fisher - freeman - halton test, fisher s exact test, and yates continuity correction test (yates adjusted chi - square) were used for the evaluation of data . Age identifier values and follow - up periods of patients are shown in table 1 . Thigh circumference of intact extremity and operated extremity of patients are shown in table 2 . Peak torque of the mean extensor and flexor muscle strength and percentage values relative to each other, are shown in tables 3, 4, and 5 . Statistically significant relationship was observed at the level of 66.0% percent between the thigh diameter difference and cybex extension 60 of patients in negative direction (while the thigh diameter difference increasing the cybex extension 60 percent decreasing) (r: 0,660; p=0.001; p<0.01). Statistically significant relationship was observed at the level of 55.0% percent between the thigh diameter difference and cybex extension 240 of patients in negative direction (while the thigh diameter difference increasing the cybex extension 240 percent decreasing) (r: 0,550; p=0.005; p<0.01) (fig . Statistically significant relationship was observed at the level of 55.0% percent between the thigh diameter difference and cybex flexion 60 of patients in negative direction (while the thigh diameter difference increasing the cybex flexion 60 percent decreasing) (r: 0,555; p=0.002; p<0.01). It was observed a relation between the thigh diameter difference and cybex flexion 240 of patients in negative direction (while the thigh diameter difference increasing the cybex flexion 240 percent decreasing) at the level of 28.1% percent and this situation was found to be statistically significant (r: 0,281; p=0.079; p>0.05) (fig . After our study, in accordance with our findings it is still possible to encounter the thigh atrophy in average 28 months after acl reconstruction surgery even under physical rehabilitation programs and appropriate follow - up . In line with earlier studies, quadriceps muscle mass is often seen as responsible for the present thigh atrophy, however negative effects of the hamstring muscle group on muscle atrophy which is determined with manual thigh circumference measurements are undeniable . As shown in previous studies, in patients with acl lesions the development of neurological dysfunction is not available in the knee flexors comparing with quadriceps muscle group . Comparative studies which was done in patients with acl lesion and in patients without any knee problems showed that there was no significant difference between the speed of flexion and torque per unit volume of the knee flexor muscle . Despite this, in the same study it was found that isokinetic torque that occurred in the knee with acl lesions at 60/sec was less than robust knee (konishi et al ., 2012). In addition, it is shown that the acl reconstruction with hamstring tendon is not effective on muscle torque power of knee flexors and flexion speed of these muscles by showing that detection of changes in the robust knee is the same . In the light of these data, it might lead to think that acl lesions does not cause neurological dysfunction in the knee flexors . In another study there was a comparison between patients who were administered to the acl reconstruction and patients who did not have any knee problem, it has been identified that muscle torque power on per unit volume of the quadriceps was significantly lower in the patients who were administered to reconstruction . In addition, quadriceps weakness is associated with the neurologic dysfunction that is developed after acl reconstruction has been shown by other researchers (hart et al ., 2010; rice et al ., 2009; snyder - mackler et al . It has been shown that muscle weakness in the quadriceps that is induced by the acl lesions is a natural result linked to the development of neurological dysfunction . In our study we have showed the loss of torque power of thigh atrophy on quadriceps at the 60/sec, 240/sec frequency even in the patients who have an appropriate rehabilitation program after acl reconstruction and under going follow - up process . Although, it has been shown in previous studies that acl lesion development in the thigh did not lead to neurological dysfunction on hamstring; in our measurements, it has been determined statistically that atrophy developed in the thigh is effective on the hamstrings muscle torque power at least as it is on quadriceps, especially 60/sec frequency . It could not be shown that this weakness is correlated to thigh atrophy as well as quadriceps at 240/sec flexion frequency . These difficulties are likely to be related to neurological dysfunction occurred in the quadriceps muscles . Due to different muscle weakness mechanisms which are developing according to the morphological structure and muscle properties, it is inevitable for the clinician to consider these changes in diagnosis and rehabilitation stages . The point that we want to emphasize in our study is; it ca nt be ignored that muscle weakness mechanisms developing in the thigh circumference vary according to the thigh muscle group and knee flexors play an important role in thigh atrophy when determining an appropriate rehabilitation program after anterior cruciate ligament reconstruction application.
Every year, it is estimated that tens of thousands of pregnant women in malaria - endemic areas are infected with plasmodium falciparum . Frequently, placental infection occurs, owing to the accumulation of p. falciparum - infected erythrocytes in the intervillous space, despite the absence of parasites in peripheral blood . The complications of malaria during pregnancy are maternal anaemia, preterm delivery, and low birth weight of newborns, which increase perinatal morbidity [1, 3, 4]. The world health organization (who) recommends intermittent preventive treatment with sulfadoxine - pyrimethamine (iptsp) during pregnancy, with at least two doses after quickening (1820 weeks) not more frequently than monthly, use of insecticide - treated bed nets (itns) and prompt treatment of clinical malaria . Intermittent preventive treatment consists of delivering a curative treatment dose of an antimalarial at predefined intervals, regardless of the parasitological status of the woman, and the efficacy of this protocol has been demonstrated in a number of malaria - endemic countries [58]. Placental plasmodium screening in the central african republic (car) in 1990 showed a rate of 37.1% in women who had been given chemoprophylaxis with chloroquine . In 2006, the ministry of health of the car adopted and implemented the new who recommendations for malaria prevention during pregnancy . The aim of the study reported here was to estimate the prevalence of malaria in thick peripheral blood smears and placental blood from a sample of women who gave birth during september 2009 at two main maternities of bangui, the capital of car . Secondly, we assessed the women's coverage rates with the three components of the who package for malaria management during the current pregnancy and identify pregnant women characteristics associated with iptsp and itns . We conducted a cross - sectional study in the two main maternity clinics of bangui, the castors health centre and the communautaire hospital, in september 2009 . The climate is tropical, and rainfall peaks are observed from april to november, and temperature ranges from 19c to 32c . The main parasite is plasmodium falciparum, and malaria transmission is perennial with peaks during the rainy season, but no data on the intensity of malaria transmission (entomological inoculation rates) is available in car . Malaria represents more than 40% of morbidity in bangui, as well as in other car areas . The castors health centre and the communautaire hospital provide antenatal and delivery services and have established programmes for the prevention of malaria and other infectious diseases, such as mother - to - child transmission of hiv infection . Each year, it is estimated that 12,000 women deliver at those centres, representing 70% of all women who deliver in bangui . All women are screened for malaria by microscopic analysis of 4% giemsa - stained thick blood smears during visits to the antenatal clinics, performed either at the same health centre or at one of the national reference biomedical laboratories (the national laboratory for clinical biology and public health and the institut pasteur de bangui), depending on each woman's choice . Antimalarial treatment is prescribed on the basis of clinical symptoms if the woman cannot afford the laboratory fees immediately . For asymptomatic women, iptsp is given free of charge as directly observed therapy during the antenatal visit . Administration of two doses is recommended for hiv - negative women and three doses for hiv - positive women . All women from whom we obtained written informed consent were eligible for the study immediately after delivery . A study midwife administered a standardized questionnaire to record sociodemographic data (age, residence area, literacy, number of gravidities, and monthly income), hiv serological status, intake of antimalarial medications (iptsp and other antimalarial prescriptions), and bed net use . Malaria prevalence of 30% at the time of childbirth was used as a proxy to calculate the sample size in this study . Thus, a number of 323 women was necessary assuming 90% power at 5% significance level . Because of lack of an ethical committee in the car, this project was reviewed and approved by an ad hoc scientific committee of the university of bangui in charge of validating scientific study protocols in the car . The study was conducted under a collaborative agreement with the university of marseille, france . Peripheral venous and placental blood from each woman was used to prepare thick blood films . Placental blood was obtained as follows: immediately after delivery, the paracentric side of the maternal placenta was cleaned with sterile water and incised, and thick blood films were prepared from a droplet collected by aspiration through a 21-gauge needle attached to a 5-ml syringe, as described previously [12, 13]. The thick smears were air - dried and stained with 4% giemsa . At each of the two study sites, an experienced microscopist immediately examined the stained smears by light microscopy (100 oil immersion) to detect asexual forms of p. falciparum malaria parasites . On peripheral blood slides, malaria parasites were counted in 200 leukocytes, and the parasite density per microlitre of blood was estimated as the number of parasites counted multiplied by 40 under the assumption of a leukocyte count of 8000/l of blood . For both types of blood film, a result was considered to be negative if no parasites were detected per 200 leukocytes . Women with a positive peripheral blood result were given antimalarial treatment (either artemether - lumefantrine or quinine, depending on a clinical evaluation and individual tolerance). All the slides were analysed twice at the biomedical laboratory of the institut pasteur de bangui . Data were double - entered into epiinfo software version 3.5.1, and the database was checked and data entry errors corrected with the epiinfo software the association between sociodemographic criteria and malaria, use of iptsp and itns was examined using the chi - squared test, and association between those variables was tested by calculating the odds ratios (ors). Overall, 328 pregnant women delivering at the two health centres were included in the study: 168 (51.2%) at the castors and 160 (48.8%) at the communautaire hospital . The mean age was 23 years (range, 1439 years), and 33.8% were aged less than twenty years . Most of those women do not have any personal monthly income (57.9%), but the majority of them have at least secondary educational status . Hiv infection had been screened for 58.2% (191/328) of the population, resulting an infection prevalence of 9.1% (15/164; 27 hiv results could not be cross - checked on the antenatal clinic cards). Sleeping daily under bed net was reported by 81.5% (95% ci, [77.385.7]) of the women, and 42.4% (95% ci, [36.848.0]) had itns . At the two study sites, checking of antenatal clinic cards showed that 93.3% (95% ci, [90.895.8]) of the women had presented at least one antenatal visit . Less than one fourth of the women (24.1%; 95% ci, [19.328.9]) had attended an antenatal clinic during the first trimester of pregnancy, while the majority (55.6%; 95% ci, [50.061.2]) had attended a clinic during the second trimester . Overall, 35.6% (95% ci, [31.140.1]) completed four antenatal visits . The distribution of first antenatal attendance according to gestational age at each study site is shown in figure 1 . Of the women who received iptsp, 75.4% (95% ci, [69.181.8]) were given curative prescriptions of other antimalarial drugs, independently of the timing of iptsp doses (figure 2). The antenatal clinic cards of 182 women (55.5%; 95% ci, [50.160.1]) showed a history of at least one curative treatment for malaria . Of these women, 27.0% had been prescribed an antimalarial drug two or three times . Although 228 antimalarial prescriptions were recorded on antenatal clinic cards during the current pregnancy, only 56 laboratory results were positive out of the total 73 blood smears analysed . The antimalarial drugs prescribed were quinine (66.7% or 152/228; 95% ci, [60.672.8]), artemisinin - based combinations (15.5%; 95% ci, [10.720.1]) and artemisinin monotherapy (18.0%; 95% ci, [13.023.0]). At least one dose of iptsp was recorded for 54.6% (95% ci, [49.260.0]) of our study population; only 30.5% (95% ci, [23.837.2]) had received at least two doses . Most of these women (78.2%; 95% ci, [72.284.3]) had received the first iptsp dose between the fourth and seventh months of pregnancy; however, 11.7% (95% ci, [7.016.4]) had received the first dose during the first trimester . Multigravid women, were less likely to use two doses of iptsp (or = 0.14; 95% ci, [0.080.24], p <0.0001) and itns (or = 0.16; 95% ci, [0.100.28], p <0.001) compared to primigravid women . Use of iptsp (two doses) was associated with lucrative activities (or = 4.20; [2.556.92], p <0.0001) and secondary or university educational status (or = 2.22; 95% ci, [1.333.72], p = 0.002). Women with secondary or university educational status were also likely to use ints (or = 1.90; 95% ci, [1.203.01], p = 0.01). Details on association analysis between sociodemographic characteristics and those preventive tools use are shown in table 1 . Overall, peripheral blood p. falciparum infection at delivery was found in 2.8% (95% ci, [1.04.6]) of peripheral blood and in 4.0% (95% ci, [2.06.0]) of placental blood . Of the women with placental malaria, 77.0% (10/13) declared not using any bed net and 53.8% (7/13) had not taken any antimalarial drug during pregnancy . Iptsp and itns use was found not to be associated with the women sociodemographical characteristics . Moreover, there is no statistically significant association between these laboratory findings and those characteristics . The prevalence of malaria among pregnant women in the car (2.8% of thick peripheral blood smears and 4.0% of placental slides) was lower than in other areas of intense malaria transmission, such as gabon, where the rates were 34.4% in maternal blood and 53.6% in placental blood films . A recent review of randomized clinical trials and surveys on the efficacy of ipt showed overall placenta - positive rates 10% . Falade and coauthors in nigeria reported that the prevalence of placental parasitaemia was 10.5% in women given iptsp and 17% in those with no chemoprophylaxis . Three years after initiation of iptsp in car, coverage with at least one dose of iptsp was slightly more than 50% . Although who recommends two doses iptsp for 80% of pregnant women, our estimate in this study was 30.5% . The low prevalence of placental malaria in our study is therefore probably due to the combination of iptsp, other antimalarial drug, and use of itns . Hence, the relatively low prevalence of malaria at delivery is not surprising in bangui . Indeed, a similar finding of malaria prevalence at the time of delivery is observed in cte d'ivoire, where vanga - bosson and coauthors report a prevalence of 4.8% of placental malaria, in an area where iptsp coverage rate (2 doses) does not exceed 50% and, in thailand, where proportion of positive results for p. falciparum in maternal blood and in placental blood were estimated at 3.0% and 3.8%, respectively . In thailand, the authors report that this relative lower prevalence was due to antimalarial treatment with artemisinin derivatives . In our study, otherwise, our findings show that more than 80% of the women slept under a bed net . Indeed, use of itns was found to reduce the incidence of uncomplicated malarial episodes in areas of stable malaria by 50% in comparison with no nets use and by 39% in comparison with untreated nets use . In our study, the lower proportion of use of iptsp and itns in multigravid women could be due to less health conscious of those women of their pregnancy . Inversely, the relative high proportion of use of iptsp and itns in women with high level of education is due to the fact that higher educational status implies more health consciousness and is a factor influencing assimilation of health education programmes . Women with salary or other personal income are also likely to be compliant with iptsp and itns, possibly because they are able to afford health care fees . The main limitation of our study is the only use of microscopic examination of blood smears . Even if, the microscopic analysis remains the standard detection of plasmodium, submicroscopic infections are common during pregnancy [21, 22]. Hence, molecular methods (polymerase chain reaction or pcr) and rapid diagnostic tests (rdts) provide finding approximately twice as many infections as microscopy [2325]. However, determination of the possible impact of these submicroscopic infections to poor birth outcomes and maternal health is critical, and pcr is not feasible routinely . To this end, microscopic examination to detect these infections is still essential, and implementation of rdts use is challenging [21, 28]. Our results indicate that, although the recommended coverage rates of pregnant women with iptsp and itns are not reached in bangui, the prevalence of the main indicator of infection, placental malaria, is relatively low . The widespread presumptive prescription and consumption of antimalarial agents could indisputably be the cause of clearance of the existing peripheral and placental plasmodium infection and decreased the risk of new infections over the pregnancy period . Indeed, symptoms suggestive of malaria are very frequent among pregnant women attending antenatal clinics, thus implying frequently unnecessary large use of antimalarial drugs . For this purpose, strengthening national malaria control activities, taking into account prompt laboratory diagnosis and sociodemographic particularities, should contribute to the achievement of high coverage rate with the who preventive package components . Otherwise, cohort studies are needed to assess the real efficacy of iptsp in preventing malaria during pregnancy . Blood samples analysis were achieved by a. manirakiza, m. moyen, d. djalle, n. madji and r. laganier . Data analysis and interpretation were achieved by a. manirakiza, j.delmont, e. serdouma, g. soula, and alain le faou.
The ability to balance both efficacy and safety is critical for patient survival and quality of life . Cancer therapy is becoming increasingly specialized, having evolved from using cytotoxic drugs on dividing cells to targeting specific molecular events involved in oncogenic proliferation . Among these targeted therapeutics tyrosine kinases are enzymes that catalyze phosphorylation of target proteins, signaling cellular processes such as growth and proliferation . Unregulated tyrosine kinase activation, therefore, can cause uncontrolled cellular proliferation, leading to cancer . Although complete inhibition of tyrosine kinases may disrupt vital cellular signaling, targeted tkis may prevent cancerous proliferation while sparing essential kinase activity [1, 2]. Linifanib is a novel receptor tki with specificity for the vascular endothelial growth factor (vegf) and platelet - derived growth factor (pdgf) receptors . It does not possess significant activity against cytosolic tyrosine kinases or serine / threonine kinases . As tumor progression can rely on both vegf and pdgf signaling, a selective inhibitor could result in high antitumor activity without interrupting other kinase signaling pathways . In clinical trials, linifanib has demonstrated anti - tumor activity in advanced solid tumors including non - small cell lung cancer (nsclc), renal cell cancer, hepatocellular cancer, colorectal cancer, and breast cancer [511]. In a double - blind, randomized phase 2 trial, the addition of linifanib to carboplatin and paclitaxel resulted in significant improvement in response rates and progression - free survival in patients with advanced nsclc . A number of drugs have been developed to target specific tyrosine kinases known to be active in certain cancers . Some tyrosine kinases are essential for cardiac function, however . As a result, a side effect of tki treatment has been development of cardiac events, such as a delay in cardiac repolarization [2, 12, 13]. Prolongation of the qt interval (duration of ventricular depolarization and subsequent repolarization) may increase the risk of torsade de pointes or other ventricular tachyarrhythmias . Although a number of tkis have been associated with qt prolongation, the majority of tkis do not lead to appreciable qt prolongation at clinical doses [1417]. Many of these studies, however, have not been conducted in oncology patients, who may be at a greater risk due to concurrent or previous therapies . It was therefore the objective of this high - precision qt study to investigate the effect of linifanib on qt prolongation in patients with advanced solid tumors . A phase 1, single dose, open - label, randomized study in subjects with advanced solid tumors was conducted on 24 patients . This study was performed in accordance with the ethical standards laid down in the declaration of helsinki . Eligibility included age> 18 years, ecog performance status scores of 01, and adequate organ function . For the assessment of ecgs, patients were randomly assigned to 2 sequences of regimens of linifanib at the maximum tolerated dose, 0.25 mg / kg, without exceeding 17.5 mg, administered orally in a two - period (day 1 and day 7) crossover fashion . A single 12-lead resting ecg was obtained within the week before day 1, or on day 1, and at study completion, or upon subject discontinuation . Triplicate ecgs were obtained serially on day-1 at the anticipated time points for subsequent dosing and before and after dosing on day 1 and day 7 (crossover period 1 and period 2, respectively). The time points for measurements were pre - dose and 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h post - dose . Pharmacokinetic plasma samples were also collected for 72 h on day 1 and day 7 . Qt, rr, pr, and qrs intervals were measured for each ecg using abbvie s validated pc - based algorithm (abbios), with standardized manual over - reading of all ecgs by trained technicians and t u morphology assessment by cardiologists . Qtc was determined using fridericia s correction method (qtcf):\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{qt}}_{f} = \frac{\text{qt}}{{\sqrt{\text{rr}}}}$$\end{document}values for the triplicate ecgs were averaged to obtain a single - interval measurement for each time point . A linear mixed - effects model was used for the analysis of the day 1 and day 7 data to evaluate the effect of linifanib on cardiac repolarization . The analysis was performed for time - matched baseline - adjusted qtcf intervals (qtcf). For assessment of the effect of linifanib, the primary endpoint was the largest time - matched difference for qtcf between drug regimens and baseline (qtcf). An intersection union test was performed at a significance level of 0.05 within the framework of the corresponding mixed - effects model . Linifanib was considered to have a negative effect on cardiac repolarization if at all time points of the ecg measurements, the mean qtcf for linifanib, did not exceed the baseline mean by 10 ms or more with statistical significance level of 0.05 . Therefore, the maximum 95% upper confidence bound for the baseline - adjusted qtcf (qtcf) must be less than 10 ms in order to demonstrate a negative qtc effect . The intersection union test required high operational and statistical precision of the data to meet the criteria for negative qt effect, since the confidence intervals would be narrower with tighter variability . Additionally, the relationship between baseline - adjusted qtcf and plasma drug concentration was explored using an exposure response analysis . The equation for the response variable qtcf (y) is:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$y = \, \mu \, + \alpha\,^ {} \,{\text{baseqtcf}} + {\text{sequence}} + {\text{hour}} + {\text{day}} + \, \beta\,^{}\, {\text{concentration}} + \, \eta_{i} + \varepsilon_{ijk}$$\end{document} the model has terms for the baseline measurement (baseqtcf), sequence (sequence), day of measurement (day), and time of measurement (hour). The random components of this model are denoted by i and ijk, with i identifying the ith subject, j identifying the day, and k identifying the time of the measurement within a day . Within the frame work of this model, the 95% upper confidence bound for the effect of the mean cmax of the linifanib dose on the qtcf was provided . If the bound is less than 10 ms, the regimen does not have a clinically relevant effect on cardiac repolarization . No subject had qtcf values greater than 500 ms, and no subject had a change greater than 60 ms from baseline . One subject had an asymptomatic qtcf change of greater than 30 ms from baseline . Among the study population, after patients received linifanib, the qtcf for the fasting regimen ranged from 4.14 to 0.64 ms, whereas the non - fasting regimen ranged from 6.03 to 1.57 ms (table 1). The maximum 95% upper confidence bound for the drug effects for linifanib was 4.30 ms . These results are below the threshold of regulatory concern as indicated in ich e14 guidance for industry . It was therefore concluded that linifanib had no effect on cardiac repolarization.table 1intersection union test results for linifanib on qtcfregimenstime point (h)qtcf meanpoint estimate95% upper confidence bounddrugbaselinelinifanib fasting regimen0.5421.8423.92.231.431422.0423.11.242.432422.1421.40.574.243422.6421.90.644.304418.1422.41.921.786415.8419.94.140.478417.5420.22.820.8510419.6420.40.583.1212420.1420.20.163.5024422.1423.51.532.14linifanib non - fasting regimen0.5419.8423.94.150.481418.3423.14.891.232417.1421.44.330.673416.0421.96.032.364413.8422.43.820.126416.9419.93.050.618416.4420.23.870.2010417.2419.41.831.9012417.3418.91.781.9224421.0422.41.572.14 qtcf interval change form baseline (qtcf) of the least squares means (msec) intersection union test results for linifanib on qtcf qtcf interval change form baseline (qtcf) of the least squares means (msec) analysis was also performed with linifanib concentration as the drug exposure variable . The mixed - effects model showed a linear relationship between changes in qtcf interval and linifanib concentration (fig . 1). The model estimated a slope of 0.01048 with a standard error of 0.006537 (p = 0.1094). This predicted a trend toward a change in qtcf interval of 3.56 ms at a concentration of 0.34 g / ml (the cmax at the maximum tolerated dose) and a 95% upper confidence bound of 7.2 ms . In addition to supporting the finding that linifanib does not significantly affect qt interval, this model may provide useful predictions about the impact of other dosing regimens on qt prolongation.fig . 1linifanib concentration versus qtcf change from baseline (qtcf) linifanib concentration versus qtcf change from baseline (qtcf) a morphological evaluation was performed for t and u waves at each ecg data collection time point . No clinically significant morphological changes in ecg, including no abnormal u waves, were observed following linifanib treatment . Isolated non - specific t wave abnormalities were seen and expected in patients who may have previously undergone cytotoxic cancer treatments . Among the study population, baseline qtcf values ranged from 360.9 to 468.6 ms . After patients received linifanib, the qtcf for the fasting regimen ranged from 4.14 to 0.64 ms, whereas the non - fasting regimen ranged from 6.03 to 1.57 ms (table 1). The maximum 95% upper confidence bound for the drug effects for linifanib was 4.30 ms . These results are below the threshold of regulatory concern as indicated in ich e14 guidance for industry . It was therefore concluded that linifanib had no effect on cardiac repolarization.table 1intersection union test results for linifanib on qtcfregimenstime point (h)qtcf meanpoint estimate95% upper confidence bounddrugbaselinelinifanib fasting regimen0.5421.8423.92.231.431422.0423.11.242.432422.1421.40.574.243422.6421.90.644.304418.1422.41.921.786415.8419.94.140.478417.5420.22.820.8510419.6420.40.583.1212420.1420.20.163.5024422.1423.51.532.14linifanib non - fasting regimen0.5419.8423.94.150.481418.3423.14.891.232417.1421.44.330.673416.0421.96.032.364413.8422.43.820.126416.9419.93.050.618416.4420.23.870.2010417.2419.41.831.9012417.3418.91.781.9224421.0422.41.572.14 qtcf interval change form baseline (qtcf) of the least squares means (msec) intersection union test results for linifanib on qtcf qtcf interval change form baseline (qtcf) of the least squares means (msec) the mixed - effects model showed a linear relationship between changes in qtcf interval and linifanib concentration (fig . 1). The model estimated a slope of 0.01048 with a standard error of 0.006537 (p = 0.1094). This predicted a trend toward a change in qtcf interval of 3.56 ms at a concentration of 0.34 g / ml (the cmax at the maximum tolerated dose) and a 95% upper confidence bound of 7.2 ms . In addition to supporting the finding that linifanib does not significantly affect qt interval, this model may provide useful predictions about the impact of other dosing regimens on qt prolongation.fig . 1linifanib concentration versus qtcf change from baseline (qtcf) linifanib concentration versus qtcf change from baseline (qtcf) a morphological evaluation was performed for t and u waves at each ecg data collection time point . No clinically significant morphological changes in ecg, including no abnormal u waves, were observed following linifanib treatment . Isolated non - specific t wave abnormalities were seen and expected in patients who may have previously undergone cytotoxic cancer treatments . Maintaining cardiac function in patients undergoing cancer treatments is a concern in the development of any new drug . Advancements in molecular medicine have provided a number of attractive targets in the tyrosine kinase family of growth and proliferation signaling enzymes . In some cases, however, these drugs can interfere with cardiac repolarization and may pose a risk to patients who have undergone or are currently on cancer therapy . The current study is one of a few to rigorously test the effect of an investigational drug on cardiac repolarization in patients with advanced tumors who are refractory to standard treatments . Analysis of the resulting data has concluded that linifanib does not pose a heightened risk for qtc prolongation in this refractory patient population . Despite a sample size of 24 subjects, the data had high operational and statistical precision as the 95% upper confidence bounds for mean differences from baseline were below the threshold of regulatory concern at all time points . Response modeling showed qtcf change was not significant at the maximum concentration for the maximum tolerated dose, which further supports a lack of qt prolongation with linifanib . There were also no significant t or u wave morphological changes as determined by trained investigators . A categorical analysis of subjects with an absolute qtcf value in excess of 500 ms or change in baseline for more than 3060 ms supports the absence of clinically significant effects . More broadly, at the time of this analysis, no significant adverse events related to abnormal cardiac repolarization were reported in this trial nor the concurrent phase 1, 2, and 3 clinical trials, representing an analysis of more than 700 linifanib - treated patients (data on file).
At prince of wales hospital, hong kong (7), from january 2007 through august 2008, influenza infection was diagnosed for> 460 hospitalized adult patients for whom acute febrile respiratory illnesses had been diagnosed . Nasopharyngeal aspiration and immunofluorescence assays (ifa) were used for rapid diagnosis of influenza a and b infection, confirmed by virus isolation . Thirteen (2.8%) patients had signs of confusion or altered consciousness, together with fever and respiratory symptoms (mean sd age 77.7 8.8 years). We studied 3 patients from whom csf was obtained for analysis, and who fulfilled the definition of influenza - associated acute encephalopathy (altered mental status> 24 hours within 5 days of influenza onset and without alternative explanation) (1,2,46). Nasopharyngeal aspirates were subjected to ifa, virus isolation, and subsequent subtyping (7). Csf specimens were subjected to virus isolation using mdck cells, and reverse transcription pcr to detect influenza virus rna by using h1/h3 subtype - specific primers . Herpes simplex virus, herpes zoster virus, and enterovirus dna / rna was detected using pcrs (technical appendix). Csf and plasma samples collected on the same day were analyzed simultaneously for the concentrations of 11 cytokines / chemokines by bead - based multiplex flow cytometry . Their assay methods and plasma reference ranges (established from> 100 healthy persons) have been described (technical appendix) (7). In csf, in patients without central nervous system (cns) disease / infection, cytokines / chemokines are either undetectable (e.g., interleukin-6 [il-6], cxcl8/il-8, cxcl10/ip-10, cxcl9/mig) or present at low levels (e.g., ccl2/mcp-1) (810). Concentrations of oseltamivir phosphate (op) and its biologically active metabolite oseltamivir carboxylate (oc) were measured in csf and plasma taken simultaneously from 1 patient who received concurrent treatment, using tandem mass spectrometry (11). The clinical and virologic findings are summarized in table 1 . All case - patients were elderly (7286 years of age), but none were known to have neuropsychiatric illness, dementia, or to be taking psychotropic medication . None had received updated influenza vaccination (6). Confusion and altered consciousness developed in patients 1 and 2 one to 2 days after the onset of fever and cough . These patients had no meningismus, focal neurologic deficit, hypotension, respiratory distress, or metabolic disturbances . Oseltamivir was given to patient 2 only when influenza a was later confirmed by nasopharnygeal aspirate / ifa; patient 1 did not receive antiviral treatment . Patient 3 had fever, severe chronic obstructive pulmonary disease exacerbation requiring noninvasive ventilatory support, complicated by acute coronary syndrome . He was given oseltamivir, 75 mg 2/day, after influenza a infection was confirmed . Agitation and confusion developed in the patient on day 34 of illness (onset after the third dose of oseltamivir), despite resolution of the patient s respiratory failure . These symptoms were followed by involuntary, tremulous movements involving all 4 limbs, while at rest and during movement . * copd, chronic obstructive pulmonary disease; ct, computed tomographic scan; npa, nasopharyngeal aspirate; csf, cerebrospinal fluid; rt - pcr, reverse transcription pcr; hsv, herpes simples virus; hzv, herpes zoster virus . In all cases, there was no hypoglycemia, and liver and renal function test results were normal . An electroencephalogram was performed and showed generalized slowing of background consistent with moderate encephalopathic change (similar to that observed in septic encephalopathy) (1,6). Findings are consistent with previous reports on adult cases of influenza - associated encephalopathy: patients are all unvaccinated, pleocytosis and cerebral imaging abnormalities (even with magnetic resonance imaging) are usually absent, and symptoms are generally self - limiting (1,6). Most reports have mentioned influenza a as a cause of encephalopathy, and more commonly subtype h3n2 (16). Despite apparently normal csf findings, high concentrations of cytokines / chemokines were detected in the csf and plasma specimens of all patients (table 2). Plasma concentrations of il-6, cxcl8/il-8, cxcl10/ip-10, ccl2/mcp-1, and cxcl9/mig were elevated at median values of 2.0, 2.8, 11.9, 3.7, and 2.1 the upper limits of their respective reference ranges (comparable to or higher than that observed in other hospitalized influenza patients) (table 2) (7). Il-6, cxcl8/il-8, cxcl10/ip-10, and ccl2/mcp-1 were consistently detected, and were elevated at median values of 2.6, 15.0, 3.4, and 20.0 the upper limits of their respective plasma reference ranges . The csf / plasma concentration ratios of cxcl8/il-8 and ccl2/mcp-1 were> 3 (median csf / plasma ratio 5.4 and 8.0, respectively). * csf, cerebrospinal fluid;, test not done due to inadequate sample; ud, undetectable (i.e., below the detection limit of the cytokine / chemokine assay). Cytokines: interleukin (il)1, il-6, il-10, il-12p70, tumor necrosis factor (tnf-). Chemokines: cxcl8/il-8, monokine induced by interferon- (ifn-) (cxcl9/mig), ifn-inducible protein-10 (cxcl10/ip-10), monocyte chemoattractant protein1 (ccl2/mcp-1), and regulated upon activation normal t cell expressed and secreted (ccl5/rantes). The assay sensitivities of il-1, il-6, il-10, il-12p70, tnf-, il8, mig, ip-10, mcp-1, rantes, and ifn- are 2.5, 3.3, 3.7, 1.9, 7.2, 0.2, 2.5, 2.8, 2.7, 1.0, and 7.1 pg / ml, respectively . 39 adult influenza patients hospitalized with cardio - respiratory complications (8), the median (interquartile range) plasma concentrations of il-6, il-8, ip-10, mcp-1, and mig were 10.6 (4.218.4), 5.4 (2.58.7), 7,043.0 (4,025.11,2381.1), 76.5 (49.5 - 97.0), and 992.1 (499.11,992.3) pg / ml, respectively . In csf, in subjects without neurologic disease / infection, these cytokines / chemokines are either undetectable or present at low levels (911). In a pediatrics influenza cohort, csf cytokine levels were substantially higher in encephalopathy cases when compared to those with febrile seizure; csf / plasma concentration was <1 (9). Csf / plasma cytokine concentration ratio consistently> 3 (3.512.1), in addition to csf cytokine concentrations being above the plasma reference ranges . For ifn-, il-12p70, tnf-, il-10, il-1 and rantes, because of their low / undetectable levels, the csf / plasma ratios were not calculated . Csf specimens from patients 1 and 2 were collected at the peak of symptoms, and before antiviral treatment (if given); csf from patient 3 was collected when persistent tremor developed 18 hours after the ninth dose of oseltamivir; the drug was stopped afterward . Simultaneous csf and plasma oc and op concentrations were determined for patient 3, as symptoms progressed at 18 h after oseltamivir . The concentrations (mean sd) of oc in duplicate csf and plasma samples were 18.3 0.9 ng / ml and 143.8 3.3 ng / ml, respectively; the csf / plasma concentration ratio was 12%13% . The op plasma concentration was 1.05 0.03 ng / ml; it was not detectable in the csf . We report 3 adults with acute encephalopathy (altered consciousness, confusion) associated with influenza . High csf and blood cytokine / chemokine (cxcl8/il-8, ccl2/mcp-1, il-6, cxcl10/ip-10) levels were detected . Influenza virus is rarely detected in the csf, and pleocytosis is often absent (1,2,46). High levels of cytokines (e.g., il-6, soluble tumor necrosis factor receptor 1) can be consistently found in csf / blood specimens, correlating with disease severity and outcomes (hyperactivated cytokine response is absent in febrile seizure associated with influenza) (24,8). We found a broader range of cytokines / chemokines being activated (7); for certain cytokines (cxcl8/il-8, ccl2/mcp-1), the csf concentrations were 3 those in plasma . Il-6, cxcl8/il-8, ccl2/mcp-1 and cxcl10/ip-10 have been shown to play pathogenic roles in cns viral infections, cerebral injury, and acute brain syndrome in susceptible patients (7,9,12). The high csf / plasma ratios suggest that for some cytokines, activation within the cns might have occurred along with respiratory - tract and systemic productions (cytokines are not detected in csf normally; (table 2) (4,710,12). Resident macrophages / monocytes, astrocytes, microglial and endothelial cells in the cns are shown to release cytokines / chemokines when stimulated by viral / influenza infection; activation mechanisms without involving overt cns invasion have been suggested (1,4,9,1214). Cytokines may cause direct neurotoxic effects, cerebral metabolism changes, or breakdown of the blood - brain - barrier (endothelial injury) to produce symptoms (14,8,1214). Whether early viral suppression by antivirals can lead to attenuation of these cytokine responses and better outcomes warrants further study (7). We measured oseltamivir concentrations because of the concerns over its neuropsychiatric side - effects in children and adolescents . However, only the active metabolite (oc) was detected in the csf of patient 3; the csf / plasma concentration ratio was 12%13% (18.3/143.8 ng / ml) at 18-hours postdose . This degree of csf penetration is similar to that observed among healthy patients, with a cmax csf / plasma concentration ratio of 3.5% (at 8 hours), and a ratio of 10% at 18 hours (concentration - time profiles for plasma / csf differ). Assuming a similar ratio, the csf op concentration would have fallen below the assay s detection limit (0.25 ng / ml) by 18 hours (11,15). The low csf drug - penetration, together with high cytokines in csf and symptom progression despite drug withdrawal suggest that the manifestations of patient 3 may have been disease - related . Further investigations on the cns effects of oseltamivir in the clinical setting are needed .. our study is limited by the small patient number and the lack of feasibility in obtaining csf for study / comparison in influenza patients without neurologic symptoms . Further studies on the clinical spectrum of influenza encephalopathy and encephalitis in adults (1,6) and their pathogenesis are indicated . In conclusion,
A 48-year - old man presented with acute lower extremity weakness and back pain developed while he was working in a crawl space hyper extending his back . Muscle strength was 4/5 in right iliopsoas and hamstrings, and 0/5 in the remaining muscles of right and all muscles of the left lower limb . Bilateral knee and ankle reflexes pinprick sensation was decreased over left leg up to t12 level, and right l3 through s2 dermatomes . Vibration and proprioception sensation were normal . At a peripheral hospital, spine mri was unremarkable on day 1 . Spine mri on day 2 showed a non - enhancing intra - axial bright t2-weighted signal preferentially involving the anterior part of the cord extending from the conus to t11 (figure 1a&b). On day 4, ncs showed normal sensory and motor studies except an unrecordable right peroneal motor response (chronic, post ankle fracture). F - waves were not obtainable over the peroneal and tibial nerves (figure 2a - c). Cerebrospinal fluid protein, glucose, cell count, cytology and flow cytometry were normal . Extensive metabolic, infectious, vasculitis, cardiac, malignancy and thrombophilia workup were unremarkable . He initially received intravenous methylprednisolone and plasma exchange for a presumed transverse myelitis . On day 10, mri showed smooth enhancement of the ventral nerve roots and conus (figure 1c&d). The diagnosis was revised to conus medullaris infarct based on the distribution and evolution of mri findings . . Left peroneal and bilateral tibial compound muscle action potential (cmap) amplitudes decreased . Left peroneal f waves reappeared with poor persistence (6.7%) and prolonged latency (64 ms). Although cmap amplitudes remained decreased, all f - waves were obtainable with a slightly prolonged latency and normal persistence (figure 2g - i). Spine mri axial (a) and sagittal (b) t2-weighted mri obtained 2 days after symptoms onset demonstrating t2 hyperintense signal within the conus extending to t11 . Axial t2-weighted (c) and contrast enhanced (d) mri obtained 10 days after symptom onset demonstrating contrast enhancement of the anterior part of the conus and nerve roots . F - waves (a - c) f responses recorded by stimulating the left peroneal and bilateral tibial nerves at day 4, (d - f) day 18, and (g - i) day 56 after disease onset . M - direct motor response; ml - minimal latency; mv - millivolt, nr - no response; ms - millisecond f - wave is not considered part of the workup in conus medullaris infarct . However, in the absence of upper motor neuron signs, especially with a normal early spine mri and absent f - wave, confusion about the diagnosis may arise . In a previous case report, f waves were absent in the hyperacute stage (first 4 hours) of anterior spinal cord infarct.2 in our patient, f waves were absent at the time of the first nerve conduction studies (ncs) (day 4) and they may have been absent from the onset . Hiersemenzel et al3 described reappearance of f waves after the stage of spinal shock in 12 patients with a traumatic paraplegia above t10 level, and attributed the early absence of f waves to reduced excitability of motor neurons at the stage of spinal shock . In our patient, the mechanism of injury was ischemia (rather than trauma) and the injury level was at the conus (lumbosacral spinal cord segments) with involvement of the motor nerve roots that showed enhancement on the follow - up mri . Recovery of left tibial f waves on day 18 coincided with development of hyperreflexia in the same leg . In contrast, at the time of reappearance of right tibial f waves on day 56, right ankle reflex was absent . Thus, we hypothesize that the absent f - waves in the acute stages of conus medullaris infarct might have been due to temporarily unexcitable ahc as a result of spinal shock, spinal cord edema, and possibly oligemia (figure 1). We would not expect f waves to be recordable in the setting of a diffuse infarction of all ahc in the spinal cord segments subserving the tested nerve . We are not aware of any reports describing f wave as a prognostic indicator of conus medullaris infarct . We postulate that persistently absent f waves, beyond the stage of spinal shock (2 - 4 weeks), in conus medullaris infarct indicates severe damage to ahc in the lumbosacral region and poor chance for renervation through collateral sprouting . All f waves had reappeared by day 56, at least 8 weeks before he regained walking . The area of infarct involved the ventral part of the spinal cord supplied by the anterior spinal artery . We suspect the mechanism of infarct was secondary to a hyperextension injury similar to the non - traumatic spinal cord infarct described in novice surfers (surfers myelopathy). In such cases, the postulated mechanisms of ischemia include transient arterial compression during a prolonged back hyperextension in the setting of poor collaterals, vasospasm or thrombosis of the artery of adamkiewicz, and avulsion of perforating blood vessels.4,5 the prognosis of surfers myelopathy varies from complete recovery to persistent paraparesis.4,5 a normal mri in the first few days does not rule out spinal cord infarct . In reported case series, the earliest changes (bright t2-weighted signals) appeared after 1 - 2 days, followed by enhancement appeared within the first week and peaked at 14 -21 days.6,7 cauda equina enhancement post spinal cord infarct has been described in a few case reports and attributed to a possible disruption of blood - nerve barrier.2,8,9 in conclusion, further studies are required to test our hypothesis that reappearance of tibial and peroneal f waves has a prognostic value in predicting walking in patients with conus medullaris infarct.
Primary dysmenorrhea is defined as a cyclic and painful cramps pelvic, occurring just before or during menstruation which deranges daily activities (1). Primary dysmenorrhea is one of the most common gynecologic disorders in young women which may affect more than half of menstruating women (2 - 4). Prostaglandin production by ovulation is the main cause of primary dysmenorrhea (5, 6). Digestive disorders including nausea, vomiting and diarrhea are the symptoms associated with primary dysmenorrhea, which are known due to intestinal spasms during menstruation (7). The prevalence of dysmenorrhea in different populations is 50 - 90% and in iran it is 74 - 86.1% (8, 9). Primary dysmenorrhea is a common cause of absenteeism from work, education, or referral to physician, which may lead to decreased efficacy of occupation and education . Although dysmenorrhea is not life threatening, it could have adverse effects on quality of life (10). In the usa, the annual economic loss of dysmenorrhea is 600 million working hours and two billion dollars (11, 12). In a study of 664 school students in egypt, about 75% of the students had dysmenorrhea, rated scanty in 55.3%, moderate in 30%, and severe in 14.7% (13). In a study on female students, the study suggested that 50% of girls believed that dysmenorrhea impairs daily activities (14). Several methods such as drugs (including oral contraceptive pills (ocp) consumption and nonsteroidal anti - inflammatory drugs [nsaids]), nonpharmacological treatments (including exercise, heat therapy, acupuncture, and trans - electrical nerve stimulation (tens)), dietary supplements (vitamins e, b, c, and ca, mg) and medicinal herbal have been used for treatment of primary dysmenorrhea (15, 16). Nausea, stomach irritation, ulcers, renal papillary necrosis, and decreased renal blood flow are the side effects of prostaglandin synthesis inhibitors (14). On the other hand, most of the young women have no tendency to use hormones to reduce pain . Today, regarding the effects of chemical drugs and the usage of herbal medicine, as well as alternative and complementary therapies in treatment of diseases, one of these herbal medicines and alternative therapies is cinnamon which has many applications in medicine, but has not been sufficiently documented . Despite of its high prevalence, dysmenorrhea has not been managed effectively . Therefore, due to the lack of comprehensive studies for treatment of digestive disorders accompanied with dysmenorrhea in iran and because of the importance of economic and social aspects of dysmenorrhea and acceptability and availability of traditional medicines, the aim of this study was to assess the effects of cinnamon on menstrual bleeding and systemic symptoms (nausea and vomiting) with primary dysmenorrhea in a sample of iranian female college students from ilam university of medical sciences (west of iran) during 2013 - 2014 . This was a quasi - experimental study performed at ilam university of medical sciences during 2013 - 2014 . The sample size was calculated using the information obtained from a pilot study with 10 patients and equation 1: z2 = 80% = 0.84 (test power) s = an estimate of the standard deviation of visual analogue scale (vas) in the groups; 1.67 was obtained in a pilot study . D = the minimum of the mean difference of vas between the groups which showed a significant difference and was obtained 1.1 . A simple random sampling design was used (figure 1). After getting a written permission from the school of nursing and midwifery, the researcher visited the students of dormitories and the study objectives were explained to them . Thereafter, from the interested students who had the inclusion criteria using simple random sampling of the number of the students, the residences were divided into two groups of placebo and cinnamon . In a randomized double - blind trial, 76 female students received placebo (capsules contain starch three times a day (tds), n = 38) or cinnamon (capsules containing 420 mg cinnamon, two capsules tds, n = 38) in 24 hours during the first three days of the menstrual cycle . The inclusion criteria were age 18 - 30, regular menstrual cycles, lack of chronic diseases, moderate primary dysmenorrhea, digestive disorder (nausea or vomiting) with primary dysmenorrhea, lack of pelvic inflammatory diseases, tumor or fibroma, lack of recent stressors, and bmi 19 - 26 . The exclusion criteria were the use of oral contraceptive pill (ocp), receiving analgesics during the study period, and medical or herbal allergy . The number of times of vomiting was counted and menstrual bleeding was assessed by counting the number of saturated pads . Pain intensity, nausea, vomiting and menstrual bleeding were monitored in the groups during the first 72 hours of cycle (first, second, and third days of menstruation). The pain severity was assessed in 1, 2, 3, 4, 8, 16, 24, 48 and 72 hours after the intervention . The nausea severity, vomiting and the amount of menstrual bleeding were assessed in 24, 48, and 72 hours after the intervention . The female college students age, menarche age, length of menstrual cycle, level and duration of pain, and age of dysmenorrhea were recorded . The study was approved by the institutional ethics committee of ilam university of medical sciences, ilam, iran, and informed consents were obtained from all the participants (ethical code/92/h/184, 13/dec/2012). In addition, this study was registered at the iranian registry of clinical trials (irct2013122114668n2). Vas rating is a standard tool for evaluation of pain severity, rated from 0 to 10.0; 0 means no pain and 10 means the maximum pain in this scale . Regarding the severity of nausea, 0 means no nausea and 10 means the maximum nausea . To determine the validity of the questionnaire, content validity was used . The questionnaire was provided to 10 faculty members of ilam university of medical sciences and was used after revision . To determine the reliability of the questionnaire, cronbach s alpha test was used . Descriptive statistics, independent t - test, chi - square test, repeated measurement, friedman test, and man - whitney were performed to analyze the results . The study was approved by the institutional ethics committee of ilam university of medical sciences, ilam, iran, and informed consents were obtained from all the participants (ethical code/92/h/184, 13/dec/2012). In addition, this study was registered at the iranian registry of clinical trials (irct2013122114668n2). Vas rating is a standard tool for evaluation of pain severity, rated from 0 to 10.0; 0 means no pain and 10 means the maximum pain in this scale . Regarding the severity of nausea, 0 means no nausea and 10 means the maximum nausea . To determine the validity of the questionnaire, content validity was used . The questionnaire was provided to 10 faculty members of ilam university of medical sciences and was used after revision . To determine the reliability of the questionnaire, cronbach s alpha test was used . Descriptive statistics, independent t - test, chi - square test, repeated measurement, friedman test, and man - whitney were performed to analyze the results . Samples characteristics were not different among the groups (p> 0.5) (table 1). According to kolmogorov - smirnov test, data distribution was normal and we used the parametric methods (p> 0.05) (table 2). Independent t - test showed that the mean pain severity score in the cinnamon group was less than the placebo group at various intervals (p <0.001) (table 3). The mean duration of pain in the cinnamon group was significantly less than the placebo group at various intervals (p <0.001) (table 4). Repeated measurement analysis showed that the mean pain score in the cinnamon group (p <0.001) and the placebo group (p = 0.001) were significantly different in various intervals . The mean amount of menstrual bleeding in the cinnamon group was significantly lower than the placebo group at various intervals (p <0.05, p <0.001) (table 5). According to friedman s test, the amount of menstrual bleeding in the cinnamon group was significantly different at various intervals (p <0.001) and not significantly different in the placebo group at various intervals (p = 0.21). The mean severity of nausea significantly decreased in the cinnamon group compared with the placebo group at various intervals (p <0.001 and p <0.05, respectively) (table 6). Repeated measurement test showed that the mean nausea score in the cinnamon group (p <0.001) and the placebo group (p = 0.03) were significantly different in various intervals . The frequencies of vomiting in the cinnamon group were less than the placebo group at various intervals (p <0.001, p <0.05) (table 6). The frequencies of vomiting in the cinnamon group (p <0.001) and the placebo group (p = 0.04) were significantly different in various intervals . Values are presented as mean sd . Values are presented as mean sd . Values are presented as no . Our results suggested that cinnamon significantly reduced pain, the amount of menstrual bleeding, nausea and vomiting in female college students . This finding was consistent with the previous studies on the effects of herbal medicines such as cumin (16) thymus vulgaris, achillea millefolium (17), fennel (18), matricaria recutita (19), rosa damascena extract (20), aromatherapy massage and zingiber officinale (1) in treatment of dysmenorrhea . Primary dysmenorrhea is caused by an increase in the synthesis and release of prostaglandins, particularly pgf2 from the uterine endometrium during the menstrual period . Uterine smooth muscle contractions cause colicky pains, spasmodic and labor - like pains in the lower abdomen and cause lower back pain which is a characteristic of dysmenorrhea . Furthermore, prostaglandin secretion causes smooth muscle contraction of gastric - intestinal tract, which can lead to nausea, vomiting and diarrhea (7, 23 - 27). Today in herbal medicine, numerous benefits have been found . Herbal medicines reduce the level of prostaglandins, have nitric oxide modulation effects, increase the levels of beta - endorphin, block calcium channels and improve circulation; thus, are effective in the treatment of dysmenorrhea (28 - 30). Cinnamon is a member of the lauraceae family, which has been widely used as a spice for thousands of years to improve the taste of foods and drinks . Indications of cinnamon in medicine include diarrhea treatment, as an astringent, germicide, antispasmodic, dyspeptic complaints, for chronic bronchitis, treatment of impotence, frigidity, dyspnea, inflammation of eye, leukorrhea, vaginitis, rheumatism, and neuralgia, as well as wounds and toothaches, cold and flu; but, has not been sufficiently documented . The oil extracted of cinnamon has anti - inflammatory activity, as a treatment for dysmenorrhea and to stop bleeding . However, toxicology trials performed with high doses demonstrated that the oil of this plant stimulated the mucous membranes and instigated hematuria (1, 5). The main component of the essential oil of cinnamon bar is cinnamaldehyde (55 - 57%) and eugenol (5 - 18%). Cinnamon contains a variety of vitamins such as vitamin a, thiamin, riboflavin, and ascorbic acid (20). In adults and adolescents, 1.5 - 4 g daily of dried bark cinnamon can be used . In this study, we used a total dose of 2.52 g daily (in three divided doses), which was effective on primary dysmenorrhea and no side effects were found with this dose . On the other hand, this was the first clinical trial on the effects of cinnamon on menstrual bleeding and systemic symptoms including nausea and vomiting due to primary dysmenorrhea in female college students in iran, which was the strength of this study . Some of the factors influencing pain intensity and other symptoms with primary dysmenorrhea such as culture, genetic and nutrition (20, 31) were uncontrollable, which were the weak points of this study . In conclusion, this research suggested that cinnamon has a significant effect on reduction of pain, menstrual bleeding, nausea and vomiting due to primary dysmenorrhea, and with respect to no reported side effects, cinnamon can be regarded as a safe and effective treatment for primary dysmenorrhea.
The european horse meat scandal of 2013, in which undeclared horse meat was found in a number of supermarket beef products, highlights the need for testing methods capable of detecting and measuring food fraud in meat . Several technologies have been explored, especially enzyme - linked immunosorbent assay (elisa) and dna - based methods . An alternative route, based on mass spectrometry, targets species - specific peptides which in turn arise from species - specific proteins . Here we outline one such peptide - based approach that offers both identification and relative quantitation of the adulterant species in a meat mixture . The protocol is framed in the context of red meats and the desire to determine the presence of one in another at the level of 1% by weight, the level considered by some to represent fraudulent food adulteration as opposed to contamination . The method relies in the first instance on identifying a protein which is nominally' the same' in all target meats . Myoglobin, the protein responsible for the red color of meat, is a good candidate since it is abundant, relatively heat tolerant and water soluble, and has been used for species determination of meat previously . The myoglobins for beef (bos taurus), pork (sus scrofa), horse (equus caballus) and lamb (ovis aries), for instance, are nominally the same, as required, but their sequences are not identical . Such groups of' similar but different' proteins, like these four myoglobins, can conveniently be described as' corresponding proteins' . The sequence differences in these four myoglobins are species - specific: for example, the full myoglobin proteins for beef and horse, p02192 and p68082 respectively, each comprise 154 amino acids with 18 sequence differences between the two . Subject to proteolysis using trypsin these proteins produce two sets of peptides, some of which are identical, and some which show one or more species - specific amino acid differences: corresponding proteins therefore give rise to corresponding peptides . The cpcp approach, therefore, seeks first to identify proteins from two or more species where these proteins exhibit limited species - specific sequence variants . Following proteolysis, corresponding proteins give rise to peptides, some of which likewise display species - specific sequence variants inherited from the parent protein . The cpcp approach can be used to compare levels of two corresponding proteins in a mixed species sample by monitoring the levels of corresponding peptides . The natural technology for the detection of known peptides is multiple reaction monitoring mass spectrometry, or mrm - ms . Species - specific peptides yield precursor ions, which along with their mass spectrometry fragment ions, are easily itemized in advance by software tools . These lists are then used to instruct the mass spectrometer to record only specific precursor plus fragment ion pairs, called transitions . A particular target peptide is therefore identified not only by its retention time in the chromatography preceding the mass spectrometer, but also by a set of transitions sharing a common precursor ion . This is a highly selective means of detecting known peptides that makes efficient use of the mass spectrometer resource . Other authors have used mass spectrometry to test for meat adulteration via peptide markers but from disparate proteins . Using the corresponding proteins, corresponding peptides (cpcp) scheme, however, means experimental conditions can be optimized, aiding identification of the species in the mixture from known species - specific transitions . In addition, corresponding proteins and peptides will generally behave similarly in the extraction, proteolysis and detection stages . Since transition peak areas are quantitative and reproducible, ratios of peak areas arising from pairs of corresponding peptides from different species provide a direct estimate of the relative quantities of two meats in a mixture . In contrast, more traditional quantitation routes exploit calibrations based on reference materials to establish absolute quantitation . Though the protocol is outlined in the context of myoglobin and meat, proteins other than myoglobin could be used for identification and relative quantitation via the cpcp strategy in meat mixtures, though potentially with modifications to the protocol . In addition the strategy is also applicable to binary mixtures of other species sharing one or more corresponding proteins . The starting point for the protocol is purified' reference' myoglobin, which for some species can be purchased but which for others must be prepared by conventional size - exclusion chromatography . The procedure for preparing reference myoglobin is not included in the protocol, but is described elsewhere . Software tools are used to list candidate peptides and transitions arising from myoglobins of interest . Each reference myoglobin is subjected to proteolysis and the resultant peptides analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (lc - esi - ms / ms) to discover which of the candidate precursor ions and transitions are most useful, and to determine the matching peptide retention times . The outcome of this stage is a revised list of target peptides with their transitions, suitable for species determination, and a list of cpcp pairs, suitable for relative quantitation . To test real meats, sample extractions are prepared then subjected to proteolysis to generate peptides both from myoglobin and other extraneous proteins . The myoglobin - based peptides are then monitored by lc - esi - ms / ms based on their listed transitions . The species present in a mixture are identified by the transition peaks associated with marker peptides . Estimates of the relative amounts of two meats in a binary mixture are calculated using ratios of transition peak areas . A set of test mixtures of pairs of meats will allow the ratio of peak areas for a given pair of transitions to be checked and calibrated against actual mixtures . Proteolysis of reference myoglobins prepare solutions of the purified reference myoglobins (range 0.2 - 0.5 mg / ml in 25 mm ammonium bicarbonate).transfer 1 ml aliquots of each sample to 2 ml centrifuge tubes.thermally denature the extracted proteins by heating the sample in a hot block at 95 c for 30 min . Cool the sample for approximately 15 min until it reaches room temperature . Add 30 mg of urea (final concentration 0.5 m) to enhance the digestion, then mix . Prepare solutions of the purified reference myoglobins (range 0.2 - 0.5 mg / ml in 25 mm ammonium bicarbonate). Thermally denature the extracted proteins by heating the sample in a hot block at 95 c for 30 min . Add 30 mg of urea (final concentration 0.5 m) to enhance the digestion, then mix . Tryptic proteolysis prepare a 1 mg / ml solution of trypsin in 25 mm ammonium bicarbonate and store on ice as required . Add a sufficient volume of trypsin such that the final enzyme activity is 420 baee (n - benzoyl - l - arginine ethyl ester hydrochloride) units / mg of extracted protein, then mix by gentle vortexing and allow to proteolyze overnight at 37 c.carry out sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds - page) to demonstrate the completeness of the proteolysis . Prepare a 1 mg / ml solution of trypsin in 25 mm ammonium bicarbonate and store on ice as required . Add a sufficient volume of trypsin such that the final enzyme activity is 420 baee (n - benzoyl - l - arginine ethyl ester hydrochloride) units / mg of extracted protein, carry out sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds - page) to demonstrate the completeness of the proteolysis . Desalting of the post - proteolysis sample dilute the sample 1:2 v: v with water.activate a polymeric reversed - phase (rp) cartridge filled with 30 mg rp material by adding 1 ml of methanol, then equilibrate the cartridge by adding 1 ml of 1% formic acid.load the sample onto the cartridge under gravity.wash with 1 ml of 5% methanol/1% formic acid under gravity.elute the peptides with 1 ml of acetonitrile / water (90:10 v: v; 0.1% formic acid) under gravity into 2 ml microcentrifuge tubes prefilled with 5 l dimethylsulphoxide (dmso).remove the solvent under vacuum at 50 c using a centrifugal evaporator for 120 min, then redissolve the residue in 250 l acetonitrile / water (3:97 v: v; 0.1% formic acid).transfer the solution to a low volume auto sampler vial . Note: samples can be stored at 4 c until ready for liquid chromatography mass spectrometry (lc / ms) analysis . Dilute the sample 1:2 v: v with water . Activate a polymeric reversed - phase (rp) cartridge filled with 30 mg rp material by adding 1 ml of methanol, then equilibrate the cartridge by adding 1 ml of 1% formic acid . Elute the peptides with 1 ml of acetonitrile / water (90:10 v: v; 0.1% formic acid) under gravity into 2 ml microcentrifuge tubes prefilled with 5 l dimethylsulphoxide (dmso). Remove the solvent under vacuum at 50 c using a centrifugal evaporator for 120 min, then redissolve the residue in 250 l acetonitrile / water (3:97 v: v; 0.1% formic acid). Transfer the solution to a low volume auto sampler vial . Note: samples can be stored at 4 c until ready for liquid chromatography mass spectrometry (lc / ms) analysis . Generation of transition lists for mrm locate the myoglobin sequences for the different meats from the uniprot database.enter the myoglobin sequences into the' target' box of the peptide and transition prediction software (e.g., skyline). If required, hover over a peptide to reveal its fragment list.click on' settings' and select' peptide settings' . Input the preferences for the digestion (i.e., trypsin) and the number of missed cleavages (0). Enter the required selection for additional parameters, in particular, the peptide length (6 - 25), n - terminal exclusions (0) and assumed amino acid modifications (none).click on' settings' and select' transition settings' . Select the preferences for the instrument type used for the lc / ms analysis.click on' export' and select' transition list' to create a spreadsheet containing the generated mrm transitions and parameters . Enter the myoglobin sequences into the' target' box of the peptide and transition prediction software (e.g., skyline). Input the preferences for the digestion (i.e., trypsin) and the number of missed cleavages (0). Enter the required selection for additional parameters, in particular, the peptide length (6 - 25), n - terminal exclusions (0) and assumed amino acid modifications (none). Click on' export' and select' transition list' to create a spreadsheet containing the generated mrm transitions and parameters . Analysis by lc / ms set up a system of binary gradient (water (a) and acetonitrile (b), each with 0.1% formic acid v: v) high performance liquid chromatograph (hplc) with auto sampler, c18 core shell hplc column (10 cm x 2.1 mm, 2.6 m particle size) connected to a triple quadrupole mass spectrometer operated in positive electrospray mode with mrm detection.in the data collection software (e.g., analyst), select' file' and' new' and click on' acquisition method' in the pop - up box then click on' ok' . Note: this opens the instrument method editor, which contains a list of the connected devices that will enable the setup of a new lc / ms method.click on' binary pump' and input the flow rate value (300 l / min) and the gradient times in the table, setting a binary gradient profile of 3% b to 30% b over 22 min, increasing to 100% b at 23 min for a 5 min wash out before returning to initial conditions and re - equilibration for a further 6 min.click on' autosampler' and insert the injection volume (5 l). Enable the' needle wash cycle' and enter the' wash time' (30 sec) and select' flush port'.click on' thermostatted column controller' and in' column oven properties' set the' left temperature' and' right temperature' (40 c).click on' mass spectrometer' and then click on' edit parameters' to enter the source gas conditions . Select the' scan type' as' mrm(mrm)' and the' polarity' as' positive' . Go to' period summary' and enter the' duration time', the total time for the lc analysis and equilibration (35 min).in the table right click and select' declustering potential (dp)' and' collision energy (cp)' to add these columns to the table . Enter the q1, q3, time (msec), i d, dp and ce values for all of the transitions, for a single meat species, created in the transition list (see step 1.4.5). Note: time (msec) refers to the dwell time, the time the mass spectrometer spends scanning each transition, the summation of which should not exceed 3 sec.save the acquisition method file (file extension .dam). This will create a single method file for each meat species in screen mode in preparation for analysis below.in the data collection software, click on' acquire' and select' equilibrate' . In the box that opens, select the required acquisition method to begin the instrument equilibration.put the sample vials in a rack in the auto sampler.click on' file' and select' new' then' acquisition batch' . In the' sample' tab select' add set' then' add samples' . Insert the number of samples to be analyzed and click on' ok' . In the' acquisition' box select the method file that will be used for the analysis from the drop down menu.in the table, select' plate code' and select the appropriate tray configuration from the drop down menu . Left click in the' plate code' column header then right click and select' fill down' . In' vial position' enter the position of each sample in the auto sampler in the rows.in' data file' enter the file name for the acquisition, then left click in the column header followed by right click and select' fill down' . In' sample name' insert the identity of each of the samples to be analyzed . Save as an acquisition batch file (file extension .dab).click on the' submit' tab then highlight the samples that need to be analyzed on the lc / ms . Note: each acquisition method will scan for the mrm transitions across the entire length of the chromatograph for a single meat species . Mass spectrometer settings for an mrm acquisition vary according to instrument type and peptide.view the generated data files using data viewing software . Click on xic (extracted ions) and in the drop down list highlight all the fragments (q3 values) for a single precursor (q1). A new pane will open that shows only the selected transitions.record the retention time (rt) for groups of concurrent transitions since these correspond to a single peptide.repeat the previous two steps for each set of transitions in order to assign the peaks to their respective peptides for each of the meat species.record the marker peptides which are suitable for providing species identification (e.g., peptide hpgdfgadaqgamtk, precursor m / z = 752, rt = 12.0 min, for horse), together with their retention times, and note which form corresponding pairs suitable for relative quantitation . Note: for example, the horse marker peptide (precursor m / z = 752) has a corresponding beef peptide, hpsdfgadaqaamsk (precursor m / z = 767, rt = 13.2 min).in order to create a single dynamic method embracing all of the meat species, in the data viewing software, for each meat species in turn, open the xic transition data for each precursor (assigned to a particular peptide in 1.5.8).zoom in on the peak cluster at the selected retention time by left - clicking and dragging the cursor underneath the cluster . Identify the most intense transitions (by right - clicking on the peak label).manually record the transitions and retention times in a spreadsheet.to enter the parameters as a new dynamic method on the lc / ms software, click on' mass spectrometer' and then click on' edit parameters' to enter the source gas conditions . Select the' scan type' as' mrm(mrm)' and the' polarity' as' positive'.go to' period summary' and enter the duration time (set as the total time for the lc analysis and equilibration). In the table right click and select' declustering potential (dp)' and' collision energy (cp)' to add these columns to the table . Note: the' time' column now refers to the expected retention time (min) for each transition.in the' edit parameters' section of the lc / ms data collection software, check the' scheduled mrm' box . Input the q1, q3, time (min), i d, dp and ce values for the transitions created in the spreadsheet (1.5.21) and save the acquisition method (file extension .dam). Note: this method typically reduces the number of mrm transitions to the 4 most intense for each peptide and scans only across the retention time window for each peptide peak, giving improved sensitivity and quality of the data . A' dynamic' method is a' guided retention time windowing' method, sometimes called scheduling . Set up a system of binary gradient (water (a) and acetonitrile (b), each with 0.1% formic acid v: v) high performance liquid chromatograph (hplc) with auto sampler, c18 core shell hplc column (10 cm x 2.1 mm, 2.6 m particle size) connected to a triple quadrupole mass spectrometer operated in positive electrospray mode with mrm detection . In the data collection software (e.g., analyst), select' file' and' new' and click on' acquisition method' in the pop - up box then click on' ok' . Note: this opens the instrument method editor, which contains a list of the connected devices that will enable the setup of a new lc / ms method . Click on' binary pump' and input the flow rate value (300 l / min) and the gradient times in the table, setting a binary gradient profile of 3% b to 30% b over 22 min, increasing to 100% b at 23 min for a 5 min wash out before returning to initial conditions and re - equilibration for a further 6 min . Enable the' needle wash cycle' and enter the' wash time' (30 sec) and select' flush port' . Click on' thermostatted column controller' and in' column oven properties' set the' left temperature' and' right temperature' (40 c). Click on' mass spectrometer' and then click on' edit parameters' to enter the source gas conditions . Select the' scan type' as' mrm(mrm)' and the' polarity' as' positive' . Go to' period summary' and enter the' duration time', the total time for the lc analysis and equilibration (35 min). In the table right click and select' declustering potential (dp)' and' collision energy (cp)' to add these columns to the table . Enter the q1, q3, time (msec), i d, dp and ce values for all of the transitions, for a single meat species, created in the transition list (see step 1.4.5). Note: time (msec) refers to the dwell time, the time the mass spectrometer spends scanning each transition, the summation of which should not exceed 3 sec . Save the acquisition method file (file extension .dam). This will create a single method file for each meat species in screen mode in preparation for analysis below . In the data collection software, click on' acquire' and select' equilibrate' . In the box that opens, select the required acquisition method to begin the instrument equilibration . Click on' file' and select' new' then' acquisition batch' . In the' sample' tab select' add set' then' add samples' . Insert the number of samples to be analyzed and click on' ok' . In the' acquisition' box select the method file that will be used for the analysis from the drop down menu . In the table, select' plate code' and select the appropriate tray configuration from the drop down menu . Left click in the' plate code' column header then right click and select' fill down' . In' vial position' enter the position of each sample in the auto sampler in the rows . In' data file' enter the file name for the acquisition, then left click in the column header followed by right click and select' fill down' . In' sample name' click on the' submit' tab then highlight the samples that need to be analyzed on the lc / ms . Note: each acquisition method will scan for the mrm transitions across the entire length of the chromatograph for a single meat species . Click on xic (extracted ions) and in the drop down list highlight all the fragments (q3 values) for a single precursor (q1). Record the retention time (rt) for groups of concurrent transitions since these correspond to a single peptide . Repeat the previous two steps for each set of transitions in order to assign the peaks to their respective peptides for each of the meat species . Record the marker peptides which are suitable for providing species identification (e.g., peptide hpgdfgadaqgamtk, precursor m / z = 752, rt = 12.0 min, for horse), together with their retention times, and note which form corresponding pairs suitable for relative quantitation . Note: for example, the horse marker peptide (precursor m / z = 752) has a corresponding beef peptide, hpsdfgadaqaamsk (precursor m / z = 767, rt = 13.2 min). In order to create a single dynamic method embracing all of the meat species, in the data viewing software, for each meat species in turn, open the xic transition data for each precursor (assigned to a particular peptide in 1.5.8). Zoom in on the peak cluster at the selected retention time by left - clicking and dragging the cursor underneath the cluster . Identify the most intense transitions (by right - clicking on the peak label). Manually record the transitions and retention times in a spreadsheet . To enter the parameters as a new dynamic method on the lc / ms software, click on' mass spectrometer' and then click on' edit parameters' to enter the source gas conditions . Select the' scan type' as' mrm(mrm)' and the' polarity' as' positive' . Go to' period summary' and enter the duration time (set as the total time for the lc analysis and equilibration). In the table right click and select' declustering potential (dp)' and' collision energy (cp)' to add these columns to the table . Note: the' time' column now refers to the expected retention time (min) for each transition . In the' edit parameters' section of the lc / ms data collection software, check the' scheduled mrm' box . Input the q1, q3, time (min), i d, dp and ce values for the transitions created in the spreadsheet (1.5.21) and save the acquisition method (file extension .dam). Note: this method typically reduces the number of mrm transitions to the 4 most intense for each peptide and scans only across the retention time window for each peptide peak, giving improved sensitivity and quality of the data . A' dynamic' method is a' guided retention time windowing' method, sometimes called scheduling . Extraction of meat mixtures using meat previously frozen then ground into a powder, prepare a range of meat mixtures by weighing respective amounts of meat (total mass of about 300 mg) into 15 ml plastic centrifuge tubes.add 4 ml of extraction buffer (0.15 m potassium chloride + 0.15 m phosphate buffer at ph 6.5). Extract on a lab shaker at room temperature for 2 hr at 250 cycles / min . Note: cycles / min refers to an oscillatory motion.transfer 2 ml of the extract into a 2 ml microcentrifuge tube . Centrifuge for 5 min at 4 c at 17,000 x g.transfer 200 l aliquots of the supernatant (reserving a small amount for protein assay, see 2.2) into 2 ml centrifuge tubes and dry using a centrifugal evaporator (pre - set program: 50 c, with no venting and 120 min duration). Using meat previously frozen then ground into a powder, prepare a range of meat mixtures by weighing respective amounts of meat (total mass of about 300 mg) into 15 ml plastic centrifuge tubes . Add 4 ml of extraction buffer (0.15 m potassium chloride + 0.15 m phosphate buffer at ph 6.5). Extract on a lab shaker at room temperature for 2 hr at 250 cycles / min . Centrifuge for 5 min at 4 c at 17,000 x g. transfer 200 l aliquots of the supernatant (reserving a small amount for protein assay, see 2.2) into 2 ml centrifuge tubes and dry using a centrifugal evaporator (pre - set program: 50 c, with no venting and 120 min duration). Protein assay transfer 7 l aliquots of the reserved supernatant (see 2.1.4) in triplicate into the wells of a 96 well plate.transfer 7 l aliquots of a series of protein standards in triplicate, range 0 - 1.0 mg / ml bovine serum albumin (bsa), to the same 96 well plate.add 200 l of coomassie plus protein assay reagent to each well.visually compare the color of the sample wells with the protein standards to check the samples are in the range of the calibration standards . If necessary, repeat with diluted sample so it becomes in range.leave the plate to stand for 3 min.burst any bubbles that have formed with a hypodermic needle.analyze the plate on the plate reader using a standard endpoint protocol at a wavelength of 595 nm.determine the protein concentration of the samples using calibration data from the protein standards . Note: this is required for calculation of the amount of trypsin used in the tryptic digest . Transfer 7 l aliquots of the reserved supernatant (see 2.1.4) in triplicate into the wells of a 96 well plate . Transfer 7 l aliquots of a series of protein standards in triplicate, range 0 - 1.0 mg / ml bovine serum albumin (bsa), to the same 96 well plate . Visually compare the color of the sample wells with the protein standards to check the samples are in the range of the calibration standards . Leave the plate to stand for 3 min . Burst any bubbles that have formed with a hypodermic needle . Analyze the plate on the plate reader using a standard endpoint protocol at a wavelength of 595 nm . Note: this is required for calculation of the amount of trypsin used in the tryptic digest . Proteolysis of meat mixtures redissolve the dried residue from step 2.1.4 in 1 ml of 25 mm ammonium bicarbonate solution . Mix well on a rotamixer.follow the protocol from step 1.1.3 to 1.3.7 . Redissolve the dried residue from step 2.1.4 in 1 ml of 25 mm ammonium bicarbonate solution . Analysis by lc / ms set up the lc / ms as previously (step 1.5.1).create a new acquisition batch as outlined previously (steps 1.5.9 - 1.5.14), selecting the acquisition method created at step at 1.5.24 that uses a dynamic lc / ms method combining all of the meat species, and acquire the data for the digested meat samples.display the full chromatogram in the data viewing software . Display the xic for each transition set in turn . Visually confirm each cluster contains the requisite number of bell - shaped peaks at the expected retention time, thereby confirming the existence of the selected peptide.perform quantitation using the data viewing software to integrate peak areas for each of the transitions of interest by double - clicking on' build quantitation method' in the navigation bar.in the' select sample' pane select the' data file' and the' sample' to be analyzed to generate an' analytes' table.click on the' integration' tab to display the first of the transitions (analytes) to be integrated.click on' analyte' box to display the drop down list of transitions . Select each transition in turn to display it and visually confirm the correct peak is selected for integration . To modify or force the integration, left click and drag the cursor over the target peak (click on the' select peak' button and click' apply'.save the workspace as a method file (.qmf). Note: this creates a quantitation method file for subsequent calculation of sample peak areas.double click' quantitation wizard' in the navigation bar . In the' select samples' window create' quantitation set' by selecting a single' data file', then one or more' available samples' . Leave with defaults, select' next' to display' select method' . From the drop down' method' box select the' integration method' file created in step 2.4.8, then select' finish' . Note: this creates a' results table', including transition peak areas arising from meat mixtures.save the' results table' (file extension .rdb), export as a text file (.txt) and open it in spreadsheet to review the data.plot graphs of the percentage (by transition peak area) of one meat in another versus the measured percentage (w / w) of the two meats for the selected mrm for selected transitions from corresponding peptides, focusing on those cases where the two fragments contain the same number of amino acids as counted from the c - terminal end . Note: identical fragments with identical fragmentation sites give optimal results.examine the plots from 2.4.11 above . Either visually, or using a trend line tool in the plotting package, identify a group of plots which are both linear and of similar gradient . Use any one or more of these cpcp plus fragment combinations for calibration in real meat samples . Note: a plot showing an unusual gradient may indicate either peptide or fragment suppression with a consequent reduction in signal strength . Create a new acquisition batch as outlined previously (steps 1.5.9 - 1.5.14), selecting the acquisition method created at step at 1.5.24 that uses a dynamic lc / ms method combining all of the meat species, and acquire the data for the digested meat samples . Display the xic for each transition set in turn . Visually confirm each cluster contains the requisite number of bell - shaped peaks at the expected retention time, thereby confirming the existence of the selected peptide . Perform quantitation using the data viewing software to integrate peak areas for each of the transitions of interest by double - clicking on' build quantitation method' in the navigation bar . In the' select sample' pane select the' data file' and the' sample' to be analyzed to generate an' analytes' table . Click on the' integration' select each transition in turn to display it and visually confirm the correct peak is selected for integration . To modify or force the integration, left click and drag the cursor over the target peak (click on the' select peak' button and click' apply' . Save the workspace as a method file (.qmf). Note: this creates a quantitation method file for subsequent calculation of sample peak areas . The' select samples' window create' quantitation set' by selecting a single' data file', then one or more' available samples' . Leave with defaults, select' next' to display' select method' . From the drop down' method' box select the' integration method' file created in step 2.4.8, then select' finish' . Note: this creates a' results table', including transition peak areas arising from meat mixtures . Save the' results table' (file extension .rdb), export as a text file (.txt) and open it in spreadsheet to review the data . Plot graphs of the percentage (by transition peak area) of one meat in another versus the measured percentage (w / w) of the two meats for the selected mrm for selected transitions from corresponding peptides, focusing on those cases where the two fragments contain the same number of amino acids as counted from the c - terminal end . Either visually, or using a trend line tool in the plotting package, identify a group of plots which are both linear and of similar gradient . Use any one or more of these cpcp plus fragment combinations for calibration in real meat samples . Note: a plot showing an unusual gradient may indicate either peptide or fragment suppression with a consequent reduction in signal strength . Extraction of proteins from target meat samples where applicable, excise extraneous non - meat material from the sample using a spatula . For example, scrape away sauce and pasta from a chilled lasagna.weigh 20 g of the meat into a metal beaker.add 100 ml of 0.15 m potassium chloride/0.15 m potassium monophosphate buffer at ph 6.5.extract the proteins by blending the meat in a high speed homogenizer for 1 min.follow the protocol from step 2.1.4 - 2.3.2 . Where applicable, excise extraneous non - meat material from the sample using a spatula . Add 100 ml of 0.15 m potassium chloride/0.15 m potassium monophosphate buffer at ph 6.5 . Extract the proteins by blending the meat in a high speed homogenizer for 1 min . Analysis of samples by lc / ms repeat step 2.4.2 to acquire data using the dynamic lc / ms method.identify the peptides from each meat myoglobin as performed in step 2.4.3.for quantitation, use quantitation software to integrate the peak areas for each transition of interest, as outlined in step 2.4.9.for identification of species in a mixture, record those marker peptides satisfying agreed criteria for numbers of transitions and signal to noise for those transitions.for quantitation, use integrated transition peak areas as agreed from step 2.4.12 and, using percentage by transition peak area, calculate the percentage of myoglobin from the two species in the mixture.use prior knowledge from the literature of likely myoglobin levels in the meats to estimate the relative w / w amounts of two meats present in the sample . For quantitation, use quantitation software to integrate the peak areas for each transition of interest, as outlined in step 2.4.9 . For identification of species in a mixture, record those marker peptides satisfying agreed criteria for numbers of transitions and signal to noise for those transitions . For quantitation, use integrated transition peak areas as agreed from step 2.4.12 and, using percentage by transition peak area, calculate the percentage of myoglobin from the two species in the mixture . Use prior knowledge from the literature of likely myoglobin levels in the meats to estimate the relative w / w amounts of two meats present in the sample . Proteolysis of reference myoglobins prepare solutions of the purified reference myoglobins (range 0.2 - 0.5 mg / ml in 25 mm ammonium bicarbonate).transfer 1 ml aliquots of each sample to 2 ml centrifuge tubes.thermally denature the extracted proteins by heating the sample in a hot block at 95 c for 30 min . Cool the sample for approximately 15 min until it reaches room temperature . Add 30 mg of urea (final concentration 0.5 m) to enhance the digestion, then mix . Prepare solutions of the purified reference myoglobins (range 0.2 - 0.5 mg / ml in 25 mm ammonium bicarbonate). Thermally denature the extracted proteins by heating the sample in a hot block at 95 c for 30 min . Add 30 mg of urea (final concentration 0.5 m) to enhance the digestion, then mix . Tryptic proteolysis prepare a 1 mg / ml solution of trypsin in 25 mm ammonium bicarbonate and store on ice as required . Add a sufficient volume of trypsin such that the final enzyme activity is 420 baee (n - benzoyl - l - arginine ethyl ester hydrochloride) units / mg of extracted protein, then mix by gentle vortexing and allow to proteolyze overnight at 37 c.carry out sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds - page) to demonstrate the completeness of the proteolysis . Prepare a 1 mg / ml solution of trypsin in 25 mm ammonium bicarbonate and store on ice as required . Add a sufficient volume of trypsin such that the final enzyme activity is 420 baee (n - benzoyl - l - arginine ethyl ester hydrochloride) units / mg of extracted protein, carry out sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds - page) to demonstrate the completeness of the proteolysis . Desalting of the post - proteolysis sample dilute the sample 1:2 v: v with water.activate a polymeric reversed - phase (rp) cartridge filled with 30 mg rp material by adding 1 ml of methanol, then equilibrate the cartridge by adding 1 ml of 1% formic acid.load the sample onto the cartridge under gravity.wash with 1 ml of 5% methanol/1% formic acid under gravity.elute the peptides with 1 ml of acetonitrile / water (90:10 v: v; 0.1% formic acid) under gravity into 2 ml microcentrifuge tubes prefilled with 5 l dimethylsulphoxide (dmso).remove the solvent under vacuum at 50 c using a centrifugal evaporator for 120 min, then redissolve the residue in 250 l acetonitrile / water (3:97 v: v; 0.1% formic acid).transfer the solution to a low volume auto sampler vial . Note: samples can be stored at 4 c until ready for liquid chromatography mass spectrometry (lc / ms) analysis . Dilute the sample 1:2 v: v with water . Activate a polymeric reversed - phase (rp) cartridge filled with 30 mg rp material by adding 1 ml of methanol, then equilibrate the cartridge by adding 1 ml of 1% formic acid . Elute the peptides with 1 ml of acetonitrile / water (90:10 v: v; 0.1% formic acid) under gravity into 2 ml microcentrifuge tubes prefilled with 5 l dimethylsulphoxide (dmso). Remove the solvent under vacuum at 50 c using a centrifugal evaporator for 120 min, then redissolve the residue in 250 l acetonitrile / water (3:97 v: v; 0.1% formic acid). Transfer the solution to a low volume auto sampler vial . Note: samples can be stored at 4 c until ready for liquid chromatography mass spectrometry (lc / ms) analysis . Generation of transition lists for mrm locate the myoglobin sequences for the different meats from the uniprot database.enter the myoglobin sequences into the' target' box of the peptide and transition prediction software (e.g., skyline). If required, hover over a peptide to reveal its fragment list.click on' settings' and select' peptide settings' . Input the preferences for the digestion (i.e., trypsin) and the number of missed cleavages (0). Enter the required selection for additional parameters, in particular, the peptide length (6 - 25), n - terminal exclusions (0) and assumed amino acid modifications (none).click on' settings' and select' transition settings' . Select the preferences for the instrument type used for the lc / ms analysis.click on' export' and select' transition list' to create a spreadsheet containing the generated mrm transitions and parameters . Enter the myoglobin sequences into the' target' box of the peptide and transition prediction software (e.g., skyline). Input the preferences for the digestion (i.e., trypsin) and the number of missed cleavages (0). Enter the required selection for additional parameters, in particular, the peptide length (6 - 25), n - terminal exclusions (0) and assumed amino acid modifications (none). Click on' export' and select' transition list' to create a spreadsheet containing the generated mrm transitions and parameters . Analysis by lc / ms set up a system of binary gradient (water (a) and acetonitrile (b), each with 0.1% formic acid v: v) high performance liquid chromatograph (hplc) with auto sampler, c18 core shell hplc column (10 cm x 2.1 mm, 2.6 m particle size) connected to a triple quadrupole mass spectrometer operated in positive electrospray mode with mrm detection.in the data collection software (e.g., analyst), select' file' and' new' and click on' acquisition method' in the pop - up box then click on' ok' . Note: this opens the instrument method editor, which contains a list of the connected devices that will enable the setup of a new lc / ms method.click on' binary pump' and input the flow rate value (300 l / min) and the gradient times in the table, setting a binary gradient profile of 3% b to 30% b over 22 min, increasing to 100% b at 23 min for a 5 min wash out before returning to initial conditions and re - equilibration for a further 6 min.click on' autosampler' and insert the injection volume (5 l). Enable the' needle wash cycle' and enter the' wash time' (30 sec) and select' flush port'.click on' thermostatted column controller' and in' column oven properties' set the' left temperature' and' right temperature' (40 c).click on' mass spectrometer' and then click on' edit parameters' to enter the source gas conditions . Select the' scan type' as' mrm(mrm)' and the' polarity' as' positive' . Go to' period summary' and enter the' duration time', the total time for the lc analysis and equilibration (35 min).in the table right click and select' declustering potential (dp)' and' collision energy (cp)' to add these columns to the table . Enter the q1, q3, time (msec), i d, dp and ce values for all of the transitions, for a single meat species, created in the transition list (see step 1.4.5). Note: time (msec) refers to the dwell time, the time the mass spectrometer spends scanning each transition, the summation of which should not exceed 3 sec.save the acquisition method file (file extension .dam). This will create a single method file for each meat species in screen mode in preparation for analysis below.in the data collection software, click on' acquire' and select' equilibrate' . In the box that opens, select the required acquisition method to begin the instrument equilibration.put the sample vials in a rack in the auto sampler.click on' file' and select' new' then' acquisition batch' . In the' sample' tab select' add set' then' add samples' . Insert the number of samples to be analyzed and click on' ok' . In the' acquisition' box select the method file that will be used for the analysis from the drop down menu.in the table, select' plate code' and select the appropriate tray configuration from the drop down menu . Left click in the' plate code' column header then right click and select' fill down' . In' vial position' enter the position of each sample in the auto sampler in the rows.in' data file' enter the file name for the acquisition, then left click in the column header followed by right click and select' fill down' . In' sample name' insert the identity of each of the samples to be analyzed . Save as an acquisition batch file (file extension .dab).click on the' submit' tab then highlight the samples that need to be analyzed on the lc / ms . Note: each acquisition method will scan for the mrm transitions across the entire length of the chromatograph for a single meat species . Mass spectrometer settings for an mrm acquisition vary according to instrument type and peptide.view the generated data files using data viewing software . Click on xic (extracted ions) and in the drop down list highlight all the fragments (q3 values) for a single precursor (q1). A new pane will open that shows only the selected transitions.record the retention time (rt) for groups of concurrent transitions since these correspond to a single peptide.repeat the previous two steps for each set of transitions in order to assign the peaks to their respective peptides for each of the meat species.record the marker peptides which are suitable for providing species identification (e.g., peptide hpgdfgadaqgamtk, precursor m / z = 752, rt = 12.0 min, for horse), together with their retention times, and note which form corresponding pairs suitable for relative quantitation . Note: for example, the horse marker peptide (precursor m / z = 752) has a corresponding beef peptide, hpsdfgadaqaamsk (precursor m / z = 767, rt = 13.2 min).in order to create a single dynamic method embracing all of the meat species, in the data viewing software, for each meat species in turn, open the xic transition data for each precursor (assigned to a particular peptide in 1.5.8).zoom in on the peak cluster at the selected retention time by left - clicking and dragging the cursor underneath the cluster . Identify the most intense transitions (by right - clicking on the peak label).manually record the transitions and retention times in a spreadsheet.to enter the parameters as a new dynamic method on the lc / ms software, click on' mass spectrometer' and then click on' edit parameters' to enter the source gas conditions . Select the' scan type' as' mrm(mrm)' and the' polarity' as' positive'.go to' period summary' and enter the duration time (set as the total time for the lc analysis and equilibration). In the table right click and select' declustering potential (dp)' and' collision energy (cp)' to add these columns to the table . Note: the' time' column now refers to the expected retention time (min) for each transition.in the' edit parameters' section of the lc / ms data collection software, check the' scheduled mrm' box . Input the q1, q3, time (min), i d, dp and ce values for the transitions created in the spreadsheet (1.5.21) and save the acquisition method (file extension .dam). Note: this method typically reduces the number of mrm transitions to the 4 most intense for each peptide and scans only across the retention time window for each peptide peak, giving improved sensitivity and quality of the data . A' dynamic' method is a' guided retention time windowing' method, sometimes called scheduling . Set up a system of binary gradient (water (a) and acetonitrile (b), each with 0.1% formic acid v: v) high performance liquid chromatograph (hplc) with auto sampler, c18 core shell hplc column (10 cm x 2.1 mm, 2.6 m particle size) connected to a triple quadrupole mass spectrometer operated in positive electrospray mode with mrm detection . In the data collection software (e.g., analyst), select' file' and' new' and click on' acquisition method' in the pop - up box then click on' ok' . Note: this opens the instrument method editor, which contains a list of the connected devices that will enable the setup of a new lc / ms method . Click on' binary pump' and input the flow rate value (300 l / min) and the gradient times in the table, setting a binary gradient profile of 3% b to 30% b over 22 min, increasing to 100% b at 23 min for a 5 min wash out before returning to initial conditions and re - equilibration for a further 6 min . Enable the' needle wash cycle' and enter the' wash time' (30 sec) and select' flush port' . Click on' thermostatted column controller' and in' column oven properties' set the' left temperature' and' right temperature' (40 c). Click on' mass spectrometer' and then click on' edit parameters' to enter the source gas conditions . Select the' scan type' as' mrm(mrm)' and the' polarity' as' positive' . Go to' period summary' and enter the' duration time', the total time for the lc analysis and equilibration (35 min). In the table right click and select' declustering potential (dp)' and' collision energy (cp)' to add these columns to the table . Enter the q1, q3, time (msec), i d, dp and ce values for all of the transitions, for a single meat species, created in the transition list (see step 1.4.5). Note: time (msec) refers to the dwell time, the time the mass spectrometer spends scanning each transition, the summation of which should not exceed 3 sec . Save the acquisition method file (file extension .dam). This will create a single method file for each meat species in screen mode in preparation for analysis below . In the data collection software, click on' acquire' and select' equilibrate' . In the box that opens, select the required acquisition method to begin the instrument equilibration . Click on' file' and select' new' then' acquisition batch' . In the' sample' tab select' add set' then' add samples' . Insert the number of samples to be analyzed and click on' ok' . In the' acquisition' box select the method file that will be used for the analysis from the drop down menu . In the table, select' plate code' and select the appropriate tray configuration from the drop down menu . Left click in the' plate code' column header then right click and select' fill down' . In' vial position' enter the position of each sample in the auto sampler in the rows . In' data file' enter the file name for the acquisition, then left click in the column header followed by right click and select' fill down' . In' sample name' click on the' submit' tab then highlight the samples that need to be analyzed on the lc / ms . Note: each acquisition method will scan for the mrm transitions across the entire length of the chromatograph for a single meat species . Click on xic (extracted ions) and in the drop down list highlight all the fragments (q3 values) for a single precursor (q1). Record the retention time (rt) for groups of concurrent transitions since these correspond to a single peptide . Repeat the previous two steps for each set of transitions in order to assign the peaks to their respective peptides for each of the meat species . Record the marker peptides which are suitable for providing species identification (e.g., peptide hpgdfgadaqgamtk, precursor m / z = 752, rt = 12.0 min, for horse), together with their retention times, and note which form corresponding pairs suitable for relative quantitation . Note: for example, the horse marker peptide (precursor m / z = 752) has a corresponding beef peptide, hpsdfgadaqaamsk (precursor m / z = 767, rt = 13.2 min). In order to create a single dynamic method embracing all of the meat species, in the data viewing software, for each meat species in turn, open the xic transition data for each precursor (assigned to a particular peptide in 1.5.8). Zoom in on the peak cluster at the selected retention time by left - clicking and dragging the cursor underneath the cluster . Identify the most intense transitions (by right - clicking on the peak label). Manually record the transitions and retention times in a spreadsheet . To enter the parameters as a new dynamic method on the lc / ms software, click on' mass spectrometer' and then click on' edit parameters' to enter the source gas conditions . Select the' scan type' as' mrm(mrm)' and the' polarity' as' positive' . Go to' period summary' and enter the duration time (set as the total time for the lc analysis and equilibration). In the table right click and select' declustering potential (dp)' and' collision energy (cp)' to add these columns to the table . Note: the' time' column now refers to the expected retention time (min) for each transition . In the' edit parameters' section of the lc / ms data collection software, check the' scheduled mrm' box . Input the q1, q3, time (min), i d, dp and ce values for the transitions created in the spreadsheet (1.5.21) and save the acquisition method (file extension .dam). Note: this method typically reduces the number of mrm transitions to the 4 most intense for each peptide and scans only across the retention time window for each peptide peak, giving improved sensitivity and quality of the data . A' dynamic' method is a' guided retention time windowing' method, sometimes called scheduling . Extraction of meat mixtures using meat previously frozen then ground into a powder, prepare a range of meat mixtures by weighing respective amounts of meat (total mass of about 300 mg) into 15 ml plastic centrifuge tubes.add 4 ml of extraction buffer (0.15 m potassium chloride + 0.15 m phosphate buffer at ph 6.5). Extract on a lab shaker at room temperature for 2 hr at 250 cycles / min . Note: cycles / min refers to an oscillatory motion.transfer 2 ml of the extract into a 2 ml microcentrifuge tube . Centrifuge for 5 min at 4 c at 17,000 x g.transfer 200 l aliquots of the supernatant (reserving a small amount for protein assay, see 2.2) into 2 ml centrifuge tubes and dry using a centrifugal evaporator (pre - set program: 50 c, with no venting and 120 min duration). Using meat previously frozen then ground into a powder, prepare a range of meat mixtures by weighing respective amounts of meat (total mass of about 300 mg) into 15 ml plastic centrifuge tubes . Add 4 ml of extraction buffer (0.15 m potassium chloride + 0.15 m phosphate buffer at ph 6.5). Extract on a lab shaker at room temperature for 2 hr at 250 cycles / min . Centrifuge for 5 min at 4 c at 17,000 x g. transfer 200 l aliquots of the supernatant (reserving a small amount for protein assay, see 2.2) into 2 ml centrifuge tubes and dry using a centrifugal evaporator (pre - set program: 50 c, with no venting and 120 min duration). Protein assay transfer 7 l aliquots of the reserved supernatant (see 2.1.4) in triplicate into the wells of a 96 well plate.transfer 7 l aliquots of a series of protein standards in triplicate, range 0 - 1.0 mg / ml bovine serum albumin (bsa), to the same 96 well plate.add 200 l of coomassie plus protein assay reagent to each well.visually compare the color of the sample wells with the protein standards to check the samples are in the range of the calibration standards . If necessary, repeat with diluted sample so it becomes in range.leave the plate to stand for 3 min.burst any bubbles that have formed with a hypodermic needle.analyze the plate on the plate reader using a standard endpoint protocol at a wavelength of 595 nm.determine the protein concentration of the samples using calibration data from the protein standards . Note: this is required for calculation of the amount of trypsin used in the tryptic digest . Transfer 7 l aliquots of the reserved supernatant (see 2.1.4) in triplicate into the wells of a 96 well plate . Transfer 7 l aliquots of a series of protein standards in triplicate, range 0 - 1.0 mg / ml bovine serum albumin (bsa), to the same 96 well plate . Visually compare the color of the sample wells with the protein standards to check the samples are in the range of the calibration standards . Leave the plate to stand for 3 min . Burst any bubbles that have formed with a hypodermic needle . Analyze the plate on the plate reader using a standard endpoint protocol at a wavelength of 595 nm . Note: this is required for calculation of the amount of trypsin used in the tryptic digest . Proteolysis of meat mixtures redissolve the dried residue from step 2.1.4 in 1 ml of 25 mm ammonium bicarbonate solution . Redissolve the dried residue from step 2.1.4 in 1 ml of 25 mm ammonium bicarbonate solution . Analysis by lc / ms set up the lc / ms as previously (step 1.5.1).create a new acquisition batch as outlined previously (steps 1.5.9 - 1.5.14), selecting the acquisition method created at step at 1.5.24 that uses a dynamic lc / ms method combining all of the meat species, and acquire the data for the digested meat samples.display the full chromatogram in the data viewing software . Visually confirm each cluster contains the requisite number of bell - shaped peaks at the expected retention time, thereby confirming the existence of the selected peptide.perform quantitation using the data viewing software to integrate peak areas for each of the transitions of interest by double - clicking on' build quantitation method' in the navigation bar.in the' select sample' pane select the' data file' and the' sample' to be analyzed to generate an' analytes' table.click on the' integration' tab to display the first of the transitions (analytes) to be integrated.click on' analyte' box to display the drop down list of transitions . Select each transition in turn to display it and visually confirm the correct peak is selected for integration . To modify or force the integration, left click and drag the cursor over the target peak (this will be highlighted in green). Click on the' select peak' button and click' apply'.save the workspace as a method file (.qmf). Note: this creates a quantitation method file for subsequent calculation of sample peak areas.double click' quantitation wizard' in the navigation bar . In the' select samples' window create' quantitation set' by selecting a single' data file', then one or more' available samples' . Select' next' to display' select settings and query' box . Leave with defaults, select' next' to display' select method' . From the drop down' method' box select the' integration method' file created in step 2.4.8, then select' finish' . Note: this creates a' results table', including transition peak areas arising from meat mixtures.save the' results table' (file extension .rdb), export as a text file (.txt) and open it in spreadsheet to review the data.plot graphs of the percentage (by transition peak area) of one meat in another versus the measured percentage (w / w) of the two meats for the selected mrm for selected transitions from corresponding peptides, focusing on those cases where the two fragments contain the same number of amino acids as counted from the c - terminal end . Note: identical fragments with identical fragmentation sites give optimal results.examine the plots from 2.4.11 above . Either visually, or using a trend line tool in the plotting package, identify a group of plots which are both linear and of similar gradient . Use any one or more of these cpcp plus fragment combinations for calibration in real meat samples . Note: a plot showing an unusual gradient may indicate either peptide or fragment suppression with a consequent reduction in signal strength . Create a new acquisition batch as outlined previously (steps 1.5.9 - 1.5.14), selecting the acquisition method created at step at 1.5.24 that uses a dynamic lc / ms method combining all of the meat species, and acquire the data for the digested meat samples . Visually confirm each cluster contains the requisite number of bell - shaped peaks at the expected retention time, thereby confirming the existence of the selected peptide . Perform quantitation using the data viewing software to integrate peak areas for each of the transitions of interest by double - clicking on' build quantitation method' in the navigation bar . In the' select sample' pane select the' data file' and the' sample' to be analyzed to generate an' analytes' table . Click on the' integration' tab to display the first of the transitions (analytes) to be integrated . Select each transition in turn to display it and visually confirm the correct peak is selected for integration . To modify or force the integration, left click and drag the cursor over the target peak (this will be highlighted in green). Note: this creates a quantitation method file for subsequent calculation of sample peak areas . The' select samples' window create' quantitation set' by selecting a single' data file', then one or more' available samples' . Leave with defaults, select' next' to display' select method' . From the drop down' method' box select the' integration method' file created in step 2.4.8, then select' finish' . Note: this creates a' results table', including transition peak areas arising from meat mixtures . Save the' results table' (file extension .rdb), export as a text file (.txt) and open it in spreadsheet to review the data . Plot graphs of the percentage (by transition peak area) of one meat in another versus the measured percentage (w / w) of the two meats for the selected mrm for selected transitions from corresponding peptides, focusing on those cases where the two fragments contain the same number of amino acids as counted from the c - terminal end . Either visually, or using a trend line tool in the plotting package, identify a group of plots which are both linear and of similar gradient . Use any one or more of these cpcp plus fragment combinations for calibration in real meat samples . Note: a plot showing an unusual gradient may indicate either peptide or fragment suppression with a consequent reduction in signal strength . Extraction of proteins from target meat samples where applicable, excise extraneous non - meat material from the sample using a spatula . For example, scrape away sauce and pasta from a chilled lasagna.weigh 20 g of the meat into a metal beaker.add 100 ml of 0.15 m potassium chloride/0.15 m potassium monophosphate buffer at ph 6.5.extract the proteins by blending the meat in a high speed homogenizer for 1 min.follow the protocol from step 2.1.4 - 2.3.2 . Where applicable, excise extraneous non - meat material from the sample using a spatula . Add 100 ml of 0.15 m potassium chloride/0.15 m potassium monophosphate buffer at ph 6.5 . Extract the proteins by blending the meat in a high speed homogenizer for 1 min . Analysis of samples by lc / ms repeat step 2.4.2 to acquire data using the dynamic lc / ms method.identify the peptides from each meat myoglobin as performed in step 2.4.3.for quantitation, use quantitation software to integrate the peak areas for each transition of interest, as outlined in step 2.4.9.for identification of species in a mixture, record those marker peptides satisfying agreed criteria for numbers of transitions and signal to noise for those transitions.for quantitation, use integrated transition peak areas as agreed from step 2.4.12 and, using percentage by transition peak area, calculate the percentage of myoglobin from the two species in the mixture.use prior knowledge from the literature of likely myoglobin levels in the meats to estimate the relative w / w amounts of two meats present in the sample . For quantitation, use quantitation software to integrate the peak areas for each transition of interest, as outlined in step 2.4.9 . For identification of species in a mixture, record those marker peptides satisfying agreed criteria for numbers of transitions and signal to noise for those transitions . For quantitation, use integrated transition peak areas as agreed from step 2.4.12 and, using percentage by transition peak area, calculate the percentage of myoglobin from the two species in the mixture . Use prior knowledge from the literature of likely myoglobin levels in the meats to estimate the relative w / w amounts of two meats present in the sample . In a single dynamic - mode mrm experiment each programmed transition is recorded separately (as detector counts per sec, cps) over a specified retention time window . Therefore, from all the data collected in one experiment, the peak intensity for each transition can be individually extracted . Then the only finite signal is for the retention time window set for that transition . Outside of the window, the signal for any one transition, for example, 752 1269 from horse (peptide monoisotopic mass 1,501.66 daltons, precursor ion m / z 751.84 daltons, charge state = 2, fragment ion y) typically has to compete only with measurement noise and not from other transition peaks that might perhaps be from other species . The output is therefore a set of clean peaks, one per transition, at a common retention time for those transitions sharing a common precursor ion . Figure 1 shows the output for the set of four transitions 752 (1269, 706, 248, 1366) for a mixture of 1% w / w horse in beef . Since the four transitions displayed are associated with horse, and are absent in samples of pure beef, lamb or pork, these peaks signify the presence of horse . Depending on robustness criteria, a set of two or more transitions each exceeding some specified signal to noise level establishes identification . This figure therefore establishes the presence of horse in the mixture of 1% w / w horse in beef . This indicates a chance match of precursor ion and a single fragment, possibly from an extraneous protein, with those expected from the system and programed into the mass spectrometer . The singular nature of the peak, and its occurrence at an unexpected retention time, is the signature of an accidental transition that can be ignored . Based on a suitable fragment, the ratio of horse to beef transition peak areas, for example, 752 1269 (horse) to 767 1299 (beef), will be proportional to the ratio of actual meats in the mixture . Figure 2 shows a plot of percentage by peak area for these two transitions versus the percentage weight for weight of horse in a mixture of horse with beef . If the percentage transition peak areas match the percentage weight for weight of meat then the slope is 1 . The slope in this plot is 1.03, indicating that, for these transitions and cpcp pair, the transition peak areas give a reliable measure of the relative amounts of the two meats in the mixture . If the horse meat in the sample was twice as rich in myoglobin as the beef then, with other factors unchanged, the slope of the line would be greater than one . The transitions are 752 (1269, 706, 248, 1366), shown in orange, black, blue and green, respectively . The four transition fragments can be denoted y, y, y and y, respectively, where y denotes counting in n amino acids from the peptide c - terminal end . An additional red line denotes the 752 1269 transition for 0% horse, 100% beef for comparison . This figure has been modified from watson et al .. please click here to view a larger version of this figure . Plot of horse in beef, as percent weight for weight, versus horse in beef as percent transition peak area . The plot uses the pair of peptides beef (767) and horse (752) and the y fragment ion for both . If a denotes peak area then the ordinate is 100ah / (ah + ab). The slope of the best fit line (r = 0.99) is 1.03 . This figure has been modified from watson et al .. please click here to view a larger version of this figure . A good target protein needs to have corresponding forms in species of interest, sufficient species - dependent sequence variation, species specificity, and exist in accessible quantities within the organisms . For assessing mixtures that have undergone processing (for example, heat treatment), a protein having a sequence relatively immune to that processing is desirable . Myoglobin is a good candidate for red meats, including cooked red meats, but is not the only possibility . Once the target protein is decided, the most critical part of the protocol is the protein proteolysis . This segment is very helpful but not essential . Although corresponding peptide pairs from two species of interest can be listed even without experiment, it is sometimes the case that a sequence difference has dramatic consequences on the digestion profile . For example, the peptide pair vlgfhg (beef) and elgfqg (horse) give an anomalous quantitation result (manifest as a gradient less than one in figure 2). This is because the latter peptide arises from a relatively suppressed k - e cleavage, causing an under - estimate of the level of horse in the mixture . Often the fragments from two corresponding peptides have identical amino acid sequences and are well - behaved, but this is not always the case and needs to be checked during method development . Additional meat species can be included, though the quality of the transition peak shape may deteriorate if too many marker peptides co - elute, effectively reducing the dwell time and ultimately degrading relative quantitation estimates . For example, horse, donkey and zebra myoglobins are identical and thus strictly speaking the method is only capable of detecting horse or donkey or zebra in beef . In some cases, even though myoglobins are not identical, some key peptides can be . For example, some lamb myoglobin - derived marker peptides also appear in goat . A complication facing this and any other protein - based quantitation method is that the protein level must be assumed constant across all species if the protein or peptide levels are to equate trivially to levels of meats in a mixture . For myoglobin and the four red meats the levels in general are species dependent, with pork exhibiting the lowest level of the four . So although ratios of transition peak areas map reliably to ratios of myoglobin, the mapping to ratio of actual meats is an estimate drawing on assumptions regarding likely sources of the meats in the mixture . The approach outlined in this work differs in a number of ways from other published contributions . A more typical route is to use proteomic methods to identify various disparate species - dependent marker peptides, in which case the markers for different species possess no particular relationship with one another . By contrast, we have selected proteins common to all species of interest up to species - dependent sequence variants . Apart from being central to our relative quantitation strategy, this has the advantage that sample preparation strategies can be optimized . In addition, such corresponding proteins might be expected to behave similarly, for example, in extraction or in commercial processing of samples such as cooking or canning . Species identification then normally proceeds via detection of disparate marker peptides, whereas in the cpcp approach species identification proceeds via detection of closely related peptides possessing typically one or two sequence differences . Finally, quantitation of proteins to estimate the percent by weight of one species in another might conventionally proceed via absolute quantitation of each protein separately based on known standards . However using the cpcp method, relative levels are estimated by comparing signal strengths of two corresponding peptides from the two species, bypassing the absolute measurement stage altogether . Since the ultimate goal is a percentage by weight of one species in another, a relative quantitation, then the cpcp is both more direct and simpler than comparing two absolute quantitation measurements . These features translate into short experimental times, anticipated to be approximately two hr using refined protocols, making the technique useful as a rapid surveillance tool in the realm of food fraud detection.
A maximum mouth opening that is smaller than the size of a complete denture can make prosthetic treatment challenging . This article describes a simple technique used to fabricate maxillary and mandibular custom sectional impression trays for making definitive impressions in patients with microstomia . Microstomia is defined as an abnormally small oral orifice.1 this disorder is described as a reduction in the oral aperture size associated with facial burns, diffuse scleroderma, traumatic injuries, and surgical reconstruction involving the orbicularis oris muscle . Microstomia can result in multiple debilitating sequelae such as inability to masticate, droolin g, speech problems due to poor articulation, impaired delivery of oral hygiene and dental care, and psychological problems secondary to facial disfigurement.28 several methods of prosthodontic treatment for microstomia patients have been presented, and numerous devices to expand the oral commissure have been described.3,912 the prosthetic rehabilitation of microstomia patients presents difficulties at all stages, from preliminary impressions to prosthesis fabrication.13 because such patients have small oral openings, using conventional methods for making definitive dental impressions and fabricating dentures may be extremely difficult . Making the accurate impressions represents the initial difficulty in the prosthetic rehabilitation of such patients . The recommended techniques for obtaining preliminary impressions for microstomia patients include the use of modeling plastic impression compound, the use of stock impression trays with heavy and light body silicone impression materials, and flexible impression trays with silicone putty . The casts obtained from these preliminary impressions are then used for making custom sectional impression trays . These trays typically consist of 2 locking devices or assemblies, 1 situated anteriorly and the other posteriorly, which join and provide stability to both the sections of the trays . After the definitive impressions are made, these trays are reassembled extraorally and are poured in dental stone to obtain definitive, working casts . Several studies have described various techniques that are used for making custom sectional impression trays.1418 different devices used for connecting the custom sectional trays include hinges,14 plastic building blocks (lego; lego systems inc, enfield, conn ., usa),15,16 orthodontic expansion screws,17 or locking levers.18 this article describes a simple, cost - effective, and time - saving method for fabricating custom sectional impression trays using easily available dual die - pins and sleeves as potential devices for interlocking the sectional trays . The locking mechanism design includes an anterior locking assembly for the maxillary and mandibular custom trays and a posterior locking assembly for only the maxillary custom tray . By using the conventional method, fabricate the maxillary and mandibular custom sectional impression trays by using autopolymerizing acrylic resin (dpi - rr, dental products of india, mumbai, india) on the preliminary casts . Make the handles of the trays (minimum dimensions with 13 mm height, 10 mm length, and 10 mm width) such that they incorporate the metal sleeves of the dual die - pins (m.r . Section both the custom impression trays at the midline by using a diamond disk (dfs, germany). Steps in the fabrication of the anterior lock assembly in the maxillary and mandibular custom sectional impression trays: - the assembly basically consists of 2 dual die - pins and 2 sleeves . Closely juxtapose the 2 sleeves such that the smaller keyway of 1 sleeve faces the larger keyway of the other sleeve . Join these sleeves by inserting the 2 dual die - pins and making the assembly a rigid joint (figure 1a d). - make a slot on the inside portion of the handles on each half of the sectioned custom trays to incorporate the sleeve (figure 2a & b). - attach the sleeves in the slots by using the autopolymerizing acrylic resin as mentioned previously (figure 3). - verify the position of the attached sleeves by inserting die - pins such that the halves juxtapose precisely in both the sectioned trays . - fabricate an anterior assembly for the mandibular sectional tray in a similar manner (figure 7). Steps in the fabrication of the posterior lock assembly in the maxillary custom sectional tray: - cut the 2 dual die - pins that are attached to sleeves halfway through their heights (figure 4) by using a carborundum disk (dentorium, new york, usa). Only the broader upper halves are used for fabricating the posterior assembly and the lower halves are discarded . - attach the half - cut sleeves on the posterior parts of the sectioned custom tray halves using acrylic resin such that the greatest dimension of each sleeve is oriented in the anteroposterior direction . Check for parallelism between the sleeves using a dental surveyor and verify the fit of the die - pins in the respective sleeves (figure 5). - fabricate an acrylic resin block (6 mm height, 10 mm width, and 4 mm longer than the distance between the 2 attached sleeves) using the autopolymerizing acrylic resin (figure 4). - transfer the respective points of the half - cut die - pin heads on the acrylic bar by using a pressure spot indicator (coltene psi, switzerland) and drill slightly oversized holes in the acrylic bar on the marked points by using a bur (261-ef023, brasseler, usa) - with the closely juxtaposed sectioned trays and the die - pins of the anterior assembly placed in position, secure the heads of the half - cut die - pins into the holes by using the autopolymerizing acrylic resin (figure 5 & figure 6). The 2-piece custom - made tray described above allows for a functional impression to be made despite the difficulties associated with microstomia . The anterior locking assembly described in this article does not require any special alteration in the conventional custom tray design . The die - pins with metal sleeves used in this technique provide a greater degree of stability and a precise union of the 2 sections of the tray . The joint can be made more rigid by activating the 2 prongs of each dual die - pin away from each other . This technique used for fabricating custom sectional impression trays does not require any special devices or complex locking joints . The only additional materials used are the dual die - pins which are commercially available at a minimum cost . It is often difficult to use conventional methods for fabricating dentures for patients with limited mouth opening . This article described a simple, time - saving, and cost - effective method used to fabricate custom sectional impression trays for making definitive impressions in patients with microstomia.
It has been roughly half a century since lejeune et al1 first described down syndrome in 1959 . While the technology employed for prenatal detection of down syndrome has expanded in leaps and bounds, the primary focus of prenatal care remains the same: to offer women the most thorough risk assessment with the least invasive procedure possible . Down syndrome is the leading cause of prenatal chromosome abnormalities, accounting for 53% of all reported chromosome conditions.2 testing strategies, guidelines, and screening options have expanded from their conception in the 1970s . At that time, any woman aged 35 years or older was considered to be of advanced maternal age, and this was the sole criteria used by the american congress of obstetricians and gynecologists to define pregnancies that should be offered amniocentesis or chorionic villus sampling . As of 2007, the american congress of obstetricians and gynecologists has defined a pregnancy as high - risk when any of the following criteria are met: family history of aneuploidy, advanced maternal age, abnormal serum screen, or abnormal ultrasound findings.3,4 while multiple screening options are currently available, the only diagnostic tests offered prenatally for down syndrome are amniocentesis and chorionic villus sampling . This review compares the risks, benefits, and limitations of all currently available prenatal screening methods for detection of down syndrome . Anatomical ultrasound has been used since the 1980s to provide health care practitioners and expectant mothers with information regarding a pregnancy.5 with advances in technology, prenatal sonography has expanded from focusing on detection of major structural abnormalities (ie, cardiac defects, hydrops, duodenal atresia, or cystic hygroma) to include detection of soft markers to assist in identifying pregnancies that are at risk of various chromosomal conditions . Bricker et al6 have defined soft markers as structural changes detected at ultrasound scan which may be transient and in themselves have little or no pathological significance, but are thought to be more commonly found in fetuses with congenital abnormalities, particularly karyotypic abnormalities.46 soft markers that have been linked to down syndrome include nuchal thickening, echogenic intracardiac focus, echogenic bowel, renal pelvic dilation, shortened long bones, absence of the nasal bone, pyelectasis, ventriculomegaly, clinodactyly, and sandal gap toe.5,7,8 in the absence of soft markers, the sensitivity of anatomical ultrasound to detect down syndrome is relatively low at 50% . However, the presence of one soft marker is associated with an increased risk of down syndrome, ie, one soft marker increases the risk by two - fold and three or more soft markers increases the risk by 100-fold.7 many scoring indices have been created to help maximize sensitivity, while decreasing false - positive rates.7,9,10 these indices incorporate the presence of structural abnormalities and/or soft markers and maternal age to provide physicians with a guideline as to who should be offered more invasive procedures . While these guidelines have assisted in determining the criteria for a positive finding, they remain limited by the quality of the ultrasound and the expertise of the sonographer . Beginning in 1984, multiple marker screening provided physicians with a means of offering an individualized risk for down syndrome without the inherent risk imposed by chorionic villus sampling or amniocentesis (table 1).11,12 this second trimester screening, performed at 1520 weeks gestation, is often referred to as the quad screen because it incorporates maternal age - related risk and four maternal serum biomarkers, ie, alpha - fetoprotein, free beta human chorionic gonadotropin, unconjugated estriol, and dimeric inhibin a levels.11 by combining maternal age with the quad screen, the detection rate is roughly 75% for down syndrome in women younger than 35 years and> 80% in women 35 years and older (with a positive screening rate of 5%).12 it was not until the late 1990s that first trimester screening was introduced as an earlier screening option for the detection of down syndrome . First trimester screening incorporates maternal age, nuchal translucency ultrasonography, and measurement of maternal serum free beta human chorionic gonadotropin and pregnancy - associated plasma protein a.3,4,12,13 collection of blood for biochemical analysis and ultrasound assessment for nuchal translucency is typically performed between 11 and 13 6/7 weeks gestation . Increased nuchal translucency, reduction in pregnancy - associated plasma protein a levels, and an increase in beta human chorionic gonadotropin can be an indication of down syndrome, and assist practitioners in identifying pregnancies at risk for the syndrome . A nuchal translucency measurement by itself has a detection rate for down syndrome of about 70% with a 5% false - positive rate, but when combined with pregnancy - associated plasma protein a and beta human chorionic gonadotropin measurements, detection rates increase to 79%90%, with a 5% false - positive screen rate.11,14 various studies have been conducted to determine the optimal time for performing the first trimester screening, with the goal of providing a maximum detection rate while still maintaining a low false - positive rate.1518 these studies suggest that earlier pregnancy - associated plasma protein a and beta human chorionic gonadotropin measurements taken at 910 weeks gestation, with nuchal translucency measurement taken at 12 weeks gestation, can increase the detection rate to 90%93%, with a 3%5% false - positive rate.15,17,18 a detection rate of 92%95% with a 3%5% false - positive rate can be achieved when pregnancy - associated plasma protein a measurements are done at 910 weeks gestation, with beta human chorionic gonadotropin and nuchal translucency measurements taken at 12 weeks gestation.15 first trimester screening gives women who receive prenatal care prior to 14 weeks gestation the ability to have information sooner than with second trimester screening . If the results reveal an increased risk of fetal aneuploidy, the woman can be offered genetic counseling with the option to choose either first - trimester chorionic villus sampling or second - trimester amniocentesis . Independent sequential screening is defined as independently performed first - trimester and second - trimester screenings, with separate individualized risk assessments given.11,19 although independent sequential screening increases the detection rate from 88%91% to 94%, it also increases the false - positive rate from 5% to 11%.11,19 thus, it is recommended that women who undergo first - trimester screening for aneuploidy should not also have second - trimester serum screening in the same pregnancy . If a higher detection rate is preferred, an integrated or sequential screening test which combines both first - trimester and second - trimester screening is suggested . Integrated screening is defined as the process by which a patient s individualized risk is calculated based on the combination of both the first - trimester and second - trimester screenings.11,19 due to the nature of this screening method, a patient s first - trimester screening results are not disclosed until second - trimester screening is performed and a combined risk based on both screenings can be calculated.11,19 there has been ethical debate regarding integrated screening because the patient s first - trimester screening results are not disclosed until the conclusion of the second - trimester screening . This precludes patients who are at high risk based on first - trimester screening from being offered chorionic villus sampling and having more options available to them . In contrast, patients undergoing stepwise sequential screening are provided with their individual risk once the results from the first - trimester screen are available.11,19 patients who screen positive during first - trimester screening are offered genetic counseling and are given the information regarding chorionic villus sampling and amniocentesis . Patients who screen negative are offered second - trimester screening and are provided with an adjusted risk number that incorporates both the first - trimester and second - trimester results . Incorporation of both the first - trimester and second - trimester screenings in this stepwise manner increases the detection rate to 94%96% while still maintaining a low false - positive rate of 5%6%.11,19 the final type of sequential screening is known as the contingent screening method . This method is similar to stepwise sequential screening in that scores are calculated based on results from both the first - trimester and second - trimester screening.11,19 however, the contingent screening method uses the first - trimester results to classify patients into three subgroups, ie, screen - positive, screen - negative, and borderline.11,19 second - trimester screening is only offered to patients who fall into either the screen - negative or the borderline group . The detection rate for this method is 94%95%, with a false - positive rate of about 5%.11,19 it has been known since the mid 1950s that fetal cells are present in the maternal circulation.20,21 however, low yield (1 fetal cell / ml of maternal blood), inability to develop an efficient enrichment process, and the tendency for fetal dna to disintegrate during chromosome extraction have inhibited utilization of these fetal cells for the development of a noninvasive prenatal test.20,2224 use of noninvasive prenatal testing targeted to fetal cells was finally abandoned when bianchi et al25 demonstrated that fetal cells could remain in the maternal circulation for decades, thus making it impossible to distinguish new fetal cells from those of previous pregnancies . It was not until 1997 when lo et al26 demonstrated the existence of cell - free fetal dna in the maternal circulation that the scientific community was presented with a new possible target for noninvasive prenatal testing . Fetal cells, theorized to be derived from the placenta, enter the maternal circulation where they undergo apoptosis . In the process, fetal dna is cleaved into small 150200 base pairs fragments, which are then released into the maternal bloodstream.20 these cell - free fetal dna fragments can be detected as early as the fourth week of gestation and reliably after the seventh week of gestation.12,20,23 unlike the longevity seen with fetal cells, cell - free fetal dna has a half - life of only 16 minutes and is cleared from the maternal circulation within 2 hours of delivery.27 the low false - positive rate as well as the prevalence of cell - free fetal dna in the maternal circulation (accounting for> 10% of all cell - free dna or 1680 fetal genomes / ml of maternal blood), made cell - free fetal dna a desirable target for noninvasive prenatal testing.12,20,23,28,29 over the next 15 years, scientists would work on developing and refining methods of detecting pregnancies at risk for aneuploidy . In october, 2011, sequenom inc (san diego, ca, usa) was the first company to make a noninvasive pre - natal test commercially available for the detection of down syndrome . To date, three companies (verinata health [redwood, ca, usa], ariosa diagnostics [san jose, ca, usa], and sequenom inc) offer a noninvasive prenatal test for the detection of trisomies 13 and 18 and down syndrome, with a fourth company (natera, san carlo, ca, usa) expected to have a commercially available noninvasive prenatal test within the next few months (figure 1). Figure 1 describes the various strategies employed by the four companies to obtain and analyze maternal and/or fetal dna from the maternal serum sample . Two companies, sequenom inc and verinata health, utilize massively parallel shotgun sequencing, commonly referred to as shotgun sequencing,3031 while the other two companies, natera and ariosa diagnostics, utilize targeted sequence analysis.3336 the main difference between these two technologies is that shotgun sequencing results in amplification of all genetic information, while targeted sequencing results in amplification of only the genetic information of interest, ie, chromosome(s). Targeted sequencing enables companies such as natera and ariosa diagnostics to perform noninvasive prenatal testing on smaller sample sizes compared with sequenom inc and verinata health . Massively parallel shotgun sequencing technology requires more reads (or dna fragments) than those required by targeted sequencing to ensure that there are enough fragments from the chromosomes of interest to provide accurate results . A major disadvantage of targeted sequencing is the inability to rule out other chromosomal conditions, such as microduplications and deletions, because they have not been selected for . While this does not seem to be a major limiting factor at this time, due to the fact that companies are not currently reporting nonvalidated findings, it may become a shortcoming in the future as companies aim to expand noninvasive prenatal testing to include other chromosomal abnormalities, such as microdeletions and microduplications . All four companies currently use variations of two methodologies for analyzing genetic data, ie, quantitative read counting or single nucleotide polymorphism detection . Three of the companies (sequenom inc, verinata health, and ariosa diagnostics) use quantitative read counting, while natera uses bioinformatic algorithms to analyze single nucleotide polymorphism data obtained by next - generation aneuploidy testing using single nucleotide polymorphisms . Quantitative read counting analyzes the number of chromosome fragments present from each chromosome or chromosomes of interest . Because the amount of genetic material from each chromosome is directly proportional to the chromosome size, companies can use a known euploid reference sample to calculate the expected proportion of genetic information from each chromosome given a euploid pregnancy.31,32,3739 for example, chromosome 21 generally accounts for 1.5% of the human genome.31,3739 any change in the actual proportions of genetic information from chromosome 21 that is 2.53.1 standard deviations from the mean is determined to be an aneuploidy.30,31,40 the benefit of this technology is that it does not require any differentiation between maternal and fetal genetic information . However, this is also a limitation resulting from the fact that, in a fetus with down syndrome, the contribution of genetic information from chromosome 21 increases from 1.5% to 2.25%, for an overall change of 0.75% . When taking into account the fact that fetal dna only represents 10% of the total dna in the sample, the overall change in the amount of chromosome 21 cell - free - dna would only increase from 1.5% to 1.575% . When the fetal fraction is below 10%, it can result in inconclusive results, or a no - call . One company using quantitative read counting, ariosa diagnostics, has aimed to address this issue by creating the forte (fraction optimized risk of trisomy evaluation) assay.33,34 this assay utilizes polymorphic and nonpolymorphic regions that are known to differ between fetal and maternal dna to determine the fetal fraction as well as the overall proportional representation of chromosomal fragments . Unlike quantitative read counting, the next - generation aneuploidy testing using single nucleotide polymorphisms system created by natera utilizes the parental support statistical algorithm to analyze the sequenced fetal cell - free dna together with maternal genetic information, paternal genetic information, and hapmap technology.35,36 this algorithm provides a series of possible hypotheses of fetal genotypes (eg, monosomy, disomy, trisomy) based on known common crossover points on the chromosome(s) of interest, parental information, fetal fraction, and fetal chromosome copy number . The sample - specific confidence interval for each hypothesis is calculated, and a hypothesis is considered confirmed when the confidence interval is above 98%.36 while the results seen from these four companies are promising (table 2), it is important to remember that noninvasive prenatal testing is for screening and is not a diagnostic tool.30,32,35,36,41 noninvasive prenatal test results group patients into three possible categories, ie, low risk of aneuploidy, high risk of aneuploidy, or no - call (undeterminable). The clinical implications for low - risk and high - risk populations are the same for noninvasive prenatal testing as they are for first - trimester screening and multiple marker screening . Individuals who are determined to be in the low - risk category would not require any further diagnostic evaluation, while those in the high - risk category are recommended to undergo diagnostic testing (ie, chorionic villus sampling or amniocentesis). Individuals who fall into the third category of no - call would require a repeat sample to be drawn . For these reasons, noninvasive prenatal testing is not intended to serve as a replacement for amniocentesis or chorionic villus sampling, but rather a methodology that would enable fewer unnecessary diagnostic procedures by detecting patients who are at high risk for aneuploidy more accurately, when compared with the detection rates for first - trimester screening and multiple marker screening . Physicians should ensure that their patients understand that these tests are not designed to circumvent an unwanted diagnostic procedure, and in actuality may result in the recommendation of such a diagnostic procedure should test results reveal a high risk for aneuploidy . While noninvasive prenatal tests do have an increased specificity and sensitivity compared with first - trimester screening or multiple marker screening, limitations do exist . Possible causes for false - positive results could be placental mosaicism, vanishing twin syndrome, or an unidentified maternal condition, such as mosaicism or cancer . Secondly, the majority of noninvasive prenatal tests are currently only offered to women with singleton pregnancies who are at high risk for down syndrome because of family history of aneuploidy, advanced maternal age, an abnormal serum screen, or abnormal ultrasound findings . With the exception of ariosa diagnostics, which has conducted validation studies for harmony in the low - risk population, it is important to note that these tests have not been validated in low - risk or multiple gestation populations, and their accuracy is unknown at this time.42 anatomical ultrasound has been used since the 1980s to provide health care practitioners and expectant mothers with information regarding a pregnancy.5 with advances in technology, prenatal sonography has expanded from focusing on detection of major structural abnormalities (ie, cardiac defects, hydrops, duodenal atresia, or cystic hygroma) to include detection of soft markers to assist in identifying pregnancies that are at risk of various chromosomal conditions . Bricker et al6 have defined soft markers as structural changes detected at ultrasound scan which may be transient and in themselves have little or no pathological significance, but are thought to be more commonly found in fetuses with congenital abnormalities, particularly karyotypic abnormalities.46 soft markers that have been linked to down syndrome include nuchal thickening, echogenic intracardiac focus, echogenic bowel, renal pelvic dilation, shortened long bones, absence of the nasal bone, pyelectasis, ventriculomegaly, clinodactyly, and sandal gap toe.5,7,8 in the absence of soft markers, the sensitivity of anatomical ultrasound to detect down syndrome is relatively low at 50% . However, the presence of one soft marker is associated with an increased risk of down syndrome, ie, one soft marker increases the risk by two - fold and three or more soft markers increases the risk by 100-fold.7 many scoring indices have been created to help maximize sensitivity, while decreasing false - positive rates.7,9,10 these indices incorporate the presence of structural abnormalities and/or soft markers and maternal age to provide physicians with a guideline as to who should be offered more invasive procedures . While these guidelines have assisted in determining the criteria for a positive finding, they remain limited by the quality of the ultrasound and the expertise of the sonographer . Beginning in 1984, multiple marker screening provided physicians with a means of offering an individualized risk for down syndrome without the inherent risk imposed by chorionic villus sampling or amniocentesis (table 1).11,12 this second trimester screening, performed at 1520 weeks gestation, is often referred to as the quad screen because it incorporates maternal age - related risk and four maternal serum biomarkers, ie, alpha - fetoprotein, free beta human chorionic gonadotropin, unconjugated estriol, and dimeric inhibin a levels.11 by combining maternal age with the quad screen, the detection rate is roughly 75% for down syndrome in women younger than 35 years and> 80% in women 35 years and older (with a positive screening rate of 5%).12 it was not until the late 1990s that first trimester screening was introduced as an earlier screening option for the detection of down syndrome . First trimester screening incorporates maternal age, nuchal translucency ultrasonography, and measurement of maternal serum free beta human chorionic gonadotropin and pregnancy - associated plasma protein a.3,4,12,13 collection of blood for biochemical analysis and ultrasound assessment for nuchal translucency is typically performed between 11 and 13 6/7 weeks gestation . Increased nuchal translucency, reduction in pregnancy - associated plasma protein a levels, and an increase in beta human chorionic gonadotropin can be an indication of down syndrome, and assist practitioners in identifying pregnancies at risk for the syndrome . A nuchal translucency measurement by itself has a detection rate for down syndrome of about 70% with a 5% false - positive rate, but when combined with pregnancy - associated plasma protein a and beta human chorionic gonadotropin measurements, detection rates increase to 79%90%, with a 5% false - positive screen rate.11,14 various studies have been conducted to determine the optimal time for performing the first trimester screening, with the goal of providing a maximum detection rate while still maintaining a low false - positive rate.1518 these studies suggest that earlier pregnancy - associated plasma protein a and beta human chorionic gonadotropin measurements taken at 910 weeks gestation, with nuchal translucency measurement taken at 12 weeks gestation, can increase the detection rate to 90%93%, with a 3%5% false - positive rate.15,17,18 a detection rate of 92%95% with a 3%5% false - positive rate can be achieved when pregnancy - associated plasma protein a measurements are done at 910 weeks gestation, with beta human chorionic gonadotropin and nuchal translucency measurements taken at 12 weeks gestation.15 first trimester screening gives women who receive prenatal care prior to 14 weeks gestation the ability to have information sooner than with second trimester screening . If the results reveal an increased risk of fetal aneuploidy, the woman can be offered genetic counseling with the option to choose either first - trimester chorionic villus sampling or second - trimester amniocentesis . Independent sequential screening is defined as independently performed first - trimester and second - trimester screenings, with separate individualized risk assessments given.11,19 although independent sequential screening increases the detection rate from 88%91% to 94%, it also increases the false - positive rate from 5% to 11%.11,19 thus, it is recommended that women who undergo first - trimester screening for aneuploidy should not also have second - trimester serum screening in the same pregnancy . If a higher detection rate is preferred, an integrated or sequential screening test which combines both first - trimester and second - trimester screening is suggested . Integrated screening is defined as the process by which a patient s individualized risk is calculated based on the combination of both the first - trimester and second - trimester screenings.11,19 due to the nature of this screening method, a patient s first - trimester screening results are not disclosed until second - trimester screening is performed and a combined risk based on both screenings can be calculated.11,19 there has been ethical debate regarding integrated screening because the patient s first - trimester screening results are not disclosed until the conclusion of the second - trimester screening . This precludes patients who are at high risk based on first - trimester screening from being offered chorionic villus sampling and having more options available to them . In contrast, patients undergoing stepwise sequential screening are provided with their individual risk once the results from the first - trimester screen are available.11,19 patients who screen positive during first - trimester screening are offered genetic counseling and are given the information regarding chorionic villus sampling and amniocentesis . Patients who screen negative are offered second - trimester screening and are provided with an adjusted risk number that incorporates both the first - trimester and second - trimester results . Incorporation of both the first - trimester and second - trimester screenings in this stepwise manner increases the detection rate to 94%96% while still maintaining a low false - positive rate of 5%6%.11,19 the final type of sequential screening is known as the contingent screening method . This method is similar to stepwise sequential screening in that scores are calculated based on results from both the first - trimester and second - trimester screening.11,19 however, the contingent screening method uses the first - trimester results to classify patients into three subgroups, ie, screen - positive, screen - negative, and borderline.11,19 second - trimester screening is only offered to patients who fall into either the screen - negative or the borderline group . The detection rate for this method is 94%95%, with a false - positive rate of about 5%.11,19 it has been known since the mid 1950s that fetal cells are present in the maternal circulation.20,21 however, low yield (1 fetal cell / ml of maternal blood), inability to develop an efficient enrichment process, and the tendency for fetal dna to disintegrate during chromosome extraction have inhibited utilization of these fetal cells for the development of a noninvasive prenatal test.20,2224 use of noninvasive prenatal testing targeted to fetal cells was finally abandoned when bianchi et al25 demonstrated that fetal cells could remain in the maternal circulation for decades, thus making it impossible to distinguish new fetal cells from those of previous pregnancies . It was not until 1997 when lo et al26 demonstrated the existence of cell - free fetal dna in the maternal circulation that the scientific community was presented with a new possible target for noninvasive prenatal testing . Fetal cells, theorized to be derived from the placenta, enter the maternal circulation where they undergo apoptosis . In the process, fetal dna is cleaved into small 150200 base pairs fragments, which are then released into the maternal bloodstream.20 these cell - free fetal dna fragments can be detected as early as the fourth week of gestation and reliably after the seventh week of gestation.12,20,23 unlike the longevity seen with fetal cells, cell - free fetal dna has a half - life of only 16 minutes and is cleared from the maternal circulation within 2 hours of delivery.27 the low false - positive rate as well as the prevalence of cell - free fetal dna in the maternal circulation (accounting for> 10% of all cell - free dna or 1680 fetal genomes / ml of maternal blood), made cell - free fetal dna a desirable target for noninvasive prenatal testing.12,20,23,28,29 over the next 15 years, scientists would work on developing and refining methods of detecting pregnancies at risk for aneuploidy . In october, 2011, sequenom inc (san diego, ca, usa) was the first company to make a noninvasive pre - natal test commercially available for the detection of down syndrome . To date, three companies (verinata health [redwood, ca, usa], ariosa diagnostics [san jose, ca, usa], and sequenom inc) offer a noninvasive prenatal test for the detection of trisomies 13 and 18 and down syndrome, with a fourth company (natera, san carlo, ca, usa) expected to have a commercially available noninvasive prenatal test within the next few months (figure 1). Figure 1 describes the various strategies employed by the four companies to obtain and analyze maternal and/or fetal dna from the maternal serum sample . Two companies, sequenom inc and verinata health, utilize massively parallel shotgun sequencing, commonly referred to as shotgun sequencing,3031 while the other two companies, natera and ariosa diagnostics, utilize targeted sequence analysis.3336 the main difference between these two technologies is that shotgun sequencing results in amplification of all genetic information, while targeted sequencing results in amplification of only the genetic information of interest, ie, chromosome(s). Targeted sequencing enables companies such as natera and ariosa diagnostics to perform noninvasive prenatal testing on smaller sample sizes compared with sequenom inc and verinata health . Massively parallel shotgun sequencing technology requires more reads (or dna fragments) than those required by targeted sequencing to ensure that there are enough fragments from the chromosomes of interest to provide accurate results . A major disadvantage of targeted sequencing is the inability to rule out other chromosomal conditions, such as microduplications and deletions, because they have not been selected for . While this does not seem to be a major limiting factor at this time, due to the fact that companies are not currently reporting nonvalidated findings, it may become a shortcoming in the future as companies aim to expand noninvasive prenatal testing to include other chromosomal abnormalities, such as microdeletions and microduplications . All four companies currently use variations of two methodologies for analyzing genetic data, ie, quantitative read counting or single nucleotide polymorphism detection . Three of the companies (sequenom inc, verinata health, and ariosa diagnostics) use quantitative read counting, while natera uses bioinformatic algorithms to analyze single nucleotide polymorphism data obtained by next - generation aneuploidy testing using single nucleotide polymorphisms . Quantitative read counting analyzes the number of chromosome fragments present from each chromosome or chromosomes of interest . Because the amount of genetic material from each chromosome is directly proportional to the chromosome size, companies can use a known euploid reference sample to calculate the expected proportion of genetic information from each chromosome given a euploid pregnancy.31,32,3739 for example, chromosome 21 generally accounts for 1.5% of the human genome.31,3739 any change in the actual proportions of genetic information from chromosome 21 that is 2.53.1 standard deviations from the mean is determined to be an aneuploidy.30,31,40 the benefit of this technology is that it does not require any differentiation between maternal and fetal genetic information . However, this is also a limitation resulting from the fact that, in a fetus with down syndrome, the contribution of genetic information from chromosome 21 increases from 1.5% to 2.25%, for an overall change of 0.75% . When taking into account the fact that fetal dna only represents 10% of the total dna in the sample, the overall change in the amount of chromosome 21 cell - free - dna would only increase from 1.5% to 1.575% . When the fetal fraction is below 10%, it can result in inconclusive results, or a no - call . One company using quantitative read counting, ariosa diagnostics, has aimed to address this issue by creating the forte (fraction optimized risk of trisomy evaluation) assay.33,34 this assay utilizes polymorphic and nonpolymorphic regions that are known to differ between fetal and maternal dna to determine the fetal fraction as well as the overall proportional representation of chromosomal fragments . Unlike quantitative read counting, the next - generation aneuploidy testing using single nucleotide polymorphisms system created by natera utilizes the parental support statistical algorithm to analyze the sequenced fetal cell - free dna together with maternal genetic information, paternal genetic information, and hapmap technology.35,36 this algorithm provides a series of possible hypotheses of fetal genotypes (eg, monosomy, disomy, trisomy) based on known common crossover points on the chromosome(s) of interest, parental information, fetal fraction, and fetal chromosome copy number . The sample - specific confidence interval for each hypothesis is calculated, and a hypothesis is considered confirmed when the confidence interval is above 98%.36 while the results seen from these four companies are promising (table 2), it is important to remember that noninvasive prenatal testing is for screening and is not a diagnostic tool.30,32,35,36,41 noninvasive prenatal test results group patients into three possible categories, ie, low risk of aneuploidy, high risk of aneuploidy, or no - call (undeterminable). The clinical implications for low - risk and high - risk populations are the same for noninvasive prenatal testing as they are for first - trimester screening and multiple marker screening . Individuals who are determined to be in the low - risk category would not require any further diagnostic evaluation, while those in the high - risk category are recommended to undergo diagnostic testing (ie, chorionic villus sampling or amniocentesis). Individuals who fall into the third category of no - call would require a repeat sample to be drawn . For these reasons, noninvasive prenatal testing is not intended to serve as a replacement for amniocentesis or chorionic villus sampling, but rather a methodology that would enable fewer unnecessary diagnostic procedures by detecting patients who are at high risk for aneuploidy more accurately, when compared with the detection rates for first - trimester screening and multiple marker screening . Physicians should ensure that their patients understand that these tests are not designed to circumvent an unwanted diagnostic procedure, and in actuality may result in the recommendation of such a diagnostic procedure should test results reveal a high risk for aneuploidy . While noninvasive prenatal tests do have an increased specificity and sensitivity compared with first - trimester screening or multiple marker screening possible causes for false - positive results could be placental mosaicism, vanishing twin syndrome, or an unidentified maternal condition, such as mosaicism or cancer . Secondly, the majority of noninvasive prenatal tests are currently only offered to women with singleton pregnancies who are at high risk for down syndrome because of family history of aneuploidy, advanced maternal age, an abnormal serum screen, or abnormal ultrasound findings . With the exception of ariosa diagnostics, which has conducted validation studies for harmony in the low - risk population, it is important to note that these tests have not been validated in low - risk or multiple gestation populations, and their accuracy is unknown at this time.42 it has long been a goal of prenatal genetic diagnosis to develop a noninvasive test that would allow for detection of aneuploidy and eliminate the need for invasive testing, such as chorionic villus sampling or amniocentesis . While this is still not currently possible, developments in noninvasive prenatal testing are the initial steps in this direction . While the scope of this review does not include other aneuploidy conditions, all of the companies discussed do offer screening for trisomy 13 and 18, with varying degrees of success . The noninvasive prenatal tests marketed by verinata health and natera also include a screen for sex chromosome abnormalities, such as turner syndrome, kleinfelter syndrome, and 47, xyy syndrome . Validation studies are currently underway for noninvasive prenatal testing in the detection of fetal aneuploidies in the general population as well as multiple gestations . Furthermore, the future of noninvasive prenatal testing may expand beyond the common aneuploidies to include other chromosomal conditions, such as microdeletions and microduplications . With the ever - expanding testing options that are now available to the expectant mother, it is imperative that physicians remain up to date on these technologies and have a clear understanding of the risks, benefits, and limitations of these technologies . By fully understanding each technology and the possible alternatives, physicians can provide their patients with all the information necessary to make an informed decision regarding medical management.
Heterogenous vancomycin intermediate staphylococcus aureus (hvisa) strains have been reported as indicators for reduced vancomycin susceptibility in s. aureus, and various studies associated its presence with vancomycin treatment failure . It has been shown that methicillin resistant s. aureus (mrsa) has the propensity to evolve into hvisa phenotype during in vitro exposure to subinhibitory concentrations of vancomycin . During the last decade, hvisas had been isolated in many countries including those in south east asia; nevertheless, in our knowledge, its emergence has not been reported in malaysia . As a pilot study, we investigated the prevalence of hvisa among mrsa strains isolated at hospital kuala lumpur (hkl) in a 3-month period and determined factors associated with its infections . Hospital kuala lumpur is the largest hospital in malaysia with the highest mrsa burden in the country . In this hospital, vancomycin is used as the standard first line treatment for mrsa infection; however, recently, its efficacy has been a subject of discussion due to several anecdotal vancomycin treatment failure cases in hospital kuala lumpur . We also wondered if some of the mrsas isolated in the hospital were actually hvisas with reduced susceptibilities to vancomycin that could not be detected by routine microbiological tests used in our hospital diagnostic laboratory . To investigate this, from 25 february to 25 may 2009, we collected a total of 320 index mrsa isolates (first mrsa isolated from the corresponding patients) and established them as strains for vancomycin resistance testing . As it is cost, time and labor consuming to perform vancomycin population analysis on all 320 strains to test for heterogenous vancomycin resistance, strains were first screened for the phenotype using glycopeptide resistance detection (grd) etest antibiotic strips (ab biodisk, sweden). After grd screening, a total of 8 strains were defined as presumptive hvisa, no visa strain was detected . Following that, to confirm the results of the grd screening interestingly, area under the curve (auc) analyses of the strains' population analysis profiles confirmed that 7 out of the 8 tested strains were hvisa (table 1), giving a prevalence rate of 2.19% . Table 1heterogenous vancomycin intermediate staphylococcus aureus strains and their corresponding patients in this study.specimen noagegenderprimary diagnosisspecimendiabetes mellitusrenal failuremalignancyadmission to icudays of hospital staydays of iv vcmon beta - lactamarea under curve ratio68214maleright hip osteomyelitisnasal swabnononoyes44 days0yes0.9058253femaleleft diabetic foot ulcerpus swabyesyesnoyes52 days14yes0.9318258maleacute ventriculitiscsfyesnonoyes98 days14yes1.0125220malegluteal sarcoma with hapsputumnonoyesyes24 days10yes0.9097871malepemphigus folliaceouspus swabyesnonono34 days7yes1.012154femaleacute encephalitis with haptracheal aspiratenononoyes32 days14yes0.9646029femalemeningo - encephalitis with hapsputumnononoyes38 days10yes0.98icu, intensive care unit; iv, intravenous; vcm, vancomycin; csf, cerebrospinal fluid; hap, hospital acquired pneumonia . Icu, intensive care unit; iv, intravenous; vcm, vancomycin; csf, cerebrospinal fluid; hap, hospital acquired pneumonia . All hvisa strains isolated in this study were hospital acquired as they were isolated from their corresponding patients after 48 hours of hospital admission . To determine factors associated with the 7 hvisa infections, demographic data of all corresponding patients of each index mrsa isolate were retrieved from medical records . Medical history of each patient such as diabetes mellitus, renal failure, malignancy, together with prescription history of vancomycin and beta - lactam antibiotics (as these were the only classes of antibiotics prescribed to the corresponding patients of the study isolates during this investigation), length of hospitalization and intensive care unit (icu) admission were recorded . Continuous variables were then assessed by independent samples t - test, while categorical variables were analyzed using pearson's chisquare . Calculations were performed using statistical package for social science (spss) 12.0 (spss inc ., chicago, usa) where a p - value of <0.05 was considered as significant . After performing multivariate linear regression, we found that icu admission (p<0.004), hospitalization of more than 14 days (p<0.014) and vancomycin administration of more than 7 days (p<0.016) were independent factors associated with hvisa infections in our group of patients . Our findings were in line with those of charles et al . In 2004, where hvisa / visa infections were found to be associated with longer antibiotic treatment periods and longer hospitalization . In a separate report it seems that patients who are severely ill, hospitalized for long durations with icu admissions might have a higher chance of developing hvisa infections . As many patients in hkl fulfill some or all of the above criteria, taking it together, we suspect that the prevalence of hvisa in hkl might be high; however, these strains are not being actively detected by the hospital diagnostics laboratory . As hvisa and mrsa with reduced vancomycin susceptibility has been reported to cause treatment failure, given the hvisa prevalence rate detected in this study, it is not surprising that vancomycin treatment failure cases among mrsa infected patients are increasing in hkl . In our study, we employed the grd test as a screening tool for hvisa before confirming the resistance with population analysis, and found that the grd etest was fairly specific with only one false positive result . In a review, howden and colleagues have reported the test's sensitivity as 9394% with a 8295% specificity for hvisa detection . Therefore, the grd might be considered a good screening tool for hvisa in hospitals where most hospitalized patients are severely ill with long hospitalization durations . Once identified as hvisa infected, optimal treatment could be prescribed to the corresponding patient to prevent vancomycin treatment failure, thereby increasing the chance of a good clinical outcome for the patient . As the strains used in this study were collected in a short span of 3 months, and that vancomycin treatment failure is on the rise in hkl, we suspect that the actual prevalence of hvisa in this hospital might be even higher . We found the grd test useful for hvisa screening, nevertheless pap - auc analysis still remains the gold standard for hvisa confirmation . A more comprehensive, case control study involving major hospitals in the country would be important to better understand the significance and distribution of hvisa in malaysian hospitals.
It exhibits broad bioactivity including inducing apoptosis of scar cells and anti - inflammatory and anticancer [3, 4] properties, which have been used in the traditional chinese medicine for treatment of hypertrophic scar, inflammation, cancer, and other diseases without obvious toxicity or side effect in clinic . Several preparions, hydrogel, lotion, injection, and liposome, for example, have been reported in the literature . We are further interested in the form of liquid crystalline nanoparticles (cubosome), for cubosome consists of a curved bicontinuous lipid bilayer extending in three dimensions and separating two congruent networks of water channels [911], which can enclose hydrophilic, amphiphilic, and hydrophobic substances ranging from low - molecular - weight drugs to proteins, peptides, amino acids, and nucleic acids . Compared to liposomes, cubosomes showed better storing stability at room temperature and could endure heat treatment [1315]. Cubosomes could exist at almost any dilution level in water and drug leakage was less concerned compared with liposome . Reported that cubosomes had a higher permeability coefficient (4.5-fold) compared to eye drops when dexamethasone was used as a model drug . Therefore, we consider that the cubosome might represent a promising vehicle containing matrine for effective ocular drug delivery . Hplc, lc / ms / ms, and esi - qtof - ms / ms methods have been used to determine matrine in samples at present [6, 17, 18]. The aim of this study is to establish and validate a simple, sensitive, and accurate hplc method to determine matrine combined in liquid crystalline nanoparticles . Glycerol monooleate (dimodan mo / d kosher, material number 116703) was kindly provided by danisco cultor (brabrand, denmark) and used as received . Poloxamer 407 (peo98pop67peo98) was a gift from basf (ludwigshafen, germany). Milli - q - grade water purified through a millipore system (elga labwater, sartorius, uk) was used throughout this study . Pbs (ph 6.8) was made according to the chinese pharmacopoeia (2010). Liquid crystal nanoparticles were prepared through the fragmentation of glycerol monooleate / poloxamer 407 bulk cubic gels . Glycerol monooleate (3 g) and poloxamer 407 (300 mg) were first melted at 60c in a hot water bath until they were homogeneous, after which matrine was added to dissolve / blend under continuous stirring . Water (6.7 ml) was then added gradually and the mixture was vortex - mixed to achieve a homogeneous state . After equilibration for 48 hours at room temperature, the cubic phase gel was formed . By adding 20 ml of water, the cubic gel was disrupted by mechanical stirring . Subsequently, the crude dispersion was fragmented for 10 min by intermittent probe sonication (jy-96 iin, ningbo scientz biotechology co., ltd, china) at 200 w energy input using a pulse mode (9-second pulses interrupted by 18-second breaks) under cooling in a 20c water bath . The resulting milky coarse dispersion was homogenized using a high - pressure homogenizer (avestin em - c3, ottawa, canada) at certain high pressures and cycles to obtain an opalescent dispersion of the cubic nanoparticles . The final dispersion of liquid crystal nanoparticles was stored at room temperature for further studies . The hplc analysis was carried out using a shimadzu system that is equipped with an lc-20at pump, spd-20a uv / vis detector connected to shimadzu spin chrome software . The chromatographic assay was performed on a reversed - phase ods - bp c18 column (5 m, 4.6 mm 250 mm) at ambient temperature 25c . The mobile phase under isocratic mode was a mixture of methanol - pbs (ph 6.8)-triethylamine (50: 50: 0.1%, v / v). The mobile phase was degassed by an ultrasonic bath and filtered with 0.45 m membrane under vacuum . All the calculations concerning the quantitative analysis were carried out by an external standard method based on peak areas . To prepare the stock solution, matrine (10 mg) was accurately weighed into 50 ml volumetric flask, made up to volume with methanol, and then the volume was adjusted to 50 ml . This solution was further diluted with methanol to yield solutions containing 100.0, 50.0, 25.0, 12.5, 6.3, 3.1, and 1.6 g / ml . The chromatogram peak area of each known concentration was calculated . Results from each analysis were subjected to regression analysis . 0.2 ml (or 200.0 mg) of the nanoparticles was accurately transferred into a 10 ml volumetric flask, dissolved, and made up to volume with methanol . Then, the sample solutions were filtered using a 0.45 m filter membrane and injected (10 l) into the hplc system three times under optimized chromatographic conditions . The method was validated in terms of parameters of specificity, linearity, sensitivity, accuracy, precision, and reproducibility according to the international conference on harmonisation . The specificity of the method was assessed by comparing chromatograms of matrine working solution, blank excipients sample without matrine, and equal concentrations samples of compound liquid crystalline nanoparticles made as the previous procedure . The linearity of the method was studied by injecting seven known concentrations of the standard in the range of 1.6200 g / ml . The sensitivity of the method was evaluated with limit of detection (lod) and limit of quantification (loq). Lod and loq were established at a signal - to - noise ratio (s / n) of 3 and 10, respectively . The accuracy of the method was tested by comparing the percent analyte recovered by the optimum method at three concentration levels (80.0, 100, and 120.0 g / ml). Intraday precision was determined by injection of standard solutions of matrine at 3 concentration levels (50, 25, and 12.5 g / ml), on the same day . The specificity was evaluated by analyzing blank excipients sample, matrine standard solution, and liquid crystalline nanoparticles samples . The retention times of matrine at a flow rate of 1.0 ml / min was 16.3 min . The calibration curves for matrine were found to be linear within the range of 1.6 to 200.0 g / ml . The regression equation was y = 10706x 2959 (r = 1.0), where y is peak area and x is the concentration (g / ml) of matrine standard solution . The correlation coefficient indicated a good linear relationship between peak area and concentration over a wide range . The lod (signal / noise ratio of 3: 1) was calculated as 1.3 10 g / ml and the loq (signal / noise ratio of 10: 1) was determined as 3.9 10 g / ml . Mean recovery for matrine at three concentration levels (80.0, 100, and 120.0 g / ml) was found to be 102.1 1.9% (rsd = 1.96%, n = 3), 102.6 1.9% (rsd = 2.94%, n = 3), 100.5 2.1% (rsd = 2.12%, n = 3), respectively . The mean concentration was 9.5 mg / ml (rsd = 1.4%, n = 3). Several mobile phase systems including methanol - water, ethanol - wate - kh2po4, and acetonitrile - ethanol - h3po4 systems have been tested in this study . However, the chromatogram of standard matrine might disappear or appear in a wide range with inaccurate calculation area (see figure 2). Matrine crystalline has 4 forms, namely,,,, and matrine . The incorrect mobile phase may change the matrine solution to nanocrystalline because of the solubility . The chromatographic method was eventually carried out using an isocratic system with a mobile phase of methanol - pbs (ph 6.8)-triethylamine (50: 50: 0.1%) applied at a flow rate of 1 ml / min with detection wavelength at 220 nm . Under these optimum mobile phase conditions, elution of analyte was completed in less than 20.0 min and retention time of matrine was 16.3 min . The method was validated according to ich guidelines with the parameters of specificity, linearity, sensitivity, accuracy, precision, and reproducibility . A simple, rapid, selective, and sensitive hplc method has been developed and validated for the determination of matrine when formulated in cubosome particles . The present study is the first report on the matrine determination combined with particle dispersion system . The method can be used for controlling the quality of the cubosome and helpful for further investigation.
However, outcomes of anatomic arthroplasty with an osteoarthritic rotator - cuff - deficient shoulder have been limited . The reverse shoulder arthroplasty (rsa) is a potential solution for shoulder osteoarthritis with deficient rotator cuff . Theoretical advantages of rsa are an increased lever arm of the deltoid muscle through a medialized center of rotation of the prosthesis (increasing deltoid efficiency), increased prosthetic stabilization through humeral lengthening (increasing deltoid tension), and decreased mechanical torque at the glenoid component (decreasing glenoid loosening) these factors are related to the indication for surgery, surgeon's experience, characteristics of the implant, characteristics of the surgical technique, type of approach, or postoperative rehabilitation, among others . Unfortunately, the analysis of outcomes of rsa depending on the type of prosthesis, type of approach, and indication for surgery has not been well reported to date . The purpose of this study was to compare the clinical and functional outcomes of rsa depending on the surgical approach, type of prosthesis, and indication for surgery through a comprehensive, systematic review of the literature . The methodology for this study was reported following the prisma statement for systematic review and meta - analysis . All human studies reporting clinical and/or functional outcomes in patients treated with primary or revision rsa were assessed for eligibility . Studies were included if they had a level of evidence between i and iv, were written in english, had a minimum of 2 years of follow - up and had a minimum sample size of 10 patients . Studies reporting complications only, nonoriginal articles, or studies with insufficient outcome data the authors are not aware of any relevant publication related to rsa before 1985, so the search was limited to this period . The keywords and search strategy employed in this study included the following: (reverse or inverse) and shoulder and (arthroplasty or replacement or prosthesis), limited to human studies published in the above - mentioned period . Thus, cinahl, ebsco - sportdiscus, and the cochrane central register of controlled trials were also used to search for relevant publications in the same period . Articles of potential interest were reviewed in detail (full text) by two authors and a decision was made regarding inclusion or exclusion . Clinical and functional outcomes were extracted from all included studies in a systematic way using a table template by one author, which was then verified by another author . In cases of disagreement between both authors with regard to study inclusion or data extraction, a reference list of all included articles was reviewed to search for potential studies not previously identified . Mean, standard deviation (sd), and range were extracted (whenever provided) in the preoperative and postoperative periods for the following variables: constant score, american shoulder and elbow society (ases) score, simple shoulder test, range of motion (rom), and satisfaction . Furthermore, relevant information regarding level of evidence, type of prosthesis (either with medialized or lateralized center of rotation), type of approach (either deltopectoral or superolateral), indication for rsa, sample size, percentage of females in the sample, follow - up, and age of patients was extracted from all studies . The pubmed search yielded 329 citations, from which 174 were clinical studies in humans that were reviewed in further detail . About 32 met inclusion criteria and additional database searches and review of the reference list from included articles yielded a final number of 35 articles included in the descriptive analysis of clinical outcomes [figure 1]. From all 174 articles assessed for eligibility, the senior author had to review four of them because of disagreement between the two authors conducting the systematic review . Literature search flow chart the 35 included studies were grouped depending on the approach and type of prosthesis: deltopectoral approach associated with prosthesis with a medialized center of rotation (dm group; n = 18 studies), lateralized (dl group; n = 8 studies), and a combination of approaches associated with a medialized prosthesis (cm group; n = 9 studies). The latter group was created because the authors employed different approaches, but clinical outcomes were not specified depending on the type of approach (all these studies employed a prosthesis with a medialized center of rotation). Prostheses with a medialized center of rotation included in this study were the following: delta iii (depuy, france), delta xtend (depuy, warsaw, in, usa), aequalis (tornier, france), smr modular shoulder system (systema multiplana randelli, lima - lto, san daniele de friuli, italy), and exactech (gainesville, fl, usa). Prostheses with a more lateralized center of rotation included in this study were the following: reverse shoulder prosthesis (djo surgical, austin, texas), and arrow anatomical shoulder system (mulhouse, france). The 35 studies included a total sample of 2049 patients with a mean (sd) percentage of females of 71.6% (13.4), age of 71.5 years (3.7), and follow - up of 43.1 months (18.8); the respective data separately in groups was, dm, 1085 patients, 73.4% (10.2), 72.4 years (3.1), and 38.1 months (8.2). Dl, 241 patients, 69.7% (12.5), 70.1 years (1.7), and 40 months (7.6) and cm group, 723 patients, 73.1% (7.5), 73 years (2.8), and 50.6 months (33.9). Clinical outcomes depending on the type of approach - type of prosthesis in the analysis of clinical outcomes depending on the indication for rsa, not all 35 studies could be included because results were not always specified by indication . The number of studies included (total subjects involved) by indications for rsa were cuff tear arthropathy 12 (581); revision of anatomic prosthesis 10 (263); failed rotator cuff repair 5 (150); fracture sequelae 4 (82); rheumatoid arthritis 3 (52); massive cuff tear 2 (68); primary osteoarthritis with degenerative cuff tear 2 (51); posttraumatic osteoarthritis 2 (59); and revision of reverse prosthesis 1 (14). Indications of rsa for tumors and acute fractures were not included due to limited data . Mean (sd) for percentage of females, age, and follow - up depending on indications was the following: cuff tear arthropathy 74% (12), 72.5 years (3.4), and 34.6 months (8), respectively; revision of anatomic prosthesis 66.5% (11.7), 68.2 years (2.7), and 38.2 months (6.6); failed rotator cuff repair 69% (19.8), 69.8 years (3.6), and 39.6 months (11.8); fracture sequelae 70% (8.1), 73.2 years (5.3), and 37.6 months (8.2); and rheumatoid arthritis 87.6% (9.8), 68.2 years (2.9), and 56 months (22.2), respectively . For massive cuff tear, primary osteoarthritis with degenerative rotator cuff, and posttraumatic osteoarthritis only the mean (range) follow - up was provided: 34 months (range 24 - 118), 38 months (range 24 - 81), and 42 months (range 24 - 97), respectively . For revision of reverse prosthesis, percentage of females, and mean (sd) of age and follow - up were 28%, 70.6 years (8.7), and 33 months (11.2), respectively . Table 2 summarizes the clinical outcomes depending on the most common indication for rsa in the included studies . The purpose of this study was to compare the clinical and functional outcomes of rsa depending on the type of prosthesis (with either medialized or lateralized center of rotation), type of approach, and indication for surgery . The principal finding of this study was that both types of prostheses clearly improved the outcomes, but lateralized prostheses had more pre - to - postoperative differences (improvement) for ases total and pain scores and external rotation compared with medialized prostheses . In addition, outcomes depending on each indication considerably improved, but those corresponding to revision of anatomic prosthesis, failed rotator cuff repair, and fracture sequelae demonstrated lower improvements compared to cuff tear arthropathy . The postoperative patient's satisfaction with surgery was very high (overall mean of 90%) in both types of prostheses and for all indications for surgery . To the best of our knowledge, this is the first systematic review aimed to investigate the clinical and functional outcomes of rsa depending on the type of prosthesis, type of approach, and an indication for surgery . Khan et al . Conducted a comprehensive, systematic review aimed to investigate the outcomes of rsa depending for cuff tear arthropathy, massive cuff tear, and rheumatoid arthritis . However, the authors only included delta ii prostheses and the review included studies up to 2010 . The present investigation found many references in the last 2 years and in addition, different type of prostheses, more indications, and a higher number of studies were analyzed . Based on the present study and on the existing literature, rsa is an excellent surgical solution with great improvements in clinical outcomes for cuff tear arthropathy, massive cuff tear, 42 failed rotator cuff repair, rheumatoid arthritis, fracture sequelae, revision of anatomic prosthesis, and revision of reverse prosthesis . In addition, both types of prostheses demonstrated excellent improvements in the postoperative period with regard to all outcomes . The fact that prostheses with lateralized center of rotation had greater improvement in ases and external rotation have to be interpreted with caution, as this study had some limitations . First and foremost, a pooled analysis of the results (meta - analysis with inferential statistics) was not possible for methodological reasons, as nearly all studies did not report the sd in the outcomes and an accurate meta - analysis could not be, therefore, conducted . In addition, most of the studies did not disclose the outcomes depending on the indications for surgery, so this parameter had to be considered separately to avoid a significant decrease in the number of studies included in the comparisons of outcomes . Thus, only the type of prosthesis and type of approach could be analyzed altogether . Second, as almost all included studies were case series, the comparison of outcomes depending on the type of prosthesis, approach, and an indication was indirect in nature with a greater potential influence of uncontrolled variables . Third, the influence of several factors potentially influencing the outcomes could not be assessed because of limited information, heterogeneity of studies, and small number of studies included for some comparisons (which would decrease even more the available data if more subgroups were done). Finally, it must be mentioned that the ases score in medialized prosthesis was only reported by one study, which may decrease the value of the comparison of this parameter between medialized and lateralized prostheses . It must be first recognized that there are a considerable number of potential factors not controlled in this analysis that may have a potential influence on the outcomes: different surgeon's experience, different rehabilitation protocols (given the multicentric nature of this study), type (eccentric or concentric) and size of glenosphere, location and orientation of the glenosphere and humeral components (inferiorly placed glenosphere, anteversion / retroversion, of the humeral component) degree of fatty infiltration of the teres minor muscle, degree of bone stock, soft tissue tensioning, status of the subscapularis muscle, humeral osteotomy angle, or previous surgery . In addition, no attempt was made to analyze data based on differences in humeral components, medialized versus lateralized, high neck angle versus low neck angle, sit - on - top versus sit inside, and cemented versus uncemented . In the present study, the influence of the type of approach on the outcomes of rsa could not be well determined . Some studies used a combination of approaches, and the outcomes were not specified depending on whether the approach was deltopectoral or superolateral . Therefore, some studies were grouped as cm to refer to studies using a combination of approaches (and a medialized prosthesis). In some ways, differences between groups dm and cm may be explained by differences in the type of approach, as a type of prosthesis in both groups has a medialized center of rotation . However, considering that the cm has a combination of approaches rather than a unique superolateral approach, no clear conclusions can be drawn regarding its influence on the outcomes of rsa . There are some studies that have found that the surgical approach does not have an influence on the outcomes of rsa . Clearly, further research is needed in this aspect to better elucidate the influence of the type of approach on the outcomes of rsa . Well - designed level i- or ii - evidence comparative studies are needed before clear conclusions can be established . Nonetheless, the clinical relevance of this research question (influence of the type of approach) may be questioned, as some surgeons may be forced to adopt a certain approach based on the surgical history of the patient or the characteristics of the patient's disorder itself . The type of prosthesis (with a medialized or lateralized center of rotation) seems to have a much more relevant influence on the outcomes of rsa . Given that many studies only employed the deltopectoral approach, the type of prosthesis was more easily isolated . Thus, the comparison between dm and dl may show the influence of the center of rotation on the outcomes . Essentially, both groups demonstrated great improvements in outcomes in the postoperative period . Unfortunately, no studies employing a lateralized center of rotation reported the constant score, so no comparisons were possible between dm and dl for this parameter . In addition, the ases score was only reported by one study in the dm group so no accurate conclusions can be drawn for this parameter . Specifically, the dl group demonstrated greater improvements in external rotation compared to the dm group . The reasons for lower improvement in external rotation in medialized prostheses have been suggested by boileau et al ., and grammont and baulot . A medialized center of rotation may imply that the humeral cup impinges the posterior neck of the scapula when the arm is at the side . In addition, as the posterior deltoid theoretically provides some external rotation when coupled with some abduction, the medialization of the center of rotation may decrease the efficacy of the posterior deltoid to assist in the external rotation . Also, the status of the teres minor may influence the degree of external rotation, but this variable was not controlled in the vast majority of studies . Conducted an interesting study in which the center of rotation of a medialized prosthesis was lateralized by placing a bone autograft from the humeral head between the base plate and the scapula . This bony lateralization of the center of rotation demonstrated good integration and the authors found 53 of external rotation and a constant score of 66 . Unfortunately, this was a case series and comparisons with medialized prostheses were only conducted based on the existing literature . The disadvantage of metallic, as opposed to bony, lateralization may be the higher torque or shear force applied to the glenoid component, which may lead to a higher rate of glenoid loosening and screw breakage witnessed . Therefore, bony lateralization was suggested to provide a benefit to external rotation without the potentially disastrous consequences of metallic lateralization . In a similar way, valenti et al . Reported the outcomes of a lateralized prosthesis and concluded that less medialization of rsa improves external rotation, thus facilitating activities of daily living of older patients . However, the authors did not compare the outcomes with a sample of patients undergoing rsa with a medialized prosthesis, so their conclusion was again based on a comparison with the existing literature . No level i- or ii - evidence studies aimed to compare the outcomes of rsa depending on the type of prosthesis were found in the literature . Most common indications for rsa were cuff tear arthropathy, revision of anatomic prosthesis, failed rotator cuff repair and fracture sequelae . This study shows that the indication for surgery may have an impact on the outcomes of rsa, which is in accordance with the existing literature . Some authors found that patients with no previous surgery undergoing rsa had higher postoperative scores in ases (total, pain, and function) and constant (total and pain) compared to patients with previous surgery . However, other authors observed no significant differences in the improvement or postoperative values of constant score, ases, simple shoulder test, visual analogue scale for pain and function, oxford shoulder score, university of california in los angeles (ucla) shoulder scale, and rom between patients with and without previous surgery . For specific indications, the present study demonstrated that cuff tear arthropathy had higher improvements in constant score (total, pain, and activity), ases score (total, pain, and function), simple shoulder test, forward flexion, and abduction compared to revision of anatomic prosthesis . Although there were no inferential statistics in the present study, these results are both in agreement and disagreement with previous studies . Found that patients with cuff tear arthropathy had significantly higher improvements in constant score compared with patients undergoing revision of the prosthesis . The authors found a higher improvement (no p value provided) in ases score and external rotation in cuff tear arthropathy compared with revision of prosthesis, but no differences (no p value provided) in forward flexion . Regarding the constant score, wall et al . Found that cuff tear arthropathy and primary osteoarthritis with degenerative rotator cuff had a higher postoperative constant score (no p value provided) compared to revision of anatomic prosthesis, massive cuff tear, and posttraumatic osteoarthritis in fact, revision of anatomic prosthesis and posttraumatic osteoarthritis had significantly worse postoperative constant score compared to the other indications . Found that patients with cuff tear arthropathy and posttraumatic osteoarthritis had higher improvements (no p value provided) in external rotation compared to revision of prosthesis, massive cuff tear, and primary osteoarthritis with degenerative rotator cuff . For forward flexion, cuff tear arthropathy and revision of anatomic prosthesis had the highest improvement (no p value provided) compared with massive cuff tear, posttraumatic osteoarthritis, and primary osteoarthritis with degenerative rotator cuff . Reported a case series in which patients underwent hemiarthroplasty, anatomic total shoulder arthroplasty, or rsa and outcomes were analyzed depending on the indication for surgery . The authors found that the primary osteoarthritis with degenerative rotator cuff and cuff tear arthropathy had a significantly higher improvement of the constant score compared to rheumatoid arthritis and avascular necrosis . Unfortunately, the number of rsa in the groups of primary osteoarthritis with degenerative rotator cuff, rheumatoid arthritis, and avascular necrosis was 2, 6, and 0, respectively . Therefore, the significant differences are likely explained by anatomic prostheses instead of rsa . Similarly, walch et al . Found that cuff tear arthropathy, primary osteoarthritis with degenerative rotator cuff, and massive cuff tear had significantly higher improvement of the constant score compared with the revision of the prosthesis and posttraumatic osteoarthritis . There is a clear need for future studies specifically comparing the use of medialized and lateralized rsa, as the present comparison was indirect in nature given that no comparative studies of this parameter have been published to date . Further clarification is needed to know to which extent there are significant differences in functional outcomes as well as in external rotation between both models . It is probable that differences on the implanted humeral side may have profound outcome implications, yet there has been no focus on this side of the joint in rsa outcome studies . In addition, the exact impact of the type of approach on the outcomes needs to be better delineated . In any further study utilizing more than one approach and type of prosthesis, furthermore, as the results of rsa may differ depending on the indications for surgery, disclosure of outcomes for indications is also warranted . Finally, there are two methodological recommendations regarding the presentation of studies to facilitate further meta - analyses . First, it is important from a methodological and statistical point of view to report the sd in all parameters collected . Most studies employing prostheses with medialized center of rotation used the constant score, whereas studies utilizing lateralized prostheses used the ases score . Other investigations employed the simple shoulder test, the oxford shoulder score, or the ucla shoulder scale . Only rom is systematically provided in the published studies, but more homogeneity is required to facilitate further meta - analyses . Both types of prostheses (with medialized and lateralized center of rotation) clearly improved all the reported outcomes, but lateralized prostheses had more improvement in external rotation compared to medialized prostheses . All outcomes of rsa implanted for all types of indications significantly improved in the postoperative period, but those corresponding to revision of anatomic prosthesis, failed rotator cuff repair, and fracture sequelae demonstrated lower improvements compared with cuff tear arthropaty . The rsa is a surgical procedure with high patient satisfaction regardless of the type of prosthesis or the indication for surgery . There is no conflict of interest or financial aid from any organization regarding the material discussed in the manuscript . Thomas w. wright, m.d . Is a consultant for exactech, inc ., gainesville, florida, and receives royalties and institutional research support on products cited to this article.
Suicide is a complex behavior which results from the complicated interaction of biological, psychological, cognitive, and environmental factors . Suicide rate and suicidal tendencies among transgender community have been reported to be high compared to general population . The suicide rate among transgender individuals in india is about 31%, and 50% of them have attempted suicide at least once before their 20 birthday and 4050 persons commit suicide every year in karnataka state alone . However, the exact prevalence of completed suicide among transgender persons remain undocumented; the gender - based discrimination has prevented them by obtaining education, livelihood, and housing because of which they are living in slums and have to resort to begging and sex work; this pitiful conditions lead them to breakdown further and end their life in suicide . The high prevalence of depression and suicidal tendencies among transgender persons seems to be highly influenced by societal stigma, lack of social support, hiv status and violence - related issues which require further thorough investigation and necessary mental health counseling, crisis management, addressing drug abuse, providing them livelihood opportunities, and so on as part of the intervention . The literature search was carried out mainly using three sources, namely, search in electronic databases (pubmed, google scholar, psycinfo, proquest), manual search (library catalog) at the library of the national institute of mental health and neuro sciences (nimhans), and gray literature (consultation with experts). To extract the related studies, the search strategies (key words) used for both electronic database and manual search are transgender and suicide, hijra and suicide, sexual minorities and suicide, transgender and self - harm, transgender and suicide prevention, the author selected 21 research studies including reports and documents as part of the search in electronic databases . These studies selected based on their relevance to the current title of the review and their availability with full text freely online . As part of the manual search (library catalog), the author could get three dissertations relevant to the title of the review, submitted to nimhans . All these studies and dissertations are published or submitted between 2005 and 2015 . As part of gray literature, the author consulted the director of karnataka state women development corporation, bengaluru, the key persons of nongovernment organizations (ngos), i.e., initiatives for development foundation (idf), sangama, samara, jeeva, and aneka . The author consulted the key persons who gave oral consent and discussed the subject and note down the key points with their permission . The discussion mainly focused on the services available at these organizations for transgender persons which may influence the suicidal behavior of the transgender community directly or indirectly . This review is part of an ongoing ph.d research program which has obtained the approval by the nimhans ethics committee, no . Nimh / do / ethics sub - committee 18 meeting/2014, dated september 19, 2014 . The suicidal behavior and suicide attempt rates are reported to be significantly high among transgender persons compared to general population across the countries . Thirty - one percent of transgender persons in india end their life by committing suicide, and 50% of them have attempted for suicide at least once before their 20 birthday; however, the exact prevalence of completed suicide among transgender persons in the country remain undocumented . Forty - one percent of the transgender persons in the united states attempt for suicide at least once in their life . In san francisco, the prevalence of attempted suicide among transgender persons is 32%, among young age (<25 years) it is 50% . Transgender persons are at higher risk for suicidal ideation and suicide attempts at virginia . Fifty percent of transgender persons in australia have attempted suicide at least once in their lives . In england, 48% of the transgender young people had attempted suicide at least once in their lives . The prevalence of suicide remains high among transgender persons irrespective of disclosing their transgender status to others and undergoing sex reassignment surgery . The self - harm behavior among sexual minority including transgender persons is equally serious and impactful as suicidality; the forms of self - harm committed by the respondents are cutting on the wrists and other areas of the arms, burning oneself, pouring gasoline on oneself but not igniting it, hanging oneself, breaking glasses, cups and other objects on one's head, fists and body, banging one's head against the wall, excessive drinking, eating and drug use, harmful sexual behavior, joining crime, street gang and violent activities to purposefully drop - out from the life and society, etc . Rejection and lack of support from the families and society, gender dysphoria associated with extreme stressful experiences, child sexual abuse, early discontinuation of schooling, forced marriages, lack of livelihood opportunities, sexual and financial exploitation by the partner and police and rowdies, and lack of legal measures for protection are some of the characteristics of transgender persons . About 62% of the transgender respondents are either have problems with their family members, or they do not have any contact with their family members hence, they are living away from their families; they left their families because of ill - treatment, being not accepted as transgender persons and being felt embarrassed to live in the community; 56% of them have discontinued their education at either primary level or secondary level; majority of the transgender persons have opt sex industry and begging for their survival; 54% of them have the habit of consuming alcohol . Fourteen percent of the transgender persons consulted mental health professionals for their gender dysphoria mostly because they were referred by the general physicians and rest of the respondents have sought help at traditional healers and transgender community leaders; 31% and 15% of the transgender persons are at high risk for tobacco and alcohol abuse, respectively, and 26% of them are have severe depression . The transgender persons are forced to go out of their family and community; they are refused from education, employment and getting a house for rent; they stay at slums and many people under the same roof; they are ill - treated at health - care centers . All the transgender persons are belong to lower socioeconomic status have high level of perceived stigma, have poor social support from family, friends and significant others, and their level of perceived stress is high . The studies have identified a number of risk factors for the high rates of suicide and suicidal behavior among transgender persons . The discrimination of the transgender persons in the society has prevented them from obtaining an education, job, and housing because of which they are living in slums or street and have to resort to begging and sex work; this pitiful conditions have lead them to breakdown further and end their life in suicide . Stigma, discrimination, and violence against transgender persons occur across multiple social and institutional contexts; they are verbally harassed, physical and sexually abused and blackmailed by the police and rowdies; rejection, hateredness, verbal and physical abuse from friends and family members, stigmatization, refusal of services, and derogatory labeling at health - care system, etc ., have lead them to lose interest in day - to - day activities; the risk of hiv and hiv status increase their psychological distress, and they express thoughts of committing suicide . The suicidality among sexual minority community is associated with poor mental health condition in forms of mental illness, psychological pain, emotion fatigue, and low self - esteem; life being hard, being confused about one's sexuality or difficulty in accepting it, not being able to disclose one's sexuality, bullying, history of forced sex, gender - based discrimination, and victimization and isolation are the other reasons for suicide among this population . Lesbian, gay, bisexual, and transgender (lgbt) assault hate crimes at the neighborhood are an additional sociocontextual risk factors for suicidal ideation and attempts among sexual - minority adolescents . Transgender persons being in adolescents and being in their early 20 s and having history of suicide attempt, those who work in the bar, entertainment and sex industries, survivors of violence perpetrated by intimate partners or family members, are potentially in higher risk for suicidality . Neither reporting the thoughts and behaviors of suicide and self - harm nor seeking help is common among sexual minorities . The psychological autopsy of the completed suicides among transgender persons has revealed that the factors such as break - up of love relationship initiated by the partner (64.3%), serious altercations with family members (14.3%), refusal of gender / sex reassignment by the family members (9.5%), financial problems (9.5%), being diagnosed with hiv positive in the past few days / weeks (2.4%) have triggered the act of suicide among the victims . The research studies have tried to explore the resiliency factors which are helping the transgender community to bounce back and continue living even with a number of hardships and adverse conditions in their day - to - day living . The transgender persons have overcome from the above - mentioned situations using at least one of the coping mechanisms or having certain personal qualities such as assertive communication, self - advocacy, spiritual coping, honesty, integrity, avoidance, physical or verbal aggression, help seeking, being future - oriented with having personal goals, being outspoken, strong, friendly, outgoing, independent, determination, etc . The transgender persons who have income of> 10,000 dollars and being educated at higher level, employed in the mainstream jobs other than sex work and begging, optimistic, having perceived social support from family, emotional stability, and child - related concerns have shown better self - esteem and resiliency level . Social support from family is found to be general protective factor which is associated with reduced risk for lifetime suicide attempts among transgender persons . The national centre for transgender equality provides information on services available for suicide prevention in the united states that includes national suicide prevention helpline (24 7, toll free), lgbt national hotline and the trevor project which provides telephonic, online, e - mail peer counseling, crisis intervention and online materials, and information about suicide and help . The most of the programs related to lgbt youth under trevor project deal with the issues such as school safety, health promotion, prevention of violence, harassment and discrimination, civil rights, peer education, emergency support, hiv and aids prevention and offer services in terms of training in life skills, enhancing peer relationships, connecting lgbt youth with supportive adults, helping parents and teachers to provide support to the lgbt youth, in - school workshops, educational materials, online educational resources for youth, and advocacy for public policy to reduce lgbt stigma . The ngos such as sangama, samara, jeeva, aneka, idf and the karnataka women development corporation of bengaluru, karnataka, the organization sahodari in tamil nadu, the humsafar trust in maharashtra, and so on organizations are providing services in terms of crisis intervention services, helpline services, clinical services, information and referral services, legal and advocacy support, drop - in - centers, alternative employment services and financial assistance, soft - skills training, self - help group formation, assistance in availing ration card, election i d card etc ., creating awareness through workshops, lobbying with media to create awareness among families to increase the acceptance of transgender children, telecasting programs through community radio, developing films and videos, screening the documentaries and films, self - esteem and resilience building services, organizing health camps to provide general health and mental health services, medical services, entertainment, competition and library services, organizing seminars, discussions, and so on services to the sexual minority community at locally . Although these services explicitly do not focus on suicide prevention, they contribute enormously in enhancing the resiliency factors and protective factors among transgender persons . The interventions and programs to enhance protective factors and resiliency factors are as important as programs for risk reduction; these interventions should be delivered through cultural competence approach and should be more lgbt inclusive which help an agency, system, or a professional to work effectively in cross - cultural situations . The suicide prevention interventions and programs for all youth can also be implemented for lgbt individuals mainly in three settings, schools (suicide awareness curricula), communities (gate - keeper training) and health - care system (screening) and crisis centres, hotlines, and risk reduction which can include restricted access to lethal means, media training, and youth life - skills training also can be part of it . Community awareness campaigns, discrimination and hate crime legislations, culturally and age appropriate suicide prevention interventions including peer - based outreach, counseling and referrals, targeting the institutions such as schools, family, community, health - care system, police and judiciary, effective treatment for symptoms of hopelessness, depression, conduct disorder, family - based interventions to enhance the support and reduce the victimization, effective intervention in addressing high rates of hiv infection, multiple and complex high - risk behavior and comorbid conditions, addressing sociocultural factors such as lgbt assault hate crimes at the neighborhood providing educational and resource materials on lgbt suicide to the lgbt organizations and encourage these organizations to consider suicide prevention at their organizations' mission and activities, all these would help in achieving increased societal acceptance of the transgender community and decreased gender - based prejudice and also in the promotion of mental health and reduction of suicidal risk among transgender community . The suicidal behavior and suicide attempt rates are reported to be significantly high among transgender persons compared to general population across the countries . Thirty - one percent of transgender persons in india end their life by committing suicide, and 50% of them have attempted for suicide at least once before their 20 birthday; however, the exact prevalence of completed suicide among transgender persons in the country remain undocumented . Forty - one percent of the transgender persons in the united states attempt for suicide at least once in their life . In san francisco, the prevalence of attempted suicide among transgender persons is 32%, among young age (<25 years) it is 50% . Transgender persons are at higher risk for suicidal ideation and suicide attempts at virginia . Fifty percent of transgender persons in australia have attempted suicide at least once in their lives . In england, 48% of the transgender young people had attempted suicide at least once in their lives . The prevalence of suicide remains high among transgender persons irrespective of disclosing their transgender status to others and undergoing sex reassignment surgery . The self - harm behavior among sexual minority including transgender persons is equally serious and impactful as suicidality; the forms of self - harm committed by the respondents are cutting on the wrists and other areas of the arms, burning oneself, pouring gasoline on oneself but not igniting it, hanging oneself, breaking glasses, cups and other objects on one's head, fists and body, banging one's head against the wall, excessive drinking, eating and drug use, harmful sexual behavior, joining crime, street gang and violent activities to purposefully drop - out from the life and society, etc . Rejection and lack of support from the families and society, gender dysphoria associated with extreme stressful experiences, child sexual abuse, early discontinuation of schooling, forced marriages, lack of livelihood opportunities, sexual and financial exploitation by the partner and police and rowdies, and lack of legal measures for protection are some of the characteristics of transgender persons . About 62% of the transgender respondents are either have problems with their family members, or they do not have any contact with their family members hence, they are living away from their families; they left their families because of ill - treatment, being not accepted as transgender persons and being felt embarrassed to live in the community; 56% of them have discontinued their education at either primary level or secondary level; majority of the transgender persons have opt sex industry and begging for their survival; 54% of them have the habit of consuming alcohol . Fourteen percent of the transgender persons consulted mental health professionals for their gender dysphoria mostly because they were referred by the general physicians and rest of the respondents have sought help at traditional healers and transgender community leaders; 31% and 15% of the transgender persons are at high risk for tobacco and alcohol abuse, respectively, and 26% of them are have severe depression . The transgender persons are forced to go out of their family and community; they are refused from education, employment and getting a house for rent; they stay at slums and many people under the same roof; they are ill - treated at health - care centers . All the transgender persons are belong to lower socioeconomic status have high level of perceived stigma, have poor social support from family, friends and significant others, and their level of perceived stress is high . The studies have identified a number of risk factors for the high rates of suicide and suicidal behavior among transgender persons . The discrimination of the transgender persons in the society has prevented them from obtaining an education, job, and housing because of which they are living in slums or street and have to resort to begging and sex work; this pitiful conditions have lead them to breakdown further and end their life in suicide . Stigma, discrimination, and violence against transgender persons occur across multiple social and institutional contexts; they are verbally harassed, physical and sexually abused and blackmailed by the police and rowdies; rejection, hateredness, verbal and physical abuse from friends and family members, stigmatization, refusal of services, and derogatory labeling at health - care system, etc ., have lead them to lose interest in day - to - day activities; the risk of hiv and hiv status increase their psychological distress, and they express thoughts of committing suicide . The suicidality among sexual minority community is associated with poor mental health condition in forms of mental illness, psychological pain, emotion fatigue, and low self - esteem; life being hard, being confused about one's sexuality or difficulty in accepting it, not being able to disclose one's sexuality, bullying, history of forced sex, gender - based discrimination, and victimization and isolation are the other reasons for suicide among this population . Lesbian, gay, bisexual, and transgender (lgbt) assault hate crimes at the neighborhood are an additional sociocontextual risk factors for suicidal ideation and attempts among sexual - minority adolescents . Transgender persons being in adolescents and being in their early 20 s and having history of suicide attempt, those who work in the bar, entertainment and sex industries, survivors of violence perpetrated by intimate partners or family members, are potentially in higher risk for suicidality . Neither reporting the thoughts and behaviors of suicide and self - harm nor seeking help is common among sexual minorities . The psychological autopsy of the completed suicides among transgender persons has revealed that the factors such as break - up of love relationship initiated by the partner (64.3%), serious altercations with family members (14.3%), refusal of gender / sex reassignment by the family members (9.5%), financial problems (9.5%), being diagnosed with hiv positive in the past few days / weeks (2.4%) have triggered the act of suicide among the victims . The research studies have tried to explore the resiliency factors which are helping the transgender community to bounce back and continue living even with a number of hardships and adverse conditions in their day - to - day living . The transgender persons have overcome from the above - mentioned situations using at least one of the coping mechanisms or having certain personal qualities such as assertive communication, self - advocacy, spiritual coping, honesty, integrity, avoidance, physical or verbal aggression, help seeking, being future - oriented with having personal goals, being outspoken, strong, friendly, outgoing, independent, determination, etc . The transgender persons who have income of> 10,000 dollars and being educated at higher level, employed in the mainstream jobs other than sex work and begging, optimistic, having perceived social support from family, emotional stability, and child - related concerns have shown better self - esteem and resiliency level . Social support from family is found to be general protective factor which is associated with reduced risk for lifetime suicide attempts among transgender persons . The national centre for transgender equality provides information on services available for suicide prevention in the united states that includes national suicide prevention helpline (24 7, toll free), lgbt national hotline and the trevor project which provides telephonic, online, e - mail peer counseling, crisis intervention and online materials, and information about suicide and help . The most of the programs related to lgbt youth under trevor project deal with the issues such as school safety, health promotion, prevention of violence, harassment and discrimination, civil rights, peer education, emergency support, hiv and aids prevention and offer services in terms of training in life skills, enhancing peer relationships, connecting lgbt youth with supportive adults, helping parents and teachers to provide support to the lgbt youth, in - school workshops, educational materials, online educational resources for youth, and advocacy for public policy to reduce lgbt stigma . The ngos such as sangama, samara, jeeva, aneka, idf and the karnataka women development corporation of bengaluru, karnataka, the organization sahodari in tamil nadu, the humsafar trust in maharashtra, and so on organizations are providing services in terms of crisis intervention services, helpline services, clinical services, information and referral services, legal and advocacy support, drop - in - centers, alternative employment services and financial assistance, soft - skills training, self - help group formation, assistance in availing ration card, election i d card etc ., creating awareness through workshops, lobbying with media to create awareness among families to increase the acceptance of transgender children, telecasting programs through community radio, developing films and videos, screening the documentaries and films, self - esteem and resilience building services, organizing health camps to provide general health and mental health services, medical services, entertainment, competition and library services, organizing seminars, discussions, and so on services to the sexual minority community at locally . Although these services explicitly do not focus on suicide prevention, they contribute enormously in enhancing the resiliency factors and protective factors among transgender persons . The interventions and programs to enhance protective factors and resiliency factors are as important as programs for risk reduction; these interventions should be delivered through cultural competence approach and should be more lgbt inclusive which help an agency, system, or a professional to work effectively in cross - cultural situations . The suicide prevention interventions and programs for all youth can also be implemented for lgbt individuals mainly in three settings, schools (suicide awareness curricula), communities (gate - keeper training) and health - care system (screening) and crisis centres, hotlines, and risk reduction which can include restricted access to lethal means, media training, and youth life - skills training also can be part of it . Community awareness campaigns, discrimination and hate crime legislations, culturally and age appropriate suicide prevention interventions including peer - based outreach, counseling and referrals, targeting the institutions such as schools, family, community, health - care system, police and judiciary, effective treatment for symptoms of hopelessness, depression, conduct disorder, family - based interventions to enhance the support and reduce the victimization, effective intervention in addressing high rates of hiv infection, multiple and complex high - risk behavior and comorbid conditions, addressing sociocultural factors such as lgbt assault hate crimes at the neighborhood providing educational and resource materials on lgbt suicide to the lgbt organizations and encourage these organizations to consider suicide prevention at their organizations' mission and activities, all these would help in achieving increased societal acceptance of the transgender community and decreased gender - based prejudice and also in the promotion of mental health and reduction of suicidal risk among transgender community . The current review covers research studies from electronic database and manual search and also supplements information with gray literature . The review has included important studies conducted across the countries and through more light on issues and situations surrounded suicidality and suicidal behavior among transgender persons, and the efforts are taken to address the same across the countries and in the indian context . The transgender community is highly vulnerable for suicidality and suicidal behavior which is a challenging phenomena for the governments and organizations globally . However, the countries like the united states are trying to address the same at national level but in the indian context, a lot of ground work should happen . The involvement of government, policy, institutions, organizations, public, along with the involvement of transgender community is required . The transgender community is one of the difficulties to reach population having its own cultural background requires understanding and interventions with culture - specific, sensitive, and transgender - inclusive approach . The review recommends the interventions to be drawn simultaneously for suicide risk reduction and enhance the protective factors and resiliency factors at the same time . This article is part ongoing ph.d research work and the study has been funded by university grants commission as part of junior research fellowship . This article is part ongoing ph.d research work and the study has been funded by university grants commission as part of junior research fellowship.
Non - small cell lung cancer (nsclc) can exhibit rearranged driver oncogenes, which are possible targets for therapy.1 the most frequently identified driver oncogenes in nsclc leading to targeted therapy are epidermal growth factor receptor (egfr) and anaplastic lymphoma kinase (alk).1,2 the prevalence of these driver oncogenes is significantly influenced by race, smoking habits, and gender.3 the frequency of egfr - mutated nsclc in asian patients is known to be higher than in caucasian patients.3 in contrast, prevalence of alk rearrangements in nsclc is reported to be similar in caucasian and asian patients.3 in recent cohort studies with afro - americans, there is no significant difference in the prevalence of egfr mutations or alk rearrangements with caucasian americans.4,5 about other races, however, less or no data are available.6,7 for obvious reasons, little is known about africa . Cancer, however, is a significant health problem in this continent, as it is estimated that there were 715,000 new cancer cases in 2008, and additional data from southern africa and northern africa already confirm that lung cancer is the leading cause of death related to cancer.8 to our knowledge, there is no literature available about the prevalence of lung cancer and rearranged driver oncogenes in sub - saharan africans . As the latter is influenced by race and smoking habits that may differ from afro - americans; we have studied the prevalence of egfr mutations and alk translocations in a case series of patients of sub - saharan african ethnicity with nsclc . We retrospectively studied all patients of sub saharan african ethnicity who have been treated for nsclc stage iv in our hospital (university hospital saint pierre) in brussels, belgium . The ethics committee of saint - pierre hospital deemed ethical approval not necessary as it was a retrospective study . Investigations were performed on biopsies obtained during flexible bronchoscopy and these samplings were immediately fixed in neutral buffered formalin and embedded in paraffin.9 driver oncogenes on biopsy specimens were researched by using the truseq amplicon cancer panel (illumina, illumina inc ., san diego, ca, usa). Not only were egfr / alk mutations investigated, but also other mutations included in the panel, such as tumor protein p53 (tp53), proto - oncogene b - raf (braf) and kirsten rat sarcoma viral oncogene homolog (kras). From july 2012 to november 2015, 6 patients of sub - saharan origin with nsclc stage iv have been treated in our hospital . Three patients originated from congo, an ex - belgian colony, 1 from djibouti, lfrom cameroon, and 1 from guinea . Egfr mutation was present in 3/6 patients and alk rearrangement was present in 1/6 patients . Two egfr mutations were common, i.e., l858r (exon 21 point mutation) and deletion exon 19.10 the significance of the third egfr mutation (t710i in exon 18) is unknown . All egfr mutations and both patients without egfr mutation and alk rearrangement were considered as light smokers (<10 pack - years). In our small case series, 4 out of 6 sub - saharan patients with nsclc had a driver oncogene, either egfr mutation or alk rearrangement . The prevalence of rearranged driver oncogenes in nsclc is known to be influenced by race, gender, and smoking status.3 differences in prevalence of egfr mutation status in nsclc between asian and caucasian patients have already been extensively studied since the early 2000s.2,3 egfr mutations (exons 1822) are present in ~30% east asian patients with lung cancer, and more specifically in 35%47% of east asian patients with lung adenocarcinoma, whereas egfr mutations are present in ~7% of caucasian patients with lung cancer, and in 13%18% of caucasian patients with lung adenocarci - noma.3,4 even within the asian population, egfr mutation status in nsclc varies.6 the east asian population is the largest asian group studied (japanese, korean, and chinese patients).3,4,6 in a large cohort of 907 indian patients, an egfr mutation was found in 23% of patients with nsclc and in 26% of lung adenocarcinomas.6 recently, egfr mutations in nsclc in afro - american patients have been investigated.4,5,11 there seems to be no difference in prevalence of egfr mutations in ncslc between caucasians and afro - americans . Yamaguchi et al described a prevalence of egfr mutations in nsclc of 18.4% in white patients and in 18.2% of black patients in their patient cohort.4 bollig - fischer et al found a prevalence of 8% of egfr mutations in both black and white patients with nsclc.11 it is not clear whether these results for afro - american patients can be extrapolated to sub - saharan african patients with nsclc . Errihani et al performed the only study, to our knowledge, on the prevalence of egfr mutation status in lung cancer patients in the african continent.7 they examined in a moroccan patient cohort the prevalence of egfr mutations in advanced lung adenocarcinoma . A prevalence of 21% egfr mutations in lung adenocarcinoma was found (29 out of 137 patients), which lies between the prevalence of egfr mutations in caucasian and asian patients with nsclc.7 it is unclear whether the results of the moroccan (north african) cohort also apply to sub saharan african patients with nsclc . Besides race, the presence of driver oncogenes is influenced by gender and smoking history.3 in this limited series, the role of gender was impossible to assess . Finally, smoking status independently influences egfr mutation status in nsclc.2,3,4 the prevalence is higher in never smokers or light smokers . In our study, 3 out of 6 patients had an egfr mutation . This incidence seems high, but interestingly, all our 6 patients were non - smokers or light smokers . In europe, the prevalence of tobacco use among adults is approximately 19% for women and 38% for men.12 in the african continent, at first sight, smoking prevalence does not seem to be different, as among males it ranges from 14% in swaziland to 40% in niger.13 however, according to a recent analysis of the american cancer society, most african countries remain in the early stages of the tobacco epidemic, with tobacco use relatively low compared to the rest of the world.14 on this basis, it is likely that the proportion of lung adenocarcinoma in non - smokers is still high and this may contribute to explaining the frequent egfr mutations in our case series . Indeed, egfr mutations in nsclc have been found in 35%40% of caucasian never smokers, and in 55%70% of asian never smokers.3,4 we believe this explanation also applies for the finding of one alk translocation in our small series . The overall prevalence of alk translocation in nsclc is thought to be 3%5%, without ethnic difference, but with a higher prevalence in non - smokers.3,4 even if these results cannot be extrapolated due to the small number of patients, the early stages of the tobacco epidemic in africa are probably associated with a high frequency of egfr mutation and alk rearrangement in nsclc . This will probably change with time, as africa is becoming a future epicenter of tobacco epidemic.13 in this small series, 4/6 patients of sub - saharan origin with nsclc presented driver oncogene rearrangements . This may be related to the fact that africa is still in the early stages of the tobacco epidemic, leading to suggest that the prevalence of driver oncogenes is high in this population . As the burden of lung cancer in sub - saharan countries is expected to rise in the next decennia,8 further investigations of nsclc subgroups in this racial group are required.
Cadaver renal transplantation is fast becoming an important modality in the management of end - stage renal disease (esrd), in india . In the present scenario it is imperative that we improve the donor pool, such that more chronic kidney disease patients are benefited . The existing renal malignancy is a contraindication for renal transplantation, but data regarding graft kidneys with suspicious solid renal masses are not widely available . A 50-year - old man with a history of road traffic accident (rta) was admitted to intensive care unit (icu) and declared brain dead . Formal consent for organ donation was obtained from the relatives and organ harvesting was done . Under the organ sharing network, our institution received the left kidney . A 36-year - old, same blood group, routine evaluation of the recipient was done . During bench dissection of the donor kidney, a 1.5 2 c - arm screening was done and no radio opaque lesion was seen . As the cadaver transplantation was done in odd hours, frozen section facilities were not available . As the lesion was found in the mid zone of the graft kidney, partial nephrectomy was not attempted and we proceeded with enucleation of the solid lesion . The nephrotomy wound was closed with absorbable sutures and the kidney was grafted to the recipient [figure 1]. Post - operative recovery was uneventful and urine output improved to 150 - 200 ml / day from post operative day two . Donor autopsy was done and no evidence of any other malignant lesions or lesions suggestive of metastasis was found . Renomedullary interstitial cell tumour, a benign lesion of the renal medulla [figure 2]. Renal imaging with a plain ct scan done two months postoperatively, found no lesion in the kidney . Although the number of live and cadaver renal transplants have increased in recent years, there continues to be a significant deficit . The increase in the donor pool is not able to match the ever increasing population with esrd and many patients die each year waiting for a suitable graft . The presence of malignancy in a renal graft is considered to be a contraindication to renal transplantation . There are many studies that have studied the use of kidneys with incidental detected renal masses in renal transplantation . Our decision in this case to go ahead with the transplantation of the graft after enucleation was due to the small size of the lesion, its grossly benign appearance, and to give the recipient a better chance of survival and quality of life . In july 2009, reported a series of five cases where <2.3 cm incidental renal masses were subjected to back table partial nephrectomy and transplanted into the matched recipients . There was no evidence of cancer - specific mortality or recurrence at a 15-month follow - up . Mchayleh et al ., in 2008, reported two cases of metastasis from renal cell carcinoma in a kidney allograft . Hence, many articles have been published regarding the use of grafts with renal malignancies . However, data on the management of benign renal masses in donor kidneys are few . Renomedullary interstitial cell tumour is a common autopsy finding in patients over 50 years, being found in about 30% of this group . These lesions are usually round to oval, pale gray to yellow with a mean size of 3 mm . Clinically symptomatic tumours are rare . The term renomedullary interstitial cell tumour was coined by lerman et al ., in 1972 . The previous terminology used was renal medullary fibroma. The finding of a discrete renal mass during bench dissection for a cadaveric renal transplant raises many questions . Forty to fifty percent of renal masses less than 2 cm in size are benign . Meticulous back table dissection and subsequent histopathological confirmation are necessary when such kidneys are transplanted . Hence, such transplant recipients should be diligently followed up to look for recurrence of renal masses and also for occurrence of metastasis . This situation also brings up the case of the need for imaging studies like a contrast - enhanced ct, for the evaluation of potential cadaveric donors . In some cases, it may not be possible to shift the brain dead donor with all supports to the ct room . Hence, at least a bedside ultrasound may add to our preoperative assessment of the kidney . Incidentalomas like in our case could have been picked up on imaging and the surgical team was better prepared to deal with the situation . Therefore, routine imaging studies like the ct or ultrasound may be advocated for all potential cadaveric donors, preoperatively . The increase in the use of imaging studies for a variety of abdominal conditions has increased the rate of detection of small clinically insignificant renal masses.
Non - alcoholic fatty liver disease (nafld) is a relevant issue in public health . It represents the most common chronic liver disease in the general population and is expected to increase in the future, as a result of an ageing population, obesity and diabetes . Nafld is the hepatic manifestation of metabolic syndrome and includes a spectrum of disease ranging from simple hepatic steatosis to non - alcoholic steatohepatitis (nash), which can progress to cirrhosis . Nafld occurs in 6095% of patients with obesity and in 2855% of patients with type 2 diabetes mellitus (t2 dm). Insulin resistance (ir), with compensatory hyperinsulinemia, plays a pathogenetic key role in nafld progression . Given its low cost, repeatability, safety and availability, ultrasound (us) is routinely the first - line imaging technique to detect liver steatosis . Central obesity, defined as a presence of excess fat in the abdominal area, is frequently associated with nafld and their coexistence in the same subjects increases the likelihood of having more advanced forms of liver disease . Liver steatosis progression is associated not only with the body mass index (bmi), but also with visceral adiposity . However, fat mass and fat distribution can be different in subjects with the same bmi . For these reasons, the body composition (bc) evaluation is necessary for assess the nutritional status and the efficacy of nutritional strategies . A valid and precise tool to measuring bc, is the dual - energy x - ray absorptiometry (dxa), an important method for studying not only osteoporosis but also the soft - tissue composition changes . The accuracy of dxa in measuring the fat mass is 98.8%, and combined with other standard anthropometric parameters, it is a reliable tool for the estimation of central fat . The adipose tissue, has been considered an energy storage organ, but over the last decade several findings have encouraged researches on its the endocrine role . Furthermore, data highlighted that central adipose tissue is a metabolic and inflammatory organ that modulates the action and the metabolism of brain, liver, muscle and cardiovascular system . Recently, the important role of adipokines, peptides synthesized from adipocytes, in the pathogenesis of ir and nafld has been discussed . In particular, adiponectin is considered an anti - inflammatory adipokine, able to reduce body fat, to improve hepatic and peripheral insulin sensitivity, and inversely associated with bmi, ir and hepatic fat accumulation . The aim of this observational study was to investigate, in overweight patients, the possible relationship between central fat mass, liver steatosis and the circulating levels of adiponectin . The study was carried out in overweight patients, referred to the division of clinical nutrition and nutrigenomics, university of rome tor vergata of rome (italy), for a nutritional evaluation, in the period january the study was performed in accordance with good clinical practice and complying with the principles laid down of declaration of helsinki . The study was approved by the appropriate ethical committee of the centre, and all patients gave their written informed consent before the recruitment . Other exclusion criteria were: insulin therapy, smoking habits, alcohol intake (> 20 g / day), hepatic virus infection, auto - immune diseases, and use of drugs known to induce liver steatosis or nash . After the initial evaluation of 52 patients, 21 (40.4%) were excluded: 5 for heart diseases, 4 for insulin therapy, 2 for hepatitis b virus infection, 6 for excessive daily alcohol intake and 4 for treatment with drugs - inducing steatosis or nash . Body weight, height, bmi, and waist circumference were measured according to standard methods . All subjects underwent a biochemical examination of fasting glucose, fasting insulin and alanine aminotransferase (alt). Fasting glucose and insulin level were used also to calculate ir, according to the homeostasis homeostasis model assessment (homa - ir). The enzyme - linked immunosorbent assay technique was used to measure fasting serum levels of adiponectin in all patients (r&d systems inc . The patients underwent us liver by a toshiba eccocee, with a convex transducer of 3.7 mhz . The results were interpreted by an investigator (la) with experience in the field . The liver images were considered normal if the texture was homogenous, exhibited fine level echoes and isoechoic compared to the renal cortex and there was adequate visualization of the hepatic vessels and diaphragm (grade 0). Criteria for determining the stage of liver steatosis, in according to the hamaguchi score, included: presence of bright echoes or increased hepato - renal contrast indicative of mild steatosis (grade 1); presence of both bright echoes and increased hepato - renal contrast as well as vessel blurring indicative of moderate steatosis (grade 2); severe steatosis was considered when in addition to the criteria for moderate steatosis, there was evidence of posterior beam attenuation and non - visualization of the diaphragm (grade 3). Bc was assessed by dxa (lunar dpx - iq; ge medical systems, milwaukee, wi), according to the standardized described procedure . Standard dxa quality control and calibration measures, were performed prior to each testing session . The test takes about twenty minutes, and the subjects remain in a supine position during the scanning . The results, were transmitted to a connected computer for further analysis, according to the manufacturer orientation . In order to show the relation between dxa parameters and us stage of nafld, we displayed graphically the evolution of these features among classes, and we assessed the statistical significance of clinical and laboratory features . Statistical differences of laboratory and clinical variables between the four groups were evaluated by the anova test . Body weight, height, bmi, and waist circumference were measured according to standard methods . All subjects underwent a biochemical examination of fasting glucose, fasting insulin and alanine aminotransferase (alt). Fasting glucose and insulin level were used also to calculate ir, according to the homeostasis homeostasis model assessment (homa - ir). The enzyme - linked immunosorbent assay technique was used to measure fasting serum levels of adiponectin in all patients (r&d systems inc . The patients underwent us liver by a toshiba eccocee, with a convex transducer of 3.7 mhz . The results were interpreted by an investigator (la) with experience in the field . The liver images were considered normal if the texture was homogenous, exhibited fine level echoes and isoechoic compared to the renal cortex and there was adequate visualization of the hepatic vessels and diaphragm (grade 0). Criteria for determining the stage of liver steatosis, in according to the hamaguchi score, included: presence of bright echoes or increased hepato - renal contrast indicative of mild steatosis (grade 1); presence of both bright echoes and increased hepato - renal contrast as well as vessel blurring indicative of moderate steatosis (grade 2); severe steatosis was considered when in addition to the criteria for moderate steatosis, there was evidence of posterior beam attenuation and non - visualization of the diaphragm (grade 3). Bc was assessed by dxa (lunar dpx - iq; ge medical systems, milwaukee, wi), according to the standardized described procedure . Standard dxa quality control and calibration measures, were performed prior to each testing session . The test takes about twenty minutes, and the subjects remain in a supine position during the scanning . The results, were transmitted to a connected computer for further analysis, according to the manufacturer orientation . In order to show the relation between dxa parameters and us stage of nafld, we displayed graphically the evolution of these features among classes, and we assessed the statistical significance of clinical and laboratory features . Statistical differences of laboratory and clinical variables between the four groups were evaluated by the anova test . Clinical and laboratory characteristics of the patients, stratified on the basis of the us stage of nafld, are reported in table i. of the 31 patients included in the study, 13 were male (41.9%). The median value of anthropometric parameters assessed were: bmi 31.45.9 and waist circumference 97.216.2 cm, with 16 patients (7 males) that presented central obesity, according to the definition of the national cholesterol education program adult treatment panel iii (ncep atp iii). Us evaluation of the liver showed that: in 8 patients (25.8%) steatosis was absent, in 9 (29%) mild (grade 1), in 6 (19.4%) moderate (grade 2), and in 8 (25.8%) severe (grade 3). The median value of alt was 33.627.3 ul-1, with four patients that reported high alt value, one without, two with mild and one with severe us liver steatosis . Of all dxa parameters analyzed, those that present a statistical significant difference between the us stages of nafld were (figure 1): abdominal fat (af,%), mean value 42.611.9 (p<0.05); abdominal tissue mass (atm: fat plus lean mass, kg), (mean value 72.5, p<0.05); abdominal fat mass (afm, kg), (mean value 3.91, p<0.05). Considering the only distribution of l2l5 fat tissue (kg), (mean value 5.21.2), we observed that the value was higher, but reached no statistical significance, in patients with moderate and severe steatosis, compared to those with the mild type . The assessment of ir by homa - ir, showed a statistically significant increase of insulin level, related to steatosis progression (p<0.05). Finally, the concentration of adiponectin (7.782.2 g / ml), was significantly lower in the advanced us stages of nafld (p<0.05). Obesity, which is often associated with ir, represents a chronic low - grade inflammatory state, characterized by elevated circulating levels of cytokines and activation of pro - inflammatory signaling pathways . In particular, ir plays a key role in the pathogenesis of nafld, causing alterations in the uptake, degradation or secretion of lipid molecules, with consequent accumulation of lipid in the hepatocytes . Nafld is usually prevalent in obese subjects, and several studies have reported that regional fat distribution associated with ir is an important factor for development and progression of liver steatosis . In our study, abdominal fat accumulation and anthropometric parameters, increased with us severity of liver steatosis and ir . In this context the first observation is that by using dxa, we quantified the regional distribution of adipose tissue and we found the association between increased central fat mass, and liver steatosis severity . This observation is in agreement with the progression of the values of bmi and circumference observed in our series . Concerning the relationship between bc and nafld, this study has shown that central fat accumulation constitutes an important determinant of liver steatosis in overweight patients, independently to bmi . Recent data document that the abdominal adipose tissue is an active endocrine organ capable to secrete a multitude of hormones, cytokines, chemokines, and enzymes, collectively known as adipokines . In particular adiponectin, leptin, resistin, visfatin and pro - inflammatory cytokines, such as tumor necrosis factor-, and interleukins, have been shown to be involved in pathogenesis and progression of nafld . The increased number of abdominal adipocytes produces a disequilibrium in adipokines secretion, which promotes liver fat accumulation, and subsequently the development of nafld . In particular, adiponectin improves muscular and hepatic insulin sensitivity, through its anti - inflammatory and anti - atherogenic activity, and its ability to decrease triglyceride synthesis and stimulate -oxidation . In fact adiponectin protects hepatocytes from triglycerides accumulation by increasing -oxidation of free fatty acid and/or decreasing de novo free fatty acid production in hepatocytes . Adiponectin levels are reduced in obese patients, and t2 dm, and the plasma concentrations are inversely related to body weight, especially to visceral adiposity . Moreover, adiponectin is inversely associated with other traditional cardiovascular risk factors, such as blood pressure, low - density lipoprotein cholesterol and triglyceride levels, and is positively related to high - density lipoprotein cholesterol levels . In our cohort, us liver steatosis progression is characterized by ir and low adiponectin serum levels, pathogenetic factors that can increase the concentrations of intra - cellular fatty acids, and may enhance oxidative stress that is the second stage in the pathogenesis of nash . Bc assessed by anthropometry and dxa, may be used as indicator of nafld severity in overweight patients . However, further studies are required to better understand not only this correlation, but also to define the pathogenetic role of central fat distribution and the changes in adipokine levels in the progression of nafld.
Osteoporosis is characterized by decreased bone strength, and it leads to increased bone fragility and susceptibility to fractures.1 postmenopausal osteoporosis and associated fractures are major public health problems across the world, especially in the people s republic of china . Osteoporosis is caused by an imbalance between bone tissue formation by osteoblasts and resorption by osteoclasts.2 osteoporosis may be prevented or treated in its early stages using basic approaches (calcium, vitamin d, diet, exercise, and smoking cessation).1 several antiosteoporotic drugs have been used widely for its more severe stages, and they have demonstrated efficacy in increasing bone mineral density (bmd) and decreasing fracture incidences . Other drugs such as bisphosphonates,3 raloxifene,4 estrogen / progestin therapy,5 and denosumab6 have also been found to be effective against osteoporosis . Menatetrenone (vitamin k2) is known to be a cofactor of -carboxylase, which converts glutamic acid residues in the osteocalcin (oc) molecule to -carboxyglutamic acid, and it is therefore essential for -carboxylation of oc.79 low vitamin k consumption is associated with a higher risk of hip fracture among older women and men.1013 a meta - analysis14 has revealed that menatetrenone therapy decreases new vertebral fractures and possibly reduces long bone fractures . Menatetrenone has been approved in japan in 1995 for treatment of postmenopausal osteoporosis; since then, it has been used widely in that country.15 hence, available published data are mostly obtained from a japanese population, which may have a different pattern of risk factors (diet, exercise, and genetics) for the disease than a chinese population . The efficacy and tolerance of menatetrenone in chinese postmenopausal osteoporotic women has not been previously investigated . This 1-year study was therefore designed to evaluate the efficacy and safety of menatetrenone for the treatment of osteoporosis in chinese postmenopausal women in comparison with alfacalcidol treatment . Since the study involved osteoporotic women, a placebo control was not used, and alfacalcidol was chosen as a positive drug control to demonstrate the noninferiority of these two drugs . The study was approved by the state food and drug administration of china (2004l03447). Women patients included in the study had to: 1) be ambulatory; 2) aged between 45 and 75 years; 3) be postmenopausal for at least 5 years; 4) have a body mass index between 18 and 30 kg / m; 5) have a lumbar spine t - score (l2l4) and/or femoral neck bmd lower than 2.0; and 6) be willing to provide written informed consent . Patients with the following criteria were excluded from the study: 1) disorders known to affect bone metabolism; 2) chronic diseases such as hyperthyroidism, hyperparathyroidism, osteomalacia, or diabetes; 3) treatment with bisphosphonates within the past year; 4) treatment with selective estrogen receptor modulators within the past 6 months; 5) treatment with activated vitamin d, calcitonin, estrogens, or androgens within the past 3 months; 6) treatment with warfarin; 7) chronic renal or liver disorders; and 8) history of malignant tumor . Patients were also excluded if one of these following laboratory tests was abnormal: 1) alkaline phosphatase (alp) elevated> 10% of upper normal limit; 2) alanine aminotransferase (alt) or aspartate aminotransferase (ast) elevated> 50% of upper normal limit; 3) serum creatinine levels> 133 mmol / l (1.5 mg / dl); and 4) fasting blood glucose> 126 mg / dl (7.0 mmol / l). This was a multicenter, randomized, double - blinded, double - dummy, noninferiority, positive drug - controlled clinical trial conducted in the following five investigational sites of the people s republic of china: department of endocrinology, peking union medical college hospital, beijing; department of obstetrics and gynecology, general hospital of the people s liberation army, beijing; metabolic bone disease and genetic research unit, department of osteoporosis and bone disease, shanghai jiao tong university affiliated sixth people s hospital, shanghai; department of geriatrics, shanghai huadong hospital, shanghai; and department of gynecology and obstetrics, beijing hospital, ministry of public health, beijing . Patients were randomized (using random number tables and sequentially numbered envelopes) in a 1:1 ratio to receive either menatetrenone (eisai co, ltd, tokyo, japan) 15 mg, three times per day (45 mg / day) (group m) or alfacalcidol (haier pharmaceutical co, ltd, qingdao, people s republic of china) 0.25 g, twice per day (0.5 g / day) (group a) for 1 year . Additionally, patients in both groups received once daily calcichew (nycomed pharma, zurich, switzerland), containing 500 mg of elemental calcium . After screening and randomization (baseline), patients were followed up every 3 months . At each visit, occurrence and nature of the adverse effects were recorded for each patient . Blood levels of calcium and phosphorus, routine blood examination, urine routine examination, kidney function (creatinine), blood urea nitrogen, liver function tests (ast and alt), and alp were tested at months 0, 6, and 12 . Biochemical markers of bone metabolism such as serum total oc and undercarboxylated oc (ucoc) were also measured at months 0, 6, and 12 . Serum oc and ucoc were measured by a central laboratory (national center for clinical laboratory, beijing, people s republic of china) using an electrochemiluminescence assay (roche diagnostics, basel, switzerland; intra - assay coefficient of variation [cv] of 2.2% and inter - assay cv of 5.9%) and an enzyme - linked immunosorbent assay (takara bio, otsu, japan; intra - assay cv of 4.2% and inter - assay cv of 4.4%). Bmds were measured at the lumbar spine and hip by dual energy x - ray absorptiometry (phantom cv <1.5%). Results at baseline and at months 6 and 12 were recorded . In order to obtain an optimal concordance between measurements from different centers, each imaging instrument was cross - calibrated using an external phantom (synarc inc, newark, ca, usa). Calibration differences between individual scanners within a scanner family (ie, lunar or hologic) were adjusted using the results of the cross - calibration phantom . Systematic differences between the lunar and hologic scanners were adjusted using in vivo cross - calibration formulae from reported research studies.16,17 lumbar and thoracic spine x - ray radiographs were taken at baseline and month 12 . Morphometric vertebral fractures were defined as a decrease of> 20% in vertebral height (minimum, 4 mm) by quantitative morphometry in radiographs.18 the primary endpoints were to evaluate the effects of treatment on bmd at the lumbar spine and hip . The secondary endpoints were to assess the effects of treatment on serum biochemical markers including serum oc, ucoc, and ucoc / oc ratio . Efficacy analysis was completed for the per - protocol set, including all patients who had undergone randomization, except for 23 patients who dropped out during the study (group m: n=108; group a: n=105). Safety analysis was performed for the safety set, and they included patients who had received at least one treatment dose, with the last available data in all subsequent measuring points (group m: n=118; group a: n=118). All follow - up measurements were compared to the baseline values, and the change rates were calculated for each patient . Due to skewed distribution, the percentage change from the baseline of bmd was presented as the median . P - values of bmd compared with the baseline within each group were based on change from baseline . Differences within and between groups were tested by the wilcoxon signed - rank test and wilcoxon rank - sum test, respectively . Statistical analyses were performed using sas v8 (sas institute inc, cary, ny, usa). The study was approved by the state food and drug administration of china (2004l03447). Women patients included in the study had to: 1) be ambulatory; 2) aged between 45 and 75 years; 3) be postmenopausal for at least 5 years; 4) have a body mass index between 18 and 30 kg / m; 5) have a lumbar spine t - score (l2l4) and/or femoral neck bmd lower than 2.0; and 6) be willing to provide written informed consent . Patients with the following criteria were excluded from the study: 1) disorders known to affect bone metabolism; 2) chronic diseases such as hyperthyroidism, hyperparathyroidism, osteomalacia, or diabetes; 3) treatment with bisphosphonates within the past year; 4) treatment with selective estrogen receptor modulators within the past 6 months; 5) treatment with activated vitamin d, calcitonin, estrogens, or androgens within the past 3 months; 6) treatment with warfarin; 7) chronic renal or liver disorders; and 8) history of malignant tumor . Patients were also excluded if one of these following laboratory tests was abnormal: 1) alkaline phosphatase (alp) elevated> 10% of upper normal limit; 2) alanine aminotransferase (alt) or aspartate aminotransferase (ast) elevated> 50% of upper normal limit; 3) serum creatinine levels> 133 mmol / l (1.5 mg / dl); and 4) fasting blood glucose> 126 mg / dl (7.0 mmol / l). This was a multicenter, randomized, double - blinded, double - dummy, noninferiority, positive drug - controlled clinical trial conducted in the following five investigational sites of the people s republic of china: department of endocrinology, peking union medical college hospital, beijing; department of obstetrics and gynecology, general hospital of the people s liberation army, beijing; metabolic bone disease and genetic research unit, department of osteoporosis and bone disease, shanghai jiao tong university affiliated sixth people s hospital, shanghai; department of geriatrics, shanghai huadong hospital, shanghai; and department of gynecology and obstetrics, beijing hospital, ministry of public health, beijing . Patients were randomized (using random number tables and sequentially numbered envelopes) in a 1:1 ratio to receive either menatetrenone (eisai co, ltd, tokyo, japan) 15 mg, three times per day (45 mg / day) (group m) or alfacalcidol (haier pharmaceutical co, ltd, qingdao, people s republic of china) 0.25 g, twice per day (0.5 g / day) (group a) for 1 year . Additionally, patients in both groups received once daily calcichew (nycomed pharma, zurich, switzerland), containing 500 mg of elemental calcium . After screening and randomization (baseline), patients were followed up every 3 months . At each visit, occurrence and nature of the adverse effects were recorded for each patient . Blood levels of calcium and phosphorus, routine blood examination, urine routine examination, kidney function (creatinine), blood urea nitrogen, liver function tests (ast and alt), and alp were tested at months 0, 6, and 12 . Biochemical markers of bone metabolism such as serum total oc and undercarboxylated oc (ucoc) were also measured at months 0, 6, and 12 . Serum oc and ucoc were measured by a central laboratory (national center for clinical laboratory, beijing, people s republic of china) using an electrochemiluminescence assay (roche diagnostics, basel, switzerland; intra - assay coefficient of variation [cv] of 2.2% and inter - assay cv of 5.9%) and an enzyme - linked immunosorbent assay (takara bio, otsu, japan; intra - assay cv of 4.2% and inter - assay cv of 4.4%). Bmds were measured at the lumbar spine and hip by dual energy x - ray absorptiometry (phantom cv <1.5%). Results at baseline and at months 6 and 12 were recorded . In order to obtain an optimal concordance between measurements from different centers, each imaging instrument was cross - calibrated using an external phantom (synarc inc, newark, ca, usa). Calibration differences between individual scanners within a scanner family (ie, lunar or hologic) were adjusted using the results of the cross - calibration phantom . Systematic differences between the lunar and hologic scanners were adjusted using in vivo cross - calibration formulae from reported research studies.16,17 lumbar and thoracic spine x - ray radiographs were taken at baseline and month 12 . Morphometric vertebral fractures were defined as a decrease of> 20% in vertebral height (minimum, 4 mm) by quantitative morphometry in radiographs.18 the primary endpoints were to evaluate the effects of treatment on bmd at the lumbar spine and hip . The secondary endpoints were to assess the effects of treatment on serum biochemical markers including serum oc, ucoc, and ucoc / oc ratio . Efficacy analysis was completed for the per - protocol set, including all patients who had undergone randomization, except for 23 patients who dropped out during the study (group m: n=108; group a: n=105). Safety analysis was performed for the safety set, and they included patients who had received at least one treatment dose, with the last available data in all subsequent measuring points (group m: n=118; group a: n=118). All follow - up measurements were compared to the baseline values, and the change rates were calculated for each patient . Due to skewed distribution, the percentage change from the baseline of bmd was presented as the median . P - values of bmd compared with the baseline within each group were based on change from baseline . Differences within and between groups were tested by the wilcoxon signed - rank test and wilcoxon rank - sum test, respectively . Statistical analyses were performed using sas v8 (sas institute inc, cary, ny, usa). A total of 236 eligible women with postmenopausal osteoporosis were enrolled in the study, and they were randomly assigned to receive menatetrenone or alfacalcidol (n=118 each). Among them, 213 patients (90.3%) completed the 1-year follow - up period, and 23 patients withdrew prematurely . In group m, ten patients withdrew (adverse event = 4, protocol violation = 1, and nonmedical reason = 5); and in group a, 13 patients withdrew (adverse event = 8, nonmedical reason = 5). Also, there were no differences in blood pressure, routine blood analyses, routine urine analyses, and liver and kidney function tests between the two groups (data not shown). Compared with baseline, bmd in group m significantly increased by 1.2% and 2.7% at the lumbar spine and trochanter, respectively, after 12 months of treatment (p<0.001). In group a, bmd significantly increased by 2.2% and 1.8% at the lumbar spine and trochanter, respectively (p<0.001). There was no significant difference between the two groups after 6 and 12 months of treatment (p>0.05) (figure 1). Figure 2 shows the effects of treatment on serum oc and ucoc levels and on the ucoc / oc ratio . In group m, after 12 months of treatment, oc and ucoc decreased by 38.7% and 82.3%, compared with baseline (p<0.001). In group a, oc and ucoc also decreased by 25.8% and 34.8%, respectively (p<0.001). The decreases in serum oc and ucoc levels were more obvious in group m than in group a (p<0.001). The ucoc / oc ratios also decreased after treatment, especially in group m (group m: p<0.001; group a: p<0.05) (figure 2). In group m, mmol / l, 2.420.12 mmol / l, and 2.440.12 mmol / l at baseline, and after 6 and 12 months of treatment, respectively . Mmol / l, 1.260.17 mmol / l, and 1.270.18 mmol / l at baseline and after 6 and 12 months of treatment, respectively . Serum alp was 75.519.4 u / l, 72.624.0 u / l, and 72.219.1 u / l at baseline, and after 6 and 12 months of treatment, respectively . There was no significant difference between the changes of serum calcium, phosphorus, and alp before and after treatment (all p>0.05). In group mmol / l, 2.430.14 mmol / l, and 2.430.13 mmol / l at baseline and after 6 and 12 months of treatment, respectively . Mmol / l, 1.280.17 mmol / l, and 1.230.18 mmol / l at baseline, and after 6 and 12 months of treatment, respectively . U / l, 73.018.6 u / l, and 70.316.5 u / l at baseline, and after 6 and 12 months of treatment, respectively . There was no significant difference between the changes of serum calcium, phosphorus, and alp before and after treatment (all p>0.05). There was no significant difference between the two groups after 6 and 12 months of treatment (all p>0.05). Two patients (1.85%, 2/108) in group m suffered new bone fractures during the 1-year observation period, which included one tibia fracture and one femoral neck fracture . In group a, four patients (3.81%, 4/105) suffered new fractures, which included three lumbar spine compressive fractures and one forearm fracture . Thirteen patients (12.04%, 13/108) in group m experienced new falls during the 1-year observation period, while ten patients (9.52%, 10/105) in group a experienced new falls . There were no significant differences in new bone fractures and falls between the two groups (p>0.05). The incidence of related adverse events was 7.7% in group m and 5.1% in group a. related adverse events reported during the study are shown in table 2 . Four patients in group m and eight patients in group a dropped out of the study due to adverse events . The majority of these adverse events were gastrointestinal symptoms and were of mild to moderate intensity . Serum alt or / and ast were mildly elevated (73 u / l, 155 u / l) in two patients in group m at month 12 . However, their liver function returned to within the normal range after 2 and 8 months, respectively . Transient hypercalcemia and leukocytopenia were observed in group m. unrelated adverse events included bone pain (1.7% in group m and 0.9% in group a) and joint pain (6.8% in group m and 3.4% in group a), which were mild and showed no difference between the two groups . No other clinically significant unexpected adverse events were reported in either group during the study . A total of 236 eligible women with postmenopausal osteoporosis were enrolled in the study, and they were randomly assigned to receive menatetrenone or alfacalcidol (n=118 each). Among them, 213 patients (90.3%) completed the 1-year follow - up period, and 23 patients withdrew prematurely . In group m, ten patients withdrew (adverse event = 4, protocol violation = 1, and nonmedical reason = 5); and in group a, 13 patients withdrew (adverse event = 8, nonmedical reason = 5). Also, there were no differences in blood pressure, routine blood analyses, routine urine analyses, and liver and kidney function tests between the two groups (data not shown). Compared with baseline, bmd in group m significantly increased by 1.2% and 2.7% at the lumbar spine and trochanter, respectively, after 12 months of treatment (p<0.001). In group a, bmd significantly increased by 2.2% and 1.8% at the lumbar spine and trochanter, respectively (p<0.001). There was no significant difference between the two groups after 6 and 12 months of treatment (p>0.05) (figure 1). Figure 2 shows the effects of treatment on serum oc and ucoc levels and on the ucoc / oc ratio . In group m, after 12 months of treatment, oc and ucoc decreased by 38.7% and 82.3%, compared with baseline (p<0.001). In group a, oc and ucoc also decreased by 25.8% and 34.8%, respectively (p<0.001). The decreases in serum oc and ucoc levels were more obvious in group m than in group a (p<0.001). The ucoc / oc ratios also decreased after treatment, especially in group m (group m: p<0.001; group a: p<0.05) (figure 2). In group m, serum calcium was 2.370.09 mmol / l, 2.420.12 mmol / l, and 2.440.12 mmol / l at baseline, and after 6 and 12 months of treatment, respectively . Mmol / l, 1.260.17 mmol / l, and 1.270.18 mmol / l at baseline and after 6 and 12 months of treatment, respectively . U / l, 72.624.0 u / l, and 72.219.1 u / l at baseline, and after 6 and 12 months of treatment, respectively . There was no significant difference between the changes of serum calcium, phosphorus, and alp before and after treatment (all p>0.05). In group mmol / l, 2.430.14 mmol / l, and 2.430.13 mmol / l at baseline and after 6 and 12 months of treatment, respectively . Mmol / l, 1.280.17 mmol / l, and 1.230.18 mmol / l at baseline, and after 6 and 12 months of treatment, respectively . U / l, 73.018.6 u / l, and 70.316.5 u / l at baseline, and after 6 and 12 months of treatment, respectively . There was no significant difference between the changes of serum calcium, phosphorus, and alp before and after treatment (all p>0.05). There was no significant difference between the two groups after 6 and 12 months of treatment (all p>0.05). Two patients (1.85%, 2/108) in group m suffered new bone fractures during the 1-year observation period, which included one tibia fracture and one femoral neck fracture . In group a, four patients (3.81%, 4/105) suffered new fractures, which included three lumbar spine compressive fractures and one forearm fracture . Thirteen patients (12.04%, 13/108) in group m experienced new falls during the 1-year observation period, while ten patients (9.52%, 10/105) in group a experienced new falls . There were no significant differences in new bone fractures and falls between the two groups (p>0.05). The incidence of related adverse events was 7.7% in group m and 5.1% in group a. related adverse events reported during the study are shown in table 2 . Four patients in group m and eight patients in group a dropped out of the study due to adverse events . The majority of these adverse events were gastrointestinal symptoms and were of mild to moderate intensity . Serum alt or / and ast were mildly elevated (73 u / l, 155 u / l) in two patients in group m at month 12 . However, their liver function returned to within the normal range after 2 and 8 months, respectively . Transient hypercalcemia and leukocytopenia were observed in group m. unrelated adverse events included bone pain (1.7% in group m and 0.9% in group a) and joint pain (6.8% in group m and 3.4% in group a), which were mild and showed no difference between the two groups . No other clinically significant unexpected adverse events were reported in either group during the study . The present study investigated the efficacy and safety of menatetrenone in chinese postmenopausal osteoporotic women . A significant increase in bmd by 1.2% and 2.7% at the lumbar spine and trochanter, respectively, from baseline and a significant decrease in oc and ucoc by 38.7% and 82.3%, respectively, from baseline were observed with menatetrenone treatment . Being the first clinical trial to investigate the effects of menatetrenone on a chinese population, the study results could provide a valid clinical basis on treating osteoporosis . Since the study involved postmenopausal osteoporotic women (bmd t - score less than 2.0), placebo control was not used due to ethical reasons . Alfacalcidol is a recognized drug in treating osteoporosis in the people s republic of china . Studies in japan showed no differences in bmd changes between menatetrenone and alfacalcidol in postmenopausal women.19 therefore, alfacalcidol was chosen as a positive drug control to demonstrate the noninferiority of these two drugs . Results showed that menatetrenone 45 mg / day could significantly improve lumbar spine and trochanter bmd after 12 months of treatment . The dose of menatetrenone (45 mg / day) used in this study is the standard dose to treat osteoporosis . As per the primary osteoporosis diagnosis and treatment guidelines of the chinese society of osteoporosis and bone mineral research, the dose of alfacalcidol in treating osteoporosis is 0.51.0 g / day.20 in order to avoid the side effect of high dose alfacalcidol such as hypercalcemia and hypercalciuria,21 a low dose of 0.5 g / day alfacalcidol was used in this study . No differences were observed between menatetrenone and alfacalcidol (1.2% versus 2.2% in the lumbar spine, and 2.7% versus 1.8% in the trochanter, respectively) with respect to changes in bmd . Some randomized controlled trials showed that menatetrenone had only a modest impact or no impact on bmd.2224 in the study by orimo et al,19 there were no differences observed in bmd between menatetrenone 45 mg / day and alfacalcidol 1 g / day in 24 and 48 weeks . Although menatetrenone has only a modest effect on bone loss, it may have the potential to prevent osteoporotic fractures . Vitamin k deficiency has been reported to contribute to the occurrence of fractures in elderly women, especially hip fractures,2528 and it is also a strong predictor of vertebral fractures in patients on hemodialysis.29 a randomized controlled trial suggested that vitamin k2 sustained lumbar bmd and reduced the incidence of vertebral fractures, with a reported reduction of 53% in patients with postmenopausal osteoporosis; this rate was similar to the rate reported with treatment with bisphosphonates.22 knapen et al24 indicated that menatetrenone improved the bone strength of the femoral neck by improving femoral neck width and maintaining the indices of compression, bending, and impact strength . These could lead to a decreased incidence of clinical fractures . In the present study, 1.9% of patients experienced a new fracture in group m during 1 year of treatment, while this proportion was 3.8% in group a; however, there was no significant difference between the two groups . Menatetrenone is thought to be involved in bone formation as an essential cofactor for -carboxylation of oc . Rapid conversion of ucoc to carboxylated oc following menatetrenone treatment was confirmed in elderly osteoporotic women with vertebral fractures.3033 the present study s results show that oc and ucoc decreased by 38.7% and 82.3%, respectively, compared with baseline another study showed that menatetrenone reduced serum ucoc levels, and a study even showed a decrease in serum ucoc levels after 1 month of therapy.34 however, the results on serum total oc levels are inconsistent . Some studies showed an increase in serum total oc levels,22,24 while another showed a decrease in serum total oc levels by vitamin k2 supplementation.35 two explanations can be proposed for this discrepancy: 1) since more oc becomes carboxylated and functional with the improvement of vitamin k2 status, a lesser amount is synthesized and released into circulation; and 2) more functional oc is bound into bones rather than circulating in the blood stream, thus lowering serum total oc levels.35,36 studies have showed that ucoc, but not total oc, is a marker predicting hip fracture in elderly women.2527 menatetrenone tolerability was also evaluated in this study . Adverse events included gastrointestinal symptoms, mild and transient hypercalcemia, leukocytopenia, and abnormalities in liver function . No severe side effects were ever reported before for menatetrenone.32,36 even in this study, menatetrenone was well tolerated by chinese postmenopausal women . Moreover, the treatment with menatetrenone is a pharmacoeconomically viable option . The sample size was not large enough, and trial duration was not long enough to evaluate the risk of fracture as the primary endpoint . However, results did show important short - term effects in bmd, oc, and ucoc levels, suggesting that these short - term changes should have an impact on the long - term risk of fractures . However, changes in bmd directly reflected the balance between bone resorption and bone deposition, and the study results showed that menatetrenone supplementation had a positive impact on bmd . After 1 year of treatment with menatetrenone, bmd at the lumbar spine and hip were significantly increased in chinese postmenopausal osteoporotic women, and that effect was comparable to alfacalcidol . These positive changes in bmd were reflected by changes in markers of bone metabolism, and menatetrenone had more effects on these markers . Thus, menatetrenone is an effective and safe choice for the treatment of postmenopausal osteoporosis in chinese women.
The online version of this article (doi:10.1007/s00259 - 012 - 2182 - 0) contains supplementary material, which is available to authorized users . Melanoma is a tumour with one of the most rapidly increasing incidence rates, especially in caucasian populations . In a decade, its incidence in the netherlands has risen by 63% to 21.7/100,000 (european standardized rate) from 13.3/100,000 in 1998 . Standard treatment for patients with palpable lymph node metastases (ajcc stage iiib) is therapeutic lymph node dissection (tlnd). . Established prognostic factors in patients with stage iiib melanoma are the number and size of metastatic nodes, and the presence of extranodal growth [2, 3]. Identification of additional prognostic factors could lead to more individualized treatment and follow - up schemes . Whole - body f - fdg pet is a sensitive screening tool in patients with high - risk melanoma, since melanomas are typically fdg - avid . The added value of fdg pet over ct with respect to diagnostic accuracy and impact on management has been established in several studies [410]. Besides improved detection of metastases, a standard clinical pet / ct scan offers the opportunity to quantify the level of glucose metabolism in tumours . Glucose metabolism, as assessed with fdg, is an epiphenomenon of cancer and seems to have prognostic value [1117]. However, the value of fdg as a prognostic biomarker is not undisputed, probably as a consequence of confounding and heterogeneity in both the clinical spectrum and pet technology . We have reported some preliminary data acquired in a retrospective setting suggesting an inverse relationship between tumour fdg uptake in patients with stage iii melanoma who receive surgery with curative intent . The present study aimed to prospectively validate these findings taking into account the above - mentioned potential confounders . From november 2003 until march 2008, all patients with palpable, histology- or cytology - proven lymph node metastases of melanoma referred to the university medical centre groningen (umcg) for fdg pet and ct examination were prospectively included in this study . Prior to study entry, all patients were considered candidates for tlnd, and they took part in a large prospective multicentre study to assess the diagnostic performance of fdg pet and multislice ct . Inclusion criteria for the present study were: stage iii after fdg pet and abdominal / thoracic ct, candidate for tlnd with curative intent, and scanned on the same fdg pet scanner with identical patient preparation, acquisition and reconstruction protocols . Follow - up was uniform in all patients and consisted of clinical evaluation of physical signs and symptoms at standard follow - up visits (every 3 months in the first year, every 4 months in the second year, twice a year in the third to fifth years, and every year in the sixth to tenth years); diagnostic tests (chest radiography, ct or fdg pet) were only indicated if distant metastases were suspected . Fdg was synthesized on - site according to the method described by hamacher et al . Using an automated synthesis module . Prior to fdg pet imaging, patients were instructed to fast for at least 6 h and to drink 1 l of water . After intravenous injection of fdg, whole - body imaging was performed in the two - dimensional mode using a siemens ecat exact hr+ scanner (siemens / cti, knoxville, tn), applying emission scans of 5 min per bed position, starting 90 min after the injection of fdg . Image reconstructions were iterative (ordered subset expectation maximization) with eight subsets and two iterations . All scans were corrected for decay, scatter, randoms and attenuation, and normalized . The postreconstruction filter (gaussian postprocessing filter) was 5 mm, yielding an estimated spatial resolution (full - width at half - maximum) of 7 mm . Fdg uptake was calculated as suv (radioactivity concentration in tissue in becquerels per cubic centimetre / injected dose in becquerels / patient body weight in grams). Three - dimensional volumes of interest were placed semiautomatically around the lymph node metastasis with the highest fdg uptake (visual assessment) using dedicated software (idl - viewer, leuven). The numerator of the suv was chosen to measure either the mean counts within an isocontour to 70% of the voxels with the highest counts, or the maximum itself, yielding suvmean or suvmax, respectively . To explore the distribution of suvmean and suvmax a kernel density plot was used, which approximates the probability density of the variable; kernel density plots have the advantage of being independent of the choice of origin, unlike histograms . The associations between suvmean / suvmax or the log - transformation (which gave the best fit) and patient characteristics were assessed using student s t - test or analysis of variance . Patients were divided into two groups (high and low suv) based on the median value of the log(suvmean) and log(suvmax) for the survival analysis . This dichotomization led to the same patient categorization for suvmax and suvmean in 95% of the patients . Since repeatability of suvmean is superior to that of suvmax [20, 21], we show the suvmean results in the analyses . Survival was analysed in terms of disease - free survival (dfs) and disease - specific survival (dss). An event was recorded for any recurrence; for dss an event was recorded if the patient died due to melanoma metastases . Multivariable models were created with age, gender, location of the lymph nodes defined as cervical region, axilla or groin, number of nodes removed during tlnd, number of lymph nodes positive on histopathology after tlnd, tumour size in the largest lymph node metastasis determined after tlnd by histopathology, extranodal growth defined as metastatic tumour which clearly extended (histologically) through the nodal capsule into the perinodal fatty tissue or tumour involvement in the hilar region with interruption of the smooth outline of the (presumed) capsule, breslow thickness and ulceration of the primary melanoma, and high or low suvmean . From november 2003 until march 2008, all patients with palpable, histology- or cytology - proven lymph node metastases of melanoma referred to the university medical centre groningen (umcg) for fdg pet and ct examination were prospectively included in this study . Prior to study entry, all patients were considered candidates for tlnd, and they took part in a large prospective multicentre study to assess the diagnostic performance of fdg pet and multislice ct . Inclusion criteria for the present study were: stage iii after fdg pet and abdominal / thoracic ct, candidate for tlnd with curative intent, and scanned on the same fdg pet scanner with identical patient preparation, acquisition and reconstruction protocols . Follow - up was uniform in all patients and consisted of clinical evaluation of physical signs and symptoms at standard follow - up visits (every 3 months in the first year, every 4 months in the second year, twice a year in the third to fifth years, and every year in the sixth to tenth years); diagnostic tests (chest radiography, ct or fdg pet) were only indicated if distant metastases were suspected . Fdg was synthesized on - site according to the method described by hamacher et al . Using an automated synthesis module . Prior to fdg pet imaging, patients were instructed to fast for at least 6 h and to drink 1 l of water . After intravenous injection of fdg, whole - body imaging was performed in the two - dimensional mode using a siemens ecat exact hr+ scanner (siemens / cti, knoxville, tn), applying emission scans of 5 min per bed position, starting 90 min after the injection of fdg . Image reconstructions were iterative (ordered subset expectation maximization) with eight subsets and two iterations . All scans were corrected for decay, scatter, randoms and attenuation, and normalized . The postreconstruction filter (gaussian postprocessing filter) was 5 mm, yielding an estimated spatial resolution (full - width at half - maximum) of 7 mm . Fdg uptake was calculated as suv (radioactivity concentration in tissue in becquerels per cubic centimetre / injected dose in becquerels / patient body weight in grams). Three - dimensional volumes of interest were placed semiautomatically around the lymph node metastasis with the highest fdg uptake (visual assessment) using dedicated software (idl - viewer, leuven). The numerator of the suv was chosen to measure either the mean counts within an isocontour to 70% of the voxels with the highest counts, or the maximum itself, yielding suvmean or suvmax, respectively . To explore the distribution of suvmean and suvmax a kernel density plot was used, which approximates the probability density of the variable; kernel density plots have the advantage of being independent of the choice of origin, unlike histograms . The associations between suvmean / suvmax or the log - transformation (which gave the best fit) and patient characteristics were assessed using student s t - test or analysis of variance . Patients were divided into two groups (high and low suv) based on the median value of the log(suvmean) and log(suvmax) for the survival analysis . This dichotomization led to the same patient categorization for suvmax and suvmean in 95% of the patients . Since repeatability of suvmean is superior to that of suvmax [20, 21], we show the suvmean results in the analyses . Survival was analysed in terms of disease - free survival (dfs) and disease - specific survival (dss). An event was recorded for any recurrence; for dss an event was recorded if the patient died due to melanoma metastases . Multivariable models were created with age, gender, location of the lymph nodes defined as cervical region, axilla or groin, number of nodes removed during tlnd, number of lymph nodes positive on histopathology after tlnd, tumour size in the largest lymph node metastasis determined after tlnd by histopathology, extranodal growth defined as metastatic tumour which clearly extended (histologically) through the nodal capsule into the perinodal fatty tissue or tumour involvement in the hilar region with interruption of the smooth outline of the (presumed) capsule, breslow thickness and ulceration of the primary melanoma, and high or low suvmean . There were slightly more women: 41 women (51.2%), 39 men (48.8%). Lymph node metastases were located in the groin (55.0%), axilla (32.5%) or cervical region (12.5%). The median number of removed nodes was 16 (range 748) and the median number of positive nodes was 2 (range 119). The tumour size in the lymph node ranged from 0.5 to 7.0 cm with a median of 3.1 cm . In 25 patients (31.2%) extranodal growth was recorded.table 1patient and tumour characteristics of the 80 patients with palpable, histology- or cytology - proven lymph node metastases of melanomacharacteristicvaluegender, n (%) men39 (48.8)women41 (51.2)age (years), n (%) <50 25 (31.2)506532 (40.0)>6523 (28.8)primary melanomalocation, n (%) upper extremities9 (11.2)lower extremities34 (42.5)trunk29 (36.3)head and neck6 (7.5)unknown primary2 (2.5)breslow thickness (mm), n (%) 1.010 (12.5)1.02.027 (33.8)2.041 (51.2)unknown primary2 (2.5)ulceration, n (%) no63 (78.8)yes15 (18.7)unknown primary2 (2.5)lymph node metastaseslocation, n (%) cervical region10 (12.5)axilla26 (32.5)groin44 (55.0)removed nodes, median (range)16 (748)positive nodes, median (range)2 (119)tumour size, median (range)3.1 (0.57.0)extranodal growth, n (%) no55 (68.8)yes25 (31.2) patient and tumour characteristics of the 80 patients with palpable, histology- or cytology - proven lymph node metastases of melanoma as shown in fig . 1, the suv values were not normally distributed (no gaussian distribution). A log - transformation of the suvmean and suvmax values provided the best fit to a gaussian distribution . The log - transformed suvmean and suvmax values were not associated with gender (p = 0.3 and p = 0.4), age (p = 0.2 and p = 0.1), location of the lymph nodes (p = 0.2 for both), number of removed nodes (p = 0.4 for both), number of positive nodes (p = 0.1 for both) or extranodal growth (p = 0.6 for both), respectively . Suv was, however, associated with size of the melanoma metastasis in the lymph node (p <0.001).fig . 1distributions of the suvmean and suvmax values in comparison with the normal (gaussian) distributions distributions of the suvmean and suvmax values in comparison with the normal (gaussian) distributions during the follow - up (median 3.0 years, range: 0.38.2 years, in all patients; 5.5 years, 3.18.2 years, in patients without an event), 55 patients (68.8%) developed a recurrence . Overall, 49 patients (61.3%) died as a result of the recurrent disease and 6 patients with recurrence were still alive at the end of the study . The median suvmean (used for further analysis) was 6.49 (iqr 4.510.9) and the median log(suvmean) was 1.86 . The 5-year dfs in patients with a low suvmean was 40.9% (95% ci 25.555.7%) and 24.2% (95%ci 12.438.0%) in patients with a high suvmean (p = 0.02), as shown in fig . 2 . Webtable 1 shows the univariate and multivariable analysis of dfs, and table 2 shows the analysis of dfs in relation to suv . Suvmean was associated with dfs with a hazard ratio (hr) of 1.7 (95% ci 1.03.0; p = 0.048). Other variables associated with dfs in the multivariable analysis were the number of positive nodes (hr 1.1, 95% ci 1.01.2; p = 0.02) and the presence of extranodal growth (hr 3.4, 95% ci 1.44.2; p = 0.003).fig . 2kaplan meier curves for dfs and dss in patients with a high and low suvtable 2multivariable analyses of dfs and dss for the patients with stage iii melanomavariabledfsdsshazard ratiop valuehazard ratiop valuesuvlow1 (reference)0.0481 (reference)0.1high1.74 (1.003.00)1.57 (0.862.87)adjusted for positive nodes and extranodal growth (see webtable).adjusted for sex, positive nodes and extranodal growth (see webtable).divided by the median of log(suvmean) = 1.86 . Kaplan meier curves for dfs and dss in patients with a high and low suv multivariable analyses of dfs and dss for the patients with stage iii melanoma adjusted for positive nodes and extranodal growth (see webtable). Adjusted for sex, positive nodes and extranodal growth (see webtable). The 5-year dss was 47.5% (95% ci 30.962.3) in patients with a low suvmean and 30.3% (95% ci 16.745.0) for patients with a high suvmean (p = 0.02, fig . 2). Webtable 2 shows the univariate and multivariable analysis of dss, and table 2 shows the analysis of dss in relation to suv . Suvmean was not associated with dss in the multivariable analysis (hr 1.6, 95% ci 0.92.9; p = 0.1). Female gender was associated with a better dss in the multivariable analysis (hr 0.5, 95% ci 0.30.8; p = 0.009). During the follow - up (median 3.0 years, range: 0.38.2 years, in all patients; 5.5 years, 3.18.2 years, in patients without an event), 55 patients (68.8%) developed a recurrence . Overall, 49 patients (61.3%) died as a result of the recurrent disease and 6 patients with recurrence were still alive at the end of the study . The median suvmean (used for further analysis) was 6.49 (iqr 4.510.9) and the median log(suvmean) was 1.86 . The 5-year dfs in patients with a low suvmean was 40.9% (95% ci 25.555.7%) and 24.2% (95%ci 12.438.0%) in patients with a high suvmean (p = 0.02), as shown in fig . 2 . Webtable 1 shows the univariate and multivariable analysis of dfs, and table 2 shows the analysis of dfs in relation to suv . Suvmean was associated with dfs with a hazard ratio (hr) of 1.7 (95% ci 1.03.0; p = 0.048). Other variables associated with dfs in the multivariable analysis were the number of positive nodes (hr 1.1, 95% ci 1.01.2; p = 0.02) and the presence of extranodal growth (hr 3.4, 95% ci 1.44.2; p = 0.003).fig . 2kaplan meier curves for dfs and dss in patients with a high and low suvtable 2multivariable analyses of dfs and dss for the patients with stage iii melanomavariabledfsdsshazard ratiop valuehazard ratiop valuesuvlow1 (reference)0.0481 (reference)0.1high1.74 (1.003.00)1.57 (0.862.87)adjusted for positive nodes and extranodal growth (see webtable).adjusted for sex, positive nodes and extranodal growth (see webtable).divided by the median of log(suvmean) = 1.86 . Kaplan meier curves for dfs and dss in patients with a high and low suv multivariable analyses of dfs and dss for the patients with stage iii melanoma adjusted for positive nodes and extranodal growth (see webtable). Adjusted for sex, positive nodes and extranodal growth (see webtable). The 5-year dss was 47.5% (95% ci 30.962.3) in patients with a low suvmean and 30.3% (95% ci 16.745.0) for patients with a high suvmean (p = 0.02, fig . 2). Webtable 2 shows the univariate and multivariable analysis of dss, and table 2 shows the analysis of dss in relation to suv . Suvmean was not associated with dss in the multivariable analysis (hr 1.6, 95% ci 0.92.9; p = 0.1). Female gender was associated with a better dss in the multivariable analysis (hr 0.5, 95% ci 0.30.8; p = 0.009). The present prospective study endorses the prognostic value of suv in terms of dfs in patients with stage iiib melanoma who have been optimally staged with fdg pet and ct . The use of suv as a quantitative parameter of fdg uptake has a long tradition in nuclear medicine . Although its value for predicting tumour response to therapy has become generally accepted, the clinical usefulness and applicability of suv for prognostic purposes is still under discussion . As far as the authors are aware there are two other studies (from our group) that addressed the value of suv in melanoma patients [13, 22]. The first study was a small retrospective study that showed no significant association between suvmean and survival (p = 0.11); however, dfs was significantly decreased in patients with a high suvmean (p = 0.03). There are, however, significant differences between the patients in the two databases, especially in location, number of positive nodes (more in the present study) and percentage of patients with extranodal growth (lower percentage in the present study). Furthermore, the cut - off values in both studies were data - driven and differ . In the present prospective study we confirmed the association found in the retrospective study . There was a significantly decreased dfs in patients with a high suvmean in the lymph node metastasis (p = 0.048). Dss was, however, associated with gender, as reported previously by de vries et al . . Among 10,538 melanoma patients, women had a superior survival than men even after adjustment for multiple confounding variables . Probably factors other than stage at diagnosis and location reduces mortality risk in female melanoma patients . The associations between extranodal growth and the number of nodes have been reported before: extranodal growth showed a stronger association with dfs than suvmean . Some of the primary melanoma characteristics such as breslow thickness and ulceration were not associated with survival in these patients with macrometastatic disease . Interestingly, a recent study by balch et al . Showed similar results: in patients with nodal micrometastases, multiple covariates independently predicted survival, including several primary melanoma features (thickness, mitotic rate, ulceration, and anatomic site of the primary tumour). In contrast, in patients with nodal macrometastases, primary melanoma characteristics did not predict survival . Remarkably, there was no clear significant association between tumour size and survival in the present study . Categorization of tumour size into <3 cm or 3 cm did not lead to different results (dfs hr 1.7, 95% ci 1.03.0), p = 0.1; dss unfortunately, we were not able to stratify according to tumour size . Further, larger studies should possibly involve stratification according to smaller and larger tumour in the lymph nodes . We present a single - centre study with standardized pet procedures to obtain homogeneity at that level . Since the measured suv is a function of many technical and biological factors, the absolute values are only generalizable if these are taken into account . A recent study by westerterp et al . Showed differences in suv quantification between institutes with different pet scanners . Consequently, standardization of acquisition, reconstruction and data analysis is needed for multicentre trials . When the present study was initiated, there were no guidelines on how to harmonize the results of multicentre studies using different scanners, acquisition and reconstruction protocols . However, provided that the 2010 pet guidelines for trials and clinical practice are followed, this is no longer an obstacle, and hopefully the evidence for the potential of quantitative pet in oncology will mature rapidly . In the present study we used the median value of the log - transformed suvmean for risk stratification . However, there is always change for bias if data - driven cut - off values are used . To be a practical prognostic factor in routine practice, standardization of protocols and cut - off values for suv should be agreed upon or the methodology to determine the optimal threshold for each centre should be established . As a fdg pet scan as a staging procedure prior to treatment of metastatic melanoma in regional lymph nodes is now performed, determination of suv values would provide additional information at very little extra cost . In conclusion, in patients with clinical stage iii melanoma, a high suvmean was associated with decreased dfs . If confirmed in larger studies, suvmean could potentially be used, in addition to the number of positive nodes, tumour size and extranodal growth, as a factor in deciding on adjuvant systemic treatment . This article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
A 23-year - old man (192 cm, 106.4 kg) presented to the authors for pulmonary thromboembolism and was scheduled for pulmonary artery thrombectomy . He has been diagnosed with aps after the treatment of deep vein thrombosis in his right leg at another hospital four years earlier, and he had been prescribed an anticoagulant medicine . Two weeks before the patient's hospital admission, he stopped taking the medication at his discretion . Tenderness, edema, and skin ulcers occurred in both his lower extremities . During his hospital stay, the patient developed dyspnea, and pulmonary thromboembolism was diagnosed in both his descending pulmonary arteries and lower lobe segmental and subsegmental arteries via echocardiography and chest computed tomography . The result of the lung perfusion scan that was performed was also suitable for pulmonary thromboembolism . The preoperative diluted russell's viper venom time (drvvt) result was 112.80 s (reference: 24 - 37.5 s), and the silica clotting time (sct) was prolonged to 185.6 s (reference: 23.4 - 39.2 s). The mixing test was not corrected, and the result was 112.80 s with the patient plasma, and 73.3 s with the mixed plasma . The anticardiolipin antibodies (acas) igg, and igm were positive, the antithrombin iii was 85% of the normal level, the protein c was reduced to 66%, and the protein s was reduced to 57% . Enoxaparine 120 mg was injected via the subcutaneous tissue twice a day and prednisolone was administered during the preoperative period . An abnormal coagulation profile was observed; prothrombin time / international normalized ratio (pt / inr) was 1.9, prothrombin time (pt) was 22.3 s and activated partial thromboplastin time (aptt) was 79.1 s. preoperative estimated pulmonary arterial systolic pressure (pasp) was 76 mmhg . Radial artery catheterization was performed, and midazolam (13 mg), sufentanil (150 g), and vecuronium (10 mg) were injected for induction of anesthesia . The routine monitoring devices for cardiac surgery and intravenous anesthetics were used for maintenance of anesthesia . Initial point - of - care testing (poct) results were the following: arterial oxygen tension was 201 mmhg under a 50% fraction of inspired oxygen (fio2), and plasma hematocrit was 54% . The activated coagulation time (act) was 165 s after induction and 951 s after injection of 336 mg of heparin to begin cpb . Cpb was started after insertion of the cannulas into the aorta, superior vena cava, and inferior vena cava . After the temperature was lowered to 20, pulmonary thrombectomy and endarterectomy were performed, following which intermittent total cardiopulmonary bypass using aortic - cross - clamp was conducted to clear the operating field . Forty minutes after the administration of heparin, the act was checked again and found to be 627 s. generally, such a result would be considered acceptable for a cpb operation, but concerns about the occurrence of catastrophic aps in this case, prompted the empirical administration of 100 mg of heparin via injection . The act was again measured 5 min after the injection and was found to be 1200 s. after 3 hours, the act was found to be 629 s. an additional 80 mg of heparin was thus administered, and the act was found to be 1001 s 5 min after that injection . It was decided that protamine would not be administered due to worries about the occurrence of postoperative thrombotic complications . The total injected dose of heparin was 516 mg, and the total cpb time was 248 min . At the end of the surgery the estimated blood loss (ebl) during surgery was 1,000 ml and urine output was 1,100 ml . The patient was transferred to the intensive care unit (icu) without experiencing significant bleeding . The immediate postoperative pt / inr was 1.63 and aptt was over 180 s. the goal of the postoperative anticoagulation therapy was pt / inr 3.0 with intravenous heparin infusion during the npo period and with oral warfarin after that . The patient was discharged 12 days after surgery without specific complications . A 34-year - old man (160.5 cm, 48.9 kg) the patient had been diagnosed with sle and aps after being admitted to the hospital for treatment of palpitations and dyspnea ten years earlier, and he had taken an anticoagulant medication . During the outpatient clinic follow - up, 24-hr holter monitoring revealed the increasing frequency of non - sustained atrial fibrillation . The aptt was 57.8 s and drvvt was 47.2 s (reference: 25.9 - 35.7 s). The result with patient plasma from the mixing test was 27.3 s, and the mixed plasma result was 37.6 s, while sct was 52.1 s (reference: 22.3 - 42.7 s). In addition, the patient was positive for la, negative for aca igg, and had borderline results for aca igm . Anesthesia induction and maintenance were performed as usual, and routine cardiac anesthesia monitoring was done . The act level increased from 129 s to 850 s after the injection of 150 mg of heparin . After the insertion of cannulas into the aorta, superior vena cava, and inferior vena cava, cpb was started . One hour after the administration of heparin, the act level was found to have decreased to 524 s. therefore, an additional 50 mg of heparin was injected . After the mitral annuloplasty, valvuloplasty was performed, and weaning from cpb was attempted . Due to the risk of thrombosis, a half dose (75 mg) of protamine was administered . Five minutes after the injection of protamine, the act level was found to be 220 s. the total administered dose of heparin during the surgery was 200 mg, and the total cpb time was 52 min . The immediate postoperative pt / inr was 1.39 and aptt was over 180 s. postoperative prophylactic anticoagulation was maintained with warfarin . Ten days after the operation, the patient was discharged without specific complications . A 65-year - old woman (153 cm, 62.9 kg) presented to the authors for mv leaflet mass excision . She already had percutaneous mitral valvuloplasty 10 years ago, mv replacement with tissue valve 8 years ago, and mv thrombectomy and mv replacement with mechanical valve 7 years ago . Since the final surgery, she had been on an anticoagulation regimen with warfarin . Recently she had gone to emergency room after experiencing sudden onset language disturbance, and had been diagnosed with left middle cerebral artery territory infarction . She also underwent routine follow - up transesophageal echocardiography (tee) and a mobile mass was found which was attached to the prosthetic mv leaflet . To prevent an embolic event and decrease the size of the thrombus, the patient was started on heparin several days before the surgery was scheduled . Despite heparinization the surgeon therefore considered that the patient might have a disease causing a hypercoagulable state and ordered laboratory examinations to investigate this possibility . The results of the tests were as follows: protein c (-), protein s (-), antithrombin iii 80 (80 - 120%), aca igm 15.5 mg / dl (<20), aca igg negative, la (+), drvvt 69.1 s (reference: 24 - 37.5 s), sct 137.2 (reference: 23.4 - 39.2 s), mixing test with patient plasma 137.2 s, mixing test with mixed plasma (1: 1) 99.3 s. twelve weeks later, the la test was performed again, and the patient was finally diagnosed with aps . Preoperative pt / inr was 1.37 and aptt was 59.8 s. the planned surgery was mv leaflet mass excision . The patient's left radial artery was cannulated under local anesthesia for invasive blood pressure monitoring . The patient's basal act was 113 s and 180 mg of heparin was administered for vessel cannulations . The act level increased to 742 s and cpb was begun after insertion of cannulas into the aorta, superior vena cava and inferior vena cava . Twenty minutes after the start of cpb, the act level was found to be 826 s, and an additional 60 mg of heparin was administered . The target act level during cpb was over 800 s. after 50 min, the act level had decreased to 682 s, and another 100 mg of heparin was given . At the end of cpb, the act level was 668 s, and the surgeon decided not to administer protamine, concerned about possible postoperative thrombotic complications . One hour after the cessation of cpb, the act level decreased to 577 s. the total time of cpb was 99 min, and the total administered dose of heparin was 340 mg . The immediate postoperative pt / inr was 2.93 and aptt was over 400 s. due to a bleeding tendency, 30 mg of protamine was administered to the patient . With that exception warfarin was used for prophylactic anticoagulation and the patient was discharged 9 days after the operation . A 23-year - old man (192 cm, 106.4 kg) presented to the authors for pulmonary thromboembolism and was scheduled for pulmonary artery thrombectomy . He has been diagnosed with aps after the treatment of deep vein thrombosis in his right leg at another hospital four years earlier, and he had been prescribed an anticoagulant medicine . Two weeks before the patient's hospital admission, he stopped taking the medication at his discretion . Tenderness, edema, and skin ulcers occurred in both his lower extremities . During his hospital stay, the patient developed dyspnea, and pulmonary thromboembolism was diagnosed in both his descending pulmonary arteries and lower lobe segmental and subsegmental arteries via echocardiography and chest computed tomography . The result of the lung perfusion scan that was performed was also suitable for pulmonary thromboembolism . The preoperative diluted russell's viper venom time (drvvt) result was 112.80 s (reference: 24 - 37.5 s), and the silica clotting time (sct) was prolonged to 185.6 s (reference: 23.4 - 39.2 s). The mixing test was not corrected, and the result was 112.80 s with the patient plasma, and 73.3 s with the mixed plasma . The anticardiolipin antibodies (acas) igg, and igm were positive, the antithrombin iii was 85% of the normal level, the protein c was reduced to 66%, and the protein s was reduced to 57% . Enoxaparine 120 mg was injected via the subcutaneous tissue twice a day and prednisolone was administered during the preoperative period . An abnormal coagulation profile was observed; prothrombin time / international normalized ratio (pt / inr) was 1.9, prothrombin time (pt) was 22.3 s and activated partial thromboplastin time (aptt) was 79.1 s. preoperative estimated pulmonary arterial systolic pressure (pasp) was 76 mmhg . Radial artery catheterization was performed, and midazolam (13 mg), sufentanil (150 g), and vecuronium (10 mg) were injected for induction of anesthesia . The routine monitoring devices for cardiac surgery and intravenous anesthetics were used for maintenance of anesthesia . Initial point - of - care testing (poct) results were the following: arterial oxygen tension was 201 mmhg under a 50% fraction of inspired oxygen (fio2), and plasma hematocrit was 54% . The activated coagulation time (act) was 165 s after induction and 951 s after injection of 336 mg of heparin to begin cpb . Cpb was started after insertion of the cannulas into the aorta, superior vena cava, and inferior vena cava . After the temperature was lowered to 20, pulmonary thrombectomy and endarterectomy were performed, following which intermittent total cardiopulmonary bypass using aortic - cross - clamp was conducted to clear the operating field . Forty minutes after the administration of heparin, the act was checked again and found to be 627 s. generally, such a result would be considered acceptable for a cpb operation, but concerns about the occurrence of catastrophic aps in this case, prompted the empirical administration of 100 mg of heparin via injection . The act was again measured 5 min after the injection and was found to be 1200 s. after 3 hours, the act was found to be 629 s. an additional 80 mg of heparin was thus administered, and the act was found to be 1001 s 5 min after that injection . It was decided that protamine would not be administered due to worries about the occurrence of postoperative thrombotic complications . The total injected dose of heparin was 516 mg, and the total cpb time was 248 min . At the end of the surgery the estimated blood loss (ebl) during surgery was 1,000 ml and urine output was 1,100 ml . The patient was transferred to the intensive care unit (icu) without experiencing significant bleeding . The immediate postoperative pt / inr was 1.63 and aptt was over 180 s. the goal of the postoperative anticoagulation therapy was pt / inr 3.0 with intravenous heparin infusion during the npo period and with oral warfarin after that . A 34-year - old man (160.5 cm, 48.9 kg) was presented to the authors for mitral valve (mv) replacement . The patient had been diagnosed with sle and aps after being admitted to the hospital for treatment of palpitations and dyspnea ten years earlier, and he had taken an anticoagulant medication . During the outpatient clinic follow - up, 24-hr holter monitoring revealed the increasing frequency of non - sustained atrial fibrillation . The aptt was 57.8 s and drvvt was 47.2 s (reference: 25.9 - 35.7 s). The result with patient plasma from the mixing test was 27.3 s, and the mixed plasma result was 37.6 s, while sct was 52.1 s (reference: 22.3 - 42.7 s). In addition, the patient was positive for la, negative for aca igg, and had borderline results for aca igm . Anesthesia induction and maintenance were performed as usual, and routine cardiac anesthesia monitoring was done . The act level increased from 129 s to 850 s after the injection of 150 mg of heparin . After the insertion of cannulas into the aorta, superior vena cava, and inferior vena cava, cpb was started . One hour after the administration of heparin, the act level was found to have decreased to 524 s. therefore, an additional 50 mg of heparin was injected . After the mitral annuloplasty, valvuloplasty was performed, and weaning from cpb was attempted . Due to the risk of thrombosis, a half dose (75 mg) of protamine five minutes after the injection of protamine, the act level was found to be 220 s. the total administered dose of heparin during the surgery was 200 mg, and the total cpb time was 52 min . The immediate postoperative pt / inr was 1.39 and aptt was over 180 s. postoperative prophylactic anticoagulation was maintained with warfarin . A 65-year - old woman (153 cm, 62.9 kg) presented to the authors for mv leaflet mass excision . She already had percutaneous mitral valvuloplasty 10 years ago, mv replacement with tissue valve 8 years ago, and mv thrombectomy and mv replacement with mechanical valve 7 years ago . Since the final surgery, she had been on an anticoagulation regimen with warfarin . Recently she had gone to emergency room after experiencing sudden onset language disturbance, and had been diagnosed with left middle cerebral artery territory infarction . She also underwent routine follow - up transesophageal echocardiography (tee) and a mobile mass was found which was attached to the prosthetic mv leaflet . To prevent an embolic event and decrease the size of the thrombus, the patient was started on heparin several days before the surgery was scheduled . Despite heparinization, however, the thrombus showed no change in size on tee . The surgeon therefore considered that the patient might have a disease causing a hypercoagulable state and ordered laboratory examinations to investigate this possibility . The results of the tests were as follows: protein c (-), protein s (-), antithrombin iii 80 (80 - 120%), aca igm 15.5 mg / dl (<20), aca igg negative, la (+), drvvt 69.1 s (reference: 24 - 37.5 s), sct 137.2 (reference: 23.4 - 39.2 s), mixing test with patient plasma 137.2 s, mixing test with mixed plasma (1: 1) 99.3 s. twelve weeks later, the la test was performed again, and the patient was finally diagnosed with aps . Preoperative pt / inr was 1.37 and aptt was 59.8 s. the planned surgery was mv leaflet mass excision . The patient's left radial artery was cannulated under local anesthesia for invasive blood pressure monitoring . The patient's basal act was 113 s and 180 mg of heparin was administered for vessel cannulations . The act level increased to 742 s and cpb was begun after insertion of cannulas into the aorta, superior vena cava and inferior vena cava . Twenty minutes after the start of cpb, the act level was found to be 826 s, and an additional 60 mg of heparin was administered . The target act level during cpb was over 800 s. after 50 min, the act level had decreased to 682 s, and another 100 mg of heparin was given . At the end of cpb, the act level was 668 s, and the surgeon decided not to administer protamine, concerned about possible postoperative thrombotic complications . One hour after the cessation of cpb, the act level decreased to 577 s. the total time of cpb was 99 min, and the total administered dose of heparin was 340 mg . The immediate postoperative pt / inr was 2.93 and aptt was over 400 s. due to a bleeding tendency, 30 mg of protamine was administered to the patient . With that exception warfarin was used for prophylactic anticoagulation and the patient was discharged 9 days after the operation . The three case mentioned above according to the recently revised guidelines for aps the criteria for definite aps include one or more episodes of thrombosis, a symptom related to thrombosis, and a positive antiphospholipid antibody test (apl test) result on two or more occasions that are at least 12 weeks apart . The reason for such a time interval requirement is that about 4 - 5% of healthy individuals can present positive results temporarily due to infections or drugs taken . Two important autoimmune antigens related to the pathophysiologic mechanism of aps that have been discussed are -2-glycoprotein-1 (2gpi) and prothrombin . The coupling of 2gpi and the autoimmune antibody induces blood coagulation in vivo through the signal - transporting system of the cell, but in vitro, the coagulation time is delayed by the addition or formation of negatively charged phospholipids (pl). Prothrombin (coagulation factor ii) also forms prothrombin / antiprothrombin complex, which binds with pl, delaying the coagulation time in vitro . The formation or addition of pl triggered coagulation time delay in the test (aptt and drvvt). As the apl in the patient plasma bound with pl in the normal plasma, the delay of the coagulation test was not corrected in the mixed - coagulation test . The pt is thus not useful in the detection of apl because excessive phospholipids in the form of tissue thromboplastin are added . There are two different prophylactic settings: primary thromboprophylaxis for patients who have not yet experienced a thrombotic event, and secondary thromboprophylaxis for patients who have already had a previous thrombotic event . For primary prophylaxis, patients are prescrived low dose aspirin, and for secondary prophylaxis, patients take warfarin with target pt / inr 2.0 to 3.0 . Aps patients are more vulnerable than the general public to cardiac valvular disease because if endocardial injury (mainly on the left side of the heart) due to the pressure or jet of blood flow occurs, the pl in the cell are exposed, which induces microthrombi . The valve injury thus progresses due to the fibrotic change of the microthrombi . Among the apl antibodies, the autoimmune antibody to 2gpi is frequently detected in paitents with chest pain syndrome or those who are undergoing acute coronary events . Such patients may undergo surgery using cpb, which makes the selection of an appropriate coagulation test or method particularly important . In aps patients, several factors must be considered to be able to choose which coagulation test to perform or which laboratory values to use as the guidelines for the anticoagulation therapy . This is the case because the coagulation test results can be misinterpreted in aps and because the use of cpb can induce an immunologic and a coagulative response . There has thus far been no consensus regarding which is the best method of anticoagulation is, and only a handful of cases that seem to point in the direction of any particular method have been reported . In some studies, either the standard dose (3 mg / kg) or an increased dose (sheikh et al . Recommended a heparin dose that doubles the baseline act level . The use of the act as means to measure blood coagulation is accompanied by a number of problems, namely: (1) there may be patients with increased baseline act levels; (2) half of aps patients may have thrombocytopenia; and (3) the heparin response may vary due to a patient's low level of antithrombin iii . In the cases discussed here, all three of our patients' baseline act levels were in the normal range . In addition, adequate prolongations of act levels were achieved with the standard dosage of heparin in our patients . Ducart et al . Maintained a plasma heparin level of over 2.5 iu / ml . In some cases, measurement of the antifactor xa level has been recently introduced, but there is yet no conclusion as regards which method is most effective and leads to the fewest complications . Adequate diagnostic tools and guidelines must be established according to the hospital circumstances as well as the patient's individual risk factors, and the optimal anticoagulation effect that does not increase the bleeding must be maintained . Moreover, the neutralization of heparin with protamine after the completion of cpb should be carefully done . In some reports, protamine was not used to prevent catastrophic aps . In the first case discussed here, although the act level was prolonged during both the operation and the weaning from cpb, a standard dose of heparin was administered regardless of the act result, and protamine was not injected during weaning from cpb due to worries about the occurrence of severe complications, such as catastrophic aps . In the second case, in the third case, a minimal dose of protamine (30 mg) was administered in the icu postoperatively due to a bleeding tendency . In such cases, the surgical procedure can be completed simply through the use of any of the above methods, but considering the risk of bleeding, it is reasonable to determine the appropriate dose through the continuous infusion of protamine (for example 50 mg / hr) until the dose no longer causes bleeding . The syndrome's exact cause has not yet been determined, but it is believed that operations and infections may trigger it . To prevent this, infections should be controlled through administration of appropriate antibiotics, and a parenteral anticoagulant should be taken by patients who will undergo surgery peripartum and by patients with rashes associated with sle . It has been reported that heparin administration to maintain the pt / inr level at about 3, and steroid use to prevent the release of cytokine from necrotic tissue, may help prevent infection . Taking all these factors into consideration, the anesthesiologist should use caution in interpreting the results of the coagulation panel and in preventing thrombosis during the perioperative period . Further studies are also necessary for the maintenance and monitoring of an adequate anticoagulation level, especially during cpb in aps patients.
According to the results of a nationwide survey, direct - vision internal urethrotomy (dviu) is used for most urethral strictures in the united states and the situation should be similar in korea . According to the high recurrence rate of strictures after dviu, the most cost - effective strategy for the management of short, bulbar urethral strictures is to reserve urethroplasty for patients in whom a single dviu procedure fails . Repeated dviu can increase the length and density of spongiofibrosis, thus making definitive surgical intervention more difficult . Thus, it might be more cost - effective to go straight to primary urethroplasty because most patients want a cure . For bulbar urethral strictures of 2 cm or less, excision and end - to - end anastomosis remains the ideal procedure with excellent long - term results reported [5 - 7]. Unfortunately, there have been few studies on the surgical outcomes of end - to - end anastomosis for bulbar urethral stricture in korean patients . We therefore performed a retrospective evaluation of patients who underwent bulbar end - to - end anastomosis to report our experience with the surgery and to assess the factors affecting surgical outcome . We reviewed the medical charts of 33 patients who underwent excision and end - to - end anastomosis for bulbar urethral strictures by a single surgeon and who completed at least 6 months of follow - up . The patients' records were reviewed with respect to etiology of stricture, previous treatment, preoperative evaluation, surgical findings, follow - up results, and early and late complications . Preoperative evaluation included history, physical exam, urinalysis, urine culture, uroflowmetry, and retrograde and voiding cystourethrography . The most common cause of stricture was blunt perineal trauma (straddle injury) in 18 patients (54.6%), followed by iatrogenic causes in 8 patients, idiopathic causes in 4 patients, and infection in 3 patients (table 1). About two - thirds of the patients (63.6%) underwent dviu, dilation, or multiple treatments before referral to our center (table 2). At presentation, 20 patients (60.6%) had a suprapubic cystostomy . In the remaining 13 patients with slow stream, the mean maximal flow rate (mfr) was 5.4 ml / s (range, 2.4 to 8 ml / s) the standard surgical technique of anastomotic urethroplasty was applied while the patient was positioned in a slightly hyperextended lithotomy position . After mobilization of the bulbar urethra, the area of fibrosis was completely excised and the healthy ends of the urethra were spatulated . Urethral mobilization was required, extending in some cases to the penoscrotal junction distally and perineal body proximally . Dorsal anastomosis was performed with interrupted 4 - 0 or 5 - 0 polyglactin sutures and ventral anastomosis was performed in two layers with the urethral mucosa first and then the corpus spongiosum . At the end of the procedure, a 14-fr silastic foley urethral catheter was exclusively placed and a small drain was left under the bulbospongiosus muscle for 2 to 3 days . Patients were discharged with oral antibiotics until the catheter was removed, usually after 14 days . The urethral catheter was removed when there was no extravasation on urethrography of the pericatheter . The catheter was left in place an additional 1 to 2 weeks when extravasation was present . Uroflowmetry was performed 3, 6, and 12 months after surgery in the first year and annually thereafter . Patients underwent retrograde urethrography or urethroscopy if they developed voiding symptoms, such as slow or splayed stream . Chi - square test or fisher exact test was used to assess the significance of categorical risk factors for surgical failure, and student t - test or wilcoxon rank sum test was used to assess significance in continuous factors, e.g., age or operation time . Six patients (18.2%) required corporal separation to achieve a tension - free anastomosis . Mean excised stricture length was 1.5 cm (range, 0.8 to 2.3 cm). Stricture length was less than 1 cm in 3 patients (9.1%), 1 to 2 cm in 26 patients (78.8%), and more than 2 cm in 4 patients (12.1%). The urethral catheter was removed a mean of 16.5 days (range, 13 to 24 days) postoperatively . At a mean follow - up of 42.6 months (range, 8 to 96 months), 29 of the 33 patients (87.9%) had no evidence of recurrent stricture . In one case in the success group, meatal stenosis was successfully treated with a single urethral dilation . Because there was no evidence of recurrence of bulbar stricture by retrograde urethrography or urethroscopy, the surgical outcome of this patient was classified as successful . In the success group, the mean mfr after surgery was 21.65 ml / s . Patients aged less than 50 years (n=11) showed better mfr with mean of 27.4 ml / s (range, 18 to 48 ml / s) than did those aged 50 years or more (n=18), who had a mean mfr of 18.14 ml / s (range, 12 to 47 ml / s). Six patients who had benign prostatic hyperplasia preoperatively had an mfr less than 15 ml / s . Two of these patients were treated by laser prostate surgery and the other two patients were well controlled by medication . Strictures recurred in four patients (12.1%) at a mean follow - up of 3.5 months (range, 2.5 to 4.7 months). Of the four recurrences, one patient was managed successfully by dviu, whereas the remaining three patients did not respond to dviu or dilation . These three patients underwent ventral onlay graft urethroplasty using buccal mucosa at 6, 13, and 14 months after end - to - end anastomosis, respectively . Although all patients had excellent outcomes, with good urinary stream and not requiring any intervention after the reoperation, further follow - up is needed because of the short follow - up time (range, 4 to 7 months). The recurrence rate was significantly higher in the patients with nontraumatic causes than in the patients with traumatic etiology . The stricture etiology of the four recurrent cases was iatrogenic in three patients and infection in one patient . Other variables did not affect the surgical outcome of end - to - end urethroplasty . Early complications were minor, including catheter - related infection and epididymitis that was easily treated with antibiotics in one patient each . With respect to late complications, intermittent perineal or scrotal pain bothered eight patients (24.2%) and was relieved by analgesics . Two patients complained of a decrease in ejaculatory force and volume . In the seven patients who had erectile dysfunction preoperatively all these cases had a traumatic etiology . However, no patient had new onset of erectile dysfunction postoperatively . In the bulbar urethra, many variables, such as length, severity, and location of stricture, can influence surgical outcome . The surgical technique should be selected mainly according to stricture length, but the stricture etiology and density of the spongiofibrosis tissue should also be taken into account . For the treatment of a short segmental bulbar urethral stricture (<2 cm), dviu or end - to - end urethroplasty is commonly accepted as standard therapy . When the stricture is limited in focal area, if the stricture is more than 1 cm in length, single dviu followed by end - to - end urethroplasty is commonly used as a cost - effective strategy . Dorsal or ventral onlay substitution urethroplasty using a buccal mucosa graft is currently suggested for a longer (> 2 cm) strictures, where the urethral lumen is relatively well preserved and the spongiofibrosis around the lumen is limited to 1 mm . Augmented anastomotic urethroplasty, with complete excision of the worst stricture segment, is currently recommended for strictures that cover a particularly dense and narrow area of 1 to 2 cm in length [14 - 16]. Both ventral and dorsal onlay free grafts survive well with equal success rates [14 - 16]. Short bulbar strictures are generally amenable to complete excision with primary anastomosis via a perineal incision, affording a high success rate of 95%, as reported by santucci et al . . Published their series of 260 patients with bulbar stricture who underwent end - to - end anastomosis with a mean follow - up of 50.2 months . The stricture length ranged from 0.5 to 4.5 cm (mean, 1.9 cm) and the authors reported a success rate of 98.8% . Recently, barbagli et al . Described a success rate of 90.8% in 153 patients who underwent bulbar end - to - end anastomosis with a mean follow - up of 68 months . In 2002, jezior and schlossberg summarized the surgical outcomes of excision and primary anastomosis for bulbar stricture on the basis of major series reported in the literature . These series showed a success rate of 93% in 443 patients with a range of 65% to 100% between series . In our series of 33 patients with bulbar stricture, end - to - end anastomosis had a success rate of 87.9% with a mean follow - up of 42.6 months . Many variables such as age, operation time, stricture length, previous operation history, preoperative voiding status, and etiology of stricture were evaluated as potential risk factors of recurrence . No clear consensus exists on stricture etiology and the success rate with respect to excision and end - to - end anastomosis . It is believed, however, that inflammatory strictures are more extensive, generally involving more of the urethra and corpus spongiosum, and are less likely to yield a successful result . Reported the highest failure rate in patients with strictures, which was related to prolonged indwelling catheter drainage . In our series, the stricture etiology of the four failure cases was iatrogenic in three patients and infection in one patient . Therefore, the most common cause of stricture in the surgical failure group was iatrogenic, arising after previous endoscopic surgery (n=1) or following prolonged indwelling catheter placement (n=2). Although initial postoperative retrograde urethrography findings were normal, stricture recurred in four patients at a mean follow - up of 3.5 months (range, 2.5 to 4.7 months). A typical case of recurrent stricture after excision and end - to - end anastomosis is illustrated in fig . 1 . To get the best results for end - to - end anastomosis, complete excision of unhealthy urethra and accompanying spongiofibrosis and tension - free anastomosis are essential . Failure to remove all abnormal urethra is thought to be the primary cause of surgical failure and stricture recurrence . The main cause of surgical failure in our series was also assumed to be inadequate excision of the urethral stricture . Retrograde urethrography often combined with voiding cystourethrography is a conventional preoperative tool for evaluation of the extent of urethral involvement . However, the static retrograde urethrography image can both underestimate (by as much as 50%) and overestimate the length of the stricture . Intraoperative urethrocystoscopy can be used as an adjunct to retrograde urethrography to estimate the extent of stricture . Some advocate urethral ultrasonography to accurately determine stricture length . In one study, intraoperative ultrasonography of the anterior urethra recurrence might have been prevented in our series by a wider excision of suspicious spongiofibrosis or augmented anastomotic urethroplasty . We have no clear explanation for why the patients with traumatic etiology had better surgical outcomes than did those with nontraumatic etiology . The most likely reason for the better results in the traumatic group is that spongiofibrosis developed from outside to inside, which makes it easier to identify the extent of stricture . On the contrary, spongiofibrosis propagated from inside to outside in urethral strictures of nontraumatic causes, especially those with iatrogenic or infectious causes . In addition to complete excision of abnormal urethral mucosa and spongiofibrosis, tension - free anastomosis is important for achieving the best results . The ideal stricture length for excision and end - to - end anastomosis has been a contentious issue . Guralnick and webster insisted that this operation should be limited to strictures of 1 cm or less, because excision of a 1-cm urethral segment with opposing 1-cm proximal and distal spatulations results in a 2-cm urethral shortening . In general, the best stricture length manageable by excision and primary anastomosis is 2 cm or less . However, strictures longer than 2 cm can be managed successfully in selected patients with end - to - end anastomosis [5 - 7]. Morey and kizer reported on a selected cohort of 22 patients with proximal bulbar urethral strictures longer than 2.5 cm that were treated with an extended anastomotic approach and suggested that the ability of the urethra to be reconstructed is proportional to the length and elasticity of the distal urethral segment . They reported a 91% success rate, concluding that defects up to 5 cm can be successfully excised and primarily reconstructed in select young men with proximal bulbar strictures . In our series, the majority of patients (87.9%) had an excised urethral length of 2 cm or less; no cases had a stricture length more than 2.5 cm . The literature suggests that the influence of previous treatment on surgical outcome is controversial [2,5 - 7,22]. In the recent series reported by santucci et al . And eltahawy et al ., 55% and 69.2% of the patients had failed attempts of urethroplasty or dviu, respectively . Despite this fact, furthermore, previously failed urethrotomy did not influence the long - term outcome of urethroplasty ., the only group of patients who had a lower success rate (78.6%) had undergone undergone multiple treatments (dilation, dviu, or urethroplasty), whereas the other groups (prior single or no treatment) showed similar success rates ranging from 92.1% to 100% without any statistical significance . It was also suggested that endoscopic or open urethral manipulation before anastomotic urethroplasty for posttraumatic urethral stricture has a significant impact on the outcome of urethral reconstruction . In our study, 21 of 33 patients (63.6%) underwent prior single or multiple treatments, whereas 12 patients (36.4%) had no previous treatment . Although previous treatment did not affect surgical outcome, all recurrent cases had a history of one or more dvius . Most patients feel satisfied with the surgical outcome despite some minor postoperative complications . In our series, the most frequent postoperative ejaculation disorder was decreased force of ejaculation (20%) or semen sequestration in the urethral bulb (3.3%). Yucel and baskin suggested that most likely surgical damage to the branches of the perineal nerves or bulbospongiosus muscles may have a role in determining the loss of efficient bulbar urethral contraction, thus causing difficulties in expelling semen and urine . Although the success rate of bulbar urethroplasty is high, some argued that this is the urologist's view and not necessarily the patient's view . Whereas the urologist concentrates on voiding efficiency, the patient is much more concerned with cosmetic effects and adverse effects, especially on sexual performance . Recently, a patient - reported outcome measure for urethral stricture surgery was devised and validated . A major limitation of this study was that the number of involved patients was not enough to obtain statistical significance in the multivariate analysis . For example, etiology of stricture was a significant factor that influenced recurrence after surgery, but an exact odds ratio could not be calculated because the recurrence rate of the traumatic group was 0% . Instead, we could calculate the estimated odd ratio, but the range of the confidence interval was so wide that the interpretation of the results was limited . Excision and end - to - end anastomosis for short, bulbar urethral stricture has an acceptable success rate of 87.9% with minor complications . However, strictures recurred early (less than 5 months) in four patients (12.1%). All recurrences occurred in the patients with nontraumatic causes (iatrogenic in three, infection in one patient). Therefore, careful consideration is needed when choosing a surgical procedure if the stricture etiology is nontraumatic.
An institutional review board approved this retrospective analysis, which was performed at the department of general surgery, xinhua hospital, school of medicine, shanghai jiao tong university . From january 2009 to september 2014, 220 consecutive patients with bile duct stones (152) or a common bile duct (cbd) stricture (68), who had previously undergone gastrectomy and were referred for ercp, were analyzed . The surgical approaches applied to these patients included proximal gastrectomy with esophagogastrostomy (10 patients), distal subtotal gastrectomy with billroth i (42 patients) or billroth ii (160 patients, including 25 patients with braun anastomosis), and total gastrectomy combined with roux - en - y reconstruction (8 patients) (table 1). Characteristics of the 220 patients who had previously undergone gastrectomy the procedures were performed in all patients under pharyngeal anesthesia, sedation (intramuscular 10 mg diazepam injection), and pethidine (50 mg). All patients received oxygen administered by nasal prong and were monitored by pulse oximetry and electrocardiography . Ercp was performed under fluoroscopic control using a conventional side - viewing duodenoscope with a total length of 120 cm and a working channel of 3.7 mm in diameter, which allows the use of a wide range of catheters according to the diagnostic or therapeutic objective (olympus v260; olympus medical systems, tokyo, japan). Alternatively, we used a triple - lumen retrieval balloon catheter (the extractor pro rx retrieval balloon catheter), which is capable of accepting a 0.035-inche (0.089 mm) guidewire in the open - channel guidewire lumen, while allowing simultaneous injection and inflation of the balloon in the other 2 lumens . Review of the surgical procedure notes before initiating ercp in postgastrectomy patients provided the necessary details, such as the type of reconstruction and the length of the limbs . In addition, review of the available postoperative gastrointestinal imaging studies, including upper gastrointestinal series, abdominal computed tomography examinations, and magnetic resonance imaging, also provided helpful information . Ercp in patients who had undergone proximal gastrectomy with esophagogastrostomy (fig . 1) and distal subtotal billroth i gastrectomy (fig . 2) was similar to routine ercp, because endoscopically the postsurgical anatomy appears mostly similar to the normal digestive tract . For ercp in patients who underwent distal subtotal billroth ii gastrectomy (figs . 3, 4) or total gastrectomy combined with roux - en - y reconstruction (figs . 5, 6), we facilitated successful enteroscopy with endoscope insertion using a triple - lumen retrieval balloon catheter . 7), and then a retrieval balloon was inserted over the guidewire (fig . 8). Following the injection of contrast agent, we used this retrieval balloon to explore the tract of the target limb on dynamic radiographic images (fig . The balloon was then hooked to the limb, not only to indicate the direction of the tract to guide the endoscope forward but also to facilitate the forward movement of the endoscope with fewer injuries to the intestinal wall (fig . As the balloon catheter was strongly retracted into the working channel to allow the scope to advance, the endoscope was propelled slightly forward . It should be emphasized that in ercp postgastrectomy, we relied not only on what was visible through the endoscope but also on the x - ray dynamic images from enterology . We termed the procedure retrieval balloon assisted enterography.1114 for patients with a billroth ii gastroenterostomy and braun anastomosis (fig . 11), we also used the procedure of retrieval balloon assisted enterography along the optimal route as previously reported15 (fig . Conventional side - viewing duodenoscope for ercp in patients who had undergone proximal gastrectomy with esophagogastrostomy . Conventional side - viewing duodenoscope for ercp in patients who had undergone distal subtotal billroth i gastrectomy . The procedure of retrieval balloon assisted enterography for patients who had undergone billroth ii gastroenterostomy . Conventional side - viewing duodenoscope for ercp in patients who had undergone billroth ii gastroenterostomy . The procedure of retrieval balloon assisted enterography for patients who had undergone total gastrectomy with roux - en - y reconstruction . Conventional side - viewing duodenoscope for ercp in patients who had undergone total gastrectomy with roux - en - y reconstruction . Following the injection of contrast agent, we used this retrieval balloon to explore the tract of the target limb on dynamic radiographic images . The balloon was then hooked to the limb, not only to indicate the direction of the tract to guide the endoscope forward but also to facilitate the forward movement of the endoscope with fewer injuries to the intestinal wall . Conventional side - viewing duodenoscope for ercp in patients who had undergone billroth ii gastroenterostomy with braun anastomosis . The procedure of retrieval balloon assisted enterography along the optimal enterography route for patients who had undergone billroth ii gastroenterostomy with braun anastomosis . All procedures were performed by an experienced pancreaticobiliary endoscopist (x .- f.w .) Who routinely performs> 300 to 600 ercps each year . Successful enteroscopy (endoscopic success) was defined as enterography along the correct limb and the ability to identify the papilla of vater . Diagnostic success was defined as successful duct cannulation and a successful cholangiogram leading to a diagnosis . Ercp success was defined as a successful enteroscopy with successful diagnostic and therapeutic interventions.16 post - ercp pancreatitis was defined according to cotton s criteria.17 hemorrhage was defined as bleeding requiring the local injection of hemostatic agents or clipping at the time of the procedure or a few days later . All statistical analyses were performed using the spss software, version 18.0 (spss inc ., logistic regression models were constructed by including variables that had significant univariate associations with post - ercp complications . Anova testing was constructed by including independent variables that had significant univariate associations with success rate . The procedures were performed in all patients under pharyngeal anesthesia, sedation (intramuscular 10 mg diazepam injection), and pethidine (50 mg). All patients received oxygen administered by nasal prong and were monitored by pulse oximetry and electrocardiography . Ercp was performed under fluoroscopic control using a conventional side - viewing duodenoscope with a total length of 120 cm and a working channel of 3.7 mm in diameter, which allows the use of a wide range of catheters according to the diagnostic or therapeutic objective (olympus v260; olympus medical systems, tokyo, japan). Alternatively, we used a triple - lumen retrieval balloon catheter (the extractor pro rx retrieval balloon catheter), which is capable of accepting a 0.035-inche (0.089 mm) guidewire in the open - channel guidewire lumen, while allowing simultaneous injection and inflation of the balloon in the other 2 lumens . Review of the surgical procedure notes before initiating ercp in postgastrectomy patients provided the necessary details, such as the type of reconstruction and the length of the limbs . In addition, review of the available postoperative gastrointestinal imaging studies, including upper gastrointestinal series, abdominal computed tomography examinations, and magnetic resonance imaging, also provided helpful information . Ercp in patients who had undergone proximal gastrectomy with esophagogastrostomy (fig . 1) and distal subtotal billroth i gastrectomy (fig . 2) was similar to routine ercp, because endoscopically the postsurgical anatomy appears mostly similar to the normal digestive tract . For ercp in patients who underwent distal subtotal billroth ii gastrectomy (figs . 3, 4) or total gastrectomy combined with roux - en - y reconstruction (figs . 5, 6), we facilitated successful enteroscopy with endoscope insertion using a triple - lumen retrieval balloon catheter . 7), and then a retrieval balloon was inserted over the guidewire (fig . 8). Following the injection of contrast agent, we used this retrieval balloon to explore the tract of the target limb on dynamic radiographic images (fig . The balloon was then hooked to the limb, not only to indicate the direction of the tract to guide the endoscope forward but also to facilitate the forward movement of the endoscope with fewer injuries to the intestinal wall (fig . As the balloon catheter was strongly retracted into the working channel to allow the scope to advance, the endoscope was propelled slightly forward . It should be emphasized that in ercp postgastrectomy, we relied not only on what was visible through the endoscope but also on the x - ray dynamic images from enterology . 11), we also used the procedure of retrieval balloon assisted enterography along the optimal route as previously reported15 (fig . Conventional side - viewing duodenoscope for ercp in patients who had undergone proximal gastrectomy with esophagogastrostomy . Conventional side - viewing duodenoscope for ercp in patients who had undergone distal subtotal billroth i gastrectomy . The procedure of retrieval balloon assisted enterography for patients who had undergone billroth ii gastroenterostomy . Conventional side - viewing duodenoscope for ercp in patients who had undergone billroth ii gastroenterostomy . The procedure of retrieval balloon assisted enterography for patients who had undergone total gastrectomy with roux - en - y reconstruction . Conventional side - viewing duodenoscope for ercp in patients who had undergone total gastrectomy with roux - en - y reconstruction . The retrieval balloon was inserted over the guidewire . Following the injection of contrast agent, we used this retrieval balloon to explore the tract of the target limb on dynamic radiographic images . The balloon was then hooked to the limb, not only to indicate the direction of the tract to guide the endoscope forward but also to facilitate the forward movement of the endoscope with fewer injuries to the intestinal wall . Conventional side - viewing duodenoscope for ercp in patients who had undergone billroth ii gastroenterostomy with braun anastomosis . The procedure of retrieval balloon assisted enterography along the optimal enterography route for patients who had undergone billroth ii gastroenterostomy with braun anastomosis . All procedures were performed by an experienced pancreaticobiliary endoscopist (x .- f.w .) Who routinely performs> 300 to 600 ercps each year . Successful enteroscopy (endoscopic success) was defined as enterography along the correct limb and the ability to identify the papilla of vater . Diagnostic success was defined as successful duct cannulation and a successful cholangiogram leading to a diagnosis . Ercp success was defined as a successful enteroscopy with successful diagnostic and therapeutic interventions.16 post - ercp pancreatitis was defined according to cotton s criteria.17 hemorrhage was defined as bleeding requiring the local injection of hemostatic agents or clipping at the time of the procedure or a few days later . All statistical analyses were performed using the spss software, version 18.0 (spss inc ., logistic regression models were constructed by including variables that had significant univariate associations with post - ercp complications . Anova testing was constructed by including independent variables that had significant univariate associations with success rate . The study group included 220 patients with altered gastrointestinal anatomy (77 women and 143 men; mean age, 72.2 y; range, 11 to 93 y). The indications for ercp included cbd stones (152 patients) and cbd stricture because of tumor recurrence (68 patients). The overall enterography success rate was 90.5% (199/220), and the diagnostic success and ercp success rates were both 88.6% (195/220). Among patients who underwent billroth i gastroenterostomy and proximal gastrectomy with esophagogastrostomy, endoscopic success rate was 100% (42/42), and the diagnostic success and ercp success rates were both 100% (42/42). For billroth ii gastroenterostomy, the endoscopic success rates without or with braun anastomosis were 88.9% (120/135) and 88.0% (22/25), respectively . The diagnostic success and ercp success rates for billroth ii gastroenterostomy with the duodenoscope without or with braun anastomosis were 86.7% (117/135) and 84.0% (21/25), respectively . For patients who underwent total gastrectomy with roux - en - y reconstruction, the endoscopic success rate was 62.5% (5/8), and the diagnostic success and ercp success rates were both 62.5% (5/8). Endoscopy was unsuccessful in 21 patients with a billroth ii gastroenterostomy and roux - en - y reconstruction because of failure to access the papilla due to the presence of a long afferent loop and tumor infiltration of the afferent loop . Unsuccessful diagnostic and ercp outcomes after endoscopic success occurred only in patients with billroth ii gastroenterostomy without or with braun anastomosis because of cannulation failure in 4 patients due to tumor infiltration . Factors that increased the rates of enterography success, diagnostic success, and ercp success were cbd stone, proximal gastrectomy, esophagogastrostomy, and billroth i reconstruction (table 2).the procedure - related complication rate was 5.5% (12/220), including hemorrhage (0.9%, 2/220), pancreatitis (4.1%, 9/220), and perforation (0.5%, 1/220) (table 3). One patient with a billroth ii gastroenterostomy developed afferent loop perforation, underwent laparotomy, and was discharged 2 weeks later . Two patients experienced hemorrhage at the time of ercp, which was successfully treated by the local injection of epinephrine and clipping . Factor that increased the risk of any procedure - related complication was type of previous surgery (table 4). Factors that affected the rates of enterography success, diagnostic success, and ercp success details of the ercp treatment procedures risk factors for post - ercp complication in 220 patients who had previously undergone gastrectomy ercp in patients after gastrectomy remains a challenging technique for ercp endoscopists . As in patients with normal anatomy, anterior oblique - viewing endoscopes, side - viewing endoscopes, forward - viewing gastroscopes, and multibending endoscopes have been reported in previous studies of ercp for postgastrectomy patients.1822 however, there are 3 major obstacles to overcome to successfully perform ercp when using these enteroscope variants: (1) the approach to the ampulla of vater, (2) selective bile duct cannulation, and (3) procedural reliability, including skillful technique and dedicated devices . The forward - viewing endoscope has a long - working length and permits the operator to enter the afferent loop easily and safely because of the ability to see the lumen en face . However, this approach is particularly difficult through a native ampulla because an en face view of the papilla is difficult to obtain using forward - viewing endoscopes . Moreover, optimal access to the papilla is restricted without an elevator function, and compatible devices for these enteroscopes are difficult to obtain . This lack of a cannula elevator makes it difficult to cannulate the native papilla, and the lack of dedicated devices makes it difficult to achieve therapeutic success.23,24 thus, the enterography success rate of the forward - viewing endoscope is relatively higher, but the therapeutic success rate is lower . In contrast, the side - viewing endoscope with a shorter working length has a larger working channel and a cannula elevator . However, the fact that it is impossible to see the lumen en face makes it difficult to enter the limb safely, and there are some reports of small bowel perforation associated with ercp using a side - viewing endoscope . However, the cannula elevator makes it easy to cannulate the desired duct selectively, and the larger working channel together makes it easy to achieve therapeutic success . Although the enterography success rate of the side - viewing endoscope is relatively lower, the therapeutic success rate is higher after successful enterography . In this study, therefore, 2 strategies that can improve the ercp success rate in patients after gastrectomy include improved therapeutic success with the forward - viewing endoscope25,26 and improved enterography success with the side - viewing endoscope.27,28 from our experience, the working length of the side - viewing duodenoscope is sufficiently long for almost all patients after gastrectomy . Our study also demonstrated a significantly higher therapeutic success rate after successful enterography using the side - viewing duodenoscope . Thus, our strategy for successful ercp was to improve the enterography success rate of the side - viewing duodenoscope . Among the patients who underwent billroth i gastroenterostomy and proximal gastrectomy with esophagogastrostomy, the endoscopic procedures were similar to those performed in normal digestive tracts, and the ercp success rate was 100% using the side - viewing duodenoscope in this study . Compared with patients with billroth i gastroenterostomy and esophagogastrostomy, ercp in patients with billroth ii gastrectomy and roux - en - y reconstruction is more difficult and hazardous due to the markedly altered anatomy, with the direction of approach shifted to the 6-oclock position . These alterations together with abdominal adhesions create more difficulties in the intubation of the afferent loop and the approach to the papilla along the afferent loop when using the side - viewing duodenoscope . In our technique, the guidewire of the retrieval balloon was advanced to the appropriate limb, and then a retrieval balloon was inserted over the guidewire . We used this retrieval balloon to explore the correct limb with contrast enhancement to observe the tract of the limb on the radiographic images . The balloon was then hooked to the correct limb and inflated, which not only indicated the direction of the tract to guide the endoscope forward but also facilitated the forward movement of the endoscope with fewer injuries to the intestinal wall . As the balloon catheter was strongly retracted into the working channel to allow the scope to advance, the endoscope was propelled slightly forward . By placing it within the correct limb, the retrieval balloon catheter may also be used as a guide to prevent the duodenoscope from sliding out of the correct limb and into another limb upon forward motion . After successful access of the appropriate limb is achieved, the retrieval balloon becomes visible within the tract ahead, instead of emerging from it . This is particularly important at the anastomosis site, where the correct limb must be identified . It should be emphasized that the x - ray dynamic images we observed with balloon - assisted enterography proved more helpful for ercp in patients with altered gastrointestinal anatomy . Such visualization aids the endoscopist in viewing the altered structure clearly and allows the endoscope to move more smoothly along the digestive duct, minimizing accidental injury to the intestinal wall . We termed this procedure retrieval balloon assisted enterography,11,1315 which may ensure the success of ercp using the side - viewing duodenoscope . In patients who have undergone billroth ii gastrectomy and braun anastomosis, we recommend extending the duodenoscope along the greater curvature of the stomach to the gastrojejunal anastomosis, then advancing the endoscope through the efferent loop and along this efferent loop to the braun anastomosis, whereby the middle entrance is the correct entrance to reach the papilla of vater . For patients with billroth ii gastroenterostomy and braun anastomosis, we believe that this is the optimal ercp enterography route.14,29 using these strategies, we obtained encouraging results . For billroth ii gastroenterostomy, the endoscopic success rates without or with braun anastomosis were 88.9% (120/135) and 88.0% (22/25), respectively . The diagnostic success and the ercp success rates for billroth ii gastroenterostomy with the duodenoscope without or with braun anastomosis were 86.7% (117/135) and 84.0% (21/25), respectively . For patients who underwent total gastrectomy with roux - en - y reconstruction, the endoscopic success rate was 62.5% (5/8), and the diagnostic success and ercp success rates were both 62.5% (5/8). Moreover, retrieval balloon assisted enterography using the side - viewing duodenoscope was safe for ercp in postgastrectomy patients . Only 1 patient with a billroth ii gastroenterostomy developed afferent loop perforation and underwent laparotomy . Two patients experienced hemorrhage at the time of ercp, which was successfully treated by local injection of epinephrine and clipping . Thus, we believe that retrieval balloon assisted enterography is an effective and safe method, which can improve the enterography success rate in patients with billroth ii and roux - en - y reconstruction postgastrectomy . In conclusion, the side - viewing duodenoscope is a useful instrument for performing successful ercp in patients postgastrectomy . Procedure may improve the enterography success rate in patients with billroth ii and roux - en - y reconstruction postgastrectomy . However, this study was retrospective and reflects the experience of a single center, suggesting that the reproducibility of this technique should be assessed in future prospective studies.
The incidence of failed intubation is approximately 0.05% or 1:2230 in surgical patients and approximately 0.13 - 0.35%, or 1:750 - 1:280, in the obstetric patients . The incidence of unsuspected difficult intubation is higher and is estimated to be 3% . In an updated report by the american society of anaesthesiologists (asa) task force on management of the difficult airway, a difficult airway is defined as the clinical situation in which a conventionally trained anaesthesiologist experiences difficulty with face mask ventilation of upper airway, difficult tracheal intubation or both . Failure to maintain a patent airway after induction of anaesthesia leads to irrevocable catastrophic sequelae such as brain damage or death . Anatomical malformations such as the lower jaw anomalies, chin protrusion, excessive maxillary length, limited temporomandibular joint range of motion, decreased atlanto - occipital distance and reduction of pharyngeal space and submandibular tissue compliance have been considered as causes of difficult intubation . Because of the potentially serious consequences of failed tracheal intubation, considerable attention has been focused on attempts to predict patients in whom laryngoscopy and intubation will be difficult . Ever since banister and macbeth stressed the importance of the position of the head and neck in direct laryngoscopy in order to achieve a proper alignment of the axes of the mouth, pharynx and larynx, many tests and landmarks (mallampati test, inter - incisor gap (iig), subluxation of the mandible, thyromental distance (tmd), length of mandibular rami, profile classification, chin protrusion, atlanto - occipital extension) have been introduced to predict an unanticipated difficult airway, but unfortunately these tests are not totally reliable . Despite limitations, some tests or combination of tests have been of immense value in predicting unsuspected difficult cases of endotracheal intubations . In the present study, an attempt is made to compare the diagnostic value of the upper lip bite test (ulbt) along with anatomic measurements of lateral neck radiography . After institutional ethics and research committee's approval and obtaining an informed consent, 4500 consecutive patients, asa physical status i to iii who required general anaesthesia and endotracheal intubation were studied prospectively over a 3-year period from january 2007 until december 2010 . Exclusion criteria included inability to sit, gross anatomical abnormality or recent surgery of the head and neck and patients with pregnancy or severe cardiorespiratory disorders . Anatomical factors predicting difficult intubation at direct laryngoscopy were noted and lateral radiographs were obtained from 265 patients in whom tracheal intubation proved particularly difficult and from 4235 patients in whom intubation was reasonably straightforward . A group of nine anaesthesiologists with 5 years of experience were trained during several workshops to evaluate patients in a similar way . Step 1 (primary assessment) a group of nine anaesthesiologists with 5 years of experience in anaesthesia carried out the primary assessment and evaluation as described in the protocol . Demographic data including age, sex, weight, height and body mass index were collected . The following five predictive test measurements were performed on each patient: modified mallampati test (mmt): samsoon and young's modification of the mallampati test recorded oropharyngeal structures visible upon maximal mouth opening, with the patient in the upright position . Grade 1: faucial pillars, soft palate and uvula visiblegrade 2: faucial pillars, soft palate visible, but uvula masked by the base of the tonguegrade 3: soft palate only visiblegrade 4: soft palate not visible . Tmd: distance from the thyroid cartilage to the mental prominence with the neck fully extendedsternomental distance (smd): distance measured in the seated position with the head fully extended on the neck and with the mouth closed (straight distance between the upper border of the manubrium - sterni and bony point of the mentum)horizontal length of mandible (hlm): patient seated with the head in the neutral position and straight distance from the angle of the mandible to the symphysis menti measured . Tests 2 - 4 were measured with a rigid ruleriig: distance between the upper and lower incisors, measured with the patient sitting in the neutral position and mouth maximally open with a pair of calipersulbt: class i: lower incisors biting the upper lip, making the mucosa of the upper lip totally invisible . Class iii: the lower incisors fail to bite the upper lip (ulbt class ii and iii considered as difficult intubation). Modified mallampati test (mmt): samsoon and young's modification of the mallampati test recorded oropharyngeal structures visible upon maximal mouth opening, with the patient in the upright position . Grade 1: faucial pillars, soft palate and uvula visiblegrade 2: faucial pillars, soft palate visible, but uvula masked by the base of the tonguegrade 3: soft palate only visiblegrade 4: soft palate not visible . Grade 1: faucial pillars, soft palate and uvula visible grade 2: faucial pillars, soft palate visible, but uvula masked by the base of the tongue grade 3: soft palate only visible grade 4: soft palate not visible . Tmd: distance from the thyroid cartilage to the mental prominence with the neck fully extended sternomental distance (smd): distance measured in the seated position with the head fully extended on the neck and with the mouth closed (straight distance between the upper border of the manubrium - sterni and bony point of the mentum) horizontal length of mandible (hlm): patient seated with the head in the neutral position and straight distance from the angle of the mandible to the symphysis menti measured . Tests 2 - 4 were measured with a rigid ruler iig: distance between the upper and lower incisors, measured with the patient sitting in the neutral position and mouth maximally open with a pair of calipers ulbt: class i: lower incisors biting the upper lip, making the mucosa of the upper lip totally invisible . Class iii: the lower incisors fail to bite the upper lip (ulbt class ii and iii considered as difficult intubation). The cut - off points for the predictors were determined a priori as suggested by the originators of the tests except for the smd in which the cut - off was increased from 12.5 cm to 13.5 cm after preliminary analysis of pilot data . Values below and inclusive of each cut - off point were predicted as difficult visualisation of the larynx (dvl) for the anthropometric variables . Values above the cut - off point were predicted as easy - visualisation of the larynx (evl). Dvl was predicted with mmt iii or iv, tmd <6.5 cm; smd <13.5 cm; hlm <9.0 cm; iig <4.0 cm and ulbt class ii and iii . This was followed by step 2 (radiologic assessment) a routine lateral neck view taken with the patient in an upright, sitting or standing position and patient's shoulder on level with radiology film . The patient's neck was located at approximately 20 - 30 cm distance from the film and patient's midsagittal plane being parallel to the surface of the film . Both the shoulders were kept horizontal and the head vertical to the body . In order to maintain uniformity, all the patients were asked to look at an object located in their eye axis . The radiology beam was vertical to the film surface and the centre was located on the most prominent point of the thyroid cartilage . The radiology tube was located at 150 - 180 cm from the neck . For better quality of soft - tissue image, we used settings of 75 - 90 kv and 10 - 20 ma . Siemens model the following distances were measured in each of the radiographs: anterior depth of the mandible: distance from the tip of the mandibular central incisors to the posterior border of the mandibleeffective mandibular length . Distance between tips of lower incisors to the mid - point of the temporomandibular jointposterior depth of the mandible: perpendicular distance from the lower border of the mandible to the alveolar margin at position of the posterior border of 3 molar toothatlanto - occipital distance . The vertical distance between the occiput of the skull and the superior surface of the posterior tubercle of the atlasmandibulohyoid distance (mhd): perpendicular distance from the hyoid to the mandiblemandibular angle: angle between a line intersecting the lower border of the mandible and a perpendicular line . Anterior depth of the mandible: distance from the tip of the mandibular central incisors to the posterior border of the mandible effective mandibular length . Distance between tips of lower incisors to the mid - point of the temporomandibular joint posterior depth of the mandible: perpendicular distance from the lower border of the mandible to the alveolar margin at position of the posterior border of 3 molar tooth atlanto - occipital distance . The vertical distance between the occiput of the skull and the superior surface of the posterior tubercle of the atlas mandibulohyoid distance (mhd): perpendicular distance from the hyoid to the mandible mandibular angle: angle between a line intersecting the lower border of the mandible and a perpendicular line . Step 3 (laryngoscopic assessment) consisted of anaesthesia induction and tracheal tube insertion . After establishing standard monitoring and establishing an adequate access in the operating room and preparing the required equipment for difficult intubation management, induction of anaesthesia was performed in the supine position with 5 mg / kg of sodium thiopentone or propofol 2 mg / kg intravenously . After the disappearance of fasciculations, the patient's head was placed in the sniffing position (10 cm pillow was kept underneath the occiput of the patients). Attending anaesthesiologists not involved in the airway assessment of the patients carried out the laryngoscopy and intubation . Laryngoscopy was performed using a macintosh #4 blade to visualise the larynx and the view was classified using the cormack and lehane (cl) classification (i = vocal cords visible; ii = only posterior commissure or arytenoids visible; iii = only epiglottis visible; iv = none of the foregoing visible). Distribution of demographic data were calculated and summarised based on central statistical indices and dispersion indices . For each diagnostic test, all indices of a diagnostic test were calculated in comparison with laryngoscopic view as the gold standard . Distribution of demographic data were calculated and summarised based on central statistical indices and dispersion indices . For each diagnostic test, all indices of a diagnostic test were calculated in comparison with laryngoscopic view as the gold standard . A total of 4500 patients enrolled in our study included 1505 women and 2995 men . Demographic data of patients enrolled in study diagnostic value of ulbt based on laryngoscopic view was calculated and shown in table 2 . Table 3 reveals the statistical indices of the different diagnostic tests with the highest sensitivities obtained for ulbt, tmd and mhd respectively . Ulbt had the highest specificity and negative predictive value (npv) compared with the other tests . The positive predictive value (ppv) for all the tests had been low, but marginally high in the ulbt . The result of different predictive tests based on laryngoscopic view sensitivity, specificity, ppv and npv of different evaluation tests this is the first study where in a large sample size was used and which used all clinical, radiologic and airway risk criteria in an attempt to identify factors affecting difficult laryngoscopy and intubation . We carefully matched control subjects for age, height and weight and oropharyngeal appearance to avoid age - related anatomic differences in musculoskeletal structures . None of the patients in our study had arthritic changes of the cervical spine and all patients had a full set of teeth . They determined that an increase in the anterior and posterior depth of mandible, a decrease in the atlanto - occipital gap and c1-c2 gap and limitation of movement at the temporomandibular joint were the factors that determined whether direct laryngoscopy would be easy or difficult . They felt that difference in effective mandibular length, arching of the palate and protrusion of the upper teeth, the factors that had been claimed to be associated with difficult endotracheal intubation by cass et al . Did not play a significant role in their study . In a more recent study by bellhouse and dor consistent evidence of a relationship between difficulty in endotracheal intubation and posterior and anterior depth of mandible could not be established . The difference in findings of these studies may be explained by the fact that the investigators in the study were two otolaryngologists who used different instrumentations and techniques for direct laryngoscopy than that routinely used by anaesthesiologists to accomplish endotracheal intubation . Our finding of no significant difference in the atlanto - occipital gap, which was found to be a significant parameter by white and kander is in agreement with nichol and zuck who reported a wide variation in the atlanto - occipital distance . It would be inappropriate to speak in terms of normal and abnormal. And it is not possible to identify a critical measurement that could be used as a predictor of difficulty in intubation . Several authors have given absolute values of the radiological measurements in patients with easy or with difficult intubations . The assessments have been performed in small populations and neither the sensitivity nor specificity of the measurement as indicators of difficult intubation has been evaluated . In most of the studied tests, the npv was high meaning that the tests adequately eliminated patients with difficult intubation and hence, difficult laryngoscopic view or difficult intubation was not encountered . On the other hand, a positive test result does not always indicate difficult laryngoscopy as a low value predicts intubation difficulty when there is none; it falsely predicts intubation difficulty in a certain number of patients . This may be useful in some situations, but it becomes necessary to subject the patient to many tests of predicting difficult airway and thus obviating an unanticipated difficult intubation . Most of the tests in our study had low ppvs implying that the tests lacked the utility to forecast difficulty in intubation and these findings corroborate with other studies conducted so far . Subjecting patients to risks of radiological exposure may be a consideration, but a single exposure in select cases may be worth the effort in avoiding and predicting difficult intubation, undetected with the available tests . Lateral neck x - ray may be used confined only to cases where positive findings provide us a definite clue that a difficult intubation is in the offing . Although all the tests in this study had relatively acceptable predictive values, perhaps a combination of tests could be of value in arriving at better results . It would not be practical to recommend radiological measurements in the assessment of difficulty in intubation as screening tests . They can be of value in understanding the problems encountered during laryngoscopy and thus can help assessment in some selected patients with difficulty in intubation.
About 80% of the worldwide population use herbal products for their basic health care (primary care), such as extracts, teas and their active principles, a market estimated at us$50 billion per year . Despite the interest in molecular modeling, combinatorial chemistry and other chemical synthesis techniques by institutions and pharmaceutical industries, the natural products, particularly medicinal plants, persist as an important source of new therapeutic agents against infectious (fungal or bacterial) and cardiovascular diseases, insects, cancer, and immunomodulation [2 - 6]. Genus lippia (verbenaceae, lamiales / magnoliopsida) includes about 200 species of herbs, shrubs and small trees mainly distributed in central and south americas and in africa tropical [7 - 8]. Some lippia species are prevalent at caatinga biome, a region with approximately 1,539,000 km2, distributed in nine brazilian northeastern states, with warm and dry climate and where grows a peculiar xerophyte vegetation . In caatinga flora, there are almost 1 000 vascular plant species . Because of the extreme climate conditions most species are endemic and present particular morphological adaptations . Among the lippia species, l. microphylla cham . (syn . : l. microphylla cham . And schlecht . ; l. microphylla mart . ; lantana microphylla mart . Then, this work aimed to review its biological potentialities, emphasizing the properties of essential oils (eos). Moreover, in order to suggest a strategic plan for genus lippia and l. microphylla, main publication areas, its respective patents and institutions and authors were also analyzed to identify studies and orientate the development of pharmaceutical products . General aspects of lippia microphylla cham for a complete and reliable review, primary and secondary resources were used, including original and review articles, books and government documents written in english, portuguese or spanish . Databases searched were lilacs - bireme (databases on latin american health and biological sciences), medline / index medicus (medical literature analysis and retrieval system online), scielo (scientific electronic library online), web of science, pubmed (maintained by the national library of medicine) and science direct . A software (vantage point 7.1) associated with derwent innovation index was used to performer bibliometric analyses, data generation, and identification of quantitative scientific indicators from 1948 to the present . Therefore, it was used the following keywords: lippia, biological properties, cytotoxicity, folk use, l. microphylla, and eos . Vantage point version 7.1 is a powerful text - mining tool for discovering knowledge in search results from patent and literature databases, giving a better perspective about information and enabling to clarify relationships and find critical patterns in distinct areas of expertise . Lippia species have shown a large number of important usages in folk medicine for various diseases, particularly in the treatment of cough, bronchitis, indigestion, liver, hypertension, dysentery [12 - 14], worms, and skin diseases . Many species have promising biological activities, including antiviral, antimalarial, anti - inflammatory, analgesic, antipyretic, molluscicidal against biomphalaria glabrata, antimicrobial [19 - 25], insecticidal, and anticonvulsant properties . Compounds isolated from lippia also revealed in vitro antitumor activity on leukemia (k-562, hl-60, cem), colon (hct-116), breast (mcf-7), glioblastoma (u-251), and prostate (pc -3) cell lines [28 - 30]. Besides its medicinal properties, the leaves of the most lippia species are used for food preparation . Moreover, it is interesting to note the importance of leptotyphlops dulcis, whose main component of leaves and flowers is (+) -hernandulcine, a molecule 1000-fold sweeter than sucrose . Popularly called as alecrim - da - chapada, alecrim - de - tabuleiro, alecrim - pimenta and alecrim - do - mato in the northeast brazilian, l. microphylla is a deciduous shrub with a thin and brittle stem (up to 2 m in height), white flowers and with simple and aromatic leaves, which presents serrate margins and evident nerves with no more than 1 cm in length . Ethnopharmacological records report the use of l. microphylla leaves to treat gastrointestinal disorders and influenza, bronchitis and sinusitis during vaporization resulting from boiling water . Phytochemical studies revealed the presence of quinones and flavonoids from stem and roots ethanol extracts . Meanwhile, its aromatic volatile oils extracted by water vapor exhibit an eos rich in monoterpenes, especially cineole, and terpineol, its more likely active principles . Secondary metabolites of plants, many of them produced to protect against microorganisms and predator insects, are natural candidates for the discovery of new active products . The saturated antiseptic and balsamic vapors with cineole and other eos found in l. microphylla lighten respiratory tract mucous membranes during congestion, which explains its folk use for the treatment of influenza, cough and nasal congestion . Its compounds are capable of fluidizing bronchial secretion, facilitating expectoration, and decreasing cough reflex and refreshing breath . Due to these balsamic properties, home practices of inhalation to alleviate symptoms of respiratory diseases are considered an easy way to treat them . To prepare the inhalation, leaves (50 - 60 g) are put in boiling water, and the person inhales the fumes through a resistant funnel, being careful to heat both parts of the face where sinus are positioned . If the eo is available, it should be used 1 - 2 ml per 1 - 2 l of boiling water . Some works published previously have also highlighted the insecticidal importance of the eos presented in lippia species [37 - 40]. Eos of l. microphylla, in particular, showed significant larvicidal activity on a. aegypti larvae, with a ld50 of 75.6 ppm . Gleiser et al . Showed that lippia integrifolia and lippia junelliana oils are potent repellents against a. aegypti adult insects . Both species contained similar quantities of limonene and camphor (20.7% and 26.5%), though they differ in others such as myrcene that was detected in l. junelliana (14.1%) and methylheptenone in l. integrifolia (24.9%). Limonene is a registered active element in pesticide products used as insecticide and repellent, and cotton fabrics treated with limonene have shown repellence against anopheles mosquitoes . In addition, repellent properties against a. aegypti have been reported for limonene, camphor, and myrcene . Thus, it is possible that the higher repellence of l. junelliana compared to l. integrifolia is due to the additional repellent effect of myrcene and this compound can be responsible for the insecticidal activity found in l. microphylla, since myrcene is also present in its leaves . Oils from l. microphylla also revealed antifungal (strains of aspergillus niger, fusarium spp ., rizhopus spp ., and rhizoctonia solani) and antibacterial activities (staphylococcus aureus, shigella flexneri, escherichia coli, and streptococcus pyogenes) using gel diffusion methods . A tolerance of gram - negative bacteria to eos, such as e. coli, has been attributed to the existence of a hydrophilic outer layer, which may be blocks the infiltration of hydrophobic components throughout the cell membrane . On the other hand, the inhibitory action of natural products on mold cells involves cytoplasm granulation, plasmatic membrane disrupting and inactivation and/or synthesis inhibition of enzymes . These actions can appear isolate or simultaneously, leading to the mycelium propagation, and growth inhibition . In fact, oils rich in monoterpenic compounds are reported to exhibit high levels of antimicrobial activity . Moreover, they are probably responsible, at least in part by the antioxidant activity in l. microphylla extracts . In the verbenaceae family, chemical studies by gas chromatographic techniques of volatile constituents from l. microphylla leaves resulted in the identification of -pinene (1), sabinene (2), -pinene (3), -myrcene (4), p - cymene (5), 1,8-cineole (6), -terpinene (7), 4-terpineol (8), a - terpineol (9), anisole (10), thymol (11), and carvacrol (12) [figure 2]. Among them, the main components are 1, 8-cineol (36%), -pinene (11%), and thymol (11%). Silva et al . Identified the germacrene d (13) and bicyclogermacrene (14) [figure 2] from eos and hexanic fractions as the most common compounds in specimens collected in dry and rainy periods, while the major monoterpene was -pinene . Volatile essential oils from lippia microphylla cham analyses of the fixed constituents of the ethanolic extracts of l. microphylla roots and stems result in isolation of a flavonol glycoside (15) and four quinones, two prenylated naphthoquinone dimers (16 and 17) and two furan naphthoquinones (18 and 19) [figure 3]. Among these, microphyllaquinone (16) and a mixture of 6-methoxy- and 7-methoxy - naphtho[2,3-b]-furan-4,9-quinones (18 + 19, respectively) isolated from l. microphylla were evaluated for their cytotoxicity, using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h - tetrazolium bromide (mtt) method, which analyzes the ability of living cells to reduce the yellow dye mtt to a purple formazan product . According to santos et al ., the molecules microphyllaquinone and the mixture demonstrated cytotoxic potential against a panel of different murine and human cancer cell lines (b-16 [murine melanoma], cem [lymphocyte leukemia], hl-60 [promyelocyte leukemia], hct-8 [colon adenocarcinoma,] and mcf-7 [breast adenocarcinoma]), with ic50 values ranging from 0.69 to 3.13 g / ml [table 1]. Cytotoxicity of naphthoquinones isolated from l. microphylla and analyzed by mtt assay after 72 h exposure fixed components of lippia microphylla cham the eo of l. microphylla leaves also showed in vitro cytotoxic action on sarcoma 180 (ic50 of 100.1 [94.9 - 105.5] g / ml) and human chronic myelocyte leukemia k-562 cells (ic50 of 51.9 [47.9 - 56.3] g / ml) [table 2]. Lytic activity was not detected in normal erythrocytes from swiss mice at a concentration of 250 g / ml . After 7 days of treatment, this same oil presented in vivo antitumor action in a dose - dependent way, and tumor growth inhibition of 38.2% and 59.8% (50 and 100 mg / kg, respectively). In vitro antitumor activity of the essential oil from l. microphylla leaves determined by mtt assay after 72 h exposure interestingly, in the presence of cyclosporine a, an inhibitor of the pore formation in the mitochondrial permeability transition, it was detected reduction in the cytotoxicity on sarcoma 180 cells with this eo (ic50 of 118.3 [113.7 - 123.1] g / ml). Declining in cytotoxicity in the presence of cyclosporine a is indicative of the intrinsic pathway involvement in the mechanism of death [table 2], whereas after adding pore inhibitors, probably occurred the mitochondrial pores opening blocking, which inhibited the release of pro - apoptotic proteins and reduced the cytotoxicity of the oil . Similarly, when k-562 cells were treated with the eo in the presence of n - acetylcysteine, an antioxidant molecule and scavenger of free radicals that stimulates the biosynthesis of reduced glutathione, cytotoxicity diminution of the oil was also evidenced . Then, it was proposed that reactive oxygen species (ros) production is involved, at least partially, in the mechanism of cytotoxicity on k-562 cells, and activation of apoptotic intrinsic pathways in sarcoma 180 cells . Cytochrome c binds to apaf-1 (apoptotic protease - activating factor 1) and generate the catalytically active form of caspase-9, which activates caspase-3, the most important effector caspase that acts as an effective dnase to slice the genomic dna into nucleosomes, producing fragments of 180 - 200 base pairs, degradation of laminin and mitotic apparatus proteins and nuclear and cellular reduction and pyknosis . Releasing of the molecules due to alterations in mitochondrial permeability transition leads to the loss of cellular homeostasis, preventing atp synthesis, and increasing production of ros . Studies of certain antineoplasic agents in distinct cell lines have demonstrated that some substances execute their activity by oxidative stress caused by ros, generally occurring when the homeostasis of oxidation and reduction is altered within cells . Since ros possess strong chemical reactivity with biomolecules such as proteins and dna, this may result in dna denaturation, leading to changes in protein synthesis and cell duplication . Furthermore, it is known that ros induce activation of caspases-3 and -9 [55 - 57]. Lippia species have shown a large number of important usages in folk medicine for various diseases, particularly in the treatment of cough, bronchitis, indigestion, liver, hypertension, dysentery [12 - 14], worms, and skin diseases . Many species have promising biological activities, including antiviral, antimalarial, anti - inflammatory, analgesic, antipyretic, molluscicidal against biomphalaria glabrata, antimicrobial [19 - 25], insecticidal, and anticonvulsant properties . Compounds isolated from lippia also revealed in vitro antitumor activity on leukemia (k-562, hl-60, cem), colon (hct-116), breast (mcf-7), glioblastoma (u-251), and prostate (pc -3) cell lines [28 - 30]. Besides its medicinal properties, the leaves of the most lippia species are used for food preparation . Moreover, it is interesting to note the importance of leptotyphlops dulcis, whose main component of leaves and flowers is (+) -hernandulcine, a molecule 1000-fold sweeter than sucrose . Popularly called as alecrim - da - chapada, alecrim - de - tabuleiro, alecrim - pimenta and alecrim - do - mato in the northeast brazilian, l. microphylla is a deciduous shrub with a thin and brittle stem (up to 2 m in height), white flowers and with simple and aromatic leaves, which presents serrate margins and evident nerves with no more than 1 cm in length . Ethnopharmacological records report the use of l. microphylla leaves to treat gastrointestinal disorders and influenza, bronchitis and sinusitis during vaporization resulting from boiling water . Phytochemical studies revealed the presence of quinones and flavonoids from stem and roots ethanol extracts . Meanwhile, its aromatic volatile oils extracted by water vapor exhibit an eos rich in monoterpenes, especially cineole, and terpineol, its more likely active principles . Secondary metabolites of plants, many of them produced to protect against microorganisms and predator insects, are natural candidates for the discovery of new active products . The saturated antiseptic and balsamic vapors with cineole and other eos found in l. microphylla lighten respiratory tract mucous membranes during congestion, which explains its folk use for the treatment of influenza, cough and nasal congestion . Its compounds are capable of fluidizing bronchial secretion, facilitating expectoration, and decreasing cough reflex and refreshing breath . Due to these balsamic properties, home practices of inhalation to alleviate symptoms of respiratory diseases are considered an easy way to treat them . To prepare the inhalation, leaves (50 - 60 g) are put in boiling water, and the person inhales the fumes through a resistant funnel, being careful to heat both parts of the face where sinus are positioned . If the eo is available, it should be used 1 - 2 ml per 1 - 2 l of boiling water . Some works published previously have also highlighted the insecticidal importance of the eos presented in lippia species [37 - 40]. Eos of l. microphylla, in particular, showed significant larvicidal activity on a. aegypti larvae, with a ld50 of 75.6 ppm . Gleiser et al . Showed that lippia integrifolia and lippia junelliana oils are potent repellents against a. aegypti adult insects . Both species contained similar quantities of limonene and camphor (20.7% and 26.5%), though they differ in others such as myrcene that was detected in l. junelliana (14.1%) and methylheptenone in l. integrifolia (24.9%). Limonene is a registered active element in pesticide products used as insecticide and repellent, and cotton fabrics treated with limonene have shown repellence against anopheles mosquitoes . In addition, repellent properties against a. aegypti have been reported for limonene, camphor, and myrcene . Thus, it is possible that the higher repellence of l. junelliana compared to l. integrifolia is due to the additional repellent effect of myrcene and this compound can be responsible for the insecticidal activity found in l. microphylla, since myrcene is also present in its leaves . Oils from l. microphylla also revealed antifungal (strains of aspergillus niger, fusarium spp ., rizhopus spp ., and rhizoctonia solani) and antibacterial activities (staphylococcus aureus, shigella flexneri, escherichia coli, and streptococcus pyogenes) using gel diffusion methods . A tolerance of gram - negative bacteria to eos, such as e. coli, has been attributed to the existence of a hydrophilic outer layer, which may be blocks the infiltration of hydrophobic components throughout the cell membrane . On the other hand, the inhibitory action of natural products on mold cells involves cytoplasm granulation, plasmatic membrane disrupting and inactivation and/or synthesis inhibition of enzymes . These actions can appear isolate or simultaneously, leading to the mycelium propagation, and growth inhibition . In fact, oils rich in monoterpenic compounds are reported to exhibit high levels of antimicrobial activity . Moreover, they are probably responsible, at least in part by the antioxidant activity in l. microphylla extracts . In the verbenaceae family, genus lippia products great quantities of volatile mixtures . Chemical studies by gas chromatographic techniques of volatile constituents from l. microphylla leaves resulted in the identification of -pinene (1), sabinene (2), -pinene (3), -myrcene (4), p - cymene (5), 1,8-cineole (6), -terpinene (7), 4-terpineol (8), a - terpineol (9), anisole (10), thymol (11), and carvacrol (12) [figure 2]. Among them, the main components are 1, 8-cineol (36%), -pinene (11%), and thymol (11%). Silva et al . Identified the germacrene d (13) and bicyclogermacrene (14) [figure 2] from eos and hexanic fractions as the most common compounds in specimens collected in dry and rainy periods, while the major monoterpene was -pinene . Analyses of the fixed constituents of the ethanolic extracts of l. microphylla roots and stems result in isolation of a flavonol glycoside (15) and four quinones, two prenylated naphthoquinone dimers (16 and 17) and two furan naphthoquinones (18 and 19) [figure 3]. Among these, microphyllaquinone (16) and a mixture of 6-methoxy- and 7-methoxy - naphtho[2,3-b]-furan-4,9-quinones (18 + 19, respectively) isolated from l. microphylla were evaluated for their cytotoxicity, using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h - tetrazolium bromide (mtt) method, which analyzes the ability of living cells to reduce the yellow dye mtt to a purple formazan product . According to santos et al ., the molecules microphyllaquinone and the mixture demonstrated cytotoxic potential against a panel of different murine and human cancer cell lines (b-16 [murine melanoma], cem [lymphocyte leukemia], hl-60 [promyelocyte leukemia], hct-8 [colon adenocarcinoma,] and mcf-7 [breast adenocarcinoma]), with ic50 values ranging from 0.69 to 3.13 g / ml [table 1]. Cytotoxicity of naphthoquinones isolated from l. microphylla and analyzed by mtt assay after 72 h exposure fixed components of lippia microphylla cham the eo of l. microphylla leaves also showed in vitro cytotoxic action on sarcoma 180 (ic50 of 100.1 [94.9 - 105.5] g / ml) and human chronic myelocyte leukemia k-562 cells (ic50 of 51.9 [47.9 - 56.3] g / ml) [table 2]. Lytic activity was not detected in normal erythrocytes from swiss mice at a concentration of 250 g / ml . After 7 days of treatment, this same oil presented in vivo antitumor action in a dose - dependent way, and tumor growth inhibition of 38.2% and 59.8% (50 and 100 mg / kg, respectively). In vitro antitumor activity of the essential oil from l. microphylla leaves determined by mtt assay after 72 h exposure interestingly, in the presence of cyclosporine a, an inhibitor of the pore formation in the mitochondrial permeability transition, it was detected reduction in the cytotoxicity on sarcoma 180 cells with this eo (ic50 of 118.3 [113.7 - 123.1] g / ml). Hence, declining in cytotoxicity in the presence of cyclosporine a is indicative of the intrinsic pathway involvement in the mechanism of death [table 2], whereas after adding pore inhibitors, probably occurred the mitochondrial pores opening blocking, which inhibited the release of pro - apoptotic proteins and reduced the cytotoxicity of the oil . Similarly, when k-562 cells were treated with the eo in the presence of n - acetylcysteine, an antioxidant molecule and scavenger of free radicals that stimulates the biosynthesis of reduced glutathione, cytotoxicity diminution of the oil was also evidenced . Then, it was proposed that reactive oxygen species (ros) production is involved, at least partially, in the mechanism of cytotoxicity on k-562 cells, and activation of apoptotic intrinsic pathways in sarcoma 180 cells . Cytochrome c binds to apaf-1 (apoptotic protease - activating factor 1) and generate the catalytically active form of caspase-9, which activates caspase-3, the most important effector caspase that acts as an effective dnase to slice the genomic dna into nucleosomes, producing fragments of 180 - 200 base pairs, degradation of laminin and mitotic apparatus proteins and nuclear and cellular reduction and pyknosis . Releasing of the molecules due to alterations in mitochondrial permeability transition leads to the loss of cellular homeostasis, preventing atp synthesis, and increasing production of ros . Studies of certain antineoplasic agents in distinct cell lines have demonstrated that some substances execute their activity by oxidative stress caused by ros, generally occurring when the homeostasis of oxidation and reduction is altered within cells . Since ros possess strong chemical reactivity with biomolecules such as proteins and dna, this may result in dna denaturation, leading to changes in protein synthesis and cell duplication . Furthermore, it is known that ros induce activation of caspases-3 and -9 [55 - 57]. Before formulating the action plan for implementation of the herbal medicine, we suggest the prospection of main institutions, researchers and areas of knowledge in a defined timeline to direct the strategic planning . From this perspective, the prospecting is a manner to anticipate advances and can influence the orientation of technological trajectories . Within the context of herbal medicines, this tool allows to target search according to which has already been produced, and to establish partnerships or cooperation that leverage innovation as determined by the requirements of public and private institutions and government agencies . Using lippia as keyword to search in the web of science and derwent innovation index, when the investigation was refined for l. microphylla, the number of manuscripts decreased for 10 and patents were not found . After that, there was a vertiginous growth of manuscripts, especially in the 2000s, as seen in the figure 4 . The countries that more published about lippia were brazil, argentina, mxico and united states of america [figure 5]. Temporal evolution on the number of published articles involving the genus lippia (vantage point 7.1) world participation in publications belonging to the genus lippia (vantage point 7.1) it is important to note that this rising of articles approaching lippia species overlaps with the implantation of the national program on biodiversity, whose objectives include investments and management of funds to produce new medicines . In fact, identifying which are the most important needs and opportunities for research and development (r and d) in the future, from planned interventions in innovation systems can be an important step in established programs in brazil as the national program on medicinal plants and phytotherapics . This program, approved in 2006, aims to ensure safe access and rational use of medicinal plants and phytotherapics by the population based on the list of regulated plants by anvisa (national agency of sanitary surveillance in brazil). In relation to lippia species, l. alba (142 articles) and l. sidoides (97 articles) are the species that have received more attention [figure 6]. On the other hand, l. microphylla has a few reports about its biology and pharmacology as described above . These results are confirmed by the figure 7 that presents the areas of publications on l. microphylla, most of them concentrated in food and chemical sciences . Number of articles about the major species belonging to the genus lippia (vantage point 7.1) areas of publications about lippia microphylla (vantage point 7.1) figure 8 shows the interaction between brazilian and international institutions . This data is extremely important to identify universities involved in the study about l. microphylla, and their partnerships and cooperation in order to direct funding, define methods and technology to implement the appropriated findings . Research groups at the federal university of cear and federal university of paraba are the brazilian institutions with more articles about l. microphylla . Embrapa foundation works without partnerships or, at least, it did not publish works with other institutions yet . The academic institutions around the world are the leading centers for generation of new patentable technologies, as observed in this study . However, brazil has low competitivity and shows little effort to innovate in the area of technological inventions, probably due to some failures in the innovation system (cooperation between government, business, and institutions to promote an effective system of production and development of medicines). Then, brazil does not have a valuable protecting structure of lippia species, reflecting the lack of incentives to safeguard the technologies developed using industrial property . Institutions and their collaborations about lippia microphylla publications (vantage point 7.1) finally, in 2004, it was promulgated a brazilian law about technological innovation (number 10,973), which was regulated in 2005 (decree 5,563). This law normalizes the incentives for the involvement of scientific and technological institutions (institutos de cincia e tecnologia - ict s) in the innovation process for innovation in companies, for the independent inventor and conception of investment funds for innovation . It is the first brazilian law that deals with the relationship between universities and/or research institutions and companies with the creation of technological innovation centers (ncleos de inovao tecnolgica - nit s), helping the institutional maturity to make strategic management of intellectual property in the brazilian ict s . Despite a high brazilian scientific production numbers of patents is far from the quantity of published articles, existing an abysmal between which is published which is patented, and which would become a product or service to generate work and wealth to the country [figure 9]. Japan made its first deposit in 1990 and, currently, it is the largest holder of patents (36). Thus, these data are not consistent with the brazilian scientific production about lippia species and the number of published articles, suggesting a lack of encouragement in researches for the development of inventions involving lippia species . Moreover, the scientific production in brazil is recent (last 100 years), and is concentrated in public universities and research centers, and in honorable exceptions, in private institutions, as a result of the brazilian public policies in science and education . Certainly, this is the capital challenge in the brazilian national innovation system for the transfer of technology generated in universities and research centers to industry, in a way that new processes and products may be generated from these institutions . Wipo = world intellectual property organization; epo = european organization; usa = united states of america number of patents about genus lippia depositated from 1987 (vantage point 7.1) the growing interest in plant products with different purposes is linked, in part, to the low cost of drug production based on active principles isolated from natural products when compared to investments for synthesis in the laboratory . In the context of the herbal market evolution, estimated at us$22 billion and corresponding to 3.7% of the global market, this growth accompanies the pharmaceutical industry, considered one of the most lucrative in the world . Brazil is considered the seventh largest market, and from 2006 to 2010 grew by 14% . In 2015 hence, brazilian pharmaceutical and bio prospecting areas require constant and high investments in research and development of new products, since the natural resources have money - making value, attract investors and catch the attention to the preservation of endemic species . Lippia species are a source of remarkable bioactive substances (such as eos) with economic potential for local communities . Specifically, l. microphylla is an endemic underexploited brazilian vegetal with great medicinal properties that has gained much attention in the general population and scientific community . Then, this bio prospection helps to build perspectives of the bioactivity areas of real interest for capital investment and to give support for brazilian institutions to establish cooperation and partnerships in order to change the scientific reality, where basic and applied researches in pharmaceutical sectors are ineffective and without technological impact to create and innovate.
It is known as the fast enemy that should be treated and destroyed very fast as well . Most women do not like to hear the word cancer, and feel worried and stressed over it . It can be the beginning of learning how to fight, getting the facts, and finding hope . This is followed by lack of patient's personal control over the current treatment method and uncertainty of its outcome . Therefore, anxiety is associated with cancer; it is the most prevalent psychological symptoms perceived by cancer patients as a response to a threat, and so many patients are anxious . In one study done by ashbury et al, 77% of 913 patients within 2 years of treatment recalled experiencing anxiety . However, anxiety after cancer diagnosis is not necessarily abnormal, may not present a problem, or may even be a constructive part of dealing with problems . The most common cancer and the number one cause of cancer death amongst women in malaysia is breast cancer . If not detected and treated promptly, breast cancer can metastasize, spreading to the lymph glands and other parts of the body including the lungs, bones, and liver . Usually, cancer is named after the body part in which it originated; thus, breast cancer refers to the erratic growth and proliferation of cells that originate in the breast tissue . The term breast cancer refers to a malignant tumor that has developed from cells in the breast . The breast is composed of two main types of tissues: glandular tissues and stromal (supporting) tissues . Glandular tissues house the milk - producing glands (lobules) and the ducts (the milk passages), while stromal tissues include fatty and fibrous connective tissues of the breast . The breast is also made up of lymphatic tissue - immune system tissue that removes cellular fluids and waste . Breast cancer is characterized by the uncontrolled growth of abnormal cells in the milk - producing glands of the breast or in the passages (ducts) that deliver milk to the nipples . The early stage of breast cancer usually refers to the cancer that is confined to the fatty tissue of the breast . It may then spread to underlying tissues of the chest wall and then to other parts of the body . Furthermore, worldwide, breast cancer is the leading cause of cancer death in women, and more than one million women are diagnosed each year . In addition to that, more than 500,000 women every year die from the disease worldwide . Anxiety, tension, worry, stress, and strain are all common feelings and it is a part of our life today . Simple worry or stress will not drive us to look for specialist, but when these feelings become a chronics and interfere with our lives we need to do something and look for ways to manage it in order to function well . Anxiety can be defined as an unpleasant subjective experience associated with the perception of real threat; therefore, it is a common symptom in connection with cancer . Furthermore, it can be described as an emotional state characterized by feelings of unpleasant expectation and a sense of imminent danger . According to stark, et al . Autonomic hyper - arousal with acceleration of heart rate and respiration, tremor, sweating, muscle tension, and gastrointestinal changes are common physiological experiences . Apprehension, feeling powerless, and fearing loss of control are psychological aspects . According to kazdin, anxiety is an emotion that characterized by feelings of tension, worry, and stress as well as physiological changes such as increased blood pressure . Furthermore, medical news today defines anxiety as a general term for several disorders that cause nervousness, fear, apprehension, and worrying . These disorders affect how we feel and behave, and they can manifest real physical symptoms . Mild anxiety is vague and unsettling, while severe anxiety can be extremely debilitating, having a serious impact on daily life . . We may worry about things that might happen or have a restless night of sleep . But, people with an anxiety problem worry so much that it affects their lives in negative ways . As stated above, anxiety is one of the most dominant psychological challenges associated with cancer . In another word, patients anxiety increases once they discover that they suffer from breast cancer, they may also become more anxious as cancer spreads or treatment becomes more intense . Consequently, the level of anxiety experienced by one person with cancer may differ from the anxiety experienced by another . Many anxiety cases associated with cancer were treated from this sickness, but others were not . Therefore, psychologists need to give support and hope to breast cancer's patients; they need to help them cope with their feeling and pain . Moreover, cancer's patients may experience anxiety at different situation as while undergoing a screening test, waiting for the results, receiving a diagnosis, undergoing treatment, or anticipating a recurrence of their cancer . The anxiety associated with cancer may increase feelings of pain, interfere their ability to sleep, causes nausea and vomiting, and interfere with their quality of life . And for most patients, cancer requires facing uncertainty, worries about cancer treatment effects, fear of cancer progression and death, guilt, and spiritual questioning . A study by ashbury et al ., indicated that 77% of patients within 2 years of treatment recalled experiencing anxiety . On the other hand, anxiety after cancer diagnosis is not necessarily to be normal, understanding the nature of the anxiety in cancer patient populations is important because abnormal anxiety is troublesome the psychological wellbeing of the patients sherbourne and sheard . Interviewing some breast cancer patients reported that their anxiety is characterized by a number of typical symptoms and signs such as shivering or tremor . The level of anxiety experienced by one person may differ from the level of anxiety experienced by another . Anxiety in breast cancer patients is associated with death anxiety, fear of death as a result of their symptoms . According to pollak this type of anxiety is lower for people who have a positive sense of well - being and sense of meaning in life . In addition, evidence indicates that religious beliefs influence their level of anxiety [figure 1]. Many researchers have investigated the differences in anxiety level among women receiving different breast cancer treatments . Recent study done by lim, indicated that anxiety presents in all treatment types for breast cancer . Moreover, the anxiety level in women who underwent chemotherapy was highest before the first chemotherapy infusion, mediated by age and trait anxiety . This result confirms the needs for more research and studies on anxiety among breast cancer patients . In addition, the villadeguadarrama free article reported that the cancer - related anxiety may manifest as physical symptoms, such as rapid heartbeat, tightness in the chest or shortness of breath . The patient may also experience digestive symptoms, such as nausea, vomiting, or diarrhea . Thinking about anxiety that can lead to physical symptoms, the overall symptoms of anxiety among cancer patients include: excessive, ongoing worry and tension, an unrealistic view of problems, restlessness or a feeling of being edgy, irritability, muscle tension, headaches, sweating, difficulty concentrating, nausea, the need to go to the bathroom frequently, tiredness, trouble falling or staying asleep, trembling, and being easily startled . Symptoms of anxiety and depression have been found to be common in patients with cancer, frequently occurring around the time of diagnosis and during the period of chemotherapy . High - depression burden has been found at the time when patients experience adverse effects of chemotherapy . Significant difference in psychological distress has been found depending on age, gender, and living situation, with those living alone experiencing higher levels of distress in a sample of icelandic cancer patients during the treatment period . A study of women at high risk for breast cancer showed significantly higher levels of depressive symptoms and feelings of emotional alienation than did a standardized test group, with 27% of this population defined as having a level of psychological distress justifying psychological counseling . A second study also documented an increase in distress among first - degree relatives of breast cancer patients . An israeli study found that first - degree relatives who have physical symptoms of breast pathology respond with more emotional distress than do women of normal risk in the same situation . Patients with cancer face most of the stressors associated with diagnosis, illness, and treatment . Cancer diagnosis and treatment brings changes in patients personal paths of life, in their daily activities, work, relationships, and family roles, and it is associated with a high level of patient psychological stress . If you are really caring for someone who is having the symptoms of anxiety, encourage him or her to get help . People are differing in the way they perceive their sickness and they are also differ in the way they cope with their anxiety . Some are easily disturbed by their feeling of being anxious and other may take it as a challenge and they try to look for ways to overcome their feeling . There are many treatments for clinical depression including medicines, counseling, or a combination of both . Therefore, the diagnosis of anxiety in cancer is usually complicated by the overlap of anxiety and sickness symptoms such as cancer - related fatigue and pain . Many common symptoms of major depression were observed in cancer patients who do not endorse full depression symptoms . Different studies showed that various kinds of coping strategies are used to overcome the anxiety in different types and stages of cancer . For instance, reuter et al ., stated that patients using ineffective coping strategies have higher levels of anxiety and depression and that benefiting from social support results in a marked reduction in the levels of anxiety and depression . Moreover, the importance of social support to good mental health outcomes is well established . As well as the positive effect of good social support, the detrimental effect of negative interactions with significant others in the social environment has also become apparent in psychiatric and other conditions . Eventually, people are unique in the way they perceive and cope with the anxiety, also the way one patient uses is different from the way other uses . Undoubtedly, it is related to the personality, strength, faith, and hope patients is having toward their sickness . Worry, tension, fear, and stress are interrelated to the anxiety and depression among them . Patients are differing in the way they perceive their problem as well as the way they cope with the anxiety associate with it . The huge literatures on anxiety, its effects, coping, counseling, and mental health are evidence of the extensive belief that the way people cope is somehow linked to their belief and faith . In conclusion, anxiety has a great effect on the feeling of breast cancer patients and it leads to high level of coping mechanisms.
Neurological soft signs (nss) have long been considered one of the functional features (tsuang and faraone, 1999; tsuang et al ., 1991) and endophenotypes (chan and gottesman, 2008; chan et al ., 2010a) of schizophrenia spectrum disorders . However, most of the studies of nss have been limited to the use of clinical ratings . Instead, recent findings from both structural and functional imaging studies have shown that nss may be associated with specific brain alterations . Specifically, in individuals with psychosis, more nss have been associated with smaller volumes of the inferior frontal lobes (thomann et al ., 2009a), the pre - central gyrus (heuser et al ., 2011; mouchet - mages et al ., 2011), the global cortical sulci (dazzan et al ., 2004; gay et al ., 2013) as well as the cerebellum (ho et al ., 2004; thomann et al ., nss may be responsible for some of the observed clinical manifestations in schizophrenia (keshavan et al ., 2003; mouchet - mages et al ., 2011; schroder et al ., 1999). A recent meta - analysis of structural and functional imaging findings suggests that nss are associated with volume reductions of the pre - central gyrus, the cerebellum, the inferior frontal gyrus and the thalamus . Furthermore, the same meta - analysis also suggests that functional imaging studies support an association between nss and altered neural activation in the inferior frontal gyrus, the bilateral putamen, the cerebellum and the superior temporal gyrus in patients with schizophrenia (zhao et al .,, these findings support the presence of a dysfunction of neural circuitry that underlies the presence of these minor neurological abnormalities, which are already present at illness onset . On the other hand, empirical findings have also shown that non - psychotic first - degree relatives of schizophrenia patients exhibit a higher prevalence of nss compared to healthy controls (egan et al . A meta - analysis comparing the prevalence of nss between patients with schizophrenia, non - psychotic first - degree relatives and healthy controls indicated a mean effect size of 0.8 and 0.97 for patients and their non - psychotic first - degree relatives and for non - psychotic first - degree relatives and controls respectively (chan et al ., 2010b). These results are consistent with the argument that nss are familial in nature and segregate with the illness . However, no studies had examined the brain correlates of nss in healthy relatives of patients with schizophrenia . Furthermore, all the aforementioned neuroimaging studies in schizophrenia patients used conventional subtraction analysis and did not examine the underlying connectivity between the brain regions identified . Given that some nss, such as the fist palm (fep) task (first described by luria, see heuser et al ., 2011), involve the functional integration or regulation of areas involved in motor sequencing rather than being directly activated, a connectivity approach is more appropriate to identify the specific network involved . Only one study (rao et al ., 2008) adopted a psychophysiological interaction (ppi) (friston et al ., 1997) method to re - analyze their previous findings on the fep task in healthy volunteers (chan et al ., 2006). This new analysis showed enhanced functional connectivities between the left- and right sensorimotor cortices (smc) and the right inferior and middle prefrontal cortices during fep task performance compared to a simple palming task . These findings suggest a regulatory role of the prefrontal cortex on fep task execution . In this study, we examined whether prefrontal regions are involved in the integration or regulation of neural activity underlying motor sequencing in patients with first - episode schizophrenia and their non - psychotic first degree relatives (fdr). More specifically, we aimed to identify any frontal regions where coupling in these areas and the sensorimotor cortex (smc) significantly differed between the complex fep task and a simple control motor task using ppi analysis . We hypothesized that patients with first - episode schizophrenia and their non - psychotic fdrs would show a reduced dysfunction of the prefrontal cortex and smc while performing the fep task . Thirteen right - handed first - episode schizophrenia patients were recruited from the mental health center, peking university, beijing, for the study . Participants were recruited to the study if they met the following inclusion criteria: a) diagnosis of schizophrenia ascertained by experienced psychiatrists according to the dsm - iv (apa, 1994); b) aged 1840 years; and c) illness duration within 2 years . The exclusion criteria were: a) a history of neurological disorders; b) a lifetime history of substance abuse and c) an estimated iq lower than 70 . Current symptom severity was assessed with the positive and negative syndrome scale (kay et al ., 1987). Fourteen non - psychotic fdrs of the patients taking part in the study were also invited to participate . Potential participants were excluded if they a) met the dsm - iv criteria for substance abuse; b) were suffering from any clinically unstable medical disorder; c) had a history of head injury (past or present); and d) had an estimated iq lower than 70 . Fourteen healthy controls matched with the patients in age, gender and handedness were recruited from local universities through advertisements . The institutional review board of the institute of psychology, the chinese academy of sciences, approved the study protocol . Behavioral neurological soft signs were examined with the abridged version of the cambridge neurological inventory (cni) (chan et al ., 2009). This abridged version offers instructions for eliciting and rating a comprehensive range of nss in motor coordination, sensory integration and disinhibition . Rao et al ., 2008) (for the earliest description, please see luria, 1966; in heuser et al ., 2011). In brief, the task consisted of three right - hand motor tasks which varied in complexity . In the simple palm tapping (pt) task, participants were required to repeat only one right palm tapping in the prone position . In the intermediate complex pronation / supination (ps) task, participants were required to perform right palm tapping in the prone and supine positions alternatively . In the complex fep task, participants were required to successivley place their right hand in a fist resting position vertically (fist), a palm resting position vertically (edge), and a palm resting position horizontally (palm). A resting condition (a) without any hand movement when participants were asked to focus on the screen was used as the control baseline of the pt task, and the pt task was in turn used as the control baseline of the ps and fep tasks . Participants were asked to practice the motor actions correctly at a constant rate before entering the scanner . During scanning, their performance was monitored by the experimenter through the window of the scanner room . In the formal imaging task, participants were instructed to perform the three tasks at a similar pace throughout the entire experiment . Pt and ps tasks were executed in 1 s and the fep task was executed in 1.5 s. the experimenter monitored the task performance outside the scanner room to ensure that the participants performed the correct hand movements . First, a resting condition lasted for 20 s and then 6 s of counting backward reminded the participants to get ready for their hand movement . After backward counting, the pt or ps or fep task lasted for 40 s. one of these three hand movements would be presented on the screen . Participants were required to conduct the hand movements according to the demonstration on the screen . The sequence of the three hand movement tasks was optimized and counterbalanced within the three runs . The functional imaging data was originally acquired in a ge 3 t sigma scanner (general electric, waukesha, wi, usa) with a standard ge birdcage - type rf coil using a standard t2 * -weighted epi sequence . The epi parameters were: tr = 2 s te = 60 ms, fov = 24 24 cm, matrix = 64 64, flip angle = 60, 22 axial slices (5 mm thick/1.2 mm sp, from superior to inferior). The spatial resolution for the functional images was 3.75 3.75 6.2 mm . High - resolution anatomical images were also obtained using the standard t1-weighted sequence (66 axial slices, 2.0 mm thick / interleaved, fov = 24 24 cm, matrix = 256 256). Images were analyzed with statistical parametric mapping software (spm8, wellcome department of imaging neuroscience, london, uk) implemented in matlab 2009b (mathworks inc ., sherborn, ma, usa). Three dummy scans in the beginning of the experiment were removed automatically from the dataset . Images were registered to the first icbm 152, which was based on 152 brains and was created by the montreal neurological institute (mni) with a 2 2 2 mm resolution . In the final step of pre - processing, the images were spatially smoothed by an isotropic gaussian kernel with fwhm of 8 mm . Conventional analyses were first conducted at the individual - level using voxel - wise general linear modeling (glm) and four t - contrasts were defined between the tasks and the corresponding baselines, i.e., pt vs. rest, ps vs. rest, fep vs. rest and fep vs. pt . Data from the contrasts of the first level model in the healthy control group, the schizophrenia group and the fdr group were entered into this model . A threshold of alphasim corrected p <0.01 and cluster size larger than 15 voxels were used to identify the activations associated with each contrast . Furthermore, we used a regression model to find the regions with group difference . We set up an f contrast to find the regions with main effect of group difference . Signal change percentage was retrieved from the rois with group difference for post - hoc analysis . Ppi analysis was used to estimate functional integration during task execution under different motor complexity conditions . The left smc was determined a - priori as the reference region for the ppi analysis because the motor tasks in the present study were only completed with the right hand . This region was defined by using a sphere with a radius of 8 mm and a center at the peak activation in the left smc activation (mni coordinates = [36 28 52], from the conventional analysis of the contrast of pt vs. rest). We performed voxel - wise ppi analysis at individual - level for the left smc to see if any other brain areas connected to the smc showed a significant increase in functional coupling (the slope of regression) during the fep task compared with a control task (e.g., the pt or ps task). For each participant, the activation time course signal in the reference region (i.e., the first eigenvariate time series, adjusted by effect of interest) was extracted from the conventional glm and entered into the ppi analysis as the first regressor representing the physiological variable . A second regressor representing the motor tasks with different complexity was entered into the ppi analysis as the psychological variable . The psychophysiological interaction between task complexity and activation signal in the reference region was designated as the regressor of interest in the ppi analysis . Thereafter we performed group - level random effect analysis on the individual results using one sample t - tests for the contrast fep vs. pt . Group - level paired t - tests were conducted for the contrast fep vs. ps . Areas of significant activation were identified at a threshold of uncorrected p <0.001 and cluster size larger than 15 voxels . There was no significant difference between patients with schizophrenia, their fdrs and healthy controls in the three subscales and total score of cni . Conventional activation of the pt, ps and fep tasks in the three groups, after co - varying for age, is shown in table 3 . The left frontal parietal region was significantly activated when participants performed the pt, ps and fep tasks with their right hand . Moreover, in the pt rest contrast, activation in the left frontal parietal region, together with the left medial frontal region, were lowest in patients with schizophrenia, intermediate in the fdrs and highest in healthy controls (scz <rel <hc). We further used a regression model and conducted a post - hoc analysis to identify the regions that were different across groups both linearly and non - linearly . In the pt rest contrast, we confirmed that the left frontal precentral gyrus (30 34 64, k = 100, f(2,37) = 20.23) was linearly activated in all three groups (scz <rel <hc). The left medial frontal region (hc = rel> scz, 8 18 64, k = 136, f(2,37) = 15.59) and the left middle temporal gyrus (hc> scz = rel, 48 68 10, k = 124, f(2,37) = 11.7) were non - linearly activated in all three groups . The activations associated with task complexity were examined in the fep ps and the fep pt contrasts . In the fep pt contrast, the bilateral middle frontal regions were activated in the healthy control group . The left middle frontal region was activated in the fdr group, but there was no frontal region activation in first episode schizophrenia patients . 2 illustrates the frontal activation in both healthy controls and fdrs . In the fep ps contrast, the left middle frontal region was also activated in the fdr group, but not in the healthy control group or the schizophrenia group . The ppi analysis identified right frontal regions in which the activity showed significant activation coupling to activity in the left smc during performance of the more complex fep task relative to the simple pt or ps tasks in the healthy control group, but not in the schizophrenia group . In this study, we report for the first time the neural activation and connectivity elicited by execution of the fep task in patients with first episode schizophrenia and their healthy first degree relatives . Our main finding is that patients with first episode schizophrenia do not show, in comparison with healthy controls and their fdrs, activation of the left middle frontal gyrus in the execution of the fep versus a simpler motor task like the pt . This provides evidence of a frontal dysfunction in these patients when performing a complex motor task . The right inferior frontal gyrus was found to modulate the activation of the sensorimotor cortex with the increase in motor complexity in healthy controls . A frontal dysfunction was also demonstrated by our second main finding, suggesting that with increase in task difficulty, patients with first episode schizophrenia do not show functional connectivity between the sensorimotor cortex and the right frontal gyrus, in contrast to healthy controls . The presence of a prefrontal dysfunction in schizophrenia is supported by extensive evidence from both structural and functional neuroimaging studies . Prefrontal areas have been consistently reported as reduced in volume in patients with schizophrenia (chan et al ., 2011; dazzan et al ., furthermore, fmri studies have shown altered activation of frontal areas during executive and working memory tasks, particularly with increasing capacity demand (barch et al ., 2012; callicott et al ., 2000; perry et al ., 2001). At a functional level, patients with schizophrenia also show an excess of signs reflecting frontal release, such as abnormalities in eye movements and short - term memory deficits, which again point to a frontal cortical alteration (hyde et al ., 2007). In this study, we tested the activation of frontal areas during a complex motor task, the fep task, which has long been considered in neurological and neurocognitive studies as indicative of frontal lobe lesions (luria, 1966). Two previous fmri studies have reported that in healthy individuals, the execution of this task did not induce activation of prefrontal areas, but induced activation of other parts of the cortex, including the sensorimotor areas (chan et al ., 2006; umetsu et al ., 2002). A subsequent study, using the same ppi approach that we used in this study, showed that this finding could reflect an indirect involvement of the prefrontal cortex, which could exert an integrative and regulatory function on neural circuitry involved in complex motor sequences . We found here that in healthy individuals, the execution of the fep task, compared to a simpler motor act, was indeed associated with both direct activation of the prefrontal cortex, and greater coupling of prefrontal areas and sensorimotor cortex . Interestingly, a recent meta - analysis of the brain connectome suggests that the frontal lobe represents a hub (a region highly interconnected with other brain regions and valuable for integrative information processing and adaptive behavior) particularly affected in patients with schizophrenia (crossley et al ., 2014). There is a paucity of studies on brain activation during complex motor tasks in these patients . However, neuroimaging studies that examined gross motor coordination signs such as finger - to - thumb operation tests in these patients have reported reduced activation in the smc and the supplementary motor area (sma) in comparison to healthy controls (schrder et al ., a later similar study used pronation / supination tests and found significant reduced activation in the smc in patients with schizophrenia compared to healthy controls (schroder et al ., 1999). The lack of activation we found with a task like the fep, which requires fine motor coordination and requires executive processes like inhibition, planning and updating, could be interpreted as part of the hypofrontality hypothesis of schizophrenia reflecting a reduced function of the prefrontal cortex (callicott et al ., 2000). Response relationship of the left frontal parietal activation across patients, their fdrs and healthy controls . Here, in the pt vs. rest contrast, activation was lowest in the schizophrenia group, intermediate in the fdr group and highest in the healthy control group . Although these differences were highlighted with a motor sequence less complex than the fep, the finding suggests that at least part of the pathophysiological substrate that underlies motor difficulties in psychosis may be linked to a genetic susceptibility to schizophrenia . Indeed, deficits in motor skills have been reported extensively in individuals at risk of schizophrenia because of either genetic loading or prodromal features (blanchard et al ., 2010; first, the clinical sample size was relatively small . However, the merit of the present sample is that it included both first - episode schizophrenia patients and their non - psychotic siblings . Secondly, the use of ppi analysis may not fully detect the connectivity changes related to the performance of the fep task . However, ppi is an appropriate method to test the relationship between two simple mental activities (friston et al ., 1997). Other more sophisticated approaches, such as dynamic casual modeling, may help identify further subtle connectivity changes in the performance of the fep task . Thirdly, the use of the screen to synchronize the motor actions might induce the activation of mirror neurons . However, the contrasts between any two of the three motor actions (fep, pt, and ps) should minimize this effect by subtraction methods . Finally, we should also consider the validity of our task, since previous subtraction analyses of the fep task did not elicit activation of the frontal cortex . However, the fact that the left frontal parietal region was significantly activated when participants performed the pt, ps and fep tasks with their right hand suggests that this hand movement imaging task was valid . Notwithstanding these limitations, our findings suggest that the fep task may be a useful endophenotype of schizophrenia, as it fulfills the criteria of being familial and shows a dose previous study had demonstrated that patients with schizophrenia showed aberrant brain activation in the premotor area after a week of motor training (kodama et al ., 2001). Given this temporal stability of nss, the fact that this is an imaging endophenotype rather than a simple behavioral rating arguably enhances its precision . This task is also simpler to carry out when compared to a full nss scale . We believe that our findings add valuable information to the understanding of the origin and underlying neural mechanism of motor coordination signs . Future studies should recruit a larger sample of first - episode, preferably medication - nave schizophrenia patients for further validation of our findings.
The updb includes birth and death data of more than 7 million individuals, with some records extending back more than 12 generations . The original utah genealogy includes complete 3-generation genealogic data for the utah mormon pioneers (members of the church of jesus christ of latter - day saints) and their descendants up to 1972 . Since then, the original genealogy data have been expanded with utah vital records such as birth certificates (using father, mother, and child trios). For the analyses reported in this study, we consider only those individuals in the updb with at least 3 generations of genealogy data who are related to the original utah genealogy . Utah's founding pioneers were composed of a sizable, largely unrelated mixture of northern european populations . This population continued to have high rates of immigration for years after utah's founding in 1847 . Studies using pedigree data, migration matrices, and isonymy have all shown low levels of inbreeding and similarity to northern european populations in the founding population of utah . We analyzed more than 2.7 million individuals in the updb belonging to at least 3 generations of genealogic data and connected to the original utah genealogy . Within this population of individuals with genealogy data, the cause of death on utah dcs was coded using the icd, with the revision used (i.e., icd-6 to icd-10) depending on the decade of death . For all deaths occurring before 1956, icd-10 coding was assigned . Table 1 shows the frequency of the 4,031 pd deaths by icd revision; 2,546 of these deaths occurred in males and 1,485 in females . The majority of the deaths from pd were in more recent years; 2,407 deaths occurred after 1999 . Frequency of parkinson disease deaths by icd revision the genealogical index of familiality (gif) statistic was developed to test hypotheses concerning excess relatedness among individuals sharing a specific phenotype using the updb . The gif analysis considers all genetic relationships between cases and measures the average relatedness among all possible pairs within a set of individuals . The pairwise relatedness measure implements the malcot coefficient of kinship, defined as the probability that randomly selected homologous genes from the 2 individuals are identical by descent from a common ancestor . The case gif is defined as the average of the coefficients of kinship between all possible pairs of cases (10). The pairwise relatedness of a set of cases is compared with the expected pairwise relatedness for a group of similar individuals in the updb . One thousand sets of matched controls that also had a dc were randomly selected from the updb . To test the hypothesis of no excess relatedness among the set of pd cases identified from dcs, the case gif was compared with the empirical distribution of gif statistics estimated from 1,000 sets of matched controls . Controls were randomly selected from all individuals with genealogic data and a dc and were matched to cases by birth cohort (5 years), sex, and birthplace (utah or not). These analytical methods, including gif analysis, have been previously applied to describe the familial and genetic contribution to mortality of multiple phenotypes, including intracranial aneurysms, influenza, asthma, and amyotrophic lateral sclerosis, among others . The gif statistic can also be estimated while ignoring all close relationships (relationships closer than first cousins); this allows a test of the hypothesis that excess relatedness has been observed only for distant relationships, which are unlikely to share common risk factors or exposures . This test is termed the distant gif (dgif) test and allows determination of whether the excess familial clustering observed might be due at least in part to a genetic contribution . To estimate the rr of pd among relatives, the observed number of pd deceased relatives was compared with the expected number of pd deaths in relatives . Rr estimation is described below for the example of the rr of pd death among first - degree relatives of pd death cases . All individuals in the updb who belong to at least 3 generations of genealogy and who have a coded cause of death were assigned to 1 of 132 birth year (5 years), sex-, and birthplace - specific (utah or not) cohorts . The rate of death with pd for each cohort was estimated as the total number of individuals with pd reported as a cause of death in each cohort divided by the total number of individuals with a dc in the cohort . The expected number of first - degree relatives dying with pd was estimated by counting all relatives of probands who have a dc (by cohort, each relative counted only once regardless of how many times he or she was identified as a relative of the degree of interest), multiplying the number of deceased first - degree relatives (per cohort) by the cohort - specific rate of death with pd, and then summing over all cohorts . Rrs were estimated as the observed number of first - degree relatives with pd divided by the expected number of first - degree relatives with pd; this is an unbiased estimator of rr and is calculated similarly for different relationships . Two - tailed probabilities were calculated under the null hypothesis rr = 1.0, under the assumption that the number of observed deaths follows a poisson distribution with mean equal to the expected number of deaths; confidence intervals (cis) for the rr were calculated as described elsewhere . Using data for all ancestors of each pd case, all clusters of pd cases descending from a common ancestor can be identified . Using the same methods described for rrs, these clusters (or pedigrees) can be tested for a significant excess of pd cases by counting the observed pd deaths in the pedigree and estimating the expected number of pd deaths by applying the appropriate pd rates to all individuals in the pedigree with dc data . The updb includes birth and death data of more than 7 million individuals, with some records extending back more than 12 generations . The original utah genealogy includes complete 3-generation genealogic data for the utah mormon pioneers (members of the church of jesus christ of latter - day saints) and their descendants up to 1972 . Since then, the original genealogy data have been expanded with utah vital records such as birth certificates (using father, mother, and child trios). For the analyses reported in this study, we consider only those individuals in the updb with at least 3 generations of genealogy data who are related to the original utah genealogy . Utah's founding pioneers were composed of a sizable, largely unrelated mixture of northern european populations . This population continued to have high rates of immigration for years after utah's founding in 1847 . Studies using pedigree data, migration matrices, and isonymy have all shown low levels of inbreeding and similarity to northern european populations in the founding population of utah . We analyzed more than 2.7 million individuals in the updb belonging to at least 3 generations of genealogic data and connected to the original utah genealogy . Within this population of individuals with genealogy data, the cause of death on utah dcs was coded using the icd, with the revision used (i.e., icd-6 to icd-10) depending on the decade of death . Table 1 shows the frequency of the 4,031 pd deaths by icd revision; 2,546 of these deaths occurred in males and 1,485 in females . The majority of the deaths from pd were in more recent years; 2,407 deaths occurred after 1999 . The genealogical index of familiality (gif) statistic was developed to test hypotheses concerning excess relatedness among individuals sharing a specific phenotype using the updb . The gif analysis considers all genetic relationships between cases and measures the average relatedness among all possible pairs within a set of individuals . The pairwise relatedness measure implements the malcot coefficient of kinship, defined as the probability that randomly selected homologous genes from the 2 individuals are identical by descent from a common ancestor . The case gif is defined as the average of the coefficients of kinship between all possible pairs of cases (10). The pairwise relatedness of a set of cases is compared with the expected pairwise relatedness for a group of similar individuals in the updb . One thousand sets of matched controls that also had a dc were randomly selected from the updb . To test the hypothesis of no excess relatedness among the set of pd cases identified from dcs, the case gif was compared with the empirical distribution of gif statistics estimated from 1,000 sets of matched controls . Controls were randomly selected from all individuals with genealogic data and a dc and were matched to cases by birth cohort (5 years), sex, and birthplace (utah or not). These analytical methods, including gif analysis, have been previously applied to describe the familial and genetic contribution to mortality of multiple phenotypes, including intracranial aneurysms, influenza, asthma, and amyotrophic lateral sclerosis, among others . The gif statistic can also be estimated while ignoring all close relationships (relationships closer than first cousins); this allows a test of the hypothesis that excess relatedness has been observed only for distant relationships, which are unlikely to share common risk factors or exposures . This test is termed the distant gif (dgif) test and allows determination of whether the excess familial clustering observed might be due at least in part to a genetic contribution . To estimate the rr of pd among relatives, the observed number of pd deceased relatives was compared with the expected number of pd deaths in relatives . Rr estimation is described below for the example of the rr of pd death among first - degree relatives of pd death cases . All individuals in the updb who belong to at least 3 generations of genealogy and who have a coded cause of death were assigned to 1 of 132 birth year (5 years), sex-, and birthplace - specific (utah or not) cohorts . The rate of death with pd for each cohort was estimated as the total number of individuals with pd reported as a cause of death in each cohort divided by the total number of individuals with a dc in the cohort . The expected number of first - degree relatives dying with pd was estimated by counting all relatives of probands who have a dc (by cohort, each relative counted only once regardless of how many times he or she was identified as a relative of the degree of interest), multiplying the number of deceased first - degree relatives (per cohort) by the cohort - specific rate of death with pd, and then summing over all cohorts . Rrs were estimated as the observed number of first - degree relatives with pd divided by the expected number of first - degree relatives with pd; this is an unbiased estimator of rr and is calculated similarly for different relationships . Two - tailed probabilities were calculated under the null hypothesis rr = 1.0, under the assumption that the number of observed deaths follows a poisson distribution with mean equal to the expected number of deaths; confidence intervals (cis) for the rr were calculated as described elsewhere . Using data for all ancestors of each pd case, all clusters of pd cases descending from a common ancestor can be identified . Using the same methods described for rrs, these clusters (or pedigrees) can be tested for a significant excess of pd cases by counting the observed pd deaths in the pedigree and estimating the expected number of pd deaths by applying the appropriate pd rates to all individuals in the pedigree with dc data . We identified 4,031 individuals in the updb with a dc indicating pd (table 1). The gif test for excess relatedness for the 4,031 pd death cases the sample size, average case relatedness (case gif), mean control relatedness (mean control gif), and empirical significance for both the overall gif (gif p) and the dgif test (dgif p) are shown . Genealogical index of familiality (gif) analysis for excess relatedness for parkinson disease (pd) cases the gif analysis for the 4,031 individuals dying from pd demonstrated excess relatedness than was expected (p <0.001). The results for the dgif analysis, which ignores all close relationships while testing for an excess of relatedness, show that the excess relatedness is not only observed for close relationships but also for distant relationships (p = 0.001), which suggests that there is a genetic contribution to pd mortality, in addition to any shared nongenetic risks . Because of concerns regarding missed diagnosis or misdiagnosis on dcs, an additional analysis was performed . Assuming that more recent dc diagnoses are more accurate, only the set of pd deaths diagnosed by dc after 1999 were considered . Results for the gif analysis of these 2,407 cases are also shown in table 2 and the same conclusions are met . The gif statistic summarizes pairwise relationships; the contribution from specific close and distant relationships can be considered graphically . Figure 1 shows the distribution of the contribution to the overall gif statistic (y axis) by the pairwise genetic distance (x axis) for cases and for the average of 1,000 sets of matched dc controls . A pairwise genetic distance of 1 represents parent / offspring, 2 primarily represents siblings, 3 primarily represents uncle / niece and other avuncular relationships, 4 primarily represents first cousins, and so forth . The case gif contribution exceeds that of the average control contribution for all relationships out to a genetic distance of 8 (third cousins), showing a statistical excess of both close and distant pairwise relationships and supporting a genetic contribution, perhaps in addition to other nongenetic familial factors that may be shared among close relatives . Estimated rrs for pd death among relatives of probands dying of pd table 3 includes the type of relative, total number of relatives with dc data, the number of pd deaths observed in the relatives, the expected number of pd deaths in the relatives, the significance of the test, the estimated rr, and the 95% ci for the rr . Rrs are shown for first-, second-, and third - degree relatives and are shown separately for the 3 different types of first - degree relatives (parents, siblings, and offspring) and for spouses . Significantly elevated risk for pd death was not observed among the spouses of pd death cases . Although significantly elevated rrs in first - degree relatives might indicate a genetic contribution to risk, a considerable amount of sharing of environmental risks can also be assumed among first - degree relatives and might explain the results . The significantly elevated rrs for second- and third - degree relatives, however, provide strong support for a genetic contribution to death from pd among individuals not likely to share a common environment . Estimates of relative risk for parkinson disease rr estimates for first-, second-, and third - degree relatives of only the 2,407 pd deaths diagnosed after 1999 were all significantly increased and did not differ from the estimates in table 3 (data not shown). The gif and rr results suggest the existence of high - risk pd mortality pedigrees . Not all such clusters of pd deaths represent high - risk pedigrees, as chance clustering can occur; large numbers of pd deaths might be expected in large pedigrees . We therefore identified those clusters (pedigrees) that had a significant excess of pd deaths among the descendants ., there are more than 8,000 descendants of this founder pair; here only descending lines to all pd deaths are shown . There are 13 pd deaths observed among these descendants, but only 4.2 are expected (p = 0.0013). To test whether the cases identified with dc coding were truly affected by parkinsonism and pd, a movement disorders specialist (r.s .) Records for all available pd cases diagnosed from dcs who were also seen at least 1 time in the university of utah hospital and clinics (data from 1994) were reviewed . A total of 218 cases were reviewed; 128 of these cases (58.7% of the total) had a medical record noting a clinically confirmed diagnosis of pd and 11 had atypical or secondary parkinsonism for a total of 139/218 (63.8%). We identified 4,031 individuals in the updb with a dc indicating pd (table 1). The gif test for excess relatedness for the 4,031 pd death cases the sample size, average case relatedness (case gif), mean control relatedness (mean control gif), and empirical significance for both the overall gif (gif p) and the dgif test (dgif p) are shown . Genealogical index of familiality (gif) analysis for excess relatedness for parkinson disease (pd) cases the gif analysis for the 4,031 individuals dying from pd demonstrated excess relatedness than was expected (p <0.001). The results for the dgif analysis, which ignores all close relationships while testing for an excess of relatedness, show that the excess relatedness is not only observed for close relationships but also for distant relationships (p = 0.001), which suggests that there is a genetic contribution to pd mortality, in addition to any shared nongenetic risks . Because of concerns regarding missed diagnosis or misdiagnosis on dcs, an additional analysis was performed . Assuming that more recent dc diagnoses are more accurate, only the set of pd deaths diagnosed by dc after 1999 were considered . Results for the gif analysis of these 2,407 cases are also shown in table 2 and the same conclusions are met . The gif statistic summarizes pairwise relationships; the contribution from specific close and distant relationships can be considered graphically . Figure 1 shows the distribution of the contribution to the overall gif statistic (y axis) by the pairwise genetic distance (x axis) for cases and for the average of 1,000 sets of matched dc controls . A pairwise genetic distance of 1 represents parent / offspring, 2 primarily represents siblings, 3 primarily represents uncle / niece and other avuncular relationships, 4 primarily represents first cousins, and so forth . The case gif contribution exceeds that of the average control contribution for all relationships out to a genetic distance of 8 (third cousins), showing a statistical excess of both close and distant pairwise relationships and supporting a genetic contribution, perhaps in addition to other nongenetic familial factors that may be shared among close relatives . Estimated rrs for pd death among relatives of probands dying of pd are shown in table 3 . Table 3 includes the type of relative, total number of relatives with dc data, the number of pd deaths observed in the relatives, the expected number of pd deaths in the relatives, the significance of the test, the estimated rr, and the 95% ci for the rr . Rrs are shown for first-, second-, and third - degree relatives and are shown separately for the 3 different types of first - degree relatives (parents, siblings, and offspring) and for spouses . Significantly elevated rrs for pd death were observed in all relative groups considered . Significantly elevated risk for pd death was not observed among the spouses of pd death cases . Although significantly elevated rrs in first - degree relatives might indicate a genetic contribution to risk, a considerable amount of sharing of environmental risks can also be assumed among first - degree relatives and might explain the results . The significantly elevated rrs for second- and third - degree relatives, however, provide strong support for a genetic contribution to death from pd among individuals not likely to share a common environment . Estimates of relative risk for parkinson disease rr estimates for first-, second-, and third - degree relatives of only the 2,407 pd deaths diagnosed after 1999 were all significantly increased and did not differ from the estimates in table 3 (data not shown). The gif and rr results suggest the existence of high - risk pd mortality pedigrees . Not all such clusters of pd deaths represent high - risk pedigrees, as chance clustering can occur; large numbers of pd deaths might be expected in large pedigrees . We therefore identified those clusters (pedigrees) that had a significant excess of pd deaths among the descendants . Figure 2 shows an example high - risk pd pedigree . In the updb, there are more than 8,000 descendants of this founder pair; here only descending lines to all pd deaths are shown . There are 13 pd deaths observed among these descendants, but only 4.2 are expected (p = 0.0013). To test whether the cases identified with dc coding were truly affected by parkinsonism and pd, a movement disorders specialist (r.s .) Records for all available pd cases diagnosed from dcs who were also seen at least 1 time in the university of utah hospital and clinics (data from 1994) were reviewed . A total of 218 cases were reviewed; 128 of these cases (58.7% of the total) had a medical record noting a clinically confirmed diagnosis of pd and 11 had atypical or secondary parkinsonism for a total of 139/218 (63.8%). Familial pd cases are recognized to be due to mutations in the lrrk2, park2, park7, pink1, or snca gene, or in genes not yet identified . Variants in these genes are reported to account for about 30% of familial pd cases . Given the likely multifactorial nature of pd with multiple genes, environmental exposures, and the interactions of both, it is no surprise that most familial pd cases remain unexplained by known predisposition genes . More complete knowledge of familial clustering and risk to relatives can increase our understanding of this complex disorder . The updb resource has been analyzed for more than 30 years to define the observed familial clustering of disease, to detect evidence for a genetic contribution to disease, and to identify high - risk pedigrees informative for predisposition gene identification . This unique utah resource has enabled our population - based definition of familial clustering observed for pd and estimation of rrs for pd death among both close and distant relatives, allowing some discrimination of likely genetic contributions . Specific high - risk pedigrees have been identified, which will be informative for predisposition gene identification efforts . A study of pd in a similar resource in iceland presented evidence for familial aggregation of the subset of late - onset pd (defined as onset at age> 50 years) and provided rrs for spouses and for specific first-, second-, and third - degree relationships . There are 3 differences between the iceland study and this utah study: (1) this study used pd mortality whereas the iceland study considered pd diagnosis, (2) this study analyzed 4,031 pd cases whereas the iceland study analyzed 772 pd patients, and (3) although both populations are internally homogeneous, they are of different origin . In general, these 2 studies had very similar findings, but these and other differences may account for any differences in results reported . We present an independent analysis of familial clustering of pd in a large resource representing many generations of the utah population . This analysis of pd deaths in the homogeneous utah population shows strong support for a genetic contribution to pd, based on multiple tests for familial clustering . We did not observe many early - onset pd deaths and thus considered only 1 pd mortality phenotype with death primarily> 70 years . The pd study in iceland explored the familial aggregation of pd in an entire population . This utah study with 4,031 pd cases similarly found significant excess clustering of pd cases and significantly elevated rrs for first-, all second-, and all third - degree relatives . Pd rr estimates were higher in the icelandic study than in the utah study: rr for siblings = 6.7 (95% ci 4.39.6) and offspring = 3.2 (1.27.8) compared with the utah rrs of 1.78 (1.52.1) and 1.98 (1.42.7) for siblings and offspring, respectively . The utah study estimated rrs for all second - degree relatives at 1.44, compared with the iceland study's rr of 1.4 for the subset of second - degree relatives who were nieces and nephews . The utah study estimated rrs for all third - degree relatives at 1.10, compared with the iceland study's rr of 1.4 for the subset of third - degree relatives who were first cousins . Neither the iceland study nor the utah study estimated the rr for spouses of cases to be significantly different from 1.0 . Both analyses, 15 years apart in 2 different populations, showed strong evidence for a genetic contribution to pd . Rocca et al . Also reported on familial aggregation of pd in first - degree relatives of probands with pd who were representative of the minnesota population . An rr = 1.71 was observed (95% ci 1.112.64); relatives of probands with younger onset had higher risk (rr = 2.62, 95% ci 1.74.2). Individuals could be censored based on lack of genealogy data, failure to link to utah dc data, or death outside utah . Nevertheless, pd death rates were estimated from the updb population of individuals with a utah dc, so although the rates of pd death used may not represent population rates, there is no bias expected for the tests of hypothesis performed . An additional limitation is the use of a cause of death listed on a utah dc to diagnose pd . Pd mortality cases could have been censored based on the failure to note pd as a cause of death; similarly, but much less likely, is the possibility that a diagnosis of pd appeared on a dc in error . In addition, the validation that we performed, which was limited in ability to find all pertinent medical records, still found that 60% of the cases identified from dcs had evidence of a diagnosis of pd in their medical record . We report the results of a large population - based familial aggregation study of pd mortality . We analyzed more than 700,000 deaths of individuals who resided in utah, identifying 4,031 individuals with pd diagnosis in the dc, and showed strong support for a genetic contribution to pd mortality . The utah rr estimates provide further helpful information to caregivers, patients, family members, and researchers in the field . Finally, we have identified a rich resource of pedigrees with not only a large number of pd deaths but also a significant excess of pd deaths that are uniquely informative for current studies to identify predisposition genes . R. savica: drafting / revising the manuscript; study concept or design; analysis or interpretation of data . L.a . Cannon - albright: drafting / revising the manuscript; study concept or design; analysis or interpretation of data . S. pulst: drafting / revising the manuscript; study concept or design; analysis or interpretation of data . Dr . Cannon - albright has received research support from national cancer institute, nih, american association for cancer research, us department of defense, mayo clinic rochester, and alex's lemonade stand foundation and holds a patent for brca1, brca2, and p16 . Pulst has served on the editorial board of journal of cerebellum, neuromolecular medicine, continuum, experimental neurology, neurology: genetics, nature clinical practice neurology, and current genomics; holds patents for nucleic acids encoding ataxin-2 binding proteins, nucleic acid encoding schwannomin - binding - proteins and products related thereto, transgenic mouse expressing a polynucleotide encoding a human ataxin-2 polypeptide, methods of detecting spinocerebellar ataxia-2 nucleic acids, nucleic acid encoding spinocerebellar ataxia-2 and products related thereto, schwannomin - binding - proteins; and compositions and methods for spinocerebellar ataxia; has received publishing royalties for the ataxias (churchill livingston, 2007), genetics in neurology (aan press, 2005), genetics of movement disorders (academic press, 2003), neurogenetics (oxford university press, 2000), and molecular genetic testing in neurology, 2nd5th (aan press, 1996); has served as a consultant for ataxion therapeutics; has served on speakers' bureaus for athena diagnostics, inc . ; has received research support from national institutes of health (ro1ns33123, rc1ns068897, rc4ns073009, r21ns079852, r21ns081182) and national ataxia foundation; has received license fee payments for technology and/or inventions from cedars - sinai medical center; and has given expert testimony for hall & evans, llc (denver, co).
It could be an incidental diagnosis in adulthood or could present with obstructive symptoms from the herniated viscera . Surgical treatment consists of direct closure or mesh placement for the diaphragmatic defect, or suturing by transabdominal or transthoracic access . We report a patient with morgagni hernia who underwent a laparoscopic mesh placement with reduced port surgery (rps). An 85-year - old female presented in er with a 2-day history of upper abdominal discomfort and loss of appetite . She had never experienced the same symptoms previously, and she denied any history of abdominal trauma . Physical examination revealed no palpable mass in the upper abdomen, and local tenderness on upper abdomen was shown without any peritoneal inflammatory signs . Serum blood test showed a hyperlipidemia and mild elevation of urea nitrogen (total cholesterol, 250 mg / dl; urea nitrogen, 22.5 mg / dl). A ct scan showed a huge diaphragmatic anterior hernia with a segment of transverse colon and fat tissue migration (fig . The diagnosis of morgagni hernia was made and the patient was considered for repair of the diaphragmatic defect by the laparoscopic approach . Under general anesthesia in the lithotomy position, the sils port (covidien, tokyo, japan) was inserted into a 2.5-cm umbilical incision vertically . After inspection of the visceral space, a second port, 12 mm in size, was inserted into the left lower abdomen . The herniated bowel and fat tissues were gently pulled down with grasping forceps and placed entirely into the abdominal cavity (fig . The defect was ovoid and approximately 5 cm in size, and difficult to close by the suturing technique; therefore we performed mesh placement with gore - tex dual mesh (gore inc . Operation time and operative blood loss were 157 min and 5 ml, respectively . The patient started to eat a soft meal on day 1 after surgery and was discharged from our hospital on day 8 after surgery without any symptoms . After 6-month, 12-month, and 24-month follow - ups, the patient had no sign of recurrence of the morgagni hernia (figs . 4 and 5). Giovanni - battista morgagni first described this type of hernia in 1769 . At present, it could be an incidental diagnosis in adulthood or could present with obstructive symptoms of the herniated viscera . Surgical treatment is required to relive current symptoms or to prevent possible future complications such as strangulation ileus or incarceration . Originally, direct closure of the hernia site or mesh placement was used as the surgical options by laparotomy or thoracotomy . Recently, the laparoscopic procedure has become available to treat this type of hernia and has bought a number of advantages such as reduced pain, shorter recovery time, and cosmetic benefits . In addition, recent trends in laparoscopic procedures have been toward minimizing the number of incisions to achieve less invasiveness . Examples of such approaches are single incision laparoscopic surgery (sils) and reduced port surgery (rps). Only one report previously described morgagni hernia repair using single port access and closing the hernia site by non - absorbable suturing into extra - abdominal region . This report was very successful, however postoperative pain might be occurred because of suturing tension by lifting the interrupted sutures to close the hernia site . Some technical problems associated with sils have been reported: restriction of the working field and interference of surgical instrument . The sils technique does not rely on triangulation, which is one of the core principles of conventional laparoscopic surgery, allowing adequate operative exposure while maintaining an ergonomic position for the surgeon and assistant . Consequently, the inherent technical challenge that arises from the sils technique is that of a compromised view and locomotive field . Therefore unfavorite outcomes such as longer operative time and possibly higher complication rate have been reported in early period of sils operation . A small additional port or a reduced number of ports as with rps are modifications that may overcome these problems . In combination with the sils port and the additional port enable the performance of dual - port surgery, even for complex operation such as laparoscopic gastrectomy . This type of repair is technically easy and should have a high probability of success because of the tension free condition . Also, less postoperative pain is also expected, compared to the external knot tying technique . Thoracoscopic approach is also other option for this type of hernia as a less invasive treatment, although the benefits over conventional laparoscopic approach have not been cleared . Although the benefits of sils or rps over conventional laparoscopic surgery have not been established, either sils or rps could be the first choice for symptomatic morgagni hernia repair because of the acceptable operation time, blood loss, and clinical outcome . Basically, cosmetic benefits and less operative pain are also expected as advantages of sils or rps originating from fewer incisions . In addition, tissue trauma and port - related complications such as organ damage, adhesions, bleeding, wound infections and hernias could be decreased . For the future direction, additional this approach is safe and can be the good indication for this kind of hernia . Written informed consent was obtained from the patient for publication of this case report and accompanying images.
Essential hypertension, a progressively serious worldwide public - health challenge, represents one of the most important risk factors for myocardial infarction, stroke, endstage renal disease, and peripheral vascular disease . It has been predicted that ~20 - 60% of the inter - individual variation of blood pressure (bp) was genetically controlled . Hence, several studies have designed to find the potential hypertension - susceptibility genes for a better understanding of disease etiology . At molecular level the underlying mechanisms of hypertension are complex, involving many interacting systems such as the renin - angiotensin system, reactive oxygen species (ros), vascular inflammation and remodeling . The imbalance between prooxidants and antioxidants results in oxidative stress, which is the pathogenic outcome of oxidant overproduction that overwhelms the cellular antioxidant capacity . Strong experimental evidence indicates that oxidative stress plays an important pathophysiological role in the development of hypertension . The increased production of ros, reduced nitric oxide synthesis, and decreased bioavailability of antioxidants have been demonstrated in both experimental and human hypertension . Among the physiological antioxidants, reduced gsh scavenges ros along with regeneration of other antioxidants from their oxidized forms . Through these processes, gsh is converted to its oxidized form (gssg), which must be reduced by nadph - glutathione reductase . The cell's ability to conserve gsh levels is very important for its integrity and cellular function . Moreover, gsh is also an essential cofactor for different enzymes like glutathione s - transferases (gsts). The human cytosolic gst super family consists of at least 16 genes sub categorized into eight classes designated alpha, kappa, mu, pi, sigma, theta, zeta, and omega . Differences in susceptibility to various forms of disease and outcome have associated with polymorphisms of gstm1, gstt1, and gstp1 . Three different alleles are found at the locus of gstm1 (1p13.3) including gene deletion (gstm1 - 0) and two functional mutations (gstm1a and gstm1b). Individuals with homozygous deletions at the gstm1 and gstt1 loci (gstm1-null and gstt1-null) have no functional enzymatic activity . It has been shown that null genotypes of gsts are important factors in cell sensitivity to oxidative stress (os) and susceptibility to cardiovascular and metabolic disorders . The association between the variations in the gst activity in vivo and the susceptibility to cancer, cardiovascular, and other diseases has been studied primarily by the use of homozygous gstm1-null and gstt1-null genotypes, as indicators of a systemic lack of expression of the corresponding proteins . The present study was designed to investigate the association of gstt1 and gstm1 gene polymorphism with bp in the iranian population . This study was performed after approval of the ethical committee of isfahan university of medical sciences . A retrospective cohort study of the relationship between gst gene polymorphism and bp for the period between 2002 and 2007 was performed . Within isfahan cohort study (ics), which is a study including randomly selected participants from the community of three counties in central part of iran in the first stage of isfahan healthy heart program (ihhp), we randomly selected 72 healthy individuals with more complete information . Our subjects were healthy according to their medical history and new measurements at the time of this investigation . We excluded individuals who had: intrinsic renal disease, diabetes, history of cancer, asthma, and a self - reported history of hypertension that was corroborated by the family physician, or had coexisting illness . The bp was measured in the right upper arm with a standard sphygmomanometer in a sitting position . Systolic blood pressure (sbp) was taken at the return of arterial sounds and diastolic blood pressure (dbp) at the disappearance of sound . After 7 years, bp of each individual was again measured . As indices at 2002 related to gender, age, body mass index [bmi: (weight kg)/(height m)], sbp, dbp, total cholesterol, high - density lipoprotein (hdl) cholesterol, fasting blood glucose, cigarette smoking habit (no smoking, quit, smoking) were investigated . Dna was extracted from peripheral blood using blood mini kit (primeprep genomic dna isolation kit, genet bio inc . ). For analysis of gstt1 and gstm1 genotypes of the subjects, polymerase chain reaction (pcr) amplification was performed using the following primers: gstt1 forward primer 5-ttc ctt act ggt cct cac atc tc-3; and reverse primer 5-tca ccg gat cat ggc cag ca-3; gstm1 forward primer 5 aga cag aag agg aga aga ttc 3; and reverse primer 5 tcc aag tac ttt ggc ttc agt 3. the pcr reaction was done as previously described . We develop a bayesian structured regression model for investigating the effect of some genotypes deletion on hypertension incidence . Bayesian inference scheme uses the posterior distribution, that is, the conditional distribution of the model parameters given the observed data . In this study, we use a fully bayesian inference based on analysis of posterior distribution of the model parameters . Monte carlo markov chain (mcmc) was used for estimation and this was implemented in the freely available software winbugs (bugs project, http://www.mrc-bsu.cam.ac.uk/bugs/winbugs/). Gibbs sampler was employed for mcmc simulations, drawing successively from the full conditionals for the variance components and unknown parameters . We used the total number of 100,000 mcmc iterations with 20,000 burn in samples . Since, in general, these random numbers are correlated, only every 10 sampled parameter of the markov chain were stored . Convergence was assessed by visual inspection of the means in time - series plots but also more formally using the gelman rubin r - hat diagnostics . Our goal was showing the association between gstm1 and gstt1 genotypes deletion status and mean arterial pressure (map) changes as an indicator for hypertension adjusted by the effect of sex, age, bmi, and smoking status of patients . This study was performed after approval of the ethical committee of isfahan university of medical sciences . A retrospective cohort study of the relationship between gst gene polymorphism and bp for the period between 2002 and 2007 was performed . Within isfahan cohort study (ics), which is a study including randomly selected participants from the community of three counties in central part of iran in the first stage of isfahan healthy heart program (ihhp), we randomly selected 72 healthy individuals with more complete information . Our subjects were healthy according to their medical history and new measurements at the time of this investigation . We excluded individuals who had: intrinsic renal disease, diabetes, history of cancer, asthma, and a self - reported history of hypertension that was corroborated by the family physician, or had coexisting illness . The bp was measured in the right upper arm with a standard sphygmomanometer in a sitting position . Systolic blood pressure (sbp) was taken at the return of arterial sounds and diastolic blood pressure (dbp) at the disappearance of sound . After 7 years, bp of each individual was again measured . As indices at 2002 related to gender, age, body mass index [bmi: (weight kg)/(height m)], sbp, dbp, total cholesterol, high - density lipoprotein (hdl) cholesterol, fasting blood glucose, cigarette smoking habit (no smoking, quit, smoking) were investigated . Dna was extracted from peripheral blood using blood mini kit (primeprep genomic dna isolation kit, genet bio inc . ). For analysis of gstt1 and gstm1 genotypes of the subjects, polymerase chain reaction (pcr) amplification was performed using the following primers: gstt1 forward primer 5-ttc ctt act ggt cct cac atc tc-3; and reverse primer 5-tca ccg gat cat ggc cag ca-3; gstm1 forward primer 5 aga cag aag agg aga aga ttc 3; and reverse primer 5 tcc aag tac ttt ggc ttc agt 3. the pcr reaction was done as previously described . We develop a bayesian structured regression model for investigating the effect of some genotypes deletion on hypertension incidence . Bayesian inference scheme uses the posterior distribution, that is, the conditional distribution of the model parameters given the observed data . In this study, we use a fully bayesian inference based on analysis of posterior distribution of the model parameters . Monte carlo markov chain (mcmc) was used for estimation and this was implemented in the freely available software winbugs (bugs project, http://www.mrc-bsu.cam.ac.uk/bugs/winbugs/). Gibbs sampler was employed for mcmc simulations, drawing successively from the full conditionals for the variance components and unknown parameters . We used the total number of 100,000 mcmc iterations with 20,000 burn in samples . Since, in general, these random numbers are correlated, only every 10 sampled parameter of the markov chain were stored . Convergence was assessed by visual inspection of the means in time - series plots but also more formally using the gelman rubin r - hat diagnostics . Our goal was showing the association between gstm1 and gstt1 genotypes deletion status and mean arterial pressure (map) changes as an indicator for hypertension adjusted by the effect of sex, age, bmi, and smoking status of patients . Descriptive statistics as percentages and mean sd showed that 49% of total patients were male and 51% were female . The positive rate for smoking was 20% . The bmi and age variables as continuous variables were 29.5 13.6 and 57.5 9.9, respectively . Table 1 contains the posterior means, posterior standard deviations, and 95% credible intervals for the parameters of interest . The results showed that both the gstm1 and gstt1 genotypes deletion had a significant effect on the map increasing in our samples based on 95% credible intervals [table 1]. Posterior results for the final bayesian model for showing effective genotypes deletion on mean arterial pressure (map) adjusted by bmi, age, sex, and smoking status this study has investigated gstm1 and gstt1 gene polymorphisms in iranian population with the incidence of hypertension and demonstrated that both the gstm1 and gstt1 genotypes deletion had a significant effect on the map increasing in our sample based on 95% credible intervals . It is well established that high bp is a risk factor for coronary heart disease . Several studies show that between 30% and 40% of bp variation in a population is thought to have a genetic basis . Many studies have generally supported the idea that hypertension is associated with increased vascular oxidative stress; however, human studies were in conflict . Oxidative stress may produce and maintain hypertension via several mechanisms like, quenching of the vasodilator nitric oxide by ros such as superoxide; generation of vasoconstrictor lipid peroxidation products; and structural and functional alterations within the vasculature . The gsts are involved in the detoxification of many toxic compounds of different chemical structures in cigarette smoke, including epoxybutane, ethylene oxide, monohalomethane, and reactive metabolites of polycyclic aromatic hydrocarbons, such as benzo[a]pyrene . They act as antioxidant through inactivation of endogenous unsaturated aldehydes, quinines, epoxides, and hydroperoxide formed as secondary metabolites during the oxidative stress, thus playing a key role in protecting cell types of various origins, including vascular smooth muscle cells and endothelial cells against oxidant damage . In humans, there is a wide variation in gst activity due to genetic polymorphisms, which can result in oxidative stress potentiation and influence the individual's susceptibility to diseases, including hypertension . In our study, by using bayesian structured regression model and adjusted by the effect of sex, age, bm, i and smoking status, in subject with deletion of two classes of gsts, gstt1 and gstm1, the average of arterial pressure had increased from 2001 to 2007 . Map is a weighted average of sbp and dbp and is strong prognostic predictors of adverse cardiovascular events . Oniki et al . Observed that the risk of hypertension was significantly increased in the gsta1*b allele carriers that also had the gstm1-null genotype or both the gstm1 and gstt1-null genotypes . In addition, saadat and dadbine - pour showed an influence of gstm1 polymorphism on sbp in normotensive individuals and modulation of bp in individuals chronically exposed to natural sour gas containing sulfur compounds). Moreover, the results of tew et al . Described a higher frequency of gstm1 and gstt1-null genotypes (especially gstt1-nulls) among systemic sclerosis patients with hypertension and pulmonary involvement . Reported that the frequency of gstm1 and gstt1positive was significantly lower in essential hypertensive patients than in normotensive subjects . These observations proposed that a genetic background (gst gene polymorphisms) may contribute to the development of hypertension . It has been reported that reduction of gstm1 expression in the stroke - prone spontaneously hypertension rat contributes to increased oxidative stress . Based on experimental evidence and clinical studies that oxidative stress plays a key role in vascular damage, there has been a great interest in developing strategies that target ros in the treatment of hypertension . Therefore, the oxidative stress might be the necessary link between gsts activity and hypertension . It is worth noting that deleted polymorphisms in the gst genes may also influence the susceptibility to coronary artery disease by modulating the detoxification of genotoxicatherogens . The heterogeneity in the outcomes for the gst genes and bp could be due to extreme gene environment interactions that characterize the hypertensive phenotypes but could also be related to the difference in the selection of cases and controls . Further studies to understand the role of genetic susceptibility to oxidative stress in the development of hypertension are merited . M.a conducting the statistical analysis . N.s and s.h.j contributed to the study design, conducting the study, and approving the manuscript . In conclusion, the genetic polymorphism of gstt1 and gstm1 by adjusting effect of sex, age, bmi, and smoking status was significantly associated with the average arterial pressure increasing suggesting that the gstm1 and gstt1 genes are the candidate genes that alter bp and subsequently, the susceptibility to atherosclerosis with regard to sex, bmi, and cigarette smoking.
Metastatic melanoma in the brain is a serious event in patients with melanoma because of the poor prognosis and potential impact on quality of life . Symptomatic metastases represent the initial site of metastatic spread in 20% but may occur at any time during the course of the disease . Autopsy data have shown that up to 75% of patients who died from metastatic melanoma had brain metastases [2, 3]. Two large institutional series of 686 and 702 patients [3, 4] indicate a generally poor outcome, with the majority (up to 95%) dying directly from brain metastases . There were some differences in survival according to treatment received, being 8.9 months for surgery plus whole brain radiotherapy (wbrt), 8.7 months for surgery alone, 3.4 months for wbrt alone, and 2.1 months for supportive care . These differences are a probable reflection of patient selection based on the number of cerebral metastasis, performance status, and extent of extracranial metastasis . The radiation therapy oncology group recursive partitioning analysis (rpa) classes have been validated in melanoma . Age (> 65 year old) and the number of neurological symptoms (weakness and fatigue) are associated with poorer survival . Ulceration and location on the head and neck region are two main primary tumour characteristics that are associated with poorer survival . The number of cerebral metastases is a significant prognostic factor with better prognosis seen in single or oligometastatic disease (2 - 3 cerebral metastases). Patients with more than 3 metastases had a median survival of 3.5 months compared with 5.9 months for those with 3 or less metastases (p = 0.005). More recently, there is debate on whether it is the number of metastases or the overall intracranial tumour volume that is the relevant factor . The worst outcome is seen in patients with leptomeningeal disease . In all large cohorts of patients with melanoma brain metastases, md anderson cancer center analysed the outcomes of 743 patients with metastatic melanoma in the brain treated between 1986 and 2004 . The median survival for patients diagnosed before 1996 was 4.14 months compared with 5.92 months for patients diagnosed in 1996 or later (hr 0.75 95% ci 0.590.95, p = 0.02). The increased use of mri as a screening tool for brain metastases over time may have contributed to this improvement . In addition, earlier diagnosis of patients with lower burden, asymptomatic brain metastasis might allow for more frequent use of locally directed treatment such as stereotactic radiosurgery or surgical excision . A similar study of patients from the memorial sloan kettering cancer center noted that age> 65, presence of extracranial metastases, presence of neurologic symptoms and four or more metastases are predictors for poorer survival, although some of these features are self - predicting in that more aggressive treatment options are less likely to be offered . The management of metastatic melanoma in the brain depends on the combination of patient, tumor, and treatment factors . The dominant factor determining management has been the number of cerebral metastases . With the wider availability of stereotactic radiosurgery facilities enabling the effective treatment of multiple metastases in a single treatment session, reports increasingly suggest that the use of stereotactic radiosurgery to treat multiple metastases may have merit, particularly if there are less than 10 lesions, all under 3 cm in size and with limited oedema or mass effect [1113]. Recent data suggested that the total volume of the metastatic lesions rather than the number of metastases was the limiting factor for radiosurgery technique . Our general approach in the management of melanoma metastases in the brain is shown in figure 1 . For patients with a single or oligometastases, the management depends on the performance status, neurological status, the characteristic of the metastases (number, size, and location), and the extent of the extracranial disease . Those with more favourable characteristics should be considered for more aggressive local treatment of the individual metastasis . Surgery has a role in confirming the diagnosis especially as there is no clear relationship between the primary melanoma and the development of brain metastasis . In addition, surgical resection can also provide quick relief in symptoms associated with disease such as shunting in hydrocephalus . The prognosis is poor, with the majority succumbing to progressive intracranial metastases within months, irrespective of treatment . Application of the rtog recursive partitioning analysis to 74 patients with cerebral metastases from melanoma produced median survival of 10, 6, and 2 months, respectively, for rpa classes i iii, respectively, with a median survival of 5.5 months for the entire group . Initial management includes the use of steroids, typically 416 mg of dexamethasone per day . This usually results in rapid symptomatic, but often short - term, improvement in approximately 50% of patients . Whole brain radiation therapy may produce a small survival advantage compared with steroids alone and may allow reduction in the steroid dose . In addition to whole brain radiation therapy, surgical removal, or stereotactic radiosurgery of a dominant and symptomatic lesion should be considered . Conversely, patients with a poor performance status who have not responded to steroids may be better treated with supportive care . The technique of whole brain radiation therapy is a pair of parallel opposed lateral 6 mv photon fields . Commonly used regimens are 20 gy in 5 fractions and 30 gy in 10 fractions . For good performance patients with minimal extracranial disease, there might be an advantages for higher dose of whole brain radiation therapy based on a retrospective study . Rades et al . Compared the outcomes of 33 patients treated with 30 gy in 10 fractions with 18 patients treated with higher doses (40 gy in 20 fractions or 45 gy in 15 fractions). In the multivariate analysis, higher doses (p = 0.010), less than four brain metastases (p = 0.012), no extracranial metastases (p = 0.006), and rpa class 1 (p = 0.005) were associated with improved overall survival . In an attempt to improve survival of patients with multiple brain metastases, radiation has been combined with a variety of chemotherapy agents, including temozolomide, thalidomide, and fotemustine without much success [1518]. The most recent study is a phase 2 study combining whole brain radiation therapy (30 gy in 10 fractions) with temozolomide and thalidomide in 39 patients . The term radiosurgery was originally coined by lars leksell to describe the use of a multisource cobalt system (gamma knife) to deliver radiation to a defined target using stereotactic principle . It aims to deliver an ablative dose to the target while limiting the dose to surrounding normal tissue . The latest version of the gamma knife (perfexion) uses 192 cobalt sources arranged circumferentially in a noncoplanar fashion, permitting smaller doses to the surrounding normal brain tissue and a lower integral dose . The associated improvements in planning software and design modifications have resulted in the ability to treat multiple targets in one session . Historically, this has relied on frame - based stereotactic approaches that can accurately localise the tumour and target the beam in three - dimensional space . Options now include frameless image - guided approaches such as fixed beam intensity - modulated radiotherapy, helically delivered intensity - modulated radiotherapy (tomotherapy), and image - guided robotic radiosurgery (cyberknife). Arc - based intensity - modulated radiotherapy techniques (vmat and rapidarc) can also achieve highly conformal image - guided treatment in very short treatment times . Linear accelerator - based stereotactic radiosurgery will use a limited number of fields, usually (but not always) in a coplanar fashion . The dose of radiosurgery depends on the size of the target lesion and the location . The rtog 90 - 05 study was designed to determine the maximum tolerable dose of radiosurgery in patients with recurrent previously irradiated brain metastases (excluding lesions in the brain stem). The maximum tolerable doses of single fraction radiosurgery for patients with recurrent previously irradiated brain metastases were 24 gy, 18 gy, and 15 gy for tumors 20 mm, 2130 mm, and 3140 mm in maximum diameter, respectively . In the multivariate analysis, those who were treated on a linear accelerator (versus the gamma knife) had a 2.84 greater risk of local progression . However there are no randomised data showing clear superiority of any one stereotactic radiosurgery system . Mathieu et al . From the university of pittsburgh reviewed the experience of 244 patients with 754 melanoma metastases treated with gamma knife radiosurgery without adjuvant whole brain radiation therapy . Overall, 54 patients (30.9%) had progression of at least one metastasis after radiosurgery . Fifty - one patients (24.8%) underwent whole brain radiation therapy after radiosurgery because of the development of multiple new brain lesions . Multiple lesions and failure to provide systemic immunotherapy were predictors for the occurrence of new brain metastases, which developed in 41.7% of the patients . On multivariate analysis, the use of whole brain radiation therapy was not a factor that influenced local control or distant intracranial control (p = 0.061). A more recent update of the university of pittsburgh's experience on 333 consecutive patients with 1570 metastatic melanoma lesions treated with gamma knife radiosurgery showed the long - term local control rate was 73% and the actuarial survival rates were 70% at 3 months, 47% at 6 months, 25% at 12 months, and 10% at 24 months . About 25% of 259 patients who had followup imaging after stereotactic radiosurgery had evidence of delayed intratumoral haemorrhage . Factors associated with longer survival included controlled extracranial disease, better performance status, fewer number of brain metastases, no prior use of whole brain radiation therapy or chemotherapy, treatment with immunotherapy, and no intratumoral hemorrhage before radiosurgery . The potential morbidity of stereotactic radiosurgery includes progression or worsening of cerebral oedema symptomatic in 46% of patients within 1 - 2 weeks of treatment, seizures within 1 - 2 days in 26%, and delayed radiation necrosis in 211% [21, 24, 25]. This risk increases with prior treatment, larger volumes treated (both larger lesions and larger numbers of lesions), and larger doses delivered . The role for whole brain radiation therapy after surgery or stereotactic radiosurgery of the single or oligometastasis is controversial and there is no level 1 evidence in this scenario . The rationale of whole brain radiation therapy is to treat microscopic disease at the site of initial metastasis and elsewhere in the brain to maintain long - term cerebral control . Adjuvant systemic therapy is generally not used as the brain is considered a sanctuary site for chemotherapy although this assumption has been recently challenged by responses in the patients with b - raf mutant melanoma treated with b - raf inhibitor . However opponents of whole brain radiation therapy argue that melanoma is radioresistant and that whole brain radiation therapy can potentially cause late neurocognitive deficits . The australia and new zealand melanoma trials group (anzmtg) and trans - tasman radiation oncology group (trog) are conducting a phase 3 randomised trial to address the role of whole brain radiation therapy after local treatment of 1 to 3 melanoma metastases . Eligible patients are randomised to whole brain radiation therapy or observation after their local treatment of the brain metastasis . Is reported, clinicians will have to rely on data from other randomised trials included patients with metastatic disease from all histologies . Several randomised studies including metastasis from all histologies have provided good evidence for the use of whole brain radiation therapy after local treatment of oligometastases in terms of an improvement in intracranial control . Aoyama et al . Compared whole brain radiation therapy and stereotactic radiosurgery (65 patients) to stereotactic radiosurgery alone in patients with 14 brain metastases of any histology . Over 65% of the patients had metastatic lung cancer, while the number of melanoma patients per arm was not mentioned . The whole brain radiation therapy dose was 30 gy in 10 fractions over 2 weeks . There was no difference between the two groups with respect to overall survival, neurological toxicity, neurological functional preservation, and neurological death . The median survival time was 7.5 months with whole brain radiation therapy plus stereotactic radiosurgery compared to 8 months with stereotactic radiosurgery alone . The 12-month actuarial brain tumour local recurrence rate was 46.8% in the whole brain radiation therapy plus stereotactic radiosurgery group and 76.4% in the stereotactic radiosurgery alone group (p = 0.001). Fifty - five patients had new brain metastases at distant sites (21 in the whole brain radiation therapy plus stereotactic radiosurgery group and 34 in the stereotactic radiosurgery - alone group). The 12-month actuarial rate of developing distant brain metastases was 41.5% in the whole brain radiation therapy plus stereotactic radiosurgery group and 63.7% in the stereotactic radiosurgery - alone group (p = 0.003). The univariate analysis showed that patients with 24 metastases had a higher risk for developing distant intracranial disease than those with single metastasis (p = 0.03), but this did not reach significance on multivariate analysis (p = 0.06). More recently, eortc reported a randomized trial of 359 patients with 1 to 3 brain metastases from all solid tumours randomised to either observation or whole brain radiation therapy of 30 gy in 10 fractions after local treatment (surgery or stereotactic radiosurgery). The majority (53%) of patients had primary lung cancer and only 5% had metastatic melanoma . After surgery, at 2 years, whole brain radiation therapy significantly reduced the probability of relapse at initial sites from 59% to 27% (p = 0.001) and at distant intracranial sites from 42% to 23% (p = 0.008). After stereotactic radiosurgery, whole brain radiation therapy reduced the probability of relapse at initial sites from 31% to 19% (p = 0.04) and at distant intracranial sites from 48% to 33% (p = 0.023) at 2 years . The median progression - free survival was slightly longer in the whole brain radiation therapy arm compared with the observation arm (4.6 months versus 3.4 months; p = 0.02) but there was no difference in overall survival between the two arms . Eighty - one percent of the patients had single metastases but there was no analysis of one versus more than one metastases . This trial included neurocognitive and the quality of life assessments which have not yet been reported . There are also a number of single institution retrospective series in melanoma patients with their inherent selection biases . Reviewed the outcomes of 686 patients at the sydney melanoma unit (now the melanoma institute australia). There was no significant difference in the median survival between the 158 patients treated with surgery and whole brain radiation therapy and the 47 patients treated with surgery alone (8.9 months versus 8.7 months, p = 0.21). Sampson et al . Also reported no difference in median survival in patients treated with whole brain radiation therapy after surgery or surgery - alone patients (median survival of 9 months, p = 0.99). However, patients treated with whole brain radiation therapy were more likely to remain without neurological deficits or experience an improvement (81.7%) after completion of therapy than those who did not (57.7%, p = 0.01). Treatment was stereotactic radiosurgery alone (61 patients), stereotactic radiosurgery with whole brain radiation therapy (12 patients), and salvage stereotactic radiosurgery after whole brain radiation therapy (30 patients). The overall incidence of distant brain metastasis - free survival did not differ significantly between the group that received initial stereotactic radiosurgery alone and the group that received stereotactic radiosurgery and whole brain radiation therapy (17.6% versus 0%, p = 0.27). However this study did not have the statistical power to detect a difference in distant brain metastasis free survival . The initial number of brain lesions (single versus multiple) was the only factor with a significant effect on distant brain metastasis - free survival at 1 year: 23.5% for single metastases and 0% for multiple lesions (p <0.05). Samlowski et al . Performed a retrospective analysis of 44 melanoma patients with five or less brain metastases treated with stereotactic radiosurgery and showed that the addition of whole brain radiation therapy did not improve survival . Survival analysis showed that combined treatment of local and whole brain radiation therapy offered significantly better survival (p <0.0001). The median survival was 8.8 months for the combined therapy group, 4.8 months for the local - therapy - alone group . Md anderson reported a series of patients with solitary melanoma brain metastasis and no extracranial disease . Twenty - two patients received surgical excision and whole brain radiation therapy whole brain radiation therapy and 12 patients were treated with surgery alone . Despite the small sample size, intracranial recurrence rates favoured the combination (5/22 versus 9/12 surgery alone, p = 0.01). Median overall survival was 18 months in the combination therapy group versus 6 months for surgery alone (p = 0.002). These data argue that whole brain radiation therapy can decrease intracranial progression and may even convey a survival benefit in patients without active extracranial disease as a competing cause of death . One concern of delivering whole brain radiation therapy after local treatment of oligometastases is the potential neurological deficit . Preclinical and early clinical evidence suggests that a neural stem cell compartment in the hippocampus is central to the pathogenesis of neurocognitive deficits observed after cranial irradiation . Modern intensity - modulated radiotherapy technologies, such as helical tomotherapy and volumetric modulated arc therapy can conformally avoid the hippocampus during whole brain radiation therapy and therefore potentially reduce the risk of neurocognitive deficit . A rtog review of 371 patients with less than 10 brain metastasis from all histologies showed that only 3% of the metastatic deposit were within the 5 mm region around the hippocampus and none within the hippocampus itself . There is an ongoing clinical trial (rtog 0933) examining the effect of hippocampal avoidance whole brain radiation therapy technique on the neurocognitive function in patients with brain metastases from all histologies . The role of radiation therapy in the management is highly variable due to the natural history of the disease . To provide optimal management of the patient with melanoma although there have been no randomized trials especially in patients with melanoma brain metastasis, treatment can be guided by the application of evidence for the treatment of brain metastasis in general . A promising new approach to deliver radiation therapy while sparing the hippocampus
The maxillary sinuses develop in the 3rd month of intrauterine life . At the time of the birth, the volume of the sinus is 6 - 8 mm . The volume of the maxillary antrum increases by 2 mm in the vertical and lateral dimensions and by 3 mm in the anteroposterior dimension yearly until the age of 8 years . At the age of 10, the lower boundary of the maxillary sinus and the floor of the nasal cavity are on the same level . The incidence of maxillary sinus hypoplasia ranges between 1.5 and 10%, but some studies have reported less than this range . Many of the patients with aplasia or hypoplasia of the maxillary sinus are asymptomatic and unaware of their conditions and it is identified on routine radiographs . However, some of the patients may suffer from chronic headaches, facial pain, and voice problems . Maxillary sinus hypoplasia can lead to diagnostic problems, especially in conventional radiographs because it can be misdiagnosed as mucosal thickening in infectious disease or neoplasms involving the sinus . Also, atelectasis of the sinus due to chronic sinusitis can be diagnosed as aplasia or hypoplasia of the sinus . Computed tomography (ct) scanning and also cone beam computed tomography (cbct) and endoscopic examination of the sinus are the necessary diagnostic tools to detect the underlying abnormality . In this study, three cases with unilateral hypoplasia of the sinus, unilateral aplasia of the sinus, and bilateral aplasia of both maxillary sinuses are reported . A 68-year - old female was referred to the clinic of oral and maxillofacial radiology for cbct preparation, as pre - operative imaging for implant insertion in the maxilla . She looked healthy without any history of sinus disease such as headaches, nasal discharge, facial pain and voice abnormalities, hyposmia or anosmia, and purulent rhinorrhea . On physical examination, it was found that the right maxillary region was slightly depressed compared with the opposite side . She did not remember any history of infection or trauma in the right maxillary sinus . Cbct was performed by a gallileos set (sirona dental systems gmbh, bensheim, hessen, germany), and analyzed by sidexis - xg software with slices having an interval of 2 mm . Evaluation of coronal and axial views revealed that the maxillary sinus of the right side was completely absent in all of the cuts . Cbct: (a) coronal view shows absence of the right maxillary; (b) axial view shows aplasia of the right maxillary sinus due to the lack of any symptoms, no additional treatment was done and the patient was informed about the sinus condition for possible future symptoms . A 20-year - old female was admitted to the clinic of radiology for cbct of the maxilla as pre - operative diagnostic imaging for implant insertion . She was completely healthy without any history of headaches, nasal discharge, facial pain, voice abnormalities, hyposmia or anosmia, and purulent rhinorrhea . Also, she had no history of infection or trauma in the sinus areas . Cbct was performed and evaluation of coronal and axial views revealed that all the left sinus dimensions were less than those of right sinus, and its dimensions seemed to be only equal to half of those of right sinus [figure 2]. Cbct: (a) coronal view shows the smaller size of left maxillary sinus; (b) axial view shows the the smaller size of left maxillary sinus due to the lack of any symptoms, no additional treatment was done and the patient was informed about the sinus condition for possible future symptoms . A 12-year - old female was referred to the clinic of dentomaxillofacial radiology by her orthodontist for cbct of both mandible and maxilla . She suffered from chronic headaches, nasal discharges, and voice abnormality because of hypernasalism . On physical examination, cbct was performed and evaluation of coronal view revealed that the maxillary sinuses in both sides were absent in all slices, which was confirmed by the axial view as a bilateral aplasia of the maxillary sinuses [figure 3]. Cbct: (a) coronal view shows the bilateral absence of both maxillary sinuses; (b) axial view shows the bilateral absence of both maxillary sinuses the patient was informed about the sinus condition and was refered to an otolaryngologist for further assessment . A 68-year - old female was referred to the clinic of oral and maxillofacial radiology for cbct preparation, as pre - operative imaging for implant insertion in the maxilla . She looked healthy without any history of sinus disease such as headaches, nasal discharge, facial pain and voice abnormalities, hyposmia or anosmia, and purulent rhinorrhea . On physical examination, it was found that the right maxillary region was slightly depressed compared with the opposite side . She did not remember any history of infection or trauma in the right maxillary sinus . Cbct was performed by a gallileos set (sirona dental systems gmbh, bensheim, hessen, germany), and analyzed by sidexis - xg software with slices having an interval of 2 mm . Evaluation of coronal and axial views revealed that the maxillary sinus of the right side was completely absent in all of the cuts . Cbct: (a) coronal view shows absence of the right maxillary; (b) axial view shows aplasia of the right maxillary sinus due to the lack of any symptoms, no additional treatment was done and the patient was informed about the sinus condition for possible future symptoms . A 20-year - old female was admitted to the clinic of radiology for cbct of the maxilla as pre - operative diagnostic imaging for implant insertion . She was completely healthy without any history of headaches, nasal discharge, facial pain, voice abnormalities, hyposmia or anosmia, and purulent rhinorrhea . Also, she had no history of infection or trauma in the sinus areas . Cbct was performed and evaluation of coronal and axial views revealed that all the left sinus dimensions were less than those of right sinus, and its dimensions seemed to be only equal to half of those of right sinus [figure 2]. Cbct: (a) coronal view shows the smaller size of left maxillary sinus; (b) axial view shows the the smaller size of left maxillary sinus due to the lack of any symptoms, no additional treatment was done and the patient was informed about the sinus condition for possible future symptoms . A 12-year - old female was referred to the clinic of dentomaxillofacial radiology by her orthodontist for cbct of both mandible and maxilla . She suffered from chronic headaches, nasal discharges, and voice abnormality because of hypernasalism . On physical examination, the scar of previous surgery was seen in the philtrom region . Also, the maxillary sinus regions in both sides were depressed . Cbct was performed and evaluation of coronal view revealed that the maxillary sinuses in both sides were absent in all slices, which was confirmed by the axial view as a bilateral aplasia of the maxillary sinuses [figure 3]. Cbct: (a) coronal view shows the bilateral absence of both maxillary sinuses; (b) axial view shows the bilateral absence of both maxillary sinuses the patient was informed about the sinus condition and was refered to an otolaryngologist for further assessment . Hypoplasia of maxillary sinus is less likely than of sphenoid and frontal sinuses, which can be acquired or congenital . Some reasons have been mentioned as a cause of congenital hypoplasia or aplasia, such as: arresting of the development because of infection, injuries, and irradiation.congenital first arch syndrome.developmental anomalies such as craniosynostosis, osteodysplasia, and down syndrome . Also, some reasons are responsible for an acquired category of maxillary sinus hypoplasia, such as: trauma with deformity due to fracture or surgery in the sinus region.thalassemia and cretinism.wegener's gralauloma (inflammatory osteitis).neoplasms that cause osteitis . The sinus is fully developed with average dimensions of 34 33 25 mm . In case 3, the patient was 12 years old, which was under the age of complete development of sinus . However, by this age, the dimensions of normal sinus must be at least at the level of middle turbinate . She had a history of cleft lip and palate that was a result of disturbance in the first arch development and the congenital first arch syndrome can be a cause of aplasia of the sinus . Some of the putative roles that have been ascribed to the sinuses are as follows: air conditioning (heating and humidification), acting as an air reservoir, ventilation, aiding in olfaction, reduction in weight of the cranium, addition of resonance to the voice, insulation of the cerebrum and orbits, and participation in the formation of the cranium . Hypoplasia or aplasia of the maxillary sinus may cause symptoms such as headaches, facial pain, nasal discharge, and speaking voice problems . But the majority of patients are asymptomatic and unaware of their conditions . In this case series, two cases presented were asymptomatic, but case 3 had signs and symptoms of headaches, nasal discharge, and hypernasal speech . Maxillary sinus opacification on plain radiographs can be diagnosed as a mucosal thickening of infectious disease, tumor, or aplasia of the sinus . Cbct has the advantages of ct, in addition to requiring lower radiation dose, and is a good modality of diagnostic imaging in the evaluation of sinus conditions like aplasia or hypoplasia . Radiological diagnosis of maxillary sinus hypoplasia and aplasia helps the otolaryngologists to differentiate that from chronic sinusitis and neoplasm . It is important to diagnose these abnormalities to prevent possible complications during endoscopic sinus surgery, such as causing potential harm to the orbit.
Polycaprolactone (pcl)-1 dermal filler (ellans; aqtis medical, utrecht, the netherlands) is a soft tissue dermal filler based on pcl microspheres . The smooth and totally spherical - shaped pcl microspheres (2550 m) are homogenously suspended in a tailor - made aqueous carboxymethylcellulose (cmc) gel carrier . Pcl and cmc individually have an excellent and established biocompatibility profile and have been used successfully in numerous conformit europenne marked and us food and drug administration approved medical devices, such as dermal fillers, oral and maxillofacial surgery, wound dressing and controlled drug delivery [16]. Pcl is a totally bioresorbable, nontoxic medical polymer and is attractive for use in medical devices because of its controlled and safe bioresorption process [711]. With h - labeled pcl and c - labeled pcl implantation studies, it has been proved that pcl was completely excreted from the body [7, 8]. After treatment the cmc gel carrier is gradually resorbed by macrophages over a period of several weeks, during which the pcl microspheres will trigger a natural response of the human skin and stimulate a natural wound - healing process through neocollagenesis . The new collagen replaces the volume of the resorbed carrier . The pcl microspheres are totally smooth and spherical shaped, which has been shown to be optimal for dermal fillers [12, 13]. Busso and applebaum published a report of their experiences in mixing a calcium hydroxylapatite - based dermal filler with lidocaine for use of the soft tissue filler in treatment of the hand . The result of mixing the two components is that the treatment is less painful to the patient than the conventional hand injection, and is characterized by less swelling and bruising, with minimal posttreatment downtime . Increasing numbers of physicians are adopting and using this technique for mixing the pcl dermal filler with standard 2.0% lidocaine - hydrochloride (hcl) solutions, up to 0.19 ml of lidocaine with a 1.1 ml syringe of pcl dermal filler . Mixing 0.19 ml of 2.0% lidocaine solution with 1.1 ml of pcl dermal filler this concentration is equivalent to that found in other soft tissue fillers, such as restylane and juvederm [15, 16]. The authors suggest that adding up to 0.3% of the anesthetic agent lidocaine to the pcl - based filler will not substantially affect its characteristics, confirming the usability of this mixture in clinical practice . In this study the characterization of the physical properties of pcl dermal filler mixed with plain 2.0% lidocaine - hcl solutions and combined 2.0% lidocaine - hcl and epinephrine under various mixing condition is investigated . A range of lidocaine and lidocaine with epinephrine concentrations was mixed with the plc dermal filler to evaluate the changes in dynamic viscosity and elasticity, extrusion force, ph, and needle jam rates . Investigators also evaluated the mixtures at the front, middle, and back of each mixed syringe as a measure of mixing efficiency . The usage of the pcl dermal filler in this study is representative of the entire product line as all the relevant product characteristics are identical throughout the entire product range . The 2.0% lidocaine solution was composed of anhydrous lidocaine - hcl (20 mg / ml) (xylocaine 2.0%; astra zeneca bv, zoetermeer, the netherlands). The 2.0% lidocaine solution with epinephrine was composed of anhydrous lidocaine - hcl (20 mg / ml), epinephrine (5 g / ml) (xylocaine 2.0% with epinephrine; astra zeneca bv). A rheometer (haake rs-6000; thermo electron gmbh, karlsruhe, germany) was used to measure the dynamic viscosity and elasticity of the media . Rheology was evaluated with a titanium rotor, with a gap of 0.105 mm and tau () of 5 pa, over a frequency sweep of 0.110 hz evaluated at 0.6 hz . Extrusion force was measured by a material tester (model h1ks; tinius olsen, ltd ., extrusion force was evaluated through a 27g inch needle with an extension rate of 1 ml / min . Media ph was measured using a ph meter with a probe (ph340i, and sentix 41 probe, respectively; wtw, weilheim, germany). The ph was obtained by completely coating the glass bulb of the ph probe with media . The female - to - female luer lock connectors used to connect the mixing and media syringes were from baxa (rapid fill connector, ref . For the pcl-1 dermal filler, 1.1 ml syringes were mixed with one of four volumes of 2.0% lidocaine or 2.0% lidocaine with epinephrine solution: 0.05 ml (0.09% final lidocaine - hcl); 0.10 ml (0.17% final lidocaine - hcl); 0.14 ml (0.23% final lidocaine - hcl); 0.19 ml (0.30% final lidocaine - hcl). One milliliter mixing syringes (beckton dickinson, franklin lakes, new jersey, usa) were used to withdraw the lidocaine solution from the 20 ml vial via a 21g\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\raise0.5ex\hbox{$\scriptstyle 5 $} \kern-0.1em/\kern-0.15em \lower0.25ex\hbox{$\scriptstyle 8 $}} $$\end{document} inch needle . The push rod of the mixing syringe was drawn back to make sure all the lidocaine solution was cleared from the needle and afterwards depressed to remove all excess air . Next, the mixing syringe with lidocaine was firmly connected to a syringe of pcl dermal filler using a female - to - female luer lock connector (fig . 1).fig . 1polycaprolactone - based dermal filler mixed with lidocaine, using a female - to - female luer lock connector polycaprolactone - based dermal filler mixed with lidocaine, using a female - to - female luer lock connector lidocaine and pcl dermal filler were mixed by alternately depressing the plungers on the mixing and media syringes . Each mixing stroke was composed of one complete compression of the dermal filler syringe push rod, followed by one complete compression of the mixing syringe push rod . Push rods were compressed firmly and quickly, at approximately two compressions per second . Following mixing, the mixing syringe and luer lock connector were removed and discarded, and the lidocaine / dermal filler mixture was recapped with the original media syringe cap . The dermal filler - lidocaine blends were tested between 15 min and 120 min after mixing with lidocaine . In this study the characterization of the physical properties of pcl dermal filler mixed with plain 2.0% lidocaine - hcl solutions and combined 2.0% lidocaine - hcl and epinephrine under various mixing condition is investigated . A range of lidocaine and lidocaine with epinephrine concentrations was mixed with the plc dermal filler to evaluate the changes in dynamic viscosity and elasticity, extrusion force, ph, and needle jam rates . Investigators also evaluated the mixtures at the front, middle, and back of each mixed syringe as a measure of mixing efficiency . The usage of the pcl dermal filler in this study is representative of the entire product line as all the relevant product characteristics are identical throughout the entire product range . The 2.0% lidocaine solution was composed of anhydrous lidocaine - hcl (20 mg / ml) (xylocaine 2.0%; astra zeneca bv, zoetermeer, the netherlands). The 2.0% lidocaine solution with epinephrine was composed of anhydrous lidocaine - hcl (20 mg / ml), epinephrine (5 g / ml) (xylocaine 2.0% with epinephrine; astra zeneca bv). A rheometer (haake rs-6000; thermo electron gmbh, karlsruhe, germany) was used to measure the dynamic viscosity and elasticity of the media . Rheology was evaluated with a titanium rotor, with a gap of 0.105 mm and tau () of 5 pa, over a frequency sweep of 0.110 hz evaluated at 0.6 hz . Extrusion force was measured by a material tester (model h1ks; tinius olsen, ltd ., extrusion force was evaluated through a 27g inch needle with an extension rate of 1 ml / min . Media ph was measured using a ph meter with a probe (ph340i, and sentix 41 probe, respectively; wtw, weilheim, germany). The ph was obtained by completely coating the glass bulb of the ph probe with media . The female - to - female luer lock connectors used to connect the mixing and media syringes were from baxa (rapid fill connector, ref . For the pcl-1 dermal filler, 1.1 ml syringes were mixed with one of four volumes of 2.0% lidocaine or 2.0% lidocaine with epinephrine solution: 0.05 ml (0.09% final lidocaine - hcl); 0.10 ml (0.17% final lidocaine - hcl); 0.14 ml (0.23% final lidocaine - hcl); 0.19 ml (0.30% final lidocaine - hcl). One milliliter mixing syringes (beckton dickinson, franklin lakes, new jersey, usa) were used to withdraw the lidocaine solution from the 20 ml vial via a 21g\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\raise0.5ex\hbox{$\scriptstyle 5 $} \kern-0.1em/\kern-0.15em \lower0.25ex\hbox{$\scriptstyle 8 $}} $$\end{document} inch needle . The push rod of the mixing syringe was drawn back to make sure all the lidocaine solution was cleared from the needle and afterwards depressed to remove all excess air . Next, the mixing syringe with lidocaine was firmly connected to a syringe of pcl dermal filler using a female - to - female luer lock connector (fig . 1polycaprolactone - based dermal filler mixed with lidocaine, using a female - to - female luer lock connector polycaprolactone - based dermal filler mixed with lidocaine, using a female - to - female luer lock connector lidocaine and pcl dermal filler were mixed by alternately depressing the plungers on the mixing and media syringes . Each mixing stroke was composed of one complete compression of the dermal filler syringe push rod, followed by one complete compression of the mixing syringe push rod . Push rods were compressed firmly and quickly, at approximately two compressions per second . Following mixing, the mixing syringe and luer lock connector were removed and discarded, and the lidocaine / dermal filler mixture was recapped with the original media syringe cap . The dermal filler - lidocaine blends were tested between 15 min and 120 min after mixing with lidocaine . The extrusion force was used to determine the number of passes between syringes needed for sufficient blending of lidocaine with the pcl dermal filler . With adequate mixing of the components the difference in viscosity across all regions of the syringe is minimal and the extrusion force profile is uniform from the front to the back of the syringe . The difference in viscosity from the front to the back increased with increasing volume of lidocaine, suggesting that larger volumes of lidocaine required more mixing than small volumes . It also reflects a greater magnitude of change in physical properties with increasing concentration of lidocaine . Figure 2 shows the extrusion force of pcl dermal filler without lidocaine and zero mixing strokes, demonstrating a uniform profile from the front to the back.fig . 2extrusion force profile of polycaprolactone dermal filler without lidocaine, 0 mixing strokes extrusion force profile of polycaprolactone dermal filler without lidocaine, 0 mixing strokes five mixing passes did not provide adequate mixing for any volume tested . The extrusion force profile of pcl dermal filler mixed with 0.19 ml lidocaine with five mixing strokes can be seen in fig . 3, showing an increase in extrusion force from the front to the back of the syringe.fig . 3extrusion force profile of polycaprolactone dermal filler mixed with 0.19 ml lidocaine using a female - to - female luer lock connector, five mixing strokes extrusion force profile of polycaprolactone dermal filler mixed with 0.19 ml lidocaine using a female - to - female luer lock connector, five mixing strokes ten mixing passes provided adequate mixing for 0.05 ml of lidocaine, but not for the other volumes . The extrusion force profile of pcl dermal filler mixed with 0.19 ml lidocaine with 10 mixing strokes, shown in fig . 4, demonstrates a nonuniform extrusion profile reflecting the front - to - back inhomogeneity.fig . 4extrusion force profile of polycaprolactone dermal filler mixed with 0.19 ml lidocaine using a female - to - female luer lock connector, 10 mixing strokes extrusion force profile of polycaprolactone dermal filler mixed with 0.19 ml lidocaine using a female - to - female luer lock connector, 10 mixing strokes following 15 mixing strokes, the extrusion force was uniform from the front to the back of the syringe for all lidocaine volumes tested, even at the maximum tested volume of lidocaine . Figure 5 shows the uniform extrusion profile of pcl dermal filler mixed with 0.19 ml lidocaine with 15 mixing strokes.fig . 5extrusion force profile of polycaprolactone dermal filler mixed with 0.19 ml lidocaine using a female - to - female luer lock connector, 15 mixing strokes extrusion force profile of polycaprolactone dermal filler mixed with 0.19 ml lidocaine using a female - to - female luer lock connector, 15 mixing strokes the dynamic viscosity measures the way a fluid responds to stresses and strains . The dynamic viscosity of the pcl dermal filler decreased with increasing lidocaine concentration without or with epinephrine . Figure 6 shows the dynamic viscosity procentual difference of pcl dermal filler blended with 0.05 ml, 0.10 ml, 0.14 ml, and 0.19 ml 2.0% lidocaine solution mixed with 15 mixing strokes . No statistically significant viscosity differences were measured for lidocaine solutions with or without epinephrine.fig . 6dynamic viscosity procentual difference of polycaprolactone dermal filler mixed with various volumes of 2.0% lidocaine with and without epinephrine (15 mixing strokes, 0.6 hz) dynamic viscosity procentual difference of polycaprolactone dermal filler mixed with various volumes of 2.0% lidocaine with and without epinephrine (15 mixing strokes, 0.6 hz) even at 0.23 ml of lidocaine solution blended with the pcl dermal filler, the cmc gel viscosity / elasticity was sufficient enough to keep the pcl microspheres in suspension in time (not shown). The extrusion forces of the pcl dermal filler mixed with lidocaine were lower than those of the pcl dermal filler alone . The average extrusion force decreased with the increase in volume of lidocaine added to the 1.1 ml pcl dermal filler syringe . In fig . 7 the average extrusion forces of pcl dermal without lidocaine and pcl dermal filler blended with 0.05 ml, 0.10 ml, 0.14 ml, and 0.19 ml 2.0% lidocaine solution mixed with 15 mixing strokes are shown . The average extrusion force of pcl dermal filler without lidocaine through a 27g inch needle was 17.2 n and decreases to 13.0 n for pcl dermal filler mixed with 0.19 ml 2.0% lidocaine solution.fig . 7average extrusion force of polycaprolactone dermal filler mixed with various volumes of 2.0% lidocaine (15 mixing strokes) average extrusion force of polycaprolactone dermal filler mixed with various volumes of 2.0% lidocaine (15 mixing strokes) differences in average extrusion force for pcl dermal filler mixed with lidocaine with and without epinephrine was not statistically significant for all volumes, suggesting that epinephrine had no effect on the extrusion force . Needle jamming may occur if there is a cluster of pcl microspheres in the cmc gel carrier . No needle jams were observed in any of the extrusion tests, indicating optimal homogeneity and suspension of the pcl microspheres in the cmc gel carrier after mixing with lidocaine with or without epinephrine . The tan delta () values (the tangent of the ratio loss modulus [g] over the storage modulus [g]) provides a quantitative tool to evaluate the relative elasticity of the media . Figure 8 shows the elasticity percentage difference of pcl dermal filler blended with various volumes of 2.0% lidocaine with and without epinephrine mixed with 15 strokes . The pcl dermal filler blends were less elastic than pcl dermal filler without lidocaine solution . No statistically significant elasticity differences were measured for lidocaine solutions with or without epinephrine.fig . 8elasticity of pcl dermal filler mixed with various volumes of 2.0% lidocaine with and without epinephrine (15 mixing strokes) elasticity of pcl dermal filler mixed with various volumes of 2.0% lidocaine with and without epinephrine (15 mixing strokes) the ph was between 7.1 and 7.2 for all tested samples . The extrusion force was used to determine the number of passes between syringes needed for sufficient blending of lidocaine with the pcl dermal filler . With adequate mixing of the components the difference in viscosity across all regions of the syringe is minimal and the extrusion force profile is uniform from the front to the back of the syringe . The difference in viscosity from the front to the back increased with increasing volume of lidocaine, suggesting that larger volumes of lidocaine required more mixing than small volumes . It also reflects a greater magnitude of change in physical properties with increasing concentration of lidocaine . Figure 2 shows the extrusion force of pcl dermal filler without lidocaine and zero mixing strokes, demonstrating a uniform profile from the front to the back.fig . 2extrusion force profile of polycaprolactone dermal filler without lidocaine, 0 mixing strokes extrusion force profile of polycaprolactone dermal filler without lidocaine, 0 mixing strokes five mixing passes did not provide adequate mixing for any volume tested . The extrusion force profile of pcl dermal filler mixed with 0.19 ml lidocaine with five mixing strokes can be seen in fig . 3, showing an increase in extrusion force from the front to the back of the syringe.fig . 3extrusion force profile of polycaprolactone dermal filler mixed with 0.19 ml lidocaine using a female - to - female luer lock connector, five mixing strokes extrusion force profile of polycaprolactone dermal filler mixed with 0.19 ml lidocaine using a female - to - female luer lock connector, five mixing strokes ten mixing passes provided adequate mixing for 0.05 ml of lidocaine, but not for the other volumes . The extrusion force profile of pcl dermal filler mixed with 0.19 ml lidocaine with 10 mixing strokes, shown in fig . 4, demonstrates a nonuniform extrusion profile reflecting the front - to - back inhomogeneity.fig . 4extrusion force profile of polycaprolactone dermal filler mixed with 0.19 ml lidocaine using a female - to - female luer lock connector, 10 mixing strokes extrusion force profile of polycaprolactone dermal filler mixed with 0.19 ml lidocaine using a female - to - female luer lock connector, 10 mixing strokes following 15 mixing strokes, the extrusion force was uniform from the front to the back of the syringe for all lidocaine volumes tested, even at the maximum tested volume of lidocaine . Figure 5 shows the uniform extrusion profile of pcl dermal filler mixed with 0.19 ml lidocaine with 15 mixing strokes.fig . 5extrusion force profile of polycaprolactone dermal filler mixed with 0.19 ml lidocaine using a female - to - female luer lock connector, 15 mixing strokes extrusion force profile of polycaprolactone dermal filler mixed with 0.19 ml lidocaine using a female - to - female luer lock connector, 15 mixing strokes the dynamic viscosity of the pcl dermal filler decreased with increasing lidocaine concentration without or with epinephrine . Figure 6 shows the dynamic viscosity procentual difference of pcl dermal filler blended with 0.05 ml, 0.10 ml, 0.14 ml, and 0.19 ml 2.0% lidocaine solution mixed with 15 mixing strokes . No statistically significant viscosity differences were measured for lidocaine solutions with or without epinephrine.fig . 6dynamic viscosity procentual difference of polycaprolactone dermal filler mixed with various volumes of 2.0% lidocaine with and without epinephrine (15 mixing strokes, 0.6 hz) dynamic viscosity procentual difference of polycaprolactone dermal filler mixed with various volumes of 2.0% lidocaine with and without epinephrine (15 mixing strokes, 0.6 hz) even at 0.23 ml of lidocaine solution blended with the pcl dermal filler, the cmc gel viscosity / elasticity was sufficient enough to keep the pcl microspheres in suspension in time (not shown). The extrusion forces of the pcl dermal filler mixed with lidocaine were lower than those of the pcl dermal filler alone . The average extrusion force decreased with the increase in volume of lidocaine added to the 1.1 ml pcl dermal filler syringe . In fig . 7 the average extrusion forces of pcl dermal without lidocaine and pcl dermal filler blended with 0.05 ml, 0.10 ml, 0.14 ml, and 0.19 ml 2.0% lidocaine solution mixed with 15 mixing strokes are shown . The average extrusion force of pcl dermal filler without lidocaine through a 27g inch needle was 17.2 n and decreases to 13.0 n for pcl dermal filler mixed with 0.19 ml 2.0% lidocaine solution.fig . 7average extrusion force of polycaprolactone dermal filler mixed with various volumes of 2.0% lidocaine (15 mixing strokes) average extrusion force of polycaprolactone dermal filler mixed with various volumes of 2.0% lidocaine (15 mixing strokes) differences in average extrusion force for pcl dermal filler mixed with lidocaine with and without epinephrine was not statistically significant for all volumes, suggesting that epinephrine had no effect on the extrusion force . Needle jamming may occur if there is a cluster of pcl microspheres in the cmc gel carrier . No needle jams were observed in any of the extrusion tests, indicating optimal homogeneity and suspension of the pcl microspheres in the cmc gel carrier after mixing with lidocaine with or without epinephrine . The tan delta () values (the tangent of the ratio loss modulus [g] over the storage modulus [g]) provides a quantitative tool to evaluate the relative elasticity of the media . The elasticity of pcl dermal filler decreased with increasing concentrations of lidocaine solution . Figure 8 shows the elasticity percentage difference of pcl dermal filler blended with various volumes of 2.0% lidocaine with and without epinephrine mixed with 15 strokes . The pcl dermal filler blends were less elastic than pcl dermal filler without lidocaine solution . No statistically significant elasticity differences were measured for lidocaine solutions with or without epinephrine.fig . 8elasticity of pcl dermal filler mixed with various volumes of 2.0% lidocaine with and without epinephrine (15 mixing strokes) elasticity of pcl dermal filler mixed with various volumes of 2.0% lidocaine with and without epinephrine (15 mixing strokes) the ph was between 7.1 and 7.2 for all tested samples . Hand mixing lidocaine or lidocaine with epinephrine with the pcl dermal filler causes no significant changes to the physical properties of the original formulation . With 15 back - and - forth passes the anesthetic agent(s) can adequately be mixed into the gel resulting in a homogenous blend . The viscosity / elasticity of the gel in the pcl dermal filler mixed with 2.0% lidocaine with or without epinephrine is sufficient to keep the pcl microspheres in suspension even after 24 h. there were no needle jams, indicating that the pcl microspheres were homogenously suspended in the gel, even after mixing the dermal filler with the anesthetic agent . The ph values of the pcl dermal filler mixed with lidocaine with or without epinephrine are equivalent to those of the original dermal filler . The viscosity, elasticity, and the extrusion force of the dermal filler decrease with increasing lidocaine content . There was no statistically significant change in physical properties for lidocaine solutions with or without epinephrine . The changes in physical properties are identical for the entire product range . Mixing a lidocaine solution with the dermal filler obviates the need for nerve blocks or local infiltration, thereby reducing the treatment times and prevents tissue distortion that may be caused by injecting local anesthetics . In addition, previous studies have shown that the addition of lidocaine to collagen - based dermal fillers resulted in less swelling and bruising, potentially due to the antihistaminergic effect of lidocaine on mast cells . Similar findings have been reported for hyaluronic acid - based dermal fillers mixed with lidocaine, also showing a reduction in swelling, erythema, and bruising [16, 1921]. As lidocaine is suggested to decrease these side effects, this is also expected for the pcl - based dermal filler mixed with lidocaine . The limitations of this study are that it does not investigate the influence of lidocaine and epinephrine on the clinical safety and performance of the pcl dermal filler, and the influence of the clinical anesthetic effect of lidocaine after mixing with the pcl dermal filler . This study does not address a potential interaction of lidocaine or epinephrine with the components of the pcl dermal filler and its potential influence on anesthetic efficacy . It is expected that, because of its hydrophilicity, lidocaine will be situated in the aqueous (hydrophilic) cmc gel carrier . Predicted on the hydrophobicity of the nonporous pcl microspheres no affinity is expected between lidocaine and pcl microspheres, and there will be no driving force of the lidocaine to migrate into the pcl microspheres . This is supported by a study describing the release profile of lidocaine from pcl threads . The lidocaine release profile showed a rapid release in the first hours and completed in a few days, indicating that there is no affinity or reaction between the pcl matrix and lidocaine . It is not expected that lidocaine will bind or react with the cmc carrier, but is free to move and yield the desired anesthetic effect . Cmc is a known time - release agent for lidocaine, and there are several commercial medical products available based on cmc gel and lidocaine as an anesthetic agent . This study does not address the effect of the premixed anesthetic on the clinical efficacy of the pcl dermal filler . Physicians have reported no decrease in clinical efficacy after mixing the pcl dermal filler with lidocaine with or without epinephrine, as has also been reported for hyaluronic acid - based dermal fillers containing lidocaine (for review, see smith and cockerham). Planned (pre)clinical studies with lidocaine premixed before treatment and lidocaine incorporated in the pcl dermal filler syringe itself are aimed at confirming this . The advantages of mixing lidocaine with pcl dermal filler before treatment are lower viscosity and elasticity, lower extrusion force, providing a greater ease of molding, increased patient comfort, reduced need for nerve blocks and infiltration anesthesia, which may be attractive for both physicians and patients . Dr . De melo is an advisor of aqtis medical, and has received consultancy and speaking fees from the company . This article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
Klotho is expressed mainly in the kidneys, parathyroid glands, brain choroid plexus, and testes (2 - 4). Studies have confirmed klotho expression in other tissues, including the aorta, colon, thyroid gland, and pancreas, but the kidney remains the strongest klotho - producing organ (5). The latter functions as a co - receptor for fibroblast growth factor-23 (fgf23). The membrane - bound form, after losing its membrane domain, enters into the circulation as soluble klotho (skl), acting as a hormone with anti - aging and anti - oxidative stress properties; skl can also be directly generated by alterative splicing of the klotho transcript (2, 5). Klotho deficiency is an early biomarker for chronic kidney disease, and its upregulation could protect the kidney from fibrosis progression (6). The beneficial effect of physical activity in preventing premature mortality has been established by epidemiological studies showing that exercise may delay aging through various mechanisms . Klotho upregulates nitrous oxide (no) production and inhibits angiotensin ii - induced reactive oxygen species production within endothelial cells (7). In an epidemiological study, handgrip strength, an indicator of total body muscle strength, the purpose of this study was to determine whether plasma klotho levels are inuenced by aerobic exercise . For this purpose, plasma klotho levels were measured in a group of trained athletes . In this study, 30 healthy football players (males aged 18 22 years) participated . The controls were 28 healthy young males (age range 18 27 years). All subjects were nonsmokers and free of cardiovascular disease, as indicated by their medical history . None of the subjects took cardiovascular medications or hormone replacement therapy, and they all maintained routine eating habits . In the experimental group, klotho concentration was measured the morning after a session of afternoon training, with blood samples collected from the antecubital vein . All participants had abstained from caffeine and fasted for at least 8 h before sampling . We did not measure the level of physical activity in the control group; they were healthy young males with normal daily physical activity, but none were trained athletes . Plasma klotho concentrations were measured with the elisa technique, using a soluble klotho elisa assay kit based on the manufacturer s instructions (human klotho elisa kit; hangzhou eastbiopharm co., ltd ., hangzhou, china). The demographic characteristics and measurements of the athlete group and the controls, respectively, were as follows: age, 18 22 versus 18 27 years; body mass index, 22.3 1.4 versus 24.9 1.3 kg / mg; total cholesterol, 5.3 0.4 versus 5.7 0.3 mmol / l; triglycerides, 1.5 0.1 versus 1.7 0.15 mmol / l; serum calcium, 9.8 0.8 versus 9.7 0.6; serum phosphorus, 4.4 0.3 versus 4.93 0.34; systolic blood pressure, 117 5 versus 119 6 mmhg; diastolic blood pressure, 70 4 versus 71 3 mmhg; and plasma klotho, ng / ml 3.375 1.48 ng / ml versus 1.39 0.43 ng / ml (p <0.05). We found no significant differences between the groups for total cholesterol, triglycerides, and blood pressure . The control subjects were within close range of the previously proposed klotho concentrations for normal individuals, while the athlete group had significantly higher plasma klotho concentrations . The results of this study showed that aerobic exercise training induces an increase in plasma klotho levels . Plasma klotho levels were only measured one time, the day after exercise in the athlete group; therefore, it is not known whether this elevation continues over time . Our study population and the controls were healthy young adult males, and their serum calcium and phosphate levels were within the normal range . In the study group, we collected the blood samples the morning after the last evening exercise, so we cannot rule out the acute effect of exercise on plasma klotho levels . It has been shown that aerobic exercise training induces increased plasma klotho concentrations and decreased arterial stiffness in postmenopausal women (9). Exercise training might increase circulating klotho due to increases in peroxisome proliferator - activated receptors (ppar) and decreases in angiotensin ii type i receptor (at1r) signaling (10). Aerobic exercise - induced increases in plasma klotho concentrations could be responsible for exercise - induced decreases in arterial stiffness (11), enhancing vascular protection and ameliorating endothelin - induced arterial stiffness . Secreted klotho protects endothelial cells and smooth muscle cells through no production (12) and suppression of oxidative stress (13 - 15). No production regulates endothelial cell calcium inux (9). Transforming growth factor beta-1 (tgf-1) and endothelin-1 (et-1) receptor activation negatively affect arterial stiffness, and their levels are decreased by exercise training (16). Interestingly, in a cross - sectional study, low plasma klotho concentrations were independently associated with disability among the elderly (17). Exercise - induced increment of serum klotho could be due to increased klotho secretion or increased splicing of membrane - bound klotho (9). The kidney is the major source of skl production (18), and membrane - bound klotho is also a co - activator of fgf23, which is prominently expressed in distal convoluted tubule (dct) and proximal convoluted tubule (pct) cells; these locations are essential for its function as a phosphaturic substance (11). Klotho deficiency is an early biomarker for chronic kidney disease (ckd), and a progressive decline in urine klotho occurs with ckd progression (6, 11, 19). Endogenous klotho may inuence the processes of inammation, oxidative stress, and vascular calcication and remodeling (20). Secreted klotho directly blocks phosphate - induced dedifferentiation of vascular smooth muscle cells into osteoblast - like cells . Secreted klotho also prevents the transformation of endothelial cells to osteoblast - like cells (21, 22). Angiotensin ii downregulates renal klotho protein expression (23), and at1r blockade increases circulating klotho . Further studies are needed in order to clarify the dynamics of klotho production and secretion, and to understand the mechanisms of exercise - induced klotho secretion or shedding.
Growing recognition that resources for health care are scarce has led to broad acceptance that the evidence base should include economic as well as clinical evidence . In the uk, this is reflected in the work of the national institute for health and clinical excellence (nice) whose national guidance on health care for england and wales is explicitly informed by evidence of cost effectiveness as well as clinical effectiveness . The principal benefits from most clinical treatments are in the form of health gains to patients . While there can also be nonhealth benefits, for example, from earlier return to work or in terms of reduced burden on informal carers, these tend to be relatively small . Since an objective of all health care systems is to maximise the amount of health produced (allowing for other objectives such as equity), it is not surprising that the economic questions most commonly addressed when evaluating clinical treatments are concerned with identifying the most cost - effective ways of producing health . There is now a growing movement toward incorporating the principles of evidence - based medicine in evidence - based public health . At the end of 2005, nice announced that it was extending its remit to include guidance on the promotion of good health and the prevention of ill health thus explicitly recognising the contribution of public health in improving health . Of these, 1235 (73%) were classed as cost effectiveness (cea) or cost utility (cua) studies which assess how 68 cost effectively a public health programme produces health . Only 43 studies (2.5%) were classed as cost benefit analyses (cba)the technique of economic evaluation which addresses the broader issue of whether or not a public health programme is worthwhile . This paper considers the fundamental differences between cea / cua and cba in the context of providing an evidence base to inform public health policy . Using selected studies from the database produced by the team that undertook the review, this paper examines how information provided by cea / cua studies might lead to inefficient recommendations for public health policy . It does this by briefly reviewing the principles of the different techniques of economic evaluation, considers the fundamental differences between public health and clinical treatments in terms of their objectives, and then, on the basis of information from a selection of studies, suggests a simple method for assessing where cea / cua of public health programmes might make a misleading contribution to the economic evidence base for public health . Within health care, a cost effectiveness analysis will assess an intervention against a comparator in terms of cost per unit of health effect achieved . These units can be specific, for example, true positive cases of presymptomatic disease detected in a screening programme, or more generic, for example, life years saved . In cost utility analysis, the unit of effectiveness is the quality adjusted life year (qaly) or other single index measures which capture both life length of life and quality of life . The technique's name refers to the fact that the quality of life element is determined by the utilities, or values, attached to different health states . Thus while cua outcomes take account of preferences they can still be regarded as a form of cea since they seek to find the least cost way of producing a health - related unit of effect . Nice determined early on that, where possible, cua was the preferred form of analysis to provide the economic evidence for health care interventions . Cea / cua, however, only compare alternatives ways of pursuing a single objective in this case to maximise health gain . An unambiguous result, however, is produced only if the intervention in question is both more effective and less costly than the comparator (or vice versa). Dominant and there are no economic arguments for not adopting it over the comparator . Whether or not the higher costs are worth incurring, that is, whether additional resources should be allocated to the treatment of these patients is an allocative efficiency question which cannot be answered by cea / cua . In order to assist decision makers, nondominant results are commonly presented in the form of incremental cost effectiveness ratios (icers) which show the extra cost of achieving the extra health effects . Cost effectiveness acceptability curves (ceacs) deal with the uncertainty surrounding the estimates by showing the probability that a nondominant intervention will have an icer below a range of thresholds which represent the maximum amounts that a payer would be willing to pay for an extra unit of effect . For example, nice currently regards an icer of 30 000 per qaly as being at the upper limit for the interventions to be recommended for use in the british national health service . While the question of how much society is willing to pay for extra health benefits remains a live issue, the focus on assessing the cost effectiveness of clinical treatments through cea / cua remains the norm . Results of these analyses clearly depend on which costs, including cost savings, are included, that is, on the perspective adopted . In the uk costs (positive and negative) to other sectors are not considered at least in the primary analysis . This remains a contentious issue as exemplified in a recent nice appraisal of drugs for alzheimer's disease where a major issue was whether or not to include cost borne by informal carers and a recent editorial in the british medical journal has called for a rethink on continued use of a narrow perspective even for health service interventions . The uk faculty of public health has adopted sir donald acheson's definition of public health as the science and art of preventing disease, prolonging life and promoting health through organised efforts of society . The faculty regards the key elements of public health as being population based, emphasising collective responsibility for health, recognising the key role of the state, and emphasising partnerships with all those who contribute to the health of the population . These principles make it clear that public health programmes can produce health in ways which do not necessarily involve health professionals or involve the use of health services . While these principles are captured in most definitions of public health, the definition by allin et al . Ends with and involves mobilising local, regional, national and international resources to create conditions in which people can be healthy in contrast to producing health by treating illness, creating the conditions in which people can be healthy will often achieve important nonhealth benefits in addition to health . Evidence that such nonhealth benefits are positively valued was demonstrated in a study by cropper which examined people's preferences for different life saving programmes . When asked to choose between programmes, a belief that a programme would produce benefits in addition to life saving was shown to significantly increase the probability of that programme being preferred over another with the same life saving benefits . Cost benefit analysis (cba) is the technique of economic evaluation which addresses allocative efficiency . It explicitly addresses the question how much more or how much less of society's resources should be allocated to achieving this goal or to this type of healthcare? . Its foundations are rooted within welfare economics where the aim is to assess how social welfare is affected by a particular project . Cba does this by identifying and measuring all costs and all benefits; defined as everything of value that results (positive or negative) and regardless of who gains or loses, that is, using a societal perspective . When all gains and losses are measured in commensurate terms (i.e., money) healthcare objectives can be compared with each other or with those in other sectors of the economy . If the total value of the benefits (gains) exceeds the total value of the costs (losses), then the proposal passes the cost benefit test and total social welfare is increased by implementing the programme . Although in principle cea / cua are capable of capturing avoided costs in sectors other than health care, these analyses would still not capture the full range of benefits that would be picked up in a cba . For example, a cua of a proposed policy to reduce air pollution would be based on the narrow premise that the objective of the policy is solely to produce health . If the analysis showed an incremental cost / qaly above a predetermined threshold, even if that analysis included cost savings to sectors other than health such as reduced cleaning costs due to reduced pollution, the implication would be that resources should not be allocated to this intervention . A cba, however, which included nonhealth as well as health benefits say the value attached to breathing clean air independent of any health implications might show the same proposal to pass the cost benefit test . In this example, a policy decision taken on the basis of the cua would mean forgoing an opportunity to improve social welfare . It is, of course, possible that nonhealth benefits could be negative which reinforces the importance of not excluding them, particularly where they are significant . Although economists have developed numerous methods to assign money values to costs and benefits which do not have associated market prices, this inevitably is not an easy task which might explain why comprehensive cbas remain rare . It is well recognised that many studies which include the word cost benefit analysis in their title are, in reality, not cba studies at all . Such mistitled studies frequently use that term because they regard cost savings from avoided future illness as benefits and thus feel that their analysis has covered both sides of the cost benefit calculus . Some economic studies avoid valuation problems by simply listing the costs and consequences of any activity without aggregation . Such cost - consequences analyses (ccas) are, strictly, not economic evaluations as they cannot provide answers to either cost effectiveness or allocative efficiency questions . However, by identifying costs and consequence they can be an important aid to decision making beyond cost effectiveness ratios and a recent report from the public health research consortium in the uk has called for the intersectoral impacts of public health interventions to be presented in the form of a cost - consequences analysis . In terms of an emerging public health evidence base, there thus appears to be a problem . If the benefits from public health interventions frequently include more than just health gains then, arguably, its evidence base ought to include a smaller proportion of cost effectiveness and cost utility analyses than does the evidence base for clinical treatments . Nevertheless, as shown in the review, nearly three quarters of the economic evaluation undertaken in the area of public health have to date been cost effectiveness or cost utility studies . In order to illustrate the partial nature of the information provided from cea / cua evaluations in public health, hence the potential for inefficient health policy, the present study examined the nature and relative importance of benefits which were included in some of the economic studies which went beyond cost effectiveness or cost utility analyses in the recent review . Studies from the review were identified for possible selection if they were not classed as cost effectiveness or cost utility studies . It was evident from examination of abstracts, however, that despite their classification many of these studies had limited their analyses to health benefits alone . Omission of nonhealth benefits, even where they might be significant, is not necessarily a weakness in these studies . If the value of the health benefits alone can be shown to exceed the value of all of the costs, then the intervention passes the cost benefit test without the need to consider any nonhealth benefits . Including them would only reinforce the conclusion already reached although clearly, incomplete assessment would make comparisons of the benefit: cost ratios between programmes problematic . Of these two had been classed within the review as cba; the remainder being cca or multimethod apart from one case where no classification was given . The work by aunan et al . Evaluated the costs and benefits of implementing to reduce air pollution programme . Reduced damage to public health, building materials, and agricultural crops from reduced emissions of air pollutants the possible benefits from implementing the measures described by the national energy efficiency improvement and energy conservation program were evaluated using saved energy from various sectors: households, transportation, industry, service, energy, and agriculture . Health benefits included those from acute respiratory symptoms, chronic respiratory symptoms, infant deaths, and lung cancer . Crop loss due to so2 was seen to be a great concern for crop production . The analysis indicated that the annual benefit of improved health alone is likely to exceed the investment needed to implement the programme . Thus, the policy would pass the cost benefit even without inclusion of the significant benefits due to reduced damage to materials and crops . The work by miller et al . Modelled the potential health and economic impacts of implementing a medically prescribed heroin programme among canadian injecting drug users over 5 years . The potential impact of the programme was estimated by comparing hospitalisation and emergency use costs . Reductions in criminal activity costs accounted for fully 63% of the total reduction in costs . Other costs avoided since the implementation of the programme such as the costs of social housing, use of social services, counseling, and employment programs were identified but not included in the model . Although this study did not claim to be a cba it provides an example of an intervention whose nonhealth benefits were significantly larger than the health benefits . Had the researchers attempted a cba, the nonhealth benefits could have made the difference between the programme passing or failing the cost benefit test . The work by zeng - sui et al . Assessed the impact on enteric infectious disease by providing deep - well tap water across six villages in china . Health benefits included reductions in diarrhoea, dysentery, viral hepatitis, cholera, and reduce mortality form liver cell cancer . Nonhealth benefits included reductions in lost wages or earnings of patients and of their relatives who looked after them during the illness and the avoided costs of transportation, supplemental nutrition, and the value of gifts sent by relatives to assist towards their recuperation (but interestingly not the value of the gift to the recipient which illustrates how the gifts should have been regarded as a financial transfer rather than an economic cost avoided). For example, water - related conditions such as skin and eye infections, dermatosis, gynaecological conditions, parasitic enteric diseases, and vector - borne diseases were mentioned but not included . Other intangible benefits such as the improved service that will benefit future generations were also mentioned but not included in the analysis . Overall assessed benefits were more than double the costs . The work by guria et al . Evaluated the incremental outcomes of road safety programmes and driving campaigns enforced in new zealand and compared them with their resource costs . In addition to loss of life and reduced quality of life resulting from injury, the study also included the social costs of injuries and property damage avoided . Other benefits such as the development of a safety culture, improvement of road user behaviour, and the safety quality of vehicles were mentioned but not included . The study showed that road safety programmes aimed at reducing high - risk behaviours produced high returns . If 90% of road safety expenditure is attributed to the period of investment, then the benefit to cost ratio would be 12.3: 1 for 19931995 and 7.9: 1 for 19941996 . The work by aehyung et al . Presented a cba of the onchocerciasis (river blindness) control programme . Nonhealth benefits included additional agricultural output as a result of a more productive labour force and additional agricultural land made available through the control of onchocerciasis . Other nonhealth benefits such as the reduction of lost production time by family members when providing care and improved parenting were mentioned but not included in the study . The net present value (npv) ranged from us$485 million to us$3,792 million (1987 dollars) depending on the assumptions used . A positive npv is another way of saying that the programme passed the cost benefit test . The work by fleming et al . Estimated the costs and benefits of brief physician advice with problem drinkers in primary care settings . Health care benefits included avoided cost from the perspective of the managed care organisation, the use of equipment, personnel, emergency medical care, hospitalisations, treatments, and clinic visits . The study indicated that physician - delivered advice can reduce not only medical costs but also social costs associated with alcohol consumption . The total economic cost of the intervention was $80,210, or $205 (1993 dollars) per study patient . The study by cohen et al . Estimated the potential benefits from saving a high - risk youth by estimating the lifetime costs associated with the career criminal drug abuser and high school drop out . Antisocial behaviour of career criminals was included as an externality and thus seen as imposing as a social cost . Assessed nonhealth benefits included the avoided social costs from stolen property and lost wages . As this study did not examine the costs of interventions aimed at reducing antisocial behaviour, it was not a cba . However, the range and magnitude of the nonhealth benefits which were included in the valuation exercise illustrates what would be missed if a cea / cua study focussing solely on the health benefits had been undertaken . The work by caulkins et al . On school - based drug prevention programmes focused on reducing drug consumption, particularly cocaine, as an objective of the nation's drug control efforts . The study reported the quantity of cocaine consumed, the cost of drug use, and the social value of cocaine control . Benefits from the prevention program included reductions in the use of other drugs including marijuana, alcohol, and cigarettes . Nonhealth benefits included lower crime rates, higher productivity, and an increase in the number of pupils graduating from high school graduation . The programme was deemed to be affordable and social benefits were shown to exceed the total costs which justified implementation of the programme . The work ginsberg et al . Estimated the costs of making the wearing of bicycle helmets compulsory in israel . Benefits included resource saving from fewer head injuries in terms of hospitalisation, emergency room visits, ambulatory care, rehabilitation, long - term care, and special education . The authors called their analysis conservative as it did not consider reduced pain, worry, grief, work losses for ambulatory visits or even time off from housework as a result of bicycle injuries . Nor did we consider the intangible benefits of the lessening of anxiety concerning crashes by cyclists or by their friends and relatives . Inclusion of these additional benefits was unnecessary as the partial benefits (total = us$60.7 million) clearly exceeded total costs (us$20.1 million) without their inclusion (dollar base year not stated). In this example, the health service savings alone (us$44.2 million) were sufficient for the proposed policy to pass the cost benefit test . If, however, the results showed that the productivity gains (us$7.5 million) were needed to tip the balance, then their omission would have meant a lost opportunity to increase social welfare . Benefits included savings from hospital admissions, professional services, rehabilitation, prescriptions, home health care, and medical equipment . The study also included benefits from avoided productivity losses due to children not being able to work when they became adults if they were killed or permanently disabled and included a value for avoided pain, grief, and suffering . For example, it mentioned but did not include productivity losses from parents dealing with injured children . Nevertheless, it still showed a benefit to cost ratio of nearly 13: 1 . In this case, however, annual productivity benefits ($660 million) were nearly 3 times those of health service benefits ($230 million) (1992 dollars) which in a higher - cost programme might have made the difference between passing or failing the cost benefit test . It is evident that many public health interventions produce benefits in addition to health and in many cases these can be substantial . Some of these involve resource savings which, if regarded as negative costs could in principle be included in a cea / cue provided that a societal perspective were adopted . Others, however, for example, reductions in criminal activity, are clearly of value independent of any cost savings and these would not feature in a cost effectiveness or cost utility study . The extent to which the noninclusion of nonhealth benefits in evaluation by cea / cua represents a problem in the sense that it could potentially lead to foregone opportunities to increase social welfare might be predicted by considering where the intervention in question would sit along a continuum of intent . At one extreme of such a continuum would be public health measures whose intent was solely to produce health . For example, a policy to add folic acid to flour has been advocated with the specific intent of reducing the incidence of neural tube defects (ntd) in newborns . Where health gains are the sole objective of a public health programme then, on the same principles used within health care, they can be assessed in terms of cost effectiveness . If the addition of folic acid to flour were shown to have a low incremental cost / qaly, then this public health measure would be a cost - effective way of producing health relative to interventions within health care . Even in this example, however, there could still be a case for directly addressing allocative efficiency through cba, for example, if women of childbearing age receive immediate reassurance from the knowledge that eating fortified food reduces their risk of conceiving a baby with an ntd . Equally, making consumption of folic acid compulsory removes freedom of choice which to many could be a highly negatively valued nonhealth outcome . Moreover, although there are no rules within the methodology of economic evaluation to prevent a cua being undertaken from a broad perspectiveand there have been recent calls to do just that such evaluations remain uncommon . In the folic acid example, the cost / qaly derived from a study which adopted a health service perspective would not include savings to other agencies such as special education which in the case of children with ntd could be substantial . Further along the continuum of intent would be public health interventions where health is the primary concern but other objectives will clearly also be achieved . Thus, while a road safety intervention may be advocated primarily to reduce injury and death on the roads, it is evident that a reduction in accidents will also produce savings in terms of property damage . The study by guria et al . On road safety in new zealand included property damage in deriving its cost benefit ratios . Further still along the continuum would be public health interventions which clearly address multiple objectives . For example, illegal drug use is known to cause many social problems as well as health problems . Thus the study by caulkins et al . Included reduced crime and increased productivity among the benefits of schools - based drug prevention programmes, even taking account of intangible benefits such as an increase in the proportion of pupils graduating from high school . Another example is the onchocerciasis prevention programme examined by aehyung et al . Which, although driven by a desire to reduce the incidence of river blindness, would also free previously oncho - ridden tracts of land for settlement and cultivation . In both these examples, cuas would have given misleading information for policy . Further still along the continuum would be programmes which could be perceived as being only incidentally preventive in the sense that the effect of pursuing another policy objective would incidentally have a positive effect on health . An example here could be improvements in housing which are undertaken to provide people with more pleasant places to live but which can at the same time affect respiratory illnesses or reduce injuries . The health effects of such programme can be assessed via a health impact assessment . Ultimately almost any public policy can be seen as containing an element of public health . For example, macroeconomic policies to stimulate economic growth are clearly driven by concerns other than health, yet economic growth reduces unemployment and the relationship between unemployment and ill health is long established . Public health programmes can have nonhealth benefits which may not be captured when a cost effectiveness / cost utility approach to economic evaluation is undertaken . A preanalysis examination of where any public health intervention would be located on a continuum of intent (relative importance of health versus nonhealth benefits) could identify where evaluation by cost effectiveness or cost utility analysis might produce inappropriate conclusions for policy . Many of the public health programmes which to date have been assessed by cea / cua, in particular, those addressing smoking cessation have shown incremental cost effectiveness ratios which are far below current thresholds . In such cases, equally, where a cba is undertaken and the value of the health benefits alone is anticipated to clearly outweigh the costs, addition of nonhealth benefits will not affect the decision on whether to implement the programme and hence their inclusion is again unnecessary . Most public health programmes, however, are unlikely to allow such obvious a priori conclusions to be drawn and it is here that consideration of where the programme sits along the suggested continuum of intent will increase the likelihood that the most appropriate technique of economic evaluation will be used.
Cherubism is an inherited, fibro - osseous condition characterized by firm, painless swelling of the jaws, and was first described by jones in 1933 . Clinically cherubism is characterized by fullness of the cheeks and jaw bones that results in a round face with retraction of the lower eyelids and exposure of the sclera below the irises; the heaven look produced is suggestive of a cherub and gave rise to the name of this condition . The radiographical appearance is characterized by bilateral, multilocular radiolucent lesions, which often begin near the angle of the mandible and spread to the ramus and body of the mandible . The presence of eosinophilic, collagenous material around small capillaries in histopatholgical sections is of value in the diagnosis of cherubism . A 25-year - old male patient of non - consanguineous parents reported to our department with a complaint of bilateral painless swelling of both jaws . Later he noticed progressive increase in the swelling in his jaws which continued till it attained the present size . The size of the swelling had been stable for the past year and had not regressed or increased . On extra - oral examination, the swellings appeared ovoid in shape, with well - defined borders . The swelling was more prominent in the parasymphysis and body of the mandible and molar regions . Eyes were upturned with sclera visible below the iris giving an eye raised to heaven appearance . There was a difference in the ocular levels, right eye being at a slightly higher level [figure 1]. Obliteration of buccal sulcus of mandible was noticed in the molar regions . In the maxilla multiple swellings were seen, which were hard, sessile, non - tender, and covered with pink mucosa . Extra - orally the swellings appeared ovoid in shape, with well - defined borders . The orthopantamograph revealed multiple impacted teeth in the mandible, with multiloculated osteolytic lesions involving the entire body as well as the ramus of the mandible sparing the condyles [figure 2]. A 3-dimensional computed tomography showed multiloculated cystic lesions affecting the body and rami of the mandible and also the maxilla . The lesion caused marked expansion of the bones, with a multifocal cortical breakthrough [figure 3]. The orthopantamograph revealed multiple impacted teeth in the mandilble (red arrows) with multiloculated osteolytic lesions involving mandible and maxilla (blue arrows). 3-dimensional computed tomography scan showing multiloculated cystic lesions affecting the body and rami of the mandible (blue arrows) and maxilla with raised orbital floor (red arrows). Biopsy to obtain specimen from both the jaws in the alveolar region was performed under local anesthesia.microscopy revealed highly cellular stroma with many multinucleated giant cells . The cellular stroma showed spindle cell fibroblasts with vesicular nuclei arranged in fascicles, whorled pattern and irregularly in few areas . The giant cells were unequally distributed and were of different sizes with varied number of nuclei resembling osteoclasts . There were few blood vessels and areas of extravasated blood and minimal amount of collagen was seen . Microscopy revealed highly cellular stroma consisting of plenty of multinucleated giant cells (black arrow). The cellular stroma consists of spindle cell fibroblasts with vesicular nuclei arranged in fascicles (blue arrow). The grotesque appearance and the parent's insistence on esthetic improvement compelled the surgeon to attempt cosmetic surgical recontouring of the jaws under general anaesthesia . The entire mandible and maxilla was degloved intraorally . The exposed lesion tissue consisted of multiple locules with reddish hue and semi - hard consistency . Buccal surfaces of maxilla and mandible were decorticated and curetted as much as possible to provide optimal cosmetic improvement . Impacted and loose teeth were removed . Patient had no recurrence of the lesion during the 2-year follow - up period [figure 6]. The orthopantamograph revealed multiple impacted teeth in the mandible, with multiloculated osteolytic lesions involving the entire body as well as the ramus of the mandible sparing the condyles [figure 2]. A 3-dimensional computed tomography showed multiloculated cystic lesions affecting the body and rami of the mandible and also the maxilla . The lesion caused marked expansion of the bones, with a multifocal cortical breakthrough [figure 3]. The orthopantamograph revealed multiple impacted teeth in the mandilble (red arrows) with multiloculated osteolytic lesions involving mandible and maxilla (blue arrows). 3-dimensional computed tomography scan showing multiloculated cystic lesions affecting the body and rami of the mandible (blue arrows) and maxilla with raised orbital floor (red arrows). Biopsy to obtain specimen from both the jaws in the alveolar region was performed under local anesthesia.microscopy revealed highly cellular stroma with many multinucleated giant cells . The cellular stroma showed spindle cell fibroblasts with vesicular nuclei arranged in fascicles, whorled pattern and irregularly in few areas . The giant cells were unequally distributed and were of different sizes with varied number of nuclei resembling osteoclasts . There were few blood vessels and areas of extravasated blood and minimal amount of collagen was seen . Microscopy revealed highly cellular stroma consisting of plenty of multinucleated giant cells (black arrow). The cellular stroma consists of spindle cell fibroblasts with vesicular nuclei arranged in fascicles (blue arrow). The grotesque appearance and the parent's insistence on esthetic improvement compelled the surgeon to attempt cosmetic surgical recontouring of the jaws under general anaesthesia . The entire mandible and maxilla was degloved intraorally . The exposed lesion tissue consisted of multiple locules with reddish hue and semi - hard consistency . Buccal surfaces of maxilla and mandible were decorticated and curetted as much as possible to provide optimal cosmetic improvement . Impacted and loose teeth were removed . Patient had no recurrence of the lesion during the 2-year follow - up period [figure 6]. No cause and effect relationship with trauma, infection, or hemorrhage has ever been verified . Anderson suggested that a genetically induced biochemical abnormality stimulates the giant cell lesions characteristic of cherubism . A molecular pathogenesis of cherubism has been proposed; sh3bp2 gene mutations cause dysregulation of the msx-1 gene, which is involved in regulating mesenchymal interaction in craniofacial morphogenesis . Clinically the lesion of cherubism produces painless, typically slow expansion of the affected area of jaw . The facial expansion maybe quite mild or may result in considerable deformity . More extensive involvement may result in enlargement and widening of the alveolar bone . Progressive involvement of the anterior maxillary segment bone stretches the skin and the lower eyelid beneath the orbit, exposing the lower sclera . The developing permanent teeth in the areas of involvement may be displaced, malformed, or absent . History, clinical course, and the findings in our case corresponds to the classic features of cherubism . Irregular, multilocular, well - defined, translucent, cystic spaces causing expansion of bone and sparing only a thin layer of cortex are usually seen . The teeth may be displaced, unerupted, or appear to be floating in the cyst - like spaces . In the mandible, the inferior alveolar canal maybe displaced and lesion may involve the alveolar process, the angle and ramus . It may advance toward the incisors and obliterate the sigmoid notch, but the condyles may be spared . Periosteal new bone formation is never present . In the maxilla the lesion is similar and the maxillary antra may be completely obliterated only to become normally pneumatized as the lesion regress . Seward and hankey have proposed a grading system based on the radiographical location of the lesions in the jaws . It is as follows: grade 1: involvement of bilateral mandibular molar regions and ascending rami, mandible body, or mentis . Grade 2: involvement of bilateral maxillary tuberosities (in addition to grade 1 lesions) and diffuse mandibular involvement . Grade 3: massive involvement of the entire maxilla and mandible, except the condyles . Grade 4: involvement of both jaws, including the condyles . According to ramon and engelberg grade 4 lesions our case could possibly be classified as above grade 3 cherubism as there was huge deformity of the mandible, especially the bilateral posterolaterosuperior extension of maxillary lesions with slight elevation of orbital floor . The lesions of cherubism are not distinctive histologically and are difficult to differentiate from other giant cells containing fibro - osseous lesions, making diagnosis dependent on the clinical findings impossible . Hamner histochemically demonstrated this perivascular cuffing to be collagen protein and felt its presence was of value in the diagnosis of cherubism . Surgical treatment appears to be unnecessary for grade 1 and 2 cases, in the absence of secondary disturbances . Curettage appears to be necessary in more aggressive cases (grade 3), to reduce maxillofacial deformity that occurs after puberty . Dukart et al ., found that surgical curettage and recontouring performed during a period of rapid growth of cherubism lesions not only offer a favorable immediate result but also arrests active growth of remnant lesions while stimulating bone regeneration . The rationale of calcitonin administration is that it inhibits the osteoclastic activity of the giant cells . Radiation therapy is ineffective and contraindicated in view of the risk of osteoradionecrosis, interference with dentofacial growth and development, and the effect on future surgical procedure . In our case, the patient was an adolescent affected psychologically by his grotesque appearance, which was also a concern for his parents . Therefore, a conservative surgical curettage and recontouring of the jaws was performed under general anesthesia to address his esthetic concern . Two years of postoperative follow - up showed a sustained esthetic improvement achieved by surgery of the grade 3 lesion.
A 75-year - old man with a slightly increased serum creatinine level had developed proteinuria and hypertension in 2000 . In 2007, he noticed a gradually progressive hearing loss and repeated bloody nasal discharge resulting from a refractory sinusitis . Initial laboratory findings revealed elevated serine proteinase 3-anti - neutrophil cytoplasmic antibody (pr3-anca; 26.2 eu), blood urea nitrogen (35 mg / dl), and serum creatinine (1.62 mg / dl) levels . Because of these combined findings, he was diagnosed as having granulomatosis with polyangiitis (gpa) according to the american college of rheumatology 1990 criteria for gpa and watts algorithm [1, 2]. The birmingham vasculitis activity score 2003 was 6 . Because of the low disease activity, the slow progression and the advanced age, we did not initiate immunosuppressive therapy at this time . In october 2008, he developed fever lasting for several days . Laboratory data were as follows: c - reactive protein 11.91 mg / dl, pr3-anca 49.1 u / ml, fibrinogen 752 mg / dl, d - dimer 1.7 g / ml, and urinary protein 0.27 g / day . Activated partial thromboplastin time, prothrombin time and fibrin degradation product were within the respective normal range . Although we administered meropenem (2.0 g / day for 1 week), vancomycin (500 mg / day for 17 days) and itraconazole (200 mg / day for 20 days), the fever and c - reactive protein level did not decrease . On day 12 of admission a brain mri revealed a mass lesion in the right frontal lobe and hypertrophic mucosa in the paranasal sinuses (fig . 1a) in addition to a thickened and strongly enhanced dura mater surrounding the mass lesion (fig . However, he rapidly developed left hemiparesis and deterioration of consciousness [glasgow coma scale (gcs): e1v2m4]. A brain ct revealed that the parenchymal mass lesion had enlarged and the mucous effusion was invading the posterior wall of the right frontal sinus . Due to an impending transtentorial herniation, a decompressive hemicraniectomy and partial right frontal lobectomy were performed, which moderately improved his consciousness (gcs: e2v3m4). After the surgery, he developed bloody sputa, and a chest ct revealed an infiltrative shadow in both lungs . The biopsied brain material from the surgery revealed a granulomatous inflammation with geographic necrosis and multinucleated giant cells in the perivascular area of the thickened dura mater and leptomeninges (fig . Small vessels in the meninges were involved in the granulomatous lesions, and the lumens of the veins were often occluded . In the cerebral cortices and the white matter in these areas, hemorrhagic infarction was widely observed . After treatment with prednisolone (20 mg / day) and azathioprine (40 mg / day), the disturbance of consciousness substantially improved (gcs: e4v3m5), and the epistaxis stopped . This study was conducted with the approval of the ethics committee of niigata university graduate school of medical and dental sciences . Cns involvement such as pachymeningitis and cerebrovascular events is uncommon in gpa, reported in only 28% of cases [3, 4]. We previously clarified that leptomeningeal and parenchymal involvement in the brain were significantly more common in pr3-anca - positive hypertrophic pachymeningitis compared to myeloperoxidase anti - neutrophil cytoplasmic antibody - positive and idiopathic hypertrophic pachymeningitis in a study of 36 patients (including this case). Based on the study, mri findings showed an enhancement of both the pachymeninges and leptomeninges . Moreover, the present case had the extension of the mass lesion with the granulomatous inflammation, and the venous obstruction resulted in severe edema and hemorrhagic infarction . Emergency decompressive craniectomy and partial lobectomy for cerebral infarction with gpa likely contributed to our patient's survival . To our knowledge, this is the first report successfully managed with surgical decompressive craniectomy.
For large scale global analysis, hela s3 cells were prefractionated using custom 2d - le platform, comprised of sief coupled to multiplexed gelfree . Hela s3, h1299, b16f10 cells, and mitochondrial membrane proteins were also fractionated using the custom gelfree device alone (no sief). After separation, detergent and salt were removed, and the fractions were injected into nanocapillary rplc columns for elution into a 12 tesla ltq ftms for online detection and fragmentation . The ms raw files were processed with in - house software called crawler to assign masses . Using this program, determination of both the intact masses and the corresponding fragment masses were performed and these data were searched against a human proteome database . Extensive statistical workups were also performed using several fdr estimation approaches (with decoy databases both concatenated and not). A final q - value procedure is described in detail (methods), with the data above reported using a 5% instantaneous fdr (i.e., q - value) cutoff at the protein level (supplementary fig.
A 59-year - old man presented with liver cirrhosis and esophageal varix bleeding . A ct scan (lightspeed qx / i, general electric co., milwaukee, wi, usa) with contrast media was performed and it incidentally revealed a horseshoe kidney with a well - enhanced isthmus and a 3-cm enhancing mass in the left part (fig . The tumor was limited to the kidney, but its extension into the renal pelvis was not definite . Digital subtraction angiography (dsa) via the right femoral artery route was performed under local anesthesia just before the embolization procedure . The dsa of the aorta showed that the horseshoe kidney was fed by a five - vessel supply that consisted of both the two normal main renal arteries, two aberrant vessels that were originating from the aorta and they entered both sides of the isthmus (fig . 1b) that entered the left side of the isthmus and it originated from the left common iliac artery . The dsa of the left renal artery demonstrated hypervascular tumor staining that was supplied by the anterior inferior segmental branch of the left main renal artery (fig . Although the portion of the tumor that had invaded the collecting systems could not be completely excluded, the decision was made to perform organ - preserving surgery or simple enucleation . Catheterization was performed via a transfemoral approach with the use of the standard coaxial technique . A 5-f end - hole catheter (cobra catheter, cook, bloomington, in, usa) was introduced over a 0.035-inch guide wire (termo; radifocus, tokyo, japan) to the left main renal artery . The feeding vessel to the tumor was catheterized with the use of a 3-f microcatheter (renegade; boston scientific, watertown, ma, usa) and it was embolized superselectively with contour (355 - 500 microns, boston scientific international, la garenne colombes cedex, france) (fig . The contour granules were slowly and carefully injected (to prevent reflux of the particles) under fluoroscopic guidance before embolization with the use of a 0.018-inch - diameter tornado microcoil (3 mm to 2 mm, cook). A postembolization angiography shows a successful segmental embolization of the anterior inferior segment of the left part kidney, including the tumor (fig . For the patient to undergo an effective and comfortable intervention, analgesic (midazolam 2 mg, i.v . ; roche, fontenay - sous - bois, france) and sedative (fentanyl 60 micrograms, i.v . ; co., hwasung, korea) were administered just before the dsa to achieve moderate sedation . Under general anesthesia the tumor site was a mild brown color and it was easily identified at the left side of the horseshoe kidney because of the previous renal artery embolization . The tumor was enucleated by repeated cuts with the use of an electrosurgical generator (valleylab inc ., boulder, co, usa). The parenchymal bleeding was easily controlled by suture because of the previous renal artery embolization . Upon examination of the gross specimen, the tumor showed as a well - circumscribed, bright yellow, solid mass measuring about 3 cm at its greatest diameter (fig . The tumor was confined to the kidney and it proved to be a renal cell carcinoma, grade 2 (fig . 1f). A follow - up ct scan with contrast media 33 days after the operation showed a parenchymal defect at the previous tumor site with some postoperative change (fig . The horseshoe kidney is probably the most common of all renal fusion anomalies (3). This anomaly consists of two distinct renal masses lying vertically on either side of the midline; the masses are connected at their respective lower poles by a parenchymatous or fibrous isthmus that crosses the midplane of the body (3). Most of the malignant tumors arising in horseshoe kidneys are renal cell carcinomas, but transitional cell carcinomas, squamous cell carcinomas, wilm's tumors, lymphomas, carcinoid tumors and sarcomas have also been reported (4, 5). It has been stated that the occurrence of renal cell carcinoma in horseshoe kidneys is no higher than in non - fused kidneys, but that the incidence of transitional cell carcinoma in horseshoe kidneys is higher, and this is conceivably due to the presence of chronic urinary tract infections (4). The blood supply to the horseshoe kidney can be quite variable (3). In 30% of the cases, it consists of one renal artery for each kidney (6), but the blood supply may be atypical, with duplicate or even triplicate renal arteries supplying one or both kidneys (3). The isthmus and adjacent parenchymal masses may receive a branch from each main renal artery, or they may have their own arterial supply originating from the aorta either above or below the level of the isthmus (3). Not infrequently, this area is supplied by branches from the inferior mesenteric artery, the common or external iliac arteries, or the sacral arteries (7). In this case, the isthmus was receiving two arterial supplies that originated from the aorta at the level of the isthmus, and there was an additional arterial supply from the left common iliac artery (fig . An aberrant vascular supply is one of the major anatomic features in horseshoe kidneys; thus, the vascular supply cannot be easily predicted on the surgical field . This is especially true when preoperative renal artery embolization is necessary and a part of the organ has to be removed due to malignant disease while the maximal amount of functioning renal tissue needs to be preserved . Radical nephrectomy is the standard therapy for renal cell carcinoma (8). In cases of neoplasm in a horseshoe kidney, however, there is a place for limited resection or heminephrectomy, with special attention being paid to the abnormal arteries and the renal pelvis (8). Preoperative superselective renal artery embolization helps to prevent excessive bleeding complications during organ - preserving surgery, it allows the preservation of a maximum amount of functioning renal tissue and it enables easy detection of the tumor site via the discoloration; thus, simple enucleation is then feasible during the operation . In conclusion, preoperative superselective renal artery embolization can be an effective tool to facilitate organ - preserving surgery in a case of a horseshoe kidney with renal cell carcinoma.
The course of bipolar disorder is, by definition, cyclical and characterized by episodes of depression and (hypo)mania with or without mixed features, with or without - interepisodic euthymia . As the mood switch involves a change of biological rhythms, with changes in sleeping patterns constituting perhaps the most relevant one, it was thought that abnormalities in biological rhythms might have a role in the pathophysiology of the disease . From these considerations, the literature reports a relationship between biological rhythm disturbances and treatment and the onset, maintenance and remission of bipolar episodes . There is also evidence about the relevance of dys - regulation of hormones and neurotransmitters that command biological rhythms in bipolar disorders [4 - 8]. Physiological and behavioral timekeeping processes are frequently found abnormal in bipolar disorders, thus a vulnerability to alterations in biological rhythms may play a certain role in the course of the disease . Differences in the course of bipolar disorders may also be caused by genetic differences concerning regulation of biological rhythms . Moreover, biological rhythm impairment has been associated with poor functioning and quality of life . Thus, circadian rhythms are related to bipolar disorders in three different ways: (1) by considering its etiological / triggering role, (2) as a very reliable early warning sign of relapse, very useful in psychoeducation, and (3) its modification by means of psychological or drug interventions - may have a therapeutic effect . The biological rhythms interview of assessment in neuropsychiatry (brian) is an 18-items interviewer - administered instrument which aims to investigate four main areas related to circadian rhythm disturbance, namely: sleep, activities, social rhythms and eating patterns . Items are rated using a 4-point scale, (1)= no difficulty, (2)= mild difficulty, (3)= moderate difficulty, and (4) = severe difficulty . The brian scores thus range from 1 to 72, where the higher scores suggest severe circadian rhythm disturbance . To date 44 euthymic (young mania rating scale (ymrs)<6, hamilton depression rating scales (hdrs)<8 for a period of at least one month before inclusion) outpatients with a dsm - iv - tr diagnosis of bipolar disorder and 38 control subjects balanced for sex and age were recruited . The comparison group was recruited from the general population within the catchment area of cagliari, italy . The test - retest reliability measure was carried out in half (n=41) of the sample recruited randomly out of the original sample after stratification by sex and diagnosis . (antas) a semi - structured clinical interview derived in part from the structured clinical interview for dsm - iv axis i disorders (scid - i) non - patient version (scid - i / np). The control subjects were screened using the same interview to exclude current or lifetime psychiatric disorders . The study was approved by the universit europea del mediterraneo onlus ethics committee and was carried out in compliance with the helsinki declaration of 1975 (the evaluation, support and prevention unit). All subjects contacted were informed about the study and when they decided to participate they signed a consensus form . One clinical researcher recorded the socio - demographic and clinical variables of each patient, administered the italian version of the young mania rating scale (ymrs), the 17-items hamilton depression rating scale (hdrs-17) and the global assessment functioning (gaf) scale to confirm the stability of the patient's condition and overall functioning . Interviewers administering the brian and the gaf, hdrs and ymrs were blinded to each other . The brian was developed by the bipolar disorders program & inct translational medicine and by the hospital de clnicas de porto alegre, universidade federal do rio grande do sul, porto alegre, brazil . It is an interviewer - administered instrument designed for use by a trained clinician; the time frame studied refers to the last 14 days before assessment . The 18 items evaluating sleep, activities, social rhythm and eating patterns were probed for discriminant, content and construct validity . All items were rated using a 4-point scale scored 1 = not at all, 2 = seldom, 3 = sometimes, 4 = often, or in some items 1 = never, 2 = seldom, 3 = often, 4 = always . The global score was obtained when the scores of each item were added up . The score may be regrouped in five specific sections scoring sleep, activity, sociality, eating habits and rhythms . The original version was translated into italian to english, back translated and approved by two english native speakers working as researchers in the field of bipolar disorder . A more detailed description of the instrument is shown in the original paper by giglio et al . . The accuracy of the brian score in discriminating between cases and controls (discriminant validity) was measured comparing mean brian scores in the two groups with one - way anova . The test re - test reliability of the brian total score and the brian sections (sleep, activity, sociality, eating, rythms) was measured comparing to and t1 (1 week later) using both the correlation coefficient of pearson and cohen s kappa value . 44 euthymic (young mania rating scale (ymrs)<6, hamilton depression rating scales (hdrs)<8 for a period of at least one month before inclusion) outpatients with a dsm - iv - tr diagnosis of bipolar disorder and 38 control subjects balanced for sex and age were recruited . The comparison group was recruited from the general population within the catchment area of cagliari, italy . The test - retest reliability measure was carried out in half (n=41) of the sample recruited randomly out of the original sample after stratification by sex and diagnosis . (antas) a semi - structured clinical interview derived in part from the structured clinical interview for dsm - iv axis i disorders (scid - i) non - patient version (scid - i / np). The control subjects were screened using the same interview to exclude current or lifetime psychiatric disorders . The study was approved by the universit europea del mediterraneo onlus ethics committee and was carried out in compliance with the helsinki declaration of 1975 (the evaluation, support and prevention unit). All subjects contacted were informed about the study and when they decided to participate they signed a consensus form . One clinical researcher recorded the socio - demographic and clinical variables of each patient, administered the italian version of the young mania rating scale (ymrs), the 17-items hamilton depression rating scale (hdrs-17) and the global assessment functioning (gaf) scale to confirm the stability of the patient's condition and overall functioning . Interviewers administering the brian and the gaf, hdrs and ymrs were blinded to each other . The brian was developed by the bipolar disorders program & inct translational medicine and by the hospital de clnicas de porto alegre, universidade federal do rio grande do sul, porto alegre, brazil . It is an interviewer - administered instrument designed for use by a trained clinician; the time frame studied refers to the last 14 days before assessment . The 18 items evaluating sleep, activities, all items were rated using a 4-point scale scored 1 = not at all, 2 = seldom, 3 = sometimes, 4 = often, or in some items 1 = never, 2 = seldom, 3 = often, 4 = always . The score may be regrouped in five specific sections scoring sleep, activity, sociality, eating habits and rhythms . The original version was translated into italian to english, back translated and approved by two english native speakers working as researchers in the field of bipolar disorder . A more detailed description of the instrument is shown in the original paper by giglio et al . . The accuracy of the brian score in discriminating between cases and controls (discriminant validity) was measured comparing mean brian scores in the two groups with one - way anova . The test re - test reliability of the brian total score and the brian sections (sleep, activity, sociality, eating, rythms) was measured comparing to and t1 (1 week later) using both the correlation coefficient of pearson and cohen s kappa value . 44 bipolar patients (25 bd i, 19 bd ii) and 38 healthy controls were included in the study (table 1). The mean age of the sample was 44.61 + 12.64 without differences between cases and controls regarding the distribution by sex . The bipolar sample included 16 men (42.1%) and this rate was similar (16 men, 36.4%) in the control group . In the bipolar sample, 25 subjects were diagnosed as bipolar i (30.5% of the whole sample) and 19 (23.3%) as bipolar ii (table 1). Bipolar patients scored 22.2211.19 in brian against 7.135.6 in the control group (f=56.75, df 1,80,81, p<0.0001). Brian showed good accuracy in screening between bd and non - bd with quite good performance in specificity, for example at cutoff 16, with a sensitivity of 68.2, specificity was excellent (92.5). Table 2 shows the performance of brian as a screening tool for bipolar disorders at different cutoff points . Test - retest stability was measured in half of the sample randomly recruited after stratification by diagnosis and sex (8 male; age 44.83, 19 bipolar). It was found by using both the correlation coefficient of pearson and cohen s kappa value . Table 2 shows the k values (with standard error) and the r values . The pearson r values were found very high in each section and in the total score, thus indicating a correlation between the two scores with statistical significance in all measures . The k value varied from 0.47 in the sociality section to 0.80 in the sleep section . The italian version of the brian is a valid and reliable tool with psychometric properties equivalent to the original tool and has a good test - retest reliability measured with k statistics . Moreover, the results of the present study suggest that the italian version of the brian shows an interesting discriminant validity in screening for bipolar disorder . The high discriminant validity allows the obtaining of an excellent performance using the brian as a screening tool at the cutoff of 20 . From this perspective, we must consider that a screener can really be useful if it does not generate false positives and thus is capable of producing a high predictive value of a negative . In fact, if the test is inexpensive, noninvasive and easy to apply, it can lead to a second evaluation only on positives with a more accurate diagnostic tool, or if another screener is available, one that is equally inexpensive and easy to apply but with complementary performance (with high sensitivity and low specificity), they can be administered simultaneously . We must therefore take into account that the predictive value of negative evidence we found at a cutoff of 20 is noteworthy . It is to be underscored that the brian was not conceived or initially tested as a screening tool, but even then it showed acceptable screening properties . The results are of interest because of the well - known difficulties in research in developing accurate screeners for bipolar disorder due to the several existing biases which include memory biases, perception of hypomania as a non - pathological state and others . In particular, current screening tools show good sensitivity but low specificity . From this point of view the brian could be highly complementary to questionnaires such as the mood disorder questionnaire (mdq) and hypomania checklist-32 (hcl-32) and its use associated with a clinical questionnaire (more sensitive but less specific) could make the screening very effective . In all cases the result was so good as to suggest extending the possibility of using a scale of biological rhythms as screening for bipolar disorder or introducing some items on biological rhythms into well - known clinical screeners such as the mdq or hcl-32 . From this point of view we must consider that this study was conducted by comparing patients with bipolar disorder to people without psychiatric diagnoses . In practice, a screening tool should be able to discriminate especially among people with different pathologies . From this perspective it must be considered that the disturbances of biological rhythms have recently been reported not only in bipolar disorders but also in other psychiatric disorders such as major depression, stress related disorders, autism and schizophrenia [21 - 24]. However, it has been highlighted in recent literature that the weight of the dysregulation of biological rhythms in bipolar disorder is higher than in other disorders and can be described as a kind of characteristic of these disorders [25, 26]. Even on the level of genetic determinants a justification for the association between dysregulation of biological rhythms and bipolar syndromes has been found . If it is thus evident that the screening of biological rhythm dysregulation can divide people with bipolar disorder from people without a psychiatric diagnosis, it follows that this can occur during a screening of the general population . However, the extent of biological rhythm dysregulation has never been introduced as a possible screening tool at the speculative level . In the light of these psychometric results it would be interesting to design a tool that that combines the features of mixed measurement of hyperactivity and hyperergia typical of most known screens, such as the mdq and hcl, with the measurement of biological rhythms . The study indicates that the total brian score and the brian sub - section scores reach a good correlation in the two measures (t0 and t1) measured by means of pearson s correlation factor . The test re - test reliability for each section showed a k agreement from sufficient to good . Sufficient (k from 0.40 to 0.60) was shown for sociality and activity sections; good (k from 0.60 to 0.80) was shown for sleep, eating, rhythm sections and total score . The study of the regulation of biological rhythms is becoming increasingly important in the field of bipolar disorders . The role of melatonin secretion in this control is well - known and is today the objective of several studies that may contribute to better understanding the physiopathological process of bipolar disorders as the genetic component and may suggest newer therapeutic pathways . Bipolar subjects, regardless of the mood state, experience a wide variety of disruptions of biological rhythms and sleep disorders . A decreasing amount of deep sleep per night comes just before the onset of a manic episode and may come before a depressive episode . Therefore, the decrease in sleep has been identified as a predictor of manic episodes . Psychoeducational programs have shown that preventing disruptions in the circadian sleep cycle is important in maintaining a regular sleep schedule . Amongst the limitations we must consider the preliminary nature of the data, the limited sample size and the fact that the current study was designed to validate the italian version of the brian, not to test its screening properties . Screening performances need to be measured in target populations of future screening studies (ie: community samples), and/or clinical samples including euthymic patients with major depressive disorder because a differential diagnosis between bipolar depression and unipolar depression is often the most important issue in this area in clinical practice . The results must be considered an innovative preliminary report suggesting a new perspective in the development of screening tools, it can be complementary to those explored so far . Due to the relevance of monitoring biological rhythms with a valuable and reproducible instrument, brian can be a useful tool both in research and clinical practice with bipolar patients . The results also show that brian has good discriminant validity in detecting bd from healthy controls and shows good test - retest reliability . The study suggests the possibility of developing mixed screening tools by introducing items on biological rhythm dysregulation into the usual measures of mood mgc and fc participated in the design of the study, in the analysis of the data and drafted the manuscript . Mfm, ep, mp and rm participated in acquisition of data and critical revision of the manuscript.
Based on the recent definitions having more than three of the following symptoms confirm the existence of metabolic syndrome in a patient . These symptoms include hypertension, triglyceridemia, dyslipidemia, abdomen obesity, and resistance to insulin, increase of c protein and fibrinogen . Moreover, both metabolic syndrome and periodontitis coincide with systemic inflammation and glucose tolerance disorder, which indicate the common pathophysiologic pathway of these diseases . Nevertheless, there is a little information about the relation of metabolic syndrome and hemodialysis (hd). Meanwhile, periodontitis and metabolic syndrome are prevalent in the patients suffering from hd . However, there is a little information about their association . Although there are no specific signs in the mouth indicating the presence of chronic kidney disease, a whole range of changes occur in the mouth that is associated with chronic kidney disease and hd . This condition has been reported to affect the teeth, oral mucosa, bone, periodontium, salivary glands, tongue, mouth cavity, and temporomandibular joint . These patients suffer from bad taste in their mouth, xerostomia, severe periodontitis, poor dental status, no carious tooth tissue loss, and a 14% prevalence of moderate to severe periodontitis was reported among individuals> 20 years of age in the usa population . In their study, shimazaki et al . And khader et al . Separately showed that the patients suffering from metabolic syndrome show more pocket depth and attachment loss . They concluded that metabolic syndrome increases the risk of periodontitis and thus, the patients with metabolic syndrome symptoms must be examined to diagnose whether they are affected by periodontitis or not . Moreover, d'aiuto et al . And chen et al . Separately showed that severe periodontitis is associated with metabolic syndrome in hd patients . It was concluded from nesbitt's study that the volunteers with average to severe bone resorption had a higher chance to be affected by metabolic syndrome . Since metabolic syndrome is a crucial problem of the society, it is necessary to determine the at - risk individuals to control it and decrease the mortality rate . A more exact and precise understanding of the metabolic syndrome side effects and the involving mechanisms can help to control and overcome it effectively . Moreover, the on - time diagnosis of the disease inhibits its progress and increases the patient's lifetime . Based on the previous research, it seems that no study has been done to compare the healthy people and hd patients suffering from metabolic syndrome in iran . Hence, the main goal of this research was to determine the effects of the metabolic syndrome on the periodontal indices of hd patients . In this descriptive - analytical study, 75 persons were selected by the simple method, 50 of them were hd patients (for 5 years), and 25 of them were healthy people . The inclusion criteria to allow the patients to participate in the research are as follows: the patient must have more than 10 teeththe patient must not have the local alterations such as previous surgery or bone transplantation to change the results of the studythe patient must not smoke or take antibiotics from last 2 weeks onward . The patient must have more than 10 teeth the patient must not have the local alterations such as previous surgery or bone transplantation to change the results of the study the patient must not smoke or take antibiotics from last 2 weeks onward . The first group included 25 hd patients not suffering from metabolic syndrome; the second group included 25 hd patients suffering from metabolic syndrome and the third group, namely the control group of the study, included 25 healthy persons (not affected by hd and metabolic syndrome). Age, gender, and physical condition were matched between the groups . To minimize the role of the intervening factors . This study was carried out at one of the therapeutic centers of najaf abad city . At first evaluation of the metabolic syndrome was done considering the five specifications according to the definition of the national cholesterol education program society of america as follows: abdomen obesity: the size of the waist more than 102 cm for men and more than 88 for womenhypertriglyceridemia: triglyceride more than 150 mg / dlhigh - density lipoprotein cholesterol: men <40 mg / dl and women <50 mg / dlhypertension: systolic> 130 mmhg and diastolic> 85 mmhghigh plasma glucose: glucose> 110 mg / dlat least, three of the above - mentioned specifications must be at the threshold level or higher to confirm the metabolic syndrome in the patient . Then, the periodontal conditions of the selected persons were determined by radiography and the bone resorption was calculated by two periodontitis . To measure the bone resorption, digital panoramic radiography was used . The distance from the crest of the bone to the cement - enamel junction the distance more than 2 mm was considered as the bone resorption . Considering the enlargement probability of the radiographies, the manufacturer's guidelines were used to analyze the resorption value, and the exact values were obtainedafter selecting the patients, the average value of the bone resorption of the groups under study was measured and registered . Then, the gingival index was calculated by the leo index in four points . To determine the probing pocket depth, the distance from the edge of the gingiva to the depth of the gingival fissure was measured using williams probe . The bleeding index in the interproximal areas then, the periodontal indices of the patients suffering from metabolic syndrome and the persons not affected by metabolic syndrome were compared . At the end, the data were analyzed by spss20 (ibm, armonk, ny, united states of america) using mann whitney and kruskal wallis tests at the meaningful level of = 0.05 . Abdomen obesity: the size of the waist more than 102 cm for men and more than 88 for women hypertriglyceridemia: triglyceride more than 150 mg / dl high - density lipoprotein cholesterol: men <40 mg / dl and women <50 mg / dl hypertension: systolic> 130 mmhg and diastolic> 85 mmhg high plasma glucose: glucose> 110 mg / dl at least, three of the above - mentioned specifications must be at the threshold level or higher to confirm the metabolic syndrome in the patient . Then, the periodontal conditions of the selected persons were determined by radiography and the bone resorption was calculated by two periodontitis . To measure the bone resorption, digital panoramic radiography was used . The distance from the crest of the bone to the cement - enamel junction the distance more than 2 mm was considered as the bone resorption . Considering the enlargement probability of the radiographies, the manufacturer's guidelines were used to analyze the resorption value, and the exact values were obtained after selecting the patients, the average value of the bone resorption of the groups under study was measured and registered . Then, the gingival index was calculated by the leo index in four points . To determine the probing pocket depth, the distance from the edge of the gingiva to the depth of the gingival fissure was measured using williams probe . Then, the periodontal indices of the patients suffering from metabolic syndrome and the persons not affected by metabolic syndrome were compared . At the end, the data were analyzed by spss20 (ibm, armonk, ny, united states of america) using mann whitney and kruskal wallis tests at the meaningful level of = 0.05 . The mean, minimum, maximum, median and standard deviation of the bone resorbtion analysis is shown in table 1 . The mean, minimum, maximum, median, and standard deviation of the probe depth for the groups being studied in terms of mm is shown in table 2 . The mean, minimum, maximum, median and standard deviation of the bone resorbtion analysis for the studied groups the mean, minimum, maximum, median and standard deviation of the probe depth for the studied groups in figure 1, the frequency distribution of the groups under study according to the bleeding index of the gingiva around the teeth is shown . The frequency distribution of the groups under study according to the bleeding index of the gingiva around the teeth . In figure 2, as it can be seen, the mean of the bone resorption, the mean of the probing depth, the value of the gingival index, and the bleeding index of the hd patients with metabolic syndrome show the maximum levels and those of the control group show the minimum values . Since the condition of the unilateral variance test could not be provided for this study, the kruskal the obtained results showed a meaningful difference among the groups under study (p <0.001). To complete the test, the mann the results of the test showed a significant difference among the obtained indices of the groups being studied in the binary form (p <0.001) too . Periodontitis is an intensive response to the local pathogens of the gingival areas and periodontal tissues which resultsin bone resorption and tooth loss . Previous studies have shown the high frequency of periodontitis in hd patients . In the patients suffering from periodontitis all of the risk factors are interrelated in a vicious circle: whereas poor oral health may lead to both inflammation and protein - energy wasting in hd patients, numerous pathways associate the accumulation of proinflammatory cytokines with different aspects of protein - energy wasting, including anorexia, muscle loss, low anabolic hormones, increased energy expenditure, and insulin resistance . The mean of the bone resorption for hd patients suffering from metabolic syndrome was calculated as 1.99 mm, for hd patients not suffering from metabolic syndrome as 1.45 mm and for the control group (not suffering from hd and metabolic syndrome) as 0.63 mm [table 1]. The difference between the groups understudying the binary form was statistically significant (p <0.05). Hence, the maximum value of the bone resorption was observed in the group affected by both the metabolic syndrome and hd, and the minimum value was observed in the control group, including the healthy persons . Therefore, the obtained results of this study are in line with the results of schimazaki's, nesbitt et al ., chen et al ., and d'aiuto et al . Hence, it can be concluded that because of the additive effect of metabolic syndrome and hd, the mean of the bone resorption will increase in such patients . Furthermore, in this study, the means of the probe depth for the hd patients suffering from metabolic syndrome, the hd patients not suffering from metabolic syndrome and the control group including healthy persons were 2.73, 2.17, and 1.48 mm, respectively . Hence, the patients suffering from both metabolic syndrome and hd showed the maximum value for probe depth and the control group, including the healthy persons showed the minimum value for the probe depth . Thus, the results of this study are in agreement with the results of shimazaki et al . And therefore, it can be concluded that the additive effect of the metabolic syndrome and hd will increase the mean probe depth of the patients . In this study, the frequency of the gingival bleeding index showed the maximum value in the hd patient suffering from metabolic syndrome . Furthermore, the results of this study revealed that the patients suffering severely from systemic inflammation showed a higher percentage of the gingival index frequency so that the patients suffering from both hd and metabolic syndrome showed the maximum value and the control group showed the minimum value of the gingival index . The difference between the groups being studied in the binary form was statistically significant (p <0.05). Therefore, the additive effect of the metabolic syndrome and hd increased the gingival index . In regard, based on the results obtained in a study on the 4 considered specifications of the periodontal indices, it can be concluded that the more the severity of the systemic disease increases, the more the health of the periodontal tissues will decrease . So that, in this study all the periodontal indices of the hd patients suffering from metabolic syndrome were at the lowest level in comparison with other groups and the patient not suffering from metabolic syndrome as well as the control group were in the second and third positions, respectively . The causes can be attributed to the hemodialysis role to disable the patient to care for his mouth and tooth health, the same risk factors of the metabolic syndrome and periodontal disease which intensify the periodontal disease and the suppression of the immune system resulting from chronic diseases . This study was limited by some factors, including a low number of the samples and the impossibility of the prevalence calculation of metabolic syndrome in the patients suffering from periodontal disease . A cross - sectional study with a limited case number, it has a little clinical impact, so the confounding effect of glucose intolerance or overt diabetes mellitus cannot be clearly excluded in a small - scale study also panoramic is not a suitable radiographic for bone loss evaluation due to distortions and superimpositions, so a better radiographic is recommended . The results of this study revealed when the severity of chronic systemic disease increased the health of the periodontal tissues would decrease in such a manner that the hd patients suffering from metabolic syndrome showed the lowest level of health for the periodontal tissues, and the hd patients not suffering from metabolic syndrome and the control group showed the highest level of health, respectively . The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non - financial in this article . The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non - financial in this article.
Telemedicine, broadly defined as the use of medical information exchanged from one site to another via electronic communications to improve a patient s clinical health status, has been expected to improve simultaneously the cost of, quality of, and access to healthcare . There are over 80 systematic reviews on the effectiveness of telemedicine and more than 20 of them reporting conclusively that telemedicine is effective in such areas as mental health [3 - 5] and management of chronic diseases [6 - 8] including diabetes, heart failure, and elderly care . However, previous systematic reviews on the economic evaluation of telemedicine, based on study populations in the us, the uk, and australia, show there is no conclusive evidence that telemedicine interventions are cost - saving or cost - effective compared to conventional health care [11 - 14]. In many countries, it has been reported that a large number of telemedicine programs have been started on a trial basis but discontinued for larger scale implementation due to lack of sustainable financial sources [15 - 19]. This is mainly because most insurers do not reimburse new types of healthcare delivery [20 - 22]. For this reason, several studies have stated that comprehensive cost - effectiveness studies are essential in justifying insurer coverage and reimbursement for telemedicine in the future . Japan has faced a growing proportion of elderly people amidst a declining overall population . In 2014, the proportion of the population aged 65 or older was estimated to be 25.9%the highest proportion in the world . Currently, two major healthcare issues in japan are the increasing total cost of healthcare and the uneven distribution of healthcare resources . Based on the budget records issued by japan s ministry of finance, we calculated public expenditure for development and pilot trials of health information technology, including telemedicine, from 2008 to 2014the result was $1.5 billion . Despite such enormous amounts in public subsidies for telemedicine, the literature indicated serious concern about the financial sustainability of telemedicine . Among the studies reporting the effectiveness of telemedicine experiments around the country, only a small subset of these studies conducted an economic evaluation . The absence of a cohesive body of rigorous economic evaluation studies may be one of the key obstacles to the widespread adoption, proliferation, and funding of telemedicine programs [9,28 - 30]. This study s aim was to conduct a systematic review to identify and analyze the published economic evaluations on telemedicine in japan . This qualitative assessment has three objectives: (a) to identify whether, and in what areas, telemedicine was evaluated to achieve a favorable level of economic efficiency, that is, whether it was cost - saving or cost - effective in japan; (b) to assess the methodological rigorousness of the economic evaluations; and (c) to discuss future studies necessary to improve the general financial sustainability of telemedicine . This article focuses on telemedicine in broader health areas to make the results more comprehensive . Specifically, this study gathered economic evaluations of telemedicine connecting non - specialized physicians and specialized physicians, physicians and patients, and public health nurses and patients . We searched the following databases: pubmed / medline, web of science, ieee xplore, ichushi - web database (the largest japanese health database), and cinii articles (the largest japanese database in all academic fields). All peer - reviewed articles published between january 1, 2000 and december 31, 2014 in english and japanese were selected if they included any type of economic evaluation in monetary terms . The inclusion criteria regarding economic evaluation were full economic evaluation (i.e., cost - minimization analysis [cma], cost - effectiveness analysis [cea], cost - utility analysis [cua], cost - benefit analysis [cba]), cost - only analysis, or benefit - only analysis . The exclusion criteria were articles that described only effectiveness or benefit in non - monetary terms and articles available only in abstract form . Table 1 shows the different and exact combinations of keywords, relating to economic evaluation and telemedicine, used in this research . The selection process for the articles involved reading the titles and abstracts of the results obtained by one of the authors . After duplicate articles were deleted, all abstracts were read for relevance based on the inclusion and exclusion criteria . The relevant full - text articles were then obtained and thoroughly read by two authors . A classification of the telemedicine types, economic - analysis types, study designs, study populations, time frames, and the selection for the final appraisal were obtained by reading both the abstracts and the entire articles . In case of any disagreement, the two authors discussed the article and one designated author made the final decision . In order to compare a variety of studies conducted in different time periods, we made a purchasing power parity adjustment and converted all costs to 2014 us dollars using the consumer price index . We critically assessed the methodological rigorousness of the economic evaluation for each article based on the latest version of the checklist developed by drummond et al . . This checklist was chosen partly because it was one of the most widely used assessment tools in this area, e.g., this checklist s older version was known and widely used as the bmj check - list . The most recent version of this checklist, published in 2015, contains 87 items under ten headings (detailed in table 2). Assessors are asked to check either yes, no, or not applicable (n / a) for each of ten subheadings according to the relative importance subjectively conferred to each item under each subheading . This checklist concentrates on full economic evaluations, but could also be used for partial economic evaluations, reports, and commentaries on economic evaluations . If items were not applicable to a specific study, a miki akiyama and byung - kwang yoo graded three studies using the criteria in order to reach consensus on the interpretation of the criteria, and miki akiyama graded the remaining papers . When a reviewed study reported cost and benefit but did not report the benefit - to - cost ratio (bcr) [34 - 36], we made a supplemental estimation of bcr by dividing the reported financial benefit by the reported cost . The financial benefit was estimated as the reduced medical care expenditure without assigning any monetary value for an improvement in health outcome . We searched the following databases: pubmed / medline, web of science, ieee xplore, ichushi - web database (the largest japanese health database), and cinii articles (the largest japanese database in all academic fields). All peer - reviewed articles published between january 1, 2000 and december 31, 2014 in english and japanese were selected if they included any type of economic evaluation in monetary terms . The inclusion criteria regarding economic evaluation were full economic evaluation (i.e., cost - minimization analysis [cma], cost - effectiveness analysis [cea], cost - utility analysis [cua], cost - benefit analysis [cba]), cost - only analysis, or benefit - only analysis . The exclusion criteria were articles that described only effectiveness or benefit in non - monetary terms and articles available only in abstract form . Table 1 shows the different and exact combinations of keywords, relating to economic evaluation and telemedicine, used in this research . The selection process for the articles involved reading the titles and abstracts of the results obtained by one of the authors . After duplicate articles were deleted, all abstracts were read for relevance based on the inclusion and exclusion criteria . The relevant full - text articles were then obtained and thoroughly read by two authors . A classification of the telemedicine types, economic - analysis types, study designs, study populations, time frames, and the selection for the final appraisal were obtained by reading both the abstracts and the entire articles . In case of any disagreement, the two authors discussed the article and one designated author made the final decision . In order to compare a variety of studies conducted in different time periods, we made a purchasing power parity adjustment and converted all costs to 2014 us dollars using the consumer price index . We critically assessed the methodological rigorousness of the economic evaluation for each article based on the latest version of the checklist developed by drummond et al . . This checklist was chosen partly because it was one of the most widely used assessment tools in this area, e.g., this checklist s older version was known and widely used as the bmj check - list . The most recent version of this checklist, published in 2015, contains 87 items under ten headings (detailed in table 2). Assessors are asked to check either yes, no, or not applicable (n / a) for each of ten subheadings according to the relative importance subjectively conferred to each item under each subheading . This checklist concentrates on full economic evaluations, but could also be used for partial economic evaluations, reports, and commentaries on economic evaluations . If items were not applicable to a specific study, a miki akiyama and byung - kwang yoo graded three studies using the criteria in order to reach consensus on the interpretation of the criteria, and miki akiyama graded the remaining papers . When a reviewed study reported cost and benefit but did not report the benefit - to - cost ratio (bcr) [34 - 36], we made a supplemental estimation of bcr by dividing the reported financial benefit by the reported cost . The financial benefit was estimated as the reduced medical care expenditure without assigning any monetary value for an improvement in health outcome . The initial screening of the electronic databases retrieved 138 titles and abstracts; after the removal of duplicates and irrelevant titles, 64 abstracts remained for screening and 29 potentially eligible full - text articles were obtained . We excluded 12 articles after reading the full text, which were judged to meet the exclusion criteria . Two were general reviews of the situation in asia, one was a review on the methodology of economic evaluation, four were benefit analyses using the same survey data, and five papers were not peer reviewed . Eventually, 17 peer - reviewed articles containing any form of economic appraisal in monetary terms remained . Table 3 summarizes the characteristics and key findings of the selected studies . Among these studies, 12 were published in english [34 - 45] and five in japanese [27,46 - 49]. The service settings of telemedicine were categorized into two major types: those connecting physicians for specialist consultations (the md md setting) [34,35,37 - 39,49] and those connecting healthcare providers and patients [27,36,40 - 48], which were further sub - categorized into settings connecting physicians and patients at home (the md patient setting), and those connecting public health nurses and patients at home (the phnrs patient setting). Md settings included dermatological consultation, pediatric medical consultation, emergency radiotherapy for malignant spinal cord compression, real - time computed tomography (ct) scan indication, teleradiology, and telepathology . All of the 11 settings connecting healthcare providers and patients that is, the md patient setting and phnrs patient setting dealt with home - based systems with two types of functions . One function type is a stand - alone real - time video conference system for palliative care for cancer patients without continuous monitoring of vital signs . The other type, evaluated by 10 articles [27,40 - 48], monitors vital signs with equipment such as an automated sphygmomanometer and a simple electrocardiograph for home . Healthcare providers this type of system targeted lifestyle - related diseases such as high blood pressure, diabetes, stroke, and heart failure among fragile elderly patients . Regarding the types of economic evaluation, they were classified into four types; three were cba, which provide the most useful policy implication, that is, benefit measured by monetary value compared to input cost of telemedicine . Three studies were categorized as cma [35 - 37], which estimate the cost reduction by telemedicine compared to a control group both telemedicine and control groups achieved the same level of health outcome . Four studies [27,43 - 45] measured medical expenditure saved due to telemedicine, without calculating the input telemedicine cost or showing equal health outcomes . The remaining seven studies [38,39, 41,42,46,47,49] measured willingness - to - pay (wtp) for using telemedicine . Md settings, one clinical efficacy study presented detailed estimates of both the costs and benefits of telemedicine in emergency radiotherapy for malignant spinal cord compression (mscc), connecting between a university hospital and 10 rural hospitals . Since this study did not report the bcr, we calculated the ratios to range from 4.67 to 13.40 . One cma study on dermatology consultation reported cost - saving of $330 per person per week . This study s two - way sensitivity analysis indicated that the cost - saving results were sensitive to the two key parameters of travel time and consultation time . Assuming that travel time was 60 minutes and consultation time was five minutes (based on expert opinions from japanese dermatologists), we calculated the bcr to be 1.53 . This study estimated that teleconsultation contributed to cost - saving of $144 per reading by avoiding unnecessary enhanced ct scans . One survey estimated the benefits of specialist teleconsultation in pediatric emergency care for primary care physicians in rural, underserved areas . These benefits were measured by inquiring about wtp among parents of children in two rural towns, that is, $31.40 to $33.60 per emergency department visit . There were two studies conducted by one study group to estimate medical providers wtp for teleradiology and telepathology . These two studies applied different statistical methods for the same set of survey data obtained from healthcare providers across japan, who used either teleradiology or telepathology in an md md setting . Their wtp estimates were $489 to $510 for teleradiology and $1063 to $1111 for telepathology per person per year . One cma study on home - palliative care in an md patient setting reported that telemedicine could reduce the frequency of physician visits from twice to once a week . The cost - saving of home palliative care with telemedicine were estimated to be $5000 per person per year compared to conventional home care . A sensitivity analysis of this study showed their results to be sensitive to two parameters, the number of user patients and duration of service provided . One study group published two cba studies on the same type of tele - homecare service operated in different regions with different financial schemes: (a) one region where a monthly fee was charged and (b) three other regions where no fee was charged because of public subsidies . Under the former scheme, the bcr was calculated to increase monotonically over time, that is, 1.07 (6-year operation) and 1.28 (11-year operation), using wtp estimates among patients in this region . Under the latter scheme, the bcr estimates in the three regions ranged from 0.54 to 0.61, indicating that telehomecare was not cost - saving . There were four studies reporting medical expenditure saved by telehomecare connecting public health nurses and the elderly with chronic diseases in one region [27,43 - 45]. Based on the claims data, these studies showed that treatment periods were shortened by telemedicine . Their estimates of reduced medical expenditure ranged from $148 to $629 per case per year for lifestyle diseases [27,43 - 45]. However, the input costs of telehomecare were not shown in these four studies . A single research team produced three survey studies and measured patients wtp for a telehomecare system that combines vital - signs monitoring and doctor consultation . The wtp estimates were $288, $480, and $520 per case per year, depending on three analytical methods . There was only one study conducted in an urban area to estimate users wtp for home tele - health monitoring . This study, published in 2012, showed wtp as low as $109 per patient per year . Table 4 summarizes our quality assessment of reviewed studies in terms of the ten criteria of the well - established checklist from drummond et al . . There were only four studies that met more than eight criteria, and eleven studies met only three or fewer criteria . Studies of cba and cma tended to meet more criteria than studies that measured only the benefits of telemedicine . Only two studies conducted a sensitivity analysis, and hence met the two criteria of uncertainty estimates and study results including all issues of concern . Figure 2 was created to detect a potential publication bias among the reviewed papers . A bold vertical line in this figure represents a threshold bcr value of one (also called a break - even point). A plot located rightward of this threshold vertical line indicates a favorable economic efficiency level for a telehealth program evaluated in the published study . Since the plots were skewed to the right of the threshold vertical line (i.e., bcr greater than one), figure 2 implies a moderate bias toward publishing more studies with a higher bcr estimate for a tele - health intervention . It should be noted that our interpretation regarding such potential publication bias has limited validity due to the small number of publications with bcr estimates . The initial screening of the electronic databases retrieved 138 titles and abstracts; after the removal of duplicates and irrelevant titles, 64 abstracts remained for screening and 29 potentially eligible full - text articles were obtained . We excluded 12 articles after reading the full text, which were judged to meet the exclusion criteria . Two were general reviews of the situation in asia, one was a review on the methodology of economic evaluation, four were benefit analyses using the same survey data, and five papers were not peer reviewed . Eventually, 17 peer - reviewed articles containing any form of economic appraisal in monetary terms remained . Table 3 summarizes the characteristics and key findings of the selected studies . Among these studies, 12 were published in english [34 - 45] and five in japanese [27,46 - 49]. The service settings of telemedicine were categorized into two major types: those connecting physicians for specialist consultations (the md md setting) [34,35,37 - 39,49] and those connecting healthcare providers and patients [27,36,40 - 48], which were further sub - categorized into settings connecting physicians and patients at home (the md patient setting), and those connecting public health nurses and patients at home (the phnrs patient setting). Md settings included dermatological consultation, pediatric medical consultation, emergency radiotherapy for malignant spinal cord compression, real - time computed tomography (ct) scan indication, teleradiology, and telepathology . All of the 11 settings connecting healthcare providers and patients that is, the md patient setting and phnrs patient setting dealt with home - based systems with two types of functions . One function type is a stand - alone real - time video conference system for palliative care for cancer patients without continuous monitoring of vital signs . The other type, evaluated by 10 articles [27,40 - 48], monitors vital signs with equipment such as an automated sphygmomanometer and a simple electrocardiograph for home . Healthcare providers this type of system targeted lifestyle - related diseases such as high blood pressure, diabetes, stroke, and heart failure among fragile elderly patients . Regarding the types of economic evaluation, they were classified into four types; three were cba, which provide the most useful policy implication, that is, benefit measured by monetary value compared to input cost of telemedicine . Three studies were categorized as cma [35 - 37], which estimate the cost reduction by telemedicine compared to a control group both telemedicine and control groups achieved the same level of health outcome . Four studies [27,43 - 45] measured medical expenditure saved due to telemedicine, without calculating the input telemedicine cost or showing equal health outcomes . The remaining seven studies [38,39, 41,42,46,47,49] measured willingness - to - pay (wtp) for using telemedicine . Md settings, one clinical efficacy study presented detailed estimates of both the costs and benefits of telemedicine in emergency radiotherapy for malignant spinal cord compression (mscc), connecting between a university hospital and 10 rural hospitals . Since this study did not report the bcr, we calculated the ratios to range from 4.67 to 13.40 . One cma study on dermatology consultation reported cost - saving of $330 per person per week . This study s two - way sensitivity analysis indicated that the cost - saving results were sensitive to the two key parameters of travel time and consultation time . Assuming that travel time was 60 minutes and consultation time was five minutes (based on expert opinions from japanese dermatologists), we calculated the bcr to be 1.53 . This study estimated that teleconsultation contributed to cost - saving of $144 per reading by avoiding unnecessary enhanced ct scans . One survey estimated the benefits of specialist teleconsultation in pediatric emergency care for primary care physicians in rural, underserved areas . These benefits were measured by inquiring about wtp among parents of children in two rural towns, that is, $31.40 to $33.60 per emergency department visit . There were two studies conducted by one study group to estimate medical providers wtp for teleradiology and telepathology . These two studies applied different statistical methods for the same set of survey data obtained from healthcare providers across japan, who used either teleradiology or telepathology in an md md setting . Their wtp estimates were $489 to $510 for teleradiology and $1063 to $1111 for telepathology per person per year . One cma study on home - palliative care in an md patient setting reported that telemedicine could reduce the frequency of physician visits from twice to once a week . The cost - saving of home palliative care with telemedicine were estimated to be $5000 per person per year compared to conventional home care . A sensitivity analysis of this study showed their results to be sensitive to two parameters, the number of user patients and duration of service provided . One study group published two cba studies on the same type of tele - homecare service operated in different regions with different financial schemes: (a) one region where a monthly fee was charged and (b) three other regions where no fee was charged because of public subsidies . Under the former scheme, the bcr was calculated to increase monotonically over time, that is, 1.07 (6-year operation) and 1.28 (11-year operation), using wtp estimates among patients in this region . Under the latter scheme, the bcr estimates in the three regions ranged from 0.54 to 0.61, indicating that telehomecare was not cost - saving . There were four studies reporting medical expenditure saved by telehomecare connecting public health nurses and the elderly with chronic diseases in one region [27,43 - 45]. Based on the claims data, these studies showed that treatment periods were shortened by telemedicine . Their estimates of reduced medical expenditure ranged from $148 to $629 per case per year for lifestyle diseases [27,43 - 45]. A single research team produced three survey studies and measured patients wtp for a telehomecare system that combines vital - signs monitoring and doctor consultation . The wtp estimates were $288, $480, and $520 per case per year, depending on three analytical methods . There was only one study conducted in an urban area to estimate users wtp for home tele - health monitoring . This study, published in 2012, showed wtp as low as $109 per patient per year . One cma study on home - palliative care in an md patient setting reported that telemedicine could reduce the frequency of physician visits from twice to once a week . The cost - saving of home palliative care with telemedicine were estimated to be $5000 per person per year compared to conventional home care . A sensitivity analysis of this study showed their results to be sensitive to two parameters, the number of user patients and duration of service provided . Based on the reported benefit, we calculated the bcr to be 1.93 . One study group published two cba studies on the same type of tele - homecare service operated in different regions with different financial schemes: (a) one region where a monthly fee was charged and (b) three other regions where no fee was charged because of public subsidies . Under the former scheme, the bcr was calculated to increase monotonically over time, that is, 1.07 (6-year operation) and 1.28 (11-year operation), using wtp estimates among patients in this region . Under the latter scheme, the bcr estimates in the three regions ranged from 0.54 to 0.61, indicating that telehomecare was not cost - saving . There were four studies reporting medical expenditure saved by telehomecare connecting public health nurses and the elderly with chronic diseases in one region [27,43 - 45]. Based on the claims data, these studies showed that treatment periods were shortened by telemedicine . Their estimates of reduced medical expenditure ranged from $148 to $629 per case per year for lifestyle diseases [27,43 - 45]. However, the input costs of telehomecare were not shown in these four studies . A single research team produced three survey studies and measured patients wtp for a telehomecare system that combines vital - signs monitoring and doctor consultation . The wtp estimates were $288, $480, and $520 per case per year, depending on three analytical methods . There was only one study conducted in an urban area to estimate users wtp for home tele - health monitoring . This study, published in 2012, showed wtp as low as $109 per patient per year . Table 4 summarizes our quality assessment of reviewed studies in terms of the ten criteria of the well - established checklist from drummond et al . . There were only four studies that met more than eight criteria, and eleven studies met only three or fewer criteria . Studies of cba and cma tended to meet more criteria than studies that measured only the benefits of telemedicine . Only two studies conducted a sensitivity analysis, and hence met the two criteria of uncertainty estimates and study results including all issues of concern . Figure 2 was created to detect a potential publication bias among the reviewed papers . A bold vertical line in this figure represents a threshold bcr value of one (also called a break - even point). A plot located rightward of this threshold vertical line indicates a favorable economic efficiency level for a telehealth program evaluated in the published study . Since the plots were skewed to the right of the threshold vertical line (i.e., bcr greater than one), figure 2 implies a moderate bias toward publishing more studies with a higher bcr estimate for a tele - health intervention . It should be noted that our interpretation regarding such potential publication bias has limited validity due to the small number of publications with bcr estimates . A bold vertical line in this figure represents a threshold bcr value of one (also called a break - even point). A plot located rightward of this threshold vertical line indicates a favorable economic efficiency level for a telehealth program evaluated in the published study . Since the plots were skewed to the right of the threshold vertical line (i.e., bcr greater than one), figure 2 implies a moderate bias toward publishing more studies with a higher bcr estimate for a tele - health intervention . It should be noted that our interpretation regarding such potential publication bias has limited validity due to the small number of publications with bcr estimates . To our knowledge, this is the first systematic review to summarize the economic efficiency of japanese telemedicine services and to examine the methodological rigorousness of the economic evaluation studies conducted in japan . Despite the small number and the diverse quality of economic evaluations of telemedicine, most of these evaluations indicated a favorable level of economic efficiency, that is, net cost - saving, reduced medical expenditure, and/or positive wtp . Among studies on md md live consultations in hospital settings, the most economically efficient telemedicine was emergency radiotherapy consultation about malignant spinal cord compression . This study s results were consistent with another effectiveness study of spinal cord compression in canada, in terms of decreasing patient transfer and shortening treatment decision time . However, the robustness of the reported bcr magnitude (> 4.67) is uncertain due to the absence of sensitivity analyses . Md hospital setting was teledermatology consultation ($17 160 per case per year). This study addressed parameter uncertainty and indicated that whether teledermatology is cost - saving or not depends on the travel time and consultation time of physicians . These results do not contradict the literature reporting mixed results; that is, teledermatology cost is either lower or higher [52 - 55] than conventional care costs . For an md patient home setting, bcr of telemedicine - combination home palliative care was estimated to be 1.93, compared to the status quo . This cma was methodologically rigorous with regard to (a) a clear statement of the purpose and perspective of the study, (b) a detailed illustration of the cost estimation, and (c) the implementation of a sensitivity analysis . This study in japan is one of the few studies that explicitly indicated telemedicine to be cost - saving when used to supplement in - person home visits by physicians . Similar real - time, audio - visual communication services for home palliative care have been successfully used for some years in the us [56, 57] and australia, while investigation in canada and pediatric telepalliative care in australia faced difficulties . In the us, the cost - saving of tele - palliative care was reported as due to a reduced number of home visits by nurses . Regarding telehomecare among the elderly with chronic diseases, two cbas reported cost - saving when implemented in one region (bcr, 1.07 to 1.28) where patients wtp was the only outcome measured . It should be noted that an identical system deployed in three other regions with public subsidies showed less economic efficiency (bcr 0.61) due to lower wtps (37% to 71% of the similar one - region system above) and higher system costs (186% to 195% of the one - region system). The reason for the mixed bcr results can be partly attributed to the public subsidy rule . The restrictive rules of public subsidy are exemplified by the prohibition on carrying over the budget to a second year, which may have caused over - utilization of the first year cost and hence ultimately decreased bcr . The methodological rigorousness of the reviewed studies tended to deviate from the established guidelines for economic evaluations [31,32,61 - 63]. For instance, only five studies [34 - 36,40,48] clarified the complete cost measurements of installing and operating telemedicine, which should have been compared with reduced medical expenditure due to the implementation of a telemedicine strategy such as a home telecare system (reduced by $148 to $629 per case per year) [27,43 - 45] and teleradiology ($144 per ct reading). Therefore, other than these five studies, it seemed very difficult to determine the accurate economic efficiency level of telemedicine among the reviewed studies . Moreover, none of the reviewed studies implemented a cea or a cua of the telemedicine program under focus . Therefore, it is impossible to judge whether a program is reasonably cost - effective or not when it is not cost - saving . Another example of a common limitation among the reviewed studies is the absence or very limited application of a probabilistic analysis such as a monte carlo simulation in order to address the uncertainty of multiple key parameters on cost, effectiveness, and benefit, simultaneously . In addition to the small number of total publications on the topic in focus, there are even fewer studies that examined comparable types of telemedicine technology and outcomes . This limited number of studies prevented us from performing a quantitative summary of the literature, such as a meta - analysis . We believe that the following types of future studies would be useful for an international audience as well as a japanese one . To convince policymakers to initiate and continue public subsidies for telemedicine, researchers are expected to produce more methodologically rigorous empirical evidence, presented in a reader - friendly manner . Due to diverse costs and benefits depending on setting, a probabilistic analysis result is useful to report; for example, cost - saving is achieved by telemedicine in 57% of 5000 iterations in emergency departments and 37% of 1000 iterations in intensive care units . Moreover, hospital level administrators as well as policy - makers would find it easy to understand a concrete and realistic scenario; for example, treating 10 acutely ill and injured children at a rural emergency department with telemedicine will lead to annual cost - saving of $46 620 per emergency department . These decision makers would also appreciate specific benchmarks to achieve cost - saving, such as maximum allowable cost derived from break - even analyses . Since telemedicine in a probabilistic analysis is less likely to achieve cost - saving in 100% of iterations, a cea needs to be implemented to test whether telemedicine is reasonably cost - effective or not . To make cea results of telemedicine comparable with other types of health care, a future study is expected to estimate an incremental cost - effectiveness ratio (icer) with a unit of dollar value per additional quality - adjusted life year (qaly) by the us expert panel on cost - effectiveness in health and medicine [31,61 - 63]. A certain telemedicine care strategy is concluded to be cost - effective if its icer is lower than a commonly used threshold of $100 000 per qaly . These estimates concerning cea are also appreciated by policymakers and insurers who are interested in maximizing health outcomes under a fixed budget . Other than improving the methodological rigorousness of economic evaluations, our review demonstrated the importance of wtp measured among users and public subsidy rules . The measured wtp could be used as a proxy for a future user charge, which is particularly important if the wtp exceeds the actual out - of - pocket payment for telemedicine . Partly because of the difference in the surveyed populations, wtp for a similar health - monitoring telemedicine care strategy declined by 75 percentage points from 2003 to 2010 . Potential reasons for the decline include the decreased japanese purchasing power due to long - term macro - economic recession and users observations of continuously falling prices in other it products and services . Thus, further studies are needed in measuring wtp under various and detailed scenarios as well as addressing the impact of public subsidy regulation on bcr . These studies could help justify increased public financial support and hence improve the financial sustainability of telemedicine programs . In conclusion, compared to the substantial resources deployed for more than 1000 telemedicine trials in japan, very limited resources have been deployed for the economic evaluation of telemedicine trials . Although most reviewed economic evaluations reported a favorable level of economic efficiency, their methodological rigorousness tended to vary . Further economic evaluation studies in this field are needed with more advanced methodological rigorousness and expanded research scope (e.g., public subsidy regulation impacts). These future studies are expected to help convince various decision makers such as policymakers, insurers and hospital administrators to invest further resources in telemedicine . Consequently, the general financial sustainability of telemedicine is expected to improve in the long term.
The present case - nud study was conducted in a tertiary - care multispecialty teaching hospital in central india . The present study was conducted among patients with ibs or nud referred from the departments of medicine and surgery after appropriate investigations . The investigations commonly conducted included hemogram, liver function tests, kidney function tests, ultrasound abdomen, and where required, upper or lower gi endoscopy . The diagnosis is clinically made by specialist based on findings on history, examination, and investigations . For the purposes of the study, the inclusion criteria for cases were: age between 15 and 65 years, diagnosis of ibs according to rome ii criteria, and duration of illness of more than 1 year . Nuds comprised patients aged 1565 years diagnosed with nud of more than 1-year duration . Those patients having comorbid medical illnesses, having a clinical history consistent with subnormal intelligence, being already on psychotropic medications, being pregnant or nursing those patients fulfilling the inclusion and exclusion criteria were recruited in the study after obtaining informed consent . Information was obtained from both groups about the demographic characteristics such as age, gender and marital status, illness characteristics, meal habits, and dietary patterns (vegetarian or nonvegetarian). The type of ibs was classified as diarrhea - predominant, constipation - predominant, or alternating type . The meal habits were coded as regular or irregular based on the patient self - report . The diagnosis of psychiatric disorders was made in accordance to the structured clinical interview for dsm - iv axis 1 disorders (scid - i). Anxiety and depression were assessed using the hamilton anxiety rating scale (ham - a) and the hamilton depression rating scale (ham - d), respectively . The presumptive stressful life events scale (psles) was used to assess stressors in this population . Information was collected primarily from the patients and was supplemented by the data from medical records and other accompanying family members . Scid - i is a semi - structured interview for making the major dsm - iv axis i diagnoses . The clinical version (scid - cv) assists in making standardized psychiatric diagnoses using specific probe questions . The scid - cv has six self - contained modules covering mood episodes, psychotic symptoms, psychotic disorders, mood disorders, substance use disorders, and anxiety and other disorders . The questionnaire takes about 15 min to administer and had shown good reliability and validity . Ham - a is a widely used interviewer - rated scale for assessment of anxiety . It consists of 14 items, and the total scores can range from 0 to 56 . The ham - d is a scale for measuring depression that has been very commonly been used in the published literature . The total scores can range from 0 to 53, and severity of depression can be determined based on the scores . Frequencies, percentages, means, and standard deviations were used to depict demographic and clinical variables . The cases and nuds were compared using appropriate parametric and nonparametric tests (-test, student's t - test or mann whitney u - test). Multivariate logistic regression analysis was conducted to find the independent predictors of having an scid - i psychiatric diagnosis in the combined sample of cases and nuds . The regression analysis was conducted to minimize the effect of confounding variables across the cases and nuds . For all the inferential statistics, a two - tailed test was used, and a p <0.05 was considered statistically significant . The present case - nud study was conducted in a tertiary - care multispecialty teaching hospital in central india . The present study was conducted among patients with ibs or nud referred from the departments of medicine and surgery after appropriate investigations . The investigations commonly conducted included hemogram, liver function tests, kidney function tests, ultrasound abdomen, and where required, upper or lower gi endoscopy . The diagnosis is clinically made by specialist based on findings on history, examination, and investigations . For the purposes of the study, the inclusion criteria for cases were: age between 15 and 65 years, diagnosis of ibs according to rome ii criteria, and duration of illness of more than 1 year . Nuds comprised patients aged 1565 years diagnosed with nud of more than 1-year duration . Those patients having comorbid medical illnesses, having a clinical history consistent with subnormal intelligence, being already on psychotropic medications, being pregnant or nursing those patients fulfilling the inclusion and exclusion criteria were recruited in the study after obtaining informed consent . Information was obtained from both groups about the demographic characteristics such as age, gender and marital status, illness characteristics, meal habits, and dietary patterns (vegetarian or nonvegetarian). The type of ibs was classified as diarrhea - predominant, constipation - predominant, or alternating type . The meal habits were coded as regular or irregular based on the patient self - report . The diagnosis of psychiatric disorders was made in accordance to the structured clinical interview for dsm - iv axis 1 disorders (scid - i). Anxiety and depression were assessed using the hamilton anxiety rating scale (ham - a) and the hamilton depression rating scale (ham - d), respectively . The presumptive stressful life events scale (psles) was used to assess stressors in this population . Information was collected primarily from the patients and was supplemented by the data from medical records and other accompanying family members . Scid - i is a semi - structured interview for making the major dsm - iv axis i diagnoses . The clinical version (scid - cv) assists in making standardized psychiatric diagnoses using specific probe questions . The scid - cv has six self - contained modules covering mood episodes, psychotic symptoms, psychotic disorders, mood disorders, substance use disorders, and anxiety and other disorders . The questionnaire takes about 15 min to administer and had shown good reliability and validity . Ham - a is a widely used interviewer - rated scale for assessment of anxiety . It consists of 14 items, and the total scores can range from 0 to 56 . The ham - d is a scale for measuring depression that has been very commonly been used in the published literature . The total scores can range from 0 to 53, and severity of depression can be determined based on the scores . Frequencies, percentages, means, and standard deviations were used to depict demographic and clinical variables . The cases and nuds were compared using appropriate parametric and nonparametric tests (-test, student's t - test or mann whitney u - test). Multivariate logistic regression analysis was conducted to find the independent predictors of having an scid - i psychiatric diagnosis in the combined sample of cases and nuds . The regression analysis was conducted to minimize the effect of confounding variables across the cases and nuds . For all the inferential statistics, a two - tailed test was used, and a p <0.05 was considered statistically significant . The most common essential symptoms of ibs in the present sample of cases included abdominal discomfort or pain relieved with defecation (n = 29), onset associated with a change in frequency of stools (n = 25), and onset associated with a change in the form of stools (n = 20). The associated symptoms included abnormal stool form (n = 42), abnormal stool frequency (n = 41), abnormal stool passage (n = 40), bloating sensation (n = 38), and passage of mucus (n = 30). The demographic and clinical characteristics of the cases and nuds are depicted in table 1 . The cases were more likely to be of female gender (p = 0.012), married (p = 0.009), employed (p <0.001), have irregular meals (p = 0.003), and have comparatively longer duration of illness (p = 0.004). There were no significant differences in age, education, socioeconomic status, family type, residence, and food habits across the groups . Demographic and clinical characteristics of the cases and nonulcerative dyspepsias the psychiatric diagnosis, stressors, and symptoms of anxiety and depression are depicted in table 2 . Psychiatric diagnoses were more common in the ibs group than nuds (p <0.001). The most common psychiatric diagnosis was major depression, followed by somatization disorder . Having a stressor did not significantly differ between the groups, though family and financial stressors were more common in the cases of ibs . Symptoms of anxiety and depression were more common in the group of patients with ibs than nuds (p <0.001 for both the symptoms). Psychiatric diagnosis, stressors, and symptoms of anxiety and depression to nud, for the differences between the groups, binary logistic regression analysis was conducted to find the independent predictors of having a psychiatric illness . Age, gender, group status (cases vs. nuds), presence of stressors, and duration of the illness were used as covariates . The model revealed that having a diagnosis of ibs (odds ratio [or]: 21.69, 95% confidence intervals [cis] from 7.14 to 66.67) and increased age (or: 1.04, ci: 1.041.90) were independent predictors of having a psychiatric illness according to scid . The nagelkerke r of the sample was 0.460, suggesting that the model explained 46% of the variance . Demography of the sample points toward preponderance of females in the group of patients with ibs . This is similar with the previous findings of females, being more common among patients with ibs . The ibs group had a greater proportion of patients who were married and employed, probably reflecting greater adaptation to the chronic illness . The meals were comparatively irregular in patients with ibs, a factor which has been suggested to be associated with exacerbation of symptoms . About 88% of the sample had additional diagnosis using scid, which is in line with some other studies which have found very high rates of psychiatric comorbidity in patients with ibs . The disorders that were encountered frequently included depression, anxiety, somatization disorder, and substance use disorder, which is in agreement with the previous literature . The present study finds that the rates of psychiatric diagnosis were higher than patients with nud, even after controlling for other variables . Two previous studies, however, did not find differences in psychiatric morbidity between patients with ibs and peptic ulcer disease . Having a stressor did not significantly differ between the groups . Another study has reported similar findings wherein stressful life events were not significantly different between patients with ibs and nonulcer dyspepsia . Studies to the contrary exist suggesting that patients with ibs have been found to have higher stress as compared to nuds, and life stressors are associated with increased symptoms of ibs . The relationship between stress and symptoms of ibs could be bidirectional with stressors potentially worsening symptoms of ibs and symptoms of gut dysmotility leading to further health - related stress . Symptoms of anxiety and depression were quite common in the present sample of patients with ibs . All of the patients with ibs had symptoms consistent with mild anxiety, and 96% of the samples had symptoms of at least mild depression in the ibs group . This was far higher than the nud group with symptoms of at least mild anxiety and depression in 48% and 28% of the samples, respectively . A large proportion of patients with ibs have been observed to have high rates of anxiety and depression symptoms and diagnosis, consistent with the present findings . It must be remarked that severe anxiety or depression was uncommon, and most patients had mild to moderate degree of these symptoms . Many factors can potentially explain the relationship of increased rates of psychiatric illnesses in patients with ibs . First, the gut pathology and the psychiatric symptoms may have a common etiological mechanism . Gut motility is mediated by the enteric nervous system which has intricate links with the central nervous system . Hence, it is possible that biological vulnerabilities for psychiatric disorders due to their impact on cns may also be associated with gut dysmotility which manifests as ibs . Second, a pattern of psychological processes which include obsessionality and anxious preoccupation about health may predispose an individual to develop ibs subsequently . Third, symptoms of ibs may impair the work performance of the individual, leading to invalidism and subsequently leading to anxiety and depressive disorders . Probably, all the three mechanisms have a role and have been discussed in reviews elsewhere . The strengths of the study include using a comparison group to represent the rates of psychiatric disorders in context, systematic assessment for psychiatric disorders using a structured diagnostic instrument, and using patient population from a tertiary care hospital where the diagnosis of ibs is likely to be more confident based on available investigations . Some limitations of the present study should also be considered while drawing interpretations . The study reflects findings of a single center, and generalization to other centers or community requires caution . Personality factors, health - care - seeking patterns, cultural beliefs, explanations of the illness, coping, quality of life measures, and dysfunction are other related variables that were not systematically assessed in the present study and may have implications on the presentation and management of the disorder . The authors had to restrict the number of determinants studied to elicit the adequate cooperation of the patients and complete assessments without dropouts during multisession assessments . The present study adds to the limited literature of psychiatric comorbidity in patients with ibs in the south asian region . High rates of psychiatric comorbidity were encountered though stressors were not significantly higher than nuds . Anxiety and depressive symptoms were higher in cases than nuds . Further studies are required to systematically assess the impact of prompt assessment and management of psychiatric disorder in patients with ibs on the quality of life and general level of functioning.
Hepatitis e virus (hev) is an important cause of acute clinical hepatitis in endemic countries and can lead to detrimental prognostics, such as severe acute hepatitis, liver failure, chronicity in immunocompromised patients, and death in pregnant women (1, 2). Genotypes 1 and 2 are strictly human, transmitted by fecal oral route, and responsible for water - borne hev outbreaks in asia, africa, and central america . Hev genotypes 3 and 4 are found in swine and other animal species in many countries where direct and indirect zoonotic transmission in industrialized countries have been reported (3 - 6). The disease is considered emerging in many parts of the world because of the increased awareness and availability of effective diagnostics . The hev seroprevalence has been found to be elevated even in areas classified non - endemic (7). North africa is a region classified as endemic for hepatitis e. for instance, half of the population aged above five years in egypt is serologically positive for hev (8), and high hev seroprevalence was reported in tunisia, libya, and morocco (9). The first one occurred in mostaganem (northwest algeria) in 1980 and the second in tanefdour (northeast algeria) in 1986 - 1987 (10, 11). Both epidemics were traced back to contaminated water sources, and the causative pathogen was hev genotype 1 . Another outbreak of hepatitis non - a and non - b occurred in medea (northern algeria) from october 1980 to january 1981, and it affected 788 people, mostly young adults, with a mortality rate of 100% in pregnant women (12). Although the causative pathogen in this latter outbreak was not identified, it was likely to be hev given the circumstances of the occurrence and the development of the disease . . Moreover, no studies on hev or anti - hev antibodies have been conducted, and hev infection is not investigated in the hospitals when assessing acute hepatitis . Therefore, this study aimed to investigate the presence of anti - hev antibodies in northern algeria, determine the hev genotype(s) of the circulating strains, and elucidate the contamination routes (zoonotic and/or water - borne). Sample size was calculated to be 517 based on an anticipated anti - hev igg rate of 15%, a margin of error of 3%, a confidence level of 95%, and a population size of approximately 10,000 (information from the commune chiefs on the total population depending on the three health facilities). The blood samples were collected from three hospitals in central algeria: 379 samples from blood donors and 211 samples from outpatients . This study was conducted in accordance with the national ethics regulation and was approved by the research ethics committee of southeast university in nanjing . The truncated p166 capsid protein was generated from the amino acid position 452617 of the open reading frame 2 of the following hev strains: w01 (genotype 1, jx857689), mexico-14 (genotype 2, m74506), us-1 (genotype 3, af060668), and china-9829 (genotype 4, ay789225) strains, and expressed in escherichia coli (13). Briefly, the polymerase chain reaction fragment encoding aa 452 - 617 of the hev strains was inserted into the pet28a vectors (novagen, darmstadt, germany). Then, the plasmids were used to transform competent e. coli bl21 (de3) cells (promega, madison, usa). After the confirmation of the sequence of aa 452 - 617 in the plasmids by dna sequencing, the gene expression was induced . The suspension was clarified by centrifugation, and then the supernatant was loaded onto a column containing ni - nta super flow affinity resin . The column was washed, and the fusion proteins were eluted as described previously (14). The four p166 proteins were designated as p166w01, p166mex, p166us, and p166chn, and a mixture (p166mix) containing equal concentrations of each of the four p166 proteins was prepared . Sera were screened for the presence of anti - hev antibodies with a high performance assay, namely, the in - house sandwich enzyme immunoassay, according to dong et al . Briefly, microplate wells were coated with his - p166 mix and incubated at room temperature overnight . Unbound antigens were washed with 10 mm phosphate - buffered saline containing 0.05% tween 20 (pbs - t). Then, undiluted test serum was added, and the plates were incubated at 37c for 1 hour . After a washing step with pbs - t, the horseradish peroxidase (hrp)-conjugated p166 mix was added, and the plates were incubated at 37c for 1 hour . After washing, tetramethylbenzidine was added as substrate, and the plates were read using a kinetic microplate reader at a wavelength of 450 nm . All sera were tested in duplicate, and a signal / cutoff (s / co) value of 1 was considered a positive reaction . The presence of anti - hev igm antibodies was also assessed as previously described (15). Briefly, the purified p166 proteins were used as antigens to coat microplate wells . After an incubation period of 2 h at 37c, followed by three washings with pbs containing 0.05% tween 20, after three washings, the hrp - conjugated goat anti - human igm (kpl) was added to each well and incubated at 37c for 1 hour . After a final washing, the colorimetric reactions were developed using tetramethylbenzidine substrate (sigma) for 15 minutes at room temperature and stopped with 2 m h2so4 . The p166 proteins generated from the four genotypes were used as antigens, and each p166 protein was used in a separate analysis as previously described (16). Briefly, the purified his - p166 proteins were used as antigens to coat microplate wells . After an incubation period of 2 hours at 37c, followed by three washings with pbs containing 0.05% tween 20, test and control sera serial dilutions (1: 200, 1: 400, 1: 800, 1: 1600, 1: 3200, and 1: 6400) were distributed into wells and incubated for 1 h at 37c . After three washings, the hrp - conjugated goat anti - human igg (kpl) was added to each well and incubated at 37c for 1 hour . After a final washing, the colorimetric reactions were developed using tetramethylbenzidine substrate (sigma) for 15 minutes at room temperature and stopped with 2 m h2so4 . The plates were read using a kinetic micro - plate reader at a wavelength of 450 nm . P <0.05 was considered statistically significant . For the cross - genotype neutralization assay, two - way anova with bonferroni posttest was performed using graphpad prism version 5.00 for windows (graphpad software, san diego, ca, usa). Sample size was calculated to be 517 based on an anticipated anti - hev igg rate of 15%, a margin of error of 3%, a confidence level of 95%, and a population size of approximately 10,000 (information from the commune chiefs on the total population depending on the three health facilities). The blood samples were collected from three hospitals in central algeria: 379 samples from blood donors and 211 samples from outpatients . This study was conducted in accordance with the national ethics regulation and was approved by the research ethics committee of southeast university in nanjing . The truncated p166 capsid protein was generated from the amino acid position 452617 of the open reading frame 2 of the following hev strains: w01 (genotype 1, jx857689), mexico-14 (genotype 2, m74506), us-1 (genotype 3, af060668), and china-9829 (genotype 4, ay789225) strains, and expressed in escherichia coli (13). Briefly, the polymerase chain reaction fragment encoding aa 452 - 617 of the hev strains was inserted into the pet28a vectors (novagen, darmstadt, germany). Then, the plasmids were used to transform competent e. coli bl21 (de3) cells (promega, madison, usa). After the confirmation of the sequence of aa 452 - 617 in the plasmids by dna sequencing, the gene expression was induced . The suspension was clarified by centrifugation, and then the supernatant was loaded onto a column containing ni - nta super flow affinity resin . The column was washed, and the fusion proteins were eluted as described previously (14). The four p166 proteins were designated as p166w01, p166mex, p166us, and p166chn, and a mixture (p166mix) containing equal concentrations of each of the four p166 proteins was prepared . Sera were screened for the presence of anti - hev antibodies with a high performance assay, namely, the in - house sandwich enzyme immunoassay, according to dong et al . Briefly, microplate wells were coated with his - p166 mix and incubated at room temperature overnight . Unbound antigens were washed with 10 mm phosphate - buffered saline containing 0.05% tween 20 (pbs - t). Then, undiluted test serum was added, and the plates were incubated at 37c for 1 hour . After a washing step with pbs - t, the horseradish peroxidase (hrp)-conjugated p166 mix was added, and the plates were incubated at 37c for 1 hour . After washing, tetramethylbenzidine was added as substrate, and the plates were read using a kinetic microplate reader at a wavelength of 450 nm . All sera were tested in duplicate, and a signal / cutoff (s / co) value of 1 was considered a positive reaction . The presence of anti - hev igm antibodies was also assessed as previously described (15). Briefly, the purified p166 proteins were used as antigens to coat microplate wells . After an incubation period of 2 h at 37c, followed by three washings with pbs containing 0.05% tween 20, after three washings, the hrp - conjugated goat anti - human igm (kpl) was added to each well and incubated at 37c for 1 hour . After a final washing, the colorimetric reactions were developed using tetramethylbenzidine substrate (sigma) for 15 minutes at room temperature and stopped with 2 m h2so4 . The p166 proteins generated from the four genotypes were used as antigens, and each p166 protein was used in a separate analysis as previously described (16). Briefly, the purified his - p166 proteins were used as antigens to coat microplate wells . After an incubation period of 2 hours at 37c, followed by three washings with pbs containing 0.05% tween 20, test and control sera serial dilutions (1: 200, 1: 400, 1: 800, 1: 1600, 1: 3200, and 1: 6400) were distributed into wells and incubated for 1 h at 37c . After three washings, the hrp - conjugated goat anti - human igg (kpl) was added to each well and incubated at 37c for 1 hour . After a final washing, the colorimetric reactions were developed using tetramethylbenzidine substrate (sigma) for 15 minutes at room temperature and stopped with 2 m h2so4 . The plates were read using a kinetic micro - plate reader at a wavelength of 450 nm . P <0.05 was considered statistically significant . For the cross - genotype neutralization assay, two - way anova with bonferroni posttest was performed using graphpad prism version 5.00 for windows (graphpad software, san diego, ca, usa). Although a slight difference was found in the prevalence rates between males and females (table 1), statistically no significant correlation was noted between hev seroprevalence and subjects gender (p = 0.5). The results showed that majority of the positive cases were aged between 21 and 60 years old and that no positive case was found from 0 to 10 years old . However, this result must be taken with caution given the small number of sample in this group (only four). However, compared with the group of 70 years, which also had a small number of sample (n = 11), four positive cases were detected . To better appreciate these results, they are represented as percentages compared with the total number of sample in each age group . The groups of 21 - 30, 31 - 40, and 41 - 50 years almost had equal rates at 18.82%, 18.50%, and 22.37%, respectively, but these rates were significantly higher than that of the first group . The group of patients aged less than 20 years had the lowest rate (6.25%). The positivity rate continued to increase to reach a maximum of 29.76% in group 51 - 60 years to finally decrease to 25% in the last group (61 years). Logistic regression analysis showed a significant correlation between age of the patients and presence of anti - hev antibodies (p = 0.001) as shown in table 1 . Note that among the 379 blood donors (mean age 38.73, age range 18 - 65, 55.67% are men), 83 (21.9%) were diagnosed positive for anti - hev antibodies, 37 of whom were women . Testing the presence of anti - hev igm antibodies in all samples revealed only two weakly positive cases who were both blood donors (a 48-year - old woman and a 32-year - old man). In a second step, the subjects were grouped into three age groups according to whether they were born before or after the 1987 - 1988 and 1979 - 1980 outbreaks . The results showed that 24.29% of the subjects aged over 36 years (born before the first outbreak of 1978) were positive for the presence of anti - hev antibodies . This rate decreased to 17.4% in the subjects aged between 25 and 35 years (born after the 1978 outbreak and before the 1987 outbreak). The anti - hev antibody prevalence rate was only 9.9% for the subjects aged less than 25 years (born after the last outbreak). Statistically, a significant correlation was found between age and presence of anti - hev antibodies (p = 0.033). We previously assessed the immunoreactivity of anti - hev antibodies present in the serum sample collected from patients infected with different hev genotypes (genotype 1: n = 15, genotype 3: n = 12, and genotype: 4 n = 17) using p166 antigens generated from the four hev genotypes (16). The sera were serially diluted, and the anti - hev antibodies were detected by an indirect elisa . The four p166 proteins were used as antigens, and each p166 antigen was used in a separate experiment . The results revealed that the immunoreactivity of anti - hev antibodies raised against genotype 1 strains was stronger than that against the p166 antigens generated from genotypes 1 and 2 (p166w01 and p166mex) and that against the p166 antigens generated from genotypes 3 and 4 (p166us and p166chn). By contrast, the reaction of anti - hev antibodies raised against the zoonotic genotypes 3 and 4 was more significant than that against the p166 antigens generated from genotypes 3 and 4 (p166us and p166chn) as shown in figure 1 . Moreover, we exploited this immunoreactivity difference for the prediction of the hev genotypes in this serum panel collected in algeria . As expected, the detection of anti - hev igg antibodies in the positive sera using the different p166 antigens generated from the different hev genotypes revealed a variation of immunoreactivity (figure 2). The igg antibodies reacted strongly against p166w01 (generated from hev genotype 1) at all dilution titers . These results showed the obvious effects of the antigen origin on the igg - binding ability and suggested that the iggs were more likely to be raised against an hev genotype 1 strain (figure 2). A, sera of patients infected by hev genotype 1 strains; b, sera of patients infected by hev genotype 3 strains; c, sera of patients infected by hev genotype 4 strains; d, bonferroni multiple comparison test results . Each point represents mean sd; * p <0.05; * * p <0.01 and * * * p <0.001(16). The sera were serially diluted, and the anti - hev igg antibodies were assessed using the different p166 antigens . Each p166 antigen was used in a separate experiment . , , * * = p <0.05, 0.01, 0.001 when compared with p166w01 . Although a slight difference was found in the prevalence rates between males and females (table 1), statistically no significant correlation was noted between hev seroprevalence and subjects gender (p = 0.5). The results showed that majority of the positive cases were aged between 21 and 60 years old and that no positive case was found from 0 to 10 years old . However, this result must be taken with caution given the small number of sample in this group (only four). However, compared with the group of 70 years, which also had a small number of sample (n = 11), four positive cases were detected . To better appreciate these results, they are represented as percentages compared with the total number of sample in each age group . The groups of 21 - 30, 31 - 40, and 41 - 50 years almost had equal rates at 18.82%, 18.50%, and 22.37%, respectively, but these rates were significantly higher than that of the first group . The group of patients aged less than 20 years had the lowest rate (6.25%). The positivity rate continued to increase to reach a maximum of 29.76% in group 51 - 60 years to finally decrease to 25% in the last group (61 years). Logistic regression analysis showed a significant correlation between age of the patients and presence of anti - hev antibodies (p = 0.001) as shown in table 1 . Note that among the 379 blood donors (mean age 38.73, age range 18 - 65, 55.67% are men), 83 (21.9%) were diagnosed positive for anti - hev antibodies, 37 of whom were women . Testing the presence of anti - hev igm antibodies in all samples revealed only two weakly positive cases who were both blood donors (a 48-year - old woman and a 32-year - old man). In a second step, the subjects were grouped into three age groups according to whether they were born before or after the 1987 - 1988 and 1979 - 1980 outbreaks . The results showed that 24.29% of the subjects aged over 36 years (born before the first outbreak of 1978) were positive for the presence of anti - hev antibodies . This rate decreased to 17.4% in the subjects aged between 25 and 35 years (born after the 1978 outbreak and before the 1987 outbreak). The anti - hev antibody prevalence rate was only 9.9% for the subjects aged less than 25 years (born after the last outbreak). Statistically, a significant correlation was found between age and presence of anti - hev antibodies (p = 0.033). We previously assessed the immunoreactivity of anti - hev antibodies present in the serum sample collected from patients infected with different hev genotypes (genotype 1: n = 15, genotype 3: n = 12, and genotype: 4 n = 17) using p166 antigens generated from the four hev genotypes (16). The sera were serially diluted, and the anti - hev antibodies were detected by an indirect elisa . The four p166 proteins were used as antigens, and each p166 antigen was used in a separate experiment . The results revealed that the immunoreactivity of anti - hev antibodies raised against genotype 1 strains was stronger than that against the p166 antigens generated from genotypes 1 and 2 (p166w01 and p166mex) and that against the p166 antigens generated from genotypes 3 and 4 (p166us and p166chn). By contrast, the reaction of anti - hev antibodies raised against the zoonotic genotypes 3 and 4 was more significant than that against the p166 antigens generated from genotypes 3 and 4 (p166us and p166chn) as shown in figure 1 . Moreover, we exploited this immunoreactivity difference for the prediction of the hev genotypes in this serum panel collected in algeria . As expected, the detection of anti - hev igg antibodies in the positive sera using the different p166 antigens generated from the different hev genotypes revealed a variation of immunoreactivity (figure 2). The igg antibodies reacted strongly against p166w01 (generated from hev genotype 1) at all dilution titers . These results showed the obvious effects of the antigen origin on the igg - binding ability and suggested that the iggs were more likely to be raised against an hev genotype 1 strain (figure 2). A, sera of patients infected by hev genotype 1 strains; b, sera of patients infected by hev genotype 3 strains; c, sera of patients infected by hev genotype 4 strains; d, bonferroni multiple comparison test results . Each point represents mean sd; * p <0.05; * * p <0.01 and * * * p <0.001(16). The sera were serially diluted, and the anti - hev igg antibodies were assessed using the different p166 antigens . Each p166 antigen was used in a separate experiment . , , * * = p <0.05, 0.01, 0.001 when compared with p166w01 . To our knowledge, this study is the first to determine the presence and immunoreactivity of anti - hev antibodies in northern algeria and revealed a positivity rate of 20.17% . This prevalence is higher than those of the countries on the northern side of the mediterranean sea, such as france and italy (17, 18), but is relatively lower than those of neighboring countries on the southern side . In egypt, the prevalence of anti - hev antibodies reached 84.3% in pregnant women, 67.6% in rural areas, 56.4% in semi - urban areas, and 45.3% in blood donors (19 - 21). In morocco, the seroprevalence of hev is 46% in healthy people, 22% in blood donors, and 12% in pregnant women (23, 24). However, these results should be taken with caution because of the small number of subjects included in these studies . In the present study, no significant correlation was found between gender and presence of anti - hev antibodies, whereas a significant difference was found in seroprevalence among the different age groups . These results are similar to those previously reported in other studies conducted in various countries (25 - 27). This similarity is probably due to the comparable exposure of both sexes to the virus sources . However, exposure time is long in the elderly, and this long exposure increases the chances of contracting the virus, thus explaining the difference in hev prevalence among the different age groups . The presence of anti - hev antibodies in people under 25 years (9.9%) and the two cases that were weakly positive for anti - hev igm contradicted the exposure to virus during the last outbreaks (1979 - 1980 and 1987 - 1988). These circumstances explain the results and indicate clearly that hev infection is still present in algeria . Several strains of genotype 3 were isolated from humans and animals across different continents, where they cause sporadic cases mainly after the consumption of undercooked swine products . Several studies reported the isolation of hev from several other animal species (28 - 32). Except in swine, deer, rabbits, and mongooses, the distribution of genotype 3 and its dispersion throughout the world (33) raises the question of its presence in algeria . However, algeria, which is a muslim country, has no swine consumption and breeding, thus making the presence of genotype 3 unlikely . In this study, only two cases were weakly positive for anti - hev igm antibodies . According to (34), viral rna is no longer detectable at such a low rate of igm antibodies . To predict the genotype of the causative strain that infected the subjects, we exploited the immunoreactivity difference among the p166 proteins generated from the four genotypes as reported previously (16). We showed that the igg - binding ability is significantly stronger in the presence of antigens generated from the same genotype than from the genotype they were raised against . Using the same approach in this study, when the igg - positive sera were assessed by different p166 proteins, the antibodies showed a stronger immunoreactivity against p166w01, which was generated from a genotype 1 strain . Moreover, for the hev outbreaks that occurred in algeria (1979 - 1980 and 1987 - 1988), the isolated virus belonged to hev genotype 1, which contaminated the water sources after a period of intense rain (10, 11). Therefore, given the present results and the available history of hev in algeria, the presence of genotype 1 hev is clearly the most likely reason, and this genotype 1 strain(s) still causes sporadic cases . Recently, research on hepatitis e has been directed to investigate the risk of hev transmission via blood transfusions . Therefore, several studies on the seroprevalence of hepatitis e in blood donors were conducted (35 - 37). Although the positivity rates for anti - hev igm antibodies were relatively low, several cases of post - transfusion infection were reported (38 - 40). In this context, our study reveals a relatively high seroprevalence of anti - hev antibodies (21.9%) among blood donors, and only two cases weakly positive for anti - hev igm antibodies as discussed above were found . Given the high mortality rates in pregnant women and immune - compromised patients, detrimental effects will occur if these patients receive hev - contaminated blood products . However, making the screening of donated blood for the presence of hev as a mandatory test is still early, and more detailed investigations are required especially in endemic areas . In conclusion, we presented a new approach for the prediction of the genotype of hev strains circulating in a given region in seroprevalence studies using different antigens generated from the four genotypes . This pilot study on the field application of this method revealed that the sera positive for anti - hev antibodies presence reacted strongly against the antigens derived from hev genotype 1 . This finding indicates that hepatitis e in northern algeria is most likely caused by genotype 1 strains . Moreover, this study also revealed a relatively high seroprevalence of anti - hev antibodies within the targeted population in northern algeria . Therefore, to prevent future outbreaks, the management strategy of algerian clinicians in assessing acute hepatitis requires an urgent re - evaluation . Finally, this study raises several issues that require further investigation: assess the prevalence and incidence of hev infection throughout the algerian territory, identify the risk factors other than age (e.g., socioeconomic condition, working in animal breeding, working in the health sector, and co - infection with other pathogens), and evaluate the risk of transmission via blood donation.
Prostate cancer is the second most common cause of cancer death in men in the united states . In the united states, the age - adjusted number of new cases of prostate cancer was 147.8 per 100,000 men per year based on 2007~2011 data and the age - adjusted number of deaths was 23.0 per 100,000 men per year based on 2006~2010 data . According to 2008~2010 data, approximately 15.3% of men will be diagnosed with prostate cancer at some point during their lifetime, and in 2011, an estimated 2,707,821 men were living with prostate cancer in the united states.1 the most common sites of prostate cancer metastasis include the bone, lymph nodes, lungs, liver, pleura, and adrenal glands.2 rarely has metastatic prostate cancer been reported to involve the gastrointestinal tract.3,4,5,6,7,8,9,10,11 here, we report the case of a 64-year - old man with prostate cancer that had metastasized to the rectum and stomach . A 64-year - old african - american man presented with anemia (hemoglobin level of 6.8 g / dl) in september 2012 . He had a medical history of prostate cancer (gleason score of 5 + 4=9) diagnosed in june 2011, hypertension, coronary artery disease with stent placement, and insertion of bilateral percutaneous nephrostomy tubes due to hydronephrosis . The patient was being treated with flutamide and leuprolide acetate for prostate cancer since june 2011 . In september 2012, he presented with the complaint of dark - colored stools for a week . At that time, he did not experience any chest pain, shortness of breath, palpitations, dizziness, or headache, and no history of hemoptysis or hematemesis was reported . Physical examination of the patient was unremarkable except for the findings of a cachectic elderly man with pale conjunctiva and bilateral nephrostomy tubes . Laboratory data on admission revealed a hemoglobin level of 6.8 g / dl, hematocrit of 21%, platelet count of 281,000/mm, white cell count of 4,500/mm, and normal prothrombin time and activated partial thromboplastin time . Iron studies revealed a total iron level of 80 mg / dl, total iron binding capacity of 227 mg / dl, ferritin level of 357 ng / ml, and reticulocyte count of 0.9% . A complete metabolic profile showed a blood urea nitrogen level of 54 mg / dl and a creatinine level of 3.3 mg / dl . The patient had a computed tomography (ct) scan performed without contrast on his abdomen and pelvis . The ct scan revealed multiple cystic lesions in the liver, marked thickening of the wall of the stomach (fig . The patient also underwent a colonoscopy, which revealed circumferential nodularity and poor distensibility of the rectal lumen (fig . Biopsy of the rectum indicated diffuse infiltration of high - grade neoplastic cells that were positive for human prostatic acid phosphatase and prostate - specific membrane antigen . These findings were consistent with a diagnosis of metastatic, poorly differentiated carcinoma of the prostate . Since it was known that the patient had hormone - refractory prostate cancer, he was started on docetaxel and received 3 cycles until february 2013, at which point he was lost to follow - up . In may 2013, microscopic examination revealed sheets of atypical cells with prominent nucleoli and no glandular pattern within the gastric lamina propria (fig . Immunohistochemical studies demonstrated that the tumor cells were positive for prostate specific antigen (psa) and alpha - methylacyl - coenzyme a racemase (amacr, p504s) (fig . The patient's serum psa level was more than 1,000 mg / dl at that time . The bone scan, as well as the ct scan of the abdomen and pelvis, showed no metastases but showed retroperitoneal lymphadenopathy . At that time, the patient had an eastern cooperative oncology group performance status of 3 . The patient and family refused further chemotherapy for metastatic prostate cancer and opted for palliative care instead . The patient was admitted multiple times for similar complaints and was managed with supportive care . Prostate cancer is the most common noncutaneous cancer in men in the united states and is among the most commonly diagnosed cancers in many developed countries . Classic risk factors for this cancer include older age, african - american race / ethnicity, and a family history of prostate cancer . The increase in prostate cancer incidence rates in groups that have migrated from countries with low rates to countries with high rates strongly suggests the importance of environmental factors in its etiology . Kolonel12 reported that the the incidence rates of prostate cancer steadily increased in the japanese group with migration from mainland japan to hawaii, and the united states mainland in that order based on the ethnic studies . The majority of men are diagnosed with prostate cancer at an age older than 65 years, and the vast majority of prostate cancer deaths occur in this older age group . The median age at prostate cancer diagnosis is 71 years in caucasians and 69 years in african - americans in the united states . Metastatic prostate cancer has a poor prognosis and median survival time ranges from 1 to 3 years.3 prostate cancer preferentially spreads to the skeleton . More than 80% of men who die from prostate cancer are identified with bone metastases at autopsy.13 in contrast to most other cancers, prostate cancer predominantly forms osteoblastic metastases . The vertebral column, pelvis, ribs, and proximal long bones are the most common sites of skeletal metastases . Hematogenous, lymphatic, and direct infiltrations are the typical routes of spread.14 patients with prostate cancer can be anemic due to bone marrow involvement . Metastasis of prostatic carcinoma to the gastrointestinal tract is a very rare occurrence and presents a diagnostic challenge.6,7,8,9,10,11,13,14 because the prostate is richly supplied with lymphatic channels, metastasis to the gastrointestinal tract may occur via the lymphatic route . This should be taken into consideration during the work up for anemia in a patient with advanced prostate cancer, as supportive management will depend on the etiology of the anemia . In patients with suspected gastrointestinal bleeding or signs of iron deficiency anemia, upper and lower endoscopy two postmortem studies reported gastric metastasis from primary prostate cancer in 1% to 4% of cases.15,16 histologically, primary adenocarcinoma of the stomach is composed of atypical glands with cribriform (back to back) formation and a mucin - producing infiltrating growth pattern . Carcinoma in situ or high - grade dysplasia is usually found adjacent to the carcinoma . In contrast, tumor cells of metastatic carcinoma from prostate cancer usually have prominent nucleoli and no glandular formation . The prostate biomarker amacr has been used in conjunction with morphology with very high sensitivity and specificity in diagnostically challenging cases . Amacr, also known as racemase or p504s, is an enzyme identified by cdna subtraction and microarray technology . It is a sensitive and specific immunohistochemical marker that has been found to be consistently up - regulated in prostate carcinoma.17,18 treatment for metastatic prostate cancer is palliative . Several new agents have been introduced for the treatment of metastatic prostate cancer in the past two decades, with excellent disease control and good patient tolerability . The management of advanced prostate cancer with metastases to the gastrointestinal tract includes local control measures, supportive care, and treatment of the underlying cancer . If the disease progresses and hormone - refractory metastatic prostate cancer is diagnosed, alternative treatments include chemotherapy, immunotherapy with sipuleucel - t, androgen receptor antagonist drugs such as enzalutamide, and androgen synthesis inhibitors such as abiraterone . Although rare, it is important to consider the possibility of prostate carcinoma metastasizing to the gastrointestinal tract in patients presenting with gastrointestinal bleeding and a history of prostatic adenocarcinoma . It is crucial to distinguish primary gastrointestinal cancer from metastatic lesions, especially in cases of a previous history of cancer at another site, for appropriate management . This can be achieved by determining the histopathologic classification of the tumor and by immunohistochemical staining for psa.
Functional residual capacity, frc, is the lung volume at the end of a relaxed expiration to atmospheric pressure . The frc is affected by lung diseases and thus is a central measure for diagnosis, treatment and monitoring in anesthesia and intensive care . The calculation of frc from measurement of multiple breath nitrogen washout (mbnw) when abruptly changing the fraction of inspired oxygen (fio2) was introduced by darling . The method entailed the measurement of the mixed expiratory fraction of nitrogen before and after a step increase in oxygen and can be explained with the following simple example . The inspired fraction of oxygen, fio2, is 0.21 and the balance is n2, i.e. Fin2 = 0.79 . Assume dry alveolar fractions of o2 = 0.16, fao2, of n2 = 0.79, fan2, and of co2 = 0.05, faco2 . If the equilibrium between fin2 and fan2 is disrupted by having the person inhale at fio2 of 1 while exhaling into a bag, eventually all alveolar nitrogen will be transferred to the bag . Frc equals vn2 divided by the change in fraction of nitrogen that was in the lung: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{frc}} = {{{\rm{v}}{{\rm{n}}_{\rm{2}}}{\rm{bag}}} \over {\delta {{\rm{f}}_{\rm{a}}}{{\rm{n}}_{\rm{2}}}}},\delta {{\rm{f}}_{\rm{a}}}{{\rm{n}}_{\rm{2}}} = 0.79$$\end{document}. Vn2bag can be found by multiplying the bag nitrogen fraction with the total volume accumulated in the bag . With the advent of the nitrogen analyzer, the mass spectrometer and the use of high precision spirometers the washout could be followed on a breath - by - breath basis . This refinement eliminated the need for the bag and replaced the numerator in equation (1) with the expired volumes of nitrogen summed over the number of breaths . The modification is heavily reliant on the exact synchronization of analysis of fn2 and expired volume for the calculation of breath - by - breath volume of nitrogen . The example above utilized fio2 to step fan2 from 0.79 to 0 . Instead, any step change in fio2 could be used to create a corresponding step change in nitrogen . Established the ratio of functional residual air, r, divided by the sum of functional residual air and effective tidal volume, t, as dilution rate, r, describing the nitrogen clearance curve: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$r = \frac{r}{{r + t\prime}}. This factor multiplied by initial alveolar fraction of nitrogen and raised to the power of breath number allowed for the calculation of fraction of nitrogen in the expired volume of air . Fowler expanded the concept of the alveolar dilution factor to encompass three lung phases and introduced the term pulmonary nitrogen clearance delay, adding another characterization of the nitrogen clearance curve . The list has since been extended by investigators in pulmonary physiology, see [8, 9]. Recently, a modification of the mbnw method has been introduced, implicitly utilizing a first order difference equation for the calculation of frc based on mass conservation and a change in fio2 of 0.1 . In the present study, the difference equation is solved for the step response using basic digital signal processing (dsp) techniques where the step is interpreted as the nitrogen fraction change initiating the measurement . The resulting step response equation by comparing calculated clearance data to experimental and clinical washout data . Finally, we discuss technical and clinical limitations in addition to characteristics of the measurement . The first order difference equation based on mass conservation of nitrogen over one breath after a step change in inhaled n2 fraction is described in equation (3). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{v}}_{{\rm{ti}}}}{\rm {\times}} {{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}{\rm {+ frc}} \times {{\rm{f}}_{\rm{a}}}{{\rm{n}}_{\rm{2}}}_{_{n{\rm {- 1}}}}{\rm {=}} {{\rm{v}}_{{\rm{te}}}}{\rm {\times}} {{\rm{f}}_{{\rm{\bar e}}}}{{\rm{n}}_{\rm{2}}}_{_n}{\rm {+ frc \times}} {{\rm{f}}_{\rm{a}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} the step change in nitrogen occurs at breath index n = 0 . The step is from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{high}}}}$$\end{document} to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}$$\end{document} which will lead to n2 washing out . \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{\bar e}}}}{{\rm{n}}_{\rm{2}}}$$\end{document} is an average breath fraction of expired gas which includes gas from anatomical (series) dead space . \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{a}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} depends on the fraction of the immediately preceding breath \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{a}}}{{\rm{n}}_{\rm{2}}}_{_{n - 1}}$$\end{document}. Vti and vte designate inspiratory and expiratory tidal volumes . In all equations a refinement is to measure the lung volume to the exclusion of tidal anatomical dead space, vdtanat . Thus, the vte and vti of equation (3) must be replaced by the alveolar tidal volumes, vate and vati . Also, using endtidal co2 fraction, fetco2, as approximating alveolar fraction, faco2, the following equations can be used to calculate the alveolar tidal volumes: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{v}}{{\rm{a}}_{{\rm{te}}}}{\rm {=}} {{{{\rm{v}}_{\rm{t}}}{\rm{c}}{{\rm{o}}_{\rm{2}}}} \over {{{\rm{f}}_{{\rm{et}}}}{\rm{c}}{{\rm{o}}_{\rm{2}}}}}$$\end{document}, where vtco2 is the tidal excretion of co2 . \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{v}}{{\rm{a}}_{{\rm{ti}}}}{\rm {= v}}{{\rm{a}}_{{\rm{te}}}}{\rm {+}} \left ({{{{{\rm{v}}_{\rm{t}}}{\rm{c}}{{\rm{o}}_{\rm{2}}}} \over {{\rm{rq}}}}{\rm {-}} {{\rm{v}}_{\rm{t}}}{\rm{c}}{{\rm{o}}_{\rm{2}}}} \right)$$\end{document} rq is the respiratory quotient defined as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{rq =}} {{{{\rm{v}}_{\rm{t}}}{\rm{c}}{{\rm{o}}_{\rm{2}}}} \over {{{\rm{v}}_{\rm{t}}}{{\rm{o}}_{\rm{2}}}}}$$\end{document}. The value of rq depends on the composition of substrate being metabolized . For example, rq = 1 when carbohydrates are being metabolized and rq = 0.7 when fat is being metabolized . The range of rq is typically from 0.7 to 1.0 with reservation for gluconeo- and lipogenesis . A fixed value of rq of 0.8 is used in equation (5). For the purpose of accumulating the in- and expired volumes of nitrogen, the fractions of nitrogen are calculated as residue . \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}{\rm {= 1 -}} {{\rm{f}}_{\rm{i}}}{{\rm{o}}_{\rm{2}}}$$\end{document}\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}{\rm {= 1 -}} {{\rm{f}}_{{\rm{et}}}}{\rm{c}}{{\rm{o}}_{\rm{2}}}{\rm {-}} {{\rm{f}}_{{\rm{et}}}}{{\rm{o}}_{\rm{2}}}$$\end{document} substituting for alveolar volumes and endtidal fractions, equation (3) becomes: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{v}}{{\rm{a}}_{{\rm{ti}}}}{\rm {\times}} {{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}{\rm {+ frc \times}} {{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_{_{n - 1}} = {\rm{v}}{{\rm{a}}_{{\rm{te}}}}{\rm {\times}} {{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_n + {\rm{frc \times}} {{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} this is a first order difference equation . The fin2 and fetn2 are based on samples from the continuous fco2 and fo2 signals according to equations 6 and 7, see figure 1 . Vati and vate are calculated according to equations (4) and (5). 1sampling at two frequencies, the waveform sampling rate and two samples per breath sampling rate . Lower frame: the continuous sampling of fo2 (left y - axis) and fco2 (right y - axis) during a n2 washout procedure . From these tracings (enlarged in upper frame) two samples of fo2 and fco2 are sampled once during inspiration and as endtidal values for entering into equations 6 and 7 . Sampling at two frequencies, the waveform sampling rate and two samples per breath sampling rate . Lower frame: the continuous sampling of fo2 (left y - axis) and fco2 (right y - axis) during a n2 washout procedure . From these tracings (enlarged in upper frame) two samples of fo2 and fco2 are sampled once during inspiration and as endtidal values for entering into equations 6 and 7 . In the presence of alveolar (parallel) dead space, fetco2 <faco2 and, as a result, the equations include alveolar dead space . First, the frc is found by summing both sides of equation 8 over the total number of breaths, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{frc}} = {{\sum ({\rm{v}}{{\rm{a}}_{{\rm{te}}}}{\rm {\times}} {{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}{\rm {- v}}{{\rm{a}}_{{\rm{ti}}}}{\rm {\times}} {{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}})} \over {{{\rm{f}}_{{\rm{etinit}}}}{{\rm{n}}_{\rm{2}}}{\rm {-}} {{\rm{f}}_{{\rm{etlatest}}}}{{\rm{n}}_{\rm{2}}}}} = {{\sum ({\rm{v}}{{\rm{a}}_{\rm{e}}}{{\rm{n}}_{\rm{2}}}{\rm {- v}}{{\rm{a}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}})} \over {{{\rm{f}}_{{\rm{etinit}}}}{{\rm{n}}_{\rm{2}}}{\rm {-}} {{\rm{f}}_{{\rm{etlatest}}}}{{\rm{n}}_{\rm{2}}}}} = {{\sum {\rm{\delta v}}{{\rm{a}}_{\rm{n}}}_{_{\rm{2}}}} \over {{\rm{\delta}} {{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}}}$$\end{document}. Fetinitn2 denotes the initial condition at time n equals 1 which is the time sample before the step (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{etinit}}}}{{\rm{n}}_{\rm{2}}}{\rm {=}} {{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm {- 1}}}}$$\end{document}). Fetlatestn2 represents \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} when n equals the most recent breath . In general, vate, vati and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}$$\end{document} can vary with n and may be replaced by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{v}}{{\rm{a}}_{{\rm{te}}}}_n$$\end{document}, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{v}}{{\rm{a}}_{{\rm{ti}}}}_{_n}$$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} respectively . Second, equation 8 is solved for the step response using the frc calculated by equation (9) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$\eqalign {& {{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{{2_n}}} = {{{\rm{v}}{{\rm{a}}_{{\rm{ti}}}}{\rm {\times}} {{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}} \over {{\rm{v}}{{\rm{a}}_{{\rm{te}}}}{\rm {+ frc}}}} \times \left ({{{1 - {\lambda^{n + 1}}} \over {1 - \lambda}}} \right) + {\lambda^{n + 1}} \times {{\rm{f}}_{{\rm{etinit}}}}{{\rm{n}}_2} \cr & \lambda = {{{\rm{frc}}} \over {{\rm{frc + v}}{{\rm{a}}_{{\rm{te}}}}}} \cr} $$\end{document} in this solution, the values, vati, vate and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}$$\end{document} are assumed to remain constant during the washout . For a full derivation, see appendix a. this is an equation that gives the fetn2 for a particular breath without iteration and can be used to determine the applicability of a first order model . Some implications of equation 10 are immediately apparent: is related to the time constant of the n2 clearance.as the vate increases, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} decays faster consistent with a faster lung washout at larger tidal breaths.if the frc becomes large compared to the vate, then approaches 1 and there is very slow decay consistent with a small tidal breath requiring more breaths to change the concentration of a large lung volume (frc).as n, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_2}_n \to {{{\rm{v}}{{\rm{a}}_{{\rm{ti}}}} \cdot {{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{{\rm{low}}}} \over {{\rm{v}}{{\rm{a}}_{{\rm{te}}}}}}$$\end{document}. The \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} will not reach \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}$$\end{document} when rq 1 since vati vate . One would expect a good match between the measured fetn2 sequence compared to the calculated values if the system being measured is indeed first order . Is related to the time constant of the n2 clearance . As the vate increases, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} decays faster consistent with a faster lung washout at larger tidal breaths . If the frc becomes large compared to the vate, then approaches 1 and there is very slow decay consistent with a small tidal breath requiring more breaths to change the concentration of a large lung volume (frc). As n, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_2}_n \to {{{\rm{v}}{{\rm{a}}_{{\rm{ti}}}} \cdot {{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{{\rm{low}}}} \over {{\rm{v}}{{\rm{a}}_{{\rm{te}}}}}}$$\end{document}. The \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} will not reach \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}$$\end{document} when rq 1 since vati vate . One would expect a good match between the measured fetn2 sequence compared to the calculated values if the system being measured is indeed first order . The modified mbnw has been extensively validated in a metabolic lung model . In this paper, the prediction of fetn2 by the frc step response equation was validated by comparing calculated to measured values of fetn2 from washout procedures in one experimental and two clinical settings . The clinical measurements were made as part of two other studies for which the ethical committee of the medical faculty at gteborg university approved the studies and informed consent was obtained from the patients, next of kin or volunteers, respectively . Thus, they represent examples and are not the result of a selection powered to demonstrate differences between the normal and disease categories . Model . An experimental setup using an o2-consuming / co2-producing metabolic lung model previously described [10, 13]. The model was ventilated in volume controlled ventilation with a ge engstrm carestation ventilator (madison, wi, usa). Settings comprised two frcs (1.8, 2.9 l), two tidal volumes (500, 750 ml) and two steps of fio2 (0.1, 0.3) at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{\dot vc}}{{\rm{o}}_2}$$\end{document} 200 ml / min and rq 1 . Frc measurements were computed with the commercial software algorithm based on equation 9 detailed above . Patient recordings were performed either in the operating theater using ge adu/5 ventilator or in the intensive care unit (icu) using a servo 900c or servo 300 ventilator (siemens elema, solna, sweden). Subjects with assumed normal lungs were measured peroperatively and patients with diseased lungs in the intensive care unit, where positive pressure ventilation was instituted due to respiratory failure . The eigenvalues,, were calculated for one patient with acute respiratory distress syndrome (ards), one with heart failure (hf), two patients with chronic obstructive pulmonary disease (copd), and one obese patient with normal lungs (perioperative registration). Mono- vs biexponential fits were examined in one of the icu patients (copd) and in the bariatric patient . Recordings in healthy volunteers (two female and one male) spontaneously breathing while attached to a high flow cpap system via a snorkeling mouthpiece and equipped with a nose clip in supine, sitting and standing position . In patients and volunteers, respiratory monitoring and gas sampling were performed with datex - ohmeda spirometry module in an as/3 intensive care monitor . Data were collected using datex - ohmeda s/5 collect version 4 software (datex - ohmeda, helsinki, finland) and analyzed using lung monitor (plug - in to datex - ohmeda s/5 collect) or proprietary software programmed in test- point (measurement computing corporation, norton, ma, usa). Agreement between calculated and measured values of fetn2 was assessed by bland & altman analysis in terms of bias and limits of agreement.1 an error percentage, calculated according to below 30 was accepted as sign of good agreement . Calculated and measured values were analyzed by means of regression and r. patient and volunteer characteristics and results are reported in table 1 . Table 1patients and volunteers entered in evaluation of step response derivationpatients i d, sex and agedescriptionpeep (cm h20)vat (ml)eigenvaluesbl f102; fn2frc (ml)#biasupper & lower limit of agreementslope;constant030930, m, 76icu, sepsis, copd53750.790.4148070.822.13 & 3.761.15;6.49100.840.12090150.880.22346200.913045031110, m, 65icu, sepsis, copd122350.890.4185060.212.32 & 2.71.13;5.35180.900.122000.20.3031120, m, 48icu, hf102160.850.4122860.042.1 & 2.21.09;4.46150.860.113360.20.3040216, f, 42icu, ards, sle102800.720.560080080.040.7 & 0.630.93;2.7150.770.11040040415, f, 35laparoscopic, bariatric surgery04350.640.479860.081.42 & 1.260.93;3.34100.670.5950200.740.11200all patients350.2971.989 & 2.581.07;3.05volunteers i d, sex and ageco, f, 40sitting, standing0variable0.830.2250060.011.12 & 1.20.99;1.19100.880.20.32900coh, m, 29sitting0variable0.740.2140041.467.6 & 4.70.98;0.4550.830.22400100.850.452668ab, m, 38supine0variable0.770.20.3175060.351.7 & 2.41.03;1.1850.790.22184100.770.352125all volunteers160.163.5 & 3.21.02;0.97age, sex, location of measurement, peep and fn2 changes, baseline f1o2 and frc . Patients and volunteers entered in evaluation of step response derivation age, sex, location of measurement, peep and fn2 changes, baseline f1o2 and frc . The first order difference equation based on mass conservation of nitrogen over one breath after a step change in inhaled n2 fraction is described in equation (3). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{v}}_{{\rm{ti}}}}{\rm {\times}} {{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}{\rm {+ frc}} \times {{\rm{f}}_{\rm{a}}}{{\rm{n}}_{\rm{2}}}_{_{n{\rm {- 1}}}}{\rm {=}} {{\rm{v}}_{{\rm{te}}}}{\rm {\times}} {{\rm{f}}_{{\rm{\bar e}}}}{{\rm{n}}_{\rm{2}}}_{_n}{\rm {+ frc \times}} {{\rm{f}}_{\rm{a}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} the step change in nitrogen occurs at breath index n = 0 . The step is from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{high}}}}$$\end{document} to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}$$\end{document} which will lead to n2 washing out . \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{\bar e}}}}{{\rm{n}}_{\rm{2}}}$$\end{document} is an average breath fraction of expired gas which includes gas from anatomical (series) dead space . \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{a}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} depends on the fraction of the immediately preceding breath \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{a}}}{{\rm{n}}_{\rm{2}}}_{_{n - 1}}$$\end{document}. Vti and vte designate inspiratory and expiratory tidal volumes . In all equations a refinement is to measure the lung volume to the exclusion of tidal anatomical dead space, vdtanat . Thus, the vte and vti of equation (3) must be replaced by the alveolar tidal volumes, vate and vati . Also, using endtidal co2 fraction, fetco2, as approximating alveolar fraction, faco2, the following equations can be used to calculate the alveolar tidal volumes: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{v}}{{\rm{a}}_{{\rm{te}}}}{\rm {=}} {{{{\rm{v}}_{\rm{t}}}{\rm{c}}{{\rm{o}}_{\rm{2}}}} \over {{{\rm{f}}_{{\rm{et}}}}{\rm{c}}{{\rm{o}}_{\rm{2}}}}}$$\end{document}, where vtco2 is the tidal excretion of co2 . \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{v}}{{\rm{a}}_{{\rm{ti}}}}{\rm {= v}}{{\rm{a}}_{{\rm{te}}}}{\rm {+}} \left ({{{{{\rm{v}}_{\rm{t}}}{\rm{c}}{{\rm{o}}_{\rm{2}}}} \over {{\rm{rq}}}}{\rm {-}} {{\rm{v}}_{\rm{t}}}{\rm{c}}{{\rm{o}}_{\rm{2}}}} \right)$$\end{document} rq is the respiratory quotient defined as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{rq =}} {{{{\rm{v}}_{\rm{t}}}{\rm{c}}{{\rm{o}}_{\rm{2}}}} \over {{{\rm{v}}_{\rm{t}}}{{\rm{o}}_{\rm{2}}}}}$$\end{document}. The value of rq depends on the composition of substrate being metabolized . For example, rq = 1 when carbohydrates are being metabolized and rq = 0.7 when fat is being metabolized . The range of rq is typically from 0.7 to 1.0 with reservation for gluconeo- and lipogenesis . A fixed value of rq of 0.8 is used in equation (5). For the purpose of accumulating the in- and expired volumes of nitrogen, the fractions of nitrogen are calculated as residue . \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}{\rm {= 1 -}} {{\rm{f}}_{\rm{i}}}{{\rm{o}}_{\rm{2}}}$$\end{document}\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}{\rm {= 1 -}} {{\rm{f}}_{{\rm{et}}}}{\rm{c}}{{\rm{o}}_{\rm{2}}}{\rm {-}} {{\rm{f}}_{{\rm{et}}}}{{\rm{o}}_{\rm{2}}}$$\end{document} substituting for alveolar volumes and endtidal fractions, equation (3) becomes: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{v}}{{\rm{a}}_{{\rm{ti}}}}{\rm {\times}} {{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}{\rm {+ frc \times}} {{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_{_{n - 1}} = {\rm{v}}{{\rm{a}}_{{\rm{te}}}}{\rm {\times}} {{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_n + {\rm{frc \times}} {{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} this is a first order difference equation . The fin2 and fetn2 are based on samples from the continuous fco2 and fo2 signals according to equations 6 and 7, see figure 1 . Vati and vate are calculated according to equations (4) and (5). 1sampling at two frequencies, the waveform sampling rate and two samples per breath sampling rate . Lower frame: the continuous sampling of fo2 (left y - axis) and fco2 (right y - axis) during a n2 washout procedure . From these tracings (enlarged in upper frame) two samples of fo2 and fco2 are sampled once during inspiration and as endtidal values for entering into equations 6 and 7 . Sampling at two frequencies, the waveform sampling rate and two samples per breath sampling rate . Lower frame: the continuous sampling of fo2 (left y - axis) and fco2 (right y - axis) during a n2 washout procedure . From these tracings (enlarged in upper frame) two samples of fo2 and fco2 are sampled once during inspiration and as endtidal values for entering into equations 6 and 7 . In the presence of alveolar (parallel) dead space, fetco2 <faco2 and, as a result, the equations include alveolar dead space . First, the frc is found by summing both sides of equation 8 over the total number of breaths, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{frc}} = {{\sum ({\rm{v}}{{\rm{a}}_{{\rm{te}}}}{\rm {\times}} {{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}{\rm {- v}}{{\rm{a}}_{{\rm{ti}}}}{\rm {\times}} {{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}})} \over {{{\rm{f}}_{{\rm{etinit}}}}{{\rm{n}}_{\rm{2}}}{\rm {-}} {{\rm{f}}_{{\rm{etlatest}}}}{{\rm{n}}_{\rm{2}}}}} = {{\sum ({\rm{v}}{{\rm{a}}_{\rm{e}}}{{\rm{n}}_{\rm{2}}}{\rm {- v}}{{\rm{a}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}})} \over {{{\rm{f}}_{{\rm{etinit}}}}{{\rm{n}}_{\rm{2}}}{\rm {-}} {{\rm{f}}_{{\rm{etlatest}}}}{{\rm{n}}_{\rm{2}}}}} = {{\sum {\rm{\delta v}}{{\rm{a}}_{\rm{n}}}_{_{\rm{2}}}} \over {{\rm{\delta}} {{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}}}$$\end{document}. Fetinitn2 denotes the initial condition at time n equals 1 which is the time sample before the step (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{etinit}}}}{{\rm{n}}_{\rm{2}}}{\rm {=}} {{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm {- 1}}}}$$\end{document}). Fetlatestn2 represents \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} when n equals the most recent breath . In general, vate, vati and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}$$\end{document} can vary with n and may be replaced by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{v}}{{\rm{a}}_{{\rm{te}}}}_n$$\end{document}, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{v}}{{\rm{a}}_{{\rm{ti}}}}_{_n}$$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} respectively . Second, equation 8 is solved for the step response using the frc calculated by equation (9) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$\eqalign {& {{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{{2_n}}} = {{{\rm{v}}{{\rm{a}}_{{\rm{ti}}}}{\rm {\times}} {{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}} \over {{\rm{v}}{{\rm{a}}_{{\rm{te}}}}{\rm {+ frc}}}} \times \left ({{{1 - {\lambda^{n + 1}}} \over {1 - \lambda}}} \right) + {\lambda^{n + 1}} \times {{\rm{f}}_{{\rm{etinit}}}}{{\rm{n}}_2} \cr & \lambda = {{{\rm{frc}}} \over {{\rm{frc + v}}{{\rm{a}}_{{\rm{te}}}}}} \cr} $$\end{document} in this solution, the values, vati, vate and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}$$\end{document} are assumed to remain constant during the washout . For a full derivation, see appendix a. this is an equation that gives the fetn2 for a particular breath without iteration and can be used to determine the applicability of a first order model . Some implications of equation 10 are immediately apparent: is related to the time constant of the n2 clearance.as the vate increases, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} decays faster consistent with a faster lung washout at larger tidal breaths.if the frc becomes large compared to the vate, then approaches 1 and there is very slow decay consistent with a small tidal breath requiring more breaths to change the concentration of a large lung volume (frc).as n, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_2}_n \to {{{\rm{v}}{{\rm{a}}_{{\rm{ti}}}} \cdot {{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{{\rm{low}}}} \over {{\rm{v}}{{\rm{a}}_{{\rm{te}}}}}}$$\end{document}. The \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} will not reach \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}$$\end{document} when rq 1 since vati vate . One would expect a good match between the measured fetn2 sequence compared to the calculated values if the system being measured is indeed first order . Is related to the time constant of the n2 clearance . As the vate increases, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} decays faster consistent with a faster lung washout at larger tidal breaths . If the frc becomes large compared to the vate, then approaches 1 and there is very slow decay consistent with a small tidal breath requiring more breaths to change the concentration of a large lung volume (frc). As n, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_2}_n \to {{{\rm{v}}{{\rm{a}}_{{\rm{ti}}}} \cdot {{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{{\rm{low}}}} \over {{\rm{v}}{{\rm{a}}_{{\rm{te}}}}}}$$\end{document}. The \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_{\rm{2}}}_{_n}$$\end{document} will not reach \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}$$\end{document} when rq 1 since vati vate . One would expect a good match between the measured fetn2 sequence compared to the calculated values if the system being measured is indeed first order . The modified mbnw has been extensively validated in a metabolic lung model . In this paper, the prediction of fetn2 by the frc step response equation was validated by comparing calculated to measured values of fetn2 from washout procedures in one experimental and two clinical settings . The clinical measurements were made as part of two other studies for which the ethical committee of the medical faculty at gteborg university approved the studies and informed consent was obtained from the patients, next of kin or volunteers, respectively . Thus, they represent examples and are not the result of a selection powered to demonstrate differences between the normal and disease categories . Model . An experimental setup using an o2-consuming / co2-producing metabolic lung model previously described [10, 13]. The model was ventilated in volume controlled ventilation with a ge engstrm carestation ventilator (madison, wi, usa). Settings comprised two frcs (1.8, 2.9 l), two tidal volumes (500, 750 ml) and two steps of fio2 (0.1, 0.3) at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{\dot vc}}{{\rm{o}}_2}$$\end{document} 200 ml / min and rq 1 . Frc measurements were computed with the commercial software algorithm based on equation 9 detailed above . Patient recordings were performed either in the operating theater using ge adu/5 ventilator or in the intensive care unit (icu) using a servo 900c or servo 300 ventilator (siemens elema, solna, sweden). Subjects with assumed normal lungs were measured peroperatively and patients with diseased lungs in the intensive care unit, where positive pressure ventilation was instituted due to respiratory failure . The eigenvalues,, were calculated for one patient with acute respiratory distress syndrome (ards), one with heart failure (hf), two patients with chronic obstructive pulmonary disease (copd), and one obese patient with normal lungs (perioperative registration). Mono- vs biexponential fits were examined in one of the icu patients (copd) and in the bariatric patient . Recordings in healthy volunteers (two female and one male) spontaneously breathing while attached to a high flow cpap system via a snorkeling mouthpiece and equipped with a nose clip in supine, sitting and standing position . In patients and volunteers, respiratory monitoring and gas sampling were performed with datex - ohmeda spirometry module in an as/3 intensive care monitor . Data were collected using datex - ohmeda s/5 collect version 4 software (datex - ohmeda, helsinki, finland) and analyzed using lung monitor (plug - in to datex - ohmeda s/5 collect) or proprietary software programmed in test- point (measurement computing corporation, norton, ma, usa). Agreement between calculated and measured values of fetn2 was assessed by bland & altman analysis in terms of bias and limits of agreement.1 an error percentage, calculated according to below 30 was accepted as sign of good agreement . Calculated and measured values were analyzed by means of regression and r. patient and volunteer characteristics and results are reported in table 1 . Table 1patients and volunteers entered in evaluation of step response derivationpatients i d, sex and agedescriptionpeep (cm h20)vat (ml)eigenvaluesbl f102; fn2frc (ml)#biasupper & lower limit of agreementslope;constant030930, m, 76icu, sepsis, copd53750.790.4148070.822.13 & 3.761.15;6.49100.840.12090150.880.22346200.913045031110, m, 65icu, sepsis, copd122350.890.4185060.212.32 & 2.71.13;5.35180.900.122000.20.3031120, m, 48icu, hf102160.850.4122860.042.1 & 2.21.09;4.46150.860.113360.20.3040216, f, 42icu, ards, sle102800.720.560080080.040.7 & 0.630.93;2.7150.770.11040040415, f, 35laparoscopic, bariatric surgery04350.640.479860.081.42 & 1.260.93;3.34100.670.5950200.740.11200all patients350.2971.989 & 2.581.07;3.05volunteers i d, sex and ageco, f, 40sitting, standing0variable0.830.2250060.011.12 & 1.20.99;1.19100.880.20.32900coh, m, 29sitting0variable0.740.2140041.467.6 & 4.70.98;0.4550.830.22400100.850.452668ab, m, 38supine0variable0.770.20.3175060.351.7 & 2.41.03;1.1850.790.22184100.770.352125all volunteers160.163.5 & 3.21.02;0.97age, sex, location of measurement, peep and fn2 changes, baseline f1o2 and frc . Results of bland & altman analysis of agreement and regression analysis . Patients and volunteers entered in evaluation of step response derivation age, sex, location of measurement, peep and fn2 changes, baseline f1o2 and frc . Model . There was an excellent correspondence between 178 pairs of measured and calculated values of fetn2 in six washout procedures using the step response equation, see figure 2 . Bland & altman analysis showed bias of 0, upper and lower limits of agreement of 0.49 and 0.5 and error 1.3%, see figure 3 . The regression equation showed a slope of 1.0, intercept 0.12 and r 0.98 with a p <0.001 . Fn2 0.3, frc 1800 ml, vat 270 ml, eigenvalue 0.86, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{\dot vc}}{{\rm{o}}_2}$$\end{document} 187 ml / min . 3bland & altman plot of agreement between pairs of measured and calculated fetn2 according to step response derivation in metabolic lung model . Bias 0, upper and lower limits of agreement of 0.25 and 0.5 . Calculated and measured fetn2 during n2 washout in metabolic lung model . Fn2 0.3, frc 1800 ml, vat 270 ml, eigenvalue 0.86, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{\dot vc}}{{\rm{o}}_2}$$\end{document} 187 ml / min . The regression equation has a slope of 1.02 and a r of 0.999 . Bland & altman plot of agreement between pairs of measured and calculated fetn2 according to step response derivation in metabolic lung model . Bias 0, upper and lower limits of agreement of 0.25 and 0.5 . Patients . Peep levels varied from 0 to 20 cm h2o, frc ranged from 600 to 3000 ml, fetn2 from 0.1 to 0.3 and baseline fio2 from 0.2 to 0.5 . There was good agreement between 1222 pairs of measured and the calculated values of fetn2 . Bland & altman analysis showed bias of 0.3, upper and lower limits of agreement of 2 and 2.6, error 4.9%, see figure 4 . The regression equation showed a slope of 1.07, intercept 3.0, r 0.98 with a p <0.001 . At comparable peep levels in ards (ards) patient was 0.734, in patient with heart failure (hf) 0.846, in copd patients 0.842 and 0.889 (copd) and in perioperative normal adult lung (bariatric patient) 0.654 (periop). 4bland & altman plot of agreement between pairs of measured and calculated fetn2 according to step response derivation in 35 n2 washout procedures in five patients . The regression equation showed a slope of 1.07, intercept 3 and r 0.98 with a p <0.001.fig . 5 as a function of peep in five patients . Bland & altman plot of agreement between pairs of measured and calculated fetn2 according to step response derivation in 35 n2 washout procedures in five patients . The regression equation showed a slope of 1.07, intercept 3 and r 0.98 with a p <0.001 . As a function of peep in five patients . In the analysis of mono- vs. bicompartmental analysis we found a difference between mono- and biexponential fit to measured fetn2 in the icu (copd) patient whereas there was no notable difference in the bariatric patient . The number of patients and measurements preclude consideration statistical significance . Volunteers . Peep levels varied from 0, 5 to 10 cm h2o, frc ranged from 1400 to 2900 ml, fetn2 0.2 to 0.45 and baseline fio2 from 0.21 to 0.3 . There was good agreement between 524 pairs of measured and the calculated values of fetn2 using the step response equation . Bland & altman analysis showed bias of 0.16, upper and lower limits of agreement at 3.2 and 3.5 and an error 6.4% . The regression equation showed a slope of 0.97, intercept 1.9 and r 0.98 with a p <0.001 (figure 6). 6bland & altman plot of agreement between pairs of measured and calculated fetn2 according to step response derivation in 16 n2 washout procedures in three healthy volunteers . Bland & altman analysis showed bias of 0.16, lower and upper limits of agreement at 3.2 and 3.5 . The regression equation showed a slope of 0.97, intercept at 1.9 and r 0.98 with a p <0.001 . Bland & altman plot of agreement between pairs of measured and calculated fetn2 according to step response derivation in 16 n2 washout procedures in three healthy volunteers . Bland & altman analysis showed bias of 0.16, lower and upper limits of agreement at 3.2 and 3.5 . The regression equation showed a slope of 0.97, intercept at 1.9 and r 0.98 with a p <0.001 . We take as a starting point the mathematical derivation of a modified version of the multiple breath nitrogen washout method for calculation of frc . Using the result of the frc calculation, a step response equation was derived based on techniques from dsp and used to calculate the course of fetn2 during washout . These calculated values were then compared to measured data from an experimental setup, patients and volunteers . We found excellent alignment between measured and calculated nitrogen clearance using a single compartment, first order metabolic lung model . The first order model also fitted measurements in patients with various respiratory conditions and in healthy, spontaneously breathing volunteers . The eigenvalue of the step response was shown to be dependent on peep level and to be different among healthy and diseased states of the lung . The sample size, however, does not allow for any statistically significant inferences, but the results may indicate a diagnostic and prognostic method to be verified in further studies . Technical and clinical characteristics and limitations of the modified mbnw method and first order step response equation are addressed in the following . The present study is to our knowledge the first attempt at deriving the equivalent of the alveolar dilution ratio first introduced by darling by solving the underlying first order difference equation based on mass conservation of alveolar contents of n2 . The eigenvalue,, is equal to darling s r in equation (2). This ratio was revived by wagner as a term in linear programming of ventilation / volume ratios and by rossing . Wagner and rossing published their research before oppenheim and schaffer published their work on dsp, which forms the basis of this study . Using techniques made popular by the field of dsp, enables one to frame the problem in the form of a difference equation and solve for the step response . Dsp has entered the mainstream subsequent to many of the early works on mbnw . Reformulating the subject in a dsp framework the present study details nitrogen clearance as a first order process characterized by an eigenvalue, and compares actual values of fetn2 to values predicted by the step response equation . The degree of agreement confirms that the nitrogen washout is dominated by a first order process and the tools of dsp are applicable in this setting . Clinical impact and uses for . This paper provides an alternative derivation for in the more general framework of dsp . Results in the present study showed higher in healthy adult lungs and in patients with copd and lower values in patients with ards . In addition, is reminiscent of the strain relationship (vt / frc) which according to the baby lung2 the bariatric patient (see figure 5) the strain is perilously close to this limit . It may not be clinically justifiable to measure by setting peep at zero (zeep), but as the value is linearly related to peep by virtue of the lung compliance, a series measurement at peep levels above zero can be used to extrapolate to a value of corresponding to zeep . The or the strain relationship could be considered a discriminator for classifying lung states but may not be sufficient in isolation . Since has a dependency on the ventilator settings through frc (and as a corollary peep) and vate, calculation of at several tidal volumes would likely be a better discriminate . Further, these ratios in conjunction with other features such as hemodynamics would be an excellent start for a feature vector that could be used for pattern classification . The pattern classifier would be used to distinguish between different lung states and act as a guide for optimal ventilation therapy . The washout procedure posits that the o2-consumption and co2-excretion, the end - expiratory lung volume as well as hemodynamics are stable at baseline before washout and that no interventions are undertaken during the washout period which will alter this, e.g. Changes in inotropic support with inherent thermogenic effects or changes in ventilatory settings . In our formulation, rq is fixed at 0.8, although in reality rq can be between 0.7 and 1.0 depending on the metabolic state of the patient . In this context it can be shown that for a fio2 of 0.5, a fetn2 of 0.1, a rq of 0.1 and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{\dot vc}}{{\rm{o}}_2}$$\end{document} 200 ml / min the error in frc amounts to approximately 180 ml bias . Equality in equation 3 assumes that n2 is neither taken up nor excreted from blood and tissues . Estimates of tissue nitrogen uptake and excretion do, however, exist [18, 19]. The modified mbnw utilizes a fn2 of 0.1 and accordingly the volume excreted will be diminished to approximately 10 ml . However, repeating the fn2 with an identical change in opposite direction and averaging the results for frc compensates for this error . The fin2 and fetn2 are calculated as residue after subtraction of appropriate fractions of o2 and co2 and entered into the calculation of vn2 by multiplication with va . This assumes that the dead spaces of o2, co2 and n2 are identical within the clinical range of expired volume, which is supported by the findings in [20, 21]. Furthermore, feto2 and fetco2 measurements are used to approximate fao2 and faco2 although there is evidence to the contrary in the case of fetco2 [22, 23]. Typically, the fetco2 will be lower than faco2 and feto2 the modified mbnw method incorporates breath - by - breath measurement and calculation of vati, and vate allowing for variation of tidal volumes during the washout . In the step response derivation the discrepancy becomes obvious when examining frc measurements performed during spontaneous ventilation where breath - to - breath variation of inspiratory and expiratory tidal volumes in quiet breathing typically amounted to 50100 ml during the washout procedure . The potential of using from the step response solution of the difference equation to classify lung pathologies applies more to mechanical ventilation where the volumes are constant . Washout was clearly not monoexponential in the case of emphysematous (copd) lung with compartments of differing mechanical properties . A better curve fit was accomplished by a biexponential equation, which in the framework of dsp translates into a second order difference equation . The modified mbnw method based on a first order difference equation, however, will still be able to calculate the frc given the measurement period is extended to allow for slow compartments to empty in the case of copd lungs . The first order difference equation solution to a step input assumes that washout is monoexponential and deviations from this will cause increased lack of agreement between measured and calculated values . This was demonstrated in the fitting of washout data to a mono- vs. a biexponential equation in the case of the copd / icu and the perioperative bariatric patient . The present study is to our knowledge the first attempt at deriving the equivalent of the alveolar dilution ratio first introduced by darling by solving the underlying first order difference equation based on mass conservation of alveolar contents of n2 . The eigenvalue,, is equal to darling s r in equation (2). This ratio was revived by wagner as a term in linear programming of ventilation / volume ratios and by rossing . Wagner and rossing published their research before oppenheim and schaffer published their work on dsp, which forms the basis of this study . Using techniques made popular by the field of dsp, enables one to frame the problem in the form of a difference equation and solve for the step response . Dsp has entered the mainstream subsequent to many of the early works on mbnw . Reformulating the subject in a dsp framework should allow algorithms from that field to improve or extend mbnw algorithms . The present study details nitrogen clearance as a first order process characterized by an eigenvalue, and compares actual values of fetn2 to values predicted by the step response equation . The degree of agreement confirms that the nitrogen washout is dominated by a first order process and the tools of dsp are applicable in this setting . Clinical impact and uses for . The term = frc/(frc+vate) this paper provides an alternative derivation for in the more general framework of dsp . Results in the present study showed higher in healthy adult lungs and in patients with copd and lower values in patients with ards . In addition, is reminiscent of the strain relationship (vt / frc) which according to the baby lung2 the bariatric patient (see figure 5) the strain is perilously close to this limit . It may not be clinically justifiable to measure by setting peep at zero (zeep), but as the value is linearly related to peep by virtue of the lung compliance, a series measurement at peep levels above zero can be used to extrapolate to a value of corresponding to zeep . The or the strain relationship could be considered a discriminator for classifying lung states but may not be sufficient in isolation . Since has a dependency on the ventilator settings through frc (and as a corollary peep) and vate, calculation of at several tidal volumes would likely be a better discriminate . Further, these ratios in conjunction with other features such as hemodynamics would be an excellent start for a feature vector that could be used for pattern classification . The pattern classifier would be used to distinguish between different lung states and act as a guide for optimal ventilation therapy . The washout procedure posits that the o2-consumption and co2-excretion, the end - expiratory lung volume as well as hemodynamics are stable at baseline before washout and that no interventions are undertaken during the washout period which will alter this, e.g. Changes in inotropic support with inherent thermogenic effects or changes in ventilatory settings . In our formulation, rq is fixed at 0.8, although in reality rq can be between 0.7 and 1.0 depending on the metabolic state of the patient . In this context it can be shown that for a fio2 of 0.5, a fetn2 of 0.1, a rq of 0.1 and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\rm{\dot vc}}{{\rm{o}}_2}$$\end{document} 200 ml / min the error in frc amounts to approximately 180 ml bias . Equality in equation 3 assumes that n2 is neither taken up nor excreted from blood and tissues . Estimates of tissue nitrogen uptake and excretion do, however, exist [18, 19]. The modified mbnw utilizes a fn2 of 0.1 and accordingly the volume excreted will be diminished to approximately 10 ml . However, repeating the fn2 with an identical change in opposite direction and averaging the results for frc compensates for this error . The fin2 and fetn2 are calculated as residue after subtraction of appropriate fractions of o2 and co2 and entered into the calculation of vn2 by multiplication with va . This assumes that the dead spaces of o2, co2 and n2 are identical within the clinical range of expired volume, which is supported by the findings in [20, 21]. Furthermore, feto2 and fetco2 measurements are used to approximate fao2 and faco2 although there is evidence to the contrary in the case of fetco2 [22, 23]. Typically, the fetco2 will be lower than faco2 and feto2 will be higher than fao2 due to imperfect mixing and alveolar dead space . The modified mbnw method incorporates breath - by - breath measurement and calculation of vati, and vate allowing for variation of tidal volumes during the washout . In the step response derivation the discrepancy becomes obvious when examining frc measurements performed during spontaneous ventilation where breath - to - breath variation of inspiratory and expiratory tidal volumes in quiet breathing typically amounted to 50100 ml during the washout procedure . The potential of using from the step response solution of the difference equation to classify lung pathologies applies more to mechanical ventilation where the volumes are constant . Washout was clearly not monoexponential in the case of emphysematous (copd) lung with compartments of differing mechanical properties . A better curve fit was accomplished by a biexponential equation, which in the framework of dsp translates into a second order difference equation . The modified mbnw method based on a first order difference equation, however, will still be able to calculate the frc given the measurement period is extended to allow for slow compartments to empty in the case of copd lungs . The first order difference equation solution to a step input assumes that washout is monoexponential and deviations from this will cause increased lack of agreement between measured and calculated values . This was demonstrated in the fitting of washout data to a mono- vs. a biexponential equation in the case of the copd / icu and the perioperative bariatric patient . We have presented a modified mbnw method in terms of a first order difference equation which was solved for the step response of fetn2 to a step change in fio2 . A central concept of the step response equation, the eigenvalue, showed different values in healthy and diseased lungs . The mbnw difference equation has some interesting implications for the roles of serial and parallel dead spaces . The step response equation opens up research avenues within digital signal processing and the characterization of ventilatory states . The derivation of the step response solution is as follows . In equation (8), define the step response as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$a = {\rm{v}}{{\rm{a}}_{{\rm{ti}}}}{\rm {\times f}}{f_{\rm{i}}}{{\rm{n}}_2}_{_{{\rm{low}}}}$$\end{document} for n 0, e vate + frc, b = frc, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_n} = {{\rm{f}}_{{\rm{et}}}}{{\rm{n}}_2}_{_n}$$\end{document} and the initial condition as fetinitn2: utilizing a substitution technique: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$n = 0:{{\rm{f}}_0} = \left ({a + b \times {{\rm{f}}_{{\rm{etinit}}}}{\rm{n}}2} \right)/e$$\end{document}\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$n = 1:{{\rm{f}}_{\rm{1}}} = a / e + b \times \left ({\left ({a + b \times {{\rm{f}}_{{\rm{etinit}}}}{{\rm{n}}_2}} \right)/{e^2}} \right)$$\end{document}\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$n = 2:{{\rm{f}}_2} = a / e + b \times a/{e^2} + a \times {b^2}/{e^3} \times + {{\rm{f}}_{{\rm{etinit}}}}{{\rm{n}}_2} \times {b^3}/{e^3}$$\end{document} in general, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_n} = {a \over e}\mathop \sum \limits_{i = 0}^n {\left ({{b \over e}} \right)^i} + {\left ({{b \over e}} \right)^{n + 1}} \times {{\rm{f}}_{{\rm{etinit}}}}{{\rm{n}}_2}$$\end{document} let = b / e and equation 13 simplifies into: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_n} = {a \over e}\left ({{{1 - {\lambda^{n + 1}}} \over {1 - \lambda}}} \right) + {\lambda^{n + 1}} \times {{\rm{f}}_{{\rm{etinit}}}}{{\rm{n}}_2}$$\end{document} substitute back to the original variables: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\rm{f}}_n} = {{{\rm{v}}{{\rm{a}}_{{\rm{ti}}}}{\rm {\times}} {{\rm{f}}_{\rm{i}}}{{\rm{n}}_{\rm{2}}}_{_{{\rm{low}}}}} \over {{\rm{v}}{{\rm{a}}_{{\rm{te}}}} + {\rm{frc}}}} \times \left ({{{1 - {\lambda^{n + 1}}} \over {1 - \lambda}}} \right) + {\lambda^{n + 1}} \times {{\rm{f}}_{{\rm{etinit}}}}{{\rm{n}}_2};\lambda = {{{\rm{frc}}} \over {{\rm{frc + v}}{{\rm{a}}_{{\rm{te}}}}}}$$\end{document}
While the country relies heavily on its natural resources and agricultural land to provide food and livelihoods for its rapidly growing population, the inland fisheries sector is perhaps the most valuable, which officially accounts for about 12% of gross domestic product (gdp) and provides most cambodians with their key source of animal protein, calcium, and vitamin a . The pollution of the aquatic environment with both essential and nonessential elements has attracted serious concern in the recent years because they are indestructible and most of them have toxic effects on organisms . Heavy metals, particularly cadmium, lead, and arsenic constitute a significant potential health threat to human . Fish is one of the sources of protein, vitamin, and mineral, and it contains essential nutrients required in human diet . Fish has been widely accepted as a very important source of animal protein for supplementing both infants and adults diet [5, 6]. Fish drying is an age long practice of processing fish across the world to prolong its shelf - life and to conserve the quality . It is a slow process and when drying of these animal products took a long time, bacterial spoilage during the slow operation occurred . Besides, osd will not lower moisture content below about 15%, which is still too high for storage stability of food products . It also exposes products to birds, insects, and rodents and makes the products susceptible to contamination with foreign materials, such as dust and litter . The main contaminants that are likely to arise from bird droppings include fungus such as histoplasma capsulatum and cryptococcus neoformans and bacteria chlamydophila psittaci . In addition, insects and fungi that thrive in moist conditions render the products unusable . In recent times, smoking kiln and solar drying system (sds) are used to obtain product of high quality . Sds is a solar energy process that is well matched for drying of agricultural and fishery products in the tropical and subtropical countries . According to, phnom penh receives an average of 5.3 hours of full sun every day with an average of about 2,490 hours of sunshine per year . The maximum fluctuation in solar radiation volume throughout the year is relatively low and has been estimated at 17% . Many studies have reported on various methods of fish drying in tropical and subtropical countries . R. m. davies and o. a. davies reported six different types of traditional fish processing techniques in nigeria whereas an experimental study was carried out on solar tunnel dryer to dry fish . A hybrid solar drying system with diesel burner was employed to dry salted silver jewfish in johor, malaysia [13, 14], and its drying characteristic was compared under open and solar drying . The effect of traditional fish processing was investigated in terms of its nutritional value, proximate composition of raw and cooked thai freshwater and marine fish, mineral composition, and proximate analysis of dried salted molva molva l. and merluccius merluccius l. . Changes in nutritional and chemical composition of fried sardine (clupea pilchardus) were reported to be produced by microwave reheating and frozen storage . It has been observed that different drying methods and processing have different effect on nutritional composition of fish . The skin is compared to the flesh because the skin of channa striatus has been known to possess toxic and lethal components despite being edible among the local people . Bearing in mind that the quality of dried fish using different drying methods cannot be the same, as such, the objective of this study is to investigate the toxic and essential elements of striped snakehead fish (channa striata) dried using osd and sds in cambodia . Samples of the striped snakehead fish (channa striata) as shown in figure 1 were obtained from cambodia . The mean length and weight of fresh fish were about 35 cm and 600 g, respectively . The striped snakehead fish was gutted and washed before cutting them prior to be subjected to two different methods of drying (osd and sds). The striped snakehead fish was soaked in separate containers that contained a 25% (w / v) brine solution of nacl for 4 hr . A fish - to - brine ratio of 1: 4 l was used [22, 23]. Solar drying system is shown in figure 2(a), which comprised the forced - convection indirect type . The system consisted of a v - groove solar air collector, fans, electric heater, and drying chamber . The solar collector was of the back - pass v - groove which was connected in series . Experiment was done between 9:00 a.m. and 5:00 p.m. corresponding to the sunshine duration in cambodia . Drying experiment has been done on 62 kg striped snakehead . It was divided equally and then placed on 12 trays, as shown in figure 2(b). During this process the drying temperature setting in drying chamber was fixed at 50c and the flow rate was fixed at 0.07 kg / s . The data measured were air temperature (ambient temperature, air temperature inlet, and outlet of the collector), solar radiation, and air velocity, as well as the air temperature before it entered the dryer chamber, the temperature inside the dryer chamber, and the temperature of the air out of the dryer chamber . Air temperature was measured by t - type thermocouple, and the intensity of solar radiation was measured by pyranometer . The striped snakehead fish was placed on the bamboo tray in osd from 9:00 a.m. to 5:00 p.m. at night, fishes were piled in plastic bins which were kept inside until the next morning and then continued to dry in the osd . The striped snakehead fish was dried until the final moisture content reached about 40% w.b . The trace and minor elements content in samples were determined by using inductively coupled plasma mass spectrometry (icp - ms elan 9000) (perkinelmer, sciex usa). The icp - ms was set with the condition as stated in table 1 . For samples preparation, all the samples were processed using acid digestion method based on modified standard procedure . Samples were rinsed using deionized water to get rid of all the contaminant, 5 g (wt / wt) of the samples was placed in the 50 ml beaker, and 10 ml of concentrated nitric acid was added . After the mixture was evaporated to the desired volume, 2 ml of hydrogen peroxide solution was added . The process of adding hydrogen peroxide solution, heating, and cooling were repeated until the sample solution turned into clear sight . Sample solution was diluted with deionized water until the volume eventually reached 100 ml . For standard solution, multielement stock solution calibration number 3 from perkin elmer was used in this study . The aim of the present study is merely to compare the level of the toxic and essential elements in the striped snakehead fish using osd and sds methods of drying instead of analyzing the quality of the fried fish product; hence, analysis of microbiology was not yet considered . Performance of sds for striped snakehead fish is shown in table 2 . In this study, the inductive coupled plasma mass spectrometry (icp - ms) assay has been used to measure the elemental content in striped snakehead fish sample . Recoveries of trace metal contents in the present study are shown in tables 35 . A total number of 12 trace and minor elements (pb, cd, as, zn, mn, cu, cr, mg, mo, fe, ni, and se) in the dried striped snakehead fish from cambodia have been determined using icp - ms after acid digestion . The concentration of 12 elements was determined based on their classification of toxic metals (as, pb, and cd) in table 3 and nutritional trace elements (fe, mn, mg, se, mo, cu, ni, zn, and cr) in tables 4 and 5 . The test was done in triplicates with the number of samples n = 3 for each analysis . Table 3 shows the concentration of three heavy metals content in the skin and the flesh of the fish using two different drying methods . Out of these three toxic elements, as was detected at the highest concentration followed by pb and cd . As far as the skin samples are concerned, the fish subjected to osd showed higher level of as, pb, and cd (8.72 ppb, 5.72 ppb, and 0.54 ppb) compared to the samples dried by sds (7.32 ppb, 3.97 ppb, and 0.30 ppb). On the other hand, the flesh of the fish also accumulated high amount of as, pb, and cd (11.53 ppb, 3.87 ppb, and 0.31 ppb) during the process of osd compared to when sds technique (10.17 ppb, 1.43 ppb, and 0.18 ppb) was used . However, the levels of arsenic in both skin and flesh samples using both drying methods were well below the acceptable limit of 130 ppb for arsenic . The acceptable limits for pb and cd are 240 ppb and 60 ppb, respectively . As far as the skin samples are concerned, the nutritional trace elements were higher in the samples subjected to sds with concentration of between 3.41 ppb and 2,019.69 ppb compared to osd skin sample of within the range of 2.55 ppb to 1,550.55 ppb recorded for se, mn, ni, cu, cr, fe, mo, and mg as presented in table 4 . This means that the beneficial trace metals were conserved in the skin of the fried fish during the process of drying by sds technique . Generally, it was seen in table 5 that sds method of drying produced lower concentration of the beneficial trace elements (mn, se, cr, fe, mo, and mg) in the flesh of the fish samples compared to using conventional osd method of drying the fish . The levels of mn under sds and osd method were, respectively, 4.68 ppb and 4.73 ppb compared to the tolerable upper intake level (ul) for manganese in 70 kg adult at 11,000 g per day which corresponds to 157 ppb . Although low level of manganese intake is necessary for human health, exposure to high manganese level has the potential to cause neurotoxicity . As far as selenium is concerned, the sds and osd method displayed its concentration of 5.52 ppb and 5.62 ppb . According to the institute of medicine, the recommended dietary intake of selenium is 55 g per day for 70 kg adult equivalent to 0.80 ppb . The se levels in both samples are still considered within acceptable limit for consumption because the tolerable ul limit for selenium in 70 kg adult was set at 400 g per day which corresponds to 5.71 ppb . In one study, selenium is reported to reduce vulnerability to mercury toxicity in humans and has protective effect for neonates against neurotoxicity from prenatal mn exposure . Chromium was detected at, respectively, 9.16 ppb and 10.67 ppb in sds and osd dried fish which is 20 times greater than the recommended average daily intake level (adequate intake) in 70 kg adult of 35 g per day for 70 kg adult equivalent to 0.50 ppb . However, no adverse effects have been convincingly associated with excess intake of chromium from food or dietary supplements . Besides, it has been found that some fish are capable of bioaccumulating cr level nearly 100 times the concentration of cr in the water . The levels of fe under sds and osd method were, respectively, 98.80 ppb and 130.71 ppb compared with the tolerable ul for iron in 70 kg adult was set at 45,000 g per day which corresponds to 642.86 ppb . The levels of mg under sds and osd method were, respectively, 3000.58 ppb and 3231.07 ppb compared with the tolerable ul value for magnesium in 70 kg adult was set at 350,000 g per day which corresponds to 5,000 ppb . It can be deduced that the levels of mn, se, cr, fe, and mg in the muscle of striped snakehead fish using both methods of drying were generally low when compared with the ul limit values . This is not the case for the level of molybdenum which recorded 239.04 ppb and 272.04 ppb in sds and osd samples . Logically, this is not considered safe for human consumption because it is almost 100 times the tolerable ul value for mo in 70 kg adult as was set at 2,000 g per day or 28.57 ppb . Nevertheless, the extremely high level of molybdenum in fishery products will not cause any harmful effects associated with high molybdenum level in human such as gout, anemia, and symptoms of copper deficiency . This is because of the rapid renal clearance of the majority of ingested molybdenum, which will likely prevent deleterious effects in the event of high intake . It is interesting to note that when sds method was employed, the nutritious elements (mn, cr, fe, and mo) which were detected in high quantity in the skin of the fish samples (table 4) were found to be in lower amount in the flesh of the same sample of the fish (table 5). This is expected because when these important trace elements were greatly concentrated in the skin of the fish, they would not be accumulated in high concentration in the flesh of the same sample of dried fish . Ni and cu however demonstrated higher level in both the skin and flesh of the fish samples under the technique of solar - assisted drying compared to osd method . Therefore, this finding actually supported using of sds method of drying the fish because the reddish coloration of the fishes dried under this condition is due to the presence of higher content of cu in both the skin and flesh samples of solar - powered drying compared to both the skin and flesh samples of dried fishes subjected to osd . In addition, the quality of fish was also preserved in the sds method whereby no formation mold was observed after 5 days of packaging (figure 4) compared to the presence of mold formation in osd dried striped snakehead fish (figure 5). The finding from this study is important because it demonstrated that the different method of fish drying can influence their elemental contents and it is recommended that the drying method using sds has proven to contain higher content of the nutritious trace elements compared to using the conventional open drying system . However, the level of toxic heavy metals only showed slight difference between the two systems . As conclusion, solar drying system is recommended for healthier eating and longer shelf - life of dried striped snakehead fish.
Previous studies of traditional approaches to cognitive remediation in schizophrenia have been confounded by various methodological issues (see refs 11,12): small subject samples, open - label conditions, treatment- as - usual control groups, unblinded assessments, and unspecified plans for statistical analysis . Studies that avoided these issues have tended to find only small to moderate effect sizes . Nonetheless, the following findings point to some interesting next steps for the field: with the exception of verbal learning and memory, the meta - analysis by mcgurk et al found no significant heterogeneity in effect sizes on various matricsdefined cognitive domains based on either the number of hours of training or the method employed . This indicates that for the majority of cognitive domains, neither the training method, nor the amount of training (several hours to over 100 hours) has been a key moderating variable . Thus, although previous cognitive remediation approaches have provided modest nonspecific cognitive benefits, further refinement of the intervention and the use of rigorous study designs are critical next steps for the field . Meta - analyses have also shown that in verbal memory, larger effect sizes are obtained when computerized training is given in a drill - and - practice approach for a large number of hours . Significant synergy occurs when cognitive remediation is combined with a psychosocial intervention, such as vocational rehabilitation or social skills training . This indicates that appropriate cognitive training can prepare the individual with schizophrenia to benefit from ecologically meaningful learning events, and underscores the fact that optimal treatment in schizophrenia will necessitate multimodal approaches . Over the past decade, clinical neuroscience research has unequivocally demonstrated that declarative memory (those processes involved in recollection of facts and events) is impaired in schizophrenia; thus, in keeping with the meta - analysis findings discussed above, it seems clear that strategy coaching and other direct instruction methods are likely to be of only limited benefit for patients . Further, the sensory processing deficits of schizophrenia, which demonstrate a strong association with higher - order cognitive dysfunction, may confer a bottleneck in the response to behavioral interventions . A neuroscience - guided approach to cognitive training in schizophrenia should therefore take into account the following factors: the use of both implicit learning, through which skills and abilities are acquired indirectly and without direct awareness, and repetitive practice, may be crucial for maximizing patients' response to cognitive training . In addition, attention to sensory processing deficits may be necessary in order to drive an optimal response to cognitive treatments . Basic experimental work with motor skill learning and motor cortex remapping indicates that significant cortical synaptogenesis and reorganization of task - specific representations occurs after an animal reaches the flat congruent with the meta - analysis findings described earlier, this suggests that dosing and intensity of training is important: in order to drive maximally enduring and neurologically reliable cognitive gains, subjects must perform large numbers of learning trials and must train at threshold (ie, training must be individually adapted to the capacities of each learner). During learning, the brain selectively promotes both bottom - up and top - down neural activity patterns that represent meaningful stimuli and behaviors; successful learning is most efficiently driven by exercises which target all of the specific component skills of a given cognitive process . For example, intensive computerized frequency - sweep discrimination exercises markedly improve the ability of language - impaired children to recognize and respond to speech stimuli . For patients with schizophrenia, intensive training in a wide range of basic cognitive operations is likely to be necessary to improve higher - order functions (eg, it may be necessary to train the representational fidelity of early sensory data; vigilance; working memory; etc . Before achieving significant gains in executive functions). Learning - based plasticity is profoundly influenced by neuromodulatory neurotransmitters; therefore, learning trials must be designed that are closely attended by the subject and that involve a heavy reward schedule . Moreover, some of the medications currently used in schizophrenia may adversely affect the response to cognitive training strategies . We have been investigating the efficacy of a set of neuroscience - based cognitive training exercises designed with the considerations described above (software developed by posit science, inc). Subjects were randomly assigned to either 50 hours (1 hour per day, 5 days per week) of a computer games control condition, or to 50 hours of computerized training that places implicit, increasing demands on auditory perception and accurate aural speech reception . Frequency discrimination and phoneme recognition exercises targeted aspects of early auditory processing deficits of schizophrenia, which have been shown to affect higher - order cognitions such as verbal memory, reading ability, and social - emotional recognition . This psychophysical training was embedded within increasingly complex auditory and verbal working memory / verbal learning exercises that progress from simple frequency discrimination to phoneme identification and then recall of verbal instructions and narrative details . Compared with age- and education - matched subjects in the computer games control condition, we found that subjects who underwent targeted cognitive training showed significant increases in working memory, verbal learning and memory, and global cognition (figure 1, table i). These data suggest that an intensive dose of computerized cognitive training focused on auditory and verbal processing results in improved verbal learning and memory in adults with schizophrenia . Cognitive training subjects showed significantly greater improvement in verbal learning and memory measures from baseline to a 6-month follow - up assessment, indicating the durability of the cognitive training effects beyond the immediate post - training period . Furthermore, improved cognition was significantly associated with improvements in quality of life at 6 months . Serum anticholinergic activity (saa), reflecting medication - induced anticholinergic burden, showed a significant negative correlation with cognitive improvement after training . Consistent with basic science research, this suggests that the anticholinergic burden reduces the efficacy of cognitive training in driving cognitive improvement . Subjects in the cognitive training condition showed a significant increase in serum brain - derived neurotrophic factor (bdnf) levels compared with the computer games control group . This suggests that bdnf may be a peripheral biomarker for the effects of intensive cognitive training, and provides an indication of neurobiological response induced by the training . Normalization of physiological response patterns in auditory cortex within the cognitive training group but not the computer games control group - indicating that adaptive plastic changes in auditory processing systems can be induced in schizophrenia patients in response to a behavioral training intervention . Compared with age- and education - matched subjects in the computer games control condition, we found that subjects who underwent targeted cognitive training showed significant increases in working memory, verbal learning and memory, and global cognition (figure 1, table i). These data suggest that an intensive dose of computerized cognitive training focused on auditory and verbal processing results in improved verbal learning and memory in adults with schizophrenia . Cognitive training subjects showed significantly greater improvement in verbal learning and memory measures from baseline to a 6-month follow - up assessment, indicating the durability of the cognitive training effects beyond the immediate post - training period . Furthermore, improved cognition was significantly associated with improvements in quality of life at 6 months . Serum anticholinergic activity (saa), reflecting medication - induced anticholinergic burden, showed a significant negative correlation with cognitive improvement after training . Consistent with basic science research, this suggests that the anticholinergic burden reduces the efficacy of cognitive training in driving cognitive improvement . Subjects in the cognitive training condition showed a significant increase in serum brain - derived neurotrophic factor (bdnf) levels compared with the computer games control group . This suggests that bdnf may be a peripheral biomarker for the effects of intensive cognitive training, and provides an indication of neurobiological response induced by the training . Normalization of physiological response patterns in auditory cortex within the cognitive training group but not the computer games control group - indicating that adaptive plastic changes in auditory processing systems can be induced in schizophrenia patients in response to a behavioral training intervention . Although these early data using a neuroscienceinformed approach to cognitive training in schizophrenia are promising, they require replication with larger, more representative samples across multiple treatment sites . In addition, they raise many crucial questions for future studies: what are the necessary and sufficient ingredients essential for successful cognitive training in schizophrenia? What are the optimal methods, cognitive domains, and sequence of training? What is the minimum amount of cognitive training that results in meaningful cognitive improvement in patients? At what minimal frequency can training be delivered? What is the relationship between individual patient profiles at baseline (eg, genotype, biomarkers, neurocognitive profile) and their ability to realize and retain benefits from cognitive training (see, for example, refs 41 - 43)? What is the influence of commonly prescribed anticholinergic and antidopaminergic medications on cognitive training outcomes? How can we maximize the synergistic benefits of combining cognitive training with psychosocial rehabilitation treatments? Can targeted cognitive training be used to remit preexisting cognitive deficits and to promote recovery of function in young individuals who are in the very earliest phases of schizophrenia? If the promising initial findings we describe here are replicated, we will enter an exciting time for the field of schiz - ophrenia treatment, one which will require active collaborations between basic and clinical neuroscientists with expertise in neuroplasticity; researchers who perform clinical trials as well as experts in psychosocial remediation; clinical and research psychopharmacologists, and designers of computer games . We will enter a time of paradigm shift, and we will have the privilege of developing novel beneficial treatments for our patients.
Only 20 patients with deletions of 18q12.2 have been reported in the literature . In 15 of these patients, the deletions were examined with chromosome analysis, and in the remaining 5 patients molecular investigations identified deletions ranging from 3.2 to 24 mb in size . Common clinical findings included intellectual disability, behavioral disorders, seizures, eye manifestations, obesity, and mild or absent dysmorphic facial features . In recent years, the use of high - resolution cytogenetic techniques has led to the identification of numerous microdeletion syndromes, where patients with overlapping deletions share clinical features . However, the shared deletions often exceed hundreds of kb or even several mb, encompassing many genes that hamper the identification of the exact gene(s) underlying the associated phenotype . In contrast, truncation of single genes for example, by small intragenic deletions or by chromosomal translocation or inversion breakpoints can directly identify a disease - causing gene . Here we describe a patient with a prenatally detected de novo translocation involving chromosomes 12 and 18, with febrile seizures in childhood, borderline intelligence, developmental and behavioral disorders, myopia, and obesity . We characterized the rearrangement with snp array and next - generation sequencing (ngs) and found truncation of celf4, which has previously been suggested as a neurodevelopmental candidate gene . This report illustrates the utility of high - resolution genome - wide techniques in identifying neurodevelopmental genes . The patient was identified though a nation - wide study of prenatally detected de novo balanced structural rearrangements . The study was approved by the danish scientific ethics committee and written informed consent was obtained . Amniocentesis was performed due to advanced maternal age, and a de novo reciprocal translocation involving chromosomes 12 and 18 was diagnosed . The child was born by uncomplicated vaginal delivery at 40 weeks of gestation with a birth weight of 4000 g and birth length 55 cm . According to the mother, he walked independently at 12 months and there was no delay in language development . At 23 months of age owing to minor neurological deficits and difficulties with complex motor tasks, he was diagnosed with minimal brain dysfunction at age 4 . At the age of 6, he was described as aggressive, with immature and oppositional behavior, having problems with impulsivity and attention shifting, and with a lack of emotional reciprocity . At the age of 13, he was tested with weschler intelligence scale for children - revised and found to have an iq of 71, and he was diagnosed with behavioral disorder, oppositional defiant disorder, and mixed specific developmental disorder (all according to icd-10). A physical examination at the age of 25 years revealed no dysmorphic features . His height was 180 cm, weight 110 kg, and head circumference 61 cm . He was obese with a body mass index of 34; according to the mother, obesity has been present since the age of 67 years as a result of uninhibited eating behavior . At the age of 27 years, he was screened for major psychiatric disease using the mini international diagnostic interview version 5.0.0 that refers to the dsm - iv . No single diagnosis could be pinpointed . Signs of low iq and autistic behavior with indications of concrete thinking, impairment of social interaction skills, vagueness in description of others, lack of concentration over time, hyperactivity, and impulsive behavior were seen during the interview . There was significant impairment in occupational and social areas, and a general incapacity for labor market affiliation was manifest . In his youth, he was intensely preoccupied with trains and unable to develop peer relationships . Genomic dna was extracted by conventional methods from peripheral blood, and whole - genome copy number variation (cnv) analysis was performed with the affymetrix genome - wide human snp array 6.0 (affymetrix, santa clara, ca, usa). Raw intensity data files were analyzed with the genotyping console software (affymetrix) according to the manufacturer's recommendations . Mate pair libraries were prepared using the mate pair library v2 kit (illumina, san diego, ca, usa). Two- to three - kilobase pair fragments were isolated, end - repaired using a mix of natural and biotinylated dntps, blunt - end ligated using circularization ligase, and fragmented to 200400 bp . Biotinylated fragments were isolated and end - repaired and a - overhangs were added to the 3-ends . Paired - end adapters were ligated to the fragments and the library was amplified by 18 cycles of pcr . Mate pair libraries were subjected to 2 36 bases paired - end sequencing on a genome analyzer iix (illumina), following the manufacturer's protocol . Reads were aligned to a reference genome using bowtie allowing up to two mismatches in the seed region . Reads not aligning uniquely were discarded from further analysis . Paired reads aligning to different chromosomes or with unexpected strand orientation were extracted to identify potential translocation and inversion breakpoints, respectively . Breakpoints were only considered as candidates if they were confirmed by at least six independent paired reads with end - reads mapping within a 6-kb region . Predicted breakpoints were filtered against known in - house variants based on data from 30 individuals with known breakpoints . The translocation breakpoint was confirmed by pcr amplification and sanger sequencing of the breakpoint - spanning fragments . Amniocentesis was performed due to advanced maternal age, and a de novo reciprocal translocation involving chromosomes 12 and 18 was diagnosed . The child was born by uncomplicated vaginal delivery at 40 weeks of gestation with a birth weight of 4000 g and birth length 55 cm . According to the mother, he walked independently at 12 months and there was no delay in language development . At 23 months of age owing to minor neurological deficits and difficulties with complex motor tasks, he was diagnosed with minimal brain dysfunction at age 4 . At the age of 6, he was described as aggressive, with immature and oppositional behavior, having problems with impulsivity and attention shifting, and with a lack of emotional reciprocity . At the age of 13, he was tested with weschler intelligence scale for children - revised and found to have an iq of 71, and he was diagnosed with behavioral disorder, oppositional defiant disorder, and mixed specific developmental disorder (all according to icd-10). A physical examination at the age of 25 years revealed no dysmorphic features . His height was 180 cm, weight 110 kg, and head circumference 61 cm . He was obese with a body mass index of 34; according to the mother, obesity has been present since the age of 67 years as a result of uninhibited eating behavior . At the age of 27 years, he was screened for major psychiatric disease using the mini international diagnostic interview version 5.0.0 that refers to the dsm - iv . Signs of low iq and autistic behavior with indications of concrete thinking, impairment of social interaction skills, vagueness in description of others, lack of concentration over time, hyperactivity, and impulsive behavior were seen during the interview . There was significant impairment in occupational and social areas, and a general incapacity for labor market affiliation was manifest . In his youth, he was intensely preoccupied with trains and unable to develop peer relationships . Genomic dna was extracted by conventional methods from peripheral blood, and whole - genome copy number variation (cnv) analysis was performed with the affymetrix genome - wide human snp array 6.0 (affymetrix, santa clara, ca, usa). Raw intensity data files were analyzed with the genotyping console software (affymetrix) according to the manufacturer's recommendations . Mate pair libraries were prepared using the mate pair library v2 kit (illumina, san diego, ca, usa). Two- to three - kilobase pair fragments were isolated, end - repaired using a mix of natural and biotinylated dntps, blunt - end ligated using circularization ligase, and fragmented to 200400 bp . Biotinylated fragments were isolated and end - repaired and a - overhangs were added to the 3-ends . Paired - end adapters were ligated to the fragments and the library was amplified by 18 cycles of pcr . Mate pair libraries were subjected to 2 36 bases paired - end sequencing on a genome analyzer iix (illumina), following the manufacturer's protocol . Reads were aligned to a reference genome using bowtie allowing up to two mismatches in the seed region . Reads not aligning uniquely were discarded from further analysis . Paired reads aligning to different chromosomes or with unexpected strand orientation were extracted to identify potential translocation and inversion breakpoints, respectively . Breakpoints were only considered as candidates if they were confirmed by at least six independent paired reads with end - reads mapping within a 6-kb region . Predicted breakpoints were filtered against known in - house variants based on data from 30 individuals with known breakpoints . The translocation breakpoint was confirmed by pcr amplification and sanger sequencing of the breakpoint - spanning fragments . A total of 28 986 673 paired reads passed the chastity filter; 13 689 000 paired reads were aligned uniquely and 204 319 were chimeric pairs (end - reads mapping to different chromosomes). We removed non - clustering chimeric pairs leaving a total of 49 chimeric clusters genome - wide that were visually filtered against known variants . The breakpoint at chromosome 12 affected no annotated genes whereas the breakpoint at chromosome 18 truncated celf4 . The translocation breakpoint at chromosome 18 was confirmed by pcr amplification and subsequent sanger sequencing of the breakpoint - spanning fragments that revealed a small sequence of micro homology (agga), likely to facilitate the translocation (supplementary figure 1). Additionally, ngs revealed an associated inversion at chromosome 18 with breakpoints at 18q12.2 and 18q22.1 that truncated celf4 and loc643542 . Re - evaluation of the karyotype confirmed the cryptic complex rearrangement, and cnv analysis revealed small de novo deletions (<150 kb in size) corresponding to all four mapped breakpoints (supplementary table s1). Here, we report a male patient with a translocation involving chromosomes 12q and 18q with borderline iq, developmental and behavioral disorders, myopia, obesity, and a history of febrile seizures in childhood . Using snp array and ngs, we identified a cryptic complex rearrangement that truncated 2 refseq genes: the protein coding gene celf4 (also known as brunol4) at 18q12.2 and the non - coding rna loc643542 at 18q22.1 . Few of the previously reported patients with deletions of 18q12.2 were characterized sufficiently both clinically and molecularly to allow direct phenotype genotype comparisons . However, as illustrated in table 1 there is a considerable phenotypic overlap with our patient . Gilling et al reported a patient with childhood autism, severe myopia, and a de novo translocation, t(5;18)(q34;q12). Mcentagart et al described a patient with a de novo del(18)(q11.2q12.2) and a phenotype remarkably like the one reported here consisting of mild intellectual disability (iq 61), developmental delay, febrile seizures, clumsiness, behavioral difficulties including poor concentration and hyperactivity, distractibility and learning difficulties, and no facial dysmorphism . This patient was only examined with conventional karyotyping but the reported phenotype included intellectual disability, a history of fine motor problems, and seizures in childhood . Her behavior was described as impulsive, aggressive, defiant, restless, and labile autistic - like . Although celf4 was previously found disrupted in patients with overlapping phenotypes, no phenotype has been associated with the non - coding rna loc643542, thus we find it highly likely that haploinsufficiency of celf4 causes the described phenotype . Celf4 is expressed in various tissues with high expression in both fetal and adult human brain . In frog embryos (xenopus laevis), celf4 is solely expressed in the nervous system, including the optic veiscles, suggesting an important role in both brain and eye development . Furthermore, celf4 is highly expressed in mouse brain and celf4 insufficient transgenic mice develop convulsive seizures, hyperactivity, and late - onset body weight gain . Not only is celf4 conserved from drosphila to mouse and human, but the genomic landscape next to celf4 is also evolutionary conserved harboring ultra - conserved elements (uce), and it has been suggested that uces may be directly involved in neurodevelopmental disorders . We found celf4 to be disrupted in a patient with borderline iq, developmental and behavioral disorders, myopia, obesity, and a history of febrile seizures in childhood . The phenotype resembles that of patients with 18q12.2 deletions illustrating that celf4 haploinsufficiency has a key role in explaining their phenotype . This report, and the finding of celf4-deficient mice with overlapping traits, for example, seizures, hyperactivity, and obesity, adds to the growing evidence that celf4 is important for the brain development and in the disposition to obesity.
A previously healthy 36-year - old man presented with slowly progressive spastic paraparesis, impairment of vibration, and pain perception below the upper trunk associated with constipation, urinary difficulty, and sexual dysfunction . He had experienced lower abdominal pain and vomiting 7 months before the onset of these symptoms, and subsequently noticed a tingling sensation in the right leg and voiding difficulties . The neurologic examination performed at the time of his first visit to our clinic was compatible with a spinal cord lesion: there was bilateral hypesthesia below the t10 level, hyperactive knee and ankle jerks, and extensor plantar responses . T2-weighted magnetic resonance imaging (mri) of the spine showed a lesion with a high signal intensity and mild swelling that was not enhanced after the administration of contrast material in the cervical spinal cord (fig . 1). The cerebrospinal fluid (csf) was clear and colorless with 5 white blood cells/l, and the cells were mostly lymphocytes . Electrophoresis of csf was negative for the oligoclonal band, and the igg index was within the normal range . The laboratory findings for rheumatoid factor, antinuclear antibody, antineutrophil cytoplasmic antibodies, cryoglobulinemia, hepatitis b and c, and human immunodeficiency virus were negative . The erythrocyte sedimentation rate and c - reactive protein and complement levels (c3, c4, and ch50) were normal . The results for the visual evoked potential and brain mri were negative . The patient was treated with steroid pulse therapy under a diagnosis of transverse myelitis . He complained of intermittent chest discomfort when subsequently visiting our clinic, but electrocardiography, cardiac enzymes, and echocardiography findings were normal . The first steroid pulse therapy provided temporary relief from bilateral hypesthesia and paresthesia, but he still experienced spastic paraparesis with painful tonic spasm and bladder dysfunction including recurrent urinary tract infection and nonspecific anterior chest tenderness, especially in the costochondral joint area . Another two cycles of steroid pulse therapy were administered during the 2 years following the first treatment due to progression of sensory and motor symptoms . Follow - up spine mri after the second steroid pulse therapy revealed recurrence of intramedullary myelopathy (fig . Two years after the onset of myelopathy, he developed painful swelling of his costochondral joints and both knees . A tc methylene diphosphonate bone scan showed markedly increased uptake in the left costomanubrial junction and the first rib (fig . Pelvis radiographs showed ankylosis of bilateral hip joints and poorly delineated bilateral sacroiliac joints (fig . He subsequently displayed multiple pustular skin eruptions on the palms of the hands, soles of the feet, elbows, trunk (fig . Administration of methotrexate and sulfasalazine markedly reduced the patient's arthralgia and neurologic symptoms, which were stabilized by maintenance therapy with methotrexate and prednisolone . The spondyloarthropathies comprise a diverse group of inflammatory arthritis conditions that share certain genetic predisposing factors and clinical features . Recent studies have provided insight into distinct pathogenetic mechanisms underlying ankylosing spondylitis and reactive arthritis that arise from a complex interplay between genetic factors (including hla - b27) and environmental factors.2 the pathogenesis of reiter's syndrome may involve molecular mimicry between bacterial fragments in synovial fluid and the hla - b27 molecule . Most (70 - 80%) patients with reiter's syndrome are positive for hla - b27, as compared with only 6% of the general population . The arthritis may be perpetuated by the induction of cytotoxic t lymphocytes by microbial fragments in the joints, but these cytotoxic t lymphocytes have specificity for hla - b27-positive cells . The presence of hla - b27 may allow stronger or persistent microbial invasion.3 reactive arthritis usually has a self - limited course of 3 to 12 months, but up to 50% of patients experience recurrent bouts of arthritis, and 15% to 30% of them develop chronic symptoms of the disease.4 extra - articular manifestations such as ocular inflammation, enteritis, mucocutaneous lesions, urethritis, and (rarely) carditis provide essential support for a diagnosis of reactive arthritis . However, neurological complications are rare.5 there have been only a few case reports of polyneuropathy, cranial nerve palsy, or myelopathy in reiter's syndrome.6,7 whilst there were gastrointestinal symptoms in this case, stool examinations provided no laboratory evidence of preceding infection . The patient visited our clinic at 7 months after the onset of the first symptoms . The high dose of steroids administered might have inhibited a systemic inflammatory reaction . According to a previous report, approximately only 60% of such cases have evidence of previous infection detected either by serology or by cultures from urogenital or stool samples.8 nonsteroid anti - inflammatory drugs (nsaids) and sulfasalazine are effective treatments for reactive arthritis, and methotrexate can also be beneficial.9 our patient did not respond to nsaids, but methotrexate and sulfasalazine relieved his neurological symptoms and arthralgia, making them stable during a 5-year follow - up . The association between reactive arthritis and cervical myelopathy was not clear in this patient, but there were some features suggesting reiter's syndrome as a cause of the myelitis . First, the gastrointestinal symptoms that appeared before the development of cervical myelopathy might have reflected a preceding infection that initiated an autoimmune reaction, which led to myelitis and systemic inflammation . Initial urinary tract infection and costochondral tenderness at the time of the first attack of myelitis might be indicative of reiter's syndrome . Second, despite the application of steroid pulse therapy, recurrences of progressive myelopathy associated with systemic symptoms of the skin and joints, and the positivity for hla - b27 suggest other causes of myelitis . Third, both neurological symptoms and the arthritis that was resistant to the steroid pulse therapy did not recur after sulfasalazine and methotrexate treatment . In conclusion, reiter's syndrome should be considered in the differential diagnosis of cases of progressive myelopathy with multiple arthritis, urethritis, and skin lesions.
In may 2014, a pig fattening farm in southern germany (federal state of baden - wuerttemberg) that continuously houses 1,400 fattening pigs reported watery diarrhea in pigs in all age groups (feeders to slaughter animals). The first cases occurred after new feeder pigs from a large piglet producer were brought onto the farm . Fecal samples from diseased pigs were submitted to the regional laboratory for diagnosis, and coronaviruses were detected by electron microscopy (figure 1). Additionally, 3 pigs with catarrhal enteritis were euthanized; postmortem examination at the regional laboratory confirmed coronavirus infection in all 3 animals . Porcine epidemic diarrhea virus particles seen by negative - stain electron microscopy of fecal samples . Subsequently, ped was diagnosed in a private laboratory (ivd gmbh, hannover, germany) by use of a published multiplex reverse transcription quantitative pcr (7). Selected positive samples were submitted to the friedrich - loeffler - institut, isle of riems, germany, for confirmation and further virus characterization . At this institution, 2 fecal samples with high genome load, as determined by 2 independent, published (8,9) reverse transcription quantitative pcrs selective for pedv nucleocapsid (n) and spike (s) genes, were chosen for routine virologic testing and next - generation sequencing . The phylogenetic tree, based on full - length genomes, was constructed by using phyml (11) in the geneious software suite (http://www.geneious.com/) with a generalized time reversible substitution model, and the tree was supported by 1,000 bootstrapping replicates . Virus isolates were obtained after inoculation of cells of different permanent cell lines (pig kidney [pk]-15 and vero) with clinical material from the pigs . Sequencing of nucleic acids isolated directly from diagnostic samples pedv / ger / l00719/2014 and pedv / ger / l00721/2014 resulted in 2 viral genomes (table) encompassing all typical pedv coding sequences . Each sequence encodes a large replicase polyprotein, a spike (s) protein, an alphacoronavirus - specific accessory membrane protein, an envelope protein, a membrane protein, and a nucleocapsid (n) protein . * kj778615, kf272920, kf468753, kj584361, kj408801, kf650374, kf468752, kf267450, kc210147, jn825712, kc210145). #aa 632f detected in reference strain kf267450 only . Comparative analyses of the full genomes showed that the strains share a very high (99.5%) identity with the new variant oh851 (genbank accession no . A more comprehensive comparison of 21 full - length pedv genomes from different years and locations revealed lower similarities (98.7%) with currently circulating highly virulent strains from the united states and from china (technical appendix figure, panel a). In contrast, the new isolates from germany, pedv / ger / l00719/2014 and pedv / ger / l00721/2014, are less similar (97.1%) to the isolate from europe, cv777, which dates back to the 1970s (12). The nucleotide alignment of the obtained pedv genomes and the available references from the database revealed a region with high variability of the first 1,200 nt in the 5 portion of the s protein the n terminal s1 domain of the coronavirus s protein is necessary for virus attachment by interaction with host cell receptors (13) and might therefore be highly mutable . Although in - depth analysis of the deep - sequencing data for pedv / ger / l00721/2014 revealed a genetically homogenous population, this analysis for pedv / ger / l00719/2014 uncovered a mixed viral population with a total of 8 single - nucleotide variants . One nonsynonymous single - nucleotide variant (variant position g19042u, amino acid substitution s6348i) was detected in the polyprotein coding sequences . Of note, 7 single - nucleotide variants are located in the aforementioned variable region in the s protein coding sequences, 5 of which are nonsynonymous (table), thereby confirming the high variability in the n terminal part of the s protein . Because quite extensive differences (50 aa) were found between the recent n terminal s protein region of the isolates from germany and the highly virulent pedv strains from the united states and china, the isolates from germany described in this article seem to not be directly linked to the highly virulent pedv strains circulating in the united states (figure 2). In contrast, the recent isolates from germany and strain oh851 share not only high identity over the entire genome, including the highly variable 5 region of the s protein coding sequences, but also their clinical phenotype observed under field conditions . Phylogenetic analysis based on 21 full - length porcine epidemic diarrhea virus (pedv) genomes . The new strains from germany (pedv / ger / l00719/2014 and pedv / ger / l00721/2014, in boldface and italics) and the new 2014 pedv variant from the united states (oh851, in boldface) were included and compared with current circulating strains from the united states and china . Numbers above branches indicate proportions calculated from 1,000 bootstrap replicates: the scale bar represents nucleotide substitutions per site . Comparative analyses of full - length sequences revealed that the isolates from these pigs in germany show very high nucleotide similarity with strain oh851 found in the united states in 2014 . However, differences exist that distinguish the strains from germany from the highly virulent pedv strains that caused the major losses in the united states . Given the fact that pedv surveillance has been lacking in germany, we cannot exclude the possibility that the strains described here have already been circulating in europe for a longer time or were indeed recently introduced from the united states or asia to europe . Therefore, our report provides useful information about recent pedv strains in europe, but a comprehensive evaluation is still difficult because of a lack of data about additional strains . Future studies should therefore concentrate on analysis of additional pedv from different years and locations . Pairwise similarity matrix based on full - length genomes and schematic representation of the nucleotide sequence alignment of the complete spike protein coding sequences.
Multiple sequence alignments (msas) are an essential first step for a number of computational approaches such as protein secondary structure / function prediction, phylogeny inference, and many other common tasks in sequence analysis.15 several software tools for generating multiple sequence alignments are available,6 but none of them is suitable for all types of data sets.7 consequently, in order to generate a true alignment, a need rises for inspection and adjusting alignments by hand, which is a very laborious job.5 furthermore, handling hundreds of thousands of alignments is another problem in the domain of bioinformatics . Many popular alignment editors such as jalview,5 strap,8 seaview,9 mega,10 cinema,11 and base - by - base12 are available . All these tools either do not support tens of thousands of sequences (where length of each sequence is more than 2,500 base pairs) or they do not have user - friendly editing features . Jalview and strap do not work when the size of an alignment exceeds 30.01 mb . Seaview and mega can load big alignments, but the editing features provided by them involve multiple steps . Many software tools provide graphical user interfaces to reconstruct msas, but they do not allow loading multiple sequence files at the same time . Msa comparison tools such as suitemsa6 and sinicview13 permit to compare msas, but they support alignments comprising less than 1,000 sequences . Multiple formats of msas exist, but fasta is the most popular format . Presently, there is no tool that can convert the format of an alignment of unlimited size into fasta format . Many tools, such as matgat14 and sequence identity and similarity (sias),15 are available, to calculate the identity matrix but they do not support more than 1,000 sequences . This article describes ivistmsa, which is a software package of seven graphical tools for multiple sequence alignments . All tools allow the user to load tens of thousands of sequences to edit / analyze and compare them . Ivistmsa implemented a divide - and - conquer (dnc) approach to calculate efficiently consensus / conserved sequences, identity matrix, sum - of - pairs score (sps), colum score (cs), and conserved regions of two alignments . Sps of a column is calculated as the ratio of the sum of scores of all pairs of residues in every column of test alignment by sum of scores of all pairs of residues in the similar column of reference alignment . To calculate sps of the entire alignment, sum of sum of scores of each column of the test alignment cs is computed as the ratio of matched columns (between test and reference alignments) by the number of considered columns in the test alignment . The sequence file with 807 sequences named as bba0039.tfa available in rv100 folder enclosed in a zipped file named as this data set was replicated to generate an alignment with 1,614 sequences, which was replicated to generate 3,228 sequences, and so on . Replication was done in order to save time and avoid from constructing big alignments from an msa method . All tools of ivistmsa (fig . 1) are written in java programming language . Xml was used for saving the state of the work performed in msapad and msa comparator . A computing machine having core i7 3.34-ghz processor and 8-gb ram was used to write and analyze ivistmsa . Most of the tools of ivistmsa use dnc approach for performing efficient computations on msas . The dnc approach has been implemented using a power feature of multithreading provided by java programming language . Dnc approach divides an alignment horizontally into subalignments, and java threads are generated for each fragment . All java threads return results to the main thread, which computes the final value . Msapad uses dnc approach to compute consensus, conserved sequence(s), and distance matrix to construct a phylogenetic tree . Step 1 calculates number of subalignments for a given msa . By default, each alignment is divided into two subalignments . Step 2 creates a list / array for holding size of each sub - msa . Step 3 calculates the size of the first subalignment and stores it into the first position of the list . Step 5 subtracts one from the submsas variable because up to now, the size of the first sub - msa has been calculated . Variable is used to store the size of subalignments at correct positions in the list . Figure 2b shows the overall process of dividing msa into sub - msas, computations performed by each thread, and the final computation by the main thread . The alignment - rendering model has played a very important role to display, edit, and analyze very big alignments efficiently . The model sits between the alignment and its viewer and manages efficiently various types of manipulations on the alignments . These manipulations include finding a residue, all types of editing features provided by ivistmsa, changing color schemes, and loading the alignment itself . Abstracttablemodel class provided as part of the swing library of the java programming language . Results showed that, in contrast to all other alignment editors written in java programming language such as strap, jalview, and base - by - base, it can load more than 400% big alignments . Msapad allows the user to save the alignment, find a single residue or sequence name, add the sequence at the start or end of an alignment, add a sequence before or after a selected sequence, and move a sequence up or down (fig . 4b). It can find consensus and conserved sequence / regions of an alignment of 120-mb size in just 27 seconds . It allows the user to calculate a phylogenetic tree using neighbor joining% identity and blosum 62 matrix . The tree is drawn using archaeopteryx, which is an open - source software tool for displaying and analyzing a phylogenetic tree . Archaeopteryx and jmol are embedded to view and analyze phylogenetic trees and protein 3d chemical structures, respectively (fig . Its version 1.0.2 is already published in life science journal.16 data structures used in mqatv1.0.2 were improved, and it is now more efficient than qscore program (http://www.drive5.com/qscore/) and fastsp as well . The new version of mqat allows the user to visualize the conserved and nonconserved regions of the selected alignments from the main window of mqat . Figure 5a displays the main window of mqat, which allows the user to select an alignment . Results showed that the msa comparator can display conserved and nonconserved regions of two alignments comprising more than 8,000 sequences with a sequence length of 2,696 base pairs in less than 12 seconds . Msa reconstruction tool provides graphical interfaces for clustal omega, clustaw2, mafft, muscle, and biojava implementation for msas . Its unique feature is that the user can load multiple sequence files at the same time . Now the user does not need to sit before the system and wait for the completion of the process so that the next sequence file may be uploaded . Using msa reconstruction tool of ivistmsa interfaces for clustal omega, clustalw2, mafft, muscle, and biojava can be loaded by clicking the msa reconstruction drop - down list (fig . Fatsa generator can convert clustalw, msf, phylip, pir, gde, mega, and nexus formats of alignments of unlimited size into fasta format . We converted successfully an alignment of msf format comprising 102,102 sequences into fasta format in less than 1 second . The other important feature of this tool is that the user can load alignments of different formats . Fasta generator recognizes the format of an alignment automatically and converts it into fasta format . Msa i d calculator allows the user to calculate the identity matrix of an alignment . Results showed that it can calculate the identity matrix of more than 11,000 sequences with a sequence length of 2,696 base pairs in less than 100 seconds . Tree calculation tool calculates a phylogenetic tree using neighbor joining% identity and blosum 62 matrix and permits the user to draw the tree using archaeopteryx, which is an open - source software tool for drawing and analyzing a phylogenetic tree . Ivistmsa is a suite of seven interactive visual tools to generate, view, edit, and analyze msas . Presently, a lot of msa editing and analyzing tools are available . The popular and widely used tools include jalview,5 seaview,9 mega,10 strap,8 pfaat,17 base - by - base,12 and cinema.11 jalview, strap, pfaat, and base - by - base are java programs, whereas seaview, mega, and cinema are written in c++ language . Results showed that msapad loaded more than 50,000 sequences with a sequence length of 2,696 base pairs, whereas other alignment editors written in java programming language could load less than 12,000 sequences only . Alignment editors written in c++ language loaded more sequences than msapad, but their editing features are not user friendly . Msapad allows the user to edit a residue at its own position without opening a new interface, whereas all other tools allow editing of an alignment in a new interface . Msapad provides the feature to insert a sequence at any location of an alignment, whereas most of the available editing tools allow the user to insert a sequence at the end of an alignment . Similarly moving a column and sorting an alignment by column are also the unique features of msa - pad . Some of the alignment editors such as homed18 and maligned19 are no longer maintained by their authors . Several tools such as suite msa,6 sinicview,13 altavist,7 and balibase c program20 are available to compute sps and cs but they cannot process more than 1,000 sequences . Results showed that the msa comparator was 5,200% efficient as compared to balibase c program . It calculated conserved regions of two alignments comprising more than 8,000 sequences with a sequence length of 2,696 base pairs in less than 12 seconds, whereas suitemsa allowed the user to compute conserved regions of two alignments consisting of less than 1,000 sequences . Many software tools such as strap, suitemsa, pfaat, and seaview provide graphical user interfaces for generating msas, but they do not allow the user to load several sequence files simultaneously . Msa reconstruction tool allows the user to load several sequence files at a time to align them one by one using clustal omega, clustalw2, mafft, muscle, or biojava . Lot of tools and web servers such as alter21 and readseq22 are available for converting formats of msas, but they process alignments comprising a few thousand sequences . Fasta generator allows the user to convert seven msa formats (clustalw, msf, phylip, pir, gde, and nexus) into fasta format . It can generate an identity matrix of more than 500 sequences with sequence length of 2,696 base pairs, which was very time consuming . Since, sias is a web application, it was also not a good tool for calculating identity matrix of more than 1,000 sequences with a sequence length of 2,696 base pairs . Msa i d calculator is an efficient tool, which calculated the identity matrix of more than 11,000 sequences with a sequence length of 2,696 base pairs in less than 100 seconds . Comparison indicates that msapad, msa comparator, fasta generator, and msa i d calculator are more efficient than other similar tools available in the market for multiple sequence alignments . Msapad allows the user to edit and analyze 409% more data than jalview, strap, cinema, and base - by - base . Msa comparator (mqat version 2.0.1) allows the user to visualize consistent and inconsistent regions of reference and test alignments of more than 21-mb size in less than 12 seconds . Msa reconstruction tool allows a user to upload several sequence files through the graphical user interfaces of clustal omega, clustalw2, mafft, and muscle, and then align them one by one . Fasta generator converts the other seven formats of alignments of unlimited size into fasta format in a few seconds . Msa i d calculator is a tool that allows a user to calculate the identity matrix of more than 11,000 sequences with a sequence length of 2,696 base pairs in less than 100 seconds . Tree and distance matrix calculation tools generate phylogenetic tree and distance matrix, respectively, using the neighbor joining% identity and blosum 62 matrix . Ivistmsa allows scientists to view, edit, interpret, and analyze very big alignments . Project name: ivistmsa project home page: http://ivistmsa.com/ operating system(s): tested on windows but it should run on other platforms as well programming language: java 1.7 execution requirements: jdk1.7 or higher any restrictions to use by nonacademics: none
Nearly 12 200 new cases of cervical cancer are diagnosed annually in the u.s . . Cervical cancer is a leading health problem worldwide, causing approximately 40 000 deaths per year in developed nations and 234 000 in developing counties annually . The standard of care for cervical cancer requires concomitant chemotherapy with external - beam radiotherapy (ebrt) and brachytherapy (bt). The bt treatment planning technique, even for high - dose - rate treatments (hdr), has remained unchanged for decades . Utilizing 2-dimensional (2-d) orthogonal radiographs, a plan is created using the manchester system in which the radiation dose is prescribed to point a: an estimate of where the uterine artery intersects with the ureter [2, 3] irrespective of an individual patient's tumor characteristics . . 1 of reference) often occurs in a high - gradient region of the isodose distribution . However, studies of on the impact of different definitions of point a are limited to conference abstracts [69]. Definitions and potential errors of manchester point a and new point a. a) manchester point a. b) new point a the use of 2-d imaging in hdr bt is shifting towards the use of magnetic resonance imaging (mri) to acquire volumetric data of the macroscopic tumor region [10, 11]. Mri guidance allows treatments to be optimized to tumor volumes while sparing organs - at - risk (oars). Computed tomography (ct), though effective for other treatment sites, is inferior to mri in accurately distinguishing the tumor and tissues of the cervix . With the advent of 3-dimensional (3-d) imaging the prescription dose shifts away from point where previously point a would be treated to the prescription dose, the new target for 3-d image guided conformal treatments is for 90% of the high - risk clinical target volume (hr - ctv d90) to receive at least the prescribed dose . The international mri - guided, conformal bt protocol (embrace: an international study on mri - guided brachytherapy in locally advanced cervical cancer) recommends that hr - ctv d90 receives the conventional point a dose . Recent studies have reported significant correlation between dose - volume - histogram (dvh) parameters (d90 and d100) and local tumor control . However, the gynaecological european group of curietherapie and the european society for therapeutic radiology and oncology (gec - estro) still recommend the recording of conventional point a doses during 3d - image based planning, at least for a transition period . Also recommended is the investigation of correlation back to conventional dose measurements: point a dose and the volumetric - dose in ctv . Both gec - estro recommendations and the embrace protocol recommend mri - guided, conformal bt plans report point a doses even when dose is prescribed to hr - ctv d90 . However, the effect of point a definition on these correlations has not been reported until this present study . The original point a concept of the manchester system reported in 1938 has never changed: 2 cm lateral to the central canal of the uterus and 2 cm up from the mucous membrane of the lateral fornix, in the axis of the uterus . The superior aspects of the vaginal ovoids were originally used as a landmark for the vaginal fornix . In 1953, a cervical flange was proposed due to the difficulty recognizing the ovoids superior edge on radiographs, this was referred to as revised point a, although it creates a wider variation of point a locations due to the tandem placement being independent of the ovoid position . To minimize these variations, many institutions developed their own definition of point a, including the use of the superior tip of the ovoids in a sagittal radiograph as a landmark for the vaginal fornix [8, 9]. In an effort to standardize point a definitions, the american brachytherapy society (abs) introduced a new point a definition . 1):manchester point a: 2 cm superior to the tandem flange and 2 cm lateral from the center of tandem . This was the revised point a from 1953 .new point a: the new point definition as recommended by the abs . Draw a line through the center of each ovoid . (r + 2) cm superiorly along the tandem from the intersection of this line, where r is the radius of the ovoid and 2 cm lateral from the center of the tandem . Manchester point a: 2 cm superior to the tandem flange and 2 cm lateral from the center of tandem . New point a: the new point definition as recommended by the abs . Draw a line through the center of each ovoid . (r + 2) cm superiorly along the tandem from the intersection of this line, where r is the radius of the ovoid and 2 cm lateral from the center of the tandem . It should be noted that all point a definitions will be co - located when the applicator is correctly implanted . For clarity, this work will use the form manchester point a or new point a to refer to the specific definitions and point a to refer to the general treatment concept . A total of 55 hdr plans from patients with biopsy - proven cervical cancer who received radiotherapy (rt) were retrospectively studied after approval from the local institutional review board . 2d - f) from a high resolution (3.0 tesla) siemens magnetom trio 3 t mr scanner (siemens medical solutions, erlangen, germany). Only the 25 plans using mri were analyzed using dvh parameters . In addition, the 25 plans were retrospectively re - planned using conformal planning techniques . In the authors institutional protocol, the majority of patients with figo (international federation of gynecology and obstetrics) stage ib2 and above cervical cancer received ebrt, hdr bt and weekly concomitant cisplatin infusion . Bt fractionation schemes consisted of either 3 fractions of 7.5 gy or 5 fractions of 5.5 gy, with occasional minor variations . A titanium fletcher - suit - declos style tandem - and - ovoids (t&o) applicator (varian medical systems, palo alto, ca) was used for all plans, along with the brachyvision treatment planning system (version 8.9, varian medical systems, palo alto, ca). Part (a) shows coronal and sagittal radiograph images and example locations of the two points, (b) shows the full isodose set for a normalized to new point a, and (c) shows the 100% isodose lines for both point a definitions . Parts (d) through (f) show high resolution (3.0 tesla) mr images with hr - ctv and oar contours and conformal plan isodose lines all conventional, clinical plans had been optimized to generate standard pear - shaped isodose distributions using reference lines that were located 2 cm lateral from the tandem and 0.5 cm lateral from each ovoid's surface (fig . 2). The locations of manchester point a and new point a were retrospectively defined by a single person to avoid inter - physicist variation . For both point definitions, the stand pear - shaped isodose lines were re - normalized such that the point received the prescribed dose . The 25 plans using mri were also retrospectively re - planned with the intention of generating mri - guided, conformal plans (fig . 2d - f) in accordance with gec - estro recommendations [10, 11] and embrace protocol . The details of the mri scan protocols for the t&o hdr bt procedure were summarized in the previous study . An experienced physician contoured the intermediate risk ctv (ir - ctv), the high risk ctv (hr - ctv) and organs - at - risk (oar) according to gec - estro recommendations [10, 11] and embrace protocol . A conformal plan was optimized to achieve the following: hr - ctv d90 was to receive the prescription dose and d2cc (the minimum dose within the 2 cc of tissue receiving the greatest dose) of each oar was to receive less than the limits of the gec - estro recommendations [10, 11] and embrace protocol . The recommended limits of d2cc for the rectum, bladder and sigmoid colon are 75 gy, 90 gy, and 75 gy, respectively . These limits are the combined ebrt and hdr doses in terms of eqd2 (equivalent dose in 2 gy fractions of ebrt, using an / value of 3). Ebrt plan data were used during conformal hdr plan optimization procedures to apply the recommended oar limits . The hybrid - inverse optimization described in our previous work was utilized for all conformal plans (fig . 2): starting with a conventional point a plan, dose - volume objectives were set and inverse planning performed . The isodose lines and dvh measurements of the optimized plan were then checked and further inverse planning or graphical dose shaping used where necessary . Among total 55 conventional plans, the 30 plans guided by 2-d radiographs were analyzed by measuring the volume receiving 100% or more of the prescription dose (v100%), the total reference air kerma (trak), and the dose to the rectum and bladder points of icru report #38 (international commission on radiation units and measurement). The other 25 conventional plans using mri were further analyzed using dvh parameters, including the minimum dose received by 90% of the hr - ctv (d90) and the minimum dose to the hottest 2cc of each oar (d2cc). All dvh parameters were analyzed only for hdr plans no ebrt plan data were analyzed . For each measurement of a plan re - normalized using manchester point a (manchester point a plan), for example, a corresponding measurement was taken using the new point a normalized plan (new point h plan). As the focus was on systematic differences between these plans, we took the percent difference of each pair as the first step of our analysis . This was done with respect to the new point a plans such that negative results indicated a lower value in the point a plan (see fig . Average percent differences of manchester point a plans when normalized to new point a plans . Negative values indicate lower results for the manchester point a plans . The 95% confidence interval is marked the 25 conformal plans using mri were also analyzed by dvh parameters, along with the 2-d parameters v100%, trak, and rectal and bladder point doses . In conformal plans, the correlation between tumor coverage values such as hr - ctv d90, d100 or ir - ctv d90 and point a doses are of interest, since the majority of clinical outcomes are linked with these point measurements it is noted that conformal plans do not use point a to generate plans, but use hr - ctv and oar to perform volume optimization . Therefore, we quantified how the correlation is affected by different point a definitions for a given conformal plan . Specifically percent differences between manchester point a or new point a dose and tumor coverage values (d90 or d100) were taken with respect to the dose volume measurement values, with negative results indicating a lower value in the point dose (cf . 4). For all dose - volume parameter analysis (i.e. D90, d100, and d2cc), physical doses were converted to eqd2 values using / = 3 for healthy tissue and / = 10 for target volumes [10, 11]. Percent differences between manchester point a (point h) doses and prescription doses & tumor coverage metric from mri - guided, conformal plans . The original point a concept of the manchester system reported in 1938 has never changed: 2 cm lateral to the central canal of the uterus and 2 cm up from the mucous membrane of the lateral fornix, in the axis of the uterus . The superior aspects of the vaginal ovoids were originally used as a landmark for the vaginal fornix . In 1953, a cervical flange was proposed due to the difficulty recognizing the ovoids superior edge on radiographs, this was referred to as revised point a, although it creates a wider variation of point a locations due to the tandem placement being independent of the ovoid position . To minimize these variations, many institutions developed their own definition of point a, including the use of the superior tip of the ovoids in a sagittal radiograph as a landmark for the vaginal fornix [8, 9]. In an effort to standardize point a definitions, the american brachytherapy society (abs) introduced a new point a definition . 1):manchester point a: 2 cm superior to the tandem flange and 2 cm lateral from the center of tandem . This was the revised point a from 1953 .new point a: the new point definition as recommended by the abs . Draw a line through the center of each ovoid . (r + 2) cm superiorly along the tandem from the intersection of this line, where r is the radius of the ovoid and 2 cm lateral from the center of the tandem . Manchester point a: 2 cm superior to the tandem flange and 2 cm lateral from the center of tandem . New point a: the new point definition as recommended by the abs . Draw a line through the center of each ovoid . (r + 2) cm superiorly along the tandem from the intersection of this line, where r is the radius of the ovoid and 2 cm lateral from the center of the tandem . It should be noted that all point a definitions will be co - located when the applicator is correctly implanted . For clarity, this work will use the form manchester point a or new point a to refer to the specific definitions and point a to refer to the general treatment concept . A total of 55 hdr plans from patients with biopsy - proven cervical cancer who received radiotherapy (rt) were retrospectively studied after approval from the local institutional review board . 2d - f) from a high resolution (3.0 tesla) siemens magnetom trio 3 t mr scanner (siemens medical solutions, erlangen, germany). In addition, the 25 plans were retrospectively re - planned using conformal planning techniques . In the authors institutional protocol, the majority of patients with figo (international federation of gynecology and obstetrics) stage ib2 and above cervical cancer received ebrt, hdr bt and weekly concomitant cisplatin infusion . Bt fractionation schemes consisted of either 3 fractions of 7.5 gy or 5 fractions of 5.5 gy, with occasional minor variations . A titanium fletcher - suit - declos style tandem - and - ovoids (t&o) applicator (varian medical systems, palo alto, ca) was used for all plans, along with the brachyvision treatment planning system (version 8.9, varian medical systems, palo alto, ca). Part (a) shows coronal and sagittal radiograph images and example locations of the two points, (b) shows the full isodose set for a normalized to new point a, and (c) shows the 100% isodose lines for both point a definitions . Parts (d) through (f) show high resolution (3.0 tesla) mr images with hr - ctv and oar contours and conformal plan isodose lines all conventional, clinical plans had been optimized to generate standard pear - shaped isodose distributions using reference lines that were located 2 cm lateral from the tandem and 0.5 cm lateral from each ovoid's surface (fig . 2). The locations of manchester point a and new point a were retrospectively defined by a single person to avoid inter - physicist variation . For both point definitions, the stand pear - shaped isodose lines were re - normalized such that the point received the prescribed dose . The 25 plans using mri were also retrospectively re - planned with the intention of generating mri - guided, conformal plans (fig . 2d - f) in accordance with gec - estro recommendations [10, 11] and embrace protocol . The details of the mri scan protocols for the t&o hdr bt procedure were summarized in the previous study . An experienced physician contoured the intermediate risk ctv (ir - ctv), the high risk ctv (hr - ctv) and organs - at - risk (oar) according to gec - estro recommendations [10, 11] and embrace protocol . A conformal plan was optimized to achieve the following: hr - ctv d90 was to receive the prescription dose and d2cc (the minimum dose within the 2 cc of tissue receiving the greatest dose) of each oar was to receive less than the limits of the gec - estro recommendations [10, 11] and embrace protocol . The recommended limits of d2cc for the rectum, bladder and sigmoid colon are 75 gy, 90 gy, and 75 gy, respectively . These limits are the combined ebrt and hdr doses in terms of eqd2 (equivalent dose in 2 gy fractions of ebrt, using an / value of 3). Ebrt plan data were used during conformal hdr plan optimization procedures to apply the recommended oar limits . The hybrid - inverse optimization described in our previous work was utilized for all conformal plans (fig . 2): starting with a conventional point a plan, dose - volume objectives were set and inverse planning performed . The isodose lines and dvh measurements of the optimized plan among total 55 conventional plans, the 30 plans guided by 2-d radiographs were analyzed by measuring the volume receiving 100% or more of the prescription dose (v100%), the total reference air kerma (trak), and the dose to the rectum and bladder points of icru report #38 (international commission on radiation units and measurement). The other 25 conventional plans using mri were further analyzed using dvh parameters, including the minimum dose received by 90% of the hr - ctv (d90) and the minimum dose to the hottest 2cc of each oar (d2cc). All dvh parameters were analyzed only for hdr plans no ebrt plan data were analyzed . For each measurement of a plan re - normalized using manchester point a (manchester point a plan), for example, a corresponding measurement was taken using the new point a normalized plan (new point h plan). As the focus was on systematic differences between these plans, we took the percent difference of each pair as the first step of our analysis . This was done with respect to the new point a plans such that negative results indicated a lower value in the point a plan (see fig . Average percent differences of manchester point a plans when normalized to new point a plans . The 95% confidence interval is marked the 25 conformal plans using mri were also analyzed by dvh parameters, along with the 2-d parameters v100%, trak, and rectal and bladder point doses . In conformal plans, the correlation between tumor coverage values such as hr - ctv d90, d100 or ir - ctv d90 and point a doses are of interest, since the majority of clinical outcomes are linked with these point measurements . It is noted that conformal plans do not use point a to generate plans, but use hr - ctv and oar to perform volume optimization . Therefore, we quantified how the correlation is affected by different point a definitions for a given conformal plan . Specifically percent differences between manchester point a or new point a dose and tumor coverage values (d90 or d100) were taken with respect to the dose volume measurement values, with negative results indicating a lower value in the point dose (cf . 4). For all dose - volume parameter analysis (i.e. D90, d100, and d2cc), physical doses were converted to eqd2 values using / = 3 for healthy tissue and / = 10 for target volumes [10, 11]. Percent differences between manchester point a (point h) doses and prescription doses & tumor coverage metric from mri - guided, conformal plans . We found that a conventional, point a based plan is minimally affected by variations caused by implant procedure and/or patient anatomy when new point a definition was used . For any plan, there is both a left and right side point a and the normalization ensures that their average dose values equal the prescribed dose . We observed that the percent deviations of the higher single point a values with respect to the prescription doses were up to 9.5% (mean 1.7%, ci95 0.5%, p <0.001) and for new point a up to 6.7% (mean 1.5%, ci95 0.3%, p <0.001). Across the 55 plans, manchester point a was found to be mean distance of 0.6 cm from new point a (ci95 0.1 cm, max 2.1 cm, p <0.001). In other words, figure 2a shows a sample radiograph of a case with a distance of over 2 cm between different point a definitions, which caused the plotted separation in 100% isodose lines . 3: we plotted the average percent difference of measurements from plans normalized to point a with respect to corresponding measurements from those normalized to new point a. a significant (p <0.005) difference was seen between different point a definitions when v100%, trak and icru rectal and bladder point doses were considered . The biggest dosimetric impact due to point a definitions were in the v100% and trak values, both of which represent the overall amount of radiation delivered . The maximum differences between manchester point a and new point a were 20% and 12% for v100% and trak, respectively . We found that plans normalized to point a had lower dosimetric values, on average, than those normalized to new point a (fig . 3). This is because new point a was generally located superiorly to manchester point a, resulting in a larger area enclosed by the 100% isodose line for new point a plans (fig . 3 shows the mean percent differences for each ctv and oar in which all variations were less than 2% . We next considered mri - guided, conformal plans . In each of these plans, the point dose values at manchester point a and new point a were recorded and compared to the prescription dose and volumetric tumor coverage values (hr - ctv d90, d100 and ir - ctv d90) (see fig . All point a doses and prescription doses were converted to eqd2 values using / = 10 [10, 11]. Figure 4 shows the percent differences of point a doses, the prescription dose and tumor coverage values . The relationship between point a doses and volumetric tumor coverage values were not considerably changed due to point a definition . The mean changes of no more than 2% were observed on the prescription dose, hr - ctv d90 and ir - ctv d90 due the point a definition . The relationship between point a dose and hr - ctv d100 was maximally changed (i.e. Mean 9%) due to the point a definition . New point a still showed smaller variations from the prescribed dose than those of manchester point a. the variance seen between point a definitions comes from two main sources: i) imprecise implantation of the applicator and/or ii) inter - planner differences while defining either point a on 2-d radiographs or 3-d imaging datasets . The latter should be limited if all planners are well trained and has been removed from this study entirely as the same investigator performed the task in all cases . There was no such restriction, however, on the applicator implantation: this process is inherently imprecise due to visualization difficulties and patient anatomy and is the source of the deviation between point a definitions . The most recent abs guideline 2012 updated the new point a definition to first move to the midpoint between the two ovoids, then superiorly r + 2 cm along the tandem (see fig . 1 in ref . Is expected to result in a yet more robust point definition . In this study, mean deviations were found to have a minimal effect on conventional plans, as only small differences were found between plans normalized to different point definitions . This finding matches the study of eng et al . Showing less than 3% variation between manchester point a and new point a plans when considering doses to point b, icru rectum point or icru bladder point . Point b was reported as a poor surrogate for pelvic lymph node dose, so it was excluded as a dosimetric measurement in this study . We found the plans normalized to new point a had on average 2 - 3% higher trak or v100% values over those normalized to manchester point a. these findings match the results of howell et al ., showing that a plan normalized to new point a generated slightly higher doses . There is no literature describing the impact on clinical outcomes due to a 2 - 3% increase in overall dose . The variation in tumor coverage for conventional point a plans was summarized in our previous study . Here, the mean variations due to the use of different point a definition were found to be relatively small . However, we found maximal trak changes of 11 - 12% and volumetric metrics such as hr - ctv d90 . Thus, the correlation between point a doses and volumetric tumor coverage (e.g. Hr - ctv d90) can be changed up to 11 - 12% due to different point a definitions . It is recommended that the use of a new point a definition for those studies is adopted . With the development of mri - guided, conformal bt, the correlation between conventional point a doses and dose - volume parameters of hr - ctv / ir - ctv have been reported, although no phase iii clinical outcome studies between mri - guided, conformal bt and conventional point a based bt are presently available . Using the more stable new point a definition is expected to enable better clinical outcome analysis, including deeper understanding of the correlations between point a dose and dose - volume parameters of the hr - ctv.
Dacryocystorhinostomy (dcr), which has been performed for the past hundred years, is a surgical procedure by which lacrimal flow is diverted into the nasal cavity through an artificial opening made at the level of the lacrimal sac . The external approach was popularized first and became the surgery of choice for most ophthalmologists, until recently (1). Since its first description by mcdonough and meiring (2), ten years ago, endoscopic dcr has been gaining popularity, largely due to technological advances in endoscopes and other modern instruments of rhinologic surgery (3). Despite much debate, many ophthalmologists still believe that external dcr provides a higher success rate than endoscopic dcr, and consider external dcr to be the gold standard treatment for nasolacrimal duct obstruction . Though many types of endonasal approaches have been attempted, long - term success rates have not been equivalent to that achieved with external dcr, which approximates 90% (4 - 7). Endoscopic dcr with laser assistance is successful between 58 and 85 percent of the time (6,8,9), whereas endoscopic dcr procedures using other tools (eg . Drill, cold knife, punch) appear to yield slightly higher success rates (10 - 11). The most common cause of a surgical failure in endoscopic dcr is obstruction of the neo - ostium by granulation tissue or synechia that forms post - operatively (5 - 11). Most previously - described endoscopic dcr procedures involve a small opening at the medial wall of the lacrimal sac, and sacrifice nasal and lacrimal sac mucosa during the procedure . Inadequate exposure of the lacrimal sac, due to limited resection of bone and excessive and unnecessary removal or injury of surrounding nasal and lacrimal sac mucosa, and, hence, exposure of bone around a small neo - ostium, appear to contribute to obstruction of the neo - ostium by granulation tissue . This retrospective study estimates the effectiveness of a surgical procedure that is a modification of previously reported endoscopic dcr techniques . This technique exposes the lacrimal sac fully after removing the maxillary bone surrounding the sac, creates a large marsupialized lacrimal sac, and covers the exposed bone with preserved nasal mucosal flaps . At our institution, from 2002 to 2004, 42 patients (3 male and 39 female) underwent 46 endoscopic dcr (17 left sides and 29 right sides) for chronic epiphora . Patients' ages ranged from 7 to 71 yr, with a mean age of 47 . Pre - operative evaluation consisted of a standard examination that included lacrimal irrigation, conventional dacryocystography, and/or dacryoscintigraphy . The nasal cavity was examined and the need for additional nasal surgery (i.e., septoplasty, middle turbinate reduction) also was determined pre - operatively . Of the 46 primary surgery cases, fourteen cases previously had failed insertion of a nasolacrimal polyurethane stent . Etiologies of epiphora were proximal nasolacrimal duct obstruction in all cases and one case had associated inferior canaliculi obstruction . Irrigation through the punctum was performed to evaluate the patency of the neo - ostium post - operatively, and the neo - ostium was judged " wide " in size if the marsupialized sac had been well maintained in shape and size . Surgery was considered unsuccessful if the patient had one or more of the following postoperative outcomes: 1) no marked improvement of preoperative chronic epiphora, or any episode of dacryocystitis; 2) inability to irrigate the lacrimal system; and 3) nasal endoscopy revealing obstruction of the neo - ostium with granulation tissue or synechia . At our institution, dcr is performed under local or general anesthesia, as determined by considering a number of patient factors . After shrinkage of the nasal cavity by inserting a gauze packing soaked in a mixture of 1:2,000 epinephrine and 1% lidocaine, the head of the middle turbinate and the mucosa surrounding the lacrimal sac are infiltrated with a mixture of 1:200,000 epinephrine and 2% lidocaine . The patient is placed in a supine position with the head elevated 15 degrees . A zero or thirty degree, 4-mm diameter endoscope is used . 1 . In the first step, using a slit knife (angled, 2.7 mm, alcon co., cleveland, ohio, u.s.a . ), a reverse " c " shaped mucosal incision, 1010 mm, is made at the lateral nasal wall anterior and slightly superior to the insertion of the middle turbinate (fig . The posteriorly based mucosal flap is elevated backwards off the maxillary bone, extending up to the uncinate process . The maxillary bone covering the lacrimal sac then is gently drilled (curved diamond dcr bur, 15 degree, 2.9 mm, xomed co., jacksonville, florida, u.s.a .) Until the sac is widely exposed, extending to the level of the fundus (fig . Metallic lacrimal probes are passed medially through both canaliculi, and gently pushed so as to tent the sac, thus facilitating incision through the sac while precisely localizing the position of the sac lumen (fig . An incision then is made with a slit knife, avoiding injury to the sac lumen and, hence, minimizing hemorrhage . The mucosal flaps are adjusted in size to cover the denuded bone surrounding the opened sac . The lacrimal sac flaps are incised, everted, and adjusted to accurately appose the nasal mucosa . A small gel foam patch is packed lightly in the exposed sac to keep the flap anastomosis in position throughout the initial healing period (fig . 1d). A silicone bicanalicular tube (canaliculus intubation set tube, xomed co., jacksonville, florida, u.s.a .) Is positioned, except in those instances in which the sac is marsupialized widely due to prior longstanding dilatation of the sac . Light nasal packing is required unless there has been associated nasal surgery (i.e. Septoplasty). Post - operatively, each patient is prescribed antibiotics and ophthalmic drops, and followed regularly for nasal dressings . Irrigation and spray of the nasal cavity with saline are performed to prevent crust formation . At our institution, dcr is performed under local or general anesthesia, as determined by considering a number of patient factors . After shrinkage of the nasal cavity by inserting a gauze packing soaked in a mixture of 1:2,000 epinephrine and 1% lidocaine, the head of the middle turbinate and the mucosa surrounding the lacrimal sac are infiltrated with a mixture of 1:200,000 epinephrine and 2% lidocaine . The patient is placed in a supine position with the head elevated 15 degrees . A zero or thirty degree, 4-mm diameter endoscope is used . 1 . In the first step, using a slit knife (angled, 2.7 mm, alcon co., cleveland, ohio, u.s.a . ), a reverse " c " shaped mucosal incision, 1010 mm, is made at the lateral nasal wall anterior and slightly superior to the insertion of the middle turbinate (fig . The posteriorly based mucosal flap is elevated backwards off the maxillary bone, extending up to the uncinate process . The maxillary bone covering the lacrimal sac then is gently drilled (curved diamond dcr bur, 15 degree, 2.9 mm, xomed co., jacksonville, florida, u.s.a .) Until the sac is widely exposed, extending to the level of the fundus (fig . Metallic lacrimal probes are passed medially through both canaliculi, and gently pushed so as to tent the sac, thus facilitating incision through the sac while precisely localizing the position of the sac lumen (fig . An incision then is made with a slit knife, avoiding injury to the sac lumen and, hence, minimizing hemorrhage . The mucosal flaps are adjusted in size to cover the denuded bone surrounding the opened sac . The lacrimal sac flaps are incised, everted, and adjusted to accurately appose the nasal mucosa . A small gel foam patch is packed lightly in the exposed sac to keep the flap anastomosis in position throughout the initial healing period (fig . 1d). A silicone bicanalicular tube (canaliculus intubation set tube, xomed co., jacksonville, florida, u.s.a .) Is positioned, except in those instances in which the sac is marsupialized widely due to prior longstanding dilatation of the sac . Light nasal packing is required unless there has been associated nasal surgery (i.e. Septoplasty). Post - operatively, each patient is prescribed antibiotics and ophthalmic drops, and followed regularly for nasal dressings . Irrigation and spray of the nasal cavity with saline are performed to prevent crust formation . The average follow - up period varied from three to 33 months, with an average of 5.9 months . Of 46 cases, 38 cases (83%) demonstrated primary surgical success, defined as decreased or absent epiphora and an adequately patent neo - ostium . In eight cases (17%), including one case of functional obstruction, obstruction of the neo - ostium by granulation tissue or synechia was identified, all associated with persistent epiphora (table 1). Of 14 cases with a history of previously failed insertion of a nasolacrimal polyurethane stent, three showed obstruction (21%) while five showed obstruction (16%) out of the remaining 32 cases . The neo - ostium was widely patent in 35 cases (76%) (fig . 2) and " narrow but patent " in three cases (7%). Though six cases (four with wide and patent neo - ostium and two with narrow but patent neo - ostium) complained of occasional epiphora in spite of great symptom improvement postoperatively, these cases were considered surgical successes, in accordance with our prior criteria . Among the eight patients with persistent obstruction of the neo - ostium, six subsequently underwent revision procedures . All six patients who underwent revision became free of epiphora and ultimately had an adequately patent ostium that has persisted throughout the mean follow - up of six months post revision surgery . Overall, 44 of 46 cases (96%) ultimately had a successful surgical outcome . All five patients without placement of silicone tube stent after sac opening had widely patent neo - ostium without epiphora . In addition to the dcr procedure, septoplasty was performed in three cases (7%) and anterior middle turbinectomy in one case . There were no serious complications, beyond obstruction of the neo - ostium causing surgical failure . In one case, orbital fat was mildly exposed during the operation but had no influence on the postoperative surgical outcome . For dcr, the endoscopic approach has several advantages over the external approach: 1) it is less traumatic and, thus, shortens the hospital stay; 2) a facial scar is avoided; 3) there is no disruption of the medial canthal tendon, which consequently enables preservation of lacrimal pump function; 4) access to the sac is direct through the lacrimal bone, avoiding double - side dissection of the sac; and 5) it is excellent in controlling tissue and, thus, results in less trauma to the nasal mucosa . Conversely, disadvantages are: 1) the surgical field may be limited because of bleeding; 2) there is an occasional need for septoplasty or removal of the middle turbinate; and 3) there appears to be increased likelihood of granulation tissue formation, resulting in stenosis and, thereby, obstruction of the opening . Previously described, conventional endoscopic dcr techniques generally involved limited opening of the sac, yielding frequent obstruction of the neo - ostium by granulation tissue, an outcome which explains the higher failure rates . To avoid or prevent obstruction of the neo - ostium, these include complete separation of the sac from the nasolacrimal duct to divert lacrimal flow to the neo - ostium (12), use of steroids or mitomycin - c (13,14), and use of mucosal flaps after wide resection of bone surrounding the sac (15 - 17); this last approach is technically similar to our method . Our surgical technique modified the conventional endoscopic techniques in order to solve previously reported sources of surgical failure . Our modifications included the following: 1) a nasal mucosal flap was elevated to avoid unnecessary injury or removal of the mucosa; 2) we exposed the lacrimal sac as wide and as high as possible, up to the level of the fundus, by removing the bone surrounding the sac using a power drill; 3) instead of cutting off the sac wall, it was incised and everted to meet the nasal mucosa; 4) tailored mucosal flaps were created to cover all exposed bone and appose the everted lacrimal sac flap . With these techniques, we intended to make a large, epithelialized fistula, thereby potentially minimizing the formation of granulation tissue and synechia, which represent the most common causes of failure in endoscopic dcr . Our primary success rate of 83% is similar to the rates (80 - 88%) reported using other endoscopic techniques performed without the use of a mucosal flap (10,11). Other investigators who have reported using techniques similar to ours have noted slightly higher primary success rates, which may not be significantly different from ours (15 - 17). Nonetheless, considering our original intention to reduce granulation tissue formation and synechia and, thus, increase primary success by making a large marsupialized opening, our results are somewhat contrary to our expectations . First, among our 46 patients who underwent surgery, 14 previously had failed insertion of a nasolacrimal polyurethane stent; many of these 14 had developed a constricted sac with thick walls due to chronic inflammation likely caused by long - standing placement of the stent (18). In patients with a constricted sac with thick walls, though statistically not significant, these patients showed a slightly increased primary failure rate compared to other patients . Second, proper postoperative care was not delivered in all of our cases, due to inadequate follow - up . Good postoperative care is a necessity and increases the likelihood of a successful outcome . With more favorable case selection and improved postoperative care, a higher success rate might have been accomplished at the time of primary surgery . Though our primary success rate was not high enough to demonstrate advantages of our technique over others, our overall success rate after revision surgery was very high, at 96% . During revision surgery performed under local anesthesia, removal of granulation tissue or lysis of synechia which obstructed the ostium this can be attributed to the wide opening of the sac from extensive removal of surrounding bone at the time of primary surgery . These results imply that our modified technique, though it failed to achieve a higher primary success rate compared to conventional endoscopic dcr techniques, nonetheless succeeded in achieving an excellent ultimate success rate, with technically - simpler and more highly - successful revision procedures . The purposes of using silicone tubing are: 1) to maintain the opening of the neo - ostium; 2) to prevent or correct synechia of the canaliculus; and 3) to facilitate postoperative dressings (19,20). Some investigators have reported 81 - 87% success rates without using silicone tubes after endoscopic dcr, and recommend not using silicone tubing or removing it early because of granulation formation stimulated by the tubing itself (19 - 21). As suggested by the results of our study, in which widely - patent neo - ostium was achieved in all five cases without silicone tube stenting, a silicone tube can be used, as determined by the pre - operative status of the lacrimal sac and canaliculus . Complications of endoscopic dcr include re - stenosis of the opening, bleeding from the nasal cavity, orbital injury, csf leakage through a fractured ethmoid, and corneal abrasion or canaliculi erosion due to the overly - tight silicone tube placement (22). A lacrimal sump syndrome and associated recurrent infections can occur if the lower portion of the bone surrounding the sac is removed inadequately (5, 22). Our marsupialization technique opened the sac inferior to the proximal nasolacrimal duct after bone removal, thereby preventing lacrimal sump syndrome . In conclusion, the authors obtained a large marsupialized lacrimal sac with wide removal of the covering bone and use of mucosal flaps . This technique yields a good surgical result that is comparable to the results of conventional endoscopic dcr techniques . First, primary surgical failures are amenable to technically simple and highly successful surgical revisions.
Dn is one of the most serious diabetic microvascular complications and the leading cause of end - stage renal diseases (esrd); it brings about heavy social and economic burden worldwide, particularly in the developed countries . Both type 1 and type 2 diabetic patients presented indistinguishable and variable pathological changes and clinical course; the prognosis is difficult to predict because of diverse pathogenesis . Clinically, dn is characterised by different degrees of proteinuria, albuminuria, increased serum creatinine (scr), decreased glomerular filtration rate (gfr), and esrd [1, 2]. Importantly, dn also increases the risks for the development of diabetic macrovascular complications including heart attacks and strokes [3, 4]. Pathologically, dn associated histological structural changes include glomerular mesangial expansion, glomerular basement membrane (gbm) thickening, glomerular sclerosis known as kimmelstiel - wilson lesions caused by excessive extracellular matrix (ecm) proteins accumulations, and tubulointerstitial fibrosis in the advanced stages [1, 5]. Arterial hyalinosis of the afferent and efferent arterioles is often prevalently caused by endothelial dysfunction and inflammation [2, 6, 7], which will lead to glomerular hyperfiltration . In the development and progression of dn, resident kidney cells are affected by hyperglycemia: including mesangial cells, podocytes, endothelial cells, smooth muscle cells, inflammatory cells, myofibroblasts, and cells of tubular and collecting duct system . Multiple contributors including environmental and genetic factors are associated with the pathogenesis of dn, which cause metabolic, hemodynamic, and biochemical changes in the diabetic kidneys . Main pathways leading to dn include intracellular pkc activation and increased polyol pathway flux, production of reactive oxygen species (ros) and advanced glycation end products (ages), and hypertension and glomerular hyperfiltration leading to shear stress and mechanical stretch [8, 9]. Increased blood glucose activates the renin - angiotensin system (ras), tgf--smad - mapk pathway, jak - stat pathway, and g - protein signaling; aberrant expression of ecm proteins and deregulated expression of cyclin kinases and their inhibitors; transcription factor such as nf-b, proinflammatory cytokines like tnf and il-1, and toll - like receptors 4 (tlr4), which are considered to exert hemodynamic, proinflammatory, and profibrotic effects on kidney cells [8, 13]. There is cross - talk among the above - mentioned signaling pathways, which can amplify aberrant pathogenetic genes expression and lead to the progression of dn . In addition, the phenomenon of metabolic memory regulated by epigenetic mechanisms can promote these genes expressions [14, 15]. Although a lot of biochemical and molecular mechanisms and pathways have been broadly studied in the pathogenesis of dn, the undeniable fact is that the progressive incidence and prevalence of dn worldwide still exist, suggesting that more investigations will be needed in the future . Emerging evidences suggest that multiple signaling pathways activations and key transcription factors (tfs) are associated with the pathophysiology of dn, which could be influenced by epigenetically regulated mechanisms in chromatin (histones form a complex structure with dna), including dna methylation, posttranslational modifications (ptms), and noncoding rnas (ncrna), which can modulate gene expression in the cell - type - specific pattern . Core histones are subject to diverse ptms including histone lysine acetylation (hkac), histone lysine methylation (hkme), phosphorylation, ubiquitination, and sumoylation . We have implicated the roles of hkme in the pathogenesis of dn, especially in the metabolic memory phenomenon pertinent to dn [2, 16, 17]. Global acetylation alterations have been seen in a lot of human diseases including cancer and nervous system diseases, whereas the roles of hac in the pathogenesis of dn are rarely mentioned . Recently, some studies showed that hac level is linked to dn, hats and hdacs also participate in the pathogenesis of dn, and the research regarding hac and the covalent enzymes is not enough to yield a clear picture about dn so far . In this review, we describe some progress associated with the molecular mechanism underlying dn, with specific emphasis on hac and acetylation on nonhistone proteins as important regulators of gene expression in renal cell under diabetic conditions; the regulators of hac such as hats as well as hdacs in the development and progression of dn; the inhibitors of hats / hdacs in the dn pathogenesis and their therapeutic potentials for dn . Dynamic balance of histone acetylation and deacetylation can regulate gene expression, chromosome assembly, mitosis, and ptms, by altering the chromatin structure and the accessibility to tfs without affecting the sequence of dna . Hac is highly reversible and dynamic, which can be catalyzed by hats or hdacs, respectively . Hkac at n - terminal tails can facilitate gene transcription through neutralizing the positive charge of histone residues and weakening the binding of histone to negatively charged dna [22, 23]. Hkac, such as h3k9ac, h3k14ac, and h4kac, is generally linked to permissive gene expression, while histone deacetylation is often associated with chromatin condensation and gene transcriptional repression [25, 26]. Several previous studies have shown that hkac at the insulin gene promoter was specific to cells and islet - derived precursor cells, which was highly correlated with the recruitment of p300 [27, 28]. In vitro studies with hdac inhibitors (hdaci) suggested that hkac was essential in the development of pancreas . These findings cannot fully demonstrate the underlying mechanism of dn; in this review, we will discuss the current opinions of hac and nonhistone acetylation on inflammation, fibrosis, and oxidative stress in the development and progression of dn (table 1). Diabetic patients showed that levels of h3 acetylation at lysine 9 and 14 and h4 acetylation at lysine 5, 8, and 12 were increased at tnf- and cox-2 inflammatory genes promoters in human blood monocytes . Another study showed that oxidized lipids could increase h3k9/14ac at mcp-1 and il-6 gene promoters in a creb / p300-dependent manner, along with the inflammatory genes expression . Advanced dn in db / db mice underwent by uninephrectomy is specifically associated with increased acetylation of h3k9 and h3k23 . A recent study revealed that acetylation of h3k9, h3k18, and h3k23 were significantly increased in the renal cortex of akita mice, hg and nab - induced h3k9 and h3k18 acetylation was elevated in the mesangial cells also, which were associated with inflammatory factors such as mcp-1, icam-1, vcam-1, and inos expression linked to the development of dn . Thioredoxin - interacting protein (txnip) has been demonstrated to play an important role in the pathogenesis of dn . Hg - induced txnip expression was associated with the stimulation of activating h3k9ac in mcs of diverse species, which could drive the expression of proinflammatory genes predisposing to dn . Tgf-1 is established to be involved in the pathogenesis of dn, the underlying mechanism of which is still unclear . Tgf-1 treatment could increase acetylation of histone (h3k9, h3k14, and h3k27) as well as ets-1 in mouse renal glomerular mesangial cells; furthermore, acetylation of ets-1 and histone h3 was increased in glomeruli from diabetic db / db mice also, both of which can increase mir-192 expression contributing to dn . Tgf-1 treatment increased h3k9/14ac at the pai-1 and p21 promoters near smad and sp1 binding sites in rmcs, acetylation of smads was also increased [36, 37], and hg - treated rmcs exhibited increased levels of h3k9/14ac that can be blocked by tgf-1 antibodies, which played an important role in tgf-1 and hg - induced deregulated gene expression associated with hypertrophy and fibrosis linked to dn . Hg stimulation can also increase h3k9/14ac at the rage, pai-1, and mcp-1 promoters, which can be further augmented by hg+ang ii (hg / a), suggesting the key roles of h3k9/14ac in the key dn - related genes expression . Excessive h3k9/14ac levels were reported at the ctgf, pai-1, and fn-1 promoters in diabetic kidneys, which were associated with p300/cbp activation . Although there is a conflicting result in an animal study that the level of h3k9/14ac was decreased in the stz - induced type 1 diabetic rat kidney [40, 41], the majority of hac is involved in the development and progression of dn . For the past few years, the phenomenon metabolic memory has been implicated in the pathogenesis of diabetes and its complications such as dn . A study of patients from dcct conventional treatment groups showed that there was association between hba1c level and h3k9ac; hyperacetylated promoters included more than 15 genes related to the nf-b pathway and could be enriched in genes associated with diabetic complications, which may be a possible epigenetic explanation along with hkme [16, 17, 43] for metabolic memory phenomenon in humans . Endoplasmic reticulum stress (ers) is an important mechanism responsible for the pathogenesis of dn . Histone h4 acetylation levels are increased at glucose - regulated protein (grp78) promoters and decreased at c / ebp - homologous protein (chop) promoters, which are associated with renal cell apoptosis, proteinuria, and increases of scr; these results provide initial experimental evidences for understanding the mechanism of dn . Apart from hac, nonhistone proteins acetylation can also take part in the pathogenesis of dn . Fork box o4 (foxo4) transcription factor can be activated to promote podocyte apoptosis by ages through bcl2111 expression, at the same time, age - bsa can also increase foxo4 acetylation; a recent study showed that alteration of foxo4 acetylation and downregulation of sirt1 expression in dm promote podocyte apoptosis; foxo4 acetylation reduction could be a therapeutic potential for preventing diabetic podocyte loss . Enhanced nf-b acetylation level was present in both diabetic rats and hg - treated rmc leading to dn in another study, which can be dampened by 3,5-diiodothyronine (t2) involved regulation of sirt1; acetylation of nf-b p65 and stat3 was increased in both mice and human diabetic kidneys and ages induced human podocytes, suggesting their critical roles in dn . P65 acetylation was also increased by hg in rmcs, pns could protect diabetic kidney through decreasing induction of inflammatory cytokines and tgf-1 . Smad 3 acetylation has been implicated in the pathogenesis of dn recently [49, 50], overexpression of transcription factor srebp-1 induces glomerulosclerosis of dn; srebp-1a k333 acetylation by cbp is required for smad3 association and srebp-1 transcriptional activity; both smad3 and srebp-1a activation regulates tgf-1 transcriptional responses associated with dn, srebp-1 inhibition could be a novel therapeutic strategy for dn . Nephrin acetylation in diabetic podocytopathy has seldom been addressed before, a recent study showed that nephrin acetylation was reduced in stz - induced diabetic mice kidney; increasing mir-29a may protect diabetic podocytopathy by modulating nephrin acetylation . Type a hats (nuclear) exist in nucleus, including (1) gnat (gcn5) family such as gcn5, p / caf, and elp3, (2) myst (hmof / myst1, hbo1/myst2, moz / myst3, morf / myst4, and tip60) family, (3) p300/cbp, (4) basal tf family (tfiiic and taf1), and (5) nrcf family, src, and actr / ncoa3, which can acetylate nucleosomal histones and other chromatin - associated proteins, while type b hats are cytoplasmic and acetylate newly synthesized histones . Hkac is generally mediated by hats including p300, cbp, p / caf, and tip60, which is associated with gene activation via adding acetyl groups . In addition, hats can also regulate gene expression through acetylation of nonhistone proteins such as smads, p53, sp1, and nf-b . Among the studies of hats and their links with dn development, in vitro and in vivo studies showed that hats cbp and p / caf recruitment was increased under diabetic conditions, which led to upregulated hkac at inflammatory genes promoters continent with the gene expression [30, 53]. It was implicated that p300 played important roles in oxidative stress - induced parp and nf-b signaling in hg - treated endothelial cells and diabetic kidneys [5355]; further study showed that hg upregulated p300, which increased hac at promoters of key ecm protein fn, as well as vasoactive factors such as et-1 and vegf in endothelial cells . Another study showed that tgf-1 increased h3k9/14ac by recruiting the hats p300 and cbp; tgf-1 treatment also increased association of p300 with smad2/3 and sp1, cotransfection experiments showed that p300 and cbp, but not p / caf, upregulated transcriptional activity of pai-1 and p21 promoters and increased tgf-1-induced gene expression . On the contrary, inhibition of cbp and p300 by overexpressing dominant - negative mutants p / caf was found sharply increased in the renal cortex of akita mice, while gcn5 was significantly decreased in the hg group, suggesting that the inflammatory genes expressions were related to dn . In vivo and in vitro results of another report showed that p / caf was closely related to h3k18ac levels at inflammatory molecules icam-1 and mcp-1 promoters, which could be a potential therapeutic agent for inflammation - related renal diseases including dn . All the data implied that hats have critical roles in acetylating both histones and nonhistone proteins in the pathogenesis of dn; these results point to the necessity of further studies on the hats activity in the development of dn, which may be therapeutic targets in the future . In preclinical trials, small - molecule hats inhibitors have been shown to sensitize cancer cells to ionizing irradiation . Curcumin, the p300/cbp inhibitor, extracted from rhizomes of turmeric curcuma longa, which was supposed to be a new target molecule for treating cns disorders and cancer [61, 62], was firstly reported to prevent the development of dn involved in the changes of ptms of histone h3 including acetylation and phosphorylation and the changes in hsp-27 and p38 expression in diabetic rats . Curcumin could also prevent hg - induced key ecm genes and vasoactive factors (enos and et-1) expression levels associated with dn in endothelial cells; it was able to reverse the upregulation of vasoactive factors, tgf-1 and ecm protein fn in stz - induced diabetic kidneys, which was associated with p300 and nf-b activity changes . Curcumin was also found to reverse hg - induced cytokines (il-6, tnf-, and mcp-1) production in human monocytes via epigenetic changes involving nf-b, but dietary curcumin failed to decrease albuminuria either before or after diabetes induction . Curcumin analogue, c66, has been demonstrated to significantly and persistently prevent renal injury and dysfunction in diabetic mice via downregulation of jnk activation and consequent suppression of diabetes - related increases in p300/cbp expression and histone acetylation (h3k9/14ac). In a recent study, c646, a novel p300/cbp specific inhibitor, has been declared to specifically suppress the growth of cbp - deficient hematopoietic and lung cancer cells in vivo and in vitro . In another in vitro study, histone h3ac activated tgf-1/smad3 pathway during emt of human peritoneal mesothelial cells; c646 could reverse the mesenchymal phenotype transition . C646 was also reported reversing acetylation involved in hg - induced txnip expression leading to dn . To date, 18 hdacs have been identified in humans and divided into 4 distinct classes based on their homology to yeast hdac, in which class i (hdac1, 2, 3, and 8), class ii including iia (hdac4, 5, 7, and 9) and iib (hdac6 and 10), and class iv (hdac11) have structurally similar zinc - dependent active sites, whereas class iii, sirtuins (sirts1 - 7), are zinc - independent but require cofactor nicotinamide adenine dinucleotide (nad). Hdacs can remove acetyl groups from conserved lysine residues and nonhistone proteins and generally act as corepressors with some exceptions . Most research related to the epigenetics of dn has focused on hac; different classes of hdacs are involved in distinct pathways that engaged in the pathogenesis of dn . Overexpression of hdac1 and hdac5 blocked tgf-1-induced gene expression, whereas inhibition of hdacs upregulated h3k9/14ac and gene expression, further supporting the key inhibitory roles of hdacs in tgf-1-induced gene expression . A recent study showed that hdac1 was significantly decreased in the renal cortex of akita mice, while the levels of hdac2 in akita and wt mice were unchanged, and hdac1 was significantly decreased in hg - cultured hbzy-1 cell, which can upregulate diabetes-, hg-, and nab - induced histone hyperacetylation leading to inflammatory factors elevation associated with dn . Glomerular sclerosis is also a core characteristic of dn resulting from excessive ecm deposition in the glomerular mesangium and the loss of glomerular epithelial cells, followed by aberrant fibrosis in the glomerular structure . Hdac2 activity was markedly increased in the kidneys of type 1 and type 2 murine models and tgf-1 treated nrk52-e cells, which played an important role in the development of dn . Knockdown of hdac2 in cell culture reduced ecm components accumulation, further implicating the role of hdac2 in the fibrosis . Oxidative stress is also of the view to play an important role in regulating fibrosis in dn; a potent oxidative stress inducer h2o2 can increase hdac2 levels, which may be an underlying mechanism in the pathogenesis of dn . Hdac4 is regarded as a contributor to podocyte injury in type 1 and type 2 diabetic models and diabetic patients and could suppress autophagy related with podocyte injury in dn by deacetylating stat1, suggesting that hdac4 is important to accelerate dn in epigenetic and nonepigenetic mechanisms [70, 71]. Sirts have been shown to be involved in diverse cellular processes such as insulin secretion, cell cycle, and apoptosis . Dysfunction of sirt1 may contribute to abnormal cancer metabolism, cancer stemness, neurological disorders, obesity, and diabetes . A previous study showed that decreased sirt1 level in diabetic kidney and intermittent fasting (if) prevents this decrease; sirt1-dependent deacetylation is thought to mediate p53 expression and activation, which could play a renoprotective effect of if in diabetes . Another report showed that resveratrol could prevent decreased sirt1 and increased p53 expression in diabetic kidney, which could be responsible for preventing apoptosis in type 1 diabetic kidney . Resveratrol has also been demonstrated to reduce oxidative stress and maintain mitochondrial function related with sirt1 activation in hg - treated mcs and db / db diabetic mice [75, 76]. Sirt1 in proximal tubules (pt) has been reported to attenuate diabetic albuminuria by suppressing the overexpression of tight junction protein claudin-1 via hypermethylation of the claudin-1 gene in podocytes [77, 78]. Another previous report showed that sirt1 could inhibit tgf-1-induced glomerular mesangial cell apoptosis via smad7 deacetylation, and overexpression of sirt1 attenuated ros - induced apoptosis in mesangial cells through p53 deacetylation and provided a new therapeutic strategy for kidney glomerular diseases; tsg has been proven to protect dn through inhibiting tgf-1 expression partially mediated by sirt1 activation . Conditional sirt1 deletion in podocytes of diabetic db / db mice developed more acetylation of nf-b p65 and stat3, proteinuria, and kidney injury compared with db / db mice without sirt1 deletion, suggesting the protective roles of sirt1 in tfs acetylation on dn . Dietary restriction was reported to ameliorate dn through regulation of the autophagy via restoration of sirt1 in diabetic fa / fa rats . The beneficial effects of sirt1 on age - associated dn correlate with the activation of nrf2/are antioxidative pathway [83, 84]. All the findings suggested the possibility of sirt1 as the target of treatment in dn [8587]. Taken together, these studies highlight important and different roles of hdacs in the pathways, and most of them are beneficial, suggesting hdacs will be the targets for the prevention of dn despite the fact that further studies are needed . The present hdacis include both natural and synthetic compounds and are subdivided into 5 categories: short - chain fatty acids, cyclic peptides, benzamides, electrophilic ketones, and small - molecule hydroxamic - acid - derived compounds [52, 88]. Hdacis are regarded as potential anticancer agents and are promising for the treatment of a lot of diseases such as inflammation and neurological diseases . Recently, hdacis have been identified as a novel class of potential therapeutic agents for dn . Here we list some progress of hdacis applied in the treatment of dn regarding antifibrotic, anti - inflammatory, and antioxidative effects . Nevertheless, most of the hdacis are nonselective and target both nuclear histones and cytoplasmic nonhistone proteins . It was found that millimolar concentrations of n - butyrate induce accumulations of acetylated histones in cells in the 1970s and inhibited deacetylation [72, 90, 91]. Sodium butyrate (nab, a nonselective inhibitor of hdacs), a short - chain fatty acid, can upregulate hac levels, promote tumor cell senescence and apoptosis, and inhibit tumor cell proliferation . Nab was used as animal feed additive and played a major role in the treatment of neurodegenerative conditions . In vivo, it was reported that nab could not only decrease blood glucose, creatinine, and urea but also ameliorate histological changes, fibrosis, apoptosis, and dna damage in the kidneys of juvenile diabetic rats . Saha (suberoylanilide hydroxamic acid, vorinostat), a nonselective hdaci, designed and synthesized as a hybrid polar compound that can strongly induce erythroid differentiation [72, 93], is orally bioavailable and clinically applicable . Saha can reduce albuminuria, glomerular hypertrophy, and glomerular type iv collagen deposition through an enos - dependent mechanism, without affecting blood pressure or blood glucose concentration . Indeed, another study showed that saha attenuated early renal enlargement in stz - induced diabetic rats, which is supposed to be mediated partly through downregulating egfr . These results indicated the key role of saha in attenuating fibrosis and oxidative damage in dn . Trichostatin a (tsa), the natural product isolated from a streptomyces strain, originally identified as an antifungal antibiotic, was discovered to have potent hdac inhibition activity in 1990 . Tsa was reported to act as an agent in preventing dn in diabetic rats, by blocking tgf-1-induced ecm accumulation and emt in diabetic kidneys as well as in renal epithelial cells; knockdown of hdac2 had similar effect of tsa treatment mediated by ros . Valproic acid (vpa), a broad - spectrum hdaci, is a first - line drug used for the treatment of epilepsy and migraine . Vpa treatment alleviated renal injury and fibrosis in stz - induced diabetic kidney by preventing myofibroblast activation and fibrogenesis through hdac4/5/7 inhibition in a dose - dependent manner, vpa has also been proven to ameliorate the podocyte and renal injuries by facilitating autophagy and inactivation of nf-b / inos pathway . A recent study showed that vpa can attenuate renal injury in a rat model of dn, by upregulating the histone h4 acetylation levels at the promoter of grp78 and downregulating the histone h4 acetylation at the promoter of chop . To our knowledge, at the time of the present review, the molecular implications of hdacis were identified in the treatment of dn, and the development of selective hdacis in preventing dn may be part of the most prevalent areas in the drug discovery . Recent research has concentrated on histone modifications to provide a reliable theoretical basis for clinical treatment . A comprehensive understanding of hac mechanisms can give rise of novel therapeutic options for dn . Increasing in vitro and in vivo evidences implicated that reversible histone and nonhistone acetylation play important roles in the pathogenesis of dn, suggesting that hac regulation could be promising therapeutic targets for dn . Hats and a small number of hdacs provide a central mechanism for regulating gene expression and cellular signaling events in dn (table 2). Experimental evidences suggest that hats / hdacs inhibitors and a large number of hdacs can delay the development and progression of dn (tables 2 and 3). Hats inhibitor curcumin and its analogue c66 could protect renal injuries in diabetic patients and diabetic animal models; apelin-13 and esculetin treatment could be innovative therapeutic agents for dn via regulation of hac also [33, 40, 41]. Continued research is needed to better understand the roles of hac in the process of dn, the modifiers and the mechanism that regulate them, and address the curative potential of more selective hats inhibitors and hdaci in treating dn.
Life of the human being is intertwined with many changes and transformation from the clotting of the semen to the death and is divided into different stages according to specific criteria ., adolescents expand their eligibility bases, and gradually the responsibility of health care would be relegated to them by the parents . Furthermore, the peers individuals have a more important role for the adolescents compared with the childhood period . The peers group provides a strong support for the adolescent and creates a sense of belongingness and power in them . Therefore, it has an intense effect on the behavior and performance of the adolescents . The occurrence of an acute disease in this period leads to the creation of disturbance in the natural perfection process in the adolescence period . Among these acute diseases, asthma is the most common disease in childhood, which is considered as the forerunner of acute diseases and simply can cause disability among the children . This disease is an important reason for the children's absence from the school due to admission into the hospitals . The prevalence of children's asthma all over the world is increasing due to numerous stressful factors, despite noticeable progressions in the field of disease controlling and access to different medications for its treatment . It can be envisaged as an epidemic disease, which has major effects on the health and the economical conditions of the society . The prevalence of this disease during the last few decades in the united states has been 16% among the children under 5 years old and nearly 74% among the adults . The percentage of the students with asthma in iran has been estimated as 10% . In recent years, due to various life problems in iran and the increased stress rate, the disease of asthma has been increased in the country like in most other societies . Considering the high prevalence rate of this disease, training is one of the important methods for promoting the hygiene and health of the students through conducting different training projects by relying on their participation in different hygienic activities . Moreover, caring through holding the training programs for the peer adolescents with acute diseases is one of the effective measures in order to train, empower, and cultivate the willpower in adolescents and also can help to change the risk - taking behaviors and decrease the side effects . Recent conducted studies have shown the effectiveness of training peers in decreasing the side effects of the acute diseases e.g. Choudhury et al . In 2009 investigated the effect of training the peers in preventing side effects of the cardiovascular diseases resulting from type ii diabetes . In addition, another study, which was conducted by mahat et al . In 2008 in new jersey, titles the effect of training peer adolescents who are susceptible to risk - taking sexual behaviors and preventing aids; the findings indicated an obvious increase in decreasing these types of behaviors among the adolescents . Since it is the most common reason for the frequent hospitalizations and school absence due to lack of information about the intensity of the disease and due to treatment among the asthmatic students and the peers of the adolescents with this disease can assist them better at the time of asthma attack because of having the possibility for an easy availability and a better trainability from the peer group and consequently decrease the incidence of side effects; therefore, this study aimed to assess the effect of conducting training programs for high school students on the performance of the peers with asthma . This study was of a quase - experimental kind, conducted before and after intervention . The instruments for gathering information the questionnaire contain 5 demographic items, 14 knowledge measurement items, and a performance measurement checklist with 14 questions . Having obtained necessary letters of permission from isfahan education organization, district 3, those high schools in which students with asthma were being taught were selected . Then, two female high schools, one for test group and the other for control group were chosen after convincing high school principals and achieving letters of consent from those students willing to participate; 40 second - grade high school students were easily included in the study in each high school . The knowledge questionnaire and performance checklist developed by the researcher were presented to some professors of and specialists on asthma and allergy, and statistics advisors to decide their validity and the necessary amendments were made after collecting ideas and suggestions . In order to calculate reliability, the researcher, during a pilot study, presented the knowledge measurement to 10 qualified people, who were not going to be included by the study to fill them out . Furthermore, the researcher, through an oral interview, tried to completed performance checklist and measured knowledge questionnaires and performance checklists cronbach as 87% and 89%, respectively . Having homogenized the two groups demographically, the pre - test was conducted, and knowledge questionnaire was answered by the two groups measure performance, and through simulating an asthma attack, the researcher completed related checklist by observing activate of peers during attack . Having examined pre - test results and determined the matters required for education, the training intervention for test group was conducted by the researcher through lecturing, group discussion, collective methods, and activating accompanied by questioning . The members of test group were firstly divided into two equal subgroups, and training program (four 60-minute training sessions held at 9 - 10 and 10 - 11 am) was performed for both subgroups while high school personnel were acting as coordinators . During first session, different types of illness and its clinical symptoms were presented via lecture and questioning after acquainting students with asthma definition . During second session, the content of the first session was firstly gone through, and some questions about first session's ideas were asked and students replied while discussing in 5-member circle . Then, content of the second session, including asthma attack reasons and its preventive methods, were offended by giving lecture and questioning . As the third session started, questioning and group discussion were adopted, and it was followed by presenting this session's content including athletic asthma and its preventing methods . Training of required activities and assistance in case of attack was presented to students providing individual students with mdi devices simultaneously, showing the pictures through powerpoint, and imitating by the researcher herself . Furthermore, the indirect training was offered during fourth session and through presenting researcher - developed booklets . After a one - month break following the last training session, knowledge questionnaires and performance checklists were again filled out by both the groups, and the data was analyzed by spss 16 software . The t - paired test, independent - t test, mann - whitney test, and dependent - t test were utilized for comparing examined variables before and one month after intervention in both test subgroups, the scores average between the two subgroups at one point in time educational level and people's age average in both subgroups, respectively . Statistically p was held significant at <0.05 . The average of an age was 16 in both groups examined in this study, and there was no difference between two groups . 82.5% of fathers of students in both groups had under diploma education, 5% of them in test group and 5 - 7% of them in control group enjoyed academic education, and the two groups were not statistically different (p = 0.95). Furthermore, 87.5% of test group's mothers and 75% of control group's mothers had under diploma studies; besides 0% of test groups mothers and 5% of control groups therefore, there was no significant difference between the two groups (p = 0.13). The occupation of a large portion of fathers and mothers of the two group's members was self - employed and housewife, respectively the comparison of peoples knowledge and performance scores average, before and one month after training for both groups, is presented in the following table, showing there is no significant difference between the scores before and one month after training in control group . However, the difference is significant in test group (p <0.001). As observed in [table 1], the average of knowledge and performance scores of the two groups have no significant difference, but the given difference will grow significant after training (p <0.001). The comparison of mean of examined variables scores of test and group before and after training the results indicated that the amount of knowledge of peers of those students suffering from asthma would grow significantly after performing training intervention . The results of a study by pit erg et al ., aiming at examining the effect of training of peers of students with asthma, indicated that the mean of student's knowledge scores of test group increased significantly one month after training intervention . The peers of students with asthma could assist them during attacks regarding study's results, helping the patient and family's fear and stress device, and consequently prevent from creating difficulties facing learning process and incurring high expenses for treatment . In a study by smith et al ., on training the peers of students with asthma, the level of knowledge and attitude of students in test group increased after training intervention compared to control group . In addition, the attitude level of students in test group showed a significant difference compared to before intervention, confirming the present study's findings and showing its ability to increase students knowledge on asthma . 's study on training risky behaviors to adolescents peers predisposed to aids has reported similar findings, indicating that the training peers of students with chronic illnesses could consequently preventing from heightening their conditions . The results of piggy et al ., study conducted in the u.s ., have reported similar findings in case of diabetes . Have also reported that training of the peers of those students suffering from diabetes helped their athletic performance improve . In another study by nazar et al ., entitled an examination of training effect on student's nutritional behaviors, it was found that the amount of knowledge and nutritional performance of students had improved significantly after training . In a study by frail et al ., on performance of students with obesity, a significant difference was found in students behaviors and selection level of foods and meals after performing training program compared to before it ., in a study aimed at examining the effect of training peers of adolescents, predisposed to sexual risky behaviors in zambia, found that the knowledge of test group students on using condom in prevention aids increased one month after training . Moreover, test group students attitude on using preventive instruments in order to prevent aids increased while no difference was observed in control group . Kirby et al ., in their systematic examination, have reported in effectiveness of schools curricula and peers training on prevention from hiv . In a study on assessment of peers training effect on adolescents regarding aids preventing behaviors and sexual illness in cameroon, found that there is a significant correlation between training by peers and knowledge enhancement in the case of new control aptiva methods and sexual illness symptoms . The results of mohammadzadeh et al . Showed that the mean knowledge score of teachers was (12 2.3). This means that the mean knowledge score was intermediate . Because of adolescents tendency toward peers and creating popularity in group, its consequent social support produced by this group, training student, in particular by peers of students with chronic illnesses, could prevent from stress and depression among affected students and be some strategy to enhance their health . Since the trainings were offered during student's classes, there was some interruption between class hours and training time; the researcher could decrease the limitation greatly in collaboration with high school principle . The average of an age was 16 in both groups examined in this study, and there was no difference between two groups . 82.5% of fathers of students in both groups had under diploma education, 5% of them in test group and 5 - 7% of them in control group enjoyed academic education, and the two groups were not statistically different (p = 0.95). Furthermore, 87.5% of test group's mothers and 75% of control group's mothers had under diploma studies; besides 0% of test groups mothers and 5% of control groups mothers had academic studies . Therefore, there was no significant difference between the two groups (p = 0.13). The occupation of a large portion of fathers and mothers of the two group's members was self - employed and housewife, respectively the comparison of peoples knowledge and performance scores average, before and one month after training for both groups, is presented in the following table, showing there is no significant difference between the scores before and one month after training in control group . However, the difference is significant in test group (p <0.001). As observed in [table 1], the average of knowledge and performance scores of the two groups have no significant difference, but the given difference will grow significant after training (p <0.001). The comparison of mean of examined variables scores of test and group before and after training the results indicated that the amount of knowledge of peers of those students suffering from asthma would grow significantly after performing training intervention . The results of a study by pit erg et al ., aiming at examining the effect of training of peers of students with asthma, indicated that the mean of student's knowledge scores of test group increased significantly one month after training intervention . The peers of students with asthma could assist them during attacks regarding study's results, helping the patient and family's fear and stress device, and consequently prevent from creating difficulties facing learning process and incurring high expenses for treatment . In a study by smith et al ., on training the peers of students with asthma, the level of knowledge and attitude of students in test group increased after training intervention compared to control group . In addition, the attitude level of students in test group showed a significant difference compared to before intervention, confirming the present study's findings and showing its ability to increase students knowledge on asthma . 's study on training risky behaviors to adolescents peers predisposed to aids has reported similar findings, indicating that the training peers of students with chronic illnesses could consequently preventing from heightening their conditions . The results of piggy et al ., study conducted in the u.s ., have reported similar findings in case of diabetes . Have also reported that training of the peers of those students suffering from diabetes helped their athletic performance improve . In another study by nazar et al ., entitled an examination of training effect on student's nutritional behaviors, it was found that the amount of knowledge and nutritional performance of students had improved significantly after training . In a study by frail et al ., on performance of students with obesity, a significant difference was found in students behaviors and selection level of foods and meals after performing training program compared to before it ., in a study aimed at examining the effect of training peers of adolescents, predisposed to sexual risky behaviors in zambia, found that the knowledge of test group students on using condom in prevention aids increased one month after training . Moreover, test group students attitude on using preventive instruments in order to prevent aids increased while no difference was observed in control group . Kirby et al ., in their systematic examination, have reported in effectiveness of schools curricula and peers training on prevention from hiv . In a study on assessment of peers training effect on adolescents regarding aids preventing behaviors and sexual illness in cameroon, found that there is a significant correlation between training by peers and knowledge enhancement in the case of new control aptiva methods and sexual illness symptoms . The results of mohammadzadeh et al . Showed that the mean knowledge score of teachers was (12 2.3). This means that the mean knowledge score was intermediate . Because of adolescents tendency toward peers and creating popularity in group, its consequent social support produced by this group, training student, in particular by peers of students with chronic illnesses, could prevent from stress and depression among affected students and be some strategy to enhance their health since the trainings were offered during student's classes, there was some interruption between class hours and training time; the researcher could decrease the limitation greatly in collaboration with high school principle . Since training of peers of students with asthma has been effective, helping the knowledge increase and performance enhance, the performance of this kind of program is suggested in the case of other chronic illness.
The chia (salvia hispanica) seed was used as an offering to the aztec gods, and, because of its religious use, it essentially disappeared for 500 years . This is an annual herbaceous plant belonging to the lamiaceae or labiatae family . In pre - columbian times, it was one of the basic foods of several central american civilizations, less important than corn and beans, but more important than amaranth . The chemical composition reports contents of protein (1525%), fats (3033%), carbohydrates (2641%), dietary fiber (1830%), and ash (4 - 5%). Chia seeds have been investigated and recommended due to their high levels of proteins, antioxidants, dietary fiber, vitamins, and minerals but particularly due to their oil content with the highest proportion of -linolenic acid (-3) compared to other natural sources known to date . Chia seeds contain up to 39% of oil, which has the highest known content of -linolenic acid, up to 68% . Chia seed gum has the potential for industrial use because of its slimy properties, evident even at very low concentration, and because the plant, native to america, grows well in semiarid regions that have few practical plants . Chia gum begins to emerge from seeds as soon as they are placed in water . The exudate is either partially cross - linked or is bound to the seed surface, since it is not easily separated from the seed . Separation can be accomplished by strong stirring, preferably in the presence of sand to aid in dislodgment or cleavage of insolubilizing bonds . For research purposes, gum has been removed by extraction of seeds with a 6 m urea solution . Chia gum is composed of -d - xylopyranosyl, -d - glucopyranosyl, and 4-o - methyl--d - glucopyranosyluronic acid unit in the ratio 2: 1: 1 . The polysaccharide seems to consist of a repeating unit . Extracted gum has a slimy, mucilaginous character at very low concentrations, giving it wide potential use in a variety of industrial applications, especially in certain foods and food preparations . The objective of the present study was to determine the physicochemical properties of fatted and defatted gums from chia (salvia hispanica) seeds . Chia (s. hispanica l.) seeds were obtained in the yucatan state of mexico . Reagents were of analytical grade and purchased from j. t. baker (phillipsburg, nj, usa), sigma (sigma chemical co., st . Louis, mo, usa), merck (darmstadt, germany), and bio - rad (bio - rad laboratories, inc . Seeds of chia were submitted to gum extraction with water at a 1: 20 ratio (w / v) for 30 min and at a 50c temperature . After that, the suspension was milled in a mixer and then it was boiled again at 50c under stirring for 15 min . The crude mixture, containing water, gum, and seeds, was centrifuged at 9460 g at 15c for 3 h. the recovered gum (fcg) was dried at 40c for 24 hours and milled . One portion of the recovered gum was partly defatted (pdcg) in a soxhlet . Standard aoac procedures were used to determine nitrogen (method 954.01), fat (method 920.39), ash (method 925.09), crude fiber (method 962.09), and moisture (method 925.09) contents in the fatted and defatted chia gums . Approximate water absorption capacity was first determined by weighing out 0.1 g (d.b .) Of sample, adding water until saturation (approximately 5 ml), and centrifuging at 2000 g for 10 min in a beckman gs-15r centrifuge . Approximate water absorption capacity was calculated by dividing the increase in sample weight (g) by the quantity of water needed to complete original sample weight to 15 g. water absorption capacity (wabc) was then determined by placing samples in four tubes, adding different quantities of water to bracket the measurement (1.5 and 0.5 ml water above original weight and 1.5 and 0.5 ml water below; one in each tube), agitating vigorously in a vortex for 2 min, and centrifuging at 2000 g for 10 min in a beckman gs-15r centrifuge . Average water absorbed was calculated and the wabc was calculated, expressed as g water absorbed per g of sample . Briefly, 0.1 g (d.b .) Of sample was placed in an equilibrium microenvironment at 98% relative humidity, generated by placing 20 ml of saturated potassium sulfate saline solution in tightly sealed glass flasks and placing these in desiccators at 25c . The sample was left in the microenvironment until reaching constant weight (72 h). Briefly, 0.1 g (d.b .) Of sample was weighed and then stirred into 20 ml of distilled water or corn oil (mazola, cpi international) for one minute . These fibrous suspensions were then centrifuged at 2200 g for 30 min and the supernatant volume was measured . Water - holding capacity was expressed as g of water held per g of sample, and oil - holding capacity was expressed as g of oil held per g of gum . Corn oil density was 0.92 g / ml . Apparent viscosity was evaluated using an adaptation of the li and chang method, using a brookfield viscometer model dv - ii (brookfield engineering lab ., stoughton, ma) with spindle 27 (for small samples) and share rate range from 2.5 to 100 rpm at 25c . The samples were dispersed in water to 0.5, 1.0, 1.5, 2.0, and 2.5% (w / v, db). The results were expressed in pa.s and data was fixed to an ostwald - de waele model to determine the consistency index (k) and flow behavior index (n). This percentage was lower than reported by sciarini et al . In gleditsia triacanthos seeds (11.934.16%). However, the chia gum's yield was higher than reported by oomah et al . In flaxseed (3.68%). The proximal composition (table 1) showed that pdcg registered a higher content of protein, ash, and nfe than fcg . The fiber content of fcg (28.96%) was similar to that reported by vazquez - ovando et al . In a fiber - rich fraction of chia seeds (29.56%). The moisture content of both gums was similar to that reported by kader et al . In acacia glomerosa (9.09%) but lower than reported in guar gum (10.36%), xanthan gum (11.08%), and gleditsia triacanthos (14.08%). With respect to the nfe content, both gums registered lower values than reported by vazquez - ovando et al . In a fiber - rich fraction of chia seeds (34.52%). However, the ash content of fcg and pdcg was higher than that reported by kader et al . In arabic gum (acacia senegal, 3.6%) although lower than reported by sciarini et al . In xanthan gum (9.35%). The protein content of both gums was higher than registered in corn gum (5.1%) and mesquite gum (5.8%), this last one with important emulsifying properties attributed to its protein content according to bosquez . In this respect, establish that hydrocolloids rich in protein, such as gelatin, arabic gum, and mesquite, are good stabilizers because they have sufficient hydrophobic groups to act as bonding points as well as hydrophilic groups that reduce surface tension in a liquid - liquid or liquid - gas interface . On the other hand, yadav et al . Establish that the lipid content in the gums may also play an important role in stabilization of oil - water emulsions . However, bosquez established that carbohydrates avoid flocculation and coalescence of oil droplets to extend in the aqueous solution . These findings suggest that fcg and pdcg could act as good emulsifiers and stabilizers in the food industry . Water absorption capacity is indicative of a structure's aptitude to spontaneously absorb water when placed in contact with a constantly moist surface or when immersed in water . Water adsorption capacity is the ability of a structure to spontaneously adsorb water when exposed to an atmosphere of constant relative humidity . Wabc was higher in fcg (44.08 g / g of sample) than pdcg (36.2 g / g of sample). The high values of wabc obtained here could be due to the proteins present in the gums, which would have a large number of exposed hydrophilic sites interacting with water . The wabc of fcg and pdcg was higher than reported by vzquez - ovando et al . In a fiber - rich fraction of chia seeds (11.73 g / g of sample), who establish that fiber content is an important factor in the increment of this property for its capacity to form gels and to hold water; this justifies the higher value of wabc in fcg . On the other hand, pdcg (0.84 g / g of sample) registered a higher value of wadc than fcg (0.27 g / g of sample). The wadc of dcg was also higher than registered by vzquez - ovando et al . In a fiber - rich fraction of chia seeds (0.3 g / g of sample), similar to that reported in carrots (0.82 g / g of sample) but lower than the value registered in beet bagasse (1.58 g / g of sample). However, whc of both gums was higher than that reported by vzquez - ovando et al . And baquero and bermdez in a fiber - rich fraction of chia seeds (15.41 g / g of fiber) and passion fruit peel (8.7 g / g of fiber), respectively . A similar behavior was observed with orange waste (7.658.23 g / g of fiber). Soluble fiber and the denaturalized proteins may have increased the whc of both gums, thus enhancing the swelling ability, an important function of proteins in preparation of viscous foods such as soups, gravies, dough, and baked products . On the other hand, fcg showed a higher ohc than pdcg, which might be related to its higher value of fat . However, both gums registered higher ohc values than those registered in guar and xanthan gum (46 g oil / g fiber) although similar to that reported in arabic gum (8 - 9 g oil / g fiber). This functional property has been attributed to the physical entrapment of oil for molecules such as lipids and proteins . For the above mentioned, the ohc registered in chia gums could be due to protein and fat contents as well as factors as particle size and the absence of hemicellulose . Chia gum seems to possess an adequate fat absorption capacity, allowing it to play an important role in food processing, since fat acts on flavor retainers and increases the mouth feel of foods . Both gums showed a non - newtonian behavior where viscosity presented a relation directly proportional to the concentration and inversely proportional to the shear rate . The maximum viscosity reached between both gums was registered by pdcg (55.4 pas) at 2.5% . In general, pdcg registered a higher viscosity profile than fcg suggesting that the fat content was the principal factor that generated this behavior . At this respect, report that gums with higher oil absorption as arabic gum show less viscosity (2.34 pas at 3.8%). According to table 2, the rheological behavior of the fcg and pdcg dispersions was a shear thinning or pseudoplastic type due to registered values of n <1 . The results suggest the use at low concentrations of dcg in products as yoghurts, sauces, toppings, and pastries among others that require high viscosity, whilst fcg could be used in sauces, mayonnaises, and meat products as emulsifying and stabilizer . The results obtained here show that chia gums present interesting physicochemical properties for the food industry . The partly defatted chia gum showed a very good ability to water holding (110.5 g / g); however, their ability of oil holding (11.67 g / g) and water absorption (36.26 g / g) was minor compared to the fatted chia gum, which provided a greater retention of oil holding (25.79 g / g) and water absorption (44.08 g / g). Rheological behavior of gums was shear thinning or pseudoplastic type . From a functional point of view, chia gum also is an important food ingredient due its emulsifier and stabilizer potentials.
Bacterial pathogens can develop antibiotic resistance either by mutations, or by the acquisition of antibiotic resistance genes from other microorganisms through horizontal gene transfer (hgt). Since bacterial pathogens were (presumably) susceptible to antibiotics at the time of the development of these compounds, it is reasonable to think that resistance genes have been acquired from non - pathogenic microorganisms . Indeed, the analysis of escherichia coli plasmids from bacterial strains isolated before and after the use of antibiotics for therapy demonstrated that the plasmid families were similar, but incorporated resistance genes after the antibiotic era (datta and hughes, 1983). Since the resistance genes did not originate in bacterial pathogens, the sources for these genes would be environmental microorganisms (martinez et al ., 2009a; davies and davies, 2010). Indeed, the fact that most antibiotics currently used in clinics originated in environmental microorganisms (waksman and woodruff, 1940) led to the proposal that the origin of resistance genes are the antibiotic - producing organisms, where resistance genes may play an auto - protective role (benveniste and davies, 1973; davies, 1997). Recent work indicates that indeed antibiotic - producing environmental microorganisms harbor a large number of resistance genes that could be potentially transferred to human pathogens (dcosta et al ., 2006). Nevertheless, the presence of resistance genes in the environment is not confined to antibiotic producers (aminov, 2009). For instance, the quinolone resistance gene qnra originated in the water - borne bacteria shewanella algae, which is not known to produce an antibiotic (poirel et al ., 2005). Given that quinolones are synthetic drugs, the existence of these determinants indicate the antibiotic resistance genes can have disparate functions in their original hosts, in such a way that the universe of potential resistance genes that can be incorporated into mobile genetic elements is even larger than predicted from the analysis of antibiotic producers . Support for this statement is the finding of genes that contribute to intrinsic resistance in different bacterial species (fajardo et al ., 2008;, 2009; alvarez - ortega et al ., 2010; liu et al ., 2010) and functional metagenomic analyses indicating that the wide dissemination of a large number of resistance genes (dcosta et al ., 2006; sommer et al ., 2009) in all analyzed ecosystems (including the human gut) whether or not contaminated by human activities . It would be expected that this diversity of resistance genes in microorganisms, that can confer an antibiotic resistant phenotype on their transfer to a new host might be mirrored by a large variability of resistance genes, acquired by hgt, in human pathogens . However, the number of different resistance determinants found among human bacterial pathogens is low in comparison to those present in the different metagenomes . This indicates that the transfer of a resistance gene from its original host to a human pathogen might be constrained by different bottlenecks, as discussed in this review . The existence of resistance genes in natural ecosystems, even those without any record of pollution by antibiotics was reported more than four decades ago (gardner et al ., 1969). However, detailed studies on this topic are more recent . In the last years an increasing number of studies of the presence of resistance genes in non - clinical ecosystems have been published . Briefly, two different methodologies are applied; one is the search for any potential gene that confers resistance on expression in a heterologous host by using functional genomic techniques (dcosta et al ., 2006, 2011; the other is the search for resistance genes already present in human pathogens, usually by pcr, in metagenomic dna (koike et al ., 2007). Whereas, in the first analysis the purpose is to characterize any gene that can cause resistance if transferred and hence study the potential natural resistome of the studied ecosystem, the second type of study analyses contamination by resistance genes already acquired by human pathogen . Functional metagenomics serves to define novel mechanisms of resistance (potentiality, see martinez et al ., 2007), but predicting whether such mechanisms will be transferred to human pathogens is not obvious (see below). Indeed, the fact that the origin of the antibiotic resistance genes currently present in human pathogens is known in only a few cases indicates that defining the environmental resistome is a needed but not sufficient condition for predicting the emergence of resistance . It is important to note however that the finding of novel mechanisms of resistance can be a valuable tool for the design of antibiotic modifications before resistance arises (wright, 2007; martinez et al ., 2011). The analysis of the presence in different ecosystems (contaminated and pristine) of genes that have been already acquired by human pathogens would provide information on the stability of these elements, the reservoirs and the factors that enrich their presence in nature . These studies can be used to evaluate the risks for human health from pollution of natural ecosystems by antibiotic resistance determinants, together with antibiotics that serve as selectors of resistance themselves (martinez, 2008, 2009). This knowledge might serve for the identification of intervention strategies to reduce the impact of anthropogenic activities on the enrichment of resistance elements, already present in mobile genetic elements (mges), in natural (non - clinical) ecosystems (baquero et al ., 2008). The relevance that farming and transport of food - borne animals or pets (guardabassi et al ., 2004; aarestrup, 2005; moreno et al ., 2008), as well as the transport of goods (ruiz et al ., 2000), or human migration (kumarasamy et al ., 2010) may have for the dissemination or resistance is well known . For these processes, procedures for tracking the presence and dissemination of resistance genes more difficult will be the implementation of such studies for analyzing the role of wild animals in the spread of resistance (gilliver et al ., 1999; livermore et al ., 2001; allen et al ., 2010). Important in this respect is the finding of resistance in migratory birds that can disseminate both antibiotic resistance determinants and infective resistant bacteria all over the world (middleton and ambrose, 2005; steele et al ., 2005; simoes et al ., 2010). Between these types of studies are functional analyses on the resistance mobilome, those resistance genes that are already present on mobile elements, irrespective of whether or not they have been acquired by human pathogens . The transfer of a potential resistance gene from the chromosome of an environmental bacterium to a human pathogen requires it to be mobilizable after its capture by a translocative element and its integration in an mge . This means that once the resistance element has been incorporated in a mobile element, the possibility of its acquisition by a human pathogen can be high, especially if this element is present in the human bacterial population . Unfortunately, studies on the environmental resistance mobilome are difficult and still rare (szczepanowski et al ., 2008, 2009; the first requirement for the transfer of a resistance gene is that both the donor and the receptor share the same habitat . In the case of pathogenic bacteria, the pathogens need not co - exist with the donor, because a chain of microorganisms may link the donor and the recipient . However, since acquiring resistance genes might confer a fitness cost (see below), the establishment of a successful gene - transfer chain is possible only with positive selection for the resistance determinant . In other words, unless resistance is selected (mainly by antibiotics), it is unlikely that mges containing resistance genes will be fixed in the populations of environmental microorganisms en route to human pathogens . Since the natural concentrations of antibiotics in non - clinical ecosystems are much lower than at hospitals (davies, 2006), only in the case of pollution by antibiotics (aquaculture, waste disposal from cities, farms, or industries) a positive selection for mges containing resistance genes can be envisaged . Following this reasoning, it has been proposed that the possibility of a given resistance gene being transferred to a human pathogen will largely depend on whether the habitat where the donor micro - organism is present close to human - linked ecosystems (baquero et al ., 2009). For instance, it would be rare for resistance genes found in deep soil allocations (brown and balkwill, 2009) or at a glacial ice core (miteva et al ., 2004) would be transferred to human pathogens . In contrast, it has been suggested that ecosystems such as waste - water treatment plants or farms, where human pathogens and environmental bacteria co - exist in the presence of contaminating antibiotic residues, might be hot - spots for the acquisition of resistance genes by bacterial pathogens (baquero et al ., 2008; aminov, 2011). For those microorganisms sharing the same ecosystem, some of them are more prone to exchange genetic material than others . The organisms that can share genes, have been named as genetic exchange communities (jain et al ., 2003). As stated in (skippington and ragan, 2011), gene exchange communities can vary widely in spatial extent, taxonomic diversity, density of internal connectivity, and involvement of vector types . These communities usually share some plasmid (or transposon) types and do not possess strong restriction / modification systems that would impede the interchange of dna . As a consequence, the entrance of a resistance gene, located in a proficient mge into a well established gene exchange community might allow its spread among different organisms and consequently fixation in populations of bacterial pathogens . This spread will be modulated by specific fitness costs that preclude the stability of the gene in some bacterial species . By founder effect, we refer to the situation in which the first gene to arrive is the one to win (baquero et al ., 2009). When there are several resistance determinants with a similar substrate profile, usually one prevails once transferred to human bacterial pathogens . As we will discuss later, this situation can be the consequence of differential fitness costs, nevertheless, a certain degree of serendipity might be the basis of the successful transfer, spread and fixation of a given resistance determinant . One example of this situation is the tem-1 beta - lactamase, which, followed by shv-1 and oxa enzymes, has been the predominant plasmid - encoded beta - lactamase in enterobacteriaceae for many years (simpson et al ., 1980; medeiros, 1997). The tem-1 beta - lactamase was acquired soon after the introduction of the first generation of beta - lactams for therapy, and plasmids coding this beta - lactamase spread rapidly among bacterial pathogens . The study of several different ecosystems has shown that there exist a large number of beta - lactamases nearly everywhere, which can confer resistance to the same antibiotics as tem-1 . Antibiotic resistance genes are acquired and maintained because of the strong selective pressure of antibiotics . Once bacteria have acquired a determinant that allow them to resist antibiotics, there is not a selection pressure for replacement of the determinant already present in bacterial populations . This situation can change if the selective pressure is altered, for example when new antibiotics are launched into clinical use (livermore, 2009; salverda et al ., the introduction of beta - lactamase inhibitors and novel beta - lactams for which tem-1 presented low activity generated two different processes: (i) evolution of the tem - enzyme that most likely occurred in clinical settings when bacterial pathogens were exposed to the novel selective pressure (ii) acquisition of novel beta - lactamase coding genes by human pathogens with novel substrate profiles . It is generally assumed that the acquisition of an antibiotic resistance determinant confers a fitness cost (andersson and levin, 1999), meaning that in the absence of selection, resistant bacteria will be outcompeted by the susceptible ones . In the case of genes acquired by hgt, these costs might be the consequence of the metabolic load imposed by the replication, transcription, and translation of the novel genetic elements . If this was the unique cause of fitness costs, the disadvantage of carrying one or another resistance gene will be similar and the fitness cost would not constitute a relevant bottleneck in selecting one resistance determinant over another . However, different studies have shown that, at least on occasion, the introduction of a given resistance gene does not impose a non - specific metabolic burden but leads to specific changes in bacterial physiology . This may be the case for ampc beta - lactamase genes, which are infrequently found on salmonella plasmids unless the plasmid also harbors the repressor of their expression (verdet et al ., 2000) or elements that compensate the biological costs associated to ampc expression (hossain et al ., 2004). It has been found that ampc alters the physiology of salmonella, decreasing its virulence and hence a differential fitness cost that decreases the probability of dissemination of specific gene among salmonella strains (morosini et al ., 2000). This example indicates that the fitness costs can be gene - specific and do not necessarily derive from a general metabolic burden . In this context, those resistance determinants conferring high fitness costs are unlikely to be fixed in bacterial populations because they would be outcompeted by other resistance determinants which lower fitness costs (martinez et al ., 2011). This reasoning must be however modified by the chances of acquiring compensatory mutations (andersson and hughes, 2011; martinez et al ., 2011). If a resistance determinant confers high fitness costs, but compensatory mutations are easily selected, the probability of being maintained in bacterial pathogens is high . In such cases, if the compensatory mutations occur in the chromosome, not in the mge, the acquisition of the resistance gene by a new host implies a new fitness cost, and as a consequence the spread of the resistance determinant will be compromised . However, it the mutation occurs in the mge, the chances for spread will be enhanced . If the acquisition of resistance confers fitness costs it is logical to suppose that resistant organisms will be outcompeted by their susceptible, fitter counterparts in the absence of selection . However, some resistant strains present no - cost (rozen et al ., 2007; balsalobre and de la campa, 2008) and even some resistance determinants can be beneficial under certain conditions (alonso et al ., 2004; maughan et al ., 2004; luo et al ., 2005; perkins and nicholson, 2008; michon et al ., finally, some fitness costs can be compensated by mutations that do not impede to keep resistance (bjorkman et al ., 1998;, 2002; paulander et al ., 2007; lind et al ., 2010; shcherbakov et al ., this indicates that reversing resistance once established can be a difficult task (andersson and hughes, 2010). Maintenance of resistance genes in habitats without a strong antibiotic pressure is favored as well by second - order selection processes . This means that the selection for one antibiotic will select for the whole array of resistance genes present in this specific mge . Furthermore, mges besides resistance genes may carry other elements such as heavy - metal resistance determinants (baker - austin et al ., 2006), or genes coding for production of siderophores, toxins, or bacteriocins (de lorenzo and martinez, 1988; clewell, 1990; herrero et al ., 2008); these can confer an ecological advantage in some ecosystems and thus co - select resistance in the absence of antibiotics . Cross - selection might also be a relevant second - order process that allows maintenance of resistance in the absence of selection . Certain resistance determinants, such as multidrug (mdr) efflux pumps confer resistance to different compounds (antibiotics, biocides, or heavy metals; martinez et al ., 2009b). This means that selection with the biocide or the heavy metal might result in cross - resistance to the antibiotic (hernandez et al . A final mechanism for the maintenance of resistance is based on the inherent systems for plasmid stability . Plasmids frequently encode toxin / antitoxin systems, which provoke death of bacteria that lose the plasmid (hayes, 2003; hayes and van melderen, 2011). If one such plasmid incorporates an antibiotic resistance determinant, the probabilities for its maintenance will be high . Taking these considerations into account, resistance genes might evade elimination (andersson and hughes, 2011) in the absence of antibiotics; indeed, resistance determinants present in human pathogens have been found on identical mges, in antibiotic - pristine habitats (pallecchi et al . Livermore et al ., 2001) and primitive human populations without any known exposure to antibiotics (grenet et al ., 2004; bartoloni et al ., these observations indicate that anthropogenic activity has enriched for a small number of resistance genes in natural ecosystems and that this type of pollution will be difficult to eradicate (salyers and amabile - cuevas, 1997; martinez, 2009). Supporting this notion, analyses of soils sampled in the netherlands from 1940 to 2008, this, in spite of the fact that restrictions on non - therapeutic use of antibiotics in agriculture and in waste management procedures have been strongly enforced (knapp et al ., 2010). Research on antibiotic resistance has been mainly focused on bacterial pathogens isolated from infections or in clinical settings . However, the fact that hgt - acquired genes originated in natural, non - clinically relevant microorganisms and that the first step in the transfer of resistance likely occurs in natural ecosystems emphasizes the need to analyze resistance in non - clinical ecosystems . Furthermore, the constant release of antibiotic resistance determinants already present in mges located in human pathogens, and in some circumstances associated with selective concentrations of antibiotics, may disrupt natural microbiota, which then serve as reservoirs for resistance genes . Non - culture based methods have demonstrated their value for the analysis of resistance in natural ecosystems . Among them, functional metagenomics provides the means to identify novel mechanisms of resistance independently of whether they will be acquired by bacterial pathogens . On the other hand, pcr analyses for specific genes serve to define reservoirs and to study elements like pollution in the dissemination and maintenance of resistance . Functional metagenomic studies indicate that very few among the resistance genes present in nature have been transferred to human pathogens . Whereas the founder effect can provide stochasticity to these acquisitions, other factors such as fitness costs, ecological connectivity, which includes the formation of gene exchange communities, are relevant bottlenecks that serve to modulate the acquisition of resistance genes by animal or human pathogens . The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Image denoising can be considered as a component of processing or as a process itself . In the first case, the image denoising is used to improve the accuracy of various image processing algorithms such as registration or segmentation . Then, the quality of the artifact correction influences performance of the procedure . In the second case this paper focuses on a new denoising method firstly introduced by buades et al . For 2d image denoising: the nonlocal (nl) means filter . We propose, to improve this filter with an automatic tuning of the filtering parameter, a blockwise implementation and a mixing of wavelet su - bands based on the approach proposed in . These contributions lead to a fully - automated method and overcome the main limitation of the classical nl - means: the computational burden . Section 4 shows the impact of our adaptations compared to different implementations of the nl - means filter and proposes a comparison with well - established methods . Many methods for image denoising have been suggested in the literature, and a complete review of them can be found in . Methods for image restoration aim at preserving the image details and local features while removing the undesirable noise . In many approaches, an initial image is progressively approximated by filtered versions which are smoother or simpler in some sense . Total variation (tv) minimization, nonlinear diffusion [46], mode filters, or regularization methods [3, 8] are among the methods of choice for noise removal . Most of these methods are based on a weighted average of the gray values of the pixels in a spatial neighborhood [9, 10]. An evolution of this approach has been presented by tomasi and manduchi who devised the bilateral filter which includes both a spatial and an intensity neighborhood . Recently, the relationships between bilateral filtering and local mode filtering, local m - estimators, and nonlinear diffusion have been established . In the context of statistical methods, the bridge between the bayesian estimators applied on a gibbs distribution, resulting with a penalty functional and averaging methods for smoothing, has also been described in . Finally, statistical averaging schemes enhanced via incorporating a variable spatial neighborhood scheme have been proposed in [1517]. The tradeoff between noise removal and image preservation is performed by tuning the filter parameters, which is not an easy task in practice . In this paper, we propose to overcome this problem with a 3d subbands wavelet mixing . As in, we have chosen to combine a multiresolution approach with the nl - means filter, which has recently shown very promising results . Recently introduced by buades et al ., the nl - means filter proposes a new approach for the denoising problem . Contrary to most denoising methods based on a local recovery paradigm, the nl - means filter is based on the idea that any periodic, textured, or natural image has redundancy, and that any voxel of the image has similar voxels that are not necessarily located in a spatial neighborhood . This new nonlocal recovery paradigm allows to improve the two most desired properties of a denoising algorithm: edge preservation and noise removal . In this section, we introduce the following notations: u: is the image, where represents the image grid, considered as cubic for the sake of simplicity and without loss of generality (\|\| = n);for the original voxelwise nl - means approach, u(xi) is the intensity observed at voxel xi, vi is the cubic search volume centered on voxel xi of size \|vi\| = (2 m + 1), m, ni is the cubic local neighborhood of xi of size \|ni\| = (2d + 1), d, u(ni) = (u (ni),, u(\|ni \|)(ni)) is the vector containing the intensities of ni (that we term patch in the following),nl(u)(xi) is the restored value of voxel xi, w(xi, xj) is the weight of voxel xj when restoring u(xi) (see figure 1(a)); for the blockwise nl - means approach, bi is the block centered on xi of size \|bi\| = (2 + 1),, u(bi) is the vector containing the intensities of the block bi, nl(u)(bi) is the vector containing the restored value of bi, w(bi, bj) is the weight of block bj when restoring the block u(bi) (see figure 1(b)), the blocks bik are centered on voxels xik which represent a subset of the image voxels, equally regularly distributed over (see figure 2),n represents the distance between the centers of the blocks bik (see figure 2). U: is the image, where represents the image grid, considered as cubic for the sake of simplicity and without loss of generality (\|\| = n); for the original voxelwise nl - means approach, u(xi) is the intensity observed at voxel xi, vi is the cubic search volume centered on voxel xi of size \|vi\| = (2 m + 1), m, ni is the cubic local neighborhood of xi of size \|ni\| = (2d + 1), d, u(ni) = (u (ni),, u(\|ni \|)(ni)) is the vector containing the intensities of ni (that we term patch in the following),nl(u)(xi) is the restored value of voxel xi, w(xi, xj) is the weight of voxel xj when restoring u(xi) (see figure 1(a)); u(xi) is the intensity observed at voxel xi, vi is the cubic search volume centered on voxel xi of size \|vi\| = (2 m + 1), m, ni is the cubic local neighborhood of xi of size \|ni\| = (2d + 1), d, u(ni) = (u (ni),, u(\|ni \|)(ni)) is the vector containing the intensities of ni (that we term patch in the following), nl(u)(xi) is the restored value of voxel xi, w(xi, xj) is the weight of voxel xj when restoring u(xi) (see figure 1(a)); for the blockwise nl - means approach, bi is the block centered on xi of size \|bi\| = (2 + 1),, u(bi) is the vector containing the intensities of the block bi, nl(u)(bi) is the vector containing the restored value of bi, w(bi, bj) is the weight of block bj when restoring the block u(bi) (see figure 1(b)), the blocks bik are centered on voxels xik which represent a subset of the image voxels, equally regularly distributed over (see figure 2),n represents the distance between the centers of the blocks bik (see figure 2). Bi is the block centered on xi of size \|bi\| = (2 + 1),, u(bi) is the vector containing the intensities of the block bi, nl(u)(bi) is the vector containing the restored value of bi, w(bi, bj) is the weight of block bj when restoring the block u(bi) (see figure 1(b)), the blocks bik are centered on voxels xik which represent a subset of the image voxels, equally regularly distributed over (see figure 2), n represents the distance between the centers of the blocks bik (see figure 2). In the classical formulation of the nl means filter, the restored intensity nl(u) (xi) of the voxel xi, is a weighted average of the voxels intensities u(xi) in the search volume vi of size (2 m + 1): (1)nl(u)(xi)=xjviw(xi, xj)u(xj), where w(xi, xj) is the weight assigned to value u(xj) to restore voxel xi . More precisely, the weight evaluates the similarity between the intensity of the local neighborhoods ni and nj centered on voxels xi and xj, such that w(xi, xj) [0, 1] and xjviw(xi, xj) = 1 (cf ., the computation of the weight is based on the euclidean distance between patches u(nj) and u(ni), defined as (2)w(xi, xj)=1zieu(ni)u(nj)22/h2, where zi is a normalization constant ensuring that jw(xi, xj) = 1, and h acts as a filtering parameter controlling the decay of the exponential function . As explained in the introduction, denoising is usually the first step of complex image processing procedures . The number and the dimensions of the data to process being continually increasing, each step of the procedures needs to be as automatic as possible . In this section, we propose an automatic tuning of the filtering parameter h first, it has been shown that the optimal smoothing parameter h is proportional to the standard deviation of the noise . Second, if we want the filter independent of the neighborhood size, the optimal h must depend on \|ni\| (see, (2)). Thus, the automatic tuning of the filtering parameter h amounts to determining the relationship h = f (, \|ni\|,), where is a constant . Firstly, the standard deviation of the noise needs to be estimated . In case of an additive white gaussian noise, this estimation can be based on pseudoresiduals i as defined in [18, 19]. For each voxel xi of the volume, let us define (3)i=67(u(xi)16xjpiu(xj)), pi being the 6-neighborhood at voxel xi and the constant 6/7 is used to ensure that e[i2]=^2 in the homogeneous areas . Thus, the standard deviation of noise ^ is computed as (4)^2=1\|3\|i3i2 . Then, in order to make the filter independent of \|ni\|, we used the euclidean distance \|\| based on the fact that, in the case of gaussian noise and with normalized l2-norm, the optimal denoising is obtained for h = 2, (2) can be written as (6)w(xi, xj)=1zieu(ni)u(nj)22/2^2\|ni\|, where only the adjusting constant needs to be manually tuned . If our estimation ^ of the standard deviation of the noise is correct, should be close to 1 . The main problem of the nl - means filter is being its computational time, a blockwise approach can be used to decrease the algorithmic complexity . Indeed, instead of denoising the image at a voxel level, entire blocks are directly restored . A blockwise implementation of the nl - means filter consists in (a) dividing the volume into blocks with overlapping supports, (b) performing nl - means - like restoration of these blocks, and (c) restoring the voxels values based on the restored values of the blocks they belong to, as follows . A partition of the volume into overlapping blocks bik of size (2 + 1) is performed, such as = kbik, under the constraint that each block bik intersects with at least one other block of the partition . These blocks are centered on voxels xik which constitute a subset of . The voxels xik are equally distributed at positions ik = (k1 n, k2 n, k3 n), (k1, k2, k3), where n represents the distance between the centers of bik . To ensure a global continuity in the denoised image, the overlapping support of blocks is nonempty: 2 n.for each block bik, an nl - means - like restoration is performed as follows: (7)nl(u)(bik)=bjvikw(bik, bj)u(bj),with w(bik, bj)=1zikeu(bik)u(bj)22/2^2\|ni\|, where zik is a normalization constant ensuring that jw(bik, bj) = 1 (see figure 1, right). For a voxel xi included in several blocks bik, several estimations of the restored intensity nl(u)(xi) the estimations given by different nl(u)(bik) for a voxel xi are stored in a vector ai . The final restored intensity of voxel xi is then defined as(8)nl(u)(xi)=1\|ai\|paiai(p), where ai(p) denotes the pth element of the vector ai . A partition of the volume into overlapping blocks bik of size (2 + 1) is performed, such as = kbik, under the constraint that each block bik intersects with at least one other block of the partition . These blocks are centered on voxels xik which constitute a subset of . The voxels xik are equally distributed at positions ik = (k1 n, k2 n, k3 n), (k1, k2, k3), where n represents the distance between the centers of bik . To ensure a global continuity in the denoised image, the overlapping support of blocks is nonempty: 2 n. for each block bik, an nl - means - like restoration is performed as follows: (7)nl(u)(bik)=bjvikw(bik, bj)u(bj),with w(bik, bj)=1zikeu(bik)u(bj)22/2^2\|ni\|, where zik is a normalization constant ensuring that jw(bik, bj) = 1 (see figure 1, right). For a voxel xi included in several blocks bik, several estimations of the restored intensity nl(u)(xi) the estimations given by different nl(u)(bik) for a voxel xi are stored in a vector ai . The final restored intensity of voxel xi is then defined as(8)nl(u)(xi)=1\|ai\|paiai(p), where ai(p) denotes the pth element of the vector ai . The main advantage of this approach is to significantly reduce the complexity of the algorithm . Indeed, for a volume of size n, the global complexity is ((2 + 1) (2 m + 1) ((n n)/n)). For instance, with n = 2, the complexity is divided by a factor 8 . In [2123], the authors have shown that neglecting the voxels / blocks with small weights (i.e., the most dissimilar patches to the current one) speeds up the filter and significantly improves the denoising results . Indeed, the selection of the most similar patches u(bj) to the current patch u(bi) to compute nl(u)(bi) can be viewed as a spatially adaptation of the patch dictionaries . As in [2123], the preselection of blocks in vi is based on the mean and the variance of u(bi) and u(bj). The selection tests are given by (9)w(bik, bj)={1zikeu(bik)u(bj)22/2^2\|ni\| if 1<u(bik)u(bj)<11, 12<var(u(bik))var(u(bj))<112,0 otherwise, where u(bik) and var(u(bik)) represent, respectively, the mean and the variance of the intensity function for the block bik centered on the voxel xik . The new parameters 0 <1 <1 and 0 <1 <1 control the level of rejection related to tests . When 1 and 1 are close to 0, there is almost no selection and the number of patches taken into account increases: thus the denoised image becomes smoother . The filter is equivalent to the classical nl - means and the computation time increases . When 1 and 1are close to 1, the selection is more severe and the number of patches taken into account decreases: the denoised image is less smoothed and the computation time decreases . This kind of selection tends to better enhance the contrast . In practice, 1 and 1 were chosen as in [21, 22]: 1 = 0.95 and 1 = 0.5 . Recently, hybrid approaches coupling the nl - means filter and a wavelet decomposition have been proposed [2, 24, 25]. In, a wavelet - based denoising of blocks is performed before the computation of the nonlocal means . The nl - means filter is performed with denoised version of blocks in order to improve the denoising result . In, the nl - means filter is applied directly on wavelet coefficients in transform domain . This approach allows a direct denoising of compressed images (such as jpeg2000) and a reduction of computational time since smaller images are processed . In, a multiresolution framework is proposed to adaptively combine the result of denoising algorithms at different space - frequency resolutions . This idea relies on the fact that a set of filtering parameters is not optimal over all the space - frequency resolutions . Thus, by combining to the transform domain the results obtained with different sets of filtering parameters, the denoising is expected to be improved . In order to improve the denoising result of the nl - means filter, we propose a multiresolution framework similar to to implicitly adapt the filtering parameters (h, \|bi\|) over the different space - frequency resolutions of the image . This adaptation is based on the fact that the size of the patches impacts the denoising properties of the nl - means filter . Indeed, the weight given to a block depends on its similarity with the block under consideration, but the similarity between the blocks depends on their sizes . Thus, given the size of the blocks, removal or preservation of image components can be favored . In the transform domain, the main features of the image correspond to low - frequency information while finer details and noise are associated to high frequencies . Nonetheless, noise is not a pure high - frequency component in most images . Noise is spanned over a certain range of frequencies in the image with mainly middle and high components . In nl - means - based restoration, large blocks and setting = 1 efficiently remove all frequencies of noise but tend to spoil the main features of the image, whereas small blocks and low smoothing parameter (= 0.5) tend to better preserve the image components but cannot completely remove all frequencies of noise . As a consequence, we propose the following workflow (see figure 3). Denoising of the original image i using two sets of filtering parameters: one adapted to the noise components removal (i.e., large blocks and = 1) and the other adapted to the image features preservation (i.e., small blocks and = 0.5). The noise is efficiently removed and, conversely, in iu, the image features are preserved.decomposing io and iu into low- and high - frequency subbands . The first level decomposition of the images is performed with a 3d discrete wavelet transform (dwt).mixing the highest - frequency subbands of io and the lowest frequency subbands of iu.reconstructing the final image by an inverse 3d dwt from the combination of the selected high and low frequencies . Denoising of the original image i using two sets of filtering parameters: one adapted to the noise components removal (i.e., large blocks and = 1) and the other adapted to the image features preservation (i.e., small blocks and = 0.5). The noise is efficiently removed and, conversely, in iu, the image features are preserved . The first level decomposition of the images is performed with a 3d discrete wavelet transform (dwt). Mixing the highest - frequency subbands of io and the lowest frequency subbands of iu . Reconstructing the final image by an inverse 3d dwt from the combination of the selected high and low frequencies . In this paper, we propose an implementation of this approach using our optimized blockwise nl - means filter and the 3d dwt daubechies-8 basis . The latter is implemented in qccpack (http://qccpack.sourceforge.net) in the form of dyadic subband pyramids . Once the original image i has been denoised using two sets of filtering parameters, a 3d dwt at the first level is performed on both io and io images . For each image, eight subbands are obtained: lll1, llh1, lhl1, hll1, lhh1, hlh1, hhl1, and hhh1 . In the eight wavelet subbands obtained with io, the frequencies corresponding to noise are efficiently removed from the high frequencies whereas the low frequencies associated to the main features are spoiled.in the eight wavelet subbands obtained with iu, the low frequencies associated to main features are efficiently preserved whereas residual frequencies corresponding to noise are present in high frequencies . In the eight wavelet subbands obtained with io, the frequencies corresponding to noise are efficiently removed from the high frequencies whereas the low frequencies associated to the main features are spoiled . In the eight wavelet subbands obtained with iu, the low frequencies associated to main features are efficiently preserved whereas residual frequencies corresponding to noise are present in high frequencies . Thus, we select the highest frequencies of io (i.e., lhh1, hlh1, hhl1, and hhh1) and the lowest frequencies of iu (i.e., lll1, llh1, lhl1, and hll1). Then, the 4 lowest subbands of iu are combined with the 4 highest subbands of io . Finally, an inverse 3d dwt is performed on these 8 selected subbands to obtain the final denoised image (see figure 3). In [21, 22], the optimal parameters for 3d mri have been estimated as = 1, m = 5, 1 = 0.95, and 1 = 0.5 . In our experiments, the two sets of parameters used to obtain iu and io were su = (u, mw, u) = (1, 3, 0, 5) and so = (o, mw, o) = (2, 3, 1). Compared to [21, 22], the size of search volume was reduced to decrease the computational time . Several sets of parameters have been tested, the mentioned numerical values are satisfying to balance the denoising performance (high psnr values) and computational burden . Finally, to decrease the computational time, this workflow is parallelized and each version is computed on different cpus or cores (see figure 3). In order to evaluate the performance of the different variants of the nl - means filter on 3d mr images, tests were performed on the brainweb database . Several images were simulated to validate the performance of the denoising on various images: (a) t1-w phantom mri for 4 levels of noise 3%, 9%, 15%, and 21% and (b) t2-w phantom mri with multiple sclerosis (ms) lesions for 4 levels of noise 3%, 9%, 15%, and 21% . A white gaussian noise was added, and the notations of brainweb are used: a noise of 3% is equivalent to (0, (3/100)), where is the value of the highest voxel intensity of the phantom (150 for t1-w and 250 for t2-w). In the following, nl - means: standard voxelwise implementation with automatic tuning of the filtering parameter h (= 1).optimized nl - means: voxelwise implementation with automatic tuning of the filtering parameter h (= 1) and voxels selection presented in . Optimized blockwise nl - means: (this filter can be freely tested at http://www.irisa.fr/visages/bench marks) blockwise implementation with automatic tuning of the filtering parameter h (= 1) and blocks selection presented in . Optimized blockwise nl - means with wavelet mixing: proposed filter based on a blockwise implementation, an automatic tuning of the filtering parameter h (= 1), a block selection, and a wavelet subbands mixing . The selected filtering parameters for the different implementations were as follows . For the nl - means and optimized nl - means filters, the parameters are those used in: d = 1, = 1, m = 5, 1 = 0.95 and 1 = 0.5.concerning the optimized blockwise nl - means filter, the sets of parameters are those used in: n = 2, = 1, = 1, m = 5, 1 = 0.95 and 1 = 0.5.finally, for the optimized blockwise nl - means with wavelet mixing filter the parameter are the following: n = 2, su = (u, mw, u) = (1, 3, 0.5), so = (o, mw, o) = (2, 3, 1), 1 = 0.95, and 1 = 0.5 . For 8-bit encoded images, the psnr is defined as follows: (10)psnr=20 log10255rmse, where rmse denotes the root mean square error estimated between the ground truth and the denoised image . For the sake of clarity, the psnr values are estimated only in the region of interest (cerebral tissues) obtained by removing the background (i.e., the label 0 of the discrete model in brainweb). Nl - means: standard voxelwise implementation with automatic tuning of the filtering parameter h (= 1). Optimized nl - means: voxelwise implementation with automatic tuning of the filtering parameter h (= 1) and voxels selection presented in . Optimized blockwise nl - means: (this filter can be freely tested at http://www.irisa.fr/visages/bench marks) blockwise implementation with automatic tuning of the filtering parameter h (= 1) and blocks selection presented in . Optimized blockwise nl - means with wavelet mixing: proposed filter based on a blockwise implementation, an automatic tuning of the filtering parameter h (= 1), a block selection, and a wavelet subbands mixing . For the nl - means and optimized nl - means filters, the parameters are those used in: d = 1, = 1, m = 5, 1 = 0.95 and 1 = 0.5 . Concerning the optimized blockwise nl - means filter, the sets of parameters are those used in: n = 2, = 1, = 1, m = 5, 1 = 0.95 and 1 = 0.5 . Finally, for the optimized blockwise nl - means with wavelet mixing filter the parameter are the following: n = 2, su = (u, mw, u) = (1, 3, 0.5), so = (o, mw, o) = (2, 3, 1), 1 = 0.95, and 1 = 0.5 . Firstly, we have experimentally verified that the optimal denoising is obtained for 1 for high levels of noise and 0.5 for low levels of noise . These results account for the error in the estimation of (^2=3.42% at 3%, ^2=7.93% at 9%, ^2=12.72% at 15%, and ^2=17.44% at 21%) (see figure 4). Table 1 shows that the blockwise approach of the nl - means filter, with and without voxels selection (see (9)), allows to drastically reduce the computational time . With a distance between the block centers corresponding to n = 2, the blockwise approach divides the timings by a factor superior to 5 (see table 1). However, the computational time reduction is balanced with a slight decrease of the psnr (see figure 5) compared to the optimized nl - means filter presented in . Our optimized blockwise nl - means with wavelet mixing allows to compensate this slight decrease of the psnr and to divide the computational by a factor 4 compared to the optimized nl - means filter . Visually, the proposed method combines the most important attributes of a denoising algorithm: edge preservation and noise removal . Figure 6 shows that our filter removes noise while keeping the integrity of ms lesions (i.e., no structure appears in the removed noise). Figure 7 focuses on the differences between the optimized blockwise nlm and the optimized blockwise nlm with wm filters . The denoising result obtained with the optimized blockwise nlm with wm filter visually preserves the edges better than the optimized blockwise nlm filter . This is also confirmed by visual inspection of the comparison with the ground truth . The images of difference between the phantom and the denoised image (see bottom of figure 7) show that less structures have been removed with the optimized blockwise nlm with wm filter . Thus, the multiresolution approach allows to better preserve the edges and to enhance the contrast between tissues . In this section, we compare the proposed method with two of the most used approaches in mri domain: the nonlinear diffusion (nld) filter r and the total variation (tv) minimization . The main difficulty to achieve this comparison is related to the tuning of smoothing parameters in order to obtain the best results for nld filter and tv minimization scheme . After quantifying the parameter space this allows us to obtain the best possible results for the nld filter and the tv minimization . For the optimized blockwise nlm with wm, the same set of parameters su = (u, mw, u) = (1, 3, 0.5) and so = (o, mw, o) = (2, 3, 1) are used for all noise levels . The automatic tuning of h adapts the smoothing to the noise level . For nld filter, the parameter k varied from 0.05 to 1 with a step of 0.05 and the number of iterations varied from 1 to 10 . For tv minimization, the parameter varied from 0.01 to 1 with a step of 0.01 and the number of iterations varied from 1 to 10 . The results obtained for a 9% of gaussian noise are presented in figure 8, but this screening was performed for the four levels of noise . It is important to underline that the results giving the best psnr are used, but these results do not necessarily give the best visual output . Actually, the best psnr value for the nld filter and tv minimization are obtained for a visually under - smoothed image since these methods tend to spoil the edges . This is explained by the fact that the optimal psnr is obtained when a good tradeoff is reached between edge preserving and noise removing . As presented in figure 9, our block - optimized nl - means with wavelet mixing filter produced the best psnr values whatever the noise level is . On average, a gain of 2.15 db the psnr value between the noisy image and the ground truth is called no processing and is used as a reference . Figure 10 shows the denoising results obtained by the nld filter, the tv minimization, and our optimized blockwise nlm with wm . The removed noise (see middle of figure 10) shows that the proposed method removes significantly less structures than nld filter or tv minimization . Finally, the comparison with the ground truth underlines that the nl - means restoration gives a result very close to the ground truth and better preserves the anatomical structure compared to nld filter and tv minimization . The t1-weighted mr images used for experiments were obtained with t1 sense 3d sequence on 3 t philips gyroscan scanner . The restoration results, presented in figure 11, show good preservation of the cerebellum . Fully automatic segmentation and quantitative analysis of such structures are still a challenge, and improving restoration schemes could greatly improve these processings . This paper presented a fully automated blockwise version of the nonlocal means filter with subbands wavelet mixing . The results on phantom shows that the proposed optimized blockwise nl - means with subbands wavelet mixing filter outperforms the classical implementation of the nl - means filter and the optimized implementation presented in [21, 22], in terms of psnr values and computational time . Compared to the classical nl - means filter, our implementation (with block selection, blockwise implementation, and wavelet subbands mixing) considerably decreases the required computational time (up to a factor of 20) and significantly increases the psnr of the denoised image . The comparison of the filtering process with and without wavelet mixing shows that the subbands mixing better preserves edges and better enhances the contrast between the tissues . This multiresolution approach allows to adapt the smoothing parameters along the frequencies by combining several denoised images . The comparison with well - established methods such as nld filter and tv minimization shows that the nl - means - based restoration produces better results . Finally, the impact of the proposed multiresolution approach based on wavelet subbands mixing should be investigated further, for instance, when combined to the nonlinear diffusion filter and the total variation minimization.
We induced diabetes in sprague - dawley rats by intraperitoneal injection of 55 mg / kg of stz in 10 mm citrate buffer (ph 4.5) after overnight fasting, or in mice by intraperitoneal injection of 100 mg / kg of stz in citrate buffer for 2 days . We confirmed diabetes by measuring blood glucose (> 250 mg / dl) 24 h after stz injection . Blood glucose at the time of surgery was 442 12 and 109 3 mg / dl for dm and ndm rats, respectively . Initial body weight of rats was 250 g and average body weight after 4 weeks was 403 10 g for ndm rats and 294 5 g for dm rats, respectively . We performed all experiments in accordance with the guidelines of the national institutes of health guide for the care and use of laboratory animals and with approval from the animal care and use committee of the joslin diabetes center . We anaesthetized the rats with pentobarbital (50 mg / kg, ip), surgically exposed the surface of the skull and slowly injected 50 l pbs, 50 l autologous whole blood (from tail vein), 15 g purified human pk (enzyme research laboratories), 30 g deactivated pk or 5 m bradykinin (calbiochem) in 50 l pbs into the brain at 1 mm anterior, 4 mm lateral and 6 mm in depth to the bregma using a 30 gauge needle . For mice, we slowly injected 10 l saline or autologous whole blood into the brain at 1 mm anterior, 2 mm lateral and 3.5 mm in depth to the bregma using a 30 gauge needle . We removed the infusion needle 10 min after injection and sealed the burr hole with bone wax . (1 g / kg / hr), [des - arg]-hoe140 (1 g / kg / hr) (sigma), 1-benzyl-1h - pyrazole-4-carboxylic acid 4-carbamimidoyl - benzylamide (asp-440) (14 g / kg / hr) or its vehicle (10% polyethylene glycol in pbs) by subcutaneous osmotic pump (alzet) into rats 1 day prior to injection . For the mixture of hoe 140 and [des - arg]-hoe140 injection, for pk - specifc antibody injection, antibody to pk (abcam, final concentration: 0.06 mg / ml) or mouse igg (final concentration: 0.06 mg / ml) was mixed with 50 l blood prior to injection . For convulxin injection, 35 ng convulxin (alexis biochemicals) was mixed with 15 g pk in 50 l pbs prior to injection . For gpvi - specific antibody injection, we injected 10 g jaq1 antibody (amfret analytics) or rat igg in 30 pbs into mice brains . For mannitol infusion experiment, we injected a bolus of 15 ml / kg of 20% mannitol intravenously (calculated plasma osmolality: 320 mosm / kg) to the rats followed by cerebral injection of autologous blood or pk . We continuously infused the rats intravenously with 20% mannitol at a rate of 15 ml / kg / h for 2 h to maintain constant osmolality . We examined cerebral hematoma area or volume at 30 min, 2 h, or 48 h after injection . We perfused the animals with saline through the left ventricle, harvested the brains, and determined hematoma areas with quantity one software (biorad). We collected blood from the vena cava of the rats and anti - coagulated with acid - citrate - dextrose buffer (9:1, v / v). Platelet - rich plasma (prp) was obtained by centrifugation of the blood at 250 g for 15 min . We then centrifuged the prp at 500 g for 10 min to obtain platelet pellets, and washed the pallets 3 times with a modified tyrode s buffer without ca (134 mm nacl, 3 mm kcl, 0.3 mm nah2po4, 2 mm mgcl2, 5 mm hepes, 5 mm glucose, 12 mm nahco3, 1 mm egta, and 3.5 mg / ml bsa). We resuspended the final pellets in tyrode s buffer with 1 mm cacl2 but no egta and adjusted to a final platelet concentration at 2x10 cells / ml . We monitored platelet aggregation using a chrono - log 680 aggregation system (chrono - log) with stirring at 37c . We activated platelets with equine type i collagen (2 g / ml, chrono - log), thrombin (0.25 u / ml, sigma), or adp (20 m, sigma), and recorded the change of transmission . Briefly, the aggregation process was carried out in 100 l volume in a 96 well flat - bottom microplate . Platelets were activated with type i collagen (2 g), collagen related peptide (crp, 10 ng, university of cambridge, uk), or convulxin (0.25 nm), and the readings were taken every 30 seconds over a 20-minute period at 405 nm wavelength . We measured the interaction between pk and collagen with a biacore 3000 system (biacore ab) using he s buffer (10 mm hepes, 150 mm nacl, 2 mm edta, ph 7.4) at 25c . Rat tail type i collagen in 10 mm sodium acetate buffer (ph 5.0) was covalently coupled to a cm5 chip using an amine coupling kit (biacore ab) according to the manufacturer's instructions . A control surface underwent the same activation and deactivation procedures in the absence of collagen . Regeneration of the collagen surface was achieved by running 10 l of 10 mm glycine (ph 2.0) through the flow cell at 10 l / min . Pk solutions at several concentrations were perfused over the immobilized collagen at a flow rate of 10 l / min for 3 min, and the resonance changes were recorded . The sensorgram of the immobilized - collagen surface was subtracted from that of the control surface, and the data thus obtained were analyzed by biaevaluation software (biacore ab). We rinsed the aorta free of blood, trimmed surrounding connective tissue and the intercostal vessels, opened longitudinally, and gently scraped the inner surface with a sterile razor blade to remove the endothelium . We washed the vessel 3 times with ice cold pbs and divided it into approximately 4 mm pieces . Care was given to assure that the exposed aorta was covered by pbs during this procedure . We incubated aorta pieces with buffer containing 300 nm pk and glucose (050 mm) for 30 min at 25c . We then washed the segments, and measured the activity of surface - bound pk . Statistical analysis was performed by one - way analysis of variance (anova) followed by bonferroni s multiple comparisons test or students t - test as appropriate . Statistically significant differences between groups were defined as p <0.05 and are indicated in the legends of the figures . The methods for generation and characterization of klkb1 mice, systemic hemostasis measurement, hemoglobin analysis, western blotting, cardiac output measurement, plasminogen activation, continuous assays of clot formation and lysis, and pk activity assay are provided in the supplementary methods online . We induced diabetes in sprague - dawley rats by intraperitoneal injection of 55 mg / kg of stz in 10 mm citrate buffer (ph 4.5) after overnight fasting, or in mice by intraperitoneal injection of 100 mg / kg of stz in citrate buffer for 2 days . We confirmed diabetes by measuring blood glucose (> 250 mg / dl) 24 h after stz injection . Blood glucose at the time of surgery was 442 12 and 109 3 mg / dl for dm and ndm rats, respectively . Initial body weight of rats was 250 g and average body weight after 4 weeks was 403 10 g for ndm rats and 294 5 g for dm rats, respectively . We performed all experiments in accordance with the guidelines of the national institutes of health guide for the care and use of laboratory animals and with approval from the animal care and use committee of the joslin diabetes center . We anaesthetized the rats with pentobarbital (50 mg / kg, ip), surgically exposed the surface of the skull and slowly injected 50 l pbs, 50 l autologous whole blood (from tail vein), 15 g purified human pk (enzyme research laboratories), 30 g deactivated pk or 5 m bradykinin (calbiochem) in 50 l pbs into the brain at 1 mm anterior, 4 mm lateral and 6 mm in depth to the bregma using a 30 gauge needle . For mice, we slowly injected 10 l saline or autologous whole blood into the brain at 1 mm anterior, 2 mm lateral and 3.5 mm in depth to the bregma using a 30 gauge needle . We removed the infusion needle 10 min after injection and sealed the burr hole with bone wax . (1 g / kg / hr), [des - arg]-hoe140 (1 g / kg / hr) (sigma), 1-benzyl-1h - pyrazole-4-carboxylic acid 4-carbamimidoyl - benzylamide (asp-440) (14 g / kg / hr) or its vehicle (10% polyethylene glycol in pbs) by subcutaneous osmotic pump (alzet) into rats 1 day prior to injection . For the mixture of hoe 140 and [des - arg]-hoe140 injection, for pk - specifc antibody injection, antibody to pk (abcam, final concentration: 0.06 mg / ml) or mouse igg (final concentration: 0.06 mg / ml) was mixed with 50 l blood prior to injection . For convulxin injection, 35 ng convulxin (alexis biochemicals) was mixed with 15 g pk in 50 l pbs prior to injection . For gpvi - specific antibody injection, we injected 10 g jaq1 antibody (amfret analytics) or rat igg in 30 pbs into mice brains . For mannitol infusion experiment, we injected a bolus of 15 ml / kg of 20% mannitol intravenously (calculated plasma osmolality: 320 mosm / kg) to the rats followed by cerebral injection of autologous blood or pk . We continuously infused the rats intravenously with 20% mannitol at a rate of 15 ml / kg / h for 2 h to maintain constant osmolality . We examined cerebral hematoma area or volume at 30 min, 2 h, or 48 h after injection . We perfused the animals with saline through the left ventricle, harvested the brains, and determined hematoma areas with quantity one software (biorad). We collected blood from the vena cava of the rats and anti - coagulated with acid - citrate - dextrose buffer (9:1, v / v). Platelet - rich plasma (prp) was obtained by centrifugation of the blood at 250 g for 15 min . We then centrifuged the prp at 500 g for 10 min to obtain platelet pellets, and washed the pallets 3 times with a modified tyrode s buffer without ca (134 mm nacl, 3 mm kcl, 0.3 mm nah2po4, 2 mm mgcl2, 5 mm hepes, 5 mm glucose, 12 mm nahco3, 1 mm egta, and 3.5 mg / ml bsa). We resuspended the final pellets in tyrode s buffer with 1 mm cacl2 but no egta and adjusted to a final platelet concentration at 2x10 cells / ml . We monitored platelet aggregation using a chrono - log 680 aggregation system (chrono - log) with stirring at 37c . We activated platelets with equine type i collagen (2 g / ml, chrono - log), thrombin (0.25 u / ml, sigma), or adp (20 m, sigma), and recorded the change of transmission . Briefly, the aggregation process was carried out in 100 l volume in a 96 well flat - bottom microplate . Platelets were activated with type i collagen (2 g), collagen related peptide (crp, 10 ng, university of cambridge, uk), or convulxin (0.25 nm), and the readings were taken every 30 seconds over a 20-minute period at 405 nm wavelength . We measured the interaction between pk and collagen with a biacore 3000 system (biacore ab) using he s buffer (10 mm hepes, 150 mm nacl, 2 mm edta, ph 7.4) at 25c . Rat tail type i collagen in 10 mm sodium acetate buffer (ph 5.0) was covalently coupled to a cm5 chip using an amine coupling kit (biacore ab) according to the manufacturer's instructions . A control surface underwent the same activation and deactivation procedures in the absence of collagen . Regeneration of the collagen surface was achieved by running 10 l of 10 mm glycine (ph 2.0) through the flow cell at 10 l / min . Pk solutions at several concentrations were perfused over the immobilized collagen at a flow rate of 10 l / min for 3 min, and the resonance changes were recorded . The sensorgram of the immobilized - collagen surface was subtracted from that of the control surface, and the data thus obtained were analyzed by biaevaluation software (biacore ab). We rinsed the aorta free of blood, trimmed surrounding connective tissue and the intercostal vessels, opened longitudinally, and gently scraped the inner surface with a sterile razor blade to remove the endothelium . We washed the vessel 3 times with ice cold pbs and divided it into approximately 4 mm pieces . Care was given to assure that the exposed aorta was covered by pbs during this procedure . We incubated aorta pieces with buffer containing 300 nm pk and glucose (050 mm) for 30 min at 25c . We then washed the segments, and measured the activity of surface - bound pk . Statistical analysis was performed by one - way analysis of variance (anova) followed by bonferroni s multiple comparisons test or students t - test as appropriate . Statistically significant differences between groups were defined as p <0.05 and are indicated in the legends of the figures . The methods for generation and characterization of klkb1 mice, systemic hemostasis measurement, hemoglobin analysis, western blotting, cardiac output measurement, plasminogen activation, continuous assays of clot formation and lysis, and pk activity assay are provided in the supplementary methods online.
The production of red blood cells follows the sequential formation, from erythroid progenitors, of proerythroblasts basophilic, polychromatophilic, and orthochromatic erythroblasts . Then the glycoprotein hormone erythropoietin (epo) positively regulates erythropoiesis by preventing apoptosis and stimulating differentiation and proliferation of erythroid progenitors and erythroblasts 2 . Epo protects cells from apoptosis by upregulating the expression of the antiapoptotic protein bcl - xl 34 . Conversely, epo deprivation results in caspase activation and apoptosis 5 . The transcription factor gata-1 is required for epo - mediated upregulation of bcl - xl 6 and expression of erythroid genes involved in erythroid differentiation 7 . In the absence of gata-1, the differentiation process is arrested at the basophilic erythroblast stage, and cells die by apoptosis . A negative feedback loop was recently described in which the ligand of fas (apo-1/cd95) expressed by mature erythroblasts activated fas - mediated cell death of immature erythroblasts 8 . Fas ligation results in the activation of caspase-8, then caspase-3, and the cleavage of gata-1, the inactivation of which leads to maturation arrest at the basophilic erythroblast stage of differentiation 9 . Altogether, these observations argued for a role of caspases in the negative regulation of erythropoiesis . Little is known about the molecular mechanisms of these changes, which include nuclear and chromatin condensation, loss of organelles, and enucleation 10 . Some of these morphological changes share similarities with features occurring during apoptosis as a consequence of caspase activation 11 . Therefore, in this report we have investigated whether caspases could play a role during erythroid differentiation . Erythroid cells were generated from cd34 progenitor cells in serum - free medium in the presence of epo (2 mu / ml) + il-3 (10 ng / ml) + stem cell factor (scf; 50 ng / ml) (epo) without or with tgf-1 (2 ng / ml) (epo + tgf-1) as previously described 12 . Throughout, the culture cell differentiation was assessed by morphological analysis of cells after cytocentrifugation and may - grnwald - giemsa coloration, and by analysis of glycophorin a and hemoglobin expression by flow cytometry and benzidine staining, respectively, as previously described 12 . Cell proliferation was assessed by counting cells every day after trypan blue dye exclusion staining 12 . Apoptosis was assessed by annexin v binding and propidium iodide (pi) staining as previously described 12 . Caspase activities were evaluated by devd - afc, ietd - afc, lehd - afc, vdvad - afc, and zeid - afc cleavage in fluorogenic assay (calbiochem) as previously described 13 . The cationic lipophilic fluorochrome 3,3-dihexyloxacarbocyanine (dioc6; sigma - aldrich) was used to measure the transmembrane potential () as described 14 . Disruption was assessed by cytometer analysis after cell labeling with dioc6(3) and pi . The variation in was also investigated by microscopic fluorescence and cytometric analysis after cell labeling with the fluorescent carbocyanine dye 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide (jc-1; alexis). To assess nuclear integrity, 5 10 cells / ml were first incubated in the presence of hoescht 33342 (sigma - aldrich) for 30 min (this first step was not performed for cytometric analysis) before being incubated with 10 g / ml jc-1 for 30 min at 37c in a humidified atmosphere of 5% co2 . Cells were then washed once with 1 pbs and resuspended in 250 l of 1 pbs . Loss of mitochondrial transmembrane potential results in green fluorescence and loss of the red fluorescence . Z - vad - fmk and devd - cmk (bachem) were resuspended in methanol and added at the indicated concentration . Anti - cd95 antibody (clone ch-11; euromedex - upstate biotechnology) was added in the culture system at the time and concentration indicated (see fig . 2 e). For detection of caspase-3 (bd pharmingen), poly(adp - ribose) polymerase (parp; boehringer mannheim), icad (inhibitor of caspase - activated dnase [cad]) (dff45; euromedex - upstate biotechnology), lamin b (provided by j. courvalin, centre national de la recherche scientifique, paris, france), acinus (provided by y. tsujimoto, osaka university medical school, suita, japan), hsp 90 (stressgen), and gata-1 (provided by i. dussanter, inserm u363, hopital cochin, paris, france), cells were lysed in laemmli buffer or in 1% sds, 10 mm tris, ph 7.4, 1 mm vanadate, 1 mm pmsf, 1 mm dithiothreitol, and 10 g / ml leupeptin, aprotinin, and pepstatin . Protein concentration was measured in the supernatant by the use of micro bca protein assay (pierce chemical co.). Erythroid cells were generated from cd34 progenitor cells in serum - free medium in the presence of epo (2 mu / ml) + il-3 (10 ng / ml) + stem cell factor (scf; 50 ng / ml) (epo) without or with tgf-1 (2 ng / ml) (epo + tgf-1) as previously described 12 . Throughout, the culture cell differentiation was assessed by morphological analysis of cells after cytocentrifugation and may - grnwald - giemsa coloration, and by analysis of glycophorin a and hemoglobin expression by flow cytometry and benzidine staining, respectively, as previously described 12 . Cell proliferation was assessed by counting cells every day after trypan blue dye exclusion staining 12 . Apoptosis was assessed by annexin v binding and propidium iodide (pi) staining as previously described 12 . Caspase activities were evaluated by devd - afc, ietd - afc, lehd - afc, vdvad - afc, and zeid - afc cleavage in fluorogenic assay (calbiochem) as previously described 13 . The cationic lipophilic fluorochrome 3,3-dihexyloxacarbocyanine (dioc6; sigma - aldrich) was used to measure the transmembrane potential () as described 14 . Disruption was assessed by cytometer analysis after cell labeling with dioc6(3) and pi . The variation in was also investigated by microscopic fluorescence and cytometric analysis after cell labeling with the fluorescent carbocyanine dye 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide (jc-1; alexis). To assess nuclear integrity, 5 10 cells / ml were first incubated in the presence of hoescht 33342 (sigma - aldrich) for 30 min (this first step was not performed for cytometric analysis) before being incubated with 10 g / ml jc-1 for 30 min at 37c in a humidified atmosphere of 5% co2 . Cells were then washed once with 1 pbs and resuspended in 250 l of 1 pbs . Loss of mitochondrial transmembrane potential results in green fluorescence and loss of the red fluorescence . Z - vad - fmk and devd - cmk (bachem) were resuspended in methanol and added at the indicated concentration . Anti - cd95 antibody (clone ch-11; euromedex - upstate biotechnology) was added in the culture system at the time and concentration indicated (see fig . 2 e). For detection of caspase-3 (bd pharmingen), poly(adp - ribose) polymerase (parp; boehringer mannheim), icad (inhibitor of caspase - activated dnase [cad]) (dff45; euromedex - upstate biotechnology), lamin b (provided by j. courvalin, centre national de la recherche scientifique, paris, france), acinus (provided by y. tsujimoto, osaka university medical school, suita, japan), hsp 90 (stressgen), and gata-1 (provided by i. dussanter, inserm u363, hopital cochin, paris, france), cells were lysed in laemmli buffer or in 1% sds, 10 mm tris, ph 7.4, 1 mm vanadate, 1 mm pmsf, 1 mm dithiothreitol, and 10 g / ml leupeptin, aprotinin, and pepstatin . Protein concentration was measured in the supernatant by the use of micro bca protein assay (pierce chemical co.). To determine whether caspases could play a role in terminal erythroid differentiation, we used a two - step amplification culture system in which normal human cd34 progenitor cells were amplified during 7 d in the presence of scf, il-3, and il-6 . Then, cd36 erythroid progenitors were isolated and cultured in the presence of scf, il-3, and epo . After 7 d of this second step of culture (day 7), all stages of erythroblast differentiation were observed (fig . 1 a), though the percentage of enucleated erythrocytes remained limited to 15% . To increase the percentage of mature erythroblasts and erythrocytes, tgf-1 (2.5 ng / ml) while inhibiting cell proliferation, tgf-1 accelerates and synchronizes erythroid differentiation with production at day 7 of 75 and 25% of mature polychromatic or orthochromatic erythroblasts and enucleated red cells, respectively 12 . Addition to culture medium from 50200 m z - vad - fmk, a permeant broad spectrum inhibitor of caspases 15, at the beginning (day 0) of cd36 cell culture, induced a dose - dependent decrease of terminal erythroid differentiation . At 150 m, z - vad - fmk completely inhibited erythroid differentiation at the basophilic erythroblast stage, before nucleus and chromatin condensation (fig . This inhibitory effect was observed both in the absence and in the presence of tgf-1 (fig . These observations suggested that caspase activation was required for erythroblast maturation after the basophilic stage of differentiation . To further explore this hypothesis, we added z - vad - fmk to cd36 cell culture medium at various times after culture initiation . When z - vad - fmk was added at day 5 of culture in the absence of tgf-1, terminal erythroid differentiation although reduced was not totally inhibited . Similarly, addition of z - vad - fmk after 3 d of culture in the presence of tgf-1, a time when most of the cells were at the end of basophilic stage of differentiation or already mature (polychromatophilic or orthochromatic erythroblasts), erythroid differentiation was no longer inhibited and enucleation occurred normally (data not shown). Addition of z - vad - fmk to the culture medium of cd36 cells also slightly reduced the number of hemoglobinized cells and their hemoglobin content, as determined by benzidine staining, in correlation with the blockade of maturation (fig . Hplc analysis of hemoglobin synthesis did not show any influence of z - vad - fmk on the pattern of synthesized hemoglobin chains (not shown). Altogether, these results suggested that caspase activation might be required for earlier phases of erythroblast differentiation, while later events such as enucleation could be caspase independent . Based on their structure and their ordering in cell death pathways, schematically, effector caspases, which include caspase-3, -6, and -7, cleave a variety of cellular substrates, while initiator caspases such as caspase-2, -8, and -9 control the activation of the former enzymes . To investigate which caspases were activated during normal erythroid differentiation, we studied the ability of cytosolic extracts from erythroid cells at various stages of differentiation to cleave fluorogenic peptide substrates 13 . Cytosolic extracts of cells cultured in the presence of epo cleaved the peptide substrate devd - afc, suggesting activation of caspase-3 and/or caspase-7 . This cleavage activity increased during the first 5 d of culture, reaching 75% of the activity measured in epo - starved cells, then decreased the last 2 d of culture (fig . 2 a). When the cells were cultured in the presence of epo + tgf-1, this devdase activity, although following the same kinetic profile with the same magnitude, occurred earlier, with a maximum at day 2 . As expected, these activities were totally inhibited by addition of z - vad - fmk (fig ., the kinetics of devdase activity were in accordance with the lack of effect of z - vad - fmk on the later stages of erythroid maturation . To determine whether devdase activity measured in cells undergoing erythroid maturation was related to caspase-3 activation, we performed immunoblot analyses . Procaspase-3 was observed to be cleaved to the active enzyme (p17 fragment; fig . Notably, while this band was clearly individualized in the positive control from epo - deprived erythroblasts, intermediate p20 and p19 bands were observed in erythroblasts cultured in the presence of epo, suggesting that procaspase-3 proteolysis was less efficient during normal erythroid maturation than during starvation - induced apoptosis . Cleavage of the 116-kd parp nuclear enzyme into a 85-kd fragment during erythroblast maturation further confirmed the activation of one or several effector caspases during the differentiation process 1318 (fig . 2 c). To emphasize the role of caspase-3, we cultured cells with 150 m of devd - cmk, an inhibitor that is more specific for caspase-3 and related caspases than z - vad - fmk . As expected, devd - cmk inhibited maturation with the same magnitude as z - vad - fmk (fig . One involves death receptors such as fas and results in the activation of the initiator caspase-8 that, in turn, activates effector caspases . This pathway is used for the negative regulation of erythropoiesis by fas ligand (fas - l) and leads to gata-1 cleavage 9 . The other pathway involves the disruption of the, resulting in opening of the permeability transition pore and the mitochondrial release of several procaspases, including procaspase-9 and -2 20 as well as apoptogenic factors 21 . These phenomena result in activation of the caspase cascade through the initiator enzyme caspase-9 . To determine whether one of these pathways was involved in caspase activation during normal erythropoiesis, we studied the ability of cell extracts to cleave in vitro the fluorogenic peptides ietd - afc, vdvad - afc, and lehd - afc that mimic caspase-8, -2, and -9 targets, respectively . While a time - dependent cleavage of lehd - afc and vdvad - afc was clearly identified (fig . 2 e), suggesting caspase-9 and -2 activation, no cleavage of ietd - afc was detected, suggesting that neither caspase-8 nor a related caspase was involved in normal erythroid differentiation (fig . Involvement of the mitochondria in the pathway leading to caspase activation during erythroid differentiation was further confirmed by using the potential sensitive dyes dioc6(3) and jc-1 that allow study of the mitochondrial transmembrane potential 14 . While the cells cultured for 2 d in the presence of epo incorporated dioc6(3), as expected from viable cells, this incorporation decreased significantly at day 4, demonstrating a disruption of the during normal erythroid differentiation (fig . At day 4, the jc-1 ratio orange - red over green fluorescence was significantly reduced (fig . Nucleus counterstaining by hoescht showed clearly that mitochondrial depolarization occurred in non apoptotic cells (fig . Reduction of the mitochondrial transmembrane potential and activation of the caspase cascade usually leads to externalization of phosphatidyl serine on the plasma membrane, dna degradation, and apoptotic cell death 21 . In our system of culture and in accordance with previous observations 22, <510% of erythroid cells exhibited features of apoptosis as assessed by annexin v binding / pi staining (fig . 4 a), and internucleosomal dna fragmentation could not be detected (data not shown). These two findings excluded the possibility that activation of caspases may favor erythroid maturation by inducing apoptosis in a subset of erythroid cells and strongly suggested that inhibition of red cell formation by z - vad - fmk was not due to a selection of immature nonapoptotic cells . In addition, the disappearance of the transcriptional factor gata-1 observed by immunoblotting in epo - deprived erythroid cells undergoing caspase - dependent apoptosis was not identified during the normal maturation of erythroblasts (fig . 4 b). Lamin b, which is highly expressed in differentiating erythroblasts 2324, forms a fibrous meshwork on the nucleoplasmic surface of the nuclear membrane . In cells undergoing apoptosis, lamin b was shown to be cleaved by caspase-6 25, an effector caspase that is activated by caspase-3 in the proteolytic cascade 26 . By using a peptide substrate (zeid - afc) that mimics the caspase-6 target site, we observed that caspase-6 is activated during erythroid maturation (fig . 5 a). Accordingly, immunoblot analyzes showed that lamin b was cleaved during the differentiation process (fig . Inhibition of caspase activation by z - vad - fmk, which blocked nuclear condensation, also inhibited lamin b cleavage, further supporting a correlation between caspase activation and morphological changes observed during erythroid maturation . Recently, the nuclear protein acinus was shown to play a role in chromatin condensation associated with apoptosis when cleaved by caspase-3 27 . This protein is devoid of endonuclease activity and so differs from dff40/cad that possesses both activities (chromatin condensation and endonuclease activities) 28 . Both acinus and dff45/icad were expressed in our cultured cells and were cleaved in epo - deprived cells undergoing apoptosis (fig ., we observed that the expression of dff45/icad remained unchanged during normal erythroid differentiation, while the native 96-kd acinus protein had almost disappeared at day 7 of cd36 cell culture in the presence of epo . The disappearance of the native acinus was related to the appearance of its active 23-kd fragment and that was prevented by z - vad - fmk (fig . In contrast, no cleavage of dff45 could be detected during erythroid differentiation (fig . Taken together, our data demonstrate that normal erythroid differentiation requires the transient activation of several caspases . These enzymes have been involved in several physiological processes, including cell death, inflammation, and t cell proliferation 29 . In erythroid cells, we show here that caspases are activated before the process of enucleation and cleave several proteins otherwise implicated in the apoptotic process such as acinus and lamin b. cleavage of these proteins may account for the nuclear structural changes associated with the maturation of erythroblasts . These changes could also influence gene expression during the last steps of the differentiation and prepare the process of enucleation . How active caspases selectively cleave some target proteins such as acinus and lamin b without cleaving other targets such as gata-1 or dff45/icad remains currently unexplained . Caspases are only transiently activated during erythroid differentiation process, suggesting that regulatory mechanisms prevent further amplification of the proteolytic process that would lead to the cell demise . Upon epo deprivation or fas activation, the transient caspase activity is amplified via either the mitochondrial / caspase-9 or the caspase-8 pathway, respectively, and lead to apoptosis . Therefore, in this report we have described a new function for caspases as enzymes critical for erythroid differentiation . Further exploration of the role of caspases in erythroid cells and other hematopoietic lineages would be of major interest for understanding the fate (differentiation versus apoptosis) of hematopoietic cells upon different physiological or pathological conditions.
Spinal cord stimulation (scs) has been used since the 1960s to treat chronic refractory pain conditions . In particular, scs has been extensively used to manage the painful symptoms related to chronic lumbosacral radiculopathy following failed back surgery syndrome and complex regional pain syndromes . Randomized controlled trials of scs for failed back surgery syndrome have reported favorable long - term results.1,2 concurrently, there has been significant technologic advancement in the equipment for scs . Specifically, implanting physicians can offer patients rechargeable implantable pulse generators, improved anchoring systems, and more stimulating lead contacts to ensure coverage for paresthesia in painful somatic regions . Thus, there is more widespread use of scs therapy and acceptance among treating physicians . Despite the positive findings and increasing experience of implanting physicians using scs therapy, complications remain a common occurrence . An analysis of the available literature by turner et al found that just over one third of patients have a complication.3 prospective data from a multicenter evaluation of scs for failed back surgery syndrome showed a similar rate of complications, even in the hands of experienced implanters.4 these adverse events are usually lead migration, infection, lead breakage, and unwanted stimulation.4,5 an allergic or immunologic/ inflammatory reaction to the system components is thought to be rare, and has been estimated by cameron to be 0.1%.5 however, the true incidence may be underestimated because clinicians may fail to include it in the differential diagnosis, attributing an inflammatory reaction of the soft tissue surrounding scs components to infection . In addition, only two articles detail the cutaneous reaction to the components of scs systems.6,7 the purpose of this paper is to detail the presentation, clinical course, and histologic findings in three patients with cutaneous reactions to spinal cord stimulator equipment components . The patient was a 61-year - old male who, despite two previous laminectomies, epidural injections, and multimodal analgesia (oxycodone / acetaminophen, cyclobenzaprine, ibuprofen), complained of severe pain secondary to left - sided chronic lumbosacral radiculopathy . The pain was 9/10 in intensity, located at his lower back on the left side with radiation down his posterior thigh, behind his knee to his lateral leg, and to the bottom of the sole of his foot . He had a past medical history of irritable bowel syndrome, squamous cell carcinoma of the skin, gastroesophageal reflux disease, and depression . Magnetic resonance imaging revealed no evidence of spinal stenosis, foraminal narrowing, or nerve root displacement, so he was implanted with an scs consisting of two eight - contact leads, two lead extensions, and a rechargeable implantable pulse generator (eon, st jude medical neuromodulation division, plano, tx, usa) following a successful trial of 7 days . He was seen one week after the operation and his wounds, which included the abdomen for the generator, flank for the extension, and back for lead insertion and anchoring, were noted to be dry with intact incisions, and no erythema, drainage, or tenderness . At one month post operation, the patient developed a left - sided rash on his flank and abdomen, with no fevers or chills (figure 1). However, he had an area of erythema / dermatitis that seemed to be localized to the area over the length of the lead extensions connecting the implantable pulse generator to the leads . It was noted that this rash appeared to be an area of reactive inflammation, as opposed to infection, and he was diagnosed with a hypersensitivity reaction to the implanted wires . He was given topical 1% hydrocortisone cream and seen 7 days later, with resolution of the rash . Forty - three days after implantation, the patient presented with complaints concerning wound drainage . The abdominal and flank incisions had dehisced, with device exposure and were draining pink, clear fluid . His white blood cell count was 6.8 and the differential was abnormal for eosinophils at 5.4%, with no bandemia or elevation in fraction of neutrophils . Because of concerns for potential early infection, urgent explantation of the entire scs device was performed . Exploration of the wounds did not reveal any purulent material, but the subcutaneous tissue directly involving the wound was frail, bled easily with manipulation, and was difficult to coagulate . Wound cultures were positive for rare coagulase negative staphylococci as well as rare staphylococcus aureus, and the patient was treated for 2 weeks with intravenous vancomycin . Clinically, however, he did not manifest with the usual clinical findings suggestive of device infection . One month later, all incisions were well healed . Because the initial reaction to the scs equipment involved the flank to the abdomen but not the back, the buttock was targeted as a potential site for the implantable pulse generator . In an attempt to rule out a hypersensitivity reaction, an in vivo allergy test using a portion of the extension lead was done (figure 3). A 4 cm lead extension piece was placed in the left buttock via a 12 gauge angiocatheter upon removal of the introducer . The extension fragment was placed into the catheter which was removed leaving the lead in the subcutaneous tissue . The puncture site was closed with 4.0 monocryl suture (ethicon inc, somerville, nj, usa). After a 2-month follow - up period one month later, the system was reimplanted, with the pulse generator pocket site now in the left buttock . Now three years following reimplantation, his pain is well managed with a combination of scs and medications . The patient was a 44-year - old gentleman with a past surgical history of decompression laminotomy, discectomy, and fusion at l5/s1 . One year following the surgery, the patient continued to have worsening low back pain, with radiation down his lateral calf into the lateral aspect of his dorsal foot . The patient described the pain as being 8/10 in severity, with an achy quality that was partially alleviated with oxycodone/ acetaminophen . He had a past history of depression and colitis, and had no known drug allergies . Magnetic resonance imaging had revealed prior posterior decompression of posterior fusion at the l5/s1 level with metallic hardware artifacts with grade 1 anterolisthesis with posterior uncovering of the disc and superimposed left paracentral protrusion . There was also enhancing tissue encasing the thecal sac and the s1 nerve roots at the l5s1 level . The patient had no sustained improvement with transforaminal epidural injections . Following a one - week trial of scs, he reported excellent relief and was subsequently implanted with dual eight - contact leads and the implantable eon pulse generator was placed in the left buttock region . After two weeks, the patient complained of increased pain from the mid back incision . On physical examination the patient was treated for a presumptive superficial wound infection with antibiotics for ten days with resolution of the pain and white count . However, despite resolution of the infection, the patient still had a skin inflammatory change that persisted for 5 weeks following implantation (figure 4). Because of poor wound healing, the decision was made to remove the dual leads, silastic anchors with associated 0 silk sutures, and the implantable pulse generator . Intraoperative frozen sections revealed acute and chronic inflammation, with a foreign body giant cell reaction (figure 5a and b). The removal of the soft inflammatory tissue in figure 4 revealed the underlying leads, and the pocket floor of the implantable pulse generator that had excess lead coils was notable for soft tissue inflammation . Interestingly, there was no fibrotic tissue deposition surrounding the leads, anchors, or the floor of the implantable pulse generator pocket . The patient was a 40-year - old right - handed female, who presented to the pain center with a diagnosis of complex regional pain syndrome affecting the right upper extremity . This condition was believed to be the result of two right shoulder operations for supraspinatus tendon tears . The pain was burning diffusely across her right shoulder, extending over her scapula and down her arm into her hand, with an intensity of 7/10 . She was allergic to nickel, oxycodone / acetaminophen, metronidazole, scopolamine, and tramadol . After multiple interventions including stellate and interscalene blocks, transcutaneous electrical nerve stimulation, physical therapy, intravenous lidocaine, and multimodal pharmacotherapy failed to bring appreciable relief, she underwent a one - week trial of scs . After reporting a greater than 50% pain intensity reduction, she had a fully implanted dual 8 contact system placed via an upper back incision at the t2/3 level, with the implantable eon pulse generator placed in the right buttock region . A small pocket for lead extensions was made on the right flank just below the bra line at the t9 level . She had reported some clear drainage from the upper back wound, but there was none noted on examination . Five weeks later she called to say that she was developing a partially opened wound at the lead insertion site as well as the lead extension site, with copious amounts of clear drainage . Examination of the wound revealed minimal erythema with no hardware exposure but it had grossly evident soft tissue inflammation . Her white blood cell count was 8.7 but she had an erythrocyte sedimentation rate of 53 and a c - reactive protein of 11.8 . Because of persistent wound inflammation and intermittent clear drainage, the leads, silastic anchors (associated 0 ethibond sutures, ethicon inc), and lead extensions were removed 7 weeks post implantation . The clinical plan was to reinsert the leads at a lower level if it was proven to be less immunoreactive using the methods described in case 1 . Figure 6a and b displays the histology of the specimen results sent from the upper back wound . The wounds healed rapidly with removal of the leads and anchors . One month after removal of the leads, the patient called to report a minor amount of bleeding from the buttock wound where the implantable pulse generator had been implanted . She was afebrile and her white blood cell count was 8.1, erythrocyte sedimentation rate was 45, and c - reactive protein was 13.8 . An inflammatory reaction of the pocket floor was noted with very friable tissue (figure 7). The fibrous floor of the pocket that is usually well formed at 3 months post - implantation was disrupted by inflammation ., all her wounds were healing well and the drainage had ceased from the buttock . Allergic or other inflammatory reactions to the components of spinal cord stimulation systems have only rarely been reported.68 a search of the literature revealed only one recent detailed case series to our knowledge, ie, a report from france describing two cases of cutaneous eruption related to spinal cord stimulators.6 the first of these cases was a foreign body type reaction to the silicone component of the neurostimulatory electrodes, with histology of the affected tissue showing a foreign body granuloma formation . A similar foreign body granuloma reaction occurred after device removal at the surgical scar, in response to silicone particles . The second case was that of a contact dermatitis reaction thought to be due to the silicone part of the stimulator, with histology of affected tissue showing a contact dermatitis pattern . Delayed hypersensitivity patch testing confirmed a specific sensitivity to silicone . While reports of inflammatory reactions to spinal cord stimulator devices are exceedingly rare, reactions to other devices, such as pacemakers and cardiac defibrillators, have been reported less rarely.9 the apparent higher rate of inflammatory reaction to cardiac rhythm management devices compared with scs may in part relate to the fact that the former has a higher implantation rate . Two types of delayed inflammatory responses might occur in response to implanted devices, ie, delayed hypersensitivity responses to a specific antigen (eg, the metal polyurethane or silicone rubber of a device) or foreign body giant cell granuloma reactions to device material(s). Delayed hypersensitiv - ity responses to an allergen are mediated by t cells and monocytes / macrophages, rather than antibodies . Contact dermatitis is a form of delayed hypersensitivity reaction to antigen at the surface of the skin, but a similar process can occur to substances inoculated intradermally or subdermally . The patient in case 1 developed such a reaction likely to the polyurethane lead extensions because the rash was along the entire length of their course . This rash was treated successfully with topical hydrocortisone, but the patient presented within 2 weeks with wound dehiscence predisposing to early infection as suggested by the cultures and inflammatory histology . We suspect that the proximity of hydrocortisone treatment to the healing wounds coupled with an inflammatory reaction likely reduced the tensile strength, leading to dehiscence . While contact dermatitis presented approximately one month after implantation, it may present as soon as 8 days, as recently reported after implantation of a peripheral nerve stimulator.10 for contact dermatitis (delayed hypersensitivity reactions to items on the surface of the skin), patch testing is a diagnostic technique that can be utilized . This involves placing a suspected substance on the surface of the skin for 48 hours, then looking for an inflammatory response of the skin at 4896 hours . While this technique can be helpful in some cases, even with contact dermatitis, there can be false negatives and false positives . There is little known about whether patch testing can predict responses to a device that is implanted subcutaneously . The immune system response that occurs with absorption of an allergen from the surface of the skin through the epidermis can be different from that which occurs when the same substance is implanted . The literature provides little insight on the reliability of patch testing to predict an inflammatory response to implanted devices . Because of the authors prior experience of false negatives on skin patch testing and the aforementioned complications of case 1, it was thought necessary to try an in vivo test of a piece of lead extension in another site for placement of the implantable pulse generator (figure 3). Interestingly, this 2-month test of implantation of the lead extension fragment in the buttock region did not result in any reaction, and the device was successfully implanted . We are unable to explain why an inflammatory dermatitis occurred in the flank / abdominal region but was absent in the back and buttock area . In addition to contact dermatitis, a delayed hypersensitivity reaction may manifest as a granuloma, as reported in the french case series . With delayed hypersensitivity reactions, an antigen is taken up by macrophages or monocytes and is then presented to t cells which can specifically recognize that antigen (ie, there is memory from previous exposure to the antigen). This leads to recruitment of further inflammatory cells to the area, including macrophages that can in turn form giant cells . Overall, this inflammatory pattern involving t cells and macrophages is referred to as a granuloma . A foreign body reaction can invoke a similar inflammatory pattern, but it is not in response to a particular immunologically recognized antigen . The inflammatory reaction in response to a foreign body starts as the body tries to respond to the foreign substance by attempting to clean the substance out from the body . Macrophages will remain at the foreign body site for an extended period of time, and recruit other inflammatory cells to the area via secretion of chemokines . Granulation tissue can form at the site of the foreign body reaction (similar to granulation tissue seen with wound healing, but in a foreign body reaction, the healing process is unable to complete itself). Therefore, the foreign body reaction consists of persistent inflammation, characterized by foreign body giant cells and granulomas seen on histology . Foreign body giant cells form when macrophages encounter a large foreign body (such as the components of an scs implant). Because the macrophage cells cannot phagocytose the foreign body, the cells fuse together to form a giant cell composed of many fused macrophages . In this case series, the histology of the affected tissue from cases 2 and 3 was that of a foreign body giant cell reaction (figure 5a and b, and figure 6a and b). In case 2, the clinical impression is that the polyurethane leads are the likely allergens, given that the inflammatory response was seen in the implantable pulse generator pocket floor where the excess lead coils are placed . The polyurethane - coated leads and extensions are even more likely to be the offending stimulus, given that the reaction was in all three wounds, including lead insertion, lead extension, and implantable pulse generator . Component materials, which include silicone rubber (eg, anchors), polyurethane (leads, extensions), titanium (implantable pulse generator), and platinum / iridium (electrodes), are part of an allergy test kit (st jude medical neuromodulation) and can be applied topically as a skin patch test.11 among the current three vendors, materials used for the components of scs devices are similar on account of regulation by the us food and drug administration . Testing was pursued because, given the location of the inflammatory reaction, it was not felt that allergy testing would alter future clinical decision - making in cases 2 and 3 . All patients who receive an implanted device develop some degree of foreign body reaction around the device . Why some patients but not others develop a substantial, pathologic, and clinically detrimental level of reaction is not fully known.12,13 interestingly, none of the three patients in this case series had a history of prior allergies to metal, rubber, or autonomic dysfunction to suggest predisposition to cutaneous reactions . In summary, delayed inflammatory responses to the components of scs devices can manifest via t cell/ monocyte - mediated delayed hypersensitivity reactions or foreign body giant cell reactions . Contact dermatitis, granuloma formation, and foreign body reactions with giant cell formation are possible in response to scs devices . The role of skin patch testing remains uncertain when testing for foreign body reactions because these occurred one month after implantation . While the lead / extension polyurethane component is suspected as the most immunogenic source in this case series, other materials of the scs device cannot be excluded . Excision of the inflamed tissue and histologic evaluation is the key for diagnosis and distinguishing between infection and inflammation . Infection may occur as a complication of poor wound healing because of an underlying inflammatory response to the component(s) of the scs device.
It is estimated that in the year 2005, 63,210 new cases of bc will be diagnosed in the united states alone . Approximately one fifth of these patients will undergo radical cystectomy (rc) and urinary diversion (ud). Rc has become an established standard of care for patients with muscle invasive bc in the united states . Despite our better understanding of pelvic anatomy and improved surgical techniques, therefore, a need persists for technological advances that would minimize intraoperative blood loss and decrease perioperative complications in patients undergoing rc . The most notable of recent advances in surgical techniques is the increased application of minimally invasive laparoscopic surgery in the management of urological disorders . The established advantages of laparoscopic surgery include decreased pain, shorter hospital stay, and decreased intraoperative blood loss compared with these things in conventional open surgery . There is still room for improvement, however, as the current laparoscopic technology is limited due to the lack of 3-dimensional visualization and poor ergonomics . Recently, the da vinci surgical robotic system (dsrs) has been added to the armamentarium of minimally invasive surgery . The dsrs adds the much needed 3-dimensional vision, 6 degrees of freedom of movement, and improved ergonomics to laparoscopic techniques . The dsrs is being increasingly used to perform complex urological procedures including radical prostatectomy, radical cystectomy, and urinary diversions . Menon et al first reported the use of the dsrs to perform rc both in men and women with decreased blood loss and operative time less than 4 hours . However, most of the published studies are limited to a small cohort of patients without comparison with the conventional open method (om). To establish the efficacy of robotic radical cystectomy, we compared the early perioperative outcomes of patients undergoing rc by either om or the robotic method (rm). This study includes a cohort of 37 consecutive patients undergoing rc by om or rm over the same time period by a single surgeon, thereby minimizing the influence of selection bias and multiple surgeons on perioperative outcome . All relevant clinical information on patients undergoing robotic urological procedures at our institution is maintained in a prospectively established microsoft access database . An institutional review board (irb) exempt status was obtained because all patient identifiers are deleted after pertinent clinical information is obtained . This study consists of 37 consecutive patients undergoing open and robotic rc since the approval of dsrs for human use in the year 2000 . The procedures were carried out at 2 teaching institutions, a university hospital and veterans administration medical center, by a single surgeon . Because the dsrs was available only at the university hospital, the option to undergo rc by rm was offered to patients at the university site with the following exclusion criteria: morbid obesity (generally body mass index>35), prior pelvic radiation, or significant medical comorbidities including pulmonary obstructive airway disease . Variables analyzed included age, sex, body mass index (bmi), operative time, postoperative analgesic use, estimated blood loss (ebl), blood transfusion rate, hospital stay, final pathology results, and early perioperative complications before discharge from the hospital . The patients, while under general anesthesia, were placed in the lithotomy and steep head down position with a nasogastric tube and urethral catheter in place . The procedure was carried out intraperitoneally following insufflation of the abdomen with co2 up to 15 mm hg pressure obtained using a veress needle . Five ports were used including a 12-mm left paraumbilical camera port, two 8-mm robot working ports placed approximately 8 cm from the camera port on either side forming a 15 to 30 angle inferiorly and 2 additional 5-mm to 12-mm working ports (one step port, u.s . Operative steps in male patients, in order, included dissection of the vas deferens on both sides leading posterior to seminal vesicles, dissection of the prerectal space posterior to the prostate, bilateral ureteral mobilization from close to the lower pole of the ipsilateral kidney to the ureterovesical junction (uvj) before clipping, division close to the uvj and tagging using 12-inch long 2 0 absorbable sutures, incision of endopelvic fascia on both sides, securing the lateral pedicles to the bladder using endovascular staplers, division of the medial umbilical ligaments to enter the retropubic space, division of deep dorsal venous complex, completely freeing up the specimen before trapping in an endocatch ii bag (u.s . Surgical, norwalk, ct) and removal of the intact specimen through an approximately 5-cm midline periumbilical or suprapubic incision . The rent in the anterior vaginal wall was closed primarily with dsrs using 2 0 vicryl sutures supported by greater omental mobilization done extracorporeally at the end of the procedure . A bilateral limited pelvic lymph node dissection was carried out intracorporeally, which included obturator and external iliac groups of lymph node . The wilcoxon rank sum test was used to compare distributions of continuous variables between the om and the rm . The patients, while under general anesthesia, were placed in the lithotomy and steep head down position with a nasogastric tube and urethral catheter in place . The procedure was carried out intraperitoneally following insufflation of the abdomen with co2 up to 15 mm hg pressure obtained using a veress needle . Five ports were used including a 12-mm left paraumbilical camera port, two 8-mm robot working ports placed approximately 8 cm from the camera port on either side forming a 15 to 30 angle inferiorly and 2 additional 5-mm to 12-mm working ports (one step port, u.s . Operative steps in male patients, in order, included dissection of the vas deferens on both sides leading posterior to seminal vesicles, dissection of the prerectal space posterior to the prostate, bilateral ureteral mobilization from close to the lower pole of the ipsilateral kidney to the ureterovesical junction (uvj) before clipping, division close to the uvj and tagging using 12-inch long 2 0 absorbable sutures, incision of endopelvic fascia on both sides, securing the lateral pedicles to the bladder using endovascular staplers, division of the medial umbilical ligaments to enter the retropubic space, division of deep dorsal venous complex, completely freeing up the specimen before trapping in an endocatch ii bag (u.s . Surgical, norwalk, ct) and removal of the intact specimen through an approximately 5-cm midline periumbilical or suprapubic incision . The rent in the anterior vaginal wall was closed primarily with dsrs using 2 0 vicryl sutures supported by greater omental mobilization done extracorporeally at the end of the procedure . A bilateral limited pelvic lymph node dissection was carried out intracorporeally, which included obturator and external iliac groups of lymph node . The wilcoxon rank sum test was used to compare distributions of continuous variables between the om and the rm . During our study period, 37 patients underwent rc, 24 (64.9%) by om and 13 (29.7%) by rm ., 6 patients underwent ileal conduit, 5 underwent ileal neobladders, and 2 underwent indiana pouch urinary diversions . In the open group, 16 patients underwent ileal conduits, 7 underwent ileal neobladders, and 1 underwent an indiana pouch urinary diversion . The bmi, age, sex, blood transfusion rate, and drop in hemoglobin were comparable between the 2 groups (table 1). Median ebl and length of hospital stay for rm were significantly lower compared with that for om (p=0.0002 and p=0.044, respectively). Operating time for rc and urinary diversion (ud) was significantly longer in the rm group (p=0.0002). There were 3 (12.5%) positive margins in the om group and none in the rm group (p=0.54). Four (16.7%) perioperative complications occurred in the om group; and 2 (15.4%) occurred in the rm group, p=1.0 (table 2). One perioperative death caused by central venous line sepsis occurred in the om group . The incidence of organ - confined (t2n0mx) and nonorgan - confined disease (t3) was 9 (37.5%) and 15 (62.5%) in the om group compared with 7 (53.8%) and 6 (46.2%) in the rm group (p=0.49). Comparison of characteristics of patients undergoing radical cystectomy by open or robotic methods early perioperative morbidities following radical cystectomy by open or robotic methods because rc is usually performed in older patients with malignancy and associated nutritional deficiencies, rc is often associated with high postoperative complication rates . In spite of several modifications to the open surgical techniques, chang et al reported median blood loss of 600 ml in a series of over 300 patients undergoing rc . The increased use of minimally invasive surgical techniques to perform major urological operations, such as radical prostatectomy and nephrectomy, over the last decade has resulted in significantly decreased intraoperative blood loss compared with their respective open methods . Several published reports establish the feasibility of safely performing robotic radical cystectomy (table 3). Comparison of reports of early perioperative outcomes following robotic radical cystectomy plnd = pelvic lymph node dissection; eplnd = extended pelvic lymph node dissection; ic = ileal conduit; onb = orthotopic neobladder; rc(p) = radical cystectomy and cystoprostatectomy; ud = urinary diversion . Our current study demonstrates a similar and significant decrease in ebl in the rm group compared with that in the om group (p=0.0002) with a consequent decrease in blood transfusion rates . In this study, 18/24 (75%) patients in the om group received blood transfusions compared with 7/13 (53.8%) in the rm group despite the fact that only 4 patients in the rm group had blood loss over 500 ml . Blood transfusion rates in this study are clearly much higher than rates of about 30% in other published studies . At our institution, blood transfusions are carried out without the benefit of well - established critical care pathways . In addition, because of the lack of prior experience in accurately estimating blood loss during robotic rc, patients received blood transfusions more readily, which accounts for the high blood transfusion rates in this series of patients . For purposes of calculation, because several patients had received intraoperative or immediate postoperative blood transfusions, the lack of significance of postoperative hemoglobin change may not accurately reflect intraoperative blood loss . The operative time for performing rc and urinary diversion was significantly longer in the rm group then in the om group . Although several of the initial cases of rc by rm lasted for more then 4 hours, rc including pelvic lymph node dissection in the last 3 patients was completed consistently in less than 4 hours . The improving operative time corroborates the published conclusions in the literature that robotic surgery can be performed efficiently with operative time comparable to that of om with increasing experience . In this study, we do not report the operative time for rc and urinary diversions separately because of lack of such data for the om cases . In spite of the minimally invasive nature of rm, the difference in hospital stay in the rm group was only marginally better although statistically significant, median of 8 days (range, 4 to 23) compared with 10 days (range, 6 to 35) in the om group (p=0.044). Following rc by rm, the specimen was removed through a small midline abdominal incision, and ud was performed extracorporeally in all patients . The patients hospital stay was directly related to the time required for return of the bowel function . Therefore, irrespective of the method of rc the return of bowel function was comparable between the 2 groups resulting in a minimal difference in hospital stays . Although we have previously reported on totally intracorporeal robot - assisted laparoscopic ileal conduit urinary diversions, we resisted our temptation to perform urinary diversions totally intracorporeally to limit operative time . The sample size is relatively small and the follow - up data are currently not mature enough to evaluate oncological outcomes . All 3 cases of positive margins occurred with om, 1 in a patient with locally advanced prostatic adenocarcinoma and the other 2 in patients with locally advanced pt3b and pt4a transitional cell carcinoma of the bladder . Although not statistically significant, almost two thirds of patients undergoing rc by om had nonorgan - confined disease compared with about half in the rm group (p=0.49), which could possibly account for the increased positive margin rate in those undergoing om . Alternatively, the dsrs provides excellent visualization by 10x magnification that facilitates meticulous dissection around the tumor, which could have contributed to decreased positive margin rates . Although, a large randomized cohort of patients comparing rc by om or rm is necessary to resolve the issue, it nevertheless raises an interesting hypothesis that rc by rm may decrease positive surgical margins . Overall perioperative complication rates were comparable between the 2 groups; 4(16.7%) and 2(15.4%) in om and rm groups, respectively (p=1.0). One perioperative death in the om group resulted from central venous line sepsis . Several studies evaluating outcomes of patients undergoing radical surgery for prostate and bladder cancer by open methods have demonstrated that the performing surgeon is an independent predictor of outcome . By comparing the perioperative outcomes using a contemporary cohort of patients undergoing rc by conventional om and rm performed by a single surgeon using our prospectively established lrusp institutional database, we minimized the influence of multiple surgeons performing the procedure at different institutions at different time periods on perioperative outcome . Therefore, our data suggest that the same surgeon familiar with the surgical techniques can perform rc by either rm or om with similar efficacy . In this study, we have not compared postoperative analgesic use between the 2 methods because several patients in the om group received epidural analgesia compared with none in the rm group . Although we focused on early perioperative complications, longer follow - up is necessary to evaluate long - term complications . Although the demographics of both groups were comparable, morbidly obese patients were not offered rm as a treatment option, which may have biased the results of this study . Furthermore, the operative time discussed in this study includes the time taken for performing urinary diversions, which limits our ability to comment specifically on the operative time for performing rc alone . Nevertheless, our study establishes the feasibility of performing rc by rm with efficacy comparable to that of conventional om . A major impetus to explore the feasibility and efficacy of robotic rc is persistently high complication rates, such as increased intraoperative blood loss in patients undergoing rc by conventional om, even at centers where a high volume of procedures are performed . Despite being a technically demanding operation, early reports of rc by rm, including the current study have demonstrated excellent perioperative outcomes including significantly decreased blood loss . Moreover, robotic pelvic surgery including radical prostatectomy is being increasingly performed, and it may only be a matter of time before rm is more widely used to perform rc . Therefore, pioneering work such as this study can contribute towards establishing the safety, efficacy, and technical standards for performing rc by rm . Radical cystectomy (rc) with urinary diversion (ud) is relatively commonly performed and arguably one of the most complex of urological operations . Rm is technically demanding, but clearly this procedure can be done with early perioperative results comparable to results of conventional open methods . Currently, the operative time is longer with rm; however, it is associated with decreased ebl and hospital stay . Longer follow - up and a larger cohort of patients are required to establish oncological outcomes.
Ischemic stroke (is) and myocardial infarction (mi) are atherosclerosis - associated complications that are the leading causes of mortality and disability all over the world . Is and mi share common features, but at the same time there are differences in many risk factors, at least quantitative, if not qualitative . Compared with myocardial infarction, stroke patients are at least 10 years older, the incidence in middle - aged men compared with women is smaller, and increasing blood pressure is more strongly associated with stroke, whereas increasing plasma cholesterol is less strongly associated with stroke . Recent studies showed that aberrant immune response evolves during both is and mi that is deleterious and contributes to cell death after the insults [3, 4, 5]. Both pathologies are characterized by expression of proinflammatory cytokines, adhesion molecules, and importantly, chemokines that orchestrate the infiltration of leukocytes into infarct area, expression of damaging agents and activation of complement system (for review see [6, 7]). Chemokines are low molecular weight polypeptides that, besides other functions, exert potent chemotactic and activating effects on specific leukocyte populations . Recently, there has been growing interest in applications of chemokine biology into clinic, including atherosclerosis . Cc chemokine ligand (ccl)2, more widely known as monocyte chemoattractant protein-1 (mcp-1), is a potent mononuclear cell attractant . It has been previously implicated in the development of both is and mi . For example, in animal experiments, high levels of mcp-1 mrna have been found in the brain 6 hours after cerebral ischemia . The maximal expression of this chemokine was observed between 12 hours and 2 days [11, 12]. Mcp-1 mrna expression was upregulated in infarcted area after mi in the same manner like in is [13, 14]. Most of the data about cytokine and chemokine involvement in pathogenesis of is and mi has been derived from animal studies . Nowadays, more attention is being paid to studies utilising human subjects . In this context, blood becomes an invaluable target for such studies, due to its relative availability . Recently, it has been shown that the levels of several cytokines are elevated in the blood of patients with is and mi [15, 16, 17, 18] we, therefore, performed the present study to provide additional data on involvement of mcp-1 in mi, and namely in is . Doing so, we wished to complement existing studies targeted at evaluation of chemokine inflammatory response in pathogenesis of these two atherosclerosis - associated complications . Forty patients with is (17 female, age [years, mean sd]: 67.5 10.5) and 64 patients with mi (8 female, age 55 8.8) were entered in the study . Patients with concurrent diseases or conditions interfering with the aim of the study, such as infections, malignancies and those on immunosuppressive drugs were excluded from study . Diagnosis of is was based on clinical history and neurological examination and was confirmed by brain ct . On ct examination, all stroke patients presented anatomically relevant hypodense areas in subcortical parts of cerebral hemispheres . Diagnosis of mi was determined from the presence of> 30 minutes of continuous chest pain, st - segment elevation> 2.0 mm on at least 2 contiguous ecg leads, and more than a 3-fold increase in serum creatine kinase levels . Regarding risk factors, 44% of mi and 50% of is patients had hypertension, 18% of mi and 9% of is patients had diabetes mellitus, 54% of mi and 18% of is patients reported themselves as current smokers, and 8% of mi and 5% of is patients reported themselves as alcohol consumers . In addition 7% of is patients previously had ischemic heart disease . Forty healthy subjects (7 female, age 52 3) free of clinical signs of infection, other systemic diseases, and negative family history of is or mi served as control population . Venous blood samples were obtained from is and mi patients within 24 hours after disease onset and once from controls . Is patients were recruited at erebouni medical center, yerevan, armenia; mi patients were diagnosed at the faculty hospital olomouc, czech republic . Protein levels in serum obtained from study participants were measured using a solid - phase sandwich enzyme - linked immunosorbent assay (mcp-1 quantikine elisa kit, r&d systems, abingdon, uk). Briefly, 100 l of duplicated samples or standards (reeombinant human mcp-1) were incubated (2 hours at room temperature) in the wells precoated with primary antihuman mcp-1 antibody . After incubation, wells were washed three times and horseradish peroxidase - conjugated polyclonal antibodies against mcp-1 were added (for 2 hours at room temperature). Finally, tetramethylbenzidine substrate solution was applied for 30 minutes and, after stopping the reactions by 2 m sulfuric acid, the absorbance was measured at 450 nm (with correction at 540 nm). The data were evaluated with kim - e software (usol, prague, czech republic); the detection limit of the mcp-1 assay was 5.0 pg / ml . Spss 11 (spss inc, chicago, ill) software was used to calculate basic statistical parameters and to perform the mann - whitney u - test to test for differences in chemokine protein levels between the mi, is, and control groups as well as for further subanalysis . Fisher's exact test was used to compare differences in qualitative parameters between is and mi groups . Spearman's rank correlation was used to evaluate relationship between age and mcp-1 levels . Data on mcp-1 levels was presented as median [interquartile range (iqr)]. Forty patients with is (17 female, age [years, mean sd]: 67.5 10.5) and 64 patients with mi (8 female, age 55 8.8) were entered in the study . Patients with concurrent diseases or conditions interfering with the aim of the study, such as infections, malignancies and those on immunosuppressive drugs were excluded from study . Diagnosis of is was based on clinical history and neurological examination and was confirmed by brain ct . On ct examination, all stroke patients presented anatomically relevant hypodense areas in subcortical parts of cerebral hemispheres . Diagnosis of mi was determined from the presence of> 30 minutes of continuous chest pain, st - segment elevation> 2.0 mm on at least 2 contiguous ecg leads, and more than a 3-fold increase in serum creatine kinase levels . Regarding risk factors, 44% of mi and 50% of is patients had hypertension, 18% of mi and 9% of is patients had diabetes mellitus, 54% of mi and 18% of is patients reported themselves as current smokers, and 8% of mi and 5% of is patients reported themselves as alcohol consumers . In addition 7% of is patients previously had ischemic heart disease . Forty healthy subjects (7 female, age 52 3) free of clinical signs of infection, other systemic diseases, and negative family history of is or mi served as control population . Venous blood samples were obtained from is and mi patients within 24 hours after disease onset and once from controls . Is patients were recruited at erebouni medical center, yerevan, armenia; mi patients were diagnosed at the faculty hospital olomouc, czech republic . Mcp-1 protein levels in serum obtained from study participants were measured using a solid - phase sandwich enzyme - linked immunosorbent assay (mcp-1 quantikine elisa kit, r&d systems, abingdon, uk). Briefly, 100 l of duplicated samples or standards (reeombinant human mcp-1) were incubated (2 hours at room temperature) in the wells precoated with primary antihuman mcp-1 antibody . After incubation, wells were washed three times and horseradish peroxidase - conjugated polyclonal antibodies against mcp-1 were added (for 2 hours at room temperature). Finally, tetramethylbenzidine substrate solution was applied for 30 minutes and, after stopping the reactions by 2 m sulfuric acid, the absorbance was measured at 450 nm (with correction at 540 nm). The data were evaluated with kim - e software (usol, prague, czech republic); the detection limit of the mcp-1 assay was 5.0 pg / ml . Spss 11 (spss inc, chicago, ill) software was used to calculate basic statistical parameters and to perform the mann - whitney u - test to test for differences in chemokine protein levels between the mi, is, and control groups as well as for further subanalysis . Fisher's exact test was used to compare differences in qualitative parameters between is and mi groups . Spearman's rank correlation was used to evaluate relationship between age and mcp-1 levels . Data on mcp-1 levels was presented as median [interquartile range (iqr)]. In this study, mcp-1 immunoreactive protein was determined in peripheral blood of patients with mi and is . Prior to chemokine analysis, the differences in risk factors among our study groups were estimated . Also, there were differences in male / female ratio (is, 42% versus mi, 12%, p <.0001) and in number of smokers (mi, 54% versus is, 18%, p <.0001). Number of sufferers from diabetes was 2-fold higher in mi group compared with is, but this difference did not reach significance (mi, 18% versus is, 9%, p>.05). The study groups did not differ with regard to hypertension and alcohol consumption . In comparison to control subjects (median [iqr]: 239 pg / ml), the levels of mcp-1 protein were elevated in patients from both study groups (figure 1). This increase was more apparent in the is group (384 pg / ml), for which also a greater degree of interindividual variability was observed than among patients with mi (360 pg / ml). While only 10/40 (25%) of control subjects had mcp-1 level higher than 314 pg / ml (75th percentile of the control group), in patients with is this proportion reached 68% (27 /40) and in mi group it was 61% (39/64). The differences between both investigated patient groups and controls, therefore, reached high significance (is versus c, p <.001; mi versus c, p <.002), while there was no difference between patients' groups themselves (is versus mi, p>.05). Further, we investigated whether mcp-1 protein is influenced by risk factors of mi and is . Serum chemokine levels were compared between patients with and without diabetes, hypertension; smokers and nonsmokers; and also groups divided based on alcohol consumption . Data analysis showed that none of the above risk factors had an influence at circulating mcp-1 levels in patients with mi and/or is (table 1). Furthermore no gender effect mcp-1 levels was apparent in both studied groups as well as in control group (is males versus females: 417 versus 253, p>.05; mi males versus females: 343 versus 397, p>.05; c males versus females: 244 versus 234, p>.05), and there was no correlation between age and the chemokine levels (is: rs = 0.009, p>.05, mi: rs = 0.06, p>.05, c: rs = 0.168, p>.05). This study aimed at the evaluation of the mcp-1 chemokine in the circulation of patients with myocardial infarction and ischemic stroke . These two atherosclerosis - associated complications share many similarities, but at the same time they have at least quantitative if not qualitative differences in their risk factors . This trend is preserved in our patients' characteristics: our is patients were older and the different incidence in males and females is less pronounced in is compared to mi . Also, is patients were more prone to hypertension than mi patients . Presence of these features, therefore, confirms that our patients' groups were properly created and reflect the actual situation with is and mi phenotypes . Our data showed almost 2-fold increase of serum mcp-1 levels in patients with mi and is compared with control group of healthy subjects . We also found that serum mcp-1 levels did not differ between is and mi patients . Finally, the levels of this chemokine were not influenced by known risk factors for these diseases such as hypertension, diabetes, smoking, or alcohol consumption . In myocardial infarction, elevation of circulating mcp-1 has been already reported [19, 20, 21] and, therefore, our findings of increased mcp-1 in our patients with mi are of a confirmatory nature . However, infrequent reports regarding this chemokine in ischemic stroke are contradictory [22, 23]. Losy and zaremba reported that blood mcp-1 levels were not increased in his group of 23 patients with is . By contrast and in line with our results, reynolds et al demonstrated elevation of the mcp-1 chemokine in the blood from a big group of more than 200 stroke patients . This discrepancy may occur due to low number of subjects included in the study by losy and zaremba and possibly also by marked variability of circulating mcp-1 levels . There are at least two mechanisms by which the observed upregulation of mcp-1 protein levels in circulation of patients with is and mi can be achieved . The main sources of mcp-1 are endothelia and macrophage like cells, which are known to play a significant role in atherosclerosis development and plaque formation . In vitro studies have revealed that endothelial cells are able to produce mcp-1 in response to ldl an important atherosclerosis triggering factor [25, 26]. Second, increased mcp-1 levels can mirror the development of inflammatory response in heart and brain . Numerous studies on animal models of stroke showed that mcp-1 and mip-1beta are two main chemokines that orchestrate infiltration of blood - derived monocytes and lymphocytes into ischemic area . The upregulation of mcp-1 mrna levels is detected in both permanent and transient models of focal ischemia [11, 12]. Moreover study by hughes et al demonstrated that mcp-1 deficiency is protective in a murine stroke model . The same processes are taking place in heart during mi [13, 14]. Furthermore, it has been demonstrated that mice lacking ccr2, the primary receptor for mcp-1, showed significant impairment of monocyte infiltration, reduction of tnf - alpha, and matrix metalloproteinases expression in infarcted area in experimental mi . Our data showed that elevation of mcp-1 in the blood is a common feature for mi and is . This may suggest that in atherosclerosis - associated complications, inflammatory response, share some similarities and are not organ - specific . This interpretation fits in with other current reports [29, 30, 31], including our recent observation of approximately 2-fold increase of mcp-1 in the blood of patients with peripheral arterial disease . Unexpectedly, in our study none of four investigated common risk factors for mi and is did not influence serum mcp-1 levels . In number of previous studies in patients with myocardial infarction, the association of mcp-1 levels with several risk factors such as hypertension and diabetes has been reported . However, these were large - population - based studies where even slight differences may be identified . For example, in the study by de lemos et al more than 2000 subjects were enrolled and thus the authors were able to identify a rather minor difference of 6% as significant . It is, therefore, possible that in our relatively small patient population of 64 mi patients, differences of similar magnitude may not be visible . Regarding patients with ischemic stroke, there have been no studies on influence of risk factors on mcp-1 levels . We, therefore, plan to further explore this phenomenon so that it can be assessed if monitoring mcp-1 serum levels can be included into the algorithm of management of is patients as an independent marker . In conclusion, our data expand previous observations showing that mcp-1 plays an important role in inflammatory response developing during myocardial infarction and ischemic stroke . Further work is, however, necessary to define more precisely the relationship between mcp-1 and clinical course of investigated diseases . Emphasis should be given to patients' stratification in order to ascertain clinical utilisation of mcp-1 measurements, namely in ischemic stroke.
Non - specific odontogenic infections are among the most frequent disorders of bacterial origin that affect individuals during the course of their life . Before antibiotic treatment was available, the mortality rate of these disorders was 1040%, but the discovery of antibiotics led to a significant improvement of this rate . However, the last 1015 years have witnessed a rebound of severe odontogenic infections caused by highly aggressive antibiotic - resistant bacteria . The main cause indicated by the literature for the development of bacteria resistant to antibiotic therapy is the incorrect or inefficient administration of antibiotics . Currently, a number of antibiotics for inflammatory dental pulp disorders or established abscesses are administered . The efficiency of these antibiotics is extremely limited because of the circulation disorders present in the inflammatory focus and, in addition, there is a high risk for inducing adverse reactions and antibiotic resistance of the bacteria involved in the development of the septic process . For this reason, we believe that it is extremely important to evaluate the way in which antibiotics are administered and their efficiency on the bacterial flora involved in the development of odontogenic suppurations, in the absence of surgery . The aim of this study is to prospectively determine the type of antibiotics used by patients with odontogenic head and neck soft tissue infections, from the point of view of their efficiency on the bacterial flora involved in the development of the septic process . The study included 10 randomly selected patients with suppurations of odontogenic origin who presented to the clinic of oral and maxillofacial surgery cluj - napoca in the period january 2014 july 2014 . The patient selection criteria were: perimaxillary soft tissue infections of odontogenic origin, disease duration of at least 5 days, antibiotic treatment duration of at least 4 days, known type and mode of administration of antibiotic medication, antibiotic treatment prescribed by the family doctor or the dentist, patient treated under continuous hospitalization, patient from which biological samples were taken for bacteriological examination and antibiogram determination, no other systemic pathology types with a possible influence on the immune response, adult patient having signed an informed consent for participation in the study . The variables monitored for each patient were: general data (age, sex), location of infection, time from onset to presentation, type of antibiotic treatment and route of administration used, bacteriological examination and antibiogram result, postoperative evolution . After the emergency admission of the patients, surgery was performed under locoregional anesthesia . After the asepsis and antisepsis of the operative field, biological samples were collected in a closed environment and were subsequently sent for bacteriological examination and antibiogram determination . The results obtained were centralized in the study data base . For the development of contingency tables, microsoft excel software was used, and the statistical interpretation of the results was performed using microsoft excel . Of the patients included in the study, 6 were males and 4 were females . The mean age of the patients was 35.4 years, with a minimum age of 22 years and a maximum age of 49 years . The mean age of female patients was 28.75 years and that of male patients 39.83 years . Regarding the location of the septic process in perimaxillary soft tissues, the majority of the infections were located in the submandibular gland area, followed by those located in the genial region (figs . 1, 2). In the case of the patients included in the study, a time period between 5 and 12 days, with a mean of 7.4 days, lapsed from the onset of the septic process to the presentation for specialized treatment . During this time period, all patients used antibiotic treatment, with a mean of 1.2 antibiotic types; 8 of the 10 patients had one antibiotic type, and 2 patients had 2 antibiotic types . 3), and the most frequent route of administration was the oral route, only in two cases the antibiotics being administered by intravenous route . The bacteriological result obtained from the purulent secretion samples collected from the septic focus evidenced microbial polymorphism (tab . I). In half of the patients included in the study, the identified bacterial flora had no sensitivity to the antibiotics 4), and in one patient in whom several bacterial strains were detected, some of these were sensitive to the antibiotic administered to the patient . After the incision and the drainage of the suppuration were performed, along with the change of the antibiotic scheme according to the antibiogram, the patients postoperative evolution was favorable . The objectives of the study were reached and the main types of antibiotics administered to patients with odontogenic septic processes complicated by perimaxillary suppurations were determined . It can be seen that the majority of the patients included in the study were young adults, which is confirmed by other literature studies . Authors analyzing extensive groups of patients evidence the fact that odontogenic infections mainly affect patients in the third decade of life, which is confirmed in this study only by female patients, male patients having a more advanced age, but without a significant difference . Of the patients included in the study, however, it cannot be concluded based on the presented data that the male sex is more frequently affected by cervical inflammatory disorders of odontogenic origin, because the patient inclusion criteria were very restrictive and the number of patients included in the study was limited . Most authors opine that there is a higher incidence of odontogenic infections among male patients, but the differences between the two sexes are extremely varied . Regarding the antibiotics prescribed to the patients included in the present study, it can be seen that more than half of the patients received amoxicillin treatment with or without beta - lactamase inhibitors . The majority of the patients took the antibiotic without beta - lactamase inhibitors, which is contrary to literature studies, which show that the main antibiotic administered for odontogenic infections is amoxicillin with beta - lactamase inhibitors . The administration of an effective antibiotic in odontogenic infections is particularly important in the attempt to limit the septic process . When the antibiotic has no effect on the main bacterial strains involved in the development of the infection and only eliminates less aggressive pathogens, the premises for extremely severe and very difficult to control infections are created . Bacteriological examination evidenced the presence of a varied bacterial flora, but in the majority of the cases, a single bacterial strain in each patient was obvious . The presence of a single bacterial strain in each patient is surprising, given the fact that at the level of the infected dental pulp or periodontal space, the main sources of bacterial flora for odontogenic suppurations, an increased number of bacterial species are concomitantly identified . Thus, it is possible that the early administration of bacterial therapy may select the majority of the bacterial species sensitive to the administered antibiotic and a single bacterial species may remain in the septic focus . This hypothesis is also supported by the antibiogram result, which evidenced no sensitivity of the identified bacteria to the administered antibiotic . Another possible cause of the identification of a single bacterial strain might be the technical limitations of microbiology laboratories or the way of collecting biological samples, which pose difficulties in identifying some bacterial strains, particularly anaerobic ones . The fact that most of the administered antibiotics were not active on the identified bacterial flora is an alarm signal . The lack of efficiency of the antibiotic on the main bacterial strains involved in the development of the septic process implicitly leads to an increase of the difficulty of treatment of these infections . Some authors indicate the use of antibiotics as a single treatment, in the absence of a preliminary bacteriological examination, as the main factor favoring the development of severe odontogenic infections such as necrotizing fasciitis . The exhaustion of the action of common antibiotics on the bacteria involved in the development of common cervical infections leads the practitioner to use niche antibiotics that should not be used under normal conditions . A limitation of this study is the fact that it does not take into account possible cases of patients with odontogenic infections who received antibiotic treatment alone and who had a favorable evolution . These cases cannot be monitored in such studies because these patients do not ask for specialized help . Most common antibiotics used as a single therapy for the treatment of cervical infections of odontogenic origin have a limited action, and the association of antibiotic treatment with surgery is recommended . The administration of antibiotics according to the bacteriological examination and antibiogram, associated with surgery, led to a favorable evolution of the patients included in this study.
The young hearts project 2000 is a cross - sectional cohort study which took place in 19992001 following a previous study, young hearts i, described in detail elsewhere . The young hearts project 2000 had a total of 2,017 participants from northern ireland, consisting of 12- and 15-year - olds from 36 nationally representative schools; the response rate was 65.4% . Eligible subjects were selected using a computer - generated random number list, divided into groups according to age and gender, resulting in approximately 500 participants in each of the four age / sex groups . Age was defined as years of age 6 months (e.g., a 12-year - old child ranges from 11.5 to 12.5 years). Inflammation markers were only measured in a subsample of the total population . In this subsample, a distinction was made between normal, overweight, and obese subjects (according to the classification by cole et al . ). The prevalence of overweight and obesity in the complete cohort was 16.2% and 14.7%, respectively . In the present study sample, all obese adolescents (n = 95) were selected and matched according to age, gender, and smoking status, with overweight and normal weight children . Because of problems in obtaining a blood sample from three of the obese adolescents, the final number of participants in each of the three groups was reduced to 92 . Written informed consent was signed by the participants and the participants' parent or guardian . For each child, the examination included a medical examination, cardiorespiratory fitness test, dietary examination, and a parental and physical activity questionnaire . The medical examination included weight (nearest 100 g, seca 770 electronic weighing scale) and height (nearest mm, holtain stadiometer) using standard techniques, with participants in light clothing and without shoes . Body mass index was calculated as weight (kg)/height (m). Peripheral venous blood (16 ml) was obtained by venepuncture in fasting state and collected into serum - z - clot activator edta - sodium or lithium heparin - coated tubes . Samples were transported in a chilled insulated container, and serum / plasma was removed after centrifugation at 100 g for 10 minutes at 4c within 4 hours from venepuncture and stored at 70c . Because cvd risk is seldom clinically defined at this age, a clustered score for biological risk factor can be used to specify subjects at risk [79].clustered cvd risk in the present study was computed using scores for mean arterial pressure (map), ldl: hdl cholesterol ratio, cardiorespiratory fitness, skinfolds, and triglyceride levels . Blood pressure was measured twice from the right arm using a hawksley random zero sphygmomanometer, with subjects sitting down quietly beforehand for at least 5 minutes . Systolic blood pressure (sb) was recorded as the mean of the two values for korotkoff phase 1, whereas the diastolic blood pressure (db) was based on the mean of the two values for korotkoff phase v (15-year - old) or phase iv (12-year - old). Total plasma - cholesterol, hdl - cholesterol, and plasma triglyceride were determined (boeringer, mannheim, germany; cobas fara automated analyzer) according to the world health organization (who) standards of quality control . Four skinfold thickness measurements (triceps, biceps, subscapula, and suprailiac; mm) were taken in duplicate on the left side of the body according to durnin and rahaman . Subjects ran a distance of 20 m at a fixed speed, and the pace was increased by .5 km / hr every minute . Physical activity was estimated by a self - administered recall questionnaire under the supervision of an exercise physiologist . The questionnaire assessed frequency, intensity, and duration of habitual activity and allocated scores out of 100 [18,2325]. Each nutritionist consulted with a similar number of participants from each group, and inter - observer reproducibility was confirmed before sampling . The mean daily caloric intake (kcals) was calculated from the 7-day recall diet history interview using the nutrition analysis software programme wisp (tinuviel software, warrington, uk), and recorded for each subject . Smoking status was determined using a self - administered questionnaire on smoking and daily number of cigarettes smoked . Plasma levels of sicam and svcam were determined using commercially available monoclonal elisa kits (diaclone, immunodiagnostic systemd, ltd) according to the recommendations of the manufacturer . Mean inter - assay coefficients of variation for sicam and svcam elisas were 4.7% and 4.2%, respectively . Plasma hscrp was quantified by a high sensitivity latex - enhanced immunoturbidimetric assay (wako chemicals gmbh) using a cobas fara automated analyzer (roche diagnostics, uk). Clustered cvd risk scores were computed using a sum of age- and gender - specific z - scores . Triglyceride, cholesterol ratio, and cardiorespiratory fitness measures were log transformed to meet the assumption of normal distribution . The existence of clustering between the biological risk factors was determined with pair - wise pearson correlation coefficients . To determine whether inflammation markers relate to clustered cvd risk independent of established lifestyle risk factors, first, univariate analyses were performed to analyze the relationship between single determinants and clustered cvd risk . Second, a multiple regression analysis with a forward selection procedure was performed in which both lifestyle risk factors and inflammation markers were included as possible determinants . A threshold significance value of 10% (p = .10) was assigned for the inclusion of variables in the final prediction model . R were used to give an indication of the quality of the final prediction model . All statistical analyses were performed in statistical package for the social sciences (spss inc, chicago, il), version 16.0 . The young hearts project 2000 is a cross - sectional cohort study which took place in 19992001 following a previous study, young hearts i, described in detail elsewhere . The young hearts project 2000 had a total of 2,017 participants from northern ireland, consisting of 12- and 15-year - olds from 36 nationally representative schools; the response rate was 65.4% . Eligible subjects were selected using a computer - generated random number list, divided into groups according to age and gender, resulting in approximately 500 participants in each of the four age / sex groups . Age was defined as years of age 6 months (e.g., a 12-year - old child ranges from 11.5 to 12.5 years). Inflammation markers were only measured in a subsample of the total population . In this subsample, a distinction was made between normal, overweight, and obese subjects (according to the classification by cole et al . ). The prevalence of overweight and obesity in the complete cohort was 16.2% and 14.7%, respectively . In the present study sample, all obese adolescents (n = 95) were selected and matched according to age, gender, and smoking status, with overweight and normal weight children . Because of problems in obtaining a blood sample from three of the obese adolescents, the final number of participants in each of the three groups was reduced to 92 . Written informed consent was signed by the participants and the participants' parent or guardian . For each child, the examination included a medical examination, cardiorespiratory fitness test, dietary examination, and a parental and physical activity questionnaire . The medical examination included weight (nearest 100 g, seca 770 electronic weighing scale) and height (nearest mm, holtain stadiometer) using standard techniques, with participants in light clothing and without shoes . Body mass index was calculated as weight (kg)/height (m). Peripheral venous blood (16 ml) was obtained by venepuncture in fasting state and collected into serum - z - clot activator edta - sodium or lithium heparin - coated tubes . Samples were transported in a chilled insulated container, and serum / plasma was removed after centrifugation at 100 g for 10 minutes at 4c within 4 hours from venepuncture and stored at 70c . Because cvd risk is seldom clinically defined at this age, a clustered score for biological risk factor can be used to specify subjects at risk [79].clustered cvd risk in the present study was computed using scores for mean arterial pressure (map), ldl: hdl cholesterol ratio, cardiorespiratory fitness, skinfolds, and triglyceride levels . Blood pressure was measured twice from the right arm using a hawksley random zero sphygmomanometer, with subjects sitting down quietly beforehand for at least 5 minutes . Systolic blood pressure (sb) was recorded as the mean of the two values for korotkoff phase 1, whereas the diastolic blood pressure (db) was based on the mean of the two values for korotkoff phase v (15-year - old) or phase iv (12-year - old). Total plasma - cholesterol, hdl - cholesterol, and plasma triglyceride were determined (boeringer, mannheim, germany; cobas fara automated analyzer) according to the world health organization (who) standards of quality control . Four skinfold thickness measurements (triceps, biceps, subscapula, and suprailiac; mm) were taken in duplicate on the left side of the body according to durnin and rahaman . Subjects ran a distance of 20 m at a fixed speed, and the pace was increased by .5 km / hr every minute . Physical activity was estimated by a self - administered recall questionnaire under the supervision of an exercise physiologist . The questionnaire assessed frequency, intensity, and duration of habitual activity and allocated scores out of 100 [18,2325]. Each nutritionist consulted with a similar number of participants from each group, and inter - observer reproducibility was confirmed before sampling . The mean daily caloric intake (kcals) was calculated from the 7-day recall diet history interview using the nutrition analysis software programme wisp (tinuviel software, warrington, uk), and recorded for each subject . Smoking status was determined using a self - administered questionnaire on smoking and daily number of cigarettes smoked . Plasma levels of sicam and svcam were determined using commercially available monoclonal elisa kits (diaclone, immunodiagnostic systemd, ltd) according to the recommendations of the manufacturer . Mean inter - assay coefficients of variation for sicam and svcam elisas were 4.7% and 4.2%, respectively . Plasma hscrp was quantified by a high sensitivity latex - enhanced immunoturbidimetric assay (wako chemicals gmbh) using a cobas fara automated analyzer (roche diagnostics, uk). Clustered cvd risk scores were computed using a sum of age- and gender - specific z - scores . Triglyceride, cholesterol ratio, and cardiorespiratory fitness measures were log transformed to meet the assumption of normal distribution . The existence of clustering between the biological risk factors was determined with pair - wise pearson correlation coefficients . To determine whether inflammation markers relate to clustered cvd risk independent of established lifestyle risk factors, first, univariate analyses were performed to analyze the relationship between single determinants and clustered cvd risk . Second, a multiple regression analysis with a forward selection procedure was performed in which both lifestyle risk factors and inflammation markers were included as possible determinants . A threshold significance value of 10% (p = .10) was assigned for the inclusion of variables in the final prediction model . R were used to give an indication of the quality of the final prediction model . All statistical analyses were performed in statistical package for the social sciences (spss inc, chicago, il), version 16.0 . Table 2 shows the pair - wise pearson correlation matrix for the biological risk factors . On the basis of the partial correlations, clustering of biological risk factors seems to exist, that is, all pearson correlations between the biological risk factors were positive and significant . Table 3 shows the pair - wise pearson correlation matrix for inflammation markers with univariate and clustered cvd risk variables . The best prediction model included hscrp, sicam, and svcam and showed an explained variance of 26% . To our knowledge, this is the first study to associate markers of inflammation and endothelial dysfunction with clustered cvd risk in adolescents . The main finding of this study was that these markers were strong predictors for cvd risk in adolescents, unlike lifestyle factors which were not predictive . The markers sicam and hscrp were positively related to cvd risk, whereas an inverse relation for svcam was found . First, membrane - bound icam and vcam are involved in leukocyte adhesion and migration to the vessel wall as seen in atherosclerosis . These markers, however, cannot be easily measured without invasive techniques . Therefore, soluble adhesion molecules (scam) are used as a surrogate for membrane bound levels, based on the assumption that soluble levels are indicative of the processes at the endothelium . Although the soluble markers have previously been found to be elevated in children at risk, although svcam is mainly a product of endothelial cells, sicam is produced by many other cell types throughout the body . Some studies have found that the contribution of sicam and svcam in atherosclerosis is similar, whereas others have found high expression of sicam in healthy subjects associated with cvd risk and high levels of svcam in ongoing stages of atherosclerosis and cvd outcomes [3,17,2830]. Such associations of vcam need not exist in a healthy adolescent population as currently used . The inverse relation of svcam as found in the current study could, therefore, indicate lower incidence of ongoing stages of vascular damage in this population . Limitations of the study design merit consideration . Although the study provides a representative population, the cross - sectional design of the study cannot provide information on change over time . Nevertheless, a clustered risk score is an accepted method of prediction of cvd risk . All variables used in the clustered risk were taken into account using the same weighting . Published data do not indicate that one variable should be weighted more heavily than another . Although lifestyle variables did not contribute to cvd risk prediction in this study, we should be careful with the interpretation of this result because lifestyle variables were measured by self - report . Finally, the rather large percentage of obese and overweight subjects compared with a normal population did not influence the results, that is, the final regression model remained more or less the same even when analyses were performed separately for the body mass index groups (data not shown). Results not only confirm a role for inflammation and endothelial dysfunction but also prompt further research on the mechanistic level . At this point, conclusions to redirect the prevention of cvd are preliminary and thus efforts should remain concentrated on classical risk prediction and preventive strategies.
Dental caries is a multifactorial and transmissible disease that originates mainly from the interplay of oral flora, a susceptible host, and dietary factors . When organic acids are produced by the dental biofilm bacteria, they start diffusing through the enamel and cementum . As the acid diffuses into the tooth structure, it may find calcium deficient / carbonate rich acid soluble minerals, and begins to dissolve it . When this process progresses long enough and reaches the dentin, the end result is tooth cavitation . The most common aetiological agents involved in this disease are streptococcus mutans, followed by streptococcus sobrinus, lactobacillus, and actinomyces . Consequently, caries prevention is based on the control of at least one of the mentioned causing factors . The broad - spectrum antibacterial chlorhexidine (chx) is a very commonly used agent for the prevention or control of oral diseases . Studies have demonstrated that chx is capable of arresting caries when applied to dentin . However, chx has some disadvantages, such as bacterial resistance, controversial efficacy against caries and toxicity to host tissues, motivating the investigation of alternatives . Natural products, especially food extracts, have been shown to be good anticariogenic alternatives to synthetic chemicals . The green tea polyphenol, epigallocatechin - gallate (egcg) was found to inhibit acid production from dental plaque bacteria, while showing antimicrobial activity against s. mutans . Moreover, egcg was shown to inhibit the matrix metalloproteinase activities present in the dentin (mmp-2 and mmp-9) and associated with caries progression . Many properties of restorative dental composites have been improved in the past decades, including the mechanical properties, masticatory and abrasive wear resistance, esthetics, and bonding to the enamel and dentin . Bis - gma (bisphenol glycidyl dimethacrylate) is still today the most commonly used monomer in composite resin formulations . It has very high viscosity, thus dilution with low viscosity alkoxyalkyl dimethacrylate esters, such as tegdma (triethyleneglycol dimethacrylate) is required to obtain adequate filler loading and handling characteristics . Thus, research has also been directed towards developing more hydrophobic and stable hydroxyl - free monomers with lower viscosities, such as ch3bis - gma (propoxylated bis - gma) as a replacement for tegdma in resin formulations in order to reduce resin water sorption and polymerization shrinkage . It is therefore proposed that at this stage, advances of restoratives should happen towards the development of materials with bio - active functions, such as antibacterial activity to provide therapeutic effects . It could be assumed that the long - term durability of resin - dentin bonds and restorations may benefit from drug - loaded methacrylate - based polymeric materials, capable of releasing bioactive compounds . The aim of this study was to test the antimicrobial activity of egcg when compared to chx, against the cariogenic organism s. mutans after being released by experimental dental copolymers . The null hypothesis to be tested is that different comonomers, incorporated drugs (chx or egcg) or drug ratios will not affect the growth of s. mutans . The s. mutans ua159 strain isolated from a child with active caries was used in this study . S. mutans was cultivated in brain heart infusion (bhi) broth at 37 c in air with 5% co2 . Bis - gma, tegdma, the photosensitizer camphorquinone (cq), reducing agent 2-(dimethylamino)ethyl methacrylate (dmaema), chx diacetate salt hydrate (sigma - aldrich, st . Louis, mo, usa) and egcg (cayman chemical group, mi, usa) were all used as received . The bis - gma analog, ch3bis - gma (2,2-bis[4-(2-methacryloxyprop-1-oxy)phenyl]propane) was synthesized, purified, and stored according to the reported methods (figures 1a to 1e). Briefly, bisphenol a was treated with propyleneoxide in the presence of naoh and tetrahydrofuran (thf). The reaction product was isolated and then treated with 3 mols of methacryloyl chloride per mol of product, together with triethylamine in thf solvent to obtain ch3bis - gma . The synthesized monomer was dissolved in deuterochloroform at 5% (w / v) and characterized by proton nuclear magnetic resonance (h nmr; varian unity 400, 400 mhz) and carbon nuclear magnetic resonance (c nmr; general electric gn500, 125mhz) at 25 c, providing spectra that were consistent with the expected product (figure 1b). Molecular structure of bis - gma (a), ch3bis - gma (b), tegdma (c), chx (d), and egcg (e) overnight cultures of s. mutans ua159 were diluted (1:20) into fresh bhi medium supplemented with different concentrations of drugs (chx: 1, 2, 5, 10, 15, 25 g / ml; egcg: 100, 250, 500, 1000 g / ml) and incubated at 37 c for 24 h. the cells were sonicated, serially diluted and, the minimum inhibitory concentration (mic) test was performed according to the broth microdilution method using bhi broth as previously described . Briefly, 10 cfu / ml of bacterial cells were added to a 96-well plate containing bhi medium supplemented with twofold serial dilutions of egcg or chx . Bacterial growth after 24 h was spectrophotometrically measured by using an elisa microtiter plate reader (model 3550; bio - rad laboratories, richmond, ca) at an absorbance of 490 nm (od490). Relative bacteria density percentages were calculated by using the following equation: (od490 of culture in the presence of each concentration of drug)/(od490 of culture in the absence of drug) 100 . The mic was determined as the lowest product concentration needed to ensure that the culture did not grow to over 10% of the relative bacterial cell density . The following two experimental resin formulations were prepared for this study: r1) bis - gma at 70 mol% combined with tegdma at 30 mol%, or r2) bis - gma at 70 mol% combined with ch3bis - gma at 30 mol% . Except for the control groups (no drugs added), each formulation was randomly mixed with either chx or egcg at 0.5x mic or 1x mic corresponding to drug concentrations in weight percentage, comprising a total of 10 groups (n=9). Comonomers were activated for visible light polymerization by the addition of cq and dmaema (0.2 w / w% each). Specimens were fabricated inside a cylindrical acrylic matrix with internal dimensions of 5 mm diameter x 3 mm height . Polymerization was done on both sides by a visible light curing unit (demi led, kerr co., wi, usa) for 40 s delivering uninterrupted 540 mw / cm, verified by a radiometer (model 100, demetron research co., ct, usa). Additional specimens were fabricated and the 24 h release rates of the drug loaded samples were tested spectrophotometrically as previously described . Overnight cultures of s. mutans ua159 were diluted (1:20) into 1.0 ml of fresh bhi broth followed by the addition of one resin sample per test tube . The next day, the cells were sonicated, and aliquots of 20 l of each test tube were plated on bhi agar for cfu determination . The percentage of cell survival corresponded to the number of viable cells after treatment divided by the total number of viable cells in the untreated sample . The data were shown to have normal distribution and equal variances (kolmogorov - smirnof test). The student t - test was applied to the bacterial cell viability data comparing each tested group with its respective control, and all r1 and r2 counterparts (same drug and drug ratio). One - way anova followed by tukey test was used for each resin to compare the bacterial cell viability among groups containing different drugs and ratios . Collected data were compiled and examined for relevance with the spss version 8.0 (spss inc ., the s. mutans ua159 strain isolated from a child with active caries was used in this study . S. mutans was cultivated in brain heart infusion (bhi) broth at 37 c in air with 5% co2 . Bis - gma, tegdma, the photosensitizer camphorquinone (cq), reducing agent 2-(dimethylamino)ethyl methacrylate (dmaema), chx diacetate salt hydrate (sigma - aldrich, st . Louis, mo, usa) and egcg (cayman chemical group, mi, usa) were all used as received . The bis - gma analog, ch3bis - gma (2,2-bis[4-(2-methacryloxyprop-1-oxy)phenyl]propane) was synthesized, purified, and stored according to the reported methods (figures 1a to 1e). Briefly, bisphenol a was treated with propyleneoxide in the presence of naoh and tetrahydrofuran (thf). The reaction product was isolated and then treated with 3 mols of methacryloyl chloride per mol of product, together with triethylamine in thf solvent to obtain ch3bis - gma . The synthesized monomer was dissolved in deuterochloroform at 5% (w / v) and characterized by proton nuclear magnetic resonance (h nmr; varian unity 400, 400 mhz) and carbon nuclear magnetic resonance (c nmr; general electric gn500, 125mhz) at 25 c, providing spectra that were consistent with the expected product (figure 1b). Molecular structure of bis - gma (a), ch3bis - gma (b), tegdma (c), chx (d), and egcg (e) overnight cultures of s. mutans ua159 were diluted (1:20) into fresh bhi medium supplemented with different concentrations of drugs (chx: 1, 2, 5, 10, 15, 25 g / ml; egcg: 100, 250, 500, 1000 g / ml) and incubated at 37 c for 24 h. the cells were sonicated, serially diluted and, cell viability was assessed by counting colony forming units (cfus). The minimum inhibitory concentration (mic) test was performed according to the broth microdilution method using bhi broth as previously described . Briefly, 10 cfu / ml of bacterial cells were added to a 96-well plate containing bhi medium supplemented with twofold serial dilutions of egcg or chx . Bacterial growth after 24 h was spectrophotometrically measured by using an elisa microtiter plate reader (model 3550; bio - rad laboratories, richmond, ca) at an absorbance of 490 nm (od490). Relative bacteria density percentages were calculated by using the following equation: (od490 of culture in the presence of each concentration of drug)/(od490 of culture in the absence of drug) 100 . The mic was determined as the lowest product concentration needed to ensure that the culture did not grow to over 10% of the relative bacterial cell density . The following two experimental resin formulations were prepared for this study: r1) bis - gma at 70 mol% combined with tegdma at 30 mol%, or r2) bis - gma at 70 mol% combined with ch3bis - gma at 30 mol% . Except for the control groups (no drugs added), each formulation was randomly mixed with either chx or egcg at 0.5x mic or 1x mic corresponding to drug concentrations in weight percentage, comprising a total of 10 groups (n=9). Comonomers were activated for visible light polymerization by the addition of cq and dmaema (0.2 w / w% each). Specimens were fabricated inside a cylindrical acrylic matrix with internal dimensions of 5 mm diameter x 3 mm height . Polymerization was done on both sides by a visible light curing unit (demi led, kerr co., wi, usa) for 40 s delivering uninterrupted 540 mw / cm, verified by a radiometer (model 100, demetron research co., ct, usa). Additional specimens were fabricated and the 24 h release rates of the drug loaded samples were tested spectrophotometrically as previously described . Overnight cultures of s. mutans ua159 were diluted (1:20) into 1.0 ml of fresh bhi broth followed by the addition of one resin sample per test tube . The next day, the cells were sonicated, and aliquots of 20 l of each test tube were plated on bhi agar for cfu determination . The percentage of cell survival corresponded to the number of viable cells after treatment divided by the total number of viable cells in the untreated sample . The data were shown to have normal distribution and equal variances (kolmogorov - smirnof test). The student t - test was applied to the bacterial cell viability data comparing each tested group with its respective control, and all r1 and r2 counterparts (same drug and drug ratio). One - way anova followed by tukey test was used for each resin to compare the bacterial cell viability among groups containing different drugs and ratios . Collected data were compiled and examined for relevance with the spss version 8.0 (spss inc ., chicago, il) statistical program . The mics of chx and egcg obtained using s. mutans ua159 strain were 2 g / ml and 700 g / ml, respectively . The 24 h drug release rates for r1 are: chx (0.5x mic=1.28 g / cm and 1x mic=2.31 g / cm and egcg (0.5x mic=6.37 g / cm and 1x mic=13.05 g / cm; and for r2 are: chx (0.5x mic=0.34 g / cm and 1x mic=0.82 g / cm and egcg (0.5x mic=1.69 g / cm and 1x mic=3.64 g / cm . The cell viability for drug - containing bis - gma / tegdma (r1) and bis - gma / ch3bis - gma (r2) the results demonstrated that both chx and egcg drugs retained their antimicrobial activity when integrated as part of r1 and r2 restorative materials . In each resin tested, there was no significant difference in bacterial growth inhibition between the treatment groups (chx 0.5x mic, chx 1x mic, and egcg 1x mic), with the exception of egcg at 0.5x mic that had significantly higher survival values (p<0.05). Moreover, when comparing r1 and r2 incorporated with the same drug and drug ratio, no significant difference was found within the groups except for egcg at 0.5x mic in r1 that had a significantly lower percentage of cell survival rate when compared to r2 . The initially proposed null hypothesis is therefore rejected . Effect of drug - incorporated resins on s. mutans survival percentage * anova and tukey's test; =0.05; s.d . : standard deviation; same upper case letters indicate no statistical difference within each line . The controlled release of drugs or bioactive agents from polymers by fickian diffusion, which is the spreading of solutes from regions of highest to regions of lower concentrations caused by the concentration gradient, has been the subject of research for many years . In dentistry, chx - containing resins have been successfully tested in terms of drug release and antibacterial activity . Most of these studies, however, use the agar plate method, where test samples are applied onto microorganisms inoculated at the surface of an agar plate . After appropriate incubation, the appearance and diameter of zones of growth inhibition around the test product indicate antimicrobial activity . In this study, the drug - containing resins were immersed into a bacterial suspension of the cariogenic organism s. mutans, which better mimics the physiological conditions in vivo . Moreover, these conditions allowed the resin samples to absorb water, swell, and have drugs diffused toward the surrounding solution from all surfaces . We demonstrated that polymerized r1 and r2 resins can effectively release incorporated chx and egcg, and that both drugs retain their antimicrobial activity upon incorporation into comonomers . Drug release from resins is a process affected by many factors including, monomer type, degree of conversion, crosslinking density, drug type and concentration, and extracting media . Therefore, the fact that r1 and r2 resins containing egcg at 0.5x mic is less effective at inhibiting s. mutans growth compared to the egcg at 1x mic is in part explained by the lower amount of drug diffusing through the polymer chains into the extracting nutrient - rich bhi medium . Although statistically significant only for egcg at 0.5x mic, lower cell survival rates were generally observed in response to r1 treatment groups compared to the r2 counterparts . The ch3bis - gma analog exhibits higher hydrophobicity compared to bis - gma due to the absence of hydrogen bonding in the system as a result of the -oh substitution for the -ch3 group . Conversely, tegdma is a very hydrophilic monomer due to the presence of linear ether linkages (figure 1a, 1b, 1c). As water penetrates more efficiently into the r1 matrix leading to larger expansion of voids between the polymer chains, more elutable species including the incorporated drugs the fact that the more hydrophobic resin (ch3bis - gma - based) released less amounts of drugs in the given time range of 24 h, may suggest extended release in the long term . The mic values for chx and egcg against s. mutans obtained in this study were within reference ranges; 0.25 - 4.0 g / ml for chx and 31.25 - 625 g / ml for egcg, depending on the bacterial strain and culture medium . After 24 h of incubation in the presence of drug - incorporated monomers (at sub - mic and mic), we demonstrated that both drugs were released from the test comonomers and efficiently inhibited bacterial growth; chx being the most effective . Chx is a bisguanide cationic broad - spectrum antimicrobial compound, active against gram - positive and gram - negative bacteria . Chx electrostatically binds to the negatively charged bacterial surface and then forms pores or disrupts the membrane . At low concentrations, chx has a bacteriostatic effect causing low molecular weight substances to leak out without damaging the cell irreversibly, while at high concentrations, chx causes precipitation of cytoplasm exerting a bactericidal effect . There is still a lack of sufficient data and information on the antibacterial mode of action of catechins . It is, however, recognized that these compounds have binding affinities for serum proteins, which is shown by the decrease in antibacterial activity of tea in the presence of serum . The fact that slightly higher mic values of egcg were obtained in this work compared to other studies also suggest that proteins present in the nutrient - rich bhi medium may bind to green tea catechins reducing their antimicrobial activities . This might also explain the lower growth inhibition values of egcg in comparison to chx, despite the higher egcg release results . Nevertheless, one could expect that the interactions of egcg with proteins, causing significant distortion of their tertiary structure, may also account for the malfunction of certain bacterial enzymes . (2011) demonstrated that egcg inhibited biofilm formation of s. mutans which could be attributed to the interactions of egcg with glucosyltransferase enzymes, thus disrupting the formation and integrity of the oral biofilm . (2012) also demonstrated that egcg suppresses the gtfb, gtfc, gtfd genes associated with extracellular polysaccharide formation of s. mutans . The potential anticariogenic activity of egcg in clinical service clearly requires more research, since egcg has been shown to interact with salivary proteins . (2012) demonstrated that egcg inhibited the activity of alpha - amylase by non - competitive inhibition, indicating that egcg is effective at inhibiting the formation of fermentable carbohydrates involved in caries formation . Both egcg and chx retain their antibacterial activity when incorporated into the resin matrix . Although less effective at the sub - mic level, egcg in r1 and r2 resins significantly reduced cell survival of the cariogenic organism s. mutans, suggesting a novel alternative to synthetic chemicals . The evident antibacterial activity of egcg suggest novel approaches in the development of dental restorative materials to help control dental caries, the most common infectious disease affecting humans.
In particular, musculoskeletal pain and lower back muscle injuries in nurses working in the geriatric setting are very high because of the significant number of patient transfers to wheelchairs that involve lifting [14]. It has been reported that physical therapists working in rehabilitation settings who perform 610 patients transfers per day are 2.4 times more likely to develop lower back injuries than therapists who do not perform transfers . A previous study on occupational and physical therapists showed that transferring or lifting patients was associated with 26.6% of all injuries during work - related activities . Moreover, a study on various nursing work activities showed that during the transfer of patients, the nurses' heart rate (hr) increased to approximately 125 beats / min (bpm) and they had higher levels of neuromuscular fatigue . Therefore, the transfer of patients to wheelchairs produces increased burden on the musculoskeletal and cardiovascular systems through changes in joint range of motion (rom), muscle activity, and hr . Therefore, there is a higher probability of musculoskeletal and physical strain in healthcare workers who transfer patients . Several interventions have been reported to decrease musculoskeletal injuries in healthcare workers [79]. In fact, the implementation of safe patient handling and movement policies by the nursing profession has dramatically decreased work - related injuries and chronic pain . Nurses who are more skilled in patient transfer increase the patients' perceptions of safety and comfort during transfers . Some previous literature reported the efforts to decrease musculoskeletal injuries; however, strong evidence for the effectiveness of intervention is lacking . On the other hand, following the adoption of no - lift policies, transfer robotic devices have emerged as tools that have the potential to prevent injuries in healthcare workers . Robotic lift and powered devices may decrease both the patients' effort and the clinicians' physical burden . One tool emerging from these initiatives is a battery - powered sit - to - stand transfer device that safely lifts and lowers patients between the seated and standing positions [11, 12]. Transfer of patients with disabilities who are unable to contribute their own effort depends on powered robotic devices . However, the patients' physical activity or motivation may increase if the patients can transfer themselves using these assistance devices . Recently, a new robotic wheel chair (rwc) has been developed which enables patients to be transferred directly in the sitting position using their own effort and the assistance of a healthcare worker . The manual transfer of disabled patients from the bed to a conventional wheelchair (cwc) is demanding and involves complex movements . Patient - handling tasks involved with a cwc can be classified into 3 groups: lifting of the patient, repositioning or turning from the bed towards the direction of the wheelchair, and seating the patient safely in the chair . However, the rwc involves only 1 transfer step, which is that the assistant pushes the patient sitting on a bed forward in the same position to the seat of the rwc . Therefore, the rwc may decrease the complexity of transfer and decrease physical load during transfer for healthcare workers . The purpose of this experimental study was to investigate the burden on healthcare workers by measuring rom, muscle activity, and hr during transfer of a simulated patient using either the rwc or a cwc . Ten females adults were recruited from an acute hospital and included 6 nurses and 4 rehabilitation therapists who had work experience in transferring patients (mean age: 32.2 9.3 years; range: 2347 years; body weight: 48.8 4.7 kg; height: 157.2 6.7 cm; bmi: 19.7 1.3 kg / m). Another female adult (age: 27 years; body weight: 49.0 kg; height: 153.0 cm; bmi: 20.9 kg / m) participated in the study acting as the simulated patient who was transferred from bed to the wheelchairs . The simulation was assumed to be a right hemiplegia patient whose right upper extremity was fixed in a sling . Instructions for this procedure were provided by a researcher as, please do not encourage movement of your right lower extremity during transfer to the wheelchair . All the participants provided written, informed consent, and the study was approved by the local ethics committee of the faculty of medicine, tottori university (number 2292). Three - dimensional (3d) motion analysis (myomotion analysis system; noraxon usa inc ., arizona, usa) consists of combined motion sensors, surface electromyography (emg), and synchronized video recordings . Signals of the subjects from these systems were digitally recorded (200 hz and 1500 hz and 30 hz, resp . ). In addition, the hr of each subject was measured via a wireless chest - strap electrocardiogram (ecg) monitor (dynascope; fukuda denshi co., ltd ., the motion sensor used inertial measurement units, which are widely recognized as a means to overcome the disadvantages of existing optical motion capture systems . The device can measure various kinematic parameters, such as object orientation and velocity, using accelerometers, gyroscopes, and magnetometers . The system has a measurement accuracy of 0.4 degrees for static measurements and 1.2 degrees for dynamic measurements . A standard cwc (matsunaga, co., ltd ., the arm and foot supports could swing out upwards (seat width, 40 mm; front height, 42 cm; total length, 95.5 cm; weight, 18 kg). The seat moves back and forth and has an elevating mechanism to adjust the height of the patient (width, 720 mm; length, 750 mm; minimum turning radius, 360 mm; weight, approximately 80 kg; battery, lithium - ion battery) (table 1 and figure 2). The motion sensors used for rom measurements during transfer of the simulated patient were placed on the seventh cervical, seventh thoracic, and fifth lumbar vertebrae and bilaterally on the upper arm, forearm, thigh, shank, and forefoot (figure 3). Calibration of the motion sensors was performed before the measurements using the segment model in the standing position . Emg electrodes were secured over the muscle bellies of both sides of the biceps, vastus medialis, upper back, and lower back muscles using standard techniques . Following practice, an emg signal was recorded during maximum isometric manual testing of each muscle . The signals from the motion sensors, emg, and hr were recorded simultaneously in both experimental conditions (i.e., transfer using either the rwc or the cwc), while the subjects performed the following tasks, once in each situation and in random order: (1) cwc: the subjects supported the trunk of the simulated patient sitting on a bed and lifted the patient to a standing position, converted the patient's position toward the direction of the wheelchair, and seated the patient safely; (2) rwc: the subjects, located on the right side of the simulated patient who was sitting on a bed, supported the pelvis of the patient and pushed the patient directly onto the seat of the rwc . Transfer of the simulated patient was assisted until the patient was positioned on the seat (figure 4). The rwc was located at the front of the patient with the height of the seat adjusted to the patient's sitting position using the robotic elevation system . Arizona, usa) was used to analyze the signal processing of the motion sensors, emg, and video recordings . In both experimental conditions, the onset and cessation of rom analysis, emg, and ecg were defined as the start of assistance until the end of assistance to transfer the simulated patient and were determined by visual interpretation of the video recordings . Rom of the upper extremity, trunk, and lower extremity segments were calculated during both experimental conditions . In both situations, real - time rom during patient transfer was analyzed using data obtained between 2 motion sensors; for example, right elbow - joint motion was analyzed using integrated signals of the accelerometers, gyroscopes, and magnetometers between the right upper arm and right forearm sensors, and the peak rom was identified . For each muscle, the emg data was integrated over 0.01 s intervals during each experimental condition and then normalized for each muscle's emg signal of maximum voluntary contraction (mvc) which was recorded during maximum isometric manual test . Mean muscle activity, expressed as% mvc, and mean hr were calculated during both experimental conditions . All statistical analyses were performed using spss for windows version 22 (ibm, co., ltd ., table 2 shows the comparison of the motion analyses for cwc and rwc transfer . The peak rom of both shoulder flexion and left ankle abduction during assistive transfer to the rwc were significantly lower than with the cwc . Left shoulder abduction, right shoulder rotation, and left knee flexion were significantly higher with the rwc than with the cwc . Table 3 shows the comparison of the muscle activation analysis for cwc and rwc transfer . The% mvc of the right biceps, the left upper back muscles, the left lower back muscles, and the right vastus medialis muscle were significantly lower with the rwc than with the cwc . The% mvc of the left biceps was significantly higher with the rwc than with the cwc . There was a significant difference in mean hr during transfer between the 2 conditions (rwc, 87.1 10.9 bpm versus cwc, 99.2 13.2 bpm; p = 0.006). We performed power diagnoses for the tests with relevant outcomes and checked that most of them would have sufficient statistical power, for example, shoulder flexion (left 62.4%, right 67.6%), shoulder abduction (left 98.7%, right 8.5%), upper back muscles (left 99.2%, right 10.0%), lower back muscles (left 99.9%, right 20.5%), and hr (88.6%). Therefore, our statistician considers that the sample size was valid from a statistical perspective . Our study showed that transferring a simulated patient from bed to the rwc decreased the rom on shoulder flexion, back muscle activity, and hr in the subjects compared to using a cwc . These findings suggest that the rwc may have the advantage of decreased muscle activity of the leg or back muscles during transfer compared with the cwc . This is because the rwc enables healthcare workers to push the patient forward in the sitting position; they may not have to lift the patients . In addition, transferring patients using a cwc involves 3 steps: lifting the patient, turning towards the direction of the wheelchair, and seating the patient safely in the chair . This adds complexity and requires the clinician to use more technical methods when transferring the patient . In contrast, the rwc does not involve lifting the patient, who instead can be transferred in 1 step using their own effort to push forward onto the seat of the rwc ., the patient does not need to change direction while in the standing position as is the case with the cwc . Therefore, transferring to the front using the rwc may decrease the physical load on healthcare workers who often have to perform many patient transfers during a single working day . The peak rom values in the motion analysis of the subjects during transfer to the rwc showed lower shoulder flexion and left ankle abduction compared with transfer to the cwc . Transfer of patients from bed to the rwc does not involve lifting and also the foot position while sitting is neutral . In contrast, the technique for transfer to a cwc places higher demand on the shoulder flexion required to lift the patient, and a stride standing position and lower extremity abduction to maintain standing balance . It is not necessary to increase these rom values when transferring a patient to a rwc . On the other hand, rom of left shoulder abduction, right shoulder rotation, and right knee flexion were higher with the rwc . During transfer to the rwc, healthcare workers flex their knees to transfer the patient in the sitting position compared with a standing transfer . Shoulder abduction and rotation occur during transfer to the rwc because of the need to hold the pelvis of the patients using both arms . However, pushing the patient forward in the rwc may place a burden on the upper extremities . It is therefore important to recognize the potential of shoulder abduction and rotation muscle overload during transfers using the rwc . With the rwc, lower back muscle activity and lower mean hr during transfers were observed on emg and ecg analyses . In the present study, analysis of the left back muscles during transfer from bed to the cwc showed 48%66% mvc demand in the subjects . In a previous study, emg analysis of the lower back muscles during transfer of patients to we observed similar findings in our study, with the rwc decreasing left back muscle activity to a greater extent (i.e.,% mvc of 29%35%). It is also possible that hr increases because of increased muscle activity with a cwc compared to a rwc . In fact, a previous study showed that nurses rated patient lifting, transfer, and turning as the most physically demanding activities . We suggest that this is an important advantage of the rwc compared to the cwc because of the need to decrease back muscle injury and cardiovascular stress during clinical work [4, 6]. Transfer from bed to the rwc requires healthcare workers to bend rather than rotate their trunks, without lifting the patients . This suggests that the rwc provides a simple transfer technique in which it is possible to transfer the load to the upper extremity when transferring patients with higher body weight . The clinical implication of the rwc is that it represents a motorized device for older adults who are physically frail with weakness of the lower extremities and enables them to stand and turn to the wheelchair during transfer . In addition, we hypothesize that transfer using the rwc is advantageous for patients with parkinsonism who cannot change their direction when standing . In this situation, we suggest that the rwc needs a robotic function or a sling to carry the patient in the sitting position to a seat . We believe that riding the rwc is suitable for patients with pressure ulcers on the sacral area because the rwc does not have a back support; patients are supported by the breast support and do not have pressure on the sacrum . However, there is no available evidence regarding the pressure of the breast support or the seat . The weakness of the rwc is that this device does not have a back support, although the seat leans forward so that subjects do not fall backwards on the rwc . Thus, the use of this device is limited to those who have the ability to sit or stand with assistance . Additionally, transferring from the rwc to the cwc or bed is a weak point of this device because subjects who ride on the rwc must turn around and look behind in order to return to the bed . The rwc may need a rearview mirror or an automatic navigation system to correct this problem . Therefore, the values obtained in our data analysis may not be applicable to the transfer of patients with paralysis, dysfunction of the lower extremities, or fractures . In addition, the sample size (n = 10) might not have been sufficiently large . However, we performed power diagnoses for the tests with relevant outcomes and checked that most of them would have sufficient statistical power . Also, as a result of the detailed experimental measurements that we made for all subjects, sufficient objective data for various indices and evidence that transfer of patients in the sitting positon clearly caused less physiological burden were obtained although repetitive load was not studied . The group of patients that would benefit most from transfers in the rwc and the associated implications are not addressed in the present study . Lastly, the study did not investigate muscle activity and psychological burden in the simulated patient . Shoulder flexion rom, activity of the back muscles, and hr were decreased in the subjects when transferred from bed to the rwc . The rwc enables patients to be transferred in the sitting position directly to the frontal position . It is possible that the rwc will decrease workplace injuries and lower back pain in healthcare workers . Using an assisted device to transfer patients without manual lifting has obvious benefits in healthcare and rehabilitation settings.
Deep venous thrombosis (dvt) is a common condition that affects approximately 1 in 1000 persons / year . Dvt has a prevalence of 10.5 - 11.9% in patients who are admitted in intensive care unit (icu) among indian patients . Upper limb dvt is increasingly being recognized as a clinical entity with potential for considerable morbidity . It usually refers to thrombosis of the axillary or subclavian veins, occurs spontaneously or sometimes develops as a complication of pacemaker use, long - term central venous catheter (cvc) use, or cancer . Approximately, 10% of all cases of deep - vein thrombosis involve the upper extremities, resulting in an annual incidence of 0.4 - 1 case/10,000 people . Here, we are presenting two cases who had developed upper limb dvt while resolving phase of their sepsis with reactive thrombocytosis . A 37-year - old man referred to our icu with acute respiratory distress syndrome (ards) and septic shock on the 23 day of his illness . He had h1n1 influenza viral infection, confirmed by reverse transcription polymerase chain reaction method . At admission, vitals were: heart rate 140/min, blood pressure (mean) 70 mmhg on noradrenaline infusion (20 g / min). His ventilatory requirement was positive end - expiratory pressure (peep) of 14 cm h2o and fio2 of 0.70 with pao2/fio2(p / f) ratio 160 . His laboratory parameters on admission was hemoglobin 12 g / dl, total leukocyte count (tlc) 18,000 and platelet count of 165 (10/l). Before admission, he had platelet count ranging from 83 to 163 (10/l). Lung protective ventilation was continued and prone position ventilation sessions were given to improve p / f ratios . By day 7 of icu stay his hemodynamics improved with no further need of vasopressor . During follow - up, he had thrombocytosis during 2 week (622 - 707 10/l). He was receiving dvt prophylaxis in appropriate doses . On day 14 of his stay swelling, was noted in the right upper limb . Bedside doppler ultrasonography of bilateral jugular veins revealed thrombosed right internal jugular vein (ijv) suggestive of acute thrombus with no flow along entire length of cervical ijv extending into right subclavian vein . Magnetic resonance imaging venogram confirmed the findings; and thromboelastography (teg) was done, which showed hypercoagulability [figure 1]. He was started on injection enoxaparin 60 mg subcutaneous 12 hourly, with antiplatelet (aspirin). Later on, he had an uneventful course in the icu and was discharged on oral anti - coagulant . The thrombus had recanalized with no residual limb edema and his platelet counts were back to normal . (a) the magnetic resonance venogram depicting thrombus in right internal jugular vein extending to right subclavian vein . (b) thromboelastogram showing hypercoagulability the second case is about a 29-year - old male patient who was admitted on the 12 day of his illness with acute viral encephalitis . On admission in icu, vitals were: heart rate 110/min, mean blood pressure 82 mm of hg (on noradrenaline infusion 10 g / min). He required ventilatory support due to encephalopathy, and kept on synchronized intermittent mandatory ventilation mode with peep of 6 of cm h2o . His arterial blood gas showed ph 7.34, po2100 mmhg and pco242 mmhg on fio2 of 0.4 with p / f ratios around 250 . He was fluid resuscitated along with other supportive care, which resulted in improvement in shock and stopping of vasopressor within 48 h of admission . His laboratory parameters were hemoglobin 10 g / dl, tlc 13,000, platelet 140 (10/l), creatinine 0.7 mg / dl . He continued to stay in the icu in the view of difficult weaning due to his muscle weakness . He showed persistent thrombocytosis (537 - 827 10/l) after 2 weeks of admission . On day 46 in icu swelling was noted in his right arm . Doppler of his upper limb showed evidence of thrombosis in both right subclavian vein and right ijv [figure 2]. He was started on therapeutic doses of low molecular weight heparin along with aspirin . In at follow - up after 1-month, his thrombosis gradually resolved with recanalization of the vein, decrease in limb edema, and he is still on oral anti - coagulant . (a) doppler ultrasonography showing thrombus in right internal jugular vein with no flow . A 37-year - old man referred to our icu with acute respiratory distress syndrome (ards) and septic shock on the 23 day of his illness . He had h1n1 influenza viral infection, confirmed by reverse transcription polymerase chain reaction method . At admission, vitals were: heart rate 140/min, blood pressure (mean) 70 mmhg on noradrenaline infusion (20 g / min). His ventilatory requirement was positive end - expiratory pressure (peep) of 14 cm h2o and fio2 of 0.70 with pao2/fio2(p / f) ratio 160 . His laboratory parameters on admission was hemoglobin 12 g / dl, total leukocyte count (tlc) 18,000 and platelet count of 165 (10/l). Before admission, he had platelet count ranging from 83 to 163 (10/l). Lung protective ventilation was continued and prone position ventilation sessions were given to improve p / f ratios . By day 7 of icu stay his hemodynamics improved with no further need of vasopressor . During follow - up, he had thrombocytosis during 2 week (622 - 707 10/l). He was receiving dvt prophylaxis in appropriate doses . On day 14 of his stay swelling, was noted in the right upper limb . Bedside doppler ultrasonography of bilateral jugular veins revealed thrombosed right internal jugular vein (ijv) suggestive of acute thrombus with no flow along entire length of cervical ijv extending into right subclavian vein . Magnetic resonance imaging venogram confirmed the findings; and thromboelastography (teg) was done, which showed hypercoagulability [figure 1]. He was started on injection enoxaparin 60 mg subcutaneous 12 hourly, with antiplatelet (aspirin). Later on, he had an uneventful course in the icu and was discharged on oral anti - coagulant . The thrombus had recanalized with no residual limb edema and his platelet counts were back to normal . (a) the magnetic resonance venogram depicting thrombus in right internal jugular vein extending to right subclavian vein . (b) thromboelastogram showing hypercoagulability the second case is about a 29-year - old male patient who was admitted on the 12 day of his illness with acute viral encephalitis . On admission in icu, vitals were: heart rate 110/min, mean blood pressure 82 mm of hg (on noradrenaline infusion 10 g / min). He required ventilatory support due to encephalopathy, and kept on synchronized intermittent mandatory ventilation mode with peep of 6 of cm h2o . His arterial blood gas showed ph 7.34, po2100 mmhg and pco242 mmhg on fio2 of 0.4 with p / f ratios around 250 . He was fluid resuscitated along with other supportive care, which resulted in improvement in shock and stopping of vasopressor within 48 h of admission . His laboratory parameters were hemoglobin 10 g / dl, tlc 13,000, platelet 140 (10/l), creatinine 0.7 mg / dl . He continued to stay in the icu in the view of difficult weaning due to his muscle weakness . He showed persistent thrombocytosis (537 - 827 10/l) after 2 weeks of admission . On day 46 in icu swelling was noted in his right arm . Doppler of his upper limb showed evidence of thrombosis in both right subclavian vein and right ijv [figure 2]. He was started on therapeutic doses of low molecular weight heparin along with aspirin . In at follow - up after 1-month, his thrombosis gradually resolved with recanalization of the vein, decrease in limb edema, and he is still on oral anti - coagulant . (a) doppler ultrasonography showing thrombus in right internal jugular vein with no flow . A 37-year - old man referred to our icu with acute respiratory distress syndrome (ards) and septic shock on the 23 day of his illness . He had h1n1 influenza viral infection, confirmed by reverse transcription polymerase chain reaction method . At admission, vitals were: heart rate 140/min, blood pressure (mean) 70 mmhg on noradrenaline infusion (20 g / min). His ventilatory requirement was positive end - expiratory pressure (peep) of 14 cm h2o and fio2 of 0.70 with pao2/fio2(p / f) ratio 160 . His laboratory parameters on admission was hemoglobin 12 g / dl, total leukocyte count (tlc) 18,000 and platelet count of 165 (10/l). Before admission, he had platelet count ranging from 83 to 163 (10/l). Lung protective ventilation was continued and prone position ventilation sessions were given to improve p / f ratios . By day 7 of icu stay his hemodynamics improved with no further need of vasopressor . During follow - up, he had thrombocytosis during 2 week (622 - 707 10/l). He was receiving dvt prophylaxis in appropriate doses . On day 14 of his stay swelling, was noted in the right upper limb . Bedside doppler ultrasonography of bilateral jugular veins revealed thrombosed right internal jugular vein (ijv) suggestive of acute thrombus with no flow along entire length of cervical ijv extending into right subclavian vein . Magnetic resonance imaging venogram confirmed the findings; and thromboelastography (teg) was done, which showed hypercoagulability [figure 1]. He was started on injection enoxaparin 60 mg subcutaneous 12 hourly, with antiplatelet (aspirin). Later on, he had an uneventful course in the icu and was discharged on oral anti - coagulant . The thrombus had recanalized with no residual limb edema and his platelet counts were back to normal . (a) the magnetic resonance venogram depicting thrombus in right internal jugular vein extending to right subclavian vein . The second case is about a 29-year - old male patient who was admitted on the 12 day of his illness with acute viral encephalitis . On admission in icu, vitals were: heart rate 110/min, mean blood pressure 82 mm of hg (on noradrenaline infusion 10 g / min). He required ventilatory support due to encephalopathy, and kept on synchronized intermittent mandatory ventilation mode with peep of 6 of cm h2o . His arterial blood gas showed ph 7.34, po2100 mmhg and pco242 mmhg on fio2 of 0.4 with p / f ratios around 250 . He was fluid resuscitated along with other supportive care, which resulted in improvement in shock and stopping of vasopressor within 48 h of admission . His laboratory parameters were hemoglobin 10 g / dl, tlc 13,000, platelet 140 (10/l), creatinine 0.7 mg / dl . He continued to stay in the icu in the view of difficult weaning due to his muscle weakness . He showed persistent thrombocytosis (537 - 827 10/l) after 2 weeks of admission . On day 46 in icu swelling was noted in his right arm . Doppler of his upper limb showed evidence of thrombosis in both right subclavian vein and right ijv [figure 2]. He was started on therapeutic doses of low molecular weight heparin along with aspirin . In at follow - up after 1-month, his thrombosis gradually resolved with recanalization of the vein, decrease in limb edema, and he is still on oral anti - coagulant . (a) doppler ultrasonography showing thrombus in right internal jugular vein with no flow . Thrombocytosis, that is, platelet count more than 450 10/l, can be primary or reactive (secondary) in etiology . Reactive thrombocytosis is common and reported in various conditions such as inflammation, postsplenectomy, hematopoietic disorder, surgery and cancer . In these conditions, elevated endogenous levels of interleukin-6 (il-6), interferon gamma (ifn-) are responsible for the thrombocytosis . Il-6 contributes to inflammatory thrombopoiesis predominantly by stimulating the hepatic production of thrombopoietin (tpo); while, ifn- has its effect on megakaryocytes growth . Furthermore, there is enhanced production of tpo, messenger ribonucleic acid and protein by stimulation of hepatocytes with il-6 . In icu patients' thrombocytosis is a commonly seen phenomenon, it's found in nearly 21% of patients admitted to an icu . Due to relatively lesser platelet count and smaller mean platelet volume, pathologic thrombus is not so common in reactive thrombocytosis in comparison to primary thrombocytosis; unless it is aggravated by underlying disease process or any condition peculiar to the patient, e.g. Arthrosclerosis . In the pathogenesis of dvt of upper extremity, presence of the indwelling catheter (cvcs, pacemaker, or defibrillator leads), cancer, surgery or trauma of the arm or shoulder are common and major risk factors . The prevalence of cvc - related upper limb dvt in cancer patients varies from 11.7% to 44% . In presented both cases, cvc were inserted in both neck vessels (ijv and subclavian vein) before diagnosis of dvt, and were not inserted after diagnosis of thrombosis and managed with catheter in the femoral vein . Presented both cases, though patients had a risk factor of having cvcs previously at the site of thrombosis; the association of increased platelet count cannot be ruled out in pathogenesis of thrombosis . In further evaluation, teg had increased maximum amplitude: 77.8 mm in the first patient and 78.1 mm in the second patient with a reference range 5166 mm . The angle was within normal limits depicting that the hypercoagulability is due to platelet and not enzyme activity . After the detection of dvt these patients were given the therapeutic doses of anti - coagulant for dvt along with anti - platelet (aspirin). There was no evidence of any more dvt or any other site of thrombosis in both these patients . Intensive care unit patient having reactive thrombocytosis during the recovery phase of sepsis must be kept in priority for screening to rule out dvt, so that timely therapeutic intervention could avoid a life - threatening pulmonary embolism.
Divided vascular lesions in the maxillofacial area into two groups: hemangioma and vascular malformations . They are more common in women than men (3:1). About 60% of hemangioma lips, tongue, and buccal mucosa are the most common sites of involvement . However, it is more likely to occur in the gingiva, mandible, palate, floor of the mouth, and parotid gland . This report introduces a rare incidence of hemangioma in the buccal fat pad (bfp) along with phlebolithiasis . The patient was a 28-year - old woman who referred with the chief complaint of a swelling and stiffness in the left cheek . From a clinical perspective on palpation, a moving mass with a stiff area was felt; and in intraoral examination, its position was felt in the anterior ramus . The only point in the patient's history was a course of laser therapy for skin rejuvenation in the left cheek and several other areas in her face . In the medical history of the lesion, there were 3 times of triamcinolone injection in the area during the last 3 years to treat the lesion by another physician . The patient said that reduction in tumor size was seen for a while after these injections . Aspiration was performed for the patient through intraoral approach, whose result was negative . Magnetic resonance imaging (mri) magnetic resonance imaging revealed a solid heterogeneous mass in the pterygopalatine fossa area with penetration and extension towards both buccal and masticator spaces on the left . The submandibular area and carotid space were normal and no abnormality was seen in the nasopharynx area . Mri revealed a solid heterogeneous mass in the pterygopalatine fossa area with penetration and extension toward both buccal and masticator spaces on the left . (b) coronal view of t1 and t2 according to the results of clinical and radiographic examinations of the treatment plan, the excisional biopsy of the studied mass was selected under complete anesthesia . Under general anesthesia, the patient underwent a surgery with intraoral access through a cut in the upper area of anterior ramus . After dissection in the upper - side direction, vascular lesion was seen in buccal extension of the bfp [figure 2]. The bfp capsule was intact and the mass had offended no soft / hard adjacent tissue . The possibility of a vascular lesion and second, for the purpose of liposuction for cosmetic goal and remove of the swelling on the patient's cheek, the vascular mass was removed along with anterior lobe, as excisional biopsy through intraoral approach and hence that we did not enter into the vascular lesion [figure 3]. The clinical swelling of the cheek was removed immediately after the surgery . In the macroscopic viewpoint, the lesion was a yellow and dark purple mass measuring 2 cm 3 cm 4 cm along with a hard nodule - like area . Microscopic results represented a vascular lesion composed of large amounts of small to large vascular structures covered with endothelial cells . View of the totally excised lesion showing hemangioma with phlebolith histopathologic view of the lesion showing vascular lesion composed of large amounts of small to large vascular structures covered with endothelial cells . Hemangiomas usually appear within a few weeks after birth and have a growth rate that exceeds the growth rate of children . In this growth phase, hemangioma will have its own characteristics: endothelial cells getting fatter along with frequent mitotic division, increased number of mast cells, and multilayer basement membrane . Following this stage, flat and inactive endothelial cells are located in a context called fibrous fatty tissue with a normal view . Sometimes, they can even involve all layers of the skin and offend the muscles . At the cellular level, hemangiomas are characterized by increased birth and death rate of endothelial cells and proliferation of mastocytes during the postnatal proliferative phase in the lesion . Derived from young proliferating hemangiomas, capillary endothelium is easily grown in cell culture mediums and forms tubes . In accumulated hemangiomas, hence, a normal hemangioma is an endothelial tumor with a very complex life cycle of cell proliferation and natural regression . . However, their clinical manifestations are not obvious sometimes until late infancy or even childhood . Phleboliths consist of a mixture of calcium carbonate and calcium phosphate salts and are thought to form when a fibrous component attaches to a developing phlebolith and becomes calcified . Radiologically, they have either a radiolucent or a radiopaque core, and repetition of this calcification causes an onion - like appearance or concentric rings . It should be noted that bfp is also a mass composed of fat tissue covered with a thin capsule membrane and is mainly located in the buccal space . Bfp has a rich blood supply and it is a proven fact that bfp has multipotential cells . However, there was no report about the incidence of hemangioma in the bfp until 1956 . Deighan and barton first pointed to the incidence of a hemangioma case with phlebolithiasis in the bfp mass in 1956 . After a review of english literature, only two cases of the incidence of hemangioma in the bfp mass were found, except the above case . Ikegami and nishijima reported the incidence of hemangioma in the bfp mass in a 23-year - old patient in 1984 . In that case, the tumor was enucleated and the presence of a cavernous hemangioma was confirmed . The last report on the incidence of hemangioma in the bfp was published by tanaka et al . In 2000 . The patient was a 3-year - old boy whose tumor was diagnosed 4 months after birth . Unlike the report of ikegami and nishijima in which the lesion surface was irregular, in this case, the lesion surface was reported smooth . However, the surface of the removed lesion was also irregular in this report . Like the case described in this report, no involvement was reported in the bfp mass adjacent areas in the previously reported cases . When radiographic examinations reveal a radiopaque lesion in the tumor, the differential diagnosis will be easier and there will be two possibilities: hemangioma or sialolithiasis of the parotid gland . However, when phlebolithiasis is not seen, preoperative diagnosis of hemangioma will be very difficult . In the report by tanaka et al ., the tumor was removed through extraoral access . However, in the extraoral access, the facial nerve is more likely to be damaged and risk of scar is present . However, tanaka et al . Suggested that they used extraoral access because they assumed the probability of large extension of the lesion . This shows the importance of careful radiographic examinations for accurate diagnosis of the lesion limits and the selection of appropriate surgical technique . Therefore, if the tumor does not have too large extension according to clinical and radiographic examinations, then intraoral access is preferred . In such cases, the use of only one single imaging modality cannot provide enough information with the physician about the diagnosis and treatment of vascular lesions . Therefore, the use of mri and computed tomography (ct) is recommended in these cases . The use of ultrasound / color doppler will also be very helpful for validating the mri and ct interpretational results . In general, the incidence of hemangioma in the bfp will be very rare, but in cases where this lesion is suspected, precise preoperative clinical and radiographic examinations are recommended.
The ink4a locus on human chromosome 9p21 encodes the p14arf tumor suppressor protein, which uses an upstream promoter and a shifted frame in exon 2 (compared to the p16 protein). The ink4a locus is often inactivated by deletion or by methylation in human melanomas, lymphomas, and other tumors . P14arf has the ability to suppress growth through multiple p53-dependent or p53-independent pathways [5 - 7]. P53 is a binding partner and ubiquitination target of hdm2 [9 - 11]. In cells that harbour wild type p53, p14arf can associate with p53 bound hdm2 forming tri - molecular complexes . In human tumor cell lines, p14arf is localized mainly to nucleoli . Nucleoli are assembled around clusters of repeated ribosomal genes, which are transcribed by rna polymerase i. the nucleoli are the sites of ribosomal biogenesis . It was suggested that p14arf targets hdm2 protein to the nucleoli, blocking the shuttling of hdm2 to the cytoplasm, and consequently enriching the nucleus with p53 protein . However, the literature data shows contradictory data about p14arf - hdm2 localization reporting that p14arf targets hdm2 to the nucleoli, hdm2 re - localizes p14arf to the nucleoplasm, or these two proteins are found in different compartments . In the present study, our goal is to verify the localization of p14arf protein and to compare it with the localization of other regulatory proteins, such as p53, hdm2, rb, p27, hsp70, and pml . We transfected p14arf, cloned into three different vectors: pbabe that did not contain any tag (pbabe - p14arf), and as a fusion with gfp at the n - terminus (gfp - p14arf) and with dsred at the c - terminus (p14arf - dsred) into mcf7, saos-2 and nih3t3 cells lines . The three different constructs produced proteins with very similar localization patterns in all the three cell lines . Pictures that were taken on mcf7 cells are used to illustrate the findings of this paper . We found that p14arf protein accumulated in the nucleoli and/or in extra - nucleolar nucleoplasmic inclusions of variable sizes (figure 1). These p14arf nuclear inclusions were negative for the nucleolar marker b23 (figure 2) and were not surrounded by perinucleolar heterochromatin . It was difficult to distinguish nucleoli from the extranucleolar inclusions using only phase contrast microscope . The p14arf nuclear inclusions were visible on the phase contrast as dark bodies in similar manner as nucleoli . We found however that staining of the perinucleolar heterochromatin with hoechst 33258 was a reliable marker to routinely discriminate between nucleoli and extranucleolar inclusions . Full - length p14arf expressed from the retroviral vector pbabe (pbabe - p14arf) is localized to the nucleoli (full arrowheads) and to extra - nucleolar inclusions of mcf7 cells (hollow arrowheads). Double staining for b23 and pbabe - p14arf shows that pbabe - p14arf forms extranucleolar inclusions as well as localizes to nucleoli in mcf7 cells . After p14arf transfection, we observed that pml bodies were re - distributed to the p14arf nuclear inclusions (figure 3). Here we demonstrated a striking difference in the properties of the nucleolar p14arf and p14arf that formed extranucleolar inclusions . Pml bodies are randomly scattered in nuclei where pbabe - p14arf exclusively localized to nucleoli (top row) but gradually associate with the p14arf positive extranucleolar inclusions in parallel with their expansion (25raw) in mcf7 cells . In order to investigate whether the p14arf nuclear inclusions could be targets of the degradation pathway, we have stained the transfected cells for the 20s core subunit of the proteasomes . The 20s subunits of the proteasomes were localized mainly to the cytoplasm of mcf7 cells . When p14arf was expressed in cells, the 20s core subunits of the proteasomes were targeted to the nucleus and co - localized with p14arf in the extra - nucleolar inclusions (figure 4). Remarkably, the proteasomes were not associated with p14arf when it was present in the nucleoli . 20s proteasomes are targeted to pbbae - p14arf positive nuclear inclusions (hollow arrowheads) but not to the nucleoli (full arrowheads) in mcf7 cells . One of the stress response proteins, hsp70, is often redistributed both to the nucleus and to the nucleolus during periods of cell stress . Here we show that in those p14arf transfected cells where p14arf accumulated in the extranucleolar inclusions hsp70 was targeted to the nucleus and showed a high grade of co - localization with p14arf extranuclear inclusions (figure 5). Hdm2 protein (red) is re - distributed to pbabe - p14arf (green) nuclear inclusions, but not to nucleoli that contain p14arf in mcf7 cells . We stained the cells for both p53 and hdm2 because these two proteins were shown to form a tri - molecular complex with p14arf protein . Neither protein was detected in nucleoli, even if p14arf was localized to the nucleoli (figures 6 and 7). Formation of the p14arf inclusions has no effect on the distribution of other nuclear proteins such as prb or p27 (data not shown). Transfection of p14arf into mcf7 cells that contain wild type p53 and hdm2 but lack endogeneous p14arf did not induce increased expression of p53 . P53 protein (red) co - localizes with pbabe - p14arf (green) in the extra - nucleolar inclusions in mcf7 cells . Pbabe - p14arf extra - nucleolar inclusions, but not the nucleoli, are the sites for the accumulation of hsp70 protein in mcf7 cells . We transfected p14arf, cloned into three different vectors: pbabe that did not contain any tag (pbabe - p14arf), and as a fusion with gfp at the n - terminus (gfp - p14arf) and with dsred at the c - terminus (p14arf - dsred) into mcf7, saos-2 and nih3t3 cells lines . The three different constructs produced proteins with very similar localization patterns in all the three cell lines . Pictures that were taken on mcf7 cells are used to illustrate the findings of this paper . We found that p14arf protein accumulated in the nucleoli and/or in extra - nucleolar nucleoplasmic inclusions of variable sizes (figure 1). These p14arf nuclear inclusions were negative for the nucleolar marker b23 (figure 2) and were not surrounded by perinucleolar heterochromatin . It was difficult to distinguish nucleoli from the extranucleolar inclusions using only phase contrast microscope . The p14arf nuclear inclusions were visible on the phase contrast as dark bodies in similar manner as nucleoli . We found however that staining of the perinucleolar heterochromatin with hoechst 33258 was a reliable marker to routinely discriminate between nucleoli and extranucleolar inclusions . Full - length p14arf expressed from the retroviral vector pbabe (pbabe - p14arf) is localized to the nucleoli (full arrowheads) and to extra - nucleolar inclusions of mcf7 cells (hollow arrowheads). Double staining for b23 and pbabe - p14arf shows that pbabe - p14arf forms extranucleolar inclusions as well as localizes to nucleoli in mcf7 cells . After p14arf transfection, we observed that pml bodies were re - distributed to the p14arf nuclear inclusions (figure 3). Here we demonstrated a striking difference in the properties of the nucleolar p14arf and p14arf that formed extranucleolar inclusions . Pml bodies are randomly scattered in nuclei where pbabe - p14arf exclusively localized to nucleoli (top row) but gradually associate with the p14arf positive extranucleolar inclusions in parallel with their expansion (25raw) in mcf7 cells . In order to investigate whether the p14arf nuclear inclusions could be targets of the degradation pathway, we have stained the transfected cells for the 20s core subunit of the proteasomes . The 20s subunits of the proteasomes were localized mainly to the cytoplasm of mcf7 cells . When p14arf was expressed in cells, the 20s core subunits of the proteasomes were targeted to the nucleus and co - localized with p14arf in the extra - nucleolar inclusions (figure 4). Remarkably, the proteasomes were not associated with p14arf when it was present in the nucleoli . 20s proteasomes are targeted to pbbae - p14arf positive nuclear inclusions (hollow arrowheads) but not to the nucleoli (full arrowheads) in mcf7 cells . One of the stress response proteins, hsp70, is often redistributed both to the nucleus and to the nucleolus during periods of cell stress . Here we show that in those p14arf transfected cells where p14arf accumulated in the extranucleolar inclusions hsp70 was targeted to the nucleus and showed a high grade of co - localization with p14arf extranuclear inclusions (figure 5). Hdm2 protein (red) is re - distributed to pbabe - p14arf (green) nuclear inclusions, but not to nucleoli that contain p14arf in mcf7 cells . We stained the cells for both p53 and hdm2 because these two proteins were shown to form a tri - molecular complex with p14arf protein . Neither protein was detected in nucleoli, even if p14arf was localized to the nucleoli (figures 6 and 7). Formation of the p14arf inclusions has no effect on the distribution of other nuclear proteins such as prb or p27 (data not shown). Transfection of p14arf into mcf7 cells that contain wild type p53 and hdm2 but lack endogeneous p14arf did not induce increased expression of p53 . P53 protein (red) co - localizes with pbabe - p14arf (green) in the extra - nucleolar inclusions in mcf7 cells . Pbabe - p14arf extra - nucleolar inclusions, but not the nucleoli, are the sites for the accumulation of hsp70 protein in mcf7 cells . P14arf protein is considered to be mainly nucleolar protein that directly interacts with hdm2 and regulates the expression of the tumor suppressor protein p53 . Experiments on cell hybrids suggested that p14arf sequesters hdm2 in the nucleolus . On the other hand it was also shown that overexpressed hdm2 protein could re - locate p14arf into the nucleoplasm in the presence of wild - type p53 . We have detected the formation of nuclear inclusions by p14arf that was expressed from three different vectors, 2472 hours after transfection . The nuclear inclusions were very similar in size, and in appearance in phase contrast microscope, to the nucleoli . The lack of b23 protein and the absence of surrounding perinuclear heterochromatin clearly indicated that the inclusions were extranucleolar structures . Pml bodies are interferon inducible, multifunctional nuclear organelles that are involved in a number of cellular processes such as antiviral defence, mhc class i dependent antigen presentation and regulation of gene expression . Pml bodies also likely play a role in the regulation of the degradation of nuclear proteins . For example mutant, misfolded influenza nucleoprotein accumulates in pml bodies in cells where proteasome mediated degradation is inhibited suggesting that pml bodies function as the nuclear analogues of the cytoplasmic aggresomes . Accumulation of mutant polyq - containing cellular proteins in polyglutamine (polyq) neurodegenerative diseases leads to the relocalization of pml bodies to the polyq inclusions . We also observed that the p14arf nuclear inclusions, but not the p14arf containing nucleoli, showed accumulation of hsp70 protein and were targeted by proteasomes . Importantly hdm2 and p53 also accumulated in p14arf inclusions but when p14arf was localized to the nucleoli, p53 or hdm2 co - localization was not detected . Our data suggests that p14arf, although it often accumulates in the nucleolus, does not sequestrate hdm2 in the nucleolus . P14arf / hdm2 complexes also contain p53 and are targeted for proteasome mediated degradation with the help of pml bodies . Overexpression of p14arf leads to the complete entrapment of hdm2 and p53 intro extranucleolar inclusion bodies . Our data show that co - localization between p53, hdm2 and p14arf occurs at extranucleolar sites . Accumulation of pml bodies and proteasomes at these sites suggest that the components of the nuclear inclusions are targeted for proteasome - mediated degradation . The following plasmids were used: full - length p14arf cdna (encoding 132 residues), cloned in pbabe vector (gift of klas g. wiman cck ki, stockholm); and full - length p14arf fused to gfp in pegfp - c1 and to dsred in pdsred2 (both clontech) in c - terminal and n - terminal positions, respectively . In this study, mcf7, a breast cancer cell line, saos-2, an osteosarcoma cell line, and nih3t3, mcf7 cells expressed wild - type p53 at low levels, hdm2 at moderate levels and had a deleted p14arf gene . Saos-2 cells had a homozygous deletion at the p53 loci and expressed moderate level of p14arf . Transfections were performed using the lipofectamine plus reagent and fugene (life technologies) according to the manufacturer's protocol . The following primary antibodies were used: mouse monoclonal antibody (mab) against b-23/nucleophosmin (npm), a kind gift of p. k. chan (baylor college of medicine, houston), mab against hdm2 ab-1, clone smp14 (dako), mab against heat shock protein 70 (hsp70) (w27) (santa cruz biotechnology), mab against pml protein (pg - m3) (santa cruz biotechnology), mab against 20s proteasome (clone hp810) (affiniti research product ltd), mab against p14arf, 240 (supernatant), a kind gift of j. bartek (danish cancer society), mab against p53 do7 (bd pharmingen), rabbit polyclonal anti - p14arf, a kind gift of k. wiman (cck, ki, stockholm). The following secondary antibodies were used: horse anti - mouse ig texas red conjugated (vector lab), rabbit anti - mouse ig fitc (dako), swine anti - rabbit fitc (dako), swine anti - rabbit tritc (dako). Bisbenzimide (hoechst 33258 from sigma) was added at a concentration of 0.4 g / ml to the secondary antibody for dna staining when needed . Cells were stained on coverslips after fixation in a mixture of methanol and acetone (1:1) at -20c . Images were captured using a das microscope leitz dm rb with a hamamatsu dual - mode cooled charge - coupled device (ccd) camera c4880 . The 3d immunofluorescence images were generated from the reconstitution of a series of de - blurred optical sections . Briefly, the images were captured using a pxl cooled ccd camera (photometrix) on a zeiss axiophot microscope equipped with z - axis motor, external shutter and excitation filter - wheel, controlled by a mac2000 lep module . The computer program, st - rfh - bin2, that controlled the image acquisition hardware and produced the digital images was developed by us (szekely, unpublished). The program captured three times 1317 images from three excitation series (rhodamine - fitc - hoechst) with focal planes 0.3 m apart . The incoming images were automatically corrected for dark current noise and optical shining through, and de - blurred using a nearest neighbour de - convolution algorithm . The resultant image stacks were projected using the maximum intensity projection algorithm producing a single - direction and stereo - pair projected images as described . The following plasmids were used: full - length p14arf cdna (encoding 132 residues), cloned in pbabe vector (gift of klas g. wiman cck ki, stockholm); and full - length p14arf fused to gfp in pegfp - c1 and to dsred in pdsred2 (both clontech) in c - terminal and n - terminal positions, respectively . In this study, mcf7, a breast cancer cell line, saos-2, an osteosarcoma cell line, and nih3t3, a mouse immortalized fibroblasts were used . Mcf7 cells expressed wild - type p53 at low levels, hdm2 at moderate levels and had a deleted p14arf gene . Saos-2 cells had a homozygous deletion at the p53 loci and expressed moderate level of p14arf . Transfections were performed using the lipofectamine plus reagent and fugene (life technologies) according to the manufacturer's protocol . The following primary antibodies were used: mouse monoclonal antibody (mab) against b-23/nucleophosmin (npm), a kind gift of p. k. chan (baylor college of medicine, houston), mab against hdm2 ab-1, clone smp14 (dako), mab against heat shock protein 70 (hsp70) (w27) (santa cruz biotechnology), mab against pml protein (pg - m3) (santa cruz biotechnology), mab against 20s proteasome (clone hp810) (affiniti research product ltd), mab against p14arf, 240 (supernatant), a kind gift of j. bartek (danish cancer society), mab against p53 do7 (bd pharmingen), rabbit polyclonal anti - p14arf, a kind gift of k. wiman (cck, ki, stockholm). The following secondary antibodies were used: horse anti - mouse ig texas red conjugated (vector lab), rabbit anti - mouse ig fitc (dako), swine anti - rabbit fitc (dako), swine anti - rabbit tritc (dako). Bisbenzimide (hoechst 33258 from sigma) was added at a concentration of 0.4 g / ml to the secondary antibody for dna staining when needed . Cells were stained on coverslips after fixation in a mixture of methanol and acetone (1:1) at -20c . Images were captured using a das microscope leitz dm rb with a hamamatsu dual - mode cooled charge - coupled device (ccd) camera c4880 . The 3d immunofluorescence images were generated from the reconstitution of a series of de - blurred optical sections . Briefly, the images were captured using a pxl cooled ccd camera (photometrix) on a zeiss axiophot microscope equipped with z - axis motor, external shutter and excitation filter - wheel, controlled by a mac2000 lep module . The computer program, st - rfh - bin2, that controlled the image acquisition hardware and produced the digital images was developed by us (szekely, unpublished). The program captured three times 1317 images from three excitation series (rhodamine - fitc - hoechst) with focal planes 0.3 m apart . The incoming images were automatically corrected for dark current noise and optical shining through, and de - blurred using a nearest neighbour de - convolution algorithm . The resultant image stacks were projected using the maximum intensity projection algorithm producing a single - direction and stereo - pair projected images as described . Ls conceived of the study, and participated in its design and coordination and together with ek drafted the manuscript all authors read and approved the final manuscript . We thank klas g. wiman for the p14arf plasmids and anti - arf antibodies and p. k. chan for anti - b23 antibodies . This work was supported by cancerfonden and by a matching grant from the concern foundation, los angeles, the cancer research institute, new york and also by ssmf (swedish society for medical research), karolinska institute and sven gard foundation.
Periodontitis is an inflammatory disease which is initiated and maintained by the gram - negative bacteria of the subgingival biofilm . Specific pathogen associated molecular patterns (pamps) and bacterial virulence factors stimulate an inflammatory host response that finally results in destruction of periodontal tissue and tooth loss . Chronic periodontitis (cp) and generalized aggressive forms of periodontitis (agp) appear to be associated with certain pathogens, including porphyromonas gingivalis, campylobacter rectus, tannerella forsythia, peptostreptococcus micro, and treponema species [3, 4]. Treponema denticola, p. gingivalis, and t. forsythia, characterized as the red complex, were strongly associated with the clinical progression of chronic periodontitis [15]. In contrast, agp was more often diagnosed in patients positive for aggregatibacter actinomycetemcomitans, but there were many individuals with agp who did not harbor this microorganism . In addition to the microbial challenge, other factors, such as genetics, environment, and host factors, play a role in the pathogenesis of these diseases [79]. Various compounds, such as cytokines, representing an important pathway of connective tissue destruction in periodontitis, have been detected in gingival crevicular fluid (gcf). Il-8, a member of the cxc chemokine family, was originally described by matsushima and oppenheim . It is the most important chemoattractant and activator of human neutrophils and an important mediator for granulocyte accumulation . Il-8 is involved in the initiation and amplification of acute inflammatory reactions and chronic inflammatory processes . Functions of il-8 are mediated through two receptors (cxcr1 and cxcr2); the expression was detected on numerous cell lineages, including neutrophils and epithelial cells . Gingival epithelial cells (gec) are capable of upregulating il8 expression rapidly in response to a. actinomycetemcomitans challenge, facilitating thus the recruitment of neutrophils as a host defense mechanism [13, 14]. Il8 expression in gec is induced by p. gingivalis and t. forsythia; il-8 production by gingival fibroblast cultures is also affected by lipopolysaccharides of p. gingivalis and p. intermedia . The il-8 levels in gingival crevicular fluid (gcf) are valuable in detecting the inflammation of periodontal tissue [1820], and periodontal therapy reduces the il-8 levels in gcf . Il-8 is encoded by the il8 gene located on chromosome 4q13 - 21 (genbank accession number m28130.1), consisting of four exons, three introns, and the proximal promoter region . Several polymorphisms have been reported in the il8 gene [2326] and some of them can regulate the il-8 production . Some of snps in the il8 gene such as 845t / c (rs2227532), 738t / c, 251a / t (rs4073, previously referred to as 353a / t), + 396t / g (rs2227307), and + 781c / t (rs2227306) have been studied in patients with agp or cp in the brazilian population [2732]. In addition, il8 251 t allele, which was associated with higher production of il-8, increased the risk of developing acute suppurative form of apical periodontitis (ap), whereas il8 251a low - producing allele was associated with chronic nonsuppurative form of ap in the colombian population . In the chinese population, il8 251a allele has been associated with decreased susceptibility to cp . The cross - sectional study in iran has also focused on the study of polymorphisms in the il8 gene but did not specify whether it was for patients with cp or agp . To date, no study analyzing allele, genotype, or haplotype frequencies of il8 gene polymorphisms in patients with periodontitis has been performed in caucasians . The aim of this study was to associate four snps in the il8 gene (rs4073, rs2227307, rs2227306, and rs2227532) and their haplotypes to cp and agp and subgingival bacterial colonization in the czech population . The study was performed with the approval of the committees for ethics of the medical faculty, masaryk university brno and st . Written informed consent was obtained from all participants before inclusion in the study, in line with the helsinki declaration . All patients were recruited from the patient pool of the periodontology department, clinic of stomatology, st . Exclusion criteria included history of cardiovascular disorders (such as coronary artery diseases or hypertension), diabetes mellitus, malignant diseases, immunodeficiency, current pregnancy, or lactation . Controls were selected from subjects referring to the clinic of stomatology for reasons other than periodontal disease (such as dental caries, orthodontic consultations, preventive dental check - ups, etc .) During the same period as patients and matched for age, gender, and smoking status . Similarly as patients, all controls had at least 20 remaining teeth and were in good general health . A total of 492 unrelated caucasian subjects of exclusively czech ethnicity from the region of south moravia were included in this case - control association study . Diagnosis of nonperiodontitis / periodontitis was based on the detailed clinical examination, medical and dental history, tooth mobility, and radiographic assessment . Probing depth (pd) and attachment loss (cal) were collected with a unc-15 periodontal probe from six sites on every tooth present . We used the index of mhlemann to evaluate decreases in alveolar bone levels.generalized cp group (n = 278): all patients with chronic periodontitis (cp) fulfilled the diagnostic criteria defined according to cal levels by the international workshop for a classification of periodontal diseases and conditions for chronic periodontitis . Inclusion criteria for patients suffering from generalized chronic periodontitis were as follows: 30% of the teeth were affected, pd was 4 mm, and the amount of cal was consistent with the presence of dental plaque.generalized agp group (n = 58): patients with aggressive periodontitis with age at disease onset <35 years, attachment loss of 4 mm or more in at least 30% of the teeth (at least three of the affected teeth were not first molars and incisors), and the severity of attachment loss being inconsistent with the amount of dental plaque were included in this study.control group (healthy / nonperiodontitis) (n = 156): controls were screened using a who probe and the cpitn (community periodontal index of treatment needs) was assessed; values of the cpitn index in controls were less than 3 . Generalized cp group (n = 278): all patients with chronic periodontitis (cp) fulfilled the diagnostic criteria defined according to cal levels by the international workshop for a classification of periodontal diseases and conditions for chronic periodontitis . Inclusion criteria for patients suffering from generalized chronic periodontitis were as follows: 30% of the teeth were affected, pd was 4 mm, and the amount of cal was consistent with the presence of dental plaque . Generalized agp group (n = 58): patients with aggressive periodontitis with age at disease onset <35 years, attachment loss of 4 mm or more in at least 30% of the teeth (at least three of the affected teeth were not first molars and incisors), and the severity of attachment loss being inconsistent with the amount of dental plaque were included in this study . Control group (healthy / nonperiodontitis) (n = 156): controls were screened using a who probe and the cpitn (community periodontal index of treatment needs) was assessed; values of the cpitn index in controls were less than 3 . In order to adjust for the effect of smoking history on periodontal disease, the subjects (patients and controls) were classified into the following groups: subjects who never smoked (referred to as nonsmokers) and subjects who were former smokers for 5 pack years or current smokers (referred to as smokers). The pack years were calculated by multiplying the number of years of smoking by the average number of cigarette packs smoked per day . Dna for genetic analysis was extracted from the peripheral blood leukocytes using standard phenol / chloroform procedures with proteinase k according to sambrook et al . . Isolation and storage of dna (working samples at concentrations of 50 ng l at 4c) as well as the genotyping of samples were conducted in the laboratory of the department of pathophysiology, faculty of medicine, masaryk university, brno, czech republic . Four snps (845c / t rs2227532, 251a / t rs4073, + 396g / t rs2227307, and + 781c / t rs2227306) in the il8 gene were genotyped using the 5 nuclease taqman assay for allelic discrimination . Individual fluorogenic taqman probes, consisting of an oligonucleotide labelled with both a fluorescent reporter dye, fam, and a quencher dye, vic, were obtained from life technologies (grand island, ny, usa). Each reaction mixture was prepared using taqman genotyping master mix (12.5 l), taqman snp genotyping assay (1.25 l), and 50 ng of genomic dna in 17.5 l of dh2o to make a 25.0 l reaction volume . Genotyping was carried out simultaneously with 88 samples on 96-well plate (+ 8 negative controls). The pcr thermocycling protocol consisted of 10 min at 95c, followed by 40 cycles of 15 s at 92c and 1 min at 60c . Each genotyping plate contained eight wells without any dna template (negative controls) and randomly selected duplicate samples (10% of plate samples). Allele genotyping from fluorescence measurements was then obtained using the abi prism 7000 sequence detection system . Sds version 1.2.3 software was used to analyze real - time and endpoint fluorescence data . Genotyping was performed by one investigator (p. b. l.) unaware of the phenotype . Subgingival bacterial colonization (aggregatibacter actinomycetemcomitans, porphyromonas gingivalis, prevotella intermedia, tannerella forsythia, treponema denticola, peptostreptococcus micros, and fusobacterium nucleatum) in subgingival pockets was investigated by the dna microarray based on a periodontal pathogen detection kit (protean ltd ., ceske budejovice, cr) in a subgroup of randomly selected subjects (n = 151 for cp, n = 21 for agp, and n = 75 for controls) before subgingival scaling . Microbial samples were collected from the deepest pocket in periodontitis patients (and from the deepest sulcus in healthy subjects) of each quadrant by inserting a sterile paper point into a base of the pocket for 20 seconds . This test determined the individual pathogens semiquantitatively as follows: () undetected, which corresponds to the number of bacteria less than 10, (+) slightly positive corresponding to the number of bacteria 10 to 10, (+ +) positive corresponding to the number of bacteria 10 to 10, and (+ + +) strongly positive, with the number of bacteria higher than 10 . Comparisons were made between allelic and genotype frequencies in the patients with chronic or aggressive form of periodontitis and control population . The significance of differences in the allele frequencies among groups was determined by fisher's exact test . Analysis was used to test for deviation of genotype distribution from hardy - weinberg equilibrium and comparison of differences in genotype combinations among groups . To examine the linkage disequilibrium (ld) between all snps, pairwise ld coefficients (d) and haplotype frequencies variations in the quantity of subgingival bacteria corresponding to the particular genotypes / alleles were tested by and fisher's exact tests . Power analysis was performed with respect to the case - control design of the study taking the incidence rate of markers and estimate of the odds ratio (or) as end - point statistical measures . Ors with corresponding 95% confidence intervals (ci) were estimated using logistic regression models, adopting age, sex, and smoking as adjusting covariates . 10.0 (statsoft inc ., tulsa, ok, usa) and spss software (spss 20.0.1, ibm corporation, 2011) the mean ages for agp patients (37.0 8.2; years sd) and healthy subjects (41.0 12.3) did not differ between the two groups . However, subjects with cp were significantly older (47.9 8.7) than the patients with agp (p <0.05). Nearly, 27% of the periodontitis patients (28.0% of cp and 26.0% of agp) and 28.0% of healthy subjects were smokers . There were no significant differences between the subjects with periodontitis and controls regarding the mean percentage of smokers and ratio of males / females (77/79 in controls, 136/142 in patients with cp, and 25/33 in patients with agp). Sample size of the study was planned in standard power calculation for case - control design of the study with the null and alternative hypotheses expressed on the basis of or . The design was prospectively optimized assuming the prevalence of examined attribute among controls to be 0.5 . The recruited sample (278 cases, 156 controls) allowed a statistically significant detection of or out of the range of 0.551.80 (alpha = 0.05, power = 0.80). In case of the agp group (58 cases, 156 controls), the statistically significantly detectable ors estimates were out of the range of 0.392.59 . Allele and genotype frequencies of all investigated il8 polymorphisms were not significantly different between the subjects with cp and/or agp and controls (p> 0.05; table 2). Considering that in the czech population, snp il8 845tt genotype occurred in 98.4% of cp patients and even 100% of controls and agp patients, we analyzed this polymorphism only in the subgroup of subjects (n = 193). Based on the previous study the haplotype analysis of these selected snps was performed in the il8 gene (251t / a rs4073, + 396t / g rs2227307, and + 781c / t rs2227306). All variants in the il8 gene were in tight linkage disequilibrium with each other to various degrees (d = 0.7931.000 in controls, d = 0.8890.951 in patients with cp, and d = 0.8911.000 in patients with agp). The complex analysis revealed differences in il8 haplotype frequencies . Specifically, the a(251)/t(+396)/t(+781) and t(251)/g(+396)/c(+781) haplotypes were significantly less frequent in patients with cp (2.0% versus 5.1%, resp ., 4.5%, p <0.05) (table 3). The decreased frequency of the tgc haplotype alleles in patients with cp was confirmed by the observation that tgc / ttc haplotype (arranged as genotypes) was less frequent in patients with cp (0.4% versus 2.6%, p <0.05, or = 0.09, 95% ci = 0.010.96). Moreover, an uncommon att / att haplotype (1.15% of the studied population) was found more, but nonsignificantly, in non - periodontitis controls (2.6% versus 0.4%, p = 0.07). There was also a nonsignificant trend in the atc / ttc haplotype association with cp (2.2% versus 0.0%, p = 0.08, table 4). F. nucleatum occurred less frequently in nonperiodontitis subjects (n = 75) positive for t allele of il8 + 396g / t variant (49.2% versus 77.8%, p <0.02; or = 0.28, 95% ci = 0.090.89) or tt genotype (21.2% versus 55.6%, p <0.05; or = 0.22, 95% ci = 0.050.91). In contrast, il8 251 t allele carriers had an increased or for individual presence of a. actinomycetemcomitans in agp (n = 21) patients (91.7% versus 40.0%, p <0.01; or = 16.5, 95% ci = 1.88145.0) and also tt genotype was more often found in a. actinomycetemcomitans presence (83.0% versus 13.3%, p <0.01; or = 32.5, 95% ci = 2.38443.2). Patients with cp (n = 151) carrying cc genotype of il8 + 781t / c variant had less frequent presence of t. forsythia in their subgingival microflora than subjects without this genotype (21.6% versus 35.1%, p <0.05; or = 0.51, 95% ci = 0.251.05). However, the relationship between periodontal bacteria and il8 gene polymorphisms must be assessed very carefully regarding small numbers of subjects in the respective subgroups . Cytokines involved in the inflammatory process, such as il8 and their genes, are important potential modifiers of individual susceptibility to agp or cp . Although none of the investigated snps in the il8 gene was individually associated with aggressive or chronic periodontitis, the patients with cp showed lower a(251)/t(+396)/t(+781) and t(251)/g(+396)/c(+781) haplotype frequencies than the controls . The association of tgc haplotype with cp was confirmed by the relationship between tgc / ttc haplotype (arranged as genotypes) and cp . These results confirm the hypothesis that haplotypes are more powerful for detecting susceptibility alleles than individual polymorphisms . Our results differ from those obtained by scarel - caminaga et al . Who associated atc / ttc and agt / tgc haplotypes with chronic periodontitis in the brazilian population for example, the frequency of the atc haplotype in the czech population was less than 3.4%, compared to 23.7% in the brazilians . In the brazilian population, some haplotypes of il8 845(t / c)/738(t / a)/353(a / t) variants showed significant association to, or protection against, cp . Of the three il8 snps, only one polymorphism was the same as in our study (i.e., snp 251 (rs4073) referred to as 353 in brazilian study). Polymorphism 845t / c (rs2227532) was also investigated in this study, but regarding a very low frequency of c allele, it was analyzed only in several individuals and therefore it was not included for any further haplotype assessment . To date, eight studies evaluating the association of il8 snps (251, + 396, and + 781) and cp or agp in different populations (mostly in brazilian but none in the caucasian population) have been performed, with contradictory results [2732, 34, 35]. Most of these studies analyzed only the individual snp and were focused on examining of cp; only andia et al . Studied the relationship between il8 251 variant and agp; however, no association was found . Similarly, kim et al . Failed to find any association between cp and allele or genotype distribution of il8 251 snp . In contrast, another study discovered a significant association between il8 251 snp and cp in nonsmokers . The il8 251ta heterozygote genotype was associated with increased levels of il8 mrna transcripts and a allele had an increased risk for developing periodontitis . This is consistent with the observation that the a allele of snp il8 251 tended to be associated with higher il-8 production in lipopolysaccharide- (lps-) stimulated human whole blood . Very recently, li et al . Found that a allele of il8 251 variant was associated with decreased susceptibility to cp in chinese population . . Found a significant difference in the genotype frequencies of il8 251a / t and + 396g / t snps between subjects with periodontitis and a control group in hamedan, iran, but did not specify whether it was for patients with cp or agp . Conversely, corbi et al . Showed that the genetic susceptibility to cp in the il8 gene was not associated with worse periodontal clinical parameters and increased il-8 concentration . With the exception of scarel - caminaga et al, no association between snp at position + 396 or + 781 and cp in the brazilian population was discovered . Several studies have examined polymorphisms in interleukins in connection with a subgingival bacterial colonization in patients with periodontitis . In this study, snps in the gene encoding il-8 were associated with the presence of pathogenic bacteria in subgingival dental plaque . The results showed that il8 251 t allele carriers had an increased or for the individual presence of a. actinomycetemcomitans in agp patients (p <0.01) and an increased or was also found for the presence of t. forsythia for t allele of il8 + 781 in cp patients (p <0.05). These data are in agreement with the notion that individual genetic susceptibility may influence the host response to infection . Nibali et al . Found association between il6 snps and a. actinomycetemcomitans, which they confirmed by the haplotype analysis . Specifically, il6 174gg genotype was associated with high (above median) counts of a. actinomycetemcomitans (both in all subjects and periodontally healthy subjects only) in indians . But nibali et al . Also suggested that only a detection of known periodontopathogenic bacteria could not discriminate different forms of periodontitis . In contrast, schulz et al . Found no evidence that snps in il1 gene cluster could be associated with subgingival colonization with a. actinomycetemcomitans and could thus be an independent risk indicator of agp . In addition, finoti et al . Observed that periodontal destruction may occur in patients who are considered to be genetically susceptible to cp with a lower microbial challenge because of the presence of the il8 atc / ttc haplotype than in patients without this haplotype . First, the major complicating factor in the study of isolated locus (such as il8) is the nature of periodontitis as a multifactorial disease in which interaction between multiple genes plays a role and each genetic polymorphism has generally only a small effect . In addition, the interaction of gene variants with environmental factors (such as bacterial pathogens), socioeconomic factors, bmi, and others not analyzed in this study, potentially affect the observed phenotype . Second, the case - control approach used is generally quite vulnerable to the population stratification, for example, due to different ethnic origin . The present sample, however, is exclusively of the czech caucasian origin, restricted to the limited geographical area populated by quite homogeneous population with low admixture . Finally, we did not measure rna expression or protein levels of il-8, therefore, we do not know the functional consequences of these polymorphisms in our subjects . In conclusion, although none of the investigated snps in the il8 gene were individually associated with periodontitis, some haplotypes can be protective against cp in the czech population . Clinical significance of these findings is low due to a very low frequency of the protective haplotypes . The individual il8 variants were associated with subgingival colonization with a. actinomycetemcomitans in agp and with t. forsythia in cp in the czech population . However, these relationships must be assessed very carefully regarding small numbers of subjects in the respective subgroups . Further studies are needed to clarify the association of these polymorphisms with periodontal diseases in other populations.
Obesity has become a major global health challenge . Although epidemiological data show that regular physical activity helps prevent obesity, cardiovascular disease, diabetes, and hypertension, the majority of adults fail to maintain physical activity at levels that can promote health . Lack of time is the most commonly cited barrier to regular exercise participation, a more time - efficient mode of exercise training has been developed . Both high - intensity interval training (hiit; involves near maximal effort at intensities between 80 and 100% of maximal heart rate) [3, 4] and low - volume sprint interval training (sit; involves all - out or supramaximal effort at the intensity of 100% of maximal oxygen uptake) [3, 4] are time - efficient training strategies that can rapidly improve cardiorespiratory fitness [5, 6], muscle metabolic adaptations [7, 8], and insulin sensitivity to resemble the changes elicited by moderate - intensity continuous training (mict). Excessive accumulation of body fat, especially intra - abdominal visceral fat, is identified as a potent independent predictor for hyperlipidemia, insulin resistance, and metabolic syndrome, whereas a slight change in the visceral adipose area / volume may significantly alter the risk profile . A growing body of evidence has demonstrated that hiit / sit could reduce body mass [5, 10], total or regional fat mass [6, 1012], and waist circumference [10, 11, 13] and increase fat - free mass [5, 6, 1012]. However, in terms of short - term intervention (e.g., 18 sessions for six weeks), it seems that sit running is effective at improving body composition in active individuals [6, 11], whereas hiit interventions showed inconsistent findings in terms of body composition, including improvement or ineffectiveness [14, 15]. From the view of the influential factors of body composition, confounding factors such as extra physical activity in addition to the training program [5, 10, 11] and/or the dietary intake of the participants [6, 8, 13, 16] given the conflicting results in previous studies [5, 17], whether the hiit intervention induces significant improvements in fat mass, lean mass, and regional fat deposits in comparison with an exercise control remains to be elucidated . Although some studies have reported that hiit protocols are enjoyable and adherent for both active male subjects and inactive normal - weight subjects, some researchers have contended that, for the largely inactive and/or obese population, the strenuous nature of sit may produce negative emotions toward exercise adherence and is likely to be a deterrent to participation [18, 20]. Given the fact that the power output of the wingate - based protocol with hard resistance (typically 7.5% of body mass) rises quickly and then decreases precipitously over the 30 s, this kind of sit training may not be suitable for the inactive female cohort . As a result little et al . Designed a low - demanding hiit model consisting of 10 60 s work bouts at 100% maximal heart rate (hr) interspersed with 60 s of recovery . Trapp et al . Established a brief hiit protocol of 8 s sprint cycling interspersed with 12 s of rest for 20 min and found that 15 weeks of hiit intervention with this protocol could improve cardiovascular fitness, body composition, and insulin resistance in young women when compared to 40 min of mict with a similar energy expenditure . Given that the young women with a higher bmi had a greater fat loss in trapp et al . 's study, we speculated that overweight / obese women and lean women may have different physiological responses to the same hiit training protocol . Hormonal abnormalities play a pathogenetic role in the development of excess body fat and are associated with metabolic diseases . A number of hormones, for example, leptin, growth hormone (gh), testosterone, cortisol, and fibroblast growth factor 21 (fgf-21), regulate lipid metabolism and affect muscle protein synthesis and muscle hypertrophy . It has been reported that acute high - intensity interval exercise may result in optimal responses on circulating testosterone, growth hormone, and cortisol in well - trained males as well as in type 1 diabetic individuals . However, it is not clear whether the hormonal responses to an acute bout of high - intensity interval exercise can be sustained after a period of training . Collectively, the purpose of this study was mainly to compare the effects of five weeks of hiit intervention or mict on body composition and blood glucose as well as the systemic hormones that may influence body composition and blood glucose in overweight and obese young women . We hypothesized that both training programs would result in similar influences on the improvements of body composition (e.g., reduce fat mass and increase lean mass) and blood glucose, while the improvements are associated with the upregulated gh and testosterone and the downregulated cortisol, leptin, and fgf-21 . Furthermore, hiit would be more time - efficient and perceived as being easier when compared with mict . The inclusion criteria were being between 18 and 30 years of age, having a classification of inactivity (defined as completion of less than 90 min of moderate - intensity exercise per week over the past six months), and being overweight or obese, defined as having a body mass index (bmi, in kgm) over 23 and a body fat percentage (%) over 30 . Volunteers who were interested in the study and met the inclusion criteria were required to complete a par - q form and a medical history questionnaire for further eligibility screening . Smokers, alcoholics, diabetics, persons with endocrine disorders, and users of oral contraceptive pills or any prescribed medications known to affect body composition or the endocrine system were excluded . Then, the subjects underwent a full physical examination to obtain clearance for undertaking vigorous exercise from a doctor . Under the assumptions of a within - subject correlation of 0.70 between the pre- and postintervention measures and a power of 0.80 with an effect size of 0.48 based on the primary outcome of vo2peak resulting from high - intensity interval training [3, 29], the sample size for the hiit group after the screening phase, 22 eligible subjects were recruited to participate in this study . They all provided written informed consent before being randomly assigned to either the hiit group (n = 11) or the mict group (n = 11). One participant in the hiit group and three participants in the mict group quit before completing the training intervention for personal reasons (figure 1). Each subject completed a five - week hiit or mict exercise intervention (four sessions per week). Before and after the training intervention, subjects underwent a body composition analysis and a vo2peak assessment, and their fasting blood samples were obtained . All pretraining and posttraining measures were conducted in the follicular stage based on the self - reported menstrual cycle survey . Because of the different menstrual cycles for the subjects, the starting time for baseline measures and training intervention were different among subjects, with the last subject starting two weeks later than the first one . However, all of the pretraining measures were taken within 48 to 144 h before the training intervention and all of the posttraining measures were taken within 48 to 144 h after the last training session . Before baseline measures, each subject visited the laboratory to sign the consent form, become familiar with all testing and training procedures, and provide a three - day diet record . Baseline measures were conducted on three different days, separated by at least 24 h, and all measures were completed at least 48 h before the training intervention . Subjects were asked to refrain from strenuous activity and caffeine for 48 h and fasted overnight (12 h) prior to the baseline blood sampling . 8 ml blood samples were collected from the cubital veins using serum separation tubes and were left to clot at room temperature for 60 min . Then, the blood samples were centrifuged at 3000 rpm for 10 min at 4c, separated for serum, and subsequently frozen at 80c until later analysis . Subjects were instructed to come to the laboratory in the morning after a fasting state (12 h). Height and weight were determined using standard methods with a stadiometer and an electronic scale (in light clothing and with no footwear) to the nearest 0.1 cm and 0.1 kg, respectively . Body mass index was calculated by dividing weight (kg) by height (m) squared . By the same operator, subjects were scanned in a supine position using a dual - energy x - ray absorptiometry scanner (norland xr-36 dxa densitometer, norland corporation, fort atkinson, ws, usa) and were analyzed with a software program (3.7.4/2.1.0; norland corporation). The instrument was calibrated daily using the phantoms provided by the manufacturer, and the value of the intra - assay coefficient of variation (cv) was 0.53% . The abdominal region referred to the area consisting of the line between the two iliac crests, the two edges of the hip, and the lateral sides of the femoral necks . The trunk region was defined as being from the lower edge of the mandibular to the upper edge of the line between the iliac crests, excluding the head and upper limbs . Lean mass and fat mass were calculated from the total and regional analysis of the whole body scan . The subjects performed a graded maximal exercise test on a computer - controlled cycle ergometer (monark 839e, sweden) to determine vo2peak and peak power output (ppo). After a two - minute warm - up at 30 w, the subjects pedaled at the initial workload of 50 w and maintained a cycling speed of 60 5 rpm, and the workload was increased by 25 w every three min until volitional exhaustion . Respiratory gases were assessed continuously using an automatic gas analyzer (meta - max 3b, cortex biophysik gmbh, leipzig, germany), and the highest oxygen consumption averaged over the final 15 s was identified as the vo2peak . Ppo was calculated according to the following formula: ppo = wcom + (t/180) 25, where wcom is the last completed workload, t is the time completed in the final unfinished workload (in seconds), 180 is the increment duration (s) in each workload, and 25 is the workload increment . Both the hiit and the mict exercise training were conducted four days per week for five weeks . Participants in the hiit group performed 60 repetitions of high - intensity interval exercise (8 s cycling and 12 s passive recovery) on a cycle ergometer (monark 874e, sweden) for 20 min . A prerecorded tape was used to coordinate the hiit intervention, and all subjects worked as hard as they could during the exercise phase . The initial resistance of the exercise phase was 1.0 kg, and once an individual could complete two consecutive sessions at the given workload, resistance would be gradually increased by increments of 0.5 kg until reaching 0.05 body weight . Hr (polar f4 m blk, finland) and ratings of perceived exertion (rpe, borg scale) were recorded before and immediately after the completion of the 8 s cycling exercise for every five intervals . During the first and last training sessions the product of all intervention sessions and the mean value of energy expenditure measured during the two mentioned sessions were regarded as the total energy expenditure of hiit . Participants in the mict group performed a continuous cycling exercise at 65% of pre - vo2peak on an ergocycle (ergometer 900pc, ergoline, germany) for 40 min; a cycling speed of 60 5 rpm would be maintained throughout each training session . With the increasing fitness indicated by a decreased hr, the energy expenditure for every training session was estimated from an individual's vo2: energy expenditure = 5.05 (kcall) vo2 (lmin) exercise time (min), whereas vo2 was determined using an equation for leg cycling ergometry: vo2 = 7.0 + 1.8 workload (kgmmin)/body weight (kg). The total energy expenditure of the mict group was calculated as the product of all intervention sessions and the energy expenditure of each session . Serum glucose was measured via the glucose oxidase method using a roche / hitachi p800 modular chemistry analyzer (roche diagnostics gmbh, mannheim, germany). Serum concentrations of testosterone, cortisol, and gh were analyzed using commercially available electrochemiluminescence immunoassay kits (roche diagnostics gmbh, mannheim, germany), whereas leptin and fgf-21 were measured using a commercial enzyme - linked immunosorbent assays (elisa) kit (abcam, cambridge, uk). The cvs were 1.1% for glucose, 4.5% for testosterone, 4.6% for cortisol, 2.9% for gh, 2.4% for leptin, and 2.6% for fgf-21 . Subjects in both the hiit and mict groups were instructed to maintain their normal eating habits and normal daily physical activities during the study period . Each subject provided a three - day diet inventory one week before and one week after the intervention as well as during the third week of the intervention . Energy intake and diet component analyses were conducted by the sports nutrition research center (national institute of sports medicine, china) using the nutrition analysis and management system . Daily physical activities were monitored using pedometers (yamax sw-200 digiwalker, japan) for three days per week for a total of seven weeks (on the weeks before and after training and every week during exercise training). Posttraining assessments were performed in the same way as described in the pretraining testing protocol and were completed within 48 to 144 h following the last training session . Blood samples were taken within 96 to 144 h after the intervention, whereas body composition and vo2peak were determined within 48 to 72 h after the last training session . Data were analyzed using pasw software (release 22.0; ibm, ny, usa). Independent - sample t - tests were performed to determine the differences in training data (hr and rpe) and energy expenditure between the two groups . A two - way mixed analysis of variance (anova) with repeated measures was used to test for main (time) and interaction effects (time group). Significant interactions or main effects were determined using tukey's honestly significant difference post hoc test . As for effect size measure of the main effect and the interaction effect, partial was considered small if <0.06 and large if> 0.14 . All results were presented as mean standard deviation (sd), and p values of <0.05 were considered significant . There were no significant differences on any measured variables between the two groups on pretraining tests . There was no significant difference in training hr between hiit and mict (164 8 bpm in the hiit group versus 160 12 bpm in the mict group; p = 0.435). However, mict is perceived to be significantly harder compared to hiit (13 1 in hiit group versus 15 1 in mict group; p = 0.042). In the first training session, the values of energy expenditure were 174 28 kcal in hiit and 301 45 kcal in mict, and the former spent less than the latter (p <0.001). Similarly, the total energy expenditure of the intervention in the hiit group (3167 549 kcal) was significantly lower than that in the mict group (6011 505 kcal; p <0.001). The daily calorie intakes (table 3) were not different within group and between groups over time (as evaluated before training, during training, and after training; p> 0.05). The proportions of macronutrient intake were approximately 50%, 35%, and 15% for carbohydrates, fat, and protein, respectively, in both groups, with no within - group or interaction differences (p> 0.05). Physical activities recorded by the pedometers had no within - group or interaction differences before (7673 1145 steps in hiit versus 8062 1367 steps in mict), during (9785 1640 steps in hiit versus 8517 791 steps in mict), and after intervention (7434 1225 steps in hiit versus 7023 849 steps in mict) (p> 0.05). After five weeks of exercise training, both hiit and mict resulted in a significant improvement in vo2peak (p = 0.006; = 0.38) and ppo (p <0.001; = 0.61). Hiit training increased vo2peak and ppo by 7.9% and 13.8%, respectively, whereas mict training increased vo2peak and ppo by 11.7% and 21.9%, respectively . There were no group differences in the magnitude of improvement in vo2peak and ppo (p> 0.05) (table 1). After the intervention, despite no significant changes in weight, bmi, total fat mass (tfm), and total body fatness (tbf) for both groups, the mict group experienced significantly decreased total lean mass (tlm) (1.7 kg or 3.9%; p = 0.011) and leg lm (0.6 kg or 3.3%, p = 0.018). Meanwhile, tlm and leg lm in the hiit group were unchanged (reduced by 0.2% and 0.1%, resp . ; p> 0.05). In the regions of the trunk and abdomen, no significant changes in lean mass, fat mass, and fatness were observed within group or between groups (table 1). Fasting glucose tended to be significantly decreased (p = 0.062; = 0.213) after the training intervention, but no group difference was found . There were no within - group or group differences in serum levels of testosterone, cortisol, the ratio of testosterone and cortisol (t / c ratio), gh, leptin, or fgf-21 (table 2). For all subjects, no significant correlations were found among the variables of the changes in aerobic capacity, body composition, and the changes in blood parameters . This study showed that five weeks of hiit, despite involving half of the time and exercise energy expenditure when compared to mict, resulted in a similar improvement in aerobic capacity but had no influence on fat mass or lean mass in the trunk or abdomen . Hiit subjects seemly lost less lean body mass and lean leg mass than did mict subjects after training . Moreover, both short - term training protocols resulted in a trend to decrease fasting serum glucose but had no effects on systemic hormones, including leptin, testosterone, cortisol, gh, and fgf-21, in overweight and obese young women . Different forms of hiit have been shown to significantly increase vo2peak [5, 6, 8, 10, 11, 13, 33] and aerobic capacity [10, 11]. The present study found that, after five weeks of this low - volume hiit protocol, the relative vo2peak and ppo were increased by 7.9% and 13.8%, respectively, consistent with an average of 7.3 4.8% increment in vo2peak reported in a meta - analysis after the wingate - based sprint interval intervention in sedentary female cohorts . In accordance with previous studies [5, 17], our study also did not find any additional effect caused by the hiit protocol when compared to mict . Moreover, previous studies have demonstrated that, using the same hiit protocol, vo2peak was improved by 15.0% (+ 5.2 mlminkg) in obese men for 12 weeks and was increased by 23.8% (+ 7.6 mlminkg) in sedentary women for 15 weeks, respectively . Although the 7.9% (+ 2.5 mlminkg) magnitude of vo2peak was relatively smaller, which might be caused by the shorter duration and differences in exercise intensity, this present study indicates that short - term training with this brief hiit protocol could also result in rapid adaptation in cardiovascular function in inactive obese young women . The possible reasons might be attributed to the upregulated mitochondrial oxidative enzyme activity [7, 8, 21, 34], the enhanced fractional muscle oxygen extraction [6, 35], and/or the increased stroke volume . Since the hr monitored during exercises were analogous (164 8 bpm in hiit versus 160 12 bpm in mict) between the two groups, the significantly lower rpe reported in the hiit group may be mainly caused by the interval exercise mode with submaximal exercise intensity, which was 89% of vo2peak during the exercise phases and 76% of vo2peak during the recovery phases according to the data measured in the first hiit session . The game - like nature of hiit, varying between short sprints and recovery intervals, may be helpful in reducing the perception of effort . Collectively, compared to mict, the present hiit protocol is a more time - efficient and easier exercise mode for improving cardiorespiratory fitness in the overweight female cohort . To our surprise, the mict group lost 1.7 kg of total lean mass and 0.6 kg of leg lean mass after training . Dxa, as a frequently used method to assess body composition, had the smallest detectable differences, at 1.39 kg and 1.30 kg for fat and lean mass in obese children, and the cvs for fat and lean mass were 1.2% and 1.1% in obese females . Based on this evidence, we estimate that an approximate 1 kg reduction in total and leg lean mass in the mict group is probably a consequence of measurement error from the dxa . Moreover, we did not detect any significant reductions in total and regional fat mass as well as fasting leptin levels following the five - week hiit training intervention . Trapp et al . Showed that 15 weeks of hiit training with a similar protocol significantly reduced resting leptin levels, and the decreases in leptin levels were positively correlated with the decreases in body weight among normal - weight females . On the contrary, a recent study demonstrated that there were no changes in fasting serum leptin despite improvement in body composition after ten weeks of high - intensity interval training in young women with polycystic ovary syndrome . However, due to the body composition, as assessed using a bioelectrical impedance analysis in their study, we believed the notion that short - term exercise training (12 weeks) does not affect leptin levels and that long - term exercise training that has reduced leptin levels is generally not independent of changes in body fat mass . Furthermore, given that fat losses were reported with the same 8 s/12 s protocol but using longer interventions (i.e., 15 weeks and 12 weeks) [5, 10], we speculated that, for a less intense hiit protocol, a longer duration is essential for accumulating measurable alterations in fat loss . There is no definite conclusion regarding whether hiit intervention improves body composition in overweight and obese individuals . Several recent studies have shown that hiit interval training reduces total fat mass [5, 10, 12] and abdominal and visceral fat mass [10, 12] and improves lean mass [5, 8, 10, 12] effectively in both obese and nonobese adults, whereas some evidence reported no changes in body composition in overweight individuals [16, 17] or in active men . However, sit, a form of supermaximal exercise intensity of a shorter duration [3, 4], seems to be more effective compared to hiit for improving body composition . A short wingate - based sit, which lasted for two weeks, has been shown to reduce abdominal and subcutaneous fat mass in sedentary overweight / obese men, reflected by decreases in waist (1.4 cm or 1.1%) and hip (1.1 cm or 1.0%) circumferences . Consistently, six weeks of running sit interventions led to significant decrement of fat mass and increment of lean mass in recreationally active men and women, and the improvements in body composition were comparable to that of mict . The similar fat losses between hiit and mict may result from the increased excess postexercise oxygen consumption (epoc) [43, 44] and/or the improved muscle oxidative capacity [7, 8, 21], though hiit had a lower total training volume . Additionally, in the present study, the training intervention demonstrated a trend toward improved fasting glucose concentrations in the obese female cohort with normal fasting glucose level . Previous studies showed that short - term hiit, and even acute hiit, can rapidly improve glucose control in prediabetic [45, 46] and type 2 diabetic patients . On the contrary, some studies reported that, when compared to baseline, short - term wingate - based hiit improved insulin sensitivity but had no substantial advantage for improving fasting blood glucose in healthy sedentary and overweight and obese men . Nybo et al . Found that 12 weeks of 20-minute high - intensity interval running per week had a similar effect of improving fasting glucose as 60-minute continuous running at 65% vo2peak in sedentary overweight and obese males . Taken together, the discrepancy in fasting blood glucose resulting from hiit may be attributable to differences in protocols, intervention durations, and initial fasting glucose levels . We did not find any changes in the basal levels of fgf-21 after the hiit or mict interventions in this population . Accumulated evidence derived primarily from animal models indicates that this novel myokine has therapeutic potential for the treatment of type 2 diabetes and has beneficial effects on metabolic disorders . Animal studies have demonstrated that both acute exercise and chronic exercise training could increase serum fgf-21 levels in rodents, and the increment is accompanied by increasing serum levels of ketone bodies, glycerol, and free fatty acids . Among the few studies examining the effects of exercise / training on fgf-21 levels in humans, it has been shown that a single bout of treadmill running exercise as well as two weeks of daily supervised training increased serum fgf-21 levels in healthy men and women . In the present study, neither exercise regimen had an effect on fasting levels of serum testosterone, cortisol, t / c ratio, and gh, indicating that short - term exercise training, even at a high intensity, cannot induce significant effects on the resting hormones in inactive overweight and obese young women . Although an acute bout of high - intensity interval exercise or sprint exercise would result in marked increment in cortisol and gh levels, previous studies have shown that the resting levels of cortisol, testosterone, and gh are unlikely to be influenced by exercise training [24, 51]. Similar to our study, the fasting levels of gh were unaffected by four to six weeks of hiit / sit in sedentary men or recreationally active males . Given that the hormonal changes respond mainly to acute exercise [24, 51], future studies should examine the acute responses of different hormones, as well as body composition, before and after hiit intervention in overweight and obese individuals . First, on the basis of sample estimation from the potential changes of vo2peak, we acknowledged that the small sample size in the present study limits the ability to draw a meaningful conclusion regarding the efficacy of hiit in improving body composition against traditional continuous exercise . Second, this study was conducted during a time of the year that people are more likely to gain weight, which started from mid - october and ended in early december . The seasonal factors may also have an influence on body composition since an average net weight gain of up to 0.5 kg in the fall and winter has been reported in a previous study . Because of this, for future studies aimed at reducing weight, seasonal factors should be taken into consideration, and a nonexercise control group is needed for interpretation of the relative results . Finally, considering the great effect of the combination of a hypoenergetic diet and exercise on weight loss and preserving muscle mass, future studies may consider managing the factors involving demographic characteristics (gender, age, menstrual cycle, etc . ), hiit modality (low and high demanding), daily physical activity (intensity and amount), and nutrition status (high- and low - protein diet) thoroughly . This would be helpful to ascertain the impact of high - intensity interval training on metabolic outcomes and the potential roles of hormone meditation during changes in overweight and obese populations . In conclusion, the present study shows that when compared to mict, short - term brief hiit intervention with 8 s of high - intensity interval cycling interspersed with 12 s of rest is a more time - efficient approach and is perceived as being easier for improving aerobic fitness and blood glucose in sedentary overweight and obese young women . Neither short - term hiit nor the mict intervention had an effect on body composition or the relevant systemic hormones.
Insects - resistant population - samples from the el mal field population of t. infestans were collected in november 2010 from infested houses in the chaco province of argentina (s2556.077 w6027.105), where vector control using pyrethroid insecticides is considered ineffective by the authorities responsible for the chagas program of chaco province . Field - collected insects were transported to the research center of pest and insecticides (cipein) laboratory and further generations of these insects were bred in the laboratory . The 50% lethal dose (ld50) obtained for deltamethrin for this population is 134 ng / insect, with a high resistance ratio of 1,031 (carvajal et al . A study performed at cipein has shown that eight years after the most recent exposure to deltamethrin, the ld50 did not vary in a population from northern argentina (germano 2013). Susceptible population - for comparison, we used a susceptible reference colony, nfs, derived from a domestic field population collected in december 2004 from santiago del estero, argentina . This laboratory colony has been maintained without the introduction of new insects from external sources . A laboratory test with nfs has shown an ld50 of 0.13 (0.11 - 0.15) ng / i for deltamethrin . This value did not differ statistically from that of the traditional laboratory - susceptible strain maintained at cipein (roca acevedo et al ., each population was kept in enclosed boxes (30 x 30 x 30 cm) at 28 1c and 50 - 60% relative humidity with a photoperiod of 12:12 h (l: d). A pigeon was provided weekly as a blood meal source (who 1994). Chemicals - technical grade imidacloprid (98%) provided by dr ehrrenstorfer (augsburg, germany) was used in topical application bioassays . Analytical grade acetone was purchased from jt baker (san pedro xalostoc, mexico). In the surface assays and spot - on bioassays, the following formulations of imidacloprid were used: 35% emulsifiable concentrates (ec) (mamboret confi and ec - chemotcnica (argentina), 70% wettable granule (wg) (bayer confidor, argentina) and 10% spot - on (bayer advantage g, argentina). Moreover, 2.5% ec deltamethrin (bayer k - othrine, argentina) was used only in surface bioassays . Topical application bioassays - t. infestans first instars (5 - 7 days old) that had been starved since eclosion were selected for toxicity tests according to the world health organization protocol (who 1994). The bioassays consisted of the topical application of 0.2 l of the insecticide diluted in acetone on the dorsal abdomen of the first instar using a 10 l hamilton syringe equipped with an automatic dispenser . In the evaluation of blood feeding, nymphs were previously fed to repletion on a pigeon and the insecticide was applied immediately after feeding . The final concentrations of imidacloprid tested ranged from 0.0025 - 0.5 mg / ml . All concentrations were replicated at least three times with a minimum of 10 insects per replicate . To calculate the ld values at p <0.05, a minimum of 30 insects per concentration was required and mortality values between 10 - 90% were observed (robertson et al . Mortality was evaluated after 24 h and also after 48 h and 72 h in the delayed toxicity assays, by placing the insects on a circular piece of filter paper (11 cm diameter) and observing their ability to walk . Only nymphs that were able to walk from the centre of the filter paper to the border evaluation of formulated insecticides - on glass - insecticides were applied to a square area (96 cm) using a 1 ml pipette and a constant flow to achieve uniform impregnation . The treated surface was dried for 24 h. each replicate consisted of a negative control group (water), a positive control group [ec 2.5% deltamethrin in water at 25 mg active ingredient (ai)/m] and one or more doses of formulated imidacloprid, ranging from 1,000 - 5,000 mg ai / m . Groups of 10 nymphs (3rd - instar nymphs aged 10 - 20 days, starved since last moult) were confined in glass rings and exposed for 1 h to the treated surfaces . After exposure, the insects were placed in clean flasks with filter paper and were maintained under the laboratory conditions described previously . Mortality was recorded after 24 h (germano et al . 2014). On filter paper - circular disks of whatman n 1 filter paper (5.5 cm diameter) the papers (area 23.75 cm) were homogeneously impregnated with 0.1 ml of the formulated insecticide . The control groups were exposed to filter paper homogeneously impregnated with 0.1 ml of pure water . After 1 h, when the solvent had evaporated, the insects (10 1st - instar nymphs per group selected as in topical application bioassays) were held in contact with the treated surface for 1 h. after this period, live insects were placed in clean flasks and were maintained under the laboratory conditions described earlier . Mortality was recorded after 24 h (rojas de arias & fournet 2002). On pigeons - selected pigeons were treated with various doses of imidacloprid advantage g. the average weight of the pigeons was 252.7 39.4 g. twenty - one pigeons were used . The experimental design included five groups (4 treated and 1 control) with a minimum of three pigeons per group . Prior to the application of the spot - on, we removed several feathers from the pigeon to produce a blind spot or arena where the insects could feed . The insecticide was applied to the base of the neck with a needle - less syringe . The insects were then exposed in the area where the spot - on formulation was applied . We have previously determined that this area was the most feasible site because the insects were removed easily without any additional disturbance to the treated pigeon . The pigeon groups were treated with 1, 5, 20 and 40 mg / ai of the formulation (i.e., = 4, 20, 80 and 160 mg / ai per kg of animal). The control group was manipulated similarly, without the addition of any insecticide, but water . Synchronised insects (1st and 3rd - instar), previously starved for 15 - 20 days, were allowed to feed for 30 min on the treated area of the pigeons . The residual effect of the drug was studied on feeding nymphs at different intervals of time (1, 7, 14 and 21 days post spot - on application) after the administration of the drug . The tests were performed by allowing the insects housed in jars containing 10 - 30 insects to feed on the pigeon . A total of 1,054 and 717 first and third - instar nymphs were used, respectively . The fed insects were transferred to clean flasks with filter paper and kept under the laboratory conditions described previously . Mortality was recorded after 24 h. statistical analysis - in the topical application and surface bioassays, mortality data were analysed using polo plus software v.2.0 . Dose - mortality data were subjected to a probit analysis to estimate the ld (ng / insect) required to kill 50% of the treated individuals (ld50). In the spot - on bioassays, all mortality data were corrected for control mortality with abbott s equation (abbott 1925). The percentage mortality was determined and transformed to arcsine square - root values for an anova . A 0.05 significance level was chosen as the criterion for biological significance among related treatments . Evaluation of delayed toxicity and influence of the blood feeding state - the ld50 values for the susceptible (s) and resistant (r) populations were 5.2 (3.4 - 7.8) and 9.2 (7.4 - 11.2) ng / insect, respectively, and did not differ up to 72 h after the initial topical application . We also studied the variation in the toxic effects of imidacloprid relative to the blood feeding condition of the insect because the cuticular distention resulting from blood feeding facilitates the penetration of the insecticide . The blood feeding condition (starvation / feeding) of the insects had no significant influence on the insecticidal activity of the imidacloprid in either population (table). Table insecticidal activity of imidacloprid over starved and fed nymphs i of triatoma infestans at 24 hpopulationblood feedingld50 confidencestate(ng / insect)limits (ng / insect)susceptiblestarved5.23.4 - 7.8fed42.4 - 7resistantstarved9.27.4 - 11.2fed10.86.4 - 19ld: lethal dose . Formulation - surfaces - four formulations of imidacloprid were tested against first and third - instar t. infestans on two different surfaces: filter paper and glass . The ecs chemotcnica and mamboret confi and the wg confidor showed no effects, either on glass against third - instar nymphs (at 1,000 - 5,000 mg ai / m) or on filter paper against first - instar nymphs (at a 100 mg / ml dose). The spot - on advantage g could not be tested on glass because it did not form a film after 24 h of drying . On filter paper, advantage g was effective, with a mortality of 100% with a 100 mg / ml dose in both susceptible and resistant populations . The lc50 obtained for advantage g on filter paper was 22.84 (14.94 - 36.89)mg / ml . In contrast, a 10 mg / ml dose of deltamethrin on filter paper caused 100% mortality in the susceptible population, whereas no mortality (0%) was found in the resistant population . Spot - on on pigeons - a first evaluation of formulations of imidacloprid on glass and filter paper showed that only the spot - on formulation was effective . Accordingly, we analysed the effect of the spot - on formulation of imidacloprid by applying different doses of the insecticide to pigeons . The applied dose of 1 mg / ai showed no lethal effects against first and third - instar t. infestans (p> 0.05). Twenty - four hours after the application of 5 mg / ai to pigeons, nymphs that had fed on the pigeons showed a higher mortality rate (49.8 1% and 40.5 18% for first and third - instar, respectively) than the control group (p <0.01). Nymphs fed seven days after a spot - on application did not show significant differences in mortality between the treated and control groups (p> 0.05). Nymphs fed 14 and 21 days after spot - on application did not show significant differences in mortality between the treated and control groups at any studied dose or nymphal stage (p> 0.05). Both first and third - instar nymphs fed on pigeons that had been treated with 20 mg or 40 mg of the formulation showed a higher mortality rate than the control group one and seven days post - treatment (p <0.01). The residual effect (7 days after treatment) was higher for 40 mg than for 20 mg (p <0.01) the lethal effect was similar against first and third - instar nymphs at all doses and time intervals (a, b in figure). Mortality of triatoma infestans fed at different intervals after the spot - on application . Bars with different letters are significantly different (p 0.05) within each day . Although the pyrethroids deltamethrin and -cia - lothrin are available, the only insecticides approved by the health service of argentina for use in the field control of t. infestans are the organophosphates fenitrothion and malathion . These insecticides were used to control t. infestans in the 70 s, but because of their high toxicity in mammals, strong odour and tendency to leave stains on the walls after application they were replaced by pyrethroids in the 80 s (schofield & dias 1999). Fenitrothion has been shown to be effective against several deltamethrin - resistant populations under laboratory and field conditions (picollo et al ., the development of pyrethroid - resistant populations has led to the re - utilisation of either malathion or fenitrothion against triatomines by the health authorities of argentina and bolivia . This is the first study of the efficacy of several formulations of imidacloprid on different surfaces against susceptible and pyrethroid - resistant t. infestans . In an attempt to characterise the toxicology of imidacloprid against t. infestans, we studied the variation in mortality through time after topical application and the influence of the blood feeding condition of the insect on this toxicity . Our results showed that the ld50 did not vary significantly up to 72 h after the initial topical application . This result could indicate that the toxicological effects of the imidacloprid remain stable through time . (2001), who found a decrease in the toxicity of imidacloprid through time in the ground beetle h. pennsylvanicus (coleoptera: carabidae) following contact exposure . The blood feeding condition (starvation / feeding) of the insects had no significant influence on the insecticidal activity of the imidacloprid . Thus, the rate of penetration associated with physicochemical modifications of the cuticle after blood feeding appears not to alter the toxic effects after topical application . We also analysed various types of commercial formulations of imidacloprid on two different surfaces . Thus, the first step, focusing on the field application of the insecticide, is to find a correct formulation of the insecticide . We found that neither the ec nor the wg formulations were effective against t. infestans nymphs . Although most surfactants and polymers are biologically inert when applied to insects, these chemicals can profoundly affect the biological activity of the pesticide when used as part of a formulation (scher 1988). Traditional spraying is highly effective inside domiciles, but it usually leaves a number of residual individuals of the vector in the peridomestic environment, as reported in the southern part of the chaco region (porcasi et al . These residual populations eventually re - colonise the domestic sites, re - establishing the domestic transmission cycle of t. cruzi (grtler et al . 1995), pyrethroid - impregnated curtains (ferral et al . 2010) and a residual paint formulated as a micro - encapsulate containing an organophosphate and a juvenile hormone analogue (alarico et al . Although t. cruzi is transmitted by several species of triatomines, animals such as dogs, cats and chickens are the main domestic reservoirs of t. cruzi in the endemic areas of chagas disease (grtler et al . Thus, reithinger et al . (2006) found that deltamethrin - impregnated dog collars reduced the survival and fecundity of exposed kissing bugs on dogs . Similarly, a spot - on formulation of fipronil applied on dogs and a pour - on formulation of cypermethrin applied on chickens have been successfully tested against t. infestans (rojas de arias & fournet 2002, amelotti et al . The imidacloprid advantage g spot - on formulation is recommended for treating cat fleas . The recommended dose ranges from 10 - 40 mg ai / kg . In this study, we tested doses from 4 - 160 mg ai / kg on pigeons . At a dose of 20 mg ai / kg, 50% of the nymphs were killed 24 h after the application . The doses of 80 and 160 mg ai / kg produced 100% mortality and had a high residual effect until seven days post - treatment . (2009) studied the efficacy in chickens of a pour - on formulation containing cypermethrin . They found that after a week of initial exposure to the insecticide at a dose of 120 mg / chicken, 53% of the treated third - instar nymphs were killed, whereas at day 14, mortality had values similar to the controls (4.9%). This finding is similar to our results because a dose of 160 mg / kg between days 7 - 14 after initial exposure produced a mortality of 58% and 8%, respectively . Rojas de arias and fournet (2002) studied the residual activity of fipronil with a contact test of the insecticide on filter paper against fifth - instar t. infestans . The authors reported a value of lc50 of 106 mg / m . Despite the subtle differences in methodology, the lc50 value of 960 mg / m of imidacloprid suggest that this compound has a high contact activity that depends on the type of formulation . Thus, an approach involving the use of the spot - on formulation might complement traditional pyrethroid spraying, which has shown a low efficacy in the elimination of t. infestans peridomestic populations . The effectiveness of imidacloprid against t. infestans is reinforced by its lower oral and dermal mammalian toxicity than fenitrothion, the current alternative to pyrethroids . For instance, the oral and dermal ld50 in rats for imidacloprid are 450 mg / kg and> 5,000 mg / kg, whereas the values for fenitrothion are 250 mg / kg and 2,500 mg / kg, respectively . Additionally, the no - observed effect level in rats is higher for a diet containing 300 mg / kg imidacloprid [based on a unit of 1 kg of body weight than for a diet containing 10 mg / kg fenitrothion (tomlin 1997)]. This concern is highly important if an insecticide must be used indoors and in a domestic environment . Because of the increasing number of populations resistant to pyrethroids and the high mammalian toxicity of fenitrothion, it is essential that other insecticidal compounds, especially those with alternative modes of action to pyrethroids and organophosphates, are rapidly made available for t. infestans control programmes in south america . This study has indicated the potential of imidacloprid in the control of chagas disease vectors . However, imidacloprid should be incorporated into an integrated pest management programme because its effectiveness is primarily restricted to domestic and peridomestic animals . Moreover, the type of formulation selected is essential in the function of the toxicokinetic and toxicodynamic processes by which the active ingredient (i.e., imidacloprid) affects the insect (t. infestans). A spot - on formulation appears to improve this interaction, resulting in increased mortality of the triatomine vector in the laboratory . Further studies are needed to test this type of formulation under semi - field or field conditions and to incorporate this formulation as a complementary strategy for triatomine control.
Streptococcus pneumoniae remains a major cause of childhood morbidity and mortality worldwide, particularlyin lower income countries . Pneumococcal diseases are the leading source of vaccine preventable deaths, mostly due to community - acquired pneumonia (cap), accounting for approximately 11% of all deaths in children under 5 years old (1). Colonization of the nasopharynx is a necessary step along the path to pneumococcal disease (pd) (2). Pneumococcal conjugate vaccines (pcv) reduce nasopharyngeal carriage of serotypes which included in the vaccine by conferring capsular - specific immunity . Experience from countries where conjugate vaccines have been introduced has shown rapid and sustained carriage reduction of vaccine serotypes (vt) following vaccination (3). The microorganism produces a plethora of virulence factors, including the polysaccharide capsule, several surface - located proteins, and the toxin pneumolysin (5, 6). The polysaccharide capsule is highly efficient in protecting the bacteria from opsonophagocytosis (7). Surface proteins of s. pneumoniae (pneumococcus) have been investigated for their role in pneumococcal pathogenicity and as candidate antigens for protein based vaccines (8). Among the surface - associated proteins, the pneumococcal surface protein a (pspa) andc (pspc) are the best characterised choline - binding proteins (6, 9). Pneumolysin is a cytoplasmic toxin released by autolysis of the cell and it is a very important virulence factor with multiple effects . The major autolytic enzyme of the pneumococcus is lyta (nacetylmuramoyl - l - alanine - amidase), which is responsible for the deoxycholate- and penicillin induced cell lysis in the stationary phase, having a great clinical importance (10). The lyta - encoded major autolysin of s. pneumoniae is a member of a widely distributed group of cell wall - degrading enzymes located in the cell envelope and postulated to play roles in avariety of physiological functions (11). Rrga is a virulence factor in a murine lung infection model and has a varied distribution among serotypes of s.pneumoniae (12). S. pneumoniae adherence was significantly enhanced by expression of an extracellular pilus composed of three subunits, rrga, rrgb and rrgc (13). Some of the pneumococcal virulence factors are potential targets for protein- based pneumococcal vaccine production . Thus, in this study the presence of three genes were detected among the isolates of s. pneumoniae . A total of 260 samples from nasopharynx of healthy children under 6 years old were taken carefully and transferred under cold condition to the laboratory . These samples were collected from children attending day care centers in different geographical areas of mashhad, iran . Both plates were incubated at 37c in an atmosphere of 5% co2 for 24 hours . The plates were examined and -haemolytic colonies suspected to be streptococcus pneumoniae were confirmed by optochin test and pcr for cpsa gene as described earlier (14). The optochin - susceptibility test was performed using a 6.5 mm diameter disc containing 5 mg optochin (oxoeid) in an atmosphere of 5% co2 . The genomic dna of the bacterial isolates were extracted by dnaase tissue kit (kiagen, tehran, iran). The presence of rrga, pspc and lyta genes were detected among confirmed s. pneumoniae isolates by three single pcr assays . Oligonucleotides which were used as primers to amplify particular sequences of s. pneumoniae amplification conditions for lyta and pspc genes were: 94 c for 2 min, 25cycles of 94 c for 10 s, 58 c for 15 s, and 72c for1 min, followed by a final extension at 72 c for 5 min . Amplification conditions for rrga gene were: 95c for 2 min, 25cycles of 95c for 30 s, 51c for 30 s, and 72c for 90 s, followed by a final extension step at 72c for 5 min . One amplicon from s. pneumoniae with the gene rrga was sequenced by macrogen company (south korea). Sequences were examined for identity with published sequence data from national center for biotechnology information (ncbi). A total of 260 samples from nasopharynx of healthy children under 6 years old were taken carefully and transferred under cold condition to the laboratory . These samples were collected from children attending day care centers in different geographical areas of mashhad, iran . Both plates were incubated at 37c in an atmosphere of 5% co2 for 24 hours . The plates were examined and -haemolytic colonies suspected to be streptococcus pneumoniae were confirmed by optochin test and pcr for cpsa gene as described earlier (14). The optochin - susceptibility test was performed using a 6.5 mm diameter disc containing 5 mg optochin (oxoeid) in an atmosphere of 5% co2 . The genomic dna of the bacterial isolates were extracted by dnaase tissue kit (kiagen, tehran, iran). The presence of rrga, pspc and lyta genes were detected among confirmed s. pneumoniae isolates by three single pcr assays . Amplification conditions for lyta and pspc genes were: 94 c for 2 min, 25cycles of 94 c for 10 s, 58 c for 15 s, and 72c for1 min, followed by a final extension at 72 c for 5 min . Amplification conditions for rrga gene were: 95c for 2 min, 25cycles of 95c for 30 s, 51c for 30 s, and 72c for 90 s, followed by a final extension step at 72c for 5 min . One amplicon from s. pneumoniae with the gene rrga was sequenced by macrogen company (south korea). Sequences were examined for identity with published sequence data from national center for biotechnology information (ncbi). Among 260 nasopharanx samples from healthy children under 6 years, 59 isolates were confirmed as s. pneumoniae . Distribution of the lyta, rrga and pspc genes among isolates of s.pneumoniae were determined . Rrga and pspc were also found in17 (28.81%), and 2 (3.38%) isolates respectively . Five patterns of these genes were seen among s. pneumoniae isolates: lyta (n=33, 55.93%), rrga (n=2, isolates, 3.38%), pspc (n=1 1.69%), lyta+rrga (n=16, 27.11%) and lyta+ rrga+ pspc (n=1, 1.69%). The amplicon represented expected sequences with more than 90% identity with published data from ncbi (genbank: ef 560634.1). The amplicon represented expected sequences with more than 90% identity with published data from ncbi (genbank: ef 560634.1). It remains unclear, however, why some children develop invasive disease, whereas in the majority of cases, colonization remains asymptomatic, a combination of bacterial virulence and host factors may be responsible (18). We studied the s. pneumoniae isolates giving special reference to identification and distribution of virulence markers such as autolysin among the isolates from nasopharynx . This exercise may help in understanding the factors contributing to the pathogenicity of s. pneumoniae . Further, there are numerous reports giving us increasing evidences on the role of lyta in pneumococcal pathogenesis suggesting that this might be more appropriate as vaccine antigen against s. pneumoniae infections . The cpsa was used as a novel genetic marker specific for identification of s. pneumoniae and to differentiate it from the closely viridans group streptococci as well as other pneumococcus - like streptococci such as s. pseudopneumoniae (19). Many virulence genes contribute to the colonization of s. pneumoniae; however, our study only demonstrates this for pspc, rrga and lyta genes . Thus, synergistic effect and correlation of these virulence factors is necessary for our future work . Our results showed that most of the isolates had lyta gene (84.74%) which has an important role in colonization . This observation is in concordance with previous report which was showed that all of the unencapsulated isolates of s. pneumoniae were negative for lyta and psaa by pcr . Detection of lyta and psaa in six encapsulated isolates for which a serotype could be determined was negative (9). False negative results were obtained by the pcr assays for these two genes may be due to mutations or sequence variation . On the other hand, lyta is essentially a rather conserved gene displaying limited genetic variation (11). In the study of whatmore, 33 out of 62 isolates of s. pneumoniae were selected to represent a diverse range in terms of serotype, clinical association, and time and place of isolation . Autolysin which was found in all strains, might appear to be a suitable target virulence, and apparently highly conserved, for inclusion in a potential vaccine (11). The pneumococcal protein lyt a is the major autolysin of s. pneumoniae, it has an important function in pathogenesis by releasing pneumolysin and plays a fundamental biological role in bacterial lysis after exposure to certain antibiotics (19). It has been reported that the lyta gene has higher specificity than the pspc for identification of s. pneumoniae (15, 20). In our study, only 2 out of 59 isolates of s. pneumoniae (3.38%) had pspc gene . Given the high sequence diversity of pspc, it is unlikely that pspc alone can be a vaccine antigen to provide protection from across different pneumococcal strains (16). It was showed that rrga is central in pilus - mediated adherence and disease, even in the absence of polymeric pilus production (13). However, it has been demonstrated that numerous protein virulence factors are involved in the pathogenesis of pneumococcal disease (21). Hence, new vaccine designs are focused on the surface proteins (e. g., pspa and pspc), cytolysin, and pneumolysin (22). We concluded that the gene cpsa was specific and highly conserved among s. pneumoniae isolates which were colonized in nasopharynx . On the other hand, we showed that lyta gene was the most frequent genes among the s. pneumoniae isolates, and combination of rrga, lyta was the most observed pattern.thus this should allow for appropriate screening of adhesin - based vaccines to prevent infections by streptococci.
Infection control is a critical aspect of dental practice, including subjects that are related to the health of dental practitioners, the dental staff and patients . Dental students must learn technical and preclinical skills before they enter the clinical environment and deliver care to patients . To this end, they use extracted human teeth to simulate, and practice different dental procedures . In this context many manufactured instructional tools such as artificial plastic blocks and teeth on manikins and models are used to teach some endodontic procedures . However, these artificial tools are used in conditions where access to extracted teeth is limited or not possible . Furthermore, these artificial blocks cannot replace the natural human teeth in examinations, education, and research . Regarding the importance of infection control and concerns echoed in the last few years, these extracted teeth have been noticed as a resource for infection . This fact prompts the investigators to evaluate the effects of disinfection / sterilization on extracted teeth . Directives by the american dental association (ada) and the center for disease control (cdc) call for thorough removal of any organisms capable of transmitting disease from no - disposable items used in patient care . By implication, these directives include those materials used in simulated preclinical education that might have come in contact with blood or saliva . These body fluids are associated with extracted teeth routinely used by dental students in educational procedures to improve their clinical skills and techniques . It is obvious that many blood - borne pathogens, including hepatitis b virus (hbv), human immunodeficiency virus (hiv) and bacterial pathogens, may be present in the pulp and radicular, and periradicular tissues of extracted human teeth . Besides, tooth preparation procedures in the laboratory are generally carried out without a liquid coolant; therefore, there is a greater risk of exposure to pathogenic organisms in the laboratory and as a result there is the risk of contagion spread via aerosols and accidental penetrating wounds that might take place during handling of dental instruments . In addition, there are problems with the use of extracted human teeth because they are grossly contaminated, difficult to sterilize because of their structure, and might be damaged or altered by sterilization procedures . The knowledge of dental students about infectious potential of extracted teeth used in preclinical practice was studied by kumar et al ., it was revealed from this study that about 90% of students know that extracted teeth are infection sources, but only 75% of them used a disinfection method to eliminate contamination from these teeth . Furthermore, most of the students used the boiling water and storing in sodium hypochlorite to sterilize these teeth . Tate and white reported that formaldehyde is the only antiseptic solution that can achieve an effective antimicrobial concentration within the pulp space . Furthermore, white and hays demonstrated the inefficacy of ethylene oxide against bacillus subtilis spores placed in the pulp chamber of extracted human molars . White et al ., showed that gamma radiation sterilizes teeth and endodontic filling materials without altering the structure and function of dentin; for complete sterilization, a dose of 173 k - rad with the help of a cesium radiation source was required . Dominici et al ., showed that only autoclaving for forty minutes at 240f and 20 psi or soaking in 10% formalin for 1 week was 100% effective in preventing microbial growth . Pantera and schuster reported that rockal solution (benzalkonium chloride) for 24 h and 3 weeks did not eliminate microorganisms in teeth . Attam et al ., reported that the chemical materials such as 2.6% sodium hypochlorite, 3% hydrogen peroxide and boiling water are not suitable and effective for disinfecting / sterilizing extracted human teeth . Since, there is no study about the knowledge, performance and attitude of dental students in relation to sterilization / disinfection methods of extracted human teeth used in preclinical courses, this study was designed to evaluate these parameters among a group of dental students of kerman dental school in iran . This descriptive cross - sectional study was carried out on fourth-, fifth- and sixth - year students at kerman dental school, iran . A self - administered questionnaire was prepared based on previous studies, which consisted of four parts (general questions, knowledge, attitude, and performance) about sterilization of extracted human teeth . In addition to questions regarding extracted teeth, the participants were asked questions about demographic data and personal information . To examine the validity of the questionnaire, it was given to 10 specialist dental practitioners who were asked to indicate their level of agreement to the question statements using a five - point rating scale (extremely appropriate, appropriate, no idea, inappropriate, extremely inappropriate). As a result of the item analysis, some test questions were modified to improve clarity, and a discussion was held with each subject to validate the items of the questionnaire and apply the necessary changes to validate the questionnaire . Overall validity of the questionnaire was 79% and the validity of each question was 75 - 89%, which was acceptable . The reliability of the questionnaire was assessed using the cronbach's alpha and gathering the replies provided by 15 students to the same questionnaire within a 15-day interval . Croenbach's coefficient for the reliability was 0.87, which was suitable for an acceptable study . After the analysis and discussion, the final questionnaire consisted of 34 questions in relation to knowledge, attitude, and performance in addition to questions about demographic data and personal information . The questionnaires were distributed among fourth-, fifth- and sixth - year students by the investigator . The goal of the study was explained and students were left alone to fill the questionnaire anonymously . To score the knowledge and performance questions, each correct response was given a score of 2; each wrong one was given a score of 0 score; and no answer was given a score of 1 . Attitude assessment questions had five possible responses (strongly disagree, disagree, neither agree nor disagree, agree, and strongly agree), where strongly agree was given a score of 5 and strongly disagree received a score of 1 . Chi - squared and fisher's exact tests were employed to compare differences in knowledge, attitudes, and performance among the dental students . Statistical analysis was performed using the spss software version 11.5 and statistical significance was defined at p 0.05 . Of 100 respondents, 60 (60%) were female and 40 (40%) were male . The mean age of the respondents was 24.1 6.9 years (a range of 22 - 47 years). The results showed 56% of the respondents had no previous formal training in sterilization of extracted teeth and other students were trained about sterilization of extracted teeth in endodontic department . 65% of the participants declared that they were not asked to sterilize the extracted teeth . Furthermore, 87% of the respondents had worked on extracted human teeth and 82% of the participants thought that they need education regarding disinfection of these teeth . No relationship was noted between gender, year of education, sterilization training of extracted teeth, and responses to general questions (p> 0.05). The total mean score for knowledge was 15.9 4.8 (15.9 2.2 and 15.3 2.8 for males and females, respectively; range = 5 - 17), with no significant differences between males and females (p = 0.28) [table 1]. Furthermore, no relationship was noted between year of education, trained about sterilization of extracted teeth and mean score for knowledge . Awareness of dental students to knowledge questions regarding the question about respondents opinion on the most appropriate methods for disinfection or sterilization of extracted teeth, 81 students chose sodium hypochlorite and 78 chose dry oven . There was a significant difference between male and female participants in their preference in relation to the use of chlorhexidine . Thirty - four students chose one method, 28 persons chose two methods, 22 chose three methods and 16 students chose more than three methods for disinfection or sterilization of extracted teeth [table 2]. Minimum time spent on disinfecting these teeth was 0 and maximum time was 60 days . Sixty students used only one method, 16 used two methods, 3 used three methods and 3 used four methods while 18 students did not use any method to disinfect the extracted teeth . Knowledge and practice of the student regarding sterilization of extracted teeth according by sex the total mean score for performance was 4.1 0.8 (3.9 1.7 and 4.3 1.1 for males and females, respectively; a range of 1 - 5), with no significant differences between males and females . The results showed that 87% of the respondents disinfected extracted teeth before working on them and 79% of the participants used mask while working on these teeth, 84% used gloves, 61% used safety glasses, and 84% use white coat . Women were more careful while handling these teeth, due to significant differences in the use of gloves while working on the extracted teeth (p = 0.014). Furthermore, no relationship was noted between year of education, trained about sterilization of extracted teeth and total mean score for performance . The total mean score for attitude was 53.1 5.2 (52.3 8.1 and 54.1 1.4 for males and females, respectively with a range of 25 - 60), with no significant differences between males and females (p = 0.35) [table 3]. Furthermore, no relationship was noted between the years of education, trained about sterilization of extracted teeth, and total mean score for attitude . Based on universal precautions discussion, all body fluids and tissues must be treated as sources of infection for hiv, hbv, and hepatitis c virus, or other blood - borne pathogens . One part of the preclinical education in dentistry is teaching different procedures on extracted human teeth, which have been in direct contact with body fluids and are therefore dangerous sources for contamination . For a long time disease transmission has been a concern in medicine and dentistry . Some potential infection sources such as saliva, blood, and body fluids are present in clinical settings and consequently, they can exist in extracted stored teeth . The occupational safety and health administration considers human teeth for the application in research and teaching purposes as potential sources of blood - borne pathogens . In the present study, the knowledge, performance, and attitudes of dental students were evaluated in relation to the use of these teeth and methods that they deemed appropriate to disinfect them . Dental students had an acceptable knowledge level regarding methods of disinfection for extracted teeth, consistent with the findings of kumar et al ., in the present study, more than half of the students did not have any education about sterilization of extracted teeth, and were not asked to disinfect these teeth while working in the laboratory; however, in the study by kumar et al ., 87.5% of the dental students in indian dental school were forced to sterilize the extracted teeth . This difference might be attributed to different educational programs that are held in two different dental schools; another reason might be that indian education is more concerned about the infection of extracted teeth because of the higher prevalence of different diseases in this country . Extracted teeth are a source of different infections; therefore, disinfection of these teeth seems essential to prevent dissemination of diseases . In order to sterilize and disinfect extracted human teeth, different methods can be used, including sodium chloramine, formalin, sodium hypochlorite, alcohol, glutaraldehyde, autoclaving, normal saline, freezing, 1:10 household bleach, ethylene oxide sterilization, and gamma radiation . In the present study, students chose dry oven and sodium hypochlorite as the best options to sterilize extracted teeth, although most of the students used boiling water, and storing in sodium hypochlorite to sterilize these teeth, which is consistent with kumar et al ., as in their study most of the students used sodium hypochlorite as the first option . Sterilization process should not alter the physical properties of dentin and enamel and hence that the operating characteristics of the shear bonding and the sense of touch will be the same as clinical conditions . There are few studies assessing the methods used for disinfection and sterilization of extracted teeth . One key factor that should be considered while working on extracted teeth is that the time duration since the extraction can change the properties of these teeth while they are still a rich source for different infections . Tate and white reported that formaldehyde is the only antiseptic solution that can achieve an effective antimicrobial concentration within the pulp space . In addition, the only disinfectant solution that penetrates the pulp chamber is 10% formalin . It can be considered as an effective antimicrobial concentration . The effect of formalin storage on apical seal integrity of obturated canals was studied by george et al ., it was shown that the rate of apical microleakage in the case group stored in formalin was much less than that in the control group . They also showed that this rate decreases for the extracted teeth in formalin in comparison to non - fixed specimens and this was significant . White and hays demonstrated the inefficiency of ethylene oxide against b. subtilis spores placed in the pulp chamber of extracted human molars . 64% of the teeth exposed to cold ethylene oxide treatment and 80% of the teeth exposed to the warm treatment still contained viable spore; therefore, ethylene oxide does not seem to be effective in eradicating infections from extracted teeth . White et al ., evaluated sterilization of extracted teeth by comparing gamma radiation with autoclaving, ethylene oxide, and dry heat . It was shown that gamma radiation sterilizes teeth and endodontic filling materials without altering the structure and function of dentin . For complete sterilization, a dose of 173 k - rad with the help of a cesium radiation source was required . Furthermore, no detectable changes were found with gamma irradiation, but all other methods introduced some detectable change in the spectra . Dominici et al ., showed that only autoclaving for forty minutes at 240f and 20 psi or soaking in 10% formalin for 1 week was 100% effective in preventing microbial growth . In addition, kumar et al ., showed that autoclaving at 121c, 15 lbs psi for 30 min and immersion in 10% formalin for seven is effective in disinfecting / sterilizing extracted human teeth and chemicals like 2.6% sodium hypochlorite, 3% hydrogen peroxide, and boiling in water are not effective in disinfecting teeth . White et al ., showed by spectroscopic observation that autoclave does not lead to color changes in the teeth, but it increases the rate of light attraction by dentin . In addition, it was found that autoclave induces some changes in the dentin mineral and organic material . The cutting characteristics of extracted teeth were investigated by parsell et al ., chandler and soares et al ., chandler showed that autoclaving produced significant softening of bovine enamel, the changes in microhardness recorded being similar to those produced by some experimental cariogenic substrates . Soares et al ., showed that the mineral and organic dentin contents were more affected in autoclaved teeth than in the specimens stored in thymol . It was reported that dentin hardness decreases by autoclaving; dentin of teeth autoclaved become softer in comparison to the control group . The ada and cdc suggest autoclaving as the best sterilization method for materials exposed to body fluids . However, teeth can be damaged or altered by the sterilization process in an autoclave . In relation to autoclaving, there is concern about its use for sterilization of extracted teeth with amalgam restorations as it may release mercury vapors in the air through autoclave exhaust and residual mercury contamination of the autoclave might occur . It is also possible that the thermal cycling may cause teeth with amalgam restorations to fracture due to differences in their coefficient of thermal expansion; therefore, autoclaving may not be a good option to sterilize extracted teeth that are going to be used for preclinical education . Pantera and schuster reported that rockal solution (benzalkonium chloride) for 24 h and 3 weeks did not eliminate microorganisms in teeth . In their study, 5.25% sodium hypochlorite failed to disinfect the teeth in 5 min, but autoclaving for 40 min at a pressure of 15 psi and a temperature of 121c destroyed all bacterial species . Attam et al ., reported that the chemical materials such as 2.6% sodium hypochlorite, 3% hydrogen peroxide and boiling water are not suitable, and effective for disinfecting / sterilizing extracted human teeth . Cdc recommends that the teeth used for educational and research purposes should be disinfected with sodium hypochlorite or liquid chemical germicides . The present investigation revealed that there is not any relation between the year of education, trained about sterilization of extracted human teeth and total mean score for performance and knowledge . Since, the students in kerman dental school are touched with extracted human teeth just at the third years of education; it is evidence that the number of the year in their education hasnt any influence on their performance and knowledge . In the field of infection control teaching, cleaning with boiling water and sodium hypochlorite are touched only, such that it does nt have any effect on the students performance and knowledge . In this research work, the students had good performance and knowledge in to sterilization / disinfection of extracted human teeth, although it seems that more education and teaching is needed for improving the quality of extracted human teeth to sterilization / disinfection . This study showed that the students under study had a positive attitude toward sterilization of these teeth but did not have a positive attitude toward using the protective accessories such as gloves and a white cloak while handling extracted teeth . Infection control measures to protect students and faculty staff are not confined to disinfection / sterilization of extracted teeth . Instrument sterilization as well as the use of gloves, eye protection, and masks should also be considered in the preclinical laboratory . Stevens reported that bacterial colonies grew on plates placed in the area of the dentists nose and mouth while performing dental procedures with an air turbine handpiece . This study showed that students have good knowledge about disinfection of extracted teeth, although some of them did not disinfect or sterilize these teeth . Therefore, we conclude that the method in which most students use to sterilize extracted teeth is not effective in practice, and more attention should be paid to teach them a suitable method to sterilize the teeth as they are working on . The results of this study indicated that knowledge, performance, and attitude of dental students in relation to sterilization / disinfection methods of extracted human teeth were good.
The pathology of rheumatoid arthritis (ra) is characterized by the proliferation of synovial cells and angiogenesis, pannus formation . Multiple cell types, including lymphocytes, dendritic cells, macrophages, and synovial fibroblasts, contribute to the chronic inflammatory responses of ra, and comprise a major portion of the invasive pannus . In addition, angiogenesis, the process of new blood vessel formation, is highly active in ra, particularly during the earliest stages of the disease [2, 3]. Newly formed vessels can maintain the chronic inflammatory state by transporting inflammatory cells to sites of synovitis, and supply nutrients and oxygen to the pannus [2, 3]. Angiogenesis is strictly regulated by many inducers and inhibitors, and a number of proangiogenic factors have been suggested to be involved in neovascularization in ra joints . These include acidic and basic fibroblast growth factors, transforming growth factor (tgf)-, angiopoietin, and placenta growth factor (pigf) in addition to vascular endothelial growth factor (vegf) [24]. The final goal of ra treatment is complete disease remission, and not symptomatic relief . At one end of the spectrum of ra treatment outcomes lie a large group of patients who do not respond to single disease - modifying antirheumatic drugs (dmards). Recent clinical trials have suggested that several biologic agents, such as tnf- blockers, rituximab, abatacept, and anakinra, are effective at retarding joint destruction and at alleviating ra activity [5, 6]. However, these biologic agents may have serious side effects, such as predispositions to tuberculosis, lymphoma, progressive multifocal leukoencephalopathy, and high cost, which limit their use . It is also a concern that abrupt stoppages or reductions in these treatments may result in a relapse of disease activity . Moreover, the pathology of ra suggests that it is unlikely that a single biologic agent that targets a specific subset of immune cells is capable of effecting cure . In this review, we integrate current knowledge concerning how angiogenesis, specifically vegf, contributes to disease exacerbations in ra . In addition, we present a new therapy for ra based on a synthetic anti - vegf hexapeptide that specifically targets the interaction between vegf and its receptor . Prospects for the development of pharmacologic regulators of placental growth factor, which is another angiogenic factor implicated in the pathogenesis of ra, also are discussed . Vegf is a dimeric glycoprotein that induces the proliferation and migration of endothelial cells to form new blood vessels, and which increases vascular permeability . Vegf plays important roles during wound healing, embryonic development, the growths of certain solid tumors, and during ascites formation . Several recent reports have demonstrated that vegf is also implicated in the pathogenesis of ra . Vegf in synovial fluids is significantly more increased in ra than in osteoarthritis [2, 13, 14], and serum levels of vegf correlate well with ra disease activity, particularly with swollen joint counts . Vegf protein and mrna are expressed by synovial macrophages and synovial fibroblasts in the synovial tissues of ra patients, and cultured synovial cells are able to secrete vegf under hypoxic conditions or when stimulated with il-1, il-6, il-17, il-18, -prostaglandin, or tgf-, or by cd40 ligation [24, 1517]. Furthermore, vegf knockout mice showed reduced pathology and synovial angiogenesis in antigen - induced models of arthritis . These findings strongly suggest that the inhibition of the angiogenic action of vegf is likely to suppress rheumatoid inflammation . Angiogenesis and inflammation are interdependent processes, and inflammatory mediators have significant effects on angiogenesis [2, 3]. Furthermore, recent studies have suggested that the reverse is also true [14, 19]. For example, chronic transgenic delivery of placental growth factor (plgf) to murine epidermis resulted in a significant increase in inflammatory response . In addition, in a previous study, we demonstrated that 165-amino acid form of vegf, vegf165, has a direct proinflammatory role in the pathogenesis of ra . In this previous study, recombinant vegf165 was found to increase the productions of tnf- and il-6 by human peripheral blood mononuclear cells (pbmc). Moreover, the synovial fluid mononuclear cells of ra patients showed a greater response to vegf165 stimulation than the pbmc of healthy controls (the major cell types that responded to vegf were monocytes). These findings suggest that vegf165 may act as a proinflammatory mediator and as an angiogenic stimulator in ra joints, and thus, they indicate that vegf is an important link between angiogenesis and the inflammatory process . A number of inflammatory cell types participate in maintaining a mutually activating network in ra joints, which leads to the establishment of a self - perpetuating cycle of autoimmunity . It has been documented that vegf165 activates endothelial cells to produce chemokines, such as mcp-1 and il-8 [20, 21], which may recruit monocytes around endothelial cells in synovial membranes, where newly employed macrophages, in addition to resident synoviocytes, can produce tnf- and il-6 when stimulated by vegf165 (as was evidenced by our work) or via cell contact with activated endothelial cells . Tnf- and il-6, in turn, further enhance the capacities of macrophages and synoviocytes to secrete vegf165, and stimulate endothelial cells to induce cell - contact - mediated macrophage activation, which generates a positive feedback - loop (figure 1). Thus, vegf165 may serve as a functional bridge between endothelial cells and macrophages / synoviocytes . In ra synovium, synovial fibroblasts proliferate abnormally and invade local environments, and in some ways exhibit the characteristics of tumor cells . Recently, we demonstrated that vegf is crucially required for the survival of rheumatoid synoviocytes . In this previous study, the ligation of recombinant vegf165 to its receptor prevented synoviocyte apoptosis induced by serum starvation or sodium nitroprusside (snp). Vegf165 rapidly triggered pakt and perk activity, and then induced bcl-2 expression in rheumatoid synoviocytes . Furthermore, vegf165 completely blocked snp - induced bcl-2 downregulation and snp - induced bax translocation from the cytosol to mitochondria . Collectively, these results suggest that vegf functions as an important synoviocyte survival factor in ra . As mentioned above, vegf165 is present at higher levels in sera, synovial fluid, and in the inflamed synovial tissues of ra patients than in those of osteoarthritis patients [13, 14]. Therefore, ra synoviocytes are more likely to be stimulated by vegf165 than osteoarthritis synoviocytes, which causes synoviocyte hyperplasia . Moreover, hyperplastic synoviocytes in ra joints secrete more vegf165, and thus, generate positive feedback that promotes their survival (figure 1). Vegf165 exerts its biological effects by binding with its receptor subtypes, that is, fms - like tyrosine kinase (flt-1), kinase insert domain - containing receptor (kdr) and neuropilin-1 (np-1). Flt-1 and kdr exhibit tyrosine kinase activity, and both are expressed in the majority of vascular endothelial cells [8, 24, 25]. Kdr is a primary mediator of endothelial cell proliferation in response to vegf165, whereas unlike kdr, flt-1 is present in inflammatory cells, such as, macrophages and monocytes [8, 24, 25]. Therefore, in addition to its proangiogenic action, flt-1 is critically involved in monocyte activation, and in addition, it also promotes the mobilization of myeloid progenitors from bone marrow to the blood [8, 24, 25]. On the other hand, np-1 has been demonstrated to function as a nontyrosine kinase receptor for vegf165, and specifically, for the heparin - binding domain of vegf165 [26, 27]. Np-1 was initially characterized as a receptor for semaphorin 3a, which mediates the guidance of neuronal cells . In endothelial cells, np-1 is also a coreceptor of vegf, and has been shown to regulate kdr - dependent angiogenesis [26, 27]. Furthermore, we previously demonstrated that np-1, rather than flt-1 or kdr, is the major vegf165 receptor in ra synoviocytes . Np-1 was found to be highly expressed in the lining layer, and on infiltrating leukocytes and endothelial cells of the rheumatoid synovium . Furthermore, the downregulation of np-1 transcripts by short interfering rna caused spontaneous synoviocyte apoptosis, which was associated with both a decrease in bcl-2 expression and an increase in bax translocation to mitochondria . In addition, more recently, we found that flt-1 is highly expressed in the sublining of leukocytes in ra synovium, and in monocytes from the pbmc of active ra patients . Furthermore, flt-1 expression levels were found to be well correlated with erythrocyte sedimentation rates, a marker of disease activity, indicating that they reflect inflammatory activity of ra . These findings suggest that chronic inflammatory milieux, such as those generated by high concentrations of proinflammatory cytokines, may upregulate flt-1 expression on ra monocytes . First, vegf165 binding to kdr may lead to an increase in angiogenesis, and thereby, the recruitment of peripheral leukocytes to inflamed synovium, which diminishes the growing burden of synoviocytes by supplying the oxygen and nutrients required for tissue metabolism . Second, via interaction with flt-1, vegf165 may directly stimulate the productions of cytochemokines, such as tnf-, il-6, mcp-1, and il-8, which are essential for the perpetuation of chronic inflammation in joints . Third, np-1 could hamper synoviocyte apoptosis upon ligation of vegf165, and thus, function as a survival factor, in an autocrine or paracrine fashion . In this manner, vegf165 would simultaneously regulate the developments of synovial inflammation, hyperplasia, and angiogenesis in ra joints (figure 1). The success of anti - vegf antibody (ab) treatment in cancer patients raises the possibility of applying antiangiogenic therapies in other diseases, such as retinopathy, ra, and other inflammatory disorders . Given the pleiotropic roles played by vegf and its receptor in ra inflammation [2, 3, 8, 24], it can be postulated that anti - vegf treatment retards chronic synovitis in several ways, as follows: (a) it may decrease nutrient supply to the tumor - like synovium; (b) inhibit leukocyte adhesion and migration by decreasing endothelial cell surface area; (c) decrease chemokine and cytokine productions by activated endothelial cells; (d) reduce the vegf - induced productions of tnf- and il-6 by monocytes / macrophages; (e) abrogate vegf - induced increases in synoviocyte survival . These different mechanisms may occur independently, but it remains to be determined which mechanism plays a dominant role in the quenching of ra inflammation . Bevacizumab, a humanized form of anti - vegf ab, was the first antiangiogenic agent approved by the fda in the us for the treatment of metastatic colon cancer, and it was also found to be beneficial for the treatment of lung and renal cell cancer [33, 34]. Although bevacizumab is generally well tolerated, it has some serious toxic effects, for example, hypertension, bleeding, and arterial thromboembolism, which occur infrequently [3234]. Currently, various different developmental approaches to inhibit vegf and its receptors are in progress . The fda approved pegaptanib, an anti - vegf rna aptamer, for the treatment of neovascular age - related macular degeneration (amd). Notably, pegaptanib was found to reduce vision loss in amd patients by about 50% during the first treatment year and to stabilize vision during the second year . Other agents which target vegf receptors, such as, chimerized anti - kdr antibody, vegf - trap, and a synthetic ribozyme of flt-1, are also undergoing phase i or ii trials for the treatment of solid tumors and cancer . The effects of vegf and of its receptor antagonists have also been tested in experimental models of ra . Neutralizing ab to vegf was found to prevent collagen - induced arthritis and to ameliorate established disease in mice, and treatment with a soluble form of flt-1 receptor significantly attenuated the severity of murine collagen - induced arthritis . Interestingly, the failure of anti - kdr ab, but not of anti - flt-1 ab, to block arthritis and atherosclerosis [24, 25, 39], indicates that anti - inflammation, rather than antiangiogenesis, may be primarily responsible for the observed effects of anti - vegf ab . Considering that flt-1 tyrosine kinase signaling promotes ra via monocyte / macrophage activation [31, 40], the selective inhibition of flt-1 may be effective at blocking vegf - induced inflammation and angiogenesis with minimal toxicity . However, no clinical trial on anti - vegf inhibitors has been undertaken in ra . Indeed, some antirheumatic drugs with well - known clinical efficacy in ra, such as cyclosporin and anti - tnf- ab, have been reported to inhibit vegf production in ra patients [41, 42]. Through the screening of positional scanning synthetic peptide libraries, we identified a soluble arginine - rich hexapeptide sequence, rrkrrr (arg - arg - lys - arg - arg - arg), which binds to vegf165, and thereby prevents it from interacting with its receptor . To increase the in vivo stability of this peptide, we changed its peptide structure from the l- to the d - form, and accordingly, were able to increase its half life to more than 24 hours, which makes the peptide more suitable for therapeutic applications . In mice, the hexapeptide rrkrrr significantly inhibited vegf - induced angiogenesis, and also retarded the growth and metastasis of colon carcinoma cells . In addition, it strongly inhibited ongoing paw inflammation in arthritic mice without apparent side effects . When compared with several known vegf antagonists, such as anti - vegf antibody and aptamer, rrkrrr is advantageous from the clinical standpoint because it is a short peptide that is easily synthesized and because it has low immunogenicity . In a similar manner, bae et al ., also found that the novel anti - flt-1 hexapeptide, gnqwfi (gly - asn - gln - trp - phe - iie), inhibited angiogenesis and tumor growth without side effects . This peptide selectively binds to flt-1, and thereby, blocks the interaction between flt-1 and vegf or plgf . Investigations on the effect of anti - flt-1 peptide gnqwfi on an experimental model of arthritis are under way . Plgf is a member of the vegf family, which was first identified in placenta, but is also known to be present in heart, lung, and joints . As a specific ligand for flt-1, plgf has potent angiogenic properties, and it also induces the growth and migration of endothelial cells [24, 25]. In addition, plgf stimulates tissue factor production and chemotaxis in monocytes, and also increases tnf-, il-1, il-6, il-8, and mcp-1 productions by normal and/or rheumatoid monocytes [31, 46], which suggests that it directly modulates the inflammatory process . Plgf concentrations were reported to be increased in ra synovial fluids, and to induce vegf production by mononuclear cells . Moreover, genetic ablation of plgf prevented the development of antibody - induced arthritis in mice suggesting the critical role of plgf in ra inflammation . Plgf exhibits functions that are distinct from those of vegf, in that it regulates the angiogenic switch during the diseased state . It was recently reported that neutralizing ab to plgf inhibits the growth and metastasis of various tumors, including those resistant to vegf inhibitors, and that it enhances the efficacies of chemotherapy and that of anti - kdr ab . Unlike anti - kdr ab, anti - plgf ab prevented the infiltration of angiogenic macrophages and severe tumor hypoxia, and thus, did not switch on the angiogenic rescue program responsible for resistance to anti - kdr ab . Furthermore, it did not cause or enhance anti - kdr ab - related side effects, such as, the inhibition of placental vascular development . Similar suppressive effects of plgf antagonist on tumor growth were observed in another study, in which an antagonistic plgf peptide (shryrlaiqlhasdsssscv) inhibited the growth and metastasis of human breast cancer xenografts . Taken together, plgf antagonists may prevent angiogenesis and tumor growth without affecting normal physiology, and thus, are ideal candidates for ra treatment . We are currently investigating whether synthetic anti - plgf peptides inhibit the severity of arthritis and angiogenesis . We and others have demonstrated that proangiogenic factors, such as vegf and plgf, exert direct proinflammatory [14, 1921, 31, 40, 45, 46] and antiapoptotic effects [23, 29]. In this regards, the developments of synovial inflammation, hyperplasia, and angiogenesis in the joints of ra patients may all be regulated by vegf . Given the importance of vegf in the pathology of ra, antiangiogenic therapies, particularly those involving an anti - flt-1 blocking agents, could when administered as a monotherapy or in combination with other biologic agents selectively ameliorate ra symptoms and reverse its fundamental pathology . The antiangiogenic peptides, rrkrrr and gnqwfi, introduced here represent a promising development in the antiangiogenic field.
Concern over the part of indoor air pollution in the damaging of human health has recently increased . People spend more than 80%90% of their time indoors, especially at home; therefore, a good indoor air quality (iaq) is crucial . There are conflicting reports on the respiratory health effects of indoor risk factor exposure, including fuel combustion, environmental tobacco smoke and allergen . Some studies reported adverse outcomes of exposure to indoor environment risk factors (1, 2), whereas, few studies reported absence of any association between adverse health effect and the similar exposure (3). Biological or chemical exposure to biomass smoke, cooking oil fumes, mold, dampness, cockroach, rodent and dust mite allergens, and environmental tobacco smoke (ets) can have adverse health effects on the adult lung function level . To illustrate, previous studies have linked exposure to mold to worse lung function in asthmatic patients (4). Allergic sensitization via cockroach and mouse allergens can be triggering mechanisms for a decrease in lung function among atopic individuals (6). Other potential indoor risk factors include active and passive smoking, positively correlated with a decline in lung function among susceptible populations (7). There is a large demand for coal for cooking and baking in households in summer . The combustion of coal and natural gas in poorly ventilated homes exposes children and adults to high levels of particulate matter and other aero contaminants . In addition, zunyi is one of china s least sunny cities with relative humidity of above 80% in summer season . Its summer has a wet, hot and gloomy climate, pre - disposed to indoor mold (or fungi) growth . For many people, the risks to respiratory health may be greater due to exposure to excessively high indoor pollutants from poorly ventilated household stoves . A better understanding of seasonal exposure in urban china and the relative contribution of behavioral and household structural factors to personal exposure is important for estimating the global disease burden attributable to indoor air pollution . This study aimed to investigate the relationship between indoor environmental risk factors and occurrence of lung function decline in adult residents in summer in zunyi city of southwest china . A cross - sectional investigation was conducted for lung function level among adult residents in zunyi, the largest city in the north of guizhou province, that has 11 inner - city areas, including laocheng (lc) road, wangli (wl) road, zhonghua (zh) road, nanmenguan (nmg) road, yanan (ya) road, zhoushuiqiao (zsq) road, zhongshan (zs) road, beijing (bj) road, shanghai (sh) road, xima (xm) road, and dalian (dl) road, covering an area of approximately 105 km, with a population of about 900000 . Eleven communities in 11 inner - city areas in zunyi were randomly selected and investigated . The first family was selected by simple random sampling of the residential address number in each community . All adult family members present at the residence who met the inclusion criteria were selected . After that, neighbors living in a residence next door who met the inclusion criteria were recruited and interviewed . If no one was at home, the interviewer returned up to three times before moving to another family next door . If the selected family refused to participate or could not be found, neighbors living in the next residence who met the inclusion criteria were recruited . This procedure was repeated for every house among the selected houses until the targeted number of participants was recruited . Inclusion criteria for the studied community were no factories / plants within the selected community . Inclusion criteria for eligible residents were 18 and above yr olds for men and women; living more than 3 yr within inner - city area in zunyi city . Lung function was examined by measuring forced vital capacity (fvc), forced expiratory volume in 1 s (fev1), fev1/fvc, and peak expiratory flow rate (pefr), according to standard guidelines using a portable electronic fgc - a+ spirometer (anhui institute of electronic science, china). The participants were asked to sit in an upright position with both feet flat on the ground . They were instructed to inhale completely, place the meter in their mouth, and to exhale with maximal force as soon as their lips were sealed around the mouthpiece, while maintaining an upright position . Based on guidelines of the american thoracic society, maneuvers were only accepted if they had low back - extrapolated volume (<5% of the fvc and <0.15 l), both the fvc and fev1 were within 0.20 l of the best effort fvc and fev1, and there was a low volume accumulated at the end of the effort . Each subject was tested on three expiratory maneuvers . To measure pm2.5 concentrations inside and outside the households, japan), which is a portable monitor based on the light - scattering principle, with a laser diode as the light source . The monitor determines the relative concentration of pm2.5 by measuring the intensity of the laser beam scattered by particles . The advantage of measuring pm2.5 relative concentration with the ld-3k fine dust monitor was that it allowed an analysis of concentration level (high or low), namely, a relative comparison (screening) and an analysis of variation with time (8). The relative concentration is reported as counts per min (cpm). To determine the cpm, the following equation was used: cpm = counts value / measuring time (in min). Each measurement was maintained for over 1 min, and three readings were taken for each measurement to calculate the mean relative concentration . There was an interval period of 1 min between the 3 measurements of pm2.5 in the kitchen, living room, or outdoors, whereas the interval period for measurements between the kitchen, living room, and outdoors was 5 min in each house . Data analysis was carried out using spss ver . 20.0 (chicago, il, usa). After ascertaining whether distributions were normally distributed, we compared continuous variables using student s two - tailed t - test, anova test, mann - whitney u test or kruskal - wallis test . A p - value of less than 0.05 was considered as level of statistical significance . T - test, anova test, or non - parametric test analysis was used to estimate the difference in pulmonary function (pefr, fvc, fev1 and fev1/fvc) among adults who exposed to various indoor exposure factors (kitchen, sleeping area characteristics and ets) in summer season . Multiple linear regression analysis was used to assess the association of kitchen, sleeping area characteristics, ets and pm2.5 exposure with pulmonary function level (pefr, fvc, fev1 and fev1/fvc), controlling for socio - demographic factors . Of these pulmonary function parameters, data analysis was conducted on the largest fvc of the two curves, the largest fev1, the ratio of the largest fev1 to the largest fvc, and the largest pefr (9). A cross - sectional investigation was conducted for lung function level among adult residents in zunyi, the largest city in the north of guizhou province, that has 11 inner - city areas, including laocheng (lc) road, wangli (wl) road, zhonghua (zh) road, nanmenguan (nmg) road, yanan (ya) road, zhoushuiqiao (zsq) road, zhongshan (zs) road, beijing (bj) road, shanghai (sh) road, xima (xm) road, and dalian (dl) road, covering an area of approximately 105 km, with a population of about 900000 . Eleven communities in 11 inner - city areas in zunyi were randomly selected and investigated . The first family was selected by simple random sampling of the residential address number in each community . All adult family members present at the residence who met the inclusion criteria were selected . After that, neighbors living in a residence next door who met the inclusion criteria were recruited and interviewed . If no one was at home, the interviewer returned up to three times before moving to another family next door . If the selected family refused to participate or could not be found, neighbors living in the next residence who met the inclusion criteria were recruited . This procedure was repeated for every house among the selected houses until the targeted number of participants was recruited . Inclusion criteria for the studied community were no factories / plants within the selected community . Inclusion criteria for eligible residents were 18 and above yr olds for men and women; living more than 3 yr within inner - city area in zunyi city . Lung function was examined by measuring forced vital capacity (fvc), forced expiratory volume in 1 s (fev1), fev1/fvc, and peak expiratory flow rate (pefr), according to standard guidelines using a portable electronic fgc - a+ spirometer (anhui institute of electronic science, china). The participants were asked to sit in an upright position with both feet flat on the ground . They were instructed to inhale completely, place the meter in their mouth, and to exhale with maximal force as soon as their lips were sealed around the mouthpiece, while maintaining an upright position . Based on guidelines of the american thoracic society, maneuvers were only accepted if they had low back - extrapolated volume (<5% of the fvc and <0.15 l), both the fvc and fev1 were within 0.20 l of the best effort fvc and fev1, and there was a low volume accumulated at the end of the effort . To measure pm2.5 concentrations inside and outside the households, we used a digital dust monitor (ld-3k; sibata scientific technology inc ., japan), which is a portable monitor based on the light - scattering principle, with a laser diode as the light source . The monitor determines the relative concentration of pm2.5 by measuring the intensity of the laser beam scattered by particles . The advantage of measuring pm2.5 relative concentration with the ld-3k fine dust monitor was that it allowed an analysis of concentration level (high or low), namely, a relative comparison (screening) and an analysis of variation with time (8). The relative concentration is reported as counts per min (cpm). To determine the cpm, the following equation was used: cpm = counts value / measuring time (in min). Each measurement was maintained for over 1 min, and three readings were taken for each measurement to calculate the mean relative concentration . There was an interval period of 1 min between the 3 measurements of pm2.5 in the kitchen, living room, or outdoors, whereas the interval period for measurements between the kitchen, living room, and outdoors was 5 min in each house . Data analysis was carried out using spss ver . 20.0 (chicago, il, usa). After ascertaining whether distributions were normally distributed, we compared continuous variables using student s two - tailed t - test, anova test, mann - whitney u test or kruskal - wallis test . A p - value of less than 0.05 was considered as level of statistical significance . T - test, anova test, or non - parametric test analysis was used to estimate the difference in pulmonary function (pefr, fvc, fev1 and fev1/fvc) among adults who exposed to various indoor exposure factors (kitchen, sleeping area characteristics and ets) in summer season . Multiple linear regression analysis was used to assess the association of kitchen, sleeping area characteristics, ets and pm2.5 exposure with pulmonary function level (pefr, fvc, fev1 and fev1/fvc), controlling for socio - demographic factors . Of these pulmonary function parameters, data analysis was conducted on the largest fvc of the two curves, the largest fev1, the ratio of the largest fev1 to the largest fvc, and the largest pefr (9). The indoor and outdoor levels of pm2.5 relative concentrations were measured in 20 selected households during the cooking period . Socio - demographic characteristics of the subjects in summer season the mean (standard deviation) age of the participants was 45.4 (16.2) yr and 54.4% were female . Only about 19.0% of the adults were over 60 yr old and the percentage of those younger than 39 yr was approximately 42.0% . The marital, educational, bmi and monthly household income status of participants did not change significantly during the two - season period . Chinese ethnic groups (han) comprised mostly of adult subjects (95.9%), followed by ethnic of minority (4.1%). Around 82.0% subjects more than half of all subjects (64.6%) had normal weight, compared with those who were under - weight (17.5%) and overweight (17.9%). Almost one - third of adult subjects (29.7%) reported familial history of asthma and asthma - related symptoms . Approximately three - fourth of adults (79.7%) had a total monthly family income of at least 1753 chinese yuan . About 20.0% of adult subjects were exposed regularly to dust or gas at work (table 1). Of the 610 subjects who performed spirometry, the adults from coal stove using families showed a significantly lower fvc, fev1 and pefr compared to adults from clean fuel stove and fix fuel stove using families (p<0.001). When subjects exposed to cooking oil fumes frequently or sometimes were compared with the subjects who seldom exposed or without such exposure, values of fvc (p=0.030), fev1/fvc (p=0.042) and pefr (p=0.029) were significantly lower in subjects who exposed to cooking oil fumes . For pulmonary function test parameters (fvc (p=0.022), fev1 (p=0.043) and pefr (p=0.017)), significant differences were observed among subjects who stated that there was no pest, subjects stated that pest haunted few of the time and subjects stated that pest haunted some - times in their houses, with median values lowest in those with pest exposure sometimes (table 2). Median values of fvc (p=0.014), fev1 (p=0.014) and pefr (p=0.017) were significantly lower among adults used mosquitoes killing spray or coil incense to expel mosquito than that among adults used mosquito net or did not use mosquito repellent . In contrast, in adults whose mattress used history more than 5 yr had a significantly higher values of fvc (p=0.023), fev1 (p=0.016), and pefr (p=0.024). Fvc (p=0.037) and fev1 (p=0.049) were significantly lower in adults who used fluffy blanket as compared with the respective non - users . Compared with the subjects did not keep pets, subjects kept pets showed significant decrease in fvc (p=0.011), fev1 (p=0.014) and pefr (p=0.012). The subjects reported the presence of mould in their bedrooms showed significant deficits in fvc (p=0.001), fev1 (p=0.006) and pefr (p=0.002) as compared with those who reported the absence of mould in their bedrooms (table 2). Pulmonary function (fvc, fev1, fev1/fvc and pefr) in adults by kitchen risk factors in summer season nonparametric test (mann - whitney u test or kruskal - wallis test), = 0.05; significant at p<0.05, significant at p<0.01, significant at p<0.001 in comparison to non - smokers, both current smokers and ex - smokers were having lower values of fvc (p<0.001), fev1 (p<0.001) and pefr (p<0.001). Compared with the subjects were not exposed to second - hand smoke, subjects who were exposed to second - hand smoke showed similar deficits in fvc (p=0.048), and pefr (p=0.039) as did the current smokers and ex - smokers (table 2). Table 3 shows that, among the 41 adult participants whose houses were monitored, no significant correlations between indoor kitchen and sleeping area as well as outdoor pm2.5 relative concentration and pulmonary function test parameters (fvc, fev1, fev1/fvc, and pefr) were observed (p>0.05). Pulmonary function levels (fvc, fev1, fev1/fvc, and pefr) in adults and its association with pm2.5 exposure level in summer season r: correlation coefficient . * significant at p<0.05; * * significant at p<0.01; * * * significant at p<0.001 fig . 1 shows that the median (25% and 75% quartile) relative concentrations in pm2.5 measured in indoor kitchen, sleeping area and outdoor were 486.0 (468.0555.5) cpm, 463.0 (440.0535.0) cpm and 459.0 (420.0489.0) cpm, respectively . When the indoor kitchen, sleeping area and outdoor concentrations of pm2.5 were compared, the pm2.5 relative concentrations were significantly higher in kitchen than in sleeping area (z=2.343, p=0.019) and outdoor (z=2.789, p=0.005). Although, the pm2.5 relative concentration was higher in sleeping area than outdoor, the difference was not significant (z=0.858, p=0.391). Pm2.5 relative concentrations in 20 monitored houses in summer season mann - whitney u test, * significant at p<0.05; * * significant at p<0.01; * * * significant at p<0.001 the indoor and outdoor levels of pm2.5 relative concentrations were measured in 20 selected households during the cooking period . Socio - demographic characteristics of the subjects in summer season the mean (standard deviation) age of the participants was 45.4 (16.2) yr and 54.4% were female . Only about 19.0% of the adults were over 60 yr old and the percentage of those younger than 39 yr was approximately 42.0% . The marital, educational, bmi and monthly household income status of participants did not change significantly during the two - season period . Chinese ethnic groups (han) comprised mostly of adult subjects (95.9%), followed by ethnic of minority (4.1%). Around 82.0% subjects more than half of all subjects (64.6%) had normal weight, compared with those who were under - weight (17.5%) and overweight (17.9%). Almost one - third of adult subjects (29.7%) reported familial history of asthma and asthma - related symptoms . Approximately three - fourth of adults (79.7%) had a total monthly family income of at least 1753 chinese yuan . About 20.0% of adult subjects were exposed regularly to dust or gas at work (table 1). Of the 610 subjects who performed spirometry, the adults from coal stove using families showed a significantly lower fvc, fev1 and pefr compared to adults from clean fuel stove and fix fuel stove using families (p<0.001). When subjects exposed to cooking oil fumes frequently or sometimes were compared with the subjects who seldom exposed or without such exposure, values of fvc (p=0.030), fev1/fvc (p=0.042) and pefr (p=0.029) were significantly lower in subjects who exposed to cooking oil fumes . For pulmonary function test parameters (fvc (p=0.022), fev1 (p=0.043) and pefr (p=0.017)), significant differences were observed among subjects who stated that there was no pest, subjects stated that pest haunted few of the time and subjects stated that pest haunted some - times in their houses, with median values lowest in those with pest exposure sometimes (table 2). Median values of fvc (p=0.014), fev1 (p=0.014) and pefr (p=0.017) were significantly lower among adults used mosquitoes killing spray or coil incense to expel mosquito than that among adults used mosquito net or did not use mosquito repellent . In contrast, in adults whose mattress used history more than 5 yr had a significantly higher values of fvc (p=0.023), fev1 (p=0.016), and pefr (p=0.024). Fvc (p=0.037) and fev1 (p=0.049) were significantly lower in adults who used fluffy blanket as compared with the respective non - users . Compared with the subjects did not keep pets, subjects kept pets showed significant decrease in fvc (p=0.011), fev1 (p=0.014) and pefr (p=0.012). The subjects reported the presence of mould in their bedrooms showed significant deficits in fvc (p=0.001), fev1 (p=0.006) and pefr (p=0.002) as compared with those who reported the absence of mould in their bedrooms (table 2). Pulmonary function (fvc, fev1, fev1/fvc and pefr) in adults by kitchen risk factors in summer season nonparametric test (mann - whitney u test or kruskal - wallis test), = 0.05; significant at p<0.05, significant at p<0.01, significant at p<0.001 in comparison to non - smokers, both current smokers and ex - smokers were having lower values of fvc (p<0.001), fev1 (p<0.001) and pefr (p<0.001). Compared with the subjects were not exposed to second - hand smoke, subjects who were exposed to second - hand smoke showed similar deficits in fvc (p=0.048), and pefr (p=0.039) as did the current smokers and ex - smokers (table 2). Table 3 shows that, among the 41 adult participants whose houses were monitored, no significant correlations between indoor kitchen and sleeping area as well as outdoor pm2.5 relative concentration and pulmonary function test parameters (fvc, fev1, fev1/fvc, and pefr) were observed (p>0.05). Pulmonary function levels (fvc, fev1, fev1/fvc, and pefr) in adults and its association with pm2.5 exposure level in summer season r: correlation coefficient . * significant at p<0.05; * * significant at p<0.01; * * * significant at p<0.001 1 shows that the median (25% and 75% quartile) relative concentrations in pm2.5 measured in indoor kitchen, sleeping area and outdoor were 486.0 (468.0555.5) cpm, 463.0 (440.0535.0) cpm and 459.0 (420.0489.0) cpm, respectively . When the indoor kitchen, sleeping area and outdoor concentrations of pm2.5 were compared, the pm2.5 relative concentrations were significantly higher in kitchen than in sleeping area (z=2.343, p=0.019) and outdoor (z=2.789, p=0.005). Although, the pm2.5 relative concentration was higher in sleeping area than outdoor, the difference was not significant (z=0.858, p=0.391). Pm2.5 relative concentrations in 20 monitored houses in summer season mann - whitney u test, * significant at p<0.05; * * significant at p<0.01; * * * significant at p<0.001 the major findings of this study are as follows: 1) subjects exposed coal fuel combustion, cooking oil fumes, pest in kitchen, mosquito repellent, fluffy blanket, pets, visible mold in bedroom and ets (active and passive smoking) tended to exhibit greater decreases in fvc, fev1 and pefr values compared with their non - exposed counterparts in summer; 2) median pm2.5 relative concentrations in kitchen, sleeping area and outdoor were 486.0cpm, 463.0cpm and 459.0cpm, respectively . Pm2.5 relative concentration in indoor kitchen and sleeping area were significant higher than outdoor in summer season . The cross - sectional questionnaire survey of people aged 18 yr provides evidence of the effects of indoor environmental factors on occurrence of lung function decline . A negative association between kitchen, sleeping area risk factors and ets exposure and a reduction in lung function in summer was revealed in zunyi . To the best of our knowledge, this is the first study to examine potential effect modifiers of indoor environmental exposure on adult lung function level in summer season in china . Our study documents lower lung function among adults exposed to cooking oil fumes (cof) compared with adults who are never or seldom exposed to cof . This association persists after controlling for the effects of socio - demographic factors, the level of pm2.5 concentration, etc . Our findings are in line with previous studies that examined the relationship between cof exposure and lung function . Raj et al . In a study to evaluate the effects of cooking fuel smoke on lung function in asymptomatic women in india demonstrated a reduction in lung function among women who were exposure to cof (10). In a community survey a semi - rural area in cameroon to assess the effects of cof exposure and lung function, compared forced respiratory volume between women using wood and women using alternative sources of energy for cooking . In that study, the authors documented a significant airflow obstruction in the wood smoke group (11). Exposure to household air pollution from coal combustion is recognized as an important cause of impaired lung function . In the present study, we observed impaired lung function among adults from coal using families . Several studies have also reported reduction in lung function in adults chronically exposed to coal fuel smoke . Our recent publication evaluated the exposure to indoor burning coal air pollution as a risk factor for pulmonary function decline in adult participants in zunyi . Exposure to coal smoke was associated with a 31.7% decrease in fvc, a 42.0% decrease in fev1, a 7.46% decrease in the fev1/fvc ratio, and a 23.1% decrease in pefr in adult residents . The slope of lung function decrease for chinese adults is approximately a 2-l decrease in fvc, a 3-l decrease in fev1, and an 8 l / s decrease in pefr per count per minute of pm2.5 exposure (1). Similar reductions in peak flow have been observed 100 indian women with asthma living in homes that used coal fuel for cooking (13). This deterioration of lung function in coal fuel users has been attributed to the fact that the amount and concentration of particulate matter and other toxic gases (e.g., so2) emitted during coal combustion while cooking are more than those emitted during combustion of lpg or cleaner fuel . Exposure to indoor microbial allergens, together with building dampness, is an important risk factor for the reduction in lung function of the occupants though the underlying mechanisms for exposure - related injury are still being investigated . Numerous studies have analyzed the relationship between cat and dog allergen exposure and effects on respiratory health in adult populations . Subjects both sensitized and exposed to high levels of sensitizing allergen (dog, cat and dust mite) had significantly lower fev1 percent predicted values (mean, 83.7% vs. 89.3%) compared with subjects not sensitized and exposed(14). Consistent with earlier reports, in this study, we observed that decreased fvc, fev1 and pefr values among adult residents were inversely related to pet exposure in their bedrooms in summer . Microbial allergens exposure has clear implications for sensitization and lung function decline through the development of bronchial hyper - responsiveness and airway inflammation in sensitized subjects; however, the underlying mechanisms responsible for the observed health effects are not well understood . Exposure to indoor environmental tobacco smoke (ets) might increase the risk of lung function decline (1618). In a denmark study of 18 to 69-yr olds, adults exposed to environmental tobacco smoke for> 5 h / d had a significantly increased risk of decreased lung function (fev(1)% predicted), compared with those not exposed (19). Eisner et al . Noted a dose - dependent effect with 10-yr cumulative ets exposure on lung function among 1057 older adults . Lifetime cumulative home and work shs exposure were associated with a greater decline of fev1 (15 ml / s; 95% ci, 29 to 1.3 ml / s and 41 ml / s; 95% ci, 55 to 28 ml / s per 10-yr cumulative exposure, respectively) (20). In this study, a significant proportion of adults reported exposure to ets, 10.9% of women are exposed to tobacco smoke compared with men (7.5%). Both current smokers and ex - smokers were having lower values of fvc, fev1 and pefr in comparison to non - smokers . Compared with the subjects not exposed to second - hand smoke, subjects exposed to second - hand smoke showed similar deficits in fvc, and pefr as did the current smokers and ex - smokers . Although, selection bias might be introduced and would mask the relationship, especially, in cross - sectional design, our results showed active smoking was associated with decrements in fev1 and pefr among current smokers and pefr among adults exposed to others cigarette smoking which was consistent with other studies from the us (20) and switzerland (21). Although, china has made progress towards achieving a smoke - free environment, there remains a high degree of exposure to ets . Collectively, those residing in inner - city areas may be affected by the health burden of tobacco use . Adult residents in zunyi have less knowledge about ets and less negative attitudes about ets, as well as smoke - free home rules are not available (22). Some limitations in this study include: 1) relatively small sample size; 2) the cross - sectional design, cannot make causal inference; 3) the lack of review about the burning coal smoke exposure time in participants due to not fully recording cooking times in the kitchens of this study; 3) some confounding factors may influence on the association between indoor environmental factors and lung function level, these factors include different location of current residence, ambient air pollution, etc . Notwithstanding the limitations, we still elaborate on the attributable risk of lung function decline due to indoor coal exposure in adult residents in summer in zunyi . We identified a number of home environmental factors associated with lung function decline among chinese adults . Cooking oil fumes, environmental tobacco smoke and coal fuel use were associated with impaired lung function among adults in summer season . Subjects exposed coal fuel combustion, cooking oil fumes, pest in kitchen, mosquito repellent, fluffy blanket, pets, visible mold in bedroom and ets (active and passive smoking) tended to exhibit greater decreases in fvc, fev1 and pefr values compared with their non - exposed counterparts . Median pm2.5 relative concentrations in kitchen, sleeping area and outdoor were 486.0cpm, 463.0cpm and 459.0cpm, respectively . The present findings suggest that public health policy for eliminating certain home exposures are needed, which could have large effects not only on public health but also on medical costs in zunyi . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc .) Have been completely observed by the authors.
The antimetabolite 5-fluorouracil (fu) is widely used in the treatment of solid tumors, including gastrointestinal, breast, head, and neck cancers . Cardiotoxicity, a rare adverse effect of 5-fu, has a reported incidence of 1.2% to 18% . The patient presented with typical angina and electrocardiographic changes suggestive of an ischemic coronary event during the continuous infusion of 5-fu . The ischemia recurred when the infusion was stopped, and was relieved by administration of nitroglycerin followed by a sublingual calcium channel blocker . An 83-year - old man who had been diagnosed with stage iiic (t4n2m0) adenocarcinoma of the ascending colon and who had undergone a right hemicolectomy was admitted to inje university seoul paik hospital to begin adjuvant chemotherapy, using the folfox4 regimen (400 mg / m 5-fu bolus infusion followed by the continuous infusion of 600 mg / m 5-fu for 22 hours on days 1 and 2; 200 mg / m leucovorin as a continuous infusion for 2 hours before 5-fu infusion on days 1 and 2; and infusion of 85 mg / m oxaliplatin on day 1). Baseline echocardiography performed 1 month before admission showed normal left ventricular systolic function and no regional wall motion abnormality . He was treated with 1,500 mg high - dose 5-fu (1,000 mg / m) per day . On the morning of the 3rd day, he developed a severe, substernal, crushing chest pain during the continuous intravenous infusion of 5-fu (cumulative dose 1,679 mg / m), which was partially relieved by administering sublingual nitroglycerin . The electrocardiogram (ecg) showed st segment elevation with a tall t wave in leads i, avl, and v4 - 6, and reciprocal st segment depression in leads v1 - 2 (fig . The troponin - i and ck - mb levels were 0.010 ng / ml (reference range, 0.1) and 3.73 ng / ml (reference range, 4.94), respectively . Severe hypokinesia of the lateral wall of the left ventricle was noted on a portable bedside echocardiogram . The 5-fu infusion was stopped, and the chest pain and electrocardiographic changes resolved after intravenous infusion of nitroglycerin at 30 g / min . Emergency coronary angiography was then performed, which revealed significant stenosis in the proximal left circumf lex coronary artery (lcx). Intracoronary nitroglycerin (200 g) was injected to exclude coronary vasospasm, but no change occurred (fig . 2). Intravenous ultrasound (ivus) showed severe luminal narrowing with a heavy concentric plaque in the proximal lcx . Percutaneous coronary intervention of the proximal lcx lesion was performed successfully with the implantation of a drug - eluting stent (3.5 16 mm; taxus, national medical center, seoul, korea) (fig . The patient was transferred to the coronary - care unit, where 8 hours later, he reported a recurrence of the anterior chest pain . The ecg also showed st segment elevation and reciprocal st changes similar to those seen in the previous ischemic events (fig . The chest pain and ecg changes persisted despite a 100 g / min nitroglycerin infusion . To rule out acute stent thrombosis, the chest pain and ecg changes were relieved after sublingual administration of 10 mg nifedipine . Repeated coronary angiography showed a widely patent stent (fig . 4). The postprocedural troponin - i and ck - mb levels were 0.010 and 4.62 ng / ml, respectively . Echocardiography performed the next day also showed the absence of the regional wall motion abnormality and normal left ventricular systolic function . We present here a case of severe cardiotoxicity mimicking acute anterolateral myocardial infarction occurring in a patient receiving 5-fu chemotherapy for adenocarcinoma of the colon . The cessation of 5-fu administration and the subsequent initiation of treatment with a sublingual calcium channel blocker and nitrate resulted in a successful outcome . Although the mechanism by which 5-fu exerts its cardiotoxic effects is unknown, the resolution of the patient's chest pain and the normalization of his ecg changes with a vasodilator strongly support the vasospastic hypothesis of 5-fu cardiotoxicity . One study postulated that 5-fu - associated cardiotoxicity is due to the uncoupling of the electromechanical mechanisms that underlie normal myocardial function, which might be mediated at the level of the cell membrane . Recently, kuzel et al . Suggested that 5-fu promotes a hypercoagulable state (e.g., coronary artery thrombosis) and observed a significant increase in fibrinopeptide a and a decrease in protein c activity during 5-fu administration . The incidence of clinically apparent 5-fu cardiotoxicity is less than 10% in patients receiving the drug . Patients with a history of coronary artery disease (cad) have a significantly increased risk of 5-fu - induced cardiotoxicity . Although our patient did not have a history of cad, a large atheromatous plaque was found on coronary angiography and ivus . Therefore, during 5-fu infusion, close, careful monitoring of patients, especially those with pre - existing cad or cad risk factors, is mandatory . Prophylactic calcium channel blockers or nitrates should be administered to patients with cad during 5-fu administration, to prevent vasospasm . One study proposed that impaired renal function is also a risk factor for 5-fu cardiotoxicity . Although it is not clear whether the cardiotoxic metabolites undergo renal excretion, the pathophysiological effect of 5-fu on the myocardium is likely to increase with decreased renal function . Thus it is necessary to clarify which patients may benefit from optimum anti - angina prophylaxis and careful, close monitoring . The incidence of 5-fu - related cardiotoxicity appears to be dependent on the dosage and delivery system . Infusion of 5-fu, which is now being used more frequently and at higher doses instead of bolus therapy, may be a significant factor in the development of 5-fu cardiotoxicity . In one study, nine patients treated with a higher - dose (> 800 mg / m) continuous infusion of 5-fu died suddenly . Interestingly, despite stopping the 5-fu, the chest pain and ecg changes recurred in our case . One series reported that 19% of the patients developed reversible angina pectoris during treatment, which lasted for up to 12 hours after cessation of the infusion . The possible mechanisms of delayed angina are the late release of potent vasoactive 5-fu metabolites, which accumulate over time due to degradation of 5-fu . Therefore, a calcium channel blocker or nitrates should be administered after stopping the 5-fu when 5-fu - induced cardiotoxicity occurs . The long - term outcome of patients with 5-fu - related cardiotoxicity has not been investigated extensively . As with our case, patel et al . Recently reported interval improvements in the left ventricular wall motion abnormalities in echocardiography performed 8 to 15 days following the initial study . When cardiotoxicity occurs, 5-fu treatment is usually discontinued due to its very high recurrence rate (90%). The re - administration of 5-fu is not recommended, and a different chemotherapy regimen should be considered . Interestingly, meydan et al . Continued 5-fu chemotherapy in one group who experienced 5-fu cardiotoxicity due to the absence of an alternative drug and found that subsequent serious, hemodynamic consequences were easily controlled with nitrate treatment . For the remaining patients, either 5-fu was removed from the combination regimen or an alternative drug was started and no cardiotoxicity developed subsequently . In summary, although 5-fu - associated cardiac toxicity is rare, it may cause angina, myocardial infarction, and even sudden death . Physicians should be aware of this potentially lethal side effect and should start the proper treatment when 5-fu cardiotoxicity develops . This case supports the vasospastic hypothesis of 5-fu cardiac toxicity, indicating that a calcium channel blocker may be effective for prevention or treatment of 5-fu cardiotoxicity.
Recent studies revealed that plants contain populations of small rnas (2025 nt) that belong to two major classes micrornas (mirnas) and endogenous short - interfering rnas (sirnas). Mirna gene transcripts adopt imperfect foldback structures and are processed by dicer - like 1 (dcl1), resulting in 2022 nt mirnas . Mature mirnas function as post - transcriptional regulators that guide either site - specific cleavage or non - degradative repression of target mrnas (1). In many cases, sirnas arise from endogenous transcripts that form dsrna structures, or that are substrates for rnai pathways . Processing of sirnas often requires other dcl proteins, such as dcl3 (11). In addition, biogenesis of several classes of endogenous sirnas requires rna - dependent rna polymerases, such as rdr2 (11). Sirna - generating loci often yield multiple, overlapping clusters of small rnas, in contrast to mirna loci that generally yield a single mirna . Endogenous sirnas arise from repetitive sequences, transposons and retroelements, genomic regions containing inverted duplications, as well as other genic and intergenic regions . A subset of sirnas also act to guide or assist formation of heterochromatin (1214). A subclass of sirnas has been shown to guide cleavage of specific target mrnas in trans, similar to mirnas . Biogenesis of trans - acting sirnas (ta - sirnas) requires dcl1 and rdr6 (15,16). In contrast to mirna genes, ta - sirna precursor transcripts do not form a foldback structure, but rather both sense and antisense small rnas are processed from perfectly complementary rna duplexes . Several small rna libraries have been constructed from arabidopsis thaliana plants with the primary goal to identify mirnas and endogenous sirnas (1722). The aim of the arabidopsis small rna project (asrp) is to analyze small rnas from different tissues and genotypes of arabidopsis, provide a public database of cloned small rna sequences and develop web - based tools to assist in analysis of small rna populations . These resources are intended to aid the identification of mirnas and mirna genes, and to enable functional analysis of sirna - producing regions of the genome . The collection represents small rna sequences from both in - house cloning projects and sequences deposited in the mirna registry (23). For sequences derived in - house, multiple small rna libraries were constructed from arabidopsis (columbia-0 ecotype) at various developmental stages, including embryos, 3-day post germination seedlings, aerial tissues (including rosette leaves and apical meristems) and inflorescences (stages 112). To genetically enrich for mirna populations, libraries were constructed from rdr2 - 1 and dcl3 - 1 mutants that have defects in the chromatin sirna pathway . . The asrp graphical user interface (gui) is composed of web pages delivered by an apache http server (http://httpd.apache.org). In addition, the server incorporates mod_perl (http://perl.apache.org) and mason (http://www.masonhq.org) to dynamically produce web pages based upon user input . The vast majority of the gui is generated by custom perl code that increasingly incorporates object - oriented coding practices to improve extensibility and re - usability of the individual software components . Bioperl (24) is used for specific tasks, such as parsing the genbank files containing the arabidopsis chromosomes . The gui interacts with a custom database backend utilizing structured query language (sql) and the open source mysql (http://www.mysql.com) database engine . Table structures and specific query statements conform to standard sql language syntax and are portable to other sql database engines . Currently, the asrp database resides on a custom - configured server managed by the redhat linux as operating system . The asrp database web interface enables users to view and analyze the small rnas in text and graphical formats . Data for each small rna is stored in mysql database tables that are easily sorted and searched . Through the web interface, users may sort and view the small rna data in the following ways: all small rnas . This page displays basic information about all unique small rnas in the database, including, if applicable, the mirna or ta - sirna name, number of loci in the arabidopsis genome, number of near predicted loci in the rice genome, number of potential mrna targets and number of times isolated . More information about a specific small rna is available by following the database number (dbe #) link.small rna clusters . This page displays clusters containing a minimum of four small rna loci, with each within 500 nt of the next small rna loci . From this page, the user can view the sequences and positions of the small rnas in each cluster in text format or the cluster can be viewed graphically in relation to the arabidopsis genome using an open access genome viewer (25).mirnas . All small rnas characterized as mirnas are displayed in a similar format as section (i) (figure 1a). The display page for each individual mirna is split into four sections; general information, arabidopsis mirna genes, predicted and validated target genes, and oryza sativa mirna genes (figure 1b). The predicted foldback structure for the pre - mirna, the flanking sequence around the mirna gene and the graphical genome view are available through links on the arabidopsis mirna genes section (figure 1c). Information about the predicted target genes, the target - mirna binding site, and the computational or experimental validity of the target - mirna binding site is displayed in the third section (figure 1b). The fourth section displays information about the small rna in o.sativa, including the predicted secondary structure of validated precursor mirnas.ta-sirnas . General information, ta - sirna - generating locus information, and predicted target genes are displayed . The user can view the information about the ta - sirnas in a manner similar to the mirna section of the database.annotated small rnas . Automated annotation programs such as repeatmasker (http://ftp.genome.washington.edu/rm/repeatmasker.html) are used to identify small rnas that originate from genomic regions of highly repetitive sequences, as well as transposons and retroelements . The user can display and sort small rnas by the specific class of annotated repeat element such as mudr or sine . This page displays basic information about all unique small rnas in the database, including, if applicable, the mirna or ta - sirna name, number of loci in the arabidopsis genome, number of near predicted loci in the rice genome, number of potential mrna targets and number of times isolated . More information about a specific small rna is available by following the database number (dbe #) link this page displays clusters containing a minimum of four small rna loci, with each within 500 nt of the next small rna loci . From this page, the user can view the sequences and positions of the small rnas in each cluster in text format or the cluster can be viewed graphically in relation to the arabidopsis genome using an open access genome viewer (25). All small rnas characterized as mirnas are displayed in a similar format as section (i) (figure 1a). The display page for each individual mirna is split into four sections; general information, arabidopsis mirna genes, predicted and validated target genes, and oryza sativa mirna genes (figure 1b). The predicted foldback structure for the pre - mirna, the flanking sequence around the mirna gene and the graphical genome view are available through links on the arabidopsis mirna genes section (figure 1c). Information about the predicted target genes, the target - mirna binding site, and the computational or experimental validity of the target - mirna binding site is displayed in the third section (figure 1b). The fourth section displays information about the small rna in o.sativa, including the predicted secondary structure of validated precursor mirnas . General information, ta - sirna - generating locus information, and predicted target genes are displayed . The user can view the information about the ta - sirnas in a manner similar to the mirna section of the database . Annotated small rnas . Automated annotation programs such as repeatmasker (http://ftp.genome.washington.edu/rm/repeatmasker.html) are used to identify small rnas that originate from genomic regions of highly repetitive sequences, as well as transposons and retroelements . The user can display and sort small rnas by the specific class of annotated repeat element such as mudr or sine . In addition to the sorting features, the web interface provides users with a variety of searching capabilities . Quick searches enable users to locate specific mirnas based on either the mirna names or the asrp database identifiers (dbe #). To search for small rnas predicted to target specific arabidopsis genes, or that originate from generic sequences, the locus identifiers (e.g. At3g60630) or user - defined fasta formatted sequences are used, respectively . Finally, users can determine if a small rna sequence is represented in the asrp database by searching the sequence against the entire population of small rnas . All small rnas in the asrp database are available through the publicly available website (http://asrp.cgrb.oregonstate.edu) or can be downloaded in fasta format from the website download page (http://asrp.cgrb.oregonstate.edu/downloads/). The asrp database was created to serve as a repository and tool to facilitate the analysis of mirnas and endogenous sirnas and their targets . To increase accessibility of the database, we are working to more completely integrate the asrp database with existing arabidopsis resources, such as tair . In addition, integration of mirnas, ta - sirnas and endogenous sirnas from the database with other research projects, such as genomic tilling microarrays and chromatin immunoprecipitation arrays (14,26), will enhance the information acquired from these experiments and further expand our understanding of small rna function . There are still many unanswered questions concerning mirnas, ta - sirnas and endogenous sirnas . The regulatory roles of mirna - target gene interaction, the regulation of mirna gene expression, and the function of sirnas in the regulation of chromatin structure and gene silencing are just a few questions currently being studied . Future plans include the integration of data from genome - scale microarray projects into the asrp database (27). The scope of the database may widen with the addition of other plant genomes, libraries or computational analysis . The inclusion of additional plant genomes will enable a more in - depth study of mirna evolution and conservation and activities of endogenous sirnas . We thank christopher m. sullivan for developing the infrastructure for the computing cluster and compiling the software technologies used for the database and heather fitzgerald for critical reading of the manuscript . The arabidopsis small rna project database is supported by a 2010 project grant from the national science foundation (mcb-0209836).
Thyroglossal duct cyst (tdc) is a common abnormality of the neck region and can occur at any age, though it is much more common in the pediatric population.1 the most definitive, efficacious management is achieved through the sistrunk procedure, a widely accepted and choice surgical technique, which effectively removes the cystic lesion and reduces recurrence rates.2 3 4 5 common postoperative complications associated with the technique include wound - related infection, pus / abscess, and hematoma / seroma (h - s) formation, with subsequent airway compromise.3 6 surgical drains are placed in patients undergoing the sistrunk procedure to prevent these postoperative complications, but hardly any studies have overtly assessed if drain placement is actually necessary . A recent case series in a pediatric population suggests that routine drain placement may not be necessary.7 as drain placement in the sistrunk procedure may often necessitate postoperative hospitalization, leading to increased cost and patient discomfort, it is important to understand if drain placement offers any substantial advantage . The rationale of our study was to assess if drain placement in the sistrunk procedure makes any significant difference in the prognosis of postoperative complications . The issue assumes significance as a focus of head and neck surgeries (including the sistrunk procedure) now is shifting toward outpatient, same - day, and ambulatory surgeries.5 8 9 studies demonstrate same - day surgery to be a safer, less costly, and reasonable alternative to admission surgery, without increased patient risk.8 9 with a general tendency to reduce hospital stays and with the sistrunk technique being increasingly performed in same - day, outpatient settings, evaluating the impact of drain placement with the technique on postoperative complications and patient morbidity is essential.5 7 with this objective, we conducted a retrospective study at our hospital to explore if same - day, outpatient sistrunk procedure performed without drain placement was a safe alternative versus the same procedure with drain placement . The study focused on postsurgical complications of the sistrunk procedure in both groups (those with or without drain placement) and evaluated whether surgical drain offered any substantial benefit in the sistrunk procedure, and if it was necessary . Records of all patients who underwent the sistrunk procedure for tdc surgery in the preceding 10 years (2004 to 2014) were retrieved, and data were searched and extracted . In total, 80 patient records of the sistrunk procedure could be retrieved and were reviewed . Certain patients were excluded to standardize the patient population and avoid any bias in the study . All those who had active infection and pus in cyst requiring incision and drainage of the cyst along with sistrunk procedure were also excluded . Patients who had incomplete surgery like removal of just the cyst without the tract including the body of the hyoid (which were documented as sistrunk but were not actually sistrunk) were also not included . Patients who in addition to sistrunk procedure also underwent other surgical procedures were excluded (e.g., those who underwent papillary carcinoma surgery and proceeded to the sistrunk procedure, or thyroidectomy / lymph node biopsies along with sistrunk procedure, among others). Moreover, cases with missing data like no mention of placement or nonplacement of drain were also not included . Records missing follow - up information or clues regarding complications after the sistrunk procedure were also not included . Based on inclusion - exclusion criteria, of 80 records, only 58 patients were deemed fit for analysis, and the remaining 22 were excluded due to either missing data or inclusion - exclusion criteria . Patients who were found suitable for analysis (n = 58) were separated into two groups: patients who did not have a drain placed (n = 38) during the sistrunk procedure, and those who had a drain placed (n = 20). Of these 58 patients, 38 were males (65.5%) and 20 (35.5%) were females . The age of the patients varied from 1 to 53 years, with the mean and median age of patients being 18.1 and 13.5 years, respectively . Overall mean age (sd) was 18.1 (14.8) y and median age, 13.5 y. all patients had same - day surgery under general anesthesia . The patients came in the morning, were operated upon, and were discharged by evening after their condition stabilized . Of the 58 patients, 13 (6 from the drain group and 7 from the no - drain group) were admitted for 1 night and discharged the next day but for reasons unrelated to complications, apparently based on patient or surgeon preference . Patient preference included those who had come from distant places and had late evening surgery, those who felt nauseous and had possibly not recovered well from anesthesia, and those who had anticipatory anxiety and reported feeling patients who were admitted due to surgeon preference included one patient who had sickle cell anemia (who did not have a drain) for optimization and another patient (who had a drain) to observe for any anticipatory h - s . Both groups of patients had homeostasis secured at time of surgery, and no major complication was noted . None of the patients developed any major complication like damage to great vessels, nerve damage, hypothyroidism, perforation of pharynx / esophagus, or injury to airway . None of the patients required any surgical exploration post sistrunk procedure, except minor wound exploration or incision and drainage of pus . Both groups of patients were reviewed, and data were extracted for various postoperative sistrunk - related complications, like presence of h - s (and if the patients required aspiration for h - s or were managed conservatively), wound infection, and pus formation . Of the 58 patients, 3 did not return for follow - up and were assumed to be doing fine . The extracted data were stored and analyzed using spss version 16 (armonk, ny, ibm corp). Pearson chi - square test was used to see the association of drain or no drain with gender, age, h - s, aspiration, wound infection, pus, and number of follow - up visits . The t test was used to compare if there was any significant difference in mean age of patients in both groups . The parametric nature of the data was accessed statistically, and normality assumption was checked before application of t test . Overall, about 10% of patients had h - s, 6.9% of whom needed aspiration for h - s and the rest were managed conservatively; 3.4% had wound infections; and 1.7% had pus collection that required wound exploration or incision and drainage . The comparative overall gross percentages of complications in both groups (drain versus no drain) did not show any significant difference (h - s, 0 versus 15.8%; aspiration needed, 0 versus 10.5%; wound infection, 5.0 versus 2.6%; pus formation, 5 versus 0%; respectively). None of the patients who were admitted overnight (patient or surgeon preference) developed any of the complications discussed . The chi - square test compared both group of patients in terms of any sistrunk procedure - related complications (h - s, aspiration required, wound infection, pus, and number of follow - up visits). The groups did not show any statistical significant difference in sistrunk - related complications in the fisher exact (two - sided) test: h - s (p = 0.08), aspiration required (p = 0.29), wound infection (p> 0.99), and pus formation (p = 0.35; table 2). The chi - square (two - sided) test also did not show any statistically significant difference in number of follow - ups for both group of patients (p = 0.81; table 2). The t test compared mean age difference between patients with and without drain, and no significant difference in mean age could be seen (p = 0.34; table 2). Abbreviations: h - s, hematoma / seroma; ns, not significant; sd, standard deviation . Note: all percent values are within group percentages . Overall mean age (sd) of participants was 18.09 14.84 . Analysis of data suggests that there is no significant difference in complications post sistrunk procedure (infection, pus / abscess, or h - s) between the drain and no - drain groups . Furthermore, no patient admitted overnight developed any of the complications discussed, which supports our methodology because the patients were admitted for reasons not related to complications and admissions were apparently due to patient and surgeon preferences . The findings of our study resonate with that of another recently published case series, which tried to ascertain if drain placement is at all necessary with the sistrunk procedure for treating tdcs in the pediatric population.7 the case series evaluated 30 consecutive pediatric patients (mean age 7.4 years) who underwent the sistrunk procedure without drain placement (study group). Importantly, the first 10 patients were hospitalized, despite no drain placement, to observe for any complications in terms of hematomas, seromas, and subsequent airway compromise . However, when no major complications were seen, the next 20 patients undergoing the sistrunk procedure without drain placement were treated with same - day, outpatient surgery . The study also had 21 age - matched controls (mean age 7.5 years) who underwent the sistrunk procedure with drain placement.7 interestingly, the study did not find any statistical difference in the complication rates between the study (no drain) and the control (drain) group (paired t test, p = 0.85). Conclusively, the author stated that surgical drain placement was not necessary in pediatric patients who underwent the sistrunk procedure.7 our study (which included both children and adults) reinforces the same finding in a pediatric population and extends the generalizability of similar outcome in the sistrunk procedure performed in an adult population . With hardly any studies exploring the need for drain placement in the sistrunk procedure, in fact, use of drains in thyroid / parathyroid surgery has been considered controversial and debatable and may be even contraindicated.10 11 it has been suggested that use of drains is not justified as drains cannot substitute for meticulous use of surgical technique and adequate hemostasis, in which noncompliance would eventually still lead to h - s . Rather, it has been contended that in noncomplicated surgeries with minimal drainage, placement of a drain could lead to a possible infection.12 13 hence, nonusage of drains in the sistrunk procedure is not only related to reduced hospital stay, less cost, and improved patient comfort, but also has implications in terms of decreased chance of postoperative infections . Though not specific to the sistrunk procedure, hurtado - lpez et al tried to analyze the actual value and effectiveness of a drain in thyroid surgery settings and found that presence or absence of drains did not affect the incidence of seroma or hematoma postsurgery.14 it is worth mentioning that although routine use of drains does not seem to be indicated in uncomplicated thyroid surgery cases, in complicated cases or when dead space is large, drains do have a use.15 the same may be applicable while using the sistrunk procedure to surgically excise and manage tdcs, and the surgeon preference for drain placement should depend on merits of its use and not on routine use in the sistrunk procedure . Due to the increasing burden of extensive head and neck surgeries requiring mandatory hospitalization, smaller surgeries like the sistrunk procedure be managed as same - day, outpatient treatment . As drain placement in the sistrunk procedure may often require in - patient admission and increased hospital stay, it should not be performed if it does not offer any considerable advantage in decreasing postoperative complications . Bratu and laberge evaluated same - day tdc surgery (which included sistrunk procedure) in 100 children in a retrospective review and found that outpatient surgery was a safe alternative to postoperative admission surgery in uncomplicated cases (no comorbidity, congenital defect, or bleeding disorders).5 interestingly, drain placement was one of the factors that prolonged the length of hospital stay in patients . Another point worth emphasizing is that 38% patients had drains placed at surgeon discretion with no further details.5 bratu and laberge also stated that outpatient, same - day surgery was safe for routine tdc excision (including the sistrunk procedure), but surgeons who were reluctant to use same - day surgery due to fear of complications needed a shift in behavior to increase outpatient tdc surgery.5 this further emphasizes the rationale of performing the sistrunk procedure as same - day, outpatient surgery and emphasizes that the unnecessary routine use of drain placement with the sistrunk procedure would necessitate admission surgery leading to waste of health resources . The fact that very few complications were observed in our study even after discharge suggests that if homeostasis is adequately achieved, drain placement in sistrunk procedure may not be necessary . However, to take care of any unprecedented postoperative hematoma and/or edema and subsequent airway compromise in patients undergoing same - day, outpatient sistrunk procedure without any drain placement, the surgeon should take all precautions . All patients with tdc having same - day sistrunk procedure should be asked to report any postoperative abnormality like swelling, purulent discharge, or fever postdischarge, as soon as possible and irrespective of the scheduled follow - up . This not only would ensure optimal utilization of health care resources to manage any adverse complication (if any arise) but also would provide cost - benefit information and improved comfort to the patient, ensuring greater patient satisfaction and better overall management of tdcs . Smaller sample size is another limitation, as complications of h - s in the sistrunk procedure are relatively rare . Due to the retrospective, observational study design, with no randomization possible, there may be a risk of selection bias . Although, to the best of our effort, we could not find any clinical or surgical variable to be associated with placement or nonplacement of a drain, its possibility cannot be completely ruled out . Furthermore, because all surgeries were performed by the consultant and residents under direct supervision of the consultant (and consultant as first assistant), there does not seem to be any performance bias . Apparently, drain placement with the sistrunk procedure does not seems to be determined by the person performing the surgery or the surgeon's expertise, and rather appears to be governed by patient and surgeon preference . However, we cannot deny it in absolute terms . A study with a larger sample population ideally, however, a randomized trial comparing the sistrunk procedure, with or without drain, should be performed to definitively understand if placing a drain is at all necessary in the sistrunk procedure . This would help create a protocol recommendation and consensus among surgeons managing tdcs, with respect to deciding whether or not a drain should be placed during the sistrunk procedure . To best of our knowledge, ours is the first study that has compared drain or no - drain sistrunk procedure across all age groups; more comparative studies are needed in both pediatric as well as nonpediatric population . Surgical placement of a drain did not seem to offer any advantage in patients undergoing the sistrunk procedure at our hospital in terms of reduced postoperative complications . Hence, there does not seem to be any apparent need for drain placement when performing the sistrunk procedure in patients with tdcs . Moreover, because no major complications were observed in surgically managing uncomplicated cases of tdcs by same - day, outpatient sistrunk procedure without drain placement, it could be considered a safe alternative to the sistrunk procedure with drain placement, which may require overnight hospitalization / admission . This would translate into better patient comfort, greater satisfaction, and reduced surgical costs in patients of tdcs undergoing the sistrunk procedure.
Cancer is the second leading cause of death worldwide, accounting for about 600,000 deaths in the united states in 2012 . Despite significant improvements in treatment, early detection remains the most important prognostic factor predicting of better outcome [24]. Current cancer screening methods, including mammography for breast cancer, colonoscopy for colon cancer, computed tomography for lung cancer, prostate - specific antigen for prostate cancer, and papanicolaou stains for cervical cancer, have demonstrated some limitations in terms of sensitivity, specificity, complexity, cost, and compliance . Serum tumor - associated antigens (taas) have been extensively studied for early cancer detection because of the simplicity and reliability of the tests used for their determination, such as western blot and enzyme - linked immunosorbent assay (elisa). Unfortunately, they are transiently secreted and rapidly eliminated from blood circulation [6, 7] and usually reach a detectable concentration only in advanced stage of the disease . Along with taas, autoantibodies are frequently detected in sera from patients affected by different types of neoplasms . This finding has been interpreted as an attempt of the immune system to block invasion and spreading of cancer cells in the organism . Circulating autoantibodies have biological and biochemical characteristics that render them particularly suitable to screen subjects at cancer risk . In fact, they may develop early in the process of tumorigenesis, when premalignant or malignant cells begin to express altered molecules as a result of cell transformation [10, 11]. In addition, they can easily be detected in the serum because of the usual high concentration and long - time stability . For these reasons, great efforts have been made in recent years to identify circulating autoantibodies directed against cancer - related proteins in order to build up tests for the early detection of neoplastic disease [1215]. In this study, we investigated the production of autoantibodies against lectin galactoside - binding soluble 3 binding protein (lgals3bp) in patients affected by different types of cancer . Lgals3bp, also known as 90k or mac-2 bp, has been largely regarded as a taa, since it is present at elevated concentrations in the blood of cancer patients and is overexpressed in the vast majority of cancer tissues . Both high serum and tumor levels of lgals3bp have been associated with a poor outcome in patients with different types of neoplasms [1821]. In fact, it can bind important molecules associated with the membrane of tumor cells, such as galectin-3, galectin-1, and 1-integrins [16, 22, 23]. Additionally, lgals3bp can interact with extracellular matrix proteins such as collagen, fibronectin, and laminin [23, 24]. Here we show that patients with different types of cancer, but not healthy subjects, develop autoantibodies against lgals3bp . This finding discloses the capability of lgals3bp to trigger a humoral immune response in cancer patients and provides the basis for further investigation on a possible use of anti - lgals3bp antibodies as biomarkers for early diagnosis of cancer . The study population consisted of 71 patients with different types of cancers, 15 gastrointestinal cancers, 13 non - small - cell lung cancers (nsclc), 12 breast cancers, 10 neuroendocrine tumors (net), 10 urogenital tract cancers, 7 melanomas, and 4 others (2 gliomas, 1 tongue cancer, and 1 osteosarcoma). Serum was collected from total blood and stored at 20c after adding 0.01% sodium azide . Human recombinant lgals3bp was immunoaffinity - purified from serum - free supernatant of human embryonic kidney ebna-293 cells (invitrogen, carlsbad, ca, usa) transfected with lgals3bp cdna . Ninety - six well microtiter plates (nalge nunc, denmark) were coated with recombinant purified lgals3bp protein (5 g / ml in pbs) at 4c overnight . The plate was saturated with 1% bsa and 0.05% tween-20 in pbs (saturation buffer) at 37c for 2 h and then incubated with 100 l of serum from healthy donors or patients at 37c for 1 h. the serum was diluted 1: 100 in saturation buffer . After 3 washes with 0.05% tween-20 in pbs (washing buffer), a second incubation was performed with 100 l of biotin - conjugated anti - human igg (sigma, st louis, mo, usa), diluted 1: 2000 in saturation buffer, at 37c for 1 h. after washing, a third incubation was performed with peroxidase - conjugated extravidin (sigma, st louis, mo, usa) diluted 1: 4000 in saturation buffer, at 37c for 45 min . After washing, 100 l of tmb substrate was added to each well and the plate was shaken at room temperature for 15 min . Eventually, the reaction was stopped by adding 100 l of 1 m h2so4/well and color revealed by reading absorbance at 450 nm in an automatic elisa reader . To measure serum concentration of lgals3bp a commercially available elisa kit (diesse, siena, italy) purified human recombinant lgals3bp (10 g / well) was separated by 8% sds - page under reducing conditions and transferred to nitrocellulose using standard procedures . Membrane was saturated in blocking buffer phosphate buffered saline with 0.05% tween-20 (pbs - t), 5% low - fat milk, 1% bsa and at 4c overnight . After washing in pbs - t, membrane was incubated with serum from controls or patients affected by cancer, diluted in an equal volume with pbs - t, at room temperature for 1 h. after washing in pbs - t, membrane was incubated with biotinylated anti - human igg (sigma, st louis, mo, usa) diluted 1: 1000 in blocking buffer at room temperature for 1 h and then with extravidin peroxidase (sigma, st louis, mo, usa) 1: 500 in blocking buffer for 30 min . To identify the presence of antibodies bound to lgals3bp, the colorimetric substrate dab (3,3-diaminobenzidine) each sample was assayed in triplicate and the mean value was used for statistical analyses . The normal upper cut - off value of anti - lgals3bp antibody in sera was set at the value of the mean + 2sd of the absorbance in 54 healthy donors . Differences in the proportion over the cut - off limit were evaluated by fisher's exact test . Patients affected by different types of cancers showed significant increased levels of lgals3bp autoantibodies (p <0.001) compared to healthy subjects (figure 1(a)). The specific binding of antibodies to lgals3bp was confirmed in western blot, where the presence of anti - lgals3bp igg was detected as two bands at about 97 kda and 66 kda, the exact size of the protein in its full length and cleaved form, respectively (figure 1(b)). Among patients, nsclc, gastrointestinal cancer, urogenital tract cancer, and net reached the highest levels of autoantibodies, while there was no significant increase in breast cancer and melanoma (table 2). Setting the normal upper cut - off limit of elisa at od 0.99 (the mean + 2sd of the absorbance in sera from healthy individuals), the assay showed a sensitivity of 33% (26/71 patients were positive) and a specificity of 98% (only 1 out of 54 controls was positive). All cancer groups, but melanoma, showed autoantibody levels significantly above the cut - off limit (table 2). As expected, the protein was significantly higher in patients with cancer compared to normal subjects (13.19 versus 6.36 g / ml, p <0.001) (table 3), but values did not correlate with the levels of autoantibodies (data not shown). Using elisa technique, we show that lgals3bp is able to elicit host immune response with igg autoantibodies production in patients affected by different types of cancer . Anti - lgals3bp igg concentrations were higher in patients with nsclc, gastrointestinal cancer, urogenital tract cancer, and net than in those with breast cancer and melanoma, but the number of patients in each subgroup was insufficient to yield a statistically reliable comparison . It is generally accepted that tumor proteins perceived as nonself by the immune system and able to trigger an immune response are often overexpressed, mutated, misfolded, or endowed with posttranslational changes, such as alterations of glycosylation and phosporylation [28, 29]. Consistently, lgals3bp may evoke autoantibody production because it is overexpressed in cancer cells, and also because it may carry posttranslational alterations in its glycidic moiety . Qualitative and quantitative changes in o- and n - glycosylation of proteins are frequent events in malignancies [30, 31] and differences in the glycosylation pattern of lgals3bp have been reported in some cancer cell lines . Finalistically, antitumor directed antibodies are generated in order to halt tumor initiation and progression . As this process initiates early in cancerogenesis, in a preclinical phase of the disease, autoantibodies production has been considered a useful biomarker for early cancer diagnosis [3235]. In this study, the serum levels of anti - lgals3bp igg detected in cancer patients were not correlated with those of lgals3bp, indicating that even small amount of the protein, as expected in the initial phase of cancer growth, may generate high concentrations of autoantibodies . This evidence suggests a possible role for anti - lgals3bp igg in the early detection of cancer . Although it is not possible to exclude that the presence of autoantibodies might affect the correct quantification of lgals3bp by elisa, the identification of anti - lgals3bp igg in western blot indicates that the epitopes recognized by these autoantibodies are different from those recognized by the antibody used in elisa . In fact, western blot performed under reducing conditions can detect only autoantibodies directed to epitopes expressed on the primary structure of lgals3bp, while the monoclonal antibody contained in the commercially available elisa kit, known as sp2, recognizes a conformational epitope shaped in the native form of the protein and, for this reason, is not suitable for western blotting . In cancer patients, autoantibodies are frequently directed against cellular proteins that play key roles in tumor progression, including molecules involved in cell cycle, signal transduction, proliferation, and apoptosis [3638]. As a consequence, the identification of the molecular target of autoantibodies might be of relevance in designing new antitumor agents . We can, therefore, speculate that lgals3bp could be a candidate for targeted therapies against cancer . In the past few years, the growing interest in autoantibodies as a possible tool for the early diagnosis of cancer and the identification of new targets for molecular therapy has made the development of high - throughput techniques such as serex (serological analysis of tumor antigens by recombinant cdna expression cloning), phage display, protein microarray, serpa (serological proteome analysis), and mapping (multiple affinity protein profiling) able to detect simultaneously multiple autoantibodies and their cognate taas . With these methods, several new targets have been identified, but collectively single antigens have shown low sensitivity and specificity to be used in clinical screening . To increase sensitivity, autoantibody diagnostic tests combining two or more taas [4143] or evaluating well - known biomarkers in combination with autoantibodies have been developed . For example, a large screening study of high - risk individuals for lung cancer has validated a test measuring autoantibody levels against a panel of six taas (p53, ny - eso-1, cage, gbu4 - 5, annexin 1, and sox2). In another study, the combination of p53 autoantibodies and ca125 levels increased sensitivity for ovarian cancer from 73.8% (ca125) to 85.7% (ca 125 plus p53 autoantibodies). The determination of anti - lgals3bp igg presented in this study showed a very high specificity (98%), but a low sensitivity (33%), comparable to that reported for autoantibodies against single taa, ranging between 10% and 30% . Therefore, our anti - lgals3bp elisa lacks sufficient sensitivity to be used in early cancer diagnosis . Nevertheless, the determination of autoantibodies against lgals3bp might be useful to increase the sensitivity of tests combining multiple autoantibodies . Preliminary results indicate that using a set of different autoantibodies combined with autoantibodies for lgals3bp will increase the sensitivity for breast cancer patients to 50% and maintain the high specificity (98%). These preliminary results should be tested for other types of cancers as well . In summary, our study demonstrated the presence of autoantibodies against lgals3bp in the serum of patients with different types of cancers . These autoantibodies may be used in developing screening tests for early - stage cancer detection.
Asthma affects about 235 million people worldwide.1 the incidence of asthma has been growing over the past 30 years due to changing environmental factors, greater awareness of this condition, and changes in diagnostic practice, particularly in the low- and middle - income countries that are least able to absorb its impact.12 fifty years ago, asthma was uncommon in nigeria; however, recent reports from different parts of nigeria have shown a prevalence of adolescent and adult asthma in excess of 10%.134 data reported from phases 1 and 3 of the international study of asthma and allergies in childhood found a rising trend in the prevalence of asthma.1 asthma causes an estimated 250,000 deaths annually (1 in 250 deaths worldwide).2 asthma is also the second leading cause of respiratory disease - related morbidity and third leading cause of respiratory disease - related emergency room visit and mortality within 24 h of admission in ekiti state, southwestern nigeria.56 smith et al . Reported that a substantial proportion of the direct costs of asthma in the usa are due to hospitalization and emergency department (ed) visits.7 several national and international thoracic societies have produced the management guidelines to standardize asthma care and achieve the goals of therapy.89 in nigeria, there is no national asthma treatment guideline1011 and most asthma care providers rely on the global initiative for asthma (gina) guideline which is a global strategy for asthma management and prevention, developed by the world health organization and the national heart, lung, and blood institute . The guideline was issued in 1995 and regularly revised to date.2 despite the effort to standardize asthma care using the gina guideline, several nigerian studies dating back to more than a decade have shown that there is under - treatment of asthma and poor adherence to the guideline by doctors.121314151617 the reduction of burden and outcome of asthma depend largely on achieving asthma control, adequate prevention, and treatment of an acute attack . It is therefore important to assess the adequacy of such treatment in our practice and plan changes to improve care; hence, the need for a clinical audit . To the best of our knowledge, there is only one study on clinical audit of acute asthma in the emergency room in nigeria, which was done in the pediatric age group14 and none in the adult population . Assessment of the quality of asthma care in developing countries is imperative because of its increasing prevalence, huge socioeconomic and psychosocial burden, and under - treatment, which leaves much room for improvement.2 this study was carried out to audit the quality of acute asthma care in two tertiary hospitals in a state in the southwestern region of nigeria and compare the clinical practice against the gina guidelines . This study was a retrospective analysis of 101 patients who presented with acute exacerbation of asthma to the hospital between november 2010 and october 2015 carried out at two tertiary hospitals in southwestern nigeria . These hospitals also provide an internship and postgraduate training programs in both internal and family medicine . The hospitals have consultant pulmonologist who runs the pulmonary clinic, resident doctors, and several other internists . In terms of infrastructure, both centers have desktop spirometers, pulse oximeters, peak flow meters, and intensive care units . In addition, they provide a combination of primary, secondary, and tertiary health - care services . This is because they have departments of family medicine and community medicine that provide primary health care (phc). Asthma patients presenting with attack were previously receiving their care in phc / private hospital in the state, some were known patients of the hospitals, while some were newly diagnosed cases presenting with acute asthma . They were seen on the first contact by casualty / medical officers, residents in family, and internal medicine undergoing emergency clerkship rotation . The attending doctor in the casualty evaluates, institutes therapy and discharges the patient when the condition has improved or referred to the internist for further treatment . The internists then referred the patient to the pulmonologist after discharge for follow - up or seek an urgent pulmonology consult depending on the severity of the disease, patients preference, and associated comorbidity . We retrieved and reviewed the medical case files of all the patients who had a clinical diagnosis of asthma in the two hospitals . The cases that had complete information and met the clinical criteria for diagnosis of acute exacerbation of asthma were studied . Nine cases of chronic obstructive pulmonary disease (copd) and one case of asthma - copd overlap syndrome (acos) were excluded from the chart review . An audit proforma was used to collect (1) sociodemographic information, (2) clinical profile: clinical features, month and season of admission, comorbid conditions, emergent investigations and asthma medications, duration of hospitalization, outcome of management, (3) standard of care recommended by gina.2 the gina guidelines specify that the initial assessment on admission should (1) include functional assessments such as spirometry, peak expiratory flow (pef) measurement, and oxygen saturation measurements with oximetry and (2) assess the severity of an asthma exacerbation using brief history and physical examination pertinent to the exacerbation and functional assessments . Such assessments are recommended to be repeated 1 h after the initial treatment and then at 12-h intervals until there is a clear response to the treatment and a decision is reached about the patient clinical state . The gina guidelines also recommended the following treatments: (1) supplemental oxygen therapy, (2) repeated administration of rapid - acting inhaled 2-agonists with or without an anticholinergic before the use of theophylline, (3) systemic glucocorticosteroids in all but the mildest exacerbations, and (4) discouraging the use of sedatives and anxiolytics . On discharge, the care of patients should include (1) a minimum 37-days course of oral corticosteroids (ocss); (2) initial or continued use of controller therapy; (3) review of inhaler technique and use of peak flow meter; (4) identification of potential triggers of exacerbations; (5) provision of a written action plan for prevention of future exacerbations; (6) encouragement to contact a physician within 1 week after discharge for a follow - up appointment . In medicolegal term, the absence of documentation implies the absence of process in data collection . Asthma was defined by the history of variable respiratory symptoms such as wheeze, shortness of breath, chest tightness, and cough and airflow limitation that has an immediate response to bronchodilator (bd) or to inhaled corticosteroids (icss) over week . In addition, presence of family history of asthma, and other allergic conditions (allergic rhinitis or eczema)2 exacerbations of asthma (asthma attacks or acute asthma) were defined as episodes of progressive increase in shortness of breath, cough, wheezing, or chest tightness, or some combination of these symptoms and decrease in lung function (pef or forced expiratory volume in 1 second [fev1]) from the patient's usual status.2 asthma - chronic obstructive pulmonary disease overlap syndrome was defined as persistent airflow limitation with several features usually associated with asthma and several features usually associated with copd.2 the data were analyzed using the statistical package for the social sciences (spss), version 17 (spss inc ., results were expressed for all cases in the audit, usually as percentages for the variables of interest . The data were analyzed using the statistical package for the social sciences (spss), version 17 (spss inc ., results were expressed for all cases in the audit, usually as percentages for the variables of interest . A total of 111 patients were admitted during the study period, of which only 101 patients met the gina diagnostic criteria . The age of the patients ranged from 16 to 85 years, with a mean age of 43 19 years . Sixty - one (60.4%) of the asthma patients were aged <45 years, and 67 (66.3%) were females with a male to female ratio of 1:2 . Fifty - one (50.5%) patients were diagnosed to have asthma for a duration <5 years . Sixty - two (61.4%) patients were admitted at night and 65 (64.4%) were admitted in the rainy (cold) season . Only 8 (7.9%) patients were regularly using ics and 69 (68.3%) were not on any medication before admission . Almost 60% of the patients were admitted and managed by the casualty officer while 16 (15.8%) were managed by the internist . The median duration of illness before the presentation was 3 days (interquartile range [iqr]; 26 days) and the median length of hospital stay was 2 days (iqr; 13 days) and the mortality was 1.0% . General characteristics of asthma patients at admission, assessment of severity of attack was performed in 33 (32.7%) patients, smoking status recorded in 30 (29.7%), attack triggering factors documented in 73 (72.3%), and level of drug adherence recorded in 18 (17.8%). The oxygen saturation of the patients at admission was measured in 5 (5.0%) and continuous or serial oxygen saturation was measured in 5 (5.0%) of the patients . Six (5.9%) patients had peak expiratory flow rate (pefr) measured at admission, and none had their post - bd pefr measured after nebulization or intravenous medication . Chest radiography was performed in 16 (15.8%) of the patients [table 2]. Assessment at admission nebulized salbutamol was the initial bd used for attack in the 76 (75.2%) of patients and the remaining had intravenous theophylline . For airway inflammation, 89 (88.1%) patients were given a corticosteroid (cs), with 29 (28.7%) receiving optimal css dose, 75 (74.3%) had intravenous corticosteroid and within 1 h, respectively . Ocs was given to 61 (60.4%) patients and 23 (22.8%) had it within 1 h of admission . Supplemental o2 was given to 31 (30.7%) and out of the 31 given oxygen, none of the oxygen administration was based on measured spo2 . Intravenous fluid was administered to 56 (55.4%) patients, 30 (29.7%) had antibiotics, while 4 (4.0%) had mucolytic agent [table 3]. Treatment practices in hospital twenty - five patients (24.8%) had their controller medication stepped / adjusted before the time of discharge, 37 (36.6%) were discharged on ics / long - acting 2-agonist combination inhaler, 10 (9.9%) were given written action, 36 (35.5%) had their inhaler technique reviewed, and 25 (24.8%) retrained on it . Specific follow - up appointment was documented in 68 (67.3%) of the discharge and 32 (31.7%) were reviewed within 30 days [table 4]. Discharge / follow - up procedures to determine the level of adherence to the 15 major recommendations of the gina guideline by their category, we stratified the doctors into three categories; these include (1) internist, (2) family physician, and (3) casualty / medical officers . In this study, we defined medical officers as doctors who have a basic medical degree, currently performing general medical duties and are not in specialist training . The pulmonologists were merged with the internists because they managed very few cases that did not allow for separate in - depth analysis . There was no significant difference among the internist, family physician, and casualty / medical officers in the level of compliance to 10 of 15 gina recommendations . There were low levels of compliance by casualty / medical officers to 4 out of 5 remaining recommendations . We also observed that the internists were significantly giving follow - up appointment than other categories of doctors [table 5]. This audit of the management of acute asthma cases admitted to the two tertiary hospitals in the southwestern region of nigeria has demonstrated marked deficiencies in standards of care measured against the gina management guidelines . At admission, less than one - third of patients had an assessment of severity, smoking status, inhaler technique, and medication adherence documented . Furthermore, the baseline and serial oxygen saturation, baseline pefr, and post - bd pefr were not documented in more than 90% of cases, respectively . Majority received nebulized salbutamol, systemic cs within 1 h, and follow - up appointment on discharge; however, only 35.7% came for follow - up within 30 days . A significant proportion of patients were not prescribed ics and given the self management plan on discharge . The clinical profile of patients showed that most of them were females and below the age of 45 years . This is in keeping with findings in other studies.617181920 female sex is a risk factor for severe asthma which is associated with increased hospital admissions . This association has been linked to several factors including menarche, menstrual cycle, pregnancy, hormone genetics, and obesity.21 women have also been associated with high level of atopy, bronchial hyperresponsiveness, and exposure to indoor pollutant.22 in the initial assessment of acute asthma admission, the gina guideline recommended history and physical examination (auscultation, use of accessory muscles, heart rate, respiratory rate, pef or fev1, oxygen saturation, arterial blood gas if patient in extremis) for assessing asthma severity to determine the site of therapy, forms of treatment, and monitoring of treatment . The assessment of severity was poor as only 32.7% were documented; it is more than 3.3% in the audit of childhood asthma in lagos,14 but it is less than 42.182.2% in other parts of the world.23 functional assessment components such as oxygen saturation and pefr were measured in 5.9% and 5.0% of the cases, respectively; these indices are comparatively <93.0% and 20% in spain24 and 96.1% and 81.0% in the uk,18 respectively . Pefr is less commonly assessed in mexico and italy and more commonly assessed in australia and france.23 none of the cases had their post - bd pefr and this is in contrast to 38.0% reported in the uk which is also a disappointing result.18 the implication of not measuring post - bd pefr is that most patients were treated and discharged without objective evaluation of airflow limitation and response to therapy . It is unimaginable that the hospitals have all the required facilities, but it was not optimally used . These include lack of knowledge and awareness of its usefulness25 and systemic problems such as delay in replacing consumables such as battery of the oximeter and spirometry and pef mouthpiece and repairing faulty components of the instruments . Smoking status was recorded in 29.7% of cases and this finding is in contrast to 91.8% reported in the uk audit.18 this is a serious omission on the part of the admitting doctors . This observation may be ascribed to physicians poor knowledge of smoking as risk factors for severe asthma and its blunting of the anti - inflammatory action of css.2627 other factor responsible for poor recording of smoking is the perception by doctors that the most women do not smoke and is not a cause of serious morbidity among women who are mostly affected by asthma.5161728 majority of the patients had their asthma triggering factors documented (72.3%) which is similar to 73.3% in a previous study.14 the level of drug adherence recorded was also disappointing as only 17.8% were documented to adhere to inhaled corticosteroids (icss). A study in nigeria has shown that the lack of controller medication adherence is strongly associated with poor asthma control.29 proper assessment of adherence would have afforded the managing team to prescribe the optimal therapy and address the cause of poor medication compliance . Majority (75.2%) of the patients received nebulized salbutamol as bd during asthma emergency; this result is an improvement on three other studies carried out in nigeria about a decade ago where most patients were given theophylline.141617 rapid - acting 2-agonists are more effective than theophylline in the management of acute asthma . Its use is associated with less severe and potentially fatal side effects.2 we also found an encouraging result in the use of css as 88.1% of the patients were given systemic cs and 77.2% were given within 1 h of admission . The rate of steroids administration in our study is similar to other studies.14161724 in terms of time of steroid administration, our data are far better than the one in the uk where 95.0% were given systemic cs, but 40.0% were given within 1 h of admission.18 the similarities in the results of this study with the previous audit might likely be due to doctors good knowledge and belief about the efficacy of cs in reversing and resolving chronic airway inflammation of asthma . In addition, ocss and intravenous corticosteroids are widely affordable and available in hospitals and private pharmacy during periods of hospital workers strike and their prescription are rarely affected by systemic or organization problems.10 systemic glucocorticosteroids speed resolution of exacerbations and should be utilized in the all, but the mildest exacerbations and the benefits are greatest in patients with life - threatening asthma and those not currently receiving steroids.2 significant benefit with systemic steroid therapy is observed within 4 h of administration.2 hence, systemic steroids take several hours to exert their anti - inflammatory effect, and therefore, prompt administration after presentation is important . Concurrently, 30.7% of the patients received supplemental oxygen; this statistic is lower than 60% reported in a spanish study.24 a multi - national study on acute asthma management, burden, and outcomes also revealed that <60% of the patients received guideline - recommended therapy with a bd, cs, and supplemental oxygen.23 oxygen administration is low in this study because there is a lack of assessment of the oximetry status and indication for acute oxygen administration during the initial assessment on admission . The poor assessment of oxygen level may be due to an unavailability of a regularly functioning pulse oximeter . There is a possibility of direct relation between knowing a patient's oxygen status and correction of hypoxemia . Furthermore, the poor administration may also be due to physician's lack of awareness of its usefulness in cases that were not life - threating or presenting with central cyanosis . Hypoxemia in asthma emergency can be detected using clinical signs, blood gas analysis, or pulse oximetry.1 cyanosis has poor sensitivity because it is possible to have hypoxemia despite the lack of cyanosis.30 systemic problems such as delay in replacing empty oxygen cylinders and repairing faulty concentrator oxygen delivery components and electrical power failure may all have roles to play in a resource - limited setting . Although, we did not evaluate the knowledge of, perception and barrier to oxygen therapy among the doctors in this study, our findings may implicate future research on oxygen therapy among health - care workers and the general populace . On discharge, nearly one out of three patients had a prescription of inhaled corticosteroid while most patients were not prescribed ics; this result is in contrast to other studies on asthma audit.1823 the use of intravenous corticosteroids is associated with good asthma control and reduced risk of asthma - related hospitalization and ed visit in nigeria.2931 furthermore, majority of the patients did not have their inhaler technique reviewed, despite recent studies showing that most patients use their inhalers incorrectly.32 also, in the uk, <50% of patients had their inhaler technique reviewed on discharge.18 the findings in our study may be due to poor awareness of the clinical consequences of improper inhaler technique . About 10% were given asthma action plan and this pattern has also been observed in other studies.183334 in a multicenter study in nigeria to determine the unmet needs in asthma treatment, 33% of patients received asthma action plan.15 this result study is a reflection of the poor partnership between asthma patient and health - care professional and poor knowledge of the asthma guidelines . Most (67.3%) of the patients were given specific follow - up appointment; however, only 35.7% were reviewed within 30 days of discharge . This is similar to 66.8% reported in the uk asthma audit.18 lack of follow - up is a common problem among asthma patients; other studies have reported low rates of follow - up visits in their patients after they were discharged from ed.3536 the frequency of follow - up visits may help to build doctor and patients partnership, to predict medication adherence, and to ensure adequate monitoring of their lung function . In assessing the doctors adherence to the guidelines, our results showed that the level of adherence was not generally satisfactory among all the categories of doctors, with the medical / casualty officers having the lowest level adherence . We also observed that the performance of doctors increased with their level of training as specialists tended to perform better than nonspecialists . This finding is comparable to other previous studies in nigeria.1215 this study has reflected the performance of two tertiary hospitals in southwestern nigeria, using the recommended standard of care, and not that of individuals, a group of professionals or specialties . This form of benchmarking has highlighted areas of deficiencies in acute asthma management that can be easily understood by medical and nonmedical staff . Poorly performing areas of the management would be examined and organizational issues as well as management protocols and clinical competence would be addressed . A second audit would be carried in 1216 months after addressing the observed deficiencies and management challenges . We are limited by some inaccuracies from the medical write - up of patients by doctors, missing medical record files, and periods of industrial unrest by health - care workers where instruments were locked up and some vital measurements could not be done . In view of the fact that this study was conducted in two tertiary hospitals, we are very cautious in generalizing result our findings to the whole country . However, this study may serve as template for future study on national asthma audit and reason for nonadherence to asthma guidelines before formulating the national asthma guideline . The result from this first audit reveals that the acute management of asthma in our setting is suboptimal and there is a low level of compliance with most recommendations of the gina guideline . This audit has implicated the need to address the nonperforming areas, knowledge gaps, clinical competence, and organizational issues before the second audit.
Studies from developed countries have shown that infections produced by resistant microorganisms are associated with increased mortality and increased cost for health care systems . Developing countries bear most of the common bacterial infection burden worldwide, but data from resource limited settings are scarce . Recent studies have predicted a shift in the burden of antimicrobial resistance from gram positive to gram negative infections . Escherichia coli is the most common cause of urinary tract infections and one of the leading cause of blood stream infection worldwide . For escherichia coli, third generation cephalosporin resistance is associated with resistance to other antimicrobials and can be used as a marker of multidrug - resistance . In europe, third generation cephalosporin resistant escherichia coli (g3crec) has shown to increase mortality and hospital stay in patients from tertiary hospitals with blood stream infection . India is a highly populated country with nearly 69% of people living in rural areas . However, the majority of these studies were performed in tertiary hospitals in urban areas, so the results of these studies may reflect local prevalence of resistant bacteria related to high antibiotic pressure in these sites . The prevalence of antibiotic resistant escherichia coli in the community of rural areas of india is not well known . The aim of this study is to describe the prevalence of g3crec and to investigate the effect of g3crec on the mortality of patients admitted to a district hospital in a rural area of india . The study was performed in the bathalapalli rural development trust hospital, a non - profit private district hospital in andhra pradesh, india . The hospital belongs to a non - governmental organization that provides free consultation and medicines at reduced cost to people of low socio - economic status . The hospital is fully computerized and, as a routine, clinical and microbiological information of the patients is introduced in a hospital database . For this study we selected cases admitted to the hospital with isolation of escherichia coli from any clinical specimen from 24 march 2011 to 18 march 2012 . Of this group, patient who met criteria for having a community - acquired isolation of escherichia coli were included in the study: isolation in the first 48 h of admission and no previous hospital admission in the last 90 days . Antimicrobial susceptibilities were performed in the microbiology laboratory of the hospital using disk diffusion testing according to recommendations of the clinical laboratory standard institute . All strains where tested for extended spectrum beta - lactamase (esbl) production by double disk synergy test . G3crec was defined when the antibiogram shown resistance to cefotaxime . The primary end - point of the study was to compare the 21-day mortality of patients who had g3crec with patient who had a cephalosporin - susceptible escherichia coli isolation . Blood, bone marrow, cerebrospinal fluid, ascitic fluid and pleural fluid were considered as normally sterile site (nss) isolations . Variables that could be related to increased mortality as age, sex, hiv infection and nss isolation were introduced in a multivariable logistic regression analysis model to assess the effect of having g3crec isolation on 21-day mortality . We used kaplan - meier survival curves to describe the mortality of patients with and without g3crec . Time was measured from the date of admission to the date of death in patients who died, or from the date of admission to the last visit date or the date of discharge, whatever occurred last . The study was performed in the bathalapalli rural development trust hospital, a non - profit private district hospital in andhra pradesh, india . The hospital belongs to a non - governmental organization that provides free consultation and medicines at reduced cost to people of low socio - economic status . The hospital is fully computerized and, as a routine, clinical and microbiological information of the patients is introduced in a hospital database . For this study we selected cases admitted to the hospital with isolation of escherichia coli from any clinical specimen from 24 march 2011 to 18 march 2012 . Of this group, patient who met criteria for having a community - acquired isolation of escherichia coli were included in the study: isolation in the first 48 h of admission and no previous hospital admission in the last 90 days . Antimicrobial susceptibilities were performed in the microbiology laboratory of the hospital using disk diffusion testing according to recommendations of the clinical laboratory standard institute . All strains where tested for extended spectrum beta - lactamase (esbl) production by double disk synergy test . G3crec was defined when the antibiogram shown resistance to cefotaxime . The primary end - point of the study was to compare the 21-day mortality of patients who had g3crec with patient who had a cephalosporin - susceptible escherichia coli isolation . Blood, bone marrow, cerebrospinal fluid, ascitic fluid and pleural fluid were considered as normally sterile site (nss) isolations . Variables that could be related to increased mortality as age, sex, hiv infection and nss isolation were introduced in a multivariable logistic regression analysis model to assess the effect of having g3crec isolation on 21-day mortality . We used kaplan - meier survival curves to describe the mortality of patients with and without g3crec . Time was measured from the date of admission to the date of death in patients who died, or from the date of admission to the last visit date or the date of discharge, whatever occurred last . During the period of the study, escherichia coli was identified in 219 specimens from 194 patients admitted to the hospital . Nitrofurantoin (only tested in urinary specimens), amikacin, chloramphenicol, meropenem and gentamicin had low levels of resistance . The proportion of specimens with g3crec was 78.1% (95% confidence interval [ci], 72.183.1). The proportion of meropenem and ciprofloxacin resistance was 11.1% (95% ci, 7.616) and 80.9% (95% ci, 75.185.6) respectively . Colistin was tested only for 17 isolations and all of them were susceptible . Compared to other sites, isolations from nss had lower levels of resistance to ciprofloxacin (p=0.019), cefotaxime (p=0.003), ceftazidime (p=0.004), cefepime (p=0.011) and piperacillin - tazobactam (p<0.001). Table 1percentage of antibiotic resistance by clinical specimen.antibioticurine (n=113)nss (n=21)pus (n=42)sputum (n=11)others (n=32)total (n=219)nitrofurantoin0.9 - ---0.9amikacin1.80.04.89.16.33.3gentamicin22.59.519.018.215.619.4chloramphenicol6.40.016.70.020.09.5ciprofloxacin86.261.973.890.981.380.9cotrimoxazole79.661.965.080.070.073.7cefotaxime85.052.471.490.975.078.1ceftazidime83.652.471.490.974.277.2cefepime82.655.073.890.975.077.6piperacillin - tazobactam100.057.170.090.077.483.9amoxicillin - clavulanate92.981.082.9100.090.689.9meropenem8.114.319.50.012.511.1nss, normally sterile site (blood, bone marrow, cerebrospinal fluid, ascitic fluid and pleural fluid). Nss, normally sterile site (blood, bone marrow, cerebrospinal fluid, ascitic fluid and pleural fluid). The proportion of patients with g3crec was 79.4% (95% ci, 73.184.4) and the global 21-day mortality was 13.4% (95% ci, 9.318.9). 21-day mortality was 5% (95% ci, 1.416.5) in patients with cephalosporin susceptible escherichia coli and 15.6% (95% ci, 10.722.1) in patients with g3crec . Among the 154 patients with g3crec, 126 (81.8%, 95% ci 75 87.1) had an esbl producing escherichia coli . Baseline characteristics of the patients and multivariable analysis of factors associated with 21-day mortality are presented in table 2 . Factors significantly related to 21-day mortality were hiv infection, isolation from a nss and isolation of g3crec . Kaplan meier survival curves of patients with and without g3crec are presented in figure 1 . Table 2baseline characteristics and factors associated with 21-day mortality.baseline characteristics n (%) 21-day mortality21-day mortality multivariable analysisno (n=168)n (%) yes (n=26)n (%) aorp - valueage (years)35.3 (2550)35 (2450)40 (32.749)1.03 (11.07)0.072female72 (37.1)65 (38.7)7 (26.9)0.78 (0.282.19)0.643hiv infection80 (41.2)59 (35.1)21 (80.8)10.89 (3.2636.42)<0.001isolation from a nss19 (9.8)13 (7.7)6 (23.1)11.96 (2.4757.97)0.002g3crec154 (79.4)130 (77.4)24 (92.3)8.24 (1.1957.28)0.033median (interquartile range); aor, adjusted odds ratio; nss, normally sterile site; g3crec, third generation cephalosporin resistant escherichia coli . Median (interquartile range); aor, adjusted odds ratio; nss, normally sterile site; g3crec, third generation cephalosporin resistant escherichia coli . Figure 1kaplan meier survival curves of patients admitted to the hospital with escherichia coli isolation . The results of this study show the high proportion of resistance to quinolones, penicillins and cephalosporins in strains of escherichia coli isolated from the community . Other indian studies have shown high levels of esbl producing escherichia coli, ranging from 63% to 79% in different settings and specimen types . However, the rise of g3crec is not an exclusive indian phenomenon . In a study that involved 31 european countries, it was predicted an exponential increase in the number of bacteraemias produced by g3crec in europe . Genes that encode third generation cephalosporin resistance are easily spread within health - care facilities, but also among travelers to countries with high levels of g3crec . G3crec is now a major public health problem in india, but we will probably see a rapid spread of g3crec in other parts of the world in the near future . Our results indicate that there is an association between isolation of g3crec and 21-day mortality in patients admitted to a district hospital in india . Not surprising, the odds ratio for mortality found in this study was higher than the odds ratios for mortality found in other studies from tertiary hospital in europe and in south korea where resources are not so limited . Although the spread g3crec affects both developed and developing countries, it will cause higher mortality in resource - limited settings because of the scarcity of laboratories capable of providing antimicrobial susceptibilities and the high cost of effective antibiotics against g3crec . Interesting, isolations from nss had lower proportion of antibiotic resistance to ciprofloxacin and beta - lactam antibiotics . It has been described that the development of antibiotic resistance by common bacteria bears a cost in terms of reduced fitness and virulence . The lower proportion of g3crec in specimens from nss could be explained by the loss of virulence of escherichia coli strains that develop resistance to these antibiotics . The spread of g3crec should have important implication for the management of urinary tract infection and suspected escherichia coli blood stream infections . The most commonly used antibiotics in non - complicated urinary tract infections (cotrimoxazole, fluoroquinolones, aminopenicillins and cephalosporins) are frequently not effective against g3crec . In our study, nitrofurantoin and chloramphenicol were more frequently susceptible than other antibiotics because they are not commonly used, so these antibiotics could be utilized for the treatment of non - complicated urinary tract infection . However, there is an important concern about the future therapeutic options for escherichia coli infections because of the scarcity of new antibiotics for treating gram negative bacteria in the pipeline . We did not collect clinical data for distinguishing infection from colonization in some of the isolations from non - sterile sites . In addition, we did not collect clinical information of present morbidities at the moment of admission . It is possible that patients with g3crec had unknown risk factors not included in the multivariable model that could modify the results of the study . However, the possibility of confounders related to having g3crec isolation is unlikely, because the study was performed in a rural area, where referrals from other health - care centers are rare, and we excluded isolations performed after 48 h of admission and patients admitted in the previous 90 days . The results of this study indicate that a high proportion of community - acquired isolations of escherichia coli are resistant to multiple antibiotics and having g3crec isolation is associated with higher risk of death in this indian setting . If these findings are confirmed by other studies in resource - limited setting, the emergence of g3crec could be a major public health threat in developing countries.
The udder is the milk - producing organ of dairy animals; hence, for optimal production, it should be healthy . Mastitis is the inflammatory response of the mammary gland (mg) tissue to physiological and metabolic changes, traumas, and allergies and, most frequently, to injuries caused by various microorganisms . Mastitis is considered the utmost threat to the dairy industry from three perspectives: economic, hygienic, and legal (eu directive 46/92, modified by directive 71/94). The intramammary inflammation (imi), accompanied by immunological and pathological changes in the mg tissue, occurs at different degrees of intensities and results in a wide range of consequences regarding physical, chemical, and often microbiological alterations of secreted milk . A wide spectrum of microorganisms, including fungi, yeast, algae, chlamydia, and viruses, have been incriminated in causing mastitis, but bacteria remain the principle causative agents of such complex [1, 2]. The major bacterial mastitis pathogens (staphylococcus aureus, streptococcus agalactiae, s. uberis, s. dysgalactiae, and coliforms) are most often responsible for clinical mastitis (cm). Meanwhile, minor pathogens (coagulase - negative staphylococci cns; streptococci other than s. agalactiae, s. uberis, and s. dysgalactiae; corynebacterium spp . ;; listeria spp . ; leptospira spp . ; yersinia spp . ; enterobacter spp . ; brucella spp . ; and mycobacterium spp .) Are typically associated with subclinical mastitis (scm) or sometimes associate clinical imis . With the exception of a few pathogens that can invade via the blood stream (e.g., brucella abortus or mycobacterium bovis), infection of the mg occurs by ascension through the ductus papillaris, the only opening of the udder to the outside world, and the pathogens pass to find an environment that is warm, moist, and nutrient - rich and thus suitable for rapid growth and multiplication . To establish a successful infection after traversing the teat end opening mg immunity depends on the complex combination and coordination of nonspecific and specific protective elements, including the anatomical features of the gland as well as cellular and humoral defence components . Nevertheless, mg immune defence varies over different stages of lactation in dairy animals and is typically depressed with exposure to stress and around drying - off and parturition, thus increasing susceptibility to mastitis [6, 7]. However, a considerable body of evidence has accumulated suggesting that mastitis is a multifactorial complex, and several management and environmental factors must interact to increase host exposure to mastitis pathogens, reduce the natural resistance of animals to disease, or aid pathogens in gaining entrance to the mg environment to cause infection [2, 4]. The primary defence mechanism of the mg is represented structurally in the teat canal [3, 8, 9], which acts as both a physical barrier and a source of antimicrobial substances . The physical barrier is provided by the smooth muscle sphincter surrounding the teat canal, which prevents escape of milk and constitutes a barricade against the entry of different pathogens by maintaining tight closure [2, 3, 9]. Normally, the healthy teat skin is coated with a protective mantle of fatty acids (fas) that slow the growth of bacterial pathogens . Additionally, the stratified squamous epithelium of the teat duct produces keratin, a waxy material lining the teat canal, which traps invading bacteria and hinders the migration of microorganisms into the gland cistern . This keratin is composed of (i) bacteriostatic fas of both esterified and nonesterified types, such as lauric, myristic, palmitoleic, and linolenic acids and (ii) fibrous proteins, which bind electrostatically to microorganisms, altering the cell wall and rendering it more susceptible to osmotic pressure changes and, thus, to lysis and death . Additionally, these cationic proteins were found to have an inhibitory effect against some pathogens as staph . Aureus and s. agalactiae, which was equal to that of proteins isolated from bovine neutrophils . The lipid content and composition of teat duct keratin have been shown to vary throughout the milking process, between lactating and dry dairy animals, and according to the severity of imi . Scm was found to not affect the lipid content of teat duct keratin, while cm was shown to be associated either with significantly higher levels of total lipids or with similar lipid composition of uninfected quarters . Additionally, the free fas in milk from clinical quarters contained fewer short - chain fas, whereas polyunsaturated fas were significantly higher . Recently, sentinel functions for the teat towards invading pathogens have been documented, as the teat canal tissue responded rapidly and intensely, with both expression of several toll - like receptors (tlrs) and production of cytokines and antimicrobial peptides [16, 17]. Damage of keratin, perhaps as a result of incorrect intramammary therapy infusion or by faulty machine milking, has been reported to increase susceptibility of the teat canal to bacterial invasion and colonisation . However, the antimicrobial effectiveness of keratin is limited [9, 21] and, despite the potent physical and chemical protection in the teat canal, there are several ways by which bacteria can penetrate the teat canal and cause imi, so much so that a number of pathogens are able to colonize the teat canal for prolonged periods, such as corynebacterium bovis, or cns . Aureus deposited a few mm inside the teat canal has also been demonstrated [2224]. Also, during milking, it is common for keratin to be flushed out with distention of the teat canal . Because the sphincter takes approximately 2 h to regain its contracted position, there is a chance for outside pathogens to enter the teat canal, causing trauma and damage to the keratin or mucous membranes lining the teat sinus [2, 21]. Additionally, during mechanical milking, microorganisms present at the teat end may be propelled into or through the teat duct into the cistern . The mg is normally protected by both innate and adaptive immune responses (irs), which coordinate and operate together to provide an optimal defence against infections . The irs also facilitate the constitutive or acute transient presence of a wide range of immune - related components in milk . The adaptive immune system (ais) responds more robustly to threats to which it has previously been exposed; however, it is slow to respond to novel threats . In contrast, the innate immune system (iis) is the first line of defence against pathogens once they have penetrated the physical barrier of the teat canal and before the ais comes into play, and it evolves into a highly effective host defence [33, 34]. This process is mediated via several intracellular signal transduction cascades that trigger an acute upregulation of several innate immune components including different leukocytes, adhesion molecules, and cytokines [3537]. The two most critical components of host innate immunity are pathogen recognition (pr) and the ability to mount a proinflammatory response, a complex interaction of cellular and molecular processes aimed at detecting and subsequently eliminating harmful pathogens [25, 34]. A wide variety of components linked to the innate ir (iir) have been identified in milk, including cellular defence components [e.g., leukocytes], components contributing to humoral defence [e.g., complement system (cs), immune - modulating factors (pro- and anti - inflammatory cytokines), lactoferrin (lf), transferrin (tf), lysozyme (lz), and components of the lactoperoxidase / myeloperoxidase systems], oligosaccharides, gangliosides, reactive oxygen species (ros), acute phase proteins (apps) (e.g., haptoglobin and serum amyloid a), ribonucleases, and a wide range of antimicrobial peptides and proteins . Many of these components originate from specialised cells that traffic to the mg [33, 34]. The ability of the iis to recognise and respond to a broad spectrum of pathogens that may or may not have been previously encountered, combined with the speed in mounting a proinflammatory response following initial pr, greatly contributes to the host's ability to control invading pathogens . Below, there is a detailed overview of the roles and mechanisms of action of some innate immune factors . . The viable leukocytes inside the mg offer some degree of cellular protection against microbial invasion through their ability to recognise microorganisms and induce a rapid inflammatory response in an attempt to resolve the imi immediately . Thus, mg - resident leukocytes likely provide a surveillance function in the uninfected gland . Also, these cells may aid in the restructuring of the mg that occurs during involution (i.e., apoptosis). In addition to microbicidal functions of phagocytosis, mg leukocytes secrete a variety of immune - related components into milk including cytokines, chemokines, ros, and antimicrobial proteins and peptides (lf, defensins, and cathelicidins). Leukocytes also assist in the repair of damaged tissue caused by shedding and renewal processes . Despite the presence of considerable numbers of immune cells in the mg environment, it has been suggested that the mg is immune - compromised when compared to the rest of the body . Moreover, the activities of all types of leukocytes in milk have been shown to be reduced compared to those in blood [28, 43, 44]. The migration of immune cells during imi plus desquamation of mg epithelia results in an increase of somatic cell count (scc) accompanied with decreased milk production according to the severity of the process [1, 45] polymorphonuclear neutrophils (pmns) constitute the second line of the iis against imi . Even under healthy conditions, pmns are permanently present inside the mg environment, and nursing or milking stimuli accompanied with milk removal were found to induce directed migration of fresh pmn into mammary tissue . Bovine neutrophils cross the mg epithelium by diapedesis without causing epithelial cell damage unless the migration is extensive, in which case both mechanical and chemical damage are possible . The neutrophil's multilobulated nucleus allows for easy and rapid migration between endothelial cells, thus arriving as the first recruited immune cell to sites of infection . Because only small numbers of mature pmns are stored in the bone marrow, the number of immature neutrophils in circulation increases as a result of mobilisation into circulation during inflammatory conditions . Several important functions are not fully developed in immature neutrophils, including those pertaining to phagocytosis, intracellular killing, and chemotaxis . Neutrophils are delineated by a plasma membrane that has a number of functionally important receptors . These include l - selectin and 2-integrin adhesion molecules, which promote the binding of pmns to endothelial cells and facilitate their migration to infected foci [39, 49, 51]. Membrane receptors for the fc portion of the igg2 and igm classes of igs and for complement components c3b and ic3b are necessary for the phagocytosis of invading bacteria [52, 53]. The activation of c3b regions on bacterial surfaces after binding with abs promotes phagocytosis and binding to cr1 and cr3 receptors on the pmn surface . Additionally, lectin - carbohydrate receptors found on neutrophil cell membranes can recognise carbohydrate - rich fimbriae of escherichia coli in the absence of specific opsonins [49, 54], resulting in a process referred to as nonopsonic phagocytosis . The primary function of pmns to engulf, phagocytose, and destroy foreign materials, including invading bacteria, occurs via two parallel systems . The first is an oxygen - dependent (respiratory burst) system that includes the production of hydroxyl and oxygen radicals . The second is an oxygen - independent system that relies on several oxygen - independent reactants such as peroxidases, lzs, hydrolytic enzymes, and lf [1, 41]. In addition to phagocytic activity, pmns also contribute to the modulation of vascular permeability and release several inflammatory mediators that play crucial roles in the coordination of innate and adaptive immune components . Furthermore, the intracellular granules of pmns contain several bactericidal peptides including defensins, enzymes (e.g., myeloperoxidase), and neutral and acidic proteases (e.g., elastase; cathepsin types b, d, and g, procathepsins) [5658], which can kill a variety of mastitis pathogens . Such proteases as well as plasmin are known to permit the chemotaxis of cells in the site of inflammation and are involved in the limitation in time of the ir (e.g., by the cleavage of some cytokines such as il-2, il-6, and il-8). The exposure of pmns to cytokines and chemoattractants causes rapid mobilization of azurophil granules (containing elastase and cathepsin g mainly) to the cell surface ., pmns can wrongly phagocytose milk fat globules, and their proteases can degrade milk casein (caseinolysis), leading to putrefaction of milk, and, together with their released hydroxyl radicals, can damage the mg epithelium which contributes to the decreased synthetic activity of the mg during imi . Once pmns perform their tasks, they undergo apoptosis, or programmed cell death, and are removed by macrophages [63, 64]. Macrophages . They constitute the predominant cell type found in milk and tissues of both healthy involuted and lactating bmgs [43, 65, 66]. In contrast to neutrophils, macrophages have large horseshoe - shaped nuclei that make their migration between endothelial cells more difficult . Macrophages contribute to induction of specific local irs through antigen (ag) processing and presentation to lymphocytes in association with mhc class ii ags [45, 6769]. Similar to pmns, macrophages can perform a variety of nonspecific functions including ingestion and phagocytosis of foreign particles, including some invading bacteria (e.g., staph . Aureus), and destroying them with proteases and ros [66, 70, 71]. Additionally, they can ingest cellular debris and accumulated milk components in involuting mgs . The phagocytic activity of macrophages can be increased in the presence of opsonic abs for specific pathogen . In cattle, mg macrophages bear receptors for igg1 and igg2 . Unlike neutrophils, macrophages possess fewer fc receptors, which decrease their phagocytic capacity . Mg macrophages are considered less effective at phagocytosis compared to blood monocytes because of indiscriminate ingestion of milk components as well as the fact that macrophage proteases can also contribute to damage of mg epithelium [62, 70, 74]. A failure of efficient killing of some mastitis pathogens (e.g., s. uberis) after engulfing and even increased intracellular multiplication of s. uberis as well as lesser stimulatory responses by ifn- to release tnf- and bactericidal products compared to blood monocytes have been also reported . However, it has been demonstrated that the bactericidal activity of mg macrophages can vary according to mg secretion, and dry - off secretion macrophages exerted higher bactericidal activities than lactational macrophages . Therefore, the ability of macrophages to secrete substances that augment local inflammatory processes, thereby inducing the migration and bactericidal activities of neutrophils, is believed to be of greater importance to nonspecific defence of the mg than their function as professional phagocytes [7, 67, 70, 75]. Lymphocytes recognise a variety of antigenic structures via membrane receptors, which define their specificity, diversity, and memory characteristics . T- and b - lymphocytes and natural killer (nk) cells are distinct lymphocyte subsets that operate in the mg (figure 1), although they differ in function and protein products . During imi, preferential trafficking of certain lymphocyte subpopulations to specific mammary tissue foci occurs [28, 76] and marked changes in milk lymphocyte count and composition during imis have been reported . Additionally, the ais response is mainly mediated by memory lymphocytes, which respond quickly to threats to which they have previously been exposed . It must be mentioned that the presence of specific lymphocyte subsets can affect the total lymphocyte function and even the whole ir . For example, the activation of cd8 + t - cells during certain bacterial imis, such as staph . Aureus, can suppress important host irs and predispose to chronic pattern of imi [78, 79]. Unfortunately, the exact roles of lymphocytes during imi and their subsets are complex and are not fully defined . Even in healthy mgs, the composition of the lymphocyte population varies during the lactation cycle [28, 76, 80]; the consequences to mg immunity are still not fully understood . Additionally, mg lymphocytes exhibit hyporesponsiveness to mitogenic, antigenic, and allogeneic stimuli compared to blood lymphocytes, possibly due to the presence of distinct lymphocyte subsets, high proportion of memory t - lymphocytes present in the mg, and/or less efficient presentation of ags by ag - presenting mg cells . In healthy bmgs, t - cells prevail in both mg secretions and parenchyma and predominantly exhibit the cd8 + phenotype, which is in contrast to the blood, where cd4 + cells are the predominant t - cell subset . Therefore, the ratio of cd4+/cd8 + t - cells is lower in milk than in blood . Cd4 + (t - helper) cells produce a variety of immunoregulatory cytokines following ag - recognition with mhc class ii molecules; and are being memory cells following ag - recognition [4, 7, 27, 81]. On the other hand, it is well established that cd8 + cells can exert either cytotoxic or suppressor functions . In coordination with major histocompatibility complex (mhc) class i molecules, cytotoxic t - cells recognise and eliminate altered self - cells via ag presentation, thus being more specific than nk cells . However, it has been suggested that their removal of damaged mammary epithelium could enhance the susceptibility of mg to infection . Suppressor t - cells are thought to play roles in control or modulation of the mgir . However, the immunoregulatory roles of cd8 + cells are also greatly dependant on lactation stage . Cells obtained from midlactation dairy cattle exhibited cytotoxic activity and mainly expressed interferon- (ifn-), whereas cd8 + lymphocytes obtained during the postpartum period exhibited no cytotoxic activity and mainly expressed interleukin 4 (il-4). Ruminants bear greater levels of t - lymphocytes in secretions and parenchyma of mg relative to blood . There are indications that t - cells can mediate cytotoxicity, similar to nk cells, with variable involvement of mhc molecules; thus, they may be able to destroy altered epithelial cells [83, 84]. T - lymphocytes preferentially migrate to particular epithelial surfaces and do not exhibit extensive recirculation . Thus, it has been indicated that t - lymphocytes play a role in antibacterial immunity and may provide a unique barrier function for mucosal microenvironments against bacterial pathogens . The wc1 subpopulation represents a minor portion of t - lymphocytic population in normal mg secretions [28, 86], but they markedly increase following parturition . Because of restricted localisation and expression of invariant ag receptors, the exact contribution of these cells to mg immunity is not fully understood . Several lines of evidence have been accumulated suggesting that these cells perform specific functions in comparison to circulating and t - cells . Recently, it has been addressed that lymphocytes exert some immunoregulatory / suppressive functions, more precisely in the wc1.1 and the wc1.2 cells . On the other hand, it has been reported that wc1 cells are not recruited to the mg during chronic imis caused by staph . One of the main roles of b - lymphocytes is to produce abs against invading pathogens . Unlike macrophages and pmns, b - lymphocytes utilise their cell surface receptors to recognise specific pathogens and then internalise, process, and present ags in the context of mhc class ii molecules to t - helper cells . Under certain conditions, b - lymphocyte differentiation can be directly stimulated by an ag such as lipopolysaccharides (lps). In contrast to t - lymphocytes, the percentages of b - lymphocytes remain fairly constant regardless of lactation stage [49, 77] or infection . Nk cells are large granular nonimmune lymphocytes that differentiate and mature in bone marrow, lymph nodes, spleen, and tonsils before passing to the circulation . Nk cells constitute the third type of cell derived from lymphoid progenitors that also generate b- and t - lymphocytes . Nk cells utilise their fc receptors to possess a cytotoxic activity critical to the iis in the absence of mhc restriction . Nk cells cause lysis of target cells through a diverse repertoire of mechanisms, including ab - dependent cell - mediated cytotoxicity, granule exocytosis, release of cytolytic factors, and receptor - mediated ag - recognition . Additionally, they secrete various toxic molecules that may initiate apoptosis in altered cells . Nk cells differ from natural killer t - cells in origin, respective effector functions, and lack of specificity for ag - recognition . However, nk cells do not require activation to kill cells that lack self - markers of mhc class i . Studies have demonstrated the capability of nk cells to kill both gram - positive (gpb) and gram - negative bacteria (gnb) and, therefore, they may be important in preventing imis [91, 92]. The differences in distribution of cellular components in mg environment between healthy and inflammatory conditions are detailed in table 1 . The distribution of leukocytes in healthy mg is somewhat variable during healthy lactating and dry periods . The percentage of pmns tends to increase during early and late lactation, while the percentage of lymphocytes decreases . Meanwhile, the proportion of macrophages is highest (68%) in the early postpartum period and lowest (21%) in late lactation . During the dry period the increase at the start of involution is most likely due to an influx of cells resulting from cessation of milk removal, or due to the concentration effect by removal of the liquid phase of the secretion . Sccs in milk from uninfected glands at the beginning of the dry period are usually higher than 1 10 cells / ml milk, but by the 7th day of the dry period this count can be as high as 2 10 cells / ml milk . Pmn counts are initially high in early involutional secretions, comprising 4080% of scc (similar to colostrum), but are reduced again from the 2nd to 4th week of the dry period and then return to lactational values in the fully involuted udder [43, 66]. Unlike in the lactation stage [43, 66] and with exception of the 1st day of the dry period in which they exhibit higher counts, macrophage concentrations are relatively low during the remaining part of early involution and in colostrum, with maximal proportions (30%) peaking by the mid - dry period and remaining constant until calving . Lymphocyte concentrations in dry secretions are approximately 30006000 times that in normal milk, and the proportions of b- and t - lymphocytes are approximately 28% and 47%, respectively, approximating proportions in peripheral blood [31, 32]. (3) distribution of cellular components in the mg environment of ovines and caprines . The milk sccs thresholds are higher in milk of small ruminants than in bovine milk . Recent studies have indicated an upper scc threshold of 2.5 10 cells / ml milk in healthy ewe's udders or more, up to 6 10 cells / ml milk . Similar to bovines, the macrophages are the predominant cell type (4684%) in milk from uninfected ewes [96, 97]. Counts of macrophages were higher in early and midlactation milk than in late lactation milk . The rest of the scs population consists of pmns (228%) and lymphocytes (1120%). Meanwhile, limited data exist on changes of leukocytes population in infected ewes' mgs . Recorded an increase of pmns percentages to 50% at a scc of 2 10 cells / ml milk and to 90% at a scc over 3 10 cells / ml milk, representing the predominant cell type at inflammatory conditions . Likewise, an increase of pmns and macrophages counts within imi of ewe's udder has been reported, whereas lymphocytes decreased . Scc of milk from uninfected goats is higher than those of milk of uninfected bovines and sheep . Unlike cow and sheep milk where macrophages are the predominant cell type, pmns comprise the major cell type in goat milk from both infected and uninfected mgs [100104]. In healthy status, pmns, macrophages, and lymphocytes comprise 4574%, 1541%, and 920% of scs population, respectively, while epithelial cells are present in low percentage (16%) [98, 103, 105, 106]. With advanced lactation, the pmns increase, manlongat et al . Explained this late - lactation rise - up on the presence of higher chemotactic activity in nonmastitic goats udder and concluded that this phenomenon was nonpathological and could play a physiologic regulatory role in mg involution . Unfortunately, very little data exist on the distribution of these cells during imi . A study by dulin et al . Reported an elevation of pmns to 7186% in infected halves, while macrophages and lymphocytes percentages are being changed to 818% and 511%, respectively . Mecs themselves are active contributors to the innate immune and inflammatory responses of mg [108, 109]. They express a range of pr receptors (prrs), most notably the tlrs [35, 36]. Additionally, the polymeric - ig receptor (pigr) expressed on the mucosal epithelium facilitates the translocation of igs, particularly iga, across the epithelium into the alveolar lumen . Upon bacterial stimulation, mecs secrete a range of innate immune effector molecules and inflammatory mediators, which contribute to attraction and recruitment of circulating leukocytes [38, 111]. It was shown that mecs secrete il-8, a potent neutrophil chemoattractant, in the presence of gpb and their exotoxins, lps from gnb or il-1 [51, 111, 112]. Mecs constitute an important source for host defence components as arachidonic acid metabolites [38, 108, 114, 115], apps, lf [111, 116], -defensins [117, 118], cathelicidins and calprotectin, and lps binding protein [bp] (lps - bp), which is involved in host recognition of the bacterial cell wall [17, 119]. Supporting results were obtained experimentally on bovine mecs, showing also their ability to express il-1, tumour necrosis factor- (tnf-), il-6, il-8, and growth related oncogene- [gro-] mrna during infection and immune stimulation [111, 114, 120, 121]. Mg epithelium may exhibit protective and phagocytic functions via the ingestion and possible digestion of phagocytosed microbes and milk components, including fat globules and casein micelles, through the formation of pseudopodia . Experimental studies showed that glutaraldehyde - killed streptococci, staphylococci, and e. coli were phagocytosed by milk secretory cells . Moreover, many peptides, proteins, and lipids which are involved in host defence and shown to have antibacterial properties (including xanthine oxidase and sphingolipids) were found in fat globule membranes, which originate from the apical membrane of the mg epithelium [123, 124]. The initiation of rapid and effective iir depends mainly on recognition of the infectious agent [36, 109]. Iir of mg is initiated when prrs on the surfaces or within host cells, primarily leukocytes and mecs, bind to particular bacterial motif molecules termed pathogen / microbial - associated molecular patterns (pamps / mamps) [109, 125, 126]. Such prrs belong to three different families, namely, the tlr, nucleotide - binding oligomerization domain- (nod-) like receptors (nlr) 1 - 2, and retinoic acid inducible gene-1- (rig-1-) like receptors, and each of these receptors recognizes a set of bacterial motifs [17, 35, 36, 109]. Activation of these prrs initiates a signalling transduction cascade in which nuclear factor-b plays a pivotal role in coordinating multiple signals and directing expression of effector response genes, including cytokines, as well as orchestrating both the local and the systemic immune responses [35, 120, 128130]. In this context, it was not surprising that the expression of prrs increases in infected bovine mgs tissues and epithelia [17, 130135]. Till now, they are the best characterized bovine prrs and they recognize a wide range of pamps . Thirteen tlrs have been identified among mammals, 10 of which are known to occur in cattle [17, 35, 136]. For example, tlr pairs such as tlr1/2 and tlr2/6 can recognise lipopeptides or lipoproteins, whereas individual tlrs such as tlr2, tlr4, tlr5, and tlr9, respectively, are involved in sensing lipoteichoic acid (lta), lps, flagellin, and 6-base dna motif consisting of an unmethylated cpg - dinucleotide motif (cpg - dna) [35, 36, 109, 137140]. Besides recognizing lps motifs, tlr4 also can recognise bacterial - derived elastases and exoenzyme - s [141, 142]. Another important prr found on pmns and macrophages in the mg is cd14, which can bind to lps and induces the synthesis and release of tnf- . Also, the role of nod1 and nod2 receptors of mecs in sensing peptidoglycans (pgs) of gnb has been addressed [109, 144, 145]. (6) contribution of specific bacterial components to the identification by host iis and induction of irs gram - negative bacteria (gnb). Cell wall lps, or endotoxin, is central to the pathogenesis of mastitis caused by gnb . Lps is considered the most potent immunostimulant of cell wall components and is the key virulence factor eliciting clinical symptoms [36, 37]. The lps layer of the outer membrane generally contains three regions: o - specific polysaccharide chain, polysaccharide core, and lipid a. lipid a was found to be responsible for most of the pathogenic phenomena associated with gnb imis, including endotoxin shock . Recognition of lps is mediated by membrane cd14, lps - lbp, an app present in the bloodstream, and tlr on mecs (primarily tlr4) [35, 37, 64, 146]. As a consequence, initiation of acute ir results in an intense elevation of scc [109, 147], activation of different leukocytes and immune - related genes, and subsequent production of antimicrobial defence proteins and peptides (e.g., lf, lz, and lap), lipid mediators (e.g., cyclooxygenase-2 and 5-lipoxygenase) [149, 150], chemokines (e.g., cxcl5, cxcl8, and rantes) [148, 151, 152], and cytokines, especially il-6, tnf- and insulin - like growth factor-1 [35, 64, 146, 151]. Additionally, binding of soluble cd14 to lps stimulates mecs to produce leukocytic chemoattractants such as il-8 [112, 153]. Despite the principle role of lps in recognizing gnb by tlrs (tlr1/2 and tlr2/6), it has been illustrated more recently that pgs fragments of e. coli, which are known to activate the cytoplasmic nod1 receptor, could be recognized by bovine mecs and, thus, can induce inflammatory response . Although nod1 receptor is cytoplasmic and its activation requires that the agonist is transported into the host cell, it is possible that pgs fragments can reach the cytoplasm of bovine mecs following invasion by e. coli, as proven by some authors . Moreover, the expression of membrane transporters under particular circumstances including inflammation could transport pgs fragments, as was shown for muramyl - dipeptide (mdp), a potent nod2 agonist [144, 145]. Gram - positive bacteria (gpb). In contrast to gnb, for which lps is the major immunostimulatory molecule, several important compounds have been identified as immune stimulators for gpb species, including cell wall lipoproteins, lta, which is a cell wall component of the murein capsule [36, 119], and pgs in addition to secreted exotoxins . Both pg and lta have been shown to induce immune cells, including monocytes and macrophages, to produce inflammatory cytokines and chemokines [159, 160]. Pg combined with lta induced the expression of mcp-1 and a slight increase in mcp-3 chemokine expression . In vitro studies have shown that lta alone can induce expression of several cytokines such as il-1 [161, 162], il-6, il-8, and tnf- in mecs, although to a lesser extent than lps [125, 161163]. Also, lta proved to induce strongly the secretion of the chemokines cxcl1, cxcl2, cxcl3, and cxcl8, which target mainly neutrophils . The role of lta and other pamps as muramyl - dipeptide in stimulating iis is not only limited to expression of specific cytokines and chemokines, but can potentiate their subsequent effects after production . The staphylococcal lta or muramyl - dipeptide enhances the expression of immune defence genes that are induced by il-17 in mecs in vitro . However, it must also be considered that the virulence of bacterial compounds such as lps and lta may vary somewhat depending on their bacterial origin . More interestingly, lps - bp has been shown to bind lta of gpb cell wall although primarily associated with gnb infection . The induction of the gene encoding lps - bp was observed in all tissues of mg challenged by staph . Aureus, and increased concentration of lps - bp has been previously reported in milk and serum after imi with staph . Tlr2 plays a major role in the recognition of a variety of components related to gpb including lta and lipoproteins . Lta activates cells via the tlr2/tlr6 heterodimer [119, 134, 138, 139, 166], and with physical and functional interactions with tlr1 and tlr6 it allows discriminating the lipid portion of lipoproteins [36, 166]. Meanwhile, the roles of tlr1, tlr2, and tlr6 in the recognition of pg remain controversial, and it has been suggested that pg recognition occurs mainly intracellularly rather than from the extracellular compartments . Despite the principle role of tlr1 and tlr6 heterodimers with tlr2, significant increases in the expression of tlrs that recognise viral ligands (tlr3 and tlr7) were also observed in bovine mgs challenged with staph . Aureus, and a previous study has shown the role of tlr7 in recognition of gpb . Aureus and il6 treatment . Additionally, expression of intracellular receptors may be important in recognizing staph . Aureus which has the potential to invade epithelial cells [170, 171]. Lf, an iron - binding glycoprotein, was first isolated from bovine milk in 1939 . In the mg environment, it is mainly produced by the secretory epithelium and to lesser extent by pmns . Little or no expression of lf occurs in lactating alveoli, and moderate to high expression occurs in the epithelia lining the ducts and cisterns, while lf expression is absent at the proximal end of the teat canal . The regulation of lf expression in mg appears to be reciprocal to that of the other milk proteins . Although bovine colostrum contains high levels of lf (up to 5 mg / ml), these levels drop very rapidly as lactation proceeds, so that mature bovine milk normally contains 200485 g / ml lf or less [176, 177], depending on daily milk production and lactation stage . On the other hand, lf increases markedly in dry secretions, with the maximum concentrations attained after 3 - 4 weeks of involution (2030 mg / ml), nearly 100-fold greater than during lactation . The antibacterial effect of lf is enhanced by increased bicarbonates and low concentrations of the lf inhibitor, citrate, present during the dry period [25, 179, 180]. The increased lf concentration during involution strongly inhibits bacterial growth, and it has been suggested to contribute to the low number of naturally occurring imis during this early dry period . Lf contributes to mg immunity, immune modulation, and transcriptional activation of various molecules via several pathways . Principally, it exerts its bacteriostatic effect by competing with bacteria for available iron [182184] or by binding to bacterial surfaces [185, 186]. Studies have shown the ability of lf to damage the outer membrane of a broad range of gnb by interacting with the lipid a portion of lps and performing proteins in the outer membrane (porins), altering the integrity and permeability of the cell wall [185, 187, 188] and releasing lps, which sensitizes the cell to antibiotics . The binding interactions of lf to gpb are still not fully understood, although it has been shown that lf binds to specific receptors on the cell walls of several gpbspecies associated with imis, including s. uberis, s. agalactiae, and staph . Aureus [186, 191], as well as several coagulase - negative staphylococci (cns) (e.g., staph . One study showed that although the antagonistic effect of bovine lf on the adhesion and invasion of cns strains to mecs is weak, it significantly decreased intracellular replication rates . Bacteria with high iron requirements are susceptible to the bacteriostatic activities of lf . Among mastitis - causing bacteria, aureus, but streptococci are more resistant . For e. coli, it appears that igs are not required for lf to exert a potent bacteriostatic effect . Aureus and e. coli, although s. uberis challenged mg shows increased mrna expression of lf - related gene and stimulated the production of lf more than the other two organisms . In this context, some studies showed that bovine lf can enhance adhesion of s. uberis to host cells and increase invasiveness, suggesting that s. uberis has evolved to take advantage of the presence of lf [198, 199]. On the other hand, bovine lf has also been shown to inhibit many pathogenic bacteria, including listeria monocytogenes and enterotoxigenic e. coli [200, 201], and to increase the antibacterial effect of antibiotics synergistically against antibiotic - resistant gpb . As a major component of the specific granules of pmns, lf additionally contributes to both hydrogen peroxide - dependent and hydrogen peroxide - independent bacterial killing and promotes the adhesion and aggregation of pmns to the endothelial surface . Another aspect of lf's antibacterial activity is based on activation of the cs via the alternative pathway . Lf may also be important in ag - processing by cells of the reticuloendothelial system and in ab production . Additionally, lf increases nk cells activities and amplifies the inflammatory response and stimulates the phagocytic and cytotoxic properties of macrophages against invading pathogens [203, 205] such as staph . Aureus but still as a potent inhibitor of granulocyte - monocyte colony - stimulating factor [205, 206]. During mastitis, lf levels in lacteal secretions may increase 30-fold, corresponding to the severity of infection [111, 149, 176, 197, 207] and depending on the causative agent, as evidence has accumulated suggesting that different pathogens induce different lf - mediated responses from mecs . The dramatic increase in lf concentrations in milk during acute mastitis is consistent with the role of lf as an acute phase response (apr) protein in the mg, in accordance with the presence of apr elements in the lf gene promoter region . In experimentally induced e. coli mastitis, the mean concentration of bovine lf was 2 mg / ml, whereas in cns mastitis it was <0.2 mg / ml . The expression of lf by mecs in vitro has been shown to be greater upon exposure to s. uberis isolated from acute mastitis compared to s. uberis isolated from chronic mastitis . Based on the strong association between lf concentrations and mastitis occurrence, combined with the antibacterial properties of lf, it has been suggested that bovine milk lf plays an important role in defence against e. coli if concentrations exceed 200 g / ml milk [185, 188, 210], while it has little effect against other major pathogens such as staph . Tf is another iron - bp in the milk of dairy ruminants, although it is present at low concentrations . The concentration of tf ranges from 1.07 mg / ml in colostrum to 0.020.04 mg / ml in milk of third week postpartum compared to 4 - 5 mg / ml in serum [213, 214]. In contrast to rodents, pigs, and rabbits, which synthesise tf in the mgs at higher concentrations, tf in the milk of dairy ruminants is not synthesised in the udder and instead comes from blood serum, from transcytosis in the normal gland, and through exudation of plasma during mastitis . Like lf, tf can damage the cell membranes of gnb with the release of lps, thereby altering outer membrane permeability . During experimental e. coli imis in dairy cows, tf concentrations were found to rise even before lf elevation, reaching 1 mg / ml in milk and paralleling the concentrations of serum albumin . Lz (n - acetylmuramyl hydrolase) is one of the components of antibacterial system in milk [4, 216, 217]. Lz has inhibitory or lytic activity mainly against gpb and to lesser extent against gnb by cleaving the 1,4-glycosidic bond between n - acetylmuramic acid and n - acetyl - d - glucosamine residues in pg, thereby disrupting the cell wall [4, 177]. However, milk lz alone is not a significant component of the bmg defence, and only a few mastitis - causing bacteria are killed by lz . Nonetheless, lz can synergize with abs, complement, and lf [4, 25]. For example, the binding of cationic lf to the lta of gpb renders staphylococci more susceptible to lz [4, 218]. In healthy conditions, lz concentration of milk shows wide variation among species and is influenced by several factors such as the period of lactation, health, age, and the parity of animals [217, 219]. After parturition, the lz concentration shows successive increase, reaching the peak (0.72 mg / l milk) at the 7th day, and then begins to decrease after the 2nd week postpartum . Nevertheless, bovine and buffalo milk contain averages of only 0.0004 and 0.000152 g lz / l milk, respectively, compared to 10 mg lz/100 ml in human milk . A substantial rise (1050-fold) of lysosomal activity of milk has been recorded during mastitis among different dairy species [149, 217, 222, 223]. However, buffalo may exhibit thousandfold greater lz activity and moderately raised sccs in milk without showing signs of mastitis . Lz in milk may be derived from blood or locally synthesized, and during imi leucocytes appear to be the source of lz . Next to xanthine oxidase, lactoperoxidase is the most abundant enzyme in milk, constituting 0.5% of the total whey proteins (30 mg / l) [225, 226], and nearly similar concentration is present in colostrum [226, 227]. As for many other indigenous enzymes, the level of lactoperoxidase in milk increases with mastitis . Locally synthesised lactoperoxidase, in the presence of thiocyanate of hepatic origin and hydrogen peroxide of either bacterial or endogenous origins, can exert antibacterial properties against both gpb and gnb via the generation of activated oxygen products like hypothiocyanate, a reactive metabolite formed from the oxidation of thiocyanate that promotes bactericidal activity of phagocytes [5, 177]. It has been hypothesized that lactoperoxidase may have a synergistic antimicrobial function with lingual antimicrobial peptide (lap), one of the host defence peptides, in mgs of dairy cows . It is mainly located in the primary granules of neutrophils, and together with peroxide and halide it has an important role in the oxygen - dependent antimicrobial system of neutrophils and thus in defence against microorganisms [231, 232]. It catalyses the same peroxidase reaction as lactoperoxidase and additionally catalyses the oxidation of chloride, the product of which provides the bactericidal activity of this system . In vitro, this system has been shown to be potent against major common udder pathogens such as staph . Unfortunately, the antibacterial properties attributed to this system are only relevant during the dry period, whereas they were found to be completely inhibited with lactation, mainly due to milk proteins . Additionally, the levels of thiocyanate in udder are dependent on the specific dietary composition, and the low oxygen tension of the mg can inhibit the production of hydrogen peroxide, thus limiting the effectiveness of this antimicrobial system against different pathogens incriminated in mastitis . Complement system (cs). Complement is a collection of proteins that are produced in plasma mainly by liver as well as tissue macrophages and monocytes and for c3 a local synthesis in the mg was suggested . In support of the assumption of a local synthesis, experimental staph . Aureus and e. coli imis induced an increase of c3 mrna - expression in mecs . Complement components elicit their biological activities through complement receptors located on a variety of cells [7, 134, 233]. The cs is central to iis because it is intimately involved in initiation and control of inflammation, opsonisation of bacterial surfaces, attraction and recruitment of phagocytes (chemoattractants) (e.g., c3a and c5a cleavage fragments), recognition and ingestion of microorganisms by phagocytes (e.g., c3 and c4), and the killing of microorganisms, either directly or through cooperation with phagocytic cells [53, 134, 233235]. Nevertheless, it was also gradually appreciated that different proteins of the cs can influence the mgir and constitute an important bridge between iis and ais [53, 235, 236]. The lowest concentrations of complement are observed in the milk of healthy mgs during lactation, and higher levels are observed during late lactation period, in colostrum, and in mammary secretions obtained during involution, presumably due to the mobilisation of complement components by transudation from blood [237240]. The alternative pathway (ap) was found to be the sole complement pathway operating under these healthy conditions, while the classical pathway (cp) is not functional due to lack or lowered presence of c1q component compared to blood [53, 233, 241]. The ap operates with two consequences that are greatly involved in recruitment and activation of phagocytes, mainly pmns: (1) deposition of opsonic c3b and c3bi on bacteria and (2) generation of the proinflammatory fragment c5a [75, 234, 241, 242]. However, the milk from noninflamed mg is generally devoid of significant haemolytic and bactericidal complement - mediated activities, especially during the midlactation period [240, 241, 243, 244], due to strong anticomplement activity of milk on complement mediated hemolysis and the absence of the c1q component required for activation of the cp [5, 177, 244], except for some healthy periods of exerting elevated complement concentrations, where these activities exist in a weak but significant manner [237, 238, 240]. Nevertheless, this inhibitory activity does not involve c3b / c3bi deposition on bacteria or the generation of c5a by the ap . Unfortunately, the lack of haemolytic activity in bovine normal milk in the absence of inflammation adversely affects a very important function of the cs, opsonisation of bacteria by cs components, mainly c3 . However, it has been shown a noteworthy deposition of c3 complement fragments from neat milk of non inflamed mg on some particular udder bacteria, as mastitis - causing staph . Aureus, and s. agalactiae even in mid - lactating period by the activation of the ap . In addition, an enhanced chemiluminescence response of pmns against invading pathogens was noticed [53, 245]. On the other hand, the production of extracellular fibrinogen - bp by staph . Aureus was found to inhibit complement activation by blocking c3 deposition on the bacterial surface . In contrast, the highest concentrations of complement are observed in mastitic milk, presumably due to the mobilisation of complement components by transudation from blood [233, 238, 239]. Relative to the increase in complement concentrations during imi after recruiting plasma components, both bactericidal and haemolytic activities of cs are increased in inflamed mg, and the intensities of these activities correlate with intensity of the ir [233, 247, 248]. Gnb (e.g., e. coli) are sensitive to complement lytic action, while some gpb (e.g., staph . Aureus) are resistant, although all bacteria show susceptibility to the opsonizing action of c3b and c3bi fragments after activation of the ap [53, 233, 241, 247, 248]. Most cytokines have more than one function and often have redundant effects with other cytokines . Because of the high affinity of their receptors, cytokines are highly potent and can elicit biological responses even at femtomolar to nanomolar concentrations . Numerous cytokines (e.g., tnf-, ifn-, gm - csf, il-8, and il-12) have been detected in normal udders [251, 252], but during imi a complex upregulation of specific cytokines occurs depending on several factors . Cytokines act at both local and systemic levels during onset, progression, and resolution of inflammation [253, 254]. They provide relatively short - range communications between cellular immune components, thus linking the innate and adaptive immune branches, and this short communication range is important to limit their effects to the appropriate cells . Although cytokines play an essential role in the host response to infection, they can also have deleterious effects . Thus, there is a fine balance between the positive and negative effects of cytokines on the host that is dictated by the duration, amount, and location of their expression . A more detailed explanation of the roles of specific cytokines, chemokines, and growth factors in mg during imis is illustrated in table 2 . Due to their important contributions to the inflammatory process, several studies have illustrated cytokines benefits in immunotherapy of mastitis via enhancing mg immunity (e.g., interferons, mainly ifn-, il-2) [257261], their contributions to control or prevention / immunisation against mastitis pathogens especially e. coli or staph . Aureus (e.g., g - gsf, gm - csf, il-2, and ifn-) [262264], and their potentiating effects on response to treatment with antibiotics (e.g., il-1, il-2, and ifn-) [262, 265269]. The efficacy of recombinant cytokines (e.g., recombinant bovine il-2 [rboil-2]) in accelerating the involution of mg during dry period, and thus reducing the time in which the mg is particularly susceptible to infection, has been addressed [270, 271]. Intramammary infusion of il-2 elicits a considerable increase in scc, which is dominated by macrophages and plasma cells producing igg1, igg2, iga, and igm . On the contrary, the immunotherapeutic properties of rboil-1 are masked by the domination of proinflammatory nature of il-1 [251, 271, 272]. Chemokines are important molecules involved in migration and recruiting leukocytes into mg during imi, besides being involved in several immunoregulatory and inflammatory processes [39, 51, 151, 161]. According to arrangement of conserved n - terminal cysteine motifs, chemokines are grouped into 4 families: c, cc, cxc (subdivided into elr and elr), and cx3c . Members that contain the motif (elr) are potent chemoattractants for neutrophils and promoters of angiogenesis, whereas those that do not contain the motif (elr) are potent chemoattractants for mononuclear cells [151, 161]. Representatives of the elr cxc chemokines are structurally similar, including il-8/cxcl8 and ena-78/cxcl5 . Chemokines target neutrophils by interacting with one (e.g., cxcl1, cxcl2, and cxcl3) or two (e.g., cxcl8) receptors, cxcr1 and cxcr2, which are expressed by neutrophils of several species including cattle . Several molecules which mediate leukocytic trafficking are expressed in the mg tissues and mecs in response to lta from gpb (e.g., cxcl1, cxcl2, cxcl3, and cxcl8) or lps from gnb (e.g., rantes, cxcl5, cxlx8, mcp-1, mcp-2, and mcp-3) and can be also detected in milk [39, 51, 127, 135, 151, 152, 161, 165, 275]. The remarkable induction of chemokine gene expression by the epithelial cell lends strong support to its role in stimulating migration of leukocytes into the mg [39, 63]. Host defence peptides (hdps) are a large family of innate immune effector molecules . They are predominantly synthesised in pmns and epithelial cells [5658, 132, 276] and have been shown to be important in the resolution of local infection through both antimicrobial and immune - regulatory properties . Defensins are an important family of hdps in cattle owing to variable bactericidal properties [57, 276] and are considered as effector arm of iis as well as representing a putative link between iis and ais [58, 117, 132, 277]. Several -defensins, including lap, tracheal antimicrobial peptide (tap), and bovine neutrophil -defensins 1, 4, and 5 (defb1, defb4, and defb5), are expressed in mg tissues in both a constitutive and an inducible manner, or even excreted in milk, in response to bacterial challenge [17, 57, 117, 118, 131, 132, 150, 278]. Also, an increase in lap mrna expression in the bovine alveolar tissue at 192 h after milking upon involution has been declared . A broad spectrum of antimicrobial activities has been demonstrated for several bovine -defensins, in particular against several species that cause mastitis as staph . Pneumoniae, and ps . Aeruginosa [57, 118, 279]. The specific or adaptive immune system [ais] recognises specific determinants of a pathogen mainly via abs molecules, macrophages, and several lymphoid populations, which subsequently facilitate selective elimination [7, 27]. Because of the memory function of certain lymphocytes, specific irs can be augmented by repeated exposure to a pathogen . Immunoglobulins (igs) are the most important specific soluble humoral factors in adaptive immune defence, linking various parts of the cellular and humoral immune system, and they constitute the main component of the ais present in colostrum and milk [33, 280]. They are able to prevent adhesion of microbes to tissues, inhibit bacterial metabolism, agglutinate bacteria, augment opsonisation and phagocytosis of bacteria, kill bacteria through activation of complement - mediated bacteriolytic reactions, and neutralize toxins and viruses [281, 282]. Igs account for up to 7080% of the total protein content in colostrum (20150 g / l) to confer passive immunity to newborns, whereas in milk they account for only 1 - 2% of total protein (0.51 g / l) [31, 226, 227, 247]. However, ig concentrations in the bmg vary during the lactation cycle, and an increase occurs at the end of lactation igs in milk may be blood - derived or may be produced in situ by ag - activated plasma cells, which traffic to the udder from the blood [77, 284] mediated by chemokines produced locally during imi . The mg plays an active role in regulating the levels of different igs present in colostrum and milk, although the mammary epithelium itself does not synthesise igs . The majority of igs are transported into mammary secretions via specialised receptors (selective receptor - mediated intracellular route). There are four different classes of igs that play dominant roles in mg defence against bacterial pathogens: igg1, igg2, igm, and iga (table 3). Functionally, igg1, igg2, and igm act as opsonins and facilitate phagocytosis by pmns and macrophages [49, 247], while iga is thought to play roles in toxin neutralisation and bacterial agglutination, thereby hindering bacterial spread and colonisation [247, 284]. Bovine colostrum contains igg1, iga, and igm in concentrations exceeding those of blood . The colostrum / blood ratios for igg1, iga, and igm are approximately 4: 1, 13: 1, and 2: 1, respectively . The most abundant ig class in bovine milk and colostrum is igg1 [287289], while igg2 increases substantially during inflammatory states . In contrast, iga and igm are present at much lower concentrations in healthy bmgs [286, 290]. As mentioned, both innate and adaptive irs are coordinating and operating together in very complicated pathways to provide the optimal defence against infections . Pr and ag presentation by innate immune components initiates a proinflammatory response with quantitative and qualitative changes of different immune components in a complex manner . Different cytokines and chemokines appear to play essential roles in this process by acting through their variable immunoregulatory roles, thus coordinating mgir . Once bacteria contact leukocytes in the milk or the lining mg epithelium accompanied by exerting various virulence mechanisms and liberating toxins, irritation or even damage to mg epithelium and, thereby inflammatory products from damaged epithelium induce locally located leukocytes and healthy mg epithelium to release several chemoattractants for the migration and recruitment of both bone marrow and circulating immune cells into the mg environment, mainly neutrophils [39, 63, 151, 255, 291, 292]. Proinflammatory cytokines (il-1, il-6, and il-17) as well as il-8 and tnf- are the main effectors to initiate the inflammatory responses at both local and systemic levels [121, 162, 291, 293, 294]. They act in collaboration with tgf-, gm - csf, and several chemotactic factors (e.g., c3a and c5a complement fragments, leukotriene b4, paf, eicosanoids [as prostaglandin - f2], oxygen radicals, and apps) to potently trigger circulation - into - mg migration of neutrophils via induction of vascular endothelial adhesion molecules expression (mainly for e- and p - selectins), thereby promoting neutrophil transendothelial migration to the infected foci [291, 295, 296]. As a consequence, enhanced expression and adhesiveness of another neutrophil adhesion molecule, mac-1 (known also as cd11b / cd18), occur, which allows neutrophils to bind tightly to activated endothelium in collaboration with another endothelial adhesion molecule, icam-1 . This adhesive interaction allows neutrophils to migrate along the endothelial surface and into mg tissues up a concentration gradient of chemoattractants; one of the most potent with long - lasting effect is il-8 [75, 256, 291, 292, 297]. It is thus clear that the migration of immune cells to mg is not a random process and a collaboration of several molecules, chemoattractants, selectins, and integrins is greatly needed to regulate chemotaxis . Il-17 has been suggested to enhance leukocytic recruitment into mg via regulating il-8 expression and enhancing expression of several chemokines targeting not only neutrophils (cxcl3 and cxcl8) but also mononuclear leucocytes (ccl2, ccl20) [121, 162, 294]. Leukocytes that freshly migrated express greater numbers of cell surface receptors for igs and complement and are more phagocytic than their counterparts in blood . Stimulation of microbicidal activities of various leukocytes located inside infected tissues is mainly regulated by certain proinflammatory cytokines (table 2). The activation status and enhancing functions of neutrophils are stimulated mainly by il-1, il-8, ifn-, tnf-, and g - csf; macrophages by il-12, m - csf, and gm - csf; and nk cells by il-2 and il-12 . Meanwhile, b - lymphocyte differentiation is driven mainly by il-2 and il-6 [27, 256, 298305]. Systematically, several physiologic responses occur as a result of imi: (1) generation of febrile response [293, 296, 301, 306, 307], (2) alterations in metabolism and gene regulation in the liver, resulting in elevation of apps levels as well as serum cortisol levels, and (3) changes in vascular permeability, tone, and activation [257, 293, 296, 309]. Some cytokines such as tnf-, il-1, and il-6 are responsible for generation of febrile response, and the latter one specifically contributes to the great extent for regulation of the apr through the synthesis of app . Il-17 greatly synergizes to generation of inflammatory reactions via enhancing production of il-6 [121, 162], il-8, and gro and the expression of inflammatory cytokines tnf- and il-1 (table 3). Likewise, tgf- has been shown to have a potential role in mediating iir and promoting inflammation by upregulating the production of prostaglandins and synergistically enhancing the effects of il-1 and tnf- [311313]. Additionally, tgf- has the ability to directly stimulate il-8 and to induce expression of antimicrobial peptides . Ags from invading mastitis - causing bacteria are processed mainly within macrophages and b - lymphocytes and appear on the membranes in association with mhc class i or ii; thus they can be recognised by different lymphocytes [27, 45, 6769]. Ifn- greatly contributes to upregulating of the mhc - i expression and mhc - ii ag presentation, thus increasing cytotoxic t - cell recognition for foreign peptides, and inducing cd4 + t - cell activation [256, 303]. Upon recognition of ag - mhc class ii on b - lymphocytes or macrophages, cd4 + cells are activated and produce cytokines that have roles in the activation and polarisation of b- and t - lymphocytes, macrophages, and various other cells that participate in the ir [4, 7, 27, 81]. Depending on the repertoire of cytokines produced, the t - helper cell response can facilitate either a cell - mediated (th1 type) or a humoral (th2 type) ir . Il-2 and ifn- are the major cytokines secreted by th1 cells, and they stimulate cellular responses against intracellular pathogens . In contrast, il-4, il-5, and il-10 are secreted by th2 lymphocytes; these cytokines promote humoral immunity and regulate both macrophage functions and the activity of cytokine production [27, 316]. On the other hand, inflammatory - inducer ifn- and regulatory il-4 are the main cytokines produced by cd8 + cytotoxic and cd8 + suppressor t - cells, respectively . Inflammatory cytokines produced by t - cells in turn induce the proliferation and differentiation of the b - lymphocytes into either ab - producing plasma cells or memory cells [27, 49], and some of them are responsible for increasing fc receptors for igg2 . Synergistically, activated macrophages release chemotactic signals for neutrophils, thereby amplifying the inflammatory response . Macrophages secrete prostaglandins and leukotrienes that augment local inflammatory processes [75, 317] as well as specific cytokines that are known to regulate t - cell differentiation, mainly il-12 . Regulation of polarising t - helper subsets into either th1 or th2 is the main axis on which some regulatory cytokines (il-4 and il-12) work . Il-12 contributes to the ir by favouring the polarising cd4 + t - cells towards th1 responses and enhancing the generation of cytotoxic - ifn- producing cd8 + cells and also acts as a growth factor for nk cells and an inducer of their cytotoxic activities [254, 318, 320]. Thus, it contributes to the production of ifn- from lymphocytes as well as nk cells [254, 318]. In contrast to il-12, il-4 favours the development of th2 subsets and exerts a clear inhibitory effect on ifn- production . Compared to the anti - inflammatory il-10 cytokine, the inhibitory effect of il-4 on monokine synthesis is lesser . Based on the effects of il-4 and il-12 on polarisation of t - cell subsets, the early preference expressed in the ir is greatly dependent on the balance between il-12 and il-4 . Resolution of the imi is mediated by upregulation of several inflammatory - antagonist cytokines, including il-10, and tgf-, and in corporation to anti - inflammatory effects elicited by il-6 and il-4 . Il-10 is the most potent contributor to this process as it downregulates both the generation of all subtypes of t - helper cells and the production of proinflammatory cytokines, chemokines, and eicosanoids by monocytes, macrophages, and neutrophils [85, 253, 291, 324]. Il-10 potently inhibits the ability of macrophages to stimulate th1 cells to produce cytokines, principally ifn-, and has an inhibitory effect on lps - induced production of il-1, il-6, and tnf- by macrophage cell lines . In cooperation with il-6, il-10 also upregulates il-1 receptor antagonist and soluble tnf receptors, impairing the ability of the proinflammatory cytokines il-1 and tnf-, respectively, to exert their effects . In contrast, il-10 does not inhibit cytokine production by b - lymphocytes nor does it affect the ability of different phagocytes to stimulate cytokine production by th2 cells . Like il-10, the major role of tgf- is to suppress the irs, although some proinflammatory properties have been reported [325, 326]. The anti - inflammatory role of tgf- is exerted through its ability to (1) inhibit macrophage production of chemokines, proinflammatory cytokines, nitric oxide, and ros; (2) limit ifn- production; (3) increase expression of the il-1 receptor antagonist; and (4) enhance macrophage clearance of bacteria and cellular debris [325, 326]. The repair of damaged mg epithelium is mainly mediated by tgf-, which promotes epithelial proliferation and tissue remodelling . Tgf-, on the other hand, promotes extracellular matrix deposition, fibrosis, and scarring . Thus, restoring healthy structure / homeostasis and scar formation is controlled by the balance between the two tgf types . During the whole process, altered cells are mainly removed by macrophages and cytotoxic t - cells, which recognise and eliminate altered self - cells via ag presentation, with the help of t - cells and nk cells, which mediate cytotoxicity with variable involvement of mhc molecules [27, 83, 84]. In addition to investigating the pathogen virulence mechanisms and the resulting histopathological changes, study of the immunological profile of the mg against a particular pathogen will help provide a better understanding of the nature, rate of development, and severity of mastitis caused by such pathogen and is considered a prerequisite to the development of novel and effective diagnostics and therapeutics . The sensitivity and responsiveness of the mg in terms of specific immune factors varies greatly against different bacteria [37, 131, 134, 165, 196, 275, 329331] and their associated toxins [125, 148, 332, 333]. Thus, the high sensitivity of the mg to some mastitis pathogens results in a robust ir, invoking an acute response to infection and likely predisposing to rapid elimination of the invading bacterium with proper host immunity and animal management . Aureus and some cns may result in subclinical or chronic imis as a result of poor responsiveness of mg immunity . In attempt to understand the pathogenesis of imis caused by different bacterial species, several studies have assessed the mammary irs towards particular mastitis pathogens, as shown in table 4 . Unfortunately, most studies regarding mammary irs towards particular pathogens in bovines have focused on staph . Most bacterial species causing coliform mastitis elicit a marked acute inflammatory response in comparison to staph . However, the iir varies among different mastitis - causative species . A strong tnf- response to lps was found to be central to the earliest initiation of mgirs and in the development of pyrexia associated with coliform mastitis, endotoxic shock in per acute form [127, 334, 335], leukopenia in peripheral blood, and concurrent increases in milk leukocytes [62, 336, 337]. The powerful chemotaxis and recruitment of leukocytes, mainly pmns, and robust production of a wide variety of cytokines reflect the mg's sensitivity to and response against e. coli compared to staph . Aureus [114, 127, 131, 165, 239, 307, 311, 329, 338, 339]. Aureus bacteria were used to stimulate isolated mecs, expression of tnf-, il-1, il-6, and il-8 was greater in cells stimulated by e. coli . Experimental studies conducted on ovines revealed similar results regarding mgir towards e. coli, and increases in leukocyte recruitment (mainly pmns) and proinflammatory cytokine levels (including il-1, il-8, and tnf- [255, 340]) have been reported in response to either e. coli or its endotoxin . Occasional increases in gm - csf and ifn- have also been shown [255, 340]. These data explain why e. coli imis follow acute form and why these imis may resolve spontaneously within a short period as declared in previous studies [341, 342]. Depending on the levels of chemoattractants and proinflammatory, inflammatory, and regulatory cytokines, the iir is also robust towards kl . Aeruginosa, reflecting the strong mgir towards these bacteria . Against s. marcescens, however, the mgir is comparatively modest [337, 343]. The number of bacteria isolated from mgs of s. marcescens - infected cows as well as sccs dropped precipitously 24 h and 48 h following infection (pi), respectively, which could reveal elimination of bacterium by mg immune system . Though several studies reported strong systemic responses and clinical signs in animals infected with several species of gnb [343345], the accurate investigations focused on the iir towards gnb other than e. coli are considered rare and mostly experimental . Further in vivo and in vitro studies are required . Aeruginosa infection in humans have revealed that secretion of exotoxin a, exoenzyme s, and elastase by such bacterium inhibits monocyte and neutrophil chemotaxis and respiratory burst, thus altering the ir [346, 347]. Unlike the case with e. coli, mgir against staph . Aureus was found to be insufficient to eliminate the bacterium, allowing persistence of infection and eventually leading to subclinical or chronic patterns of imi . Aureus imi induced strongly il-8 and tnf- gene expression in the mg tissue as well as strong activation of nf-b in mecs and triggered a rapid early expression of -defensin, tlr2, and tlr4 in the inoculated mg and lymph nodes, while impaired proinflammatory activation was paralleled by a complete lack of nf-b activation in mecs challenged by staph . Aureus or lta, and only expression of -defensin occurred later than 48 h in inoculated quarters with staph . Aureus . In a contradictory study, although all 10 tlrs' and nod 1 - 2 expression was upregulated in mg tissues challenged with staph . Aureus, with tlr8 having the least expression in comparison to the other prrs, immunohistochemistry analysis of tissues from both staph . This variability in the expression of prrs could be attributed to different strains, but in all conditions how the ir of mg towards staph . Aureus is being translated remains as a crucial point . In the last study, expression of proinflammatory cytokines (il6, il17a, and il8) and anti - inflammatory cytokine (il10) meanwhile, the production of these cytokines varied among studies (table 4), which reveal the complexity of mgir towards staph . Aureus and illustrate that mgir could be modulated due to pathogen factors suppressing the production of these cytokines . Reduced expression and induction of some inflammatory cytokines, including tnf- by lta, the principle immune - stimulator of gram - positive cell wall [17, 125, 126, 163], impaired activation of nf-b and reduced expression and production of chemokines (il-8 and rantes) [134, 165], involved in recruiting leukocytes, which may reflect why the sccs are not elevated in mgs challenged by staph . It has been hypothesized that decreased expression of immune - modulator -1 acid glycoprotein in the alveolar region of mg experimentally challenged with staph . Aureus may inhibit the early recruitment of neutrophils to the mg and could be a result of modulation of the host's ir by the pathogen in order to enhance survival . Also, since it has been suggested that tgf- was found to block the tlr signalling, the expression of tgf- in imi caused by staph . Additionally, various studies have shown that staphylococcal enterotoxins (sea, seb, sec, and toxic shock syndrome toxin-1) act as super ags by activating specific types of t - lymphocytes (mainly cd8 + suppressors) and stimulating release of specific cytokines [332, 333, 349]. The presence of high numbers of suppressor cd8 + t - cells compared to cd4 + t - cells significantly suppresses lymphocyte irs and recruitment [78, 86]; and in addition to unstable expression and release of inflammatory inducers (il-1, il-8, and tnf-) [17, 86, 114, 131, 134, 165, 239, 329], compromised expression and release of inflammatory cytokines (depressed il-2 and c5a levels) [17, 86, 114, 239, 350] and unstable release of anti - inflammatory il-10 could greatly reflect and provide explanation for the suppressive nature of mastitis - causative staph . Aureus and why imis caused by such bacterium do not usually undergo resolution and follow subclinical or chronic patterns with persistence of the pathogen . In addition to causing a marked increase in scc, cns can persist similar to staph . Unfortunately, few studies have investigated the bovine mgir against cns, and the majority were conducted in ovines or investigated only few aspects of mgirs . In both bovines and ovines [355, 356], the imis caused by staph . Simulans were associated with a decline in leukocyte counts for a short period after initiation of the inflammatory process and the absence of a marked systemic cytokine response . However, some proinflammatory cytokines, including il-1, il-8, and tnf-, were elevated in milk [354356]. These observations likely reflect the unsuccessful combat of mg against the invading bacterium and that the sensitivity or responsiveness of mg to inflammatory signals decreased as infection progressed . In experimentally induced ovine imi by staph . Epidermidis, counts of leukocyte subsets (including cd4, cd8, wc1, and mhcii) temporarily decreased and then subsequently increased, while the expression of some adhesion molecules (cd11b and cd18) on pmns decreased after 24 h . Chromogenes as measured by systemic signs, scc, milk yield, bacterial counts, and some inflammatory indicators (including enzymatic activity and app levels), but cellular and other soluble factors of mg immunity have not been studied . Xylosus have been shown to cause cellular responses in both ovines [358, 359] and caprine udders, as indicated by increased scc and leukocyte counts in milk and severe infiltration of mg tissues with mononuclear cells and neutrophils on histopathological investigations . Unfortunately, few studies have focused on mgirs against streptococci, despite their substantial contribution to mastitis . To our knowledge dysgalactiae, and few studies were conducted on s. uberis [196, 209, 336, 337, 362364]. Although not completely comprehensive, mgir towards s. dysgalactiae subsp . Dysgalactiae in one study was represented by increased expression of tlr4 plus release of various cytokines (il-1 and tnf-). Most experimental challenge studies showed that mgir against s. uberis was not sufficient to allow successful elimination of the bacterium, although increased expression and production of several inflammatory mediators and antimicrobial components as il-1, il-8, il-10, il-12, ifn-, tnf-, scd14, lps - bp, c5a, and lf have been declared during imis caused by s. uberis . In s. uberis - experimentally infected cows, both numbers of bacteria in milk and sccs remain highly elevated for long time pi, compared to s. marcescens infected cows . Neither the influx of pmns into mg infected with s. uberis [336, 337, 365] nor intracellular engulfment by macrophages [70, 366], have resulted in effective reduction in the number of bacteria, and in contrast intracellular replication of s. uberis inside macrophages increased . Additionally, it has been accumulated that mgir towards s. uberis is very complex, and different strains of s. uberis can elicit different irs . Some studies showed that strain - specific pathogenicity greatly modulates the ir, implying that pathogen factors rather than host factors play an important role in modification of mgir [209, 364]. Contradictory results have been obtained in different study when a strain of s. uberis used to induce cm in vivo failed to cause a change in the mrna levels of the immune - related genes by bovine mecs in culture, suggesting that the expression of immune - related genes by mecs may be initiated by host factors and not s. uberis . However, in the same study, challenging bovine mecs with different s. uberis strains resulted in an increase in the mrna expression of a subset of the immune - related genes measured . Also, mgirs towards different strains of s. uberis isolated from different imi cases of different intensities varied . Expression of il-1 and il-8 from mecs in vitro has been shown to be greater with exposure to living and heat - inactivated s. uberis isolated from acute mastitis than s. uberis isolated from chronic mastitis . More interestingly, a strain of s. uberis that induced acute mastitis in vivo caused twofold and fourfold higher expression of il-8 and il-1, respectively, in isolated mecs in vitro than a strain isolated from a case of chronic mastitis . Similar results were obtained in a separate study, indicating that the severity of mastitis induced by different s. uberis strains in vivo can be reflected at the level of the mgir in vitro . In another in vitro study, heat - inactivated s. uberis did not trigger an ir from mecs, although inactivated staph . Aureus did, despite the fact that both bacteria are gram - positive and contain lta in their cell walls . Continued to particularity of mgir towards s. uberis, an emergence of s. uberis - specific bactericidal t - cells in the mgs of cows after infection or environmental exposure to s. uberis has been documented, suggesting that these specific cells may play a role in control of imi caused by this bacterium . To the best of our knowledge, no studies have been performed to assess the mgir of bovines to the major contagious bacterium s. agalactiae . In a study of s. agalactiae imi in mice, the ir manifested as a massive infiltration of mg by pmns and the release of il-1, il-6, and tnf- in the first 72 h pi; these cytokine levels decreased concurrently with increased levels of il-12 and il-10 . Results obtained from different studies investigated the mgir towards different mastitis pathogens, demonstrating the complexity of the mgir to an infecting pathogen and indicating that a coordinated response exists between the resident, recruited, and inducible immune factors . In recent years, there has been considerable expansion of our knowledge concerning host mg immune defence against bacterial infections . This defence involves sophisticated mechanisms for detecting various invading bacteria and combating them by the innate and acquired irs . To improve dairy animal resistance against imis, further investigation concerning mg immunology should focus on the following: (1) enhancement of immune functions or at least the maintenance of these functions at normal levels under various lactating and nonlactating conditions, especially during periods of immune suppression; (2) clarifying the roles of specific mammary immune cells, primarily lymphocytes, and in particular the roles of nk cells and cells, which are not fully defined; (3) in vivo and in vitro investigation of mgirs against certain common bacteria in bovines, including s. uberis, s. dysgalactiae, s. agalactiae, coliforms other than e. coli, and cns because most research studies concerning mgirs have focused on staph . Aureus and e. coli, as most studies using other pathogens have involved experiments in ovines and focused on cytokine levels only without detailing the cellular responses; (4) clarifying the roles of certain chemokines as rantes and cytokines such as il-17, tgf, and csf in mg, as well as lf effect against gpb because its role is not clearly understood; and (5) changes of leukocytes population in mgs of ovines and caprines during imis.
Stearoyl - coa desaturase (scd1) is a rate - limiting enzyme that catalyzes the synthesis of monounsaturated fatty acids, which are components of triglycerides, wax esters, cholesteryl esters and membrane phospholipids 1 . The gene is highly expressed in white adipose tissue, brown adipose tissue, meibomian gland, harderian and preputial gland under normal dietary conditions 2 . However, this gene is also highly expressed in skeletal muscle from extremely obese humans 3 and from obese insulin - resistant zucker diabetic fatty rats 4 . In the human study, the authors observed that elevated scd1 expression is associated with decreased fatty acid oxidation, increased triacylglycerol synthesis and increased monounsaturation of muscle saturated fatty acids . In the rat study, the authors revealed impairments of glucose uptake and of different steps of the insulin signaling cascade in a muscle cell line that overexpresses scd1 and accumulates lipids . In addition, scd1-/- mice with a targeted disruption of the scd1 gene have reduced body adiposity, increased insulin sensitivity, and are resistant to diet - induced weight gain 5 . These data suggest that elevated expression of scd1 in skeletal muscle contributes to abnormal lipid metabolism and progression of obesity and type 2 diabetes . However, associations of the gene with obesity and type 2 diabetes - related phenotypes in the human subjects have been reported in only two studies; therefore results are inconsistent . In 1143 elderly swedish men involved in the uppsala longitudinal study of adult men, warensj and colleagues 6 reported that subjects homozygous for the rare alleles of rs10883463, rs7849, rs2167444, and rs508384 had decreased body mass index (bmi) and waist circumference and improved insulin sensitivity . In particular, the rare allele of rs7849 demonstrated the strongest effect on both insulin sensitivity (regression coefficient (beta)=1.19, p=0.007) and waist circumference (beta=-4.4, p=0.028), corresponding to 23% higher insulin sensitivity and 4 cm less waist circumference . In contrast, liew and colleagues 7 found that there was no association detected between scd1 gene (at the allele, genotype or haplotype level) and diabetes - related traits including bmi and waist - to - hip ratio in a primary set of 608 diabetic subjects and 600 control subjects as well as in a replicate set of 350 young - onset diabetic patients and 747 controls . Therefore, we decided to pursue a cross - species study using cattle as a model organism and determine if the scd1 gene is involved in muscle fat metabolism . A wagyu x limousin reference population was used in the present study, including 6 f1 bulls, 113 f1 dams and ~250 f2 progeny . The breeding program evidence has shown that the japanese wagyu (black) breed of cattle has greater muscle fat deposition and softer fat than the limousin breed (4.5% vs. 3.1% intramuscular fat content measured for the former trait and 40.2c vs. 37.8c melting point measured for the latter trait) 11 . In particular, wagyu fat has considerably less saturated and more unsaturated fatty acids resulting in much higher unsaturated / saturated ratios (1.9) compared with the other breeds (1.0) 12 . Certainly, such a wagyu x limousin reference population should be a unique resource to examine association between scd1 gene and fat metabolism phenotypes that might provide new insights into understanding genetic aspects of obesity and type 2 diabetes . As indicated above, fat accumulation and monounsaturation of saturated fatty acids in skeletal muscle are pathobiological contributors to insulin resistance in patients with these conditions . Beef marbling score (bms), subcutaneous fat depth (sfd) and percent kidney - pelvic - heart fat (kph) were assessed by a trained evaluator . Bms was determined at the interface of the 12th and 13th ribs and was evaluated by subjective comparison of the amount of fat within the longissimus muscle with photographic standards (national livestock and meat board 1981). Sfd was recorded at the 12th rib at a point three - fourths the width of the longissimus muscle from its chine bone end . The amount of kidney, pelvic, and heart (kph) fat was estimated and recorded as a percentage of carcass weight . Briefly, the entire core of the longissimus dorsi was sampled (i.e., devoid of trim fat and extraneous muscles) by dicing the muscle into 1.0-cm cubes, freeze - dried (genesis 25 freeze dryer; the virtis co.) and then ground and homogenized using a home - style electric coffee grinder . Approximately 150 mg of dried muscle was weighed, in duplicate, into 16 125-mm screw - capped tubes that contained 1.0 mg of tridecanoic acid as an internal standard, and then subjected to direct saponification 13 . Samples were reacted with 4.0 ml of 1.18 m koh in ethanol at 90 c along with frequent vortex - mixing until the sample was completely dissolved . After about 45 min, tubes were cooled, 2.0 ml of water was added and cholesterol (chol) was extracted with 2.0 ml of hexane that contained 0.1 mg / ml of stigmasterol as an internal standard for the cholesterol assay; the hexane phase was transferred to gas liquid chromatography (glc) vials and sealed . One millilitre of concentrated hcl was added to the original tubes and fatty acids were extracted in 2.0 ml of hexane for fatty acid methyl ester (fame) preparation using methanolic hcl as a catalyst . Based on these measurements, we calculated: saturated fatty acids (sfa) = myristic + pentadecanoic + palmitic + heptadecanoic + stearic, monounsaturated fatty acids (mufa) = myristoleic + pentadecenoic + palmitoleic + heptadecenoic + oleic + vaccenic and polyunsaturated fatty acids (pufa) = linoleic + linolenic . The relative amount of sfa, mufa and pufa was defined as rsfa = (sfa / total fat in 100 g dry meat) x 100%, rmufa = (mufa / total fat in 100 g dry meat) x 100% and rpufa = (pufa / total fat in 100 g dry meat) x 100%, respectively . Three stearoyl - coa desaturase activities were estimated as r1 = (14:1/14:0) x 100%, r2 two pairs of primers (forward, 5'-aac tcc tag aga ttc ccc cac agg-3'/reverse: 5'-acc tcc att cca gtc ctg act cac-3' and forward, 5'-gca gat cta cac atc agc gca tct-3'/reverse, 5'-ttc tcc tcg gct tct ctt aca tcg-3') were designed to target the promoter region and two pairs of primers (forward, 5'- gag cag ggc agt cct aaa act caa / reverse, 5'-ggc aac ctg gct aat tct tcc tct-3' and forward, 5'- cta cca ctg tgc cac tga ctt gct-3'/reverse, 5'-aaa aag ctc aga cac agg gca atc-3') to target the 3'utr of the bovine scd1 gene . Approximately 50 ng of genomic dna each from six wagyu x limousin f1 bulls was amplified in a final volume of 10 l that contained 12.5 ng of each primer, 150 m dntps, 1.5 mm mgcl2, 50 mm kcl, 20 mm tris - hcl and 0.25 u of platinum taq polymerase (invitrogen, carlsbad, ca). The pcr conditions were carried out as follows: 94c for 2 min, 35 cycles of 94c for 30 sec, 60c for 30 sec and 72c for 30 sec, followed by a further 5 min extension at 72c . Pcr products were then sequenced on an abi 3730 sequencer in the laboratory for biotechnology and bioanalysis (washington state university) using a standard protocol and polymorphisms were identified . Three snps in the 3'utr of the bovine scd1 gene were genotyped on a sequenom iplex assay using services provided by the children's hospital oakland research institute, oakland, california . The haploview program was used to determine the degrees of hardy - weinberg equilibrium within each marker and linkage disequilibrium among different markers 14 . The phenotypic data for all measurements described above were analyzed in a fixed effects model that included the effects of year, gender, age at harvest (linear) and the genotype for each marker using the glm (general linear model) procedure of sas v9.1 (sas institute inc . Pair - wise comparisons of least squares means were performed using a protected t - test . Additionally, quantitative transmission disequilibrium test (qtdt) 15 was performed to further examine the association between markers and adjusted obesity - related phenotype data . A wagyu x limousin reference population was used in the present study, including 6 f1 bulls, 113 f1 dams and ~250 f2 progeny . The breeding program evidence has shown that the japanese wagyu (black) breed of cattle has greater muscle fat deposition and softer fat than the limousin breed (4.5% vs. 3.1% intramuscular fat content measured for the former trait and 40.2c vs. 37.8c melting point measured for the latter trait) 11 . In particular, wagyu fat has considerably less saturated and more unsaturated fatty acids resulting in much higher unsaturated / saturated ratios (1.9) compared with the other breeds (1.0) 12 . Certainly, such a wagyu x limousin reference population should be a unique resource to examine association between scd1 gene and fat metabolism phenotypes that might provide new insights into understanding genetic aspects of obesity and type 2 diabetes . As indicated above, fat accumulation and monounsaturation of saturated fatty acids in skeletal muscle are pathobiological contributors to insulin resistance in patients with these conditions . Beef marbling score (bms), subcutaneous fat depth (sfd) and percent kidney - pelvic - heart fat (kph) were assessed by a trained evaluator . Bms was determined at the interface of the 12th and 13th ribs and was evaluated by subjective comparison of the amount of fat within the longissimus muscle with photographic standards (national livestock and meat board 1981). Sfd was recorded at the 12th rib at a point three - fourths the width of the longissimus muscle from its chine bone end . The amount of kidney, pelvic, and heart (kph) fat was estimated and recorded as a percentage of carcass weight . The fatty acid composition was determined using a method described previously by rule et al . 13 . Briefly, the entire core of the longissimus dorsi was sampled (i.e., devoid of trim fat and extraneous muscles) by dicing the muscle into 1.0-cm cubes, freeze - dried (genesis 25 freeze dryer; the virtis co.) and then ground and homogenized using a home - style electric coffee grinder . Approximately 150 mg of dried muscle was weighed, in duplicate, into 16 125-mm screw - capped tubes that contained 1.0 mg of tridecanoic acid as an internal standard, and then subjected to direct saponification 13 . Samples were reacted with 4.0 ml of 1.18 m koh in ethanol at 90 c along with frequent vortex - mixing until the sample was completely dissolved . After about 45 min, tubes were cooled, 2.0 ml of water was added and cholesterol (chol) was extracted with 2.0 ml of hexane that contained 0.1 mg / ml of stigmasterol as an internal standard for the cholesterol assay; the hexane phase was transferred to gas liquid chromatography (glc) vials and sealed . One millilitre of concentrated hcl was added to the original tubes and fatty acids were extracted in 2.0 ml of hexane for fatty acid methyl ester (fame) preparation using methanolic hcl as a catalyst . Based on these measurements, we calculated: saturated fatty acids (sfa) = myristic + pentadecanoic + palmitic + heptadecanoic + stearic, monounsaturated fatty acids (mufa) = myristoleic + pentadecenoic + palmitoleic + heptadecenoic + oleic + vaccenic and polyunsaturated fatty acids (pufa) = linoleic + linolenic . The relative amount of sfa, mufa and pufa was defined as rsfa = (sfa / total fat in 100 g dry meat) x 100%, rmufa = (mufa / total fat in 100 g dry meat) x 100% and rpufa = (pufa / total fat in 100 g dry meat) x 100%, respectively . Three stearoyl - coa desaturase activities were estimated as r1 = (14:1/14:0) x 100%, r2 = (16:1/16:0) x 100% and r3 = (18:1/18:0) x 100% . Two pairs of primers (forward, 5'-aac tcc tag aga ttc ccc cac agg-3'/reverse: 5'-acc tcc att cca gtc ctg act cac-3' and forward, 5'-gca gat cta cac atc agc gca tct-3'/reverse, 5'-ttc tcc tcg gct tct ctt aca tcg-3') were designed to target the promoter region and two pairs of primers (forward, 5'- gag cag ggc agt cct aaa act caa / reverse, 5'-ggc aac ctg gct aat tct tcc tct-3' and forward, 5'- cta cca ctg tgc cac tga ctt gct-3'/reverse, 5'-aaa aag ctc aga cac agg gca atc-3') to target the 3'utr of the bovine scd1 gene . Approximately 50 ng of genomic dna each from six wagyu x limousin f1 bulls was amplified in a final volume of 10 l that contained 12.5 ng of each primer, 150 m dntps, 1.5 mm mgcl2, 50 mm kcl, 20 mm tris - hcl and 0.25 u of platinum taq polymerase (invitrogen, carlsbad, ca). The pcr conditions were carried out as follows: 94c for 2 min, 35 cycles of 94c for 30 sec, 60c for 30 sec and 72c for 30 sec, followed by a further 5 min extension at 72c . Pcr products were then sequenced on an abi 3730 sequencer in the laboratory for biotechnology and bioanalysis (washington state university) using a standard protocol and polymorphisms were identified . Three snps in the 3'utr of the bovine scd1 gene were genotyped on a sequenom iplex assay using services provided by the children's hospital oakland research institute, oakland, california . The haploview program was used to determine the degrees of hardy - weinberg equilibrium within each marker and linkage disequilibrium among different markers 14 . The phenotypic data for all measurements described above were analyzed in a fixed effects model that included the effects of year, gender, age at harvest (linear) and the genotype for each marker using the glm (general linear model) procedure of sas v9.1 (sas institute inc ., pair - wise comparisons of least squares means were performed using a protected t - test . Additionally, quantitative transmission disequilibrium test (qtdt) 15 was performed to further examine the association between markers and adjusted obesity - related phenotype data . A full - length cdna sequence of the bovine scd1gene was retrieved from the genbank database with a total of 5,108 bp including a 5'utr of 144 bp, a reading frame of 1,080 bp and a long 3'utr of 3,884 bp (nm_173959.4), respectively . However, an est sequence (dy209631) further extends the 5'utr to 323bp, thus producing a full - length cdna sequence of 5,287 bp for the bovine gene . A blast search using this full - length cdna sequence as a query obtained three genomic dna sequences (aafc02116637.1, aafc02151622.1 and aafc02181333.1) from the genbank database . Alignment between cdna and these genomic dna sequences indicated that, like human, the bovine gene has six exons (figure 1a). Four pairs of primers were designed to target both the promoter and 3' utr for mutation detection, but direct sequencing of pcr products on 6 f1 bulls revealed three snps only in the 3'utr (aafc02181333.1:g.4706c> t, g.7534g> a and g.7864c> t). Interestingly, keating and colleagues 16 also failed to identify any sequence polymorphisms in the scd1 promoter region of holstein friesian, montbeliarde, normande, norwegian red, charlois, limousin and kerry breeds of cattle . Haploview analysis revealed that these three snps in the 3'utr of the bovine scd1 gene are still segregating in the population with a r value of 55% - 86% (figure 1b). As shown in table 1, rather, the gene was significantly associated with skeletal muscle fat deposition and fatty acid composition measurements . Overall, both glm analysis and qtdt test revealed that aafc02181333.1:g.4706c> t was significantly associated with r1, r3, rsfa and rmufa; g.7534g> a with bms, r1, r3 and rsfa; and g.7864c> t with r1, r3, rsfa, rmufa and cla (p<0.05) (table 1), respectively . Doran and colleagues 17 investigated the effect of a reduced protein diet on the activity and expression of scd1 as well as on the level of total lipids and the fatty acid composition of muscle and subcutaneous adipose tissue in pigs . The authors observed that a significant effect of the diet on scd1 activity and scd1 protein expression in muscle, but not in subcutaneous adipose tissue . These data indicate that the expression of scd1 might be tissue - specific under a different regulatory mechanism between skeletal muscle and subcutaneous fat tissues . All three snps in the scd1 gene were significantly associated with the estimated stearoyl - coa desaturase activities for r1 and r3 (table 1). In particular, the g.4706c> t polymorphism showed the largest effect on r1, being 64.53% higher in cc animals than in tt homozygotes (p<0.05); while the g.7864c> t polymorphism led to the largest difference by 13.39% in r3 between the cc and tt homozygotes (p<0.05). For g.7534g> a, the differences between gg and aa animals were 21.02% in r1 and 7.61% in r3, respectively (table 1). Interestingly, alleles g.4706c, g.7534 g and g.7864c had dominant effects on the estimated scd1 activity r1 in comparsion to g.4706 t, g.7534a and g.7864 t, because the heterozygotes were almost equal to the homozygotes with the former alleles (p=0.9836). However, the inheritance mode between two alleles for each of these three polymorphic sites turned to the additive fashion for r3, because the performance of heterozygotes was close to the average between two homozygotes (p=0.9287). Unfortunately, none of these snps were associated with r2 (pglm=0.20780.7765 and pqtdt=0.20120.9227) (table 1). Furthermore, the alleles associated with high stearoyl - coa desaturase activities were also associated with increased beef marbling scores and increased amounts of monounsaturated fatty acids regardless of their statistical significance levels (table 1). Among these three polymorphic sites, g.7534g> a showed the largest effect on the former trait, being 0.87 scores higher in gg animals than in aa (p<0.05); while g.7864c> t had the biggest impact on the latter phenotype, being 1.36 units higher in cc animals than in tt animals (p<0.05). However, all these alleles associated with high scd1 activities were negatively associated with amount of saturated fatty acid in skeletal muscle (table 1). The differences in rsfa between homozygotes with the high scd1 active allele and those with the low scd1 active allele ranged from 1.87 units at g.7864c> t to 1.01 units at g.7534g> a (p<0.05). T and g.7864c> t were significantly associated with conjugated linoleic acid in the skeletal muscle based on either glm analysis or qtdt test . The difference in the trait between cc and tt was 10.59 mg/100 g dry muscle sample for the former snp (p<0.05) and 11.76 mg/100 g dry muscle sample for the latter snp (p<0.05) (table 1). Two additional features were also observed: first, alleles associated with high scd1 activities had a high amount of cla in muscle and second, the highly active alleles showed almost completely dominant effects on the phenotypes, because both cc and ct animals had very similar performances (p=0.8018) (table 1). Although glm analysis indicated significant association between g.4706c> t and chol, qtdt test failed to confirm the strong association (table 1). Our cross species study using cattle as a model organism confirmed significant associations of scd1 gene with fat metabolism in skeletal muscle, thus supporting the finding reported by warensj and colleagues 6 that the scd1 gene is related to obesity in humans . The reason for failed associations between the gene and diabetes - related traits including bmi and waist - to - hip ratio reported by liew and colleagues 7 could be simple: the selection of snps for the association study . In the former study 6, markers rs10883463, rs7849, rs2167444, and rs508384 with the significant associations are 11692bp, 15341 bp, 17482 bp and 17499 bp apart from atg (+ 1) site, while snps used in the latter study 7 are located in a range of 1057 bp to 14555 bp in relation to the atg site of the human scd1 gene . Certainly, none of the markers used in both studies overlapped or were in common . Interestingly, liew and colleagues 7 reported that genotype frequencies at the + 14301 a> c snp in the 3' utr showed borderline association (p~0.06) when evidence for linkage was taken into account . Therefore, snps near the 3'utr had a stronger likelihood to show significant associations . In our present study, these three snps are located in the 3'utr of the bovine gene and in particular, two are in the proximity of polyadenylation signal site (aataaa) (nm_173959.4). In human (nm_005063.4), cattle (nm_173959.4), pig (nm_213781.1), mouse (nm_009127.3) and rat (nm_139192.2), the scd1 gene has a long 3'utr of 3350 bp 3903 bp in comparison with a coding sequence of 1068 bp to 1080 bp . Generally speaking, however, highly conserved untranslated regions might contribute significantly to gene function in posttranscriptional control . For example, the 3' utrs may contain sequence elements that play crucial roles in transcript cleavage, polyadenylation and nuclear export, and in regulating the level of transcript and the stability of transcripts 18 . T in the bovine gene is also highly conserved in human and pig (figure 1c). A missense mutation that causes an amino acid replacement from valine (type v) to alanine (type a) the authors genotyped this marker on 1003 japanese black steers and found that the scd1 type a gene contributes to higher mufa percentage and lower melting point in intramuscular fat . Mele and colleagues 20 also tested the same marker on dairy cows and found that milk of aa cows had a greater content of cis-9 c18:1 and total monounsaturated fatty acids and a higher c14:1/c14 ratio than did milk of vv cows . The relative contribution of scd1 genotype to variation of monounsaturated fatty acids, cis-9 c18:1, and cis-9 c14:1 was 5, 4, and 7.7%, respectively . These animal data further confirmed that the scd1 gene is a critical player in fatty acid metabolism . Beef is the number one protein source in american diets and the demand for beef continues to grow . Beef is a highly nutritious and valued food, but it also contains high amounts of saturated fat as a result of biohydrogenation in the rumen 21 . For many years, fats in meat have received considerable interest because in many people's minds they not only make you fat, but they also increase the risk of a number of health problems such as heart disease, stroke, diabetes, obesity, and some cancers 22 . In fact, a diet high in saturated fats tends to increase blood cholesterol levels while diets high in unsaturated fats tend to lower blood cholesterol levels . Low - density lipoprotein (ldl) cholesterol is the " bad " cholesterol because elevated ldl levels are associated with an increased risk of coronary artery (heart) disease . In contrast, high - density lipoprotein (hdl) cholesterol is the " good " cholesterol since high hdl levels are associated with less coronary disease . Therefore, heart diseases are favorably affected by the consumption of certain unsaturated fatty acids because they lower plasma total chlolesterol and bad cholesterol levels 23 . No doubt, our present study provides evidence that it is possible to produce beef with high marbling, high amount of monounsaturated fatty acids and conjugated linoleic acid content, but with low amount of saturated fatty acids . Therefore, our findings can help beef breeders and producers produce nutritionally healthy food, thus reducing the burden of health care in the nation.
Head and neck cancer has been the seventh most common malignancy and also a major cause of morbidity and mortality, worldwide (1, 2). Squamous cell carcinoma (scc) has represented the most common histologic subtype of cancers originating from the region (3, 4). Cancers have been originating from oral cavity, larynx and pharynx comprised approximately 690,000 new cases and 4.9% of all cancer incidence through the world in 2012 (5). Unfortunately, most of the head and neck cancer patients have presented with loco - regionally advanced disease (6) and despite improvements in treatment techniques, the five years overall survival of such patients has been still poor (7) several prognostic factors have defined for head and neck cancers that could influence response to therapy and eventual outcome . These factors might be categorized as follows: (a) prognostic factors related to the primary tumor like tnm stage, malignancy grading, perineural invasion, vascular invasion, (b) prognostic factors related to the patient like age, sex, general medical condition, and (c) prognostic factors related to the treatment (8). Currently, most oncologists favor mulimodality approach in the management of head and neck cancer, has focused on organ preservation, increasingly . Surgery, with or without adjuvant (chemo) rt and definitive (chemo) rt have been acceptable managements in most primary head and neck cancers and recent trials has shown improved survival rates with such approaches (9 - 11). The present study has conducted considering the influence of genetic and geographic factors on epidemiology of head and neck cancer and lack of reliable data on its prognosis and treatment outcome in iran . We have performed a retrospective analysis of 119 patients with non - metastatic non - nasopharyngeal squamous cell carcinoma of head and neck admitted between 2008 and 2014 to our clinical oncology center (jorjani cancer center, emam hossein hospital, tehran, iran). All medical records of the included patients have investigated and with a previously prepared fact sheet the following data have collected: age, sex, weight, height, date of pathologic diagnosis of the disease, primary tumor site, clinicopathological characteristics of the primary tumor, date of last follow up, date of recurrence and death (if any). The characteristics of the study population have then described and the overall survival (os) and event free survival (efs) of the patients and their relation with demographic and clinico - pathological factors have analyzed . The categorical parameters have compared using two - sided pearson s test or fisher s exact test, as appropriate . The event free survival (efs) has defined as survival without progression, recurrence, and death . The overall survival (os) time has defined as the period from the diagnosis until death of any cause or until the date of the last follow - up, at which data point has censored . All summary statistics on time - to - event variables have estimated according to the kaplan - meier method and compared using the log - rank test . The categorical parameters have compared using two - sided pearson s test or fisher s exact test, as appropriate . The event free survival (efs) has defined as survival without progression, recurrence, and death . The overall survival (os) time has defined as the period from the diagnosis until death of any cause or until the date of the last follow - up, at which data point has censored . All summary statistics on time - to - event variables have estimated according to the kaplan - meier method and compared using the log - rank test . Spss software (version 21.0) has used for statistical analysis . A p value <0.05 has considered significant . Among the 119 studied patients, 90 were male (76%) and 29 were female (24%) with mean age of 58 years (16 to 88 years). Most patients (59% of all) had normal body mass index (bmi). Larynx was the most common primary tumor site (55% of all patients) and oral cavity had the second place subsequently . According to primary tumor and lymph node stage, most cases were t1/t2 (54%) and n0/n1 (50%), respectively . Treatment has consisted of surgical (surgery + rt / chemort) and non - surgical (rt / chemort or induction chemo + rt / chemort) modalities . The patient abbreviations: bmi, body mass index; t - stage, tumor stage; n - stage, lymph node stage . (%). With a median follow - up period of 28 months (range: 2 to 84 months), os and efs of the study patients was 61.2% and 52.4%, respectively (figures 1 and 2). Tumor stage (t - stage) was the only parameter that significantly influenced the patients os . Mean os of patients with t1/t2 tumors was higher than patients with t2/t3 tumors and this difference was statistically significant (59 months vs. 47 months, respectively: p = 0.039). In terms of efs, significant differences have observed among node stage (n - stage) and bmi classification subgroups . Patients with n2/n3 tumors had lower mean efs than patients with n0/n1 tumors (40 months vs. 55 months, respectively: p = 0.043). Interestingly, the patients with normal bmis had significantly higher mean efs compared with patients with bellow or above normal bmis (table 2 and figure 2). Patients whose primary tumor site was larynx had higher mean efs and os than patients with non - laryngeal tumors although the difference had trends toward statistically significance only in terms of efs (table 2). The correlation between survival and patient, tumor and treatment characteristics have detailed in table 2 . As shown in the table 2, surgical treatment modalities have resulted in the same prognosis as non - surgical approaches . Abbreviations: bmi, body mass index; efs, event free survival; n - stage, lymph node stage; os, overall survival; t - stage, tumor stage . Among the 119 studied patients, 90 were male (76%) and 29 were female (24%) with mean age of 58 years (16 to 88 years). Most patients (59% of all) had normal body mass index (bmi). Larynx was the most common primary tumor site (55% of all patients) and oral cavity had the second place subsequently . According to primary tumor and lymph node stage, most cases were t1/t2 (54%) and n0/n1 (50%), respectively . Treatment has consisted of surgical (surgery + rt / chemort) and non - surgical (rt / chemort or induction chemo + rt / chemort) modalities . The patient abbreviations: bmi, body mass index; t - stage, tumor stage; n - stage, lymph node stage . With a median follow - up period of 28 months (range: 2 to 84 months), os and efs of the study patients was 61.2% and 52.4%, respectively (figures 1 and 2). Tumor stage (t - stage) was the only parameter that significantly influenced the patients os . Mean os of patients with t1/t2 tumors was higher than patients with t2/t3 tumors and this difference was statistically significant (59 months vs. 47 months, respectively: p = 0.039). In terms of efs, significant differences have observed among node stage (n - stage) and bmi classification subgroups . Patients with n2/n3 tumors had lower mean efs than patients with n0/n1 tumors (40 months vs. 55 months, respectively: p = 0.043). Interestingly, the patients with normal bmis had significantly higher mean efs compared with patients with bellow or above normal bmis (table 2 and figure 2). Patients whose primary tumor site was larynx had higher mean efs and os than patients with non - laryngeal tumors although the difference had trends toward statistically significance only in terms of efs (table 2). The correlation between survival and patient, tumor and treatment characteristics have detailed in table 2 . As shown in the table 2, surgical treatment modalities have resulted in the same prognosis as non - surgical approaches . Abbreviations: bmi, body mass index; efs, event free survival; n - stage, lymph node stage; os, overall survival; t - stage, tumor stage . In the present study, patients demographic characteristics like age and sex were similar to ones in other studies (both local and in other geographic regions of the world) (1 - 3). Larynx was the most common primary site followed by oral cavity and this finding has been in concordance with most other reports (1 - 3, 12). 28 months os and efs of our patients was 61.2% and 52.4%, respectively . In a study on incidence and survival trends of head and neck squamous cell carcinoma in the netherlands, braakhuis et al . Has shown a 2-year survival rate of 72% for the patients had been diagnosed between 2007 and 2011 (13). Oropharyngeal carcinoma has constituted the significant proportion of their head and neck cancers and its relation to hpv - positivity could contribute to the better prognosis has observed in their study population . Other possible reasons for the disparity between the survival rate has observed in their study and one in our investigation could be higher socioeconomic status of their patients and better access to health care facilities that resulted in earlier diagnosis and treatment of the cancer . In another study on prognosis of the patients with head and neck cancer, dwojak et al . Has shown a survival rate of 62% for american indians at two years (14). This finding was very similar to one observed in our study and could also be explained with previously mentioned reasons . In our patients t - stage and n - stage have demonstrated to be significantly associated with survival and these findings are consistent with results of most other studies (15 - 17). As an interesting observation in the present study, patients with normal bmis had significantly higher mean efs in comparison with patients with bellow or above normal bmis . Nutrition has been a matter of concern in cancer (especially head and neck cancer) management . Obesity was one of well - known risk factors for cardio - vascular disease and has also related to an increased risk of cancer progression and death (18). On the other hand, malnutrition has shown to be a risk factor of worse prognosis in some types of cancer, including hnscc (19, 20). In a retrospective study has included 706 patients with head and neck cancer diagnosed between 2004 and 2012, takenaka et al . Has shown that bmi was a prognostic factor for survival, independent of primary site, and tumor stage . Patients with normal pre - treatment bmis had higher 5-year survival rate in comparison with underweight patients and this difference was statistically significant (62.7% vs. 32.2%, respectively p <0.001) (20). In terms of treatment modality, patients whose primary treatment has included surgery had the same prognosis as ones with non - surgical approaches (figure 1, table 2). There were some evidence in the literature that has shown similar outcomes for head and neck cancer patients treated with surgical and non - surgical approaches (21, 22). In a recently published randomized trial, iyer et al . Has shown that there have appeared to be either no difference in outcome, nor a slight advantage favoring primary surgery plus rt compared with concurrent chemort for patients with advanced, nonmetastatic squamous cell carcinoma of the head and neck (5-year os rate was 45% and 35%, respectively p = 0.262). Only in subset analysis, patients with oral cavity cancer had significantly better prognosis with surgical approaches (22). In conclusion, our study seems to be the first that investigated outcome of iranian patients with head and neck cancer and factors influencing it.
Molecular diagnostics of cancer predisposition is very important for the medical treatment of the patient and persons belonging to the high risk group . Molecular studies enable the detection of mutation carriers and release from unreasonable stress of persons from the group with increased risk of cancer occurrence . The mutation spectrums in the genes predisposing to colorectal cancer in the polish population have been described [1 - 4]. In the present work we focused on recurrent mutations of the apc gene causing fap . Familial adenomatous polyposis (fap) is characterized by the appearance of numerous polyps in the large intestine . The correlation between mutations of the apc gene and the occurrence of familial adenomatous polyposis was described in 1991 and since then, mutations of the apc gene have been investigated in research centres leading to identification of various mutation types . Mutations of the apc gene, in most cases, are small deletions or insertions with the most frequent mutations, in the greater part of the described populations, being the aaaga deletion at codon 1309 and the acaaa deletion at codon 1061 . Clinical diagnoses of fap patients were conducted in collaborating genetic centres or gastroenterology clinics in the place of residence of patients . So far, samples of dna belonging to 280 polish fap families have been collected in the dna bank of polish fap families established in 1997 at the institute of human genetics, polish academy of science in poznan . Dna was extracted from peripheral blood cells by the classical phenol purification method and entire coding sequence of apc gene was screened for mutations by pcr - hd and sscp methods in 280 probands . Dna fragments showing heteroduplex or additional pattern in sscp analysis were sequenced by direct pcr product sequencing and analyzed using a megabace 500 sequencer according to the manufacturer's specifications . We identified 72 mutations in 124 of our 280 fap families and observed eight types of recurrent mutations . The mutations and age of onset two of them were localized in exon 11 and the remaining six in the 3' part of exon 15 . The most frequent mutation, 39273931delaaaga, occurred in twenty - eight families (10%); the second one was 31833187delacaaa, occurring in eight families (2.8%); and the third most frequent mutation was 32023205deltcaa, detected in 5 families (1.7%). In our fap patients y500x occurred in four families (1.4%) while q978x was detected in three families . Each of the remaining four types of mutation occurred in two families and the frequencies of these mutations were below one percent . Fifty - four recurrent mutations identified in apc gene and age of onset in a group of 124 diagnosed polish fap families nd no data available in the human mutations database at the institute of medical genetics in cardiff, considered the most representative population in the world, seven hundred mutations are listed for the apc gene . The mutation reports describe the frequency of this mutation from 0% in northwest spain, 2.4% in the australian population, 5% in dutch, 7% in israeli to 16% in the group of italian fap patients [6 - 9]. The second most frequent mutation, 31833187delacaaa, is reported with frequency ranging from 0% in northwest spain, 1.5% in israeli populations to 8.4% in australia [6 - 9]. A study of over 100 dutch families revealed equal frequency of those two most frequent mutations (39273931delaaaga and 31833187delacaaa). The latest published report in 2005 involved the analysis of over 1000 patients . In comparison to this study, the representative study of mutation frequency in the neighbouring population indicated two times higher frequency of 39273931delaaaga, whereas a difference in frequency of 31833187del - acaaa was not observed (germany 3.8%, poland 2.7%). The frequency of 32023205deltcaa was equal (1.7%) in both populations . In worldwide comparison differences in the frequency of mutations the polish population of fap patients belongs to the group where 39273931delaaaga occurred with higher frequency, whereas the frequency of mutation 31833187del - acaaa occurred with medium frequency in comparison with other populations . The two recurrent mutations localized on exon 11 were observed only in the polish population . In our two unrelated families with 14901491inst brain tumours were observed . Another mutation (q978x) did not occur with higher frequency as described for other populations . In our fap patient group q283x, which occurs with frequency of 4.5% in uk fap patients, screening for these mutations permitted us to diagnose 19% of all families in our population but eight types of mutations constitute 43.5% of all our diagnosed families . The mutation study in our population should involve these eight mutations to improve molecular diagnostics of the apc gene . The study was financed by grant 2p05a10728 from the ministry of science and higher education.
Angiotensin converting enzyme inhibitors (acei) have been shown to decrease agv in marfan syndrome (mfs). We sought to compare the effect of -blockers and acei on aortic growth velocity (agv) in mfs . We reviewed retrospectively all data from all patients with mfs seen at arkansas children s hospital between january 1, 1976 and january 1, 2013 . Generalized least squares were used to evaluate agv over time as a function of age, medication group, and the interaction between the two . A mixed model was used to compare agv between medication groups as a function of age, medication group (none, -blocker, acei), and the interaction between the two . A total of 67 patients with confirmed mfs were identified (34/67, 51% female). Mean age at first encounter was 13 10 years, with mean follow - up of 7.6 5.8 years . There were 839 patient encounters with a median of 10 (range 242) encounters per patient . Agv was nearly normal in the -blocker group, and was less than either the acei or untreated groups . The agv was higher than normal in acei and untreated groups (p<0.001 for both). We performed a retrospective review of all patients with mfs seen at arkansas children s hospital between january 1, 1976 and january 1, 2013 . Patients with mfs were identified using multiple institutional databases including those from the echocardiography and cardiac catheterization laboratories, the cardiology clinic, all cardiothoracic surgeries, and the division of genetics . Echocardiograms were performed with the patient in the supine position using commercially available ultrasound machines (siemens acuson sequoia 512 with 10, 7, 5, and 3 mhz probes and philips ie33 with 12, 8, and 5 mhz probes). Two - dimensional measurements were made in accordance with the recommendations of the american society of echocardiography using parasternal long - axis views of the aortic annulus, aortic sinus of valsalva, sinotubular junction and ascending aorta . Measurements were made from inner edge to inner edge during ventricular systole . The decision to initiate pharmacologic therapy was primarily based on the presence of aortic measurements above the normal range reported by roman et al or accelerated progressive dilation . The selection of a pharmacologic agent and the dose were provider dependent; there were no formal algorithms . After the report from our institution by yetman et al, the use of acei as primary therapy at our institution increased . Anthropometric data were used to calculate the body surface area (bsa) at each patient encounter using the dubois formula . A normative control comparison dataset for aortic dimension and growth rate was created by using the calculated bsa of each patient with mfs at each encounter using the formula: aortic root dimension = 24.0(bsa in m) + 0.1(age) 4.3 . This normative control dataset was then compared against actual measured aortic dimensions in the patient cohort . Summary statistics were expressed as frequency and percentage for categorical variables, and as mean standard deviation for continuous variables, except for the ages of the treatment groups, which are expressed as mean with first (q1) and third (q3) quartiles . To compare aortic growth velocities between medication groups, a mixed model was developed for the aortic dimension as a function of age, medication group (none, -blocker, acei, or normative control), and the interaction between the two . A restricted cubic spline was used for age when fitting the mixed model with regard to the non - linear relationship between aortic dimension and age . A compound symmetry variance matrix was used to take into account the correlated measurements from the same patient . Additional mixed models were fitted for blood pressures and heart rates to assess their differences among three medication groups (none, -blocker, or acei). All the data were analyzed using statistical software sas 9.4 (sas institute inc ., summary statistics were expressed as frequency and percentage for categorical variables, and as mean standard deviation for continuous variables, except for the ages of the treatment groups, which are expressed as mean with first (q1) and third (q3) quartiles . To compare aortic growth velocities between medication groups, a mixed model was developed for the aortic dimension as a function of age, medication group (none, -blocker, acei, or normative control), and the interaction between the two . A restricted cubic spline was used for age when fitting the mixed model with regard to the non - linear relationship between aortic dimension and age . A compound symmetry variance matrix was used to take into account the correlated measurements from the same patient . Additional mixed models were fitted for blood pressures and heart rates to assess their differences among three medication groups (none, -blocker, or acei). All the data were analyzed using statistical software sas 9.4 (sas institute inc ., a total of 67 patients with confirmed mfs were identified (34/67, 51% female). The mean sd age at first encounter was 13 10 years, with a mean followup length of 7.6 5.8 years . There were 839 patient encounters with a median number of 10 (range 242) encounters per patient . For the patients in the untreated group, the mean age was 9.8 years (q1: 3.9 years; q3: 17.2 years), which was lower than either the -blocker or acei groups (p<0.001). For those treated with -blocker therapy, the mean age was 16.9 years (q1: 12.1 years; q3: 22.2 years). -blockers used included daily atenolol (45.9%), twice daily metoprolol (48.5%), and thrice daily propranolol (5.6%). The mean dose per patient was 0.95 0.63 mg / kg (total daily dose 47.3 23.5 mg). The mean systolic (109 16 mmhg) and diastolic (67 8 mmhg) blood pressures were not different from patients who were not treated with medication . The mean heart rate in the -blocker group (78 19 bpm) was significantly lower compared with those who were untreated (90 24 bpm, p=0.001). For those treated with acei therapy, the mean age was 16.8 years (q1: 10.7 years; q3: 24.4 years). Aceis used included daily lisinopril (12.9%), twice daily enalapril (85.5%), and thrice daily captopril (1.6%). The mean acei dose per patient was 0.22 0.1 mg / kg (total daily dose 12.7 6.9 mg). The mean systolic (113 16 vs. 106 20 mmhg, p=0.005) and diastolic (68 10 vs. 64 10 mmhg, p=0.005) blood pressures were significantly higher in the acei group compared with the untreated group . The mean heart rate in the acei group (83 17 bpm) was significantly lower compared with the untreated group (90 24 bpm, p=0.003). The systolic and diastolic blood pressures were higher in the acei group compared with the -blocker group (p=0.015 and p=0.019, respectively), and there was no difference in heart rate (p=0.696). The aortic size and agv for the three largest groups (none, acei, and -blocker) were determined (figure 1) and intergroup comparisons were made, as well as comparisons with the calculated, expected normative control aortic root dimensions (figure 2). The aortic dimensions were significantly larger in all groups compared with the normative control dataset (p<0.001). At younger ages, the aortic dimensions in the -blocker group were significantly larger than the acei and untreated groups (p<0.01; figure 1). There was no difference between the acei and untreated groups with regard to aortic dimensions at younger ages (p=0.636). Aortic growth velocity was significantly attenuated in the -blocker, and nearly approximated that in the normative control dataset . The attenuation of the agv in the acei group was less than that of the -blocker group compared with those not treated with medication . The median age at aortic root replacement was 17 years (range 935 years). Two patients experienced aortic dissections: one treated with metoprolol experienced acute dissection of the entire thoracic aorta and survived to surgery; the other was treated with enalapril and found to have an asymptomatic dissection of the aortic root . Of the 11 patients who underwent aortic surgery, pharmacologic treatment prior to surgery included: acei in 5; -blocker in 4; and no therapy in 2 . The present study demonstrates a significant attenuation of the agv in pediatric patients with mfs treated with -blocker therapy . In fact, the growth velocity was almost identical to the predicted normal growth velocity for the cohort . On average, the patients in the -blocker group began with a larger aortic dimension compared with the patients in the acei and untreated groups, and this difference resolved over time, a finding that may suggest that the early initiation of -blocker therapy should be considered in patients with mfs . This is a significant finding because aortic wall tension increases as the aortic diameter increases, which results in acceleration of aortic dilation at larger diameters . The expected result would then be that the agv should be faster in the -blocker group; however, the opposite was found in the -blocker group indicating a definite mitigating effect on the agv in patients with mfs . Recently, mueller et al demonstrated a decrease in agv in patients with mfs who were treated with either -blockers or angiotensin ii receptor blockers . However, other studies have suggested that -blocker therapy does not alter the agv or clinical outcomes in patients with mfs . Gersony et al conducted a meta - analysis of six studies available at the time, and concluded that -blocker therapy was ineffective in patients with mfs . In the original studies used to conduct their meta - analysis, most of the original authors arrived at different conclusions than gersony et al . Silverman et al showed that death was twice as common in the untreated versus the -blocker treated group . Legget et al reported that aortic complications were more common in the -blocker treatment group; however, the aortic complication group also had significantly larger aortas at the initiation of the study (p<0.0001). Further, in that study patients were dichotomized, without clear delineation as to why, to those who had -blocker therapy for 1 year versus those who had therapy for <1 year . Similar to the work of silverman et al, roman et al reported that in 113 patients with mfs those who had aortic complications had larger aortic size at presentation (p<0.005), were significantly older (p<0.01), and had significantly faster agv (p<0.05); however, -blocker therapy use only trended toward being more common in the complication group (86% vs 66%, p = non - significant). Those authors stated that the trend toward increased -blocker use in the group with complications was because they started out with larger aortas, which represents a significant selection bias . Salim et al reported that the aorta is dilated at young ages in mfs and showed that the agv was faster in those who were untreated . In that study, the 5 patients treated with -blocker therapy who underwent surgical intervention had larger aortas at the time of enrollment . In their randomized trial of propranolol therapy in mfs, shores et al reported in the propranolol group a decreased rate of aortic dilation, fewer deaths, and significantly fewer patients who reached one or more predetermined clinical end - points . Finally, tahernia reported three patients who were treated with -blocker therapy and three who were not . With a mean follow - up of 3.3 years the three untreated patients had a mean follow - up of 3 years with a mean agv of 1.4 mm / year . In the consideration of studies evaluating agv, it is imperative to consider the size of the aorta at the time of the initiation of pharmacotherapy . In accordance with the law of laplace, when wall thickness and blood pressure are stable, wall tension in the aorta increases as the aortic diameter increases, resulting in acceleration of aortic dilation at larger diameters . To make a truly meaningful comparison between therapeutic groups, either both groups have to begin with the same diameters or the group that is shown to have a decreased growth rate must have begun at a larger diameter, thus showing a diminution of agv . If the aorta is larger in one group than the other, those patients are beginning the study at a different point in the aortic disease process than the group with the smaller diameter . The present study is not subject to this concern because those patients treated with -blocker therapy began, on average, with a larger aortic diameter than either the acei group or the untreated group and were still demonstrated to have a lower agv . The difference in results between the -blocker and acei groups in the present study seem to support the hypothesis that a decrease in myocardial contractility is the explanation for decreased agv in patients with mfs treated with -blockers . In the present study, there were no clinically significant differences in blood pressure or heart rate between the -blocker and acei groups; however, the agv in the -blocker group was significantly less than either the acei or untreated groups . However, other explanations cannot be ruled out, such as heretofore - unknown pleiotropic effects of -blockers . Yetman et al demonstrated that there was a decrease in the agv and improvement in the aortic distensibility in patients with mfs treated with acei versus those treated with -blocker . The present study does not corroborate those findings . In the study by yetman et al, the subject ages and the doses of both medications were similar to those in the present study . The length of follow - up was shorter in that prior study (3.0 0.2 years) compared with the present study (7.6 5.8 years). However, a previously published and validated formula for the determination of predicted aortic size was used in combination with the anthropometric data from the present study cohort to create a predicted normal aortic growth curve, which allowed for comparison of the study measurements against a unique dataset specific to the study cohort . The patients in the non - treatment group were younger than those in either the -blocker or acei group . An older population would be expected to have an increased agv, which if anything would blunt any effect of the medication on slowing agv . . Early introduction of -blocker therapy in patients with mfs should be considered particularly even prior to the demonstration of aortic dilation.
Diabetic hand - related infections or the diabetic hand is a less commonly recognized entity when compared to diabetic foot related infections . The diabetes hand - related infections generally constitute <1% of all admissions in patients with diabetes, and it is seen almost exclusively in developing countries of the tropical world . The initial reports of diabetic hand infection (dhi) were first reported by akintewe et al . From nigeria . However, this entity has been reported much earlier in the west in the 1970s . Mcconnell and neale from usa reported 204 cases of hand infections among which 7% had diabetes mellitus as a risk factor . Subsequently, a report of 22 cases of upper extremity infection in patients with diabetes was documented . This entity was recognized variably as the tropical diabetes hand syndrome (tdhs), diabetic hand sepsis syndrome, and dhi . However, the term tdhs is commonly utilized because it appears to be more common in developing countries of the tropical world . Tdhs is defined as an acute pyogenic infection of the hand, which is characterized by a mild form of infection such as cellulites, swelling of the hand, and ulceration that progress into more fulminant infections such as necrotizing fasciitis (nf) and gangrene of the hand and upper limb . The hand infections in patients with diabetes may at times be very severe and can be associated with an increased risk of mortality due to sepsis and in cases of survival, can cause significant morbidity due to amputation and functional disability . From 1990 onward, dhi had declined in prevalence in the western world; however, it continues to be common in african and asian countries . However, currently available literature regarding the nature and pattern of dhis is still limited, owing to the relatively rare presentation of the disorder . The aim of the current study was to determine the pattern of hand infections, the microbiological profile, and the outcome of diabetic hand related infections . This is a retrospective study involving 39 cases of patients with diabetic - related hand infections who presented to the hand surgery department between 2004 and 2010 . A profoma was used to capture the data with regards to the demographic profile, predisposing factors, type of diabetic hand - related infections, surgical procedures in other centers prior to admission, delay in presentation from the onset of symptoms, pattern of surgical procedures and clinical outcomes . Data on the glycemic profile fasting plasma glucose (fpg) and glycated hemoglobin (hba1c) at the time of admission too were reviewed . Aerobic and anaerobic culture reports on wound swabs and debrided soft tissue specimens were also included in the study . Antibiotic sensitivity patterns of the individual bacterial isolates were studied . The type of diabetes and duration of diabetes was also included in the study . Diabetes hand infections in this study were classified into three major groups (a) nf, (b) tenosynovitis, and (c) abscesses . Anatomically restrained abscesses in the tenosynovium with classical kanaval signs were defined as the tenosynovitis pattern . All the other abscesses with anatomical constraints and widespread necrosis of the fascia along with or without osteitis were defined as nf . The tenosynovitis and hand abscess group were further classified into a non - necrotizing group for statistical analysis in view of the small sample size . Early surgical debridement was performed in all cases, based on the clinical condition of the diabetic hand - related infection . After initial debridement, wound management was done by the same surgeon in the ward on a daily basis . Secondary reconstruction procedures such as split thin skin grafting (stsg) from the thigh or hand itself, a regional flap cover, posterior inter - osseous artery skin flaps (fillet flap cover) or other soft tissue cover procedures were performed when the wound was suitable for grafting . An amputation was considered in the setting of nf or osteitis . Patients who had features of abscess and tenosynovitis alone with superficial tissue involvement were empirically treated with amoxicillin and clavulanate and those with features of nf with involvement of deep tissues such as muscle, fascia and tendon, or suspected osteitis were treated on piperacillin and tazobactum in the initial phase . In patients who were diagnosed to have methicillin resistant staphylococcus aureus (mrsa) infection, therapy with linezolid and rifampicin was given for a period of 3 weeks . Along with wound management, a standard diabetes care management protocol was followed for all patients . A four point glucose profile (fasting, 2 hour post breakfast, lunch, and dinner) patients were treated with either insulin or combination insulin and oral antidiabetic agents to achieve adequate glycemic control . Once the wound healed adequately and when self - dressing was possible at home, patients were discharged from the hospital . Following discharge, the patients were followed up regularly in the endocrine outpatient department (opd) for glycemic control and in the hand surgery opd for surgical follow - up and physiotherapy . Statistical analysis was performed using the spss software package (version 17, ibm corp . In armonk, study the dhi were further classified into group 1: patients with nf-23 and group 2: nonnecrotizing fasciitis (nnf)-16 which included patients with both absecss-9 and tenosynovitis-7 for stastistical analysis . The mann whitney u - test and chi - square test were used to determine the relationship between the clinical characteristics of nf patients . Multivariate analysis was performed using logistic regression to determine the correlation between various clinical characteristics with a prolonged duration of hospitalization . Statistical analysis was performed using the spss software package (version 17, ibm corp . In armonk, study the dhi were further classified into group 1: patients with nf-23 and group 2: nonnecrotizing fasciitis (nnf)-16 which included patients with both absecss-9 and tenosynovitis-7 for stastistical analysis . The mann whitney u - test and chi - square test were used to determine the relationship between the clinical characteristics of nf patients . Multivariate analysis was performed using logistic regression to determine the correlation between various clinical characteristics with a prolonged duration of hospitalization . A total of 39 patients with type 2 diabetes mellitus were included in this study among whom 24 (61.5%) were males and 15 (38.46%) were females . The mean duration of diabetes was (7.01 4.54) years (016). The mean fpg level was 313.64 86.377 mg / dl (113515 mg / dl). The dhi in this study was categorized into three groups: (1) necrotizing fasciitis (2) abscess and (3) tenosynovitis . Twenty three (58.97%) patients presented with necrotizing fasciitis (nf), 9 (23.07%) had abscess, and 7 (17.94%) had tenosynovitis . The patients with abscesses and tenosynovitis groups were further categorized into nonnecrotizing group (nnf). The images on the various types of hand infection were shown in figures 1, 2 and 3a and b. in many patients either with abscesses or tenosynovitis and nf, the infection was found to be continuous . The mean delay in presentation to hospital was 6.0 2.5 (316) days . A total of 8 (20.5%) had a history of prior surgical intervention in other centers prior to admission to our hospital . Necrotizing fasciitis in a 54-year - old man following a thorn prick injury and subsequent surgical intervention outside features suggestive of flexor tenosynovitis of left middle finger in a 41-year - old lady who presented with spontaneous onset pain, redness and swelling (a and b) flexor pollicis longus tenosynovitis which evolved into necrotizing fasciitis in a 45 years male with poor glycemic control eleven (28%) patients had a history of antecedent trauma; however, no definite predisposing factors were identified in 28 (72%). The details of predisposing factors for hand infections are shown in figure 4 . In subjects with a prior history of antecedent trauma, 9 (82%) had nf and 2 (18%) subjects hand abscesses, while in subjects without any predisposing factors, nf was seen in 14 (50%) of cases . Predisposing factors in diabetic hand infections out of 39 patients, comprehensive bacteriological data was available in only 25 patients among whom, polymicrobial infections were detected in 13 (52%), 9 (36%) had monomicrobial infection and 3 (12%) had a sterile culture . A total of 41 different bacteria among 13 different bacterial species were detected . Among the 41 bacterial isolates, gram - positive bacteria constituted about 48.78%, and gram - negative bacteria constituted about 51.21% of isolates . Among the gram - negative isolates, klebsiella and pseudomonas aeruginosa species staphylococcus infections was more common (details of the microorganisms grown in culture of the patients with dhi have been shown in table 1). Pattern of microorganisms isolates in patients with diabetic hand infections twelve (31%) patients had exclusive debridement as a surgical procedure and the wounds healed spontaneously without any reconstructive procedure for skin cover or bony stability . Ablation of the affected digits were needed in 7 (18%) of cases, of which 6 had nf . A total of 15 (38%) patients needed skin and soft tissue reconstructive procedures, among whom three patients (7%) had a regional flap cover (cross finger in two patients and a posterior interosseous arterial flap in 1 patient), 12 (31%) patients had stsg from the thigh or hand itself . Out of the seven patients with digital amputations, three patients required a fillet flap from the amputated digits for skin cover and two patients required a bony procedure to stabilize the digits . A total of 23 patients had nf and 16 had non necrotizing dhi (nnf). Among patients with nf, 17 (73.91%) were male and six (26.08%) are female . The clinical characteristics of the study subjects (necrotizing fasciitis and nnf) are shown in table 2 . Patients with nf fasciitis had a higher mean fpg level and higher mean hba1c level when compared the patients with nnf . Patients with nf had more polymicrobial infections when compared to the nnf group (85.7% vs. 14.3) with a p = 0.017 . Seven out of eight (87.5%) patients with a prior surgical intervention at other centers prior to being admitted in our hospital had nf; however, it was also found to be not significant with regard to the evolution of nf (p = 0.109). The mean delay in presentation to hospital since the initial onset of symptoms in nf patients was 6.35 2.9 days versus 5.5 1.83 days in the nnf patients group (p = 0.454). The mean duration of hospitalization in patients with nf group was longer (21.8 9.96 days) when compared to the nnf group (12.7 14.5 days) and which was found to be statistically significant (p = 0.001). A total of 39 patients with type 2 diabetes mellitus were included in this study among whom 24 (61.5%) were males and 15 (38.46%) were females . The mean duration of diabetes was (7.01 4.54) years (016). The mean fpg level was 313.64 86.377 mg / dl (113515 mg / dl). The dhi in this study was categorized into three groups: (1) necrotizing fasciitis (2) abscess and (3) tenosynovitis . Twenty three (58.97%) patients presented with necrotizing fasciitis (nf), 9 (23.07%) had abscess, and 7 (17.94%) had tenosynovitis . The patients with abscesses and tenosynovitis groups were further categorized into nonnecrotizing group (nnf). The images on the various types of hand infection were shown in figures 1, 2 and 3a and b. in many patients either with abscesses or tenosynovitis and nf, the infection was found to be continuous . The mean delay in presentation to hospital was 6.0 2.5 (316) days . A total of 8 (20.5%) had a history of prior surgical intervention in other centers prior to admission to our hospital . Necrotizing fasciitis in a 54-year - old man following a thorn prick injury and subsequent surgical intervention outside features suggestive of flexor tenosynovitis of left middle finger in a 41-year - old lady who presented with spontaneous onset pain, redness and swelling (a and b) flexor pollicis longus tenosynovitis which evolved into necrotizing fasciitis in a 45 years male with poor glycemic control eleven (28%) patients had a history of antecedent trauma; however, no definite predisposing factors were identified in 28 (72%). The details of predisposing factors for hand infections are shown in figure 4 . In subjects with a prior history of antecedent trauma, 9 (82%) had nf and 2 (18%) subjects hand abscesses, while in subjects without any predisposing factors, nf was seen in 14 (50%) of cases . Out of 39 patients, comprehensive bacteriological data was available in only 25 patients among whom, polymicrobial infections were detected in 13 (52%), 9 (36%) had monomicrobial infection and 3 (12%) had a sterile culture . A total of 41 different bacteria among 13 different bacterial species were detected . Among the 41 bacterial isolates, gram - positive bacteria constituted about 48.78%, and gram - negative bacteria constituted about 51.21% of isolates . Among the gram - negative isolates, klebsiella and pseudomonas aeruginosa species staphylococcus infections was more common (details of the microorganisms grown in culture of the patients with dhi have been shown in table 1). Twelve (31%) patients had exclusive debridement as a surgical procedure and the wounds healed spontaneously without any reconstructive procedure for skin cover or bony stability . Ablation of the affected digits were needed in 7 (18%) of cases, of which 6 had nf . A total of 15 (38%) patients needed skin and soft tissue reconstructive procedures, among whom three patients (7%) had a regional flap cover (cross finger in two patients and a posterior interosseous arterial flap in 1 patient), 12 (31%) patients had stsg from the thigh or hand itself . Out of the seven patients with digital amputations, three patients required a fillet flap from the amputated digits for skin cover and two patients required a bony procedure to stabilize the digits . A total of 23 patients had nf and 16 had non necrotizing dhi (nnf). Among patients with nf, 17 (73.91%) were male and six (26.08%) are female . The clinical characteristics of the study subjects (necrotizing fasciitis and nnf) are shown in table 2 . Patients with nf fasciitis had a higher mean fpg level and higher mean hba1c level when compared the patients with nnf . Patients with nf had more polymicrobial infections when compared to the nnf group (85.7% vs. 14.3) with a p = 0.017 . Seven out of eight (87.5%) patients with a prior surgical intervention at other centers prior to being admitted in our hospital had nf; however, it was also found to be not significant with regard to the evolution of nf (p = 0.109). The mean delay in presentation to hospital since the initial onset of symptoms in nf patients was 6.35 2.9 days versus 5.5 1.83 days in the nnf patients group (p = 0.454). The mean duration of hospitalization in patients with nf group was longer (21.8 9.96 days) when compared to the nnf group (12.7 14.5 days) and which was found to be statistically significant (p = 0.001). The current study was designed to focus on the types of hand infection, pattern of microbiological profile in patients with dhis . There were 23 patients (54%) presenting with necrotizing fasciitis; 9 (26%) with abscesses, and 7 (20%) had tenosynovitis . Among patients with abscesses, three had thumb abscess (two had felon abscess), one had a middle finger abscess, one had a first web space abscess, two had thenar, and two had mid - palmar abscess . Patients in this series, differed from other series as none of the patients presented with superficial celluitis or with fulminant spesis . In the series by pinzur et al . Two studies, one from nigeria and other from libya had a fulminant sepsis rate of 3% in patients with dhis . In our study, antecedent trauma was a predisposing factor in 28% of patients; however, in 72% of cases, there was no obvious cause . The predisposing factors for a diabetes hand - related infections varied according to the population studied; however antecedent trivial trauma, constituted about 1636% of cases in previous studies . In our study, irrespective of the gender, the risk for development of dhis was dependent upon the nature of the occupation . Had shown that dhis were more common in females due to trauma sustained during household work, such as a knife injury . In our study, the mean age group for diabetic hand - related infections was 50.89 years (mean standard deviation: 50.89 10.6). However, the range in terms of age of patients with dhis may vary from 17 to 80 years in different series . In our study, out of 39 patients, 22 had shown positive microbial cultures, 13 (52%) had polymicrobial infection and a single organism was isolated in 9 (36%) patients . In polymicrobial infections, klebsiella and staphylococcal infections reported that 52% of patients in their series were found to have polymicrobial infections and s. aureus and klebsiella were more common . Previous studies by gonzalez et al . Showed that 46% of had polymicrobial infections and 55% by kour et al . In our study, gram - negative organisms were isolated in 51.21% and gram - positive organisms in (48.78%). Similarly, kour et al . Reported that 73% of culture positive infections were due to gram - negative organisms . Among gram - positive organisms, totally, 13 (52%) had grown s. aureus and mrsa was detected in 5 (20%) patients . Out of 9 patients with monomicrobial infections, 4 patients had grown staphylococcus among which one had mrsa . Bach et al . Reported that 73% of hand infections in an urban setting were related to mrsa . In a community - based study on hand infections by wilson and rinker, the overall prevalence of mrsa infection was 64% and was 20% among diabetic subjects which was comparable to our study . Isolated gram - negative infections were seen in four patients of whom 2 had klebsiella, one had pseudomonas and one had citrobacter infection . Isolated gram - negative infections were reported in 11% by jalil et al ., and citrobacter infections were also seen in their study . In our study, 31% (n = 12) required only surgical debridement, following which the ulcer healed spontaneously without any reconstructive procedure for skin cover or bony stability . Seven (18%) of our study population required amputation of the digits to control infection, or due to gangrene of the digits . Among the group with amputation, six had nf and one had a nonviable index finger due to trauma . A study by gonzalez et al . Showed that the amputation rate was as high as 39% . The increased rate of amputation in their study was related to deep tissue infection, renal failure and infection due to gram - negative and polymicrobial infections . In francel's series, the amputation rate was found to be 100% in patients with diabetes with underlying renal transplantation . Similarly, mann and peacock reported a 35% amputation rate, and jalil et al . Reported a 16.2% amputation rate series was attributed to multiple debridement and an aggressive approach towards the management of the diabetic hand . A reconstruction procedure was performed in 18 patients with dhi patients when wound had healed that was enough for grafting . In this study, 15 (38%) patients needed skin and soft tissue reconstructive procedures . Reported that nearly half of the patients with nf required reconstructive procedures (skin and soft tissue flap). In our study, 3 (7%) patients required regional flap cover and 12 (31%) required stsg . The duration of hospitalization was 21.8 9.96 days in the group with nf, while it was 12.7 14.5 days in the with nonnecrotizing group (the duration of hospitalization was 13.4 8.3 days in the group with abscesses group and 12.63 7.3 days in the group with tenosynovitis, respectively). The important factors responsible for a lengthy duration of hospitalization were delay in presentation to hospital, poor glycemic control (a higher hba1c level), high random plasma glucose levels at admission, mrsa infections, and a prior surgical procedure in a previous institution . However, none of the parameters were found to be statistically significant, probably due to the small sample size ., the longer duration of hospitalization was associated with polymicrobial infections . In our study, polymicrobial infections were not found to be associated with an increased duration of hospitalization . A retrospective study by gonzalez et al . Showed that a longer duration of hospitalization was related to the severity of infections (deep tissue infection) and polymicrobial infections . The occupation of the patients were not evaluated, the microbiological culture reports were not available for all the patients and follow - up and long - term outcome of the patients who had undergone surgical procedures were not studied in view of the retrospective nature of the study . The status of peripheral neuropathy and peripheral vascular disease was not specifically addressed in this study . The occupation of the patients were not evaluated, the microbiological culture reports were not available for all the patients and follow - up and long - term outcome of the patients who had undergone surgical procedures were not studied in view of the retrospective nature of the study . The status of peripheral neuropathy and peripheral vascular disease was not specifically addressed in this study . Diabetes hand infections are one of the less common complications of diabetes and are frequently associated with nf and gangrene . Unrecognized trivial trauma remains the most common predisposing factor . A delay in presentation and poor glycemic control early surgical intervention, good glycemic control, and early initiation of board spectrum antibiotics are essential for the rapid healing of hand infections . This study highlights the need for increased awareness among physicians regarding diabetic hand - related infections, so that an early referral may be possible to prevent overt complications.
Patients with severe stenosis should undergo treatment even if it is well tolerated and is asymptomatic at the beginning . This is due to the possibility of dangerous complications that may emerge if timely treatment is not used.1 balloon valvuloplasty for pulmonary valve stenosis is the treatment of choice for isolated pulmonary stenosis in all childhood age groups.2 the balloon valvuloplasty procedure in treating pulmonary valve stenosis has its origins in the success of surgical valvotomy to relieve the pressure gradient developed across the stenotic pulmonary valve.3 the first catheter attempts to relieve the gradient were described by rubio and limon lason in 19544 and semb et al in 1979.5 the short - term and long - term effects of this therapy are still an area of interest for interventional cardiologists as stronger evidence is needed from studies in different settings and populations . The aim of this study was to investigate characteristics and outcomes of treating pulmonary stenosis with valvuloplasty, and to compare the results among three childhood age groups . All children under 15 years of age who had undergone pulmonary valve balloon valvuloplasty in madani heart center from 20052009 were enrolled in this study . Madani heart center is a referral subspecialty center belonging to tabriz university of medical sciences and is located in tabriz, northwest of iran . The diagnosis was based on two - dimensional echocardiography and peak - to - peak pressure gradient difference between the pulmonary artery and right ventricle . The main variables investigated included: age, sex, coincident diseases, baseline right ventricle and pulmonary artery pressure gradient, pulmonary artery - right ventricle pressure gradient at baseline and after 24 hours, remaining residual pressure, annulus size, balloon size, complications, and mortality . Percutaneous balloon valvuloplasty was performed on a standard base for any patient with transvalvular gradient> 40 mmhg . A catheter, with deflated balloon at a given size, the gradient across the pulmonary outflow was measured and location of the valve was defined using fluoroscopy and a ventriculogram in the anteroposterior and lateral projections . After conducting valvuloplasty and all measurements, the results were compared among three age groups: infants, 15 years, and 515 years . Data were entered into the computer and analyzed using ibm spss software (version 16.0; spss inc, chicago, il). Distribution, independent t - test or mann whitney u test were used to compare numeric variables . Study protocol was approved by regional committee of ethics at tabriz university of medical sciences . Noncardiac coincident diseases included two cases of bilateral tonsillar hypertrophy, one congenital cataract, one case of undescended testis, one hemophilia case, and one case of left sided inguinal hernia . Mean right ventricular pressure was 95.9 35 mmhg at the first visit and mean pulmonary artery pressure was 26.2 14.7 mmhg . Mean difference between the pulmonary artery and right ventricular pressure was 77 35 mmhg . Pulmonary valve insufficiency was observed in 52 (59.8%) patients, 36 patients having mild pulmonary valve insufficiency . Stenosis remained in 56 patients leading to a mean pressure of 40 26.4 mmhg . Two children died under treatment, one of whom was a 3.5-month - old infant who died after 3 days in intensive care unit, suffering from multiple cardiac anomalies . Balloon valvuloplasty failed in nearly one - fifth of patients in this study leading to a surgical alternative treatment . Although balloon dilatation mortality and morbidity is reported to be greater than after surgery and the recurrence rate is higher following balloon dilatation, the comparison can be problematic due to methodological issues.6 however, since the first report by kan et al,7 many studies have confirmed the safety and efficacy of pulmonary balloon valvuloplasty in infants, children, and adolescents with pulmonary valve stenosis, and it has gained much popularity . Indications for intervention in this age group include the prevention of progression of right ventricular outflow tract obstruction, right ventricular hypertrophy, and right ventricular fibrosis . Outcome results in this study, as consistent with others, pose the idea of hemodynamic mechanisms being affected after pulmonary balloon valvuloplasty . In a study by alyan et al, it was found that sympathetic overactivity and increased probrain natriuretic peptide levels were associated with the symptomatic status of patients with pulmonary stenosis and associated with a decrease in atrial pressure and probrain natriuretic peptide levels; pulmonary balloon valvuloplasty yielded a decrease in adrenergic overactivity in the patients with pulmonary stenosis.8 although not statistically significant, there were 17 failures and two cases of mortality, descriptively less frequent among children> 5 years . Failure, mortality, and complication are an inevitable part of cardiothoracic interventions.911 the presence of complications is proportional to age and such complications are mostly found in infants . Although younger patients have shown poorer prognosis after valvuloplasty, interestingly the method has been used even for a 700-g neonate with pulmonary stenosis.12 using an appropriate ratio of balloon to valve hinge point diameter is shown to optimize the chance of long - term success . 13 mean balloon diameter / annulus size ratio in the present study varied from 1.2 in older age group to 1.5 among infants . The disruption of the annulus of the pulmonary valve may lead to hemorrhage into the pericardial sac and subsequent tamponade, which is why the choice of the right diameter of balloon is so important . It is best to choose the balloon according to data obtained from echocardiography and angiocardiography.14 werynski et al studied 137 children with isolated pulmonary stenosis who underwent valvuloplasty . The balloon diameter to pulmonary valve annulus ratio was 1.3 in their study and complications were seen in 3.6% of the patients, including one case of a balloon being lodged in the iliac vein . Mild pulmonary valve insufficiency was a common finding in the patients of the present study . It is associated with the diameter of balloons used during the intervention . According to literature, the occurrence of this problem ranges 10%50% of patients with pulmonary valve stenosis who underwent surgical treatment or balloon valvuloplasty . For many years the summary data suggest that in long - term observation, serious insufficiency is not well tolerated . In a polish study, none of patients needed reintervention and in the long - term observation there was no insufficiency of the pulmonary valve> ii. But all the patients undergoing balloon valvuloplasty in that study had isolated pulmonary stenosis . New mild pulmonary insufficiency was noted in 28% after pulmonary balloon valvuloplasty in a long - term assessment by fawzy et al.15 like the present study, most studies have used echocardiographic assessments . However, doppler echocardiography tends to overestimate the transvalvular gradient of systolic pressure in mild cases of pulmonary valve stenosis, in comparison to hemodynamic assessment . The study reveals that balloon valvuloplasty can be a useful and effective treatment for pulmonary stenosis in all childhood age groups . It was found that failure may not be uncommon and can lead to a subsequent surgery . Also, recurrences of the stenosis should be expected and repeated valvuloplasty may be inevitable.
The online version of this article (doi:10.1007/s10654 - 012 - 9749 - 8) contains supplementary material, which is available to authorized users . Complications may occur during pregnancy (e.g. Hypertension, thromboembolism, diabetes) and during labor (e.g. Fetal distress, dystocia and instrumental delivery / cesarean section). It is well known that obesity reduces the likelihood of a successful result of assisted reproduction (art) [3, 4]. Miscarriages, particularly early in the pregnancy, are frequent, but the relationship between bmi and the risk of miscarriages among women in the general population is not established as both underweight and obesity have been reported to increase the risk of miscarriages [612]. Brewer and balen have recently reviewed how obesity affects adversely both conception and implantation . Time to pregnancy is longer and fecundity lower in obese women than in women at optimal weight [1316]. Some previous studies has indicated that obesity as young women is associated with ovulatory infertility and menstrual problems later in life also caffeine containing beverages may be a risk factor, although the evidence is weak . Such life style factors may confound or modify the relationship between body mass and reproductive health . The aim of this study was to investigate, in a population of 46,000 american women aged 40 and above, how bmi (both low and high) at age 20 influences the frequency of reporting miscarriages, irregular periods or failing to become pregnant even if trying to get pregnant for one straight year or more . The large number of women included facilitates the investigation of effects of underweight (body mass index <18.5 kg / m) as well as obesity (both body mass index 3032.4 kg / m and body mass index 32.5 kg / m). The women were members of the adventist church, thus a large proportion had never (thus also during the childbearing years) smoked or used alcohol . Furthermore, the consumption of caffeine containing beverages (coffee or soft drinks) was low with approximately two - thirds never consuming this or using it less frequently than once a month . Adventist church members living in the usa and canada, aged 30 years and more, were included in the adventist health study-2 (ahs-2). More than 96,000 participants completed the lifestyle questionnaire which took 13 h to complete . The adventists church encourages a healthy life style with no smoking and alcohol consumption and advises members to follow a vegetarian diet . The comprehensive self - administered questionnaire included sections for medical history, diet, physical activity, supplement use and vegetarian food consumption . Information on marital status, ethnic group and lifestyle variables like smoking and the use of alcohol and caffeine containing beverages were available . Only 0.3% of the women reported living in a common law marriage, and these women were in the stratified analyses included in the group of ever married women . The female history section included information about, among other topics, menarche, irregular menstruation and difficulties in becoming pregnant (at different points during the life of the women) and the outcome of the pregnancies (including miscarriages / stillbirths, ectopic pregnancies, elective abortion and live births), and the use of oral contraceptives . There were also simple questions about current weight and height as well as weight when aged 20 . Body mass index (bmi) was computed as weight in kilograms divided by the square of height in meters (kg / m). The three dependent variables considered in our study were ever having experienced a miscarriage, menstrual irregularities and failing to become pregnant even if trying for one straight year . The women were asked to state the number of miscarriages or stillbirths she had experienced . In the main analyses, we dichotomized this information into ever / never having experienced this pregnancy outcome . Menstrual irregularities was considered present if the women answered yes to the question have your periods ever had much reduced flow, become irregular or stopped completely for at least 6 months? Do not count during or after menopause, or when you were pregnant, or nursing a child . If the women indicated that this happened before the age of 20 only, we did not include her in the group of women with menstrual irregularities . The women answered another question about problems with becoming pregnant: did you ever try for one straight year or more to become pregnant and, during that time, not become pregnant? If the women indicated that this happened before the age of 20 only or that the only reason for the problem was that the husband had fertility problem, we did not include her in the group of women with problems becoming pregnant . Bmi was categorized into 6 groups: bmi <18.5, 18.5 bmi <20, 20 bmi <25, 25 bmi <30, 30 bmi <32.5 and bmi 32.5 . These groups are in accordance with the main groups recommended by the who for classification of underweight, normal weight, overweight and obesity (<18.5, 18.524.9, 2529.9, 30), but the present classification is more detailed . The present analyses were limited to 54,369 women who were between the age of 40 and 99 at enrolment . The following missing data led to exclusions: ever having been pregnant was missing for 183 women, information regarding marital status, a key determinant of childbearing, was missing for 1,102 women; an additional 4,727 women had missing information regarding bmi at age 20 . We also excluded 808 women with estimated bmi lower than 16.0 kg / m or higher than 60.0 kg / m as these were considered to either reflect incorrect self - reported data concerning weight or height or severe illness . In some situations (24% of the women), the information from the women was missing with regard to the three dependent variables . Thus, the number of women included in the analyses varied between 45,701 regarding information concerning irregular periods to 46,582 for information on having tried for one straight year or more to become pregnant but not having become pregnant . In addition to age when completing the questionnaire (5 year age groups) and marital status (7 groups), the following variables were considered as possible confounders of the relationship between bmi at age 20 and the three different indicators of fertility problems: ethnic group (blacks vs. other), level of education, age at menarche, extended use of oral contraceptives (here defined as having used oral contraceptives for 7 or more years both when aged 2029 and when aged 3039), parity, ever smoked and ever regularly used alcohol as well as monthly or more frequently use of caffeine containing beverages . The statistical analyses included simple cross - tabulations, analyses of variance and multiple logistic regression analyses . Stratified analyses were conducted in order to control for confounding and evaluating possible effect modification . The p values in the tables test the hypothesis of any difference according to bmi (in 6 categories) rather than a linear trend over bmi categories . In some situations, we also tested for a u - formed relationship, including also a quadratic term in the model . The mean age (standard deviation) of the women at enrollment was 59.9 (14.7). Overall, 30.6, 14.1 and 16.5% reported miscarriages, irregular periods, and problems becoming pregnant, respectively . Table 1 displays the associations between bmi at age 20 and some relevant variables which may be associated with bmi . Women who were obese at age 20 were more likely to be relatively younger when completing the lifestyle questionnaire, never to have been married, to have relatively low education, to be black, and to have early menarche and have relatively low parity . Only 1 and 6%, respectively, of the women were current users of tobacco or alcohol . Monthly use of caffeinated beverages as well as ever use of tobacco or alcohol was associated with obesity.table 1unadjusted relationships between body mass at age 20 and demographic, reproductive and life style variables . Mean values (sd) or percentagesbody mass index (kg / m) at age 20n<18.518.519.92024.92529.93032.432.5p valuenumber of women47,5496,815 (14.3)10,213 (21.5)25,866 (54.4)3,539 (7.4)506 (1.1)610 (1.3)age at enrolment47,54958.5 (11.9)59.2 (12.3)60.8 (12.9)59.4 (13.3)57.0 (12.8)54.8 (11.2)<0.0001% ever married47,54994.695.694.890.687.782.3<0.0001% with college degree47,12233.134.732.528.523.525.5<0.0001% blacks47,00230.424.223.430.433.835.9<0.0001age at menarche47,22712.9 (1.6)12.7 (1.6)12.5 (1.5)12.2 (1.6)12.1 (1.7)11.7 (1.6)<0.0001live births46,3342.3 (1.7)2.3 (1.6)2.4 (1.7)2.3 (1.8)2.1 (1.8)1.8 (1.9)<0.0001% extended oc use46,9722.12.01.81.72.62.00.33% who consume caffeinated drinks44,91232.735.735.638.544.242.0<0.0001% ever smoked47,25517.016.716.419.527.731.5<0.0001% ever used alcohol47,16136.037.336.040.649.151.5<0.0001used oral contraceptives (oc) for 7 or more years both when aged 2029 and when aged 3039 unadjusted relationships between body mass at age 20 and demographic, reproductive and life style variables . Mean values (sd) or percentages used oral contraceptives (oc) for 7 or more years both when aged 2029 and when aged 3039 after adjustments for age and marital status, those reporting ever to have experienced a miscarriage had increased odds of failing to become pregnant even if trying for one straight year; odds ratio (or) 1.51 (95% ci: 1.431.59). Increasing number of miscarriages (1, 2 and> 2) was linearly related to the odds of reporting failing to become pregnant (or = 1.35 (95% ci: 1.271.44), 1.63 (95% ci: 1.491.79) and 2.37 (95% ci: 2.122.64)), respectively compared to the risk in women with no miscarriages). Also ever experienced irregular periods was positively related to failing to become pregnant even if trying for one straight year; or = 1.72 (95% ci: 1.611.83), but there was very little relationship between the experience of a miscarriage and the likelihood of reporting irregular periods (results not shown in tables) underweight or obesity at age 20 did not have any bearing on the risk of this pregnancy outcome (p = 0.16). When adjusted for age when completing the questionnaire and marital status, the odds for a hysterectomy before the age of 40 (15% of the women indicated this) was approximately 35% higher in obese women than in women with normal weight (p = 0.003), but after additional adjustments, for education and ethnic group, this relationship was no longer statistically significant (p = 0.06) (results not shown in the table). Table 2 gives the relationships between bmi at age 20 and the likelihood of reporting at least one miscarriage, irregular periods or failing to become pregnant even if trying for one straight year . The first line represents results of analyses are adjusted for age when completing the questionnaire and marital status, the next two lines when adjusted for an increasing number of possible confounders.table 2relationships between body mass index at age 20 and the likelihood of having experienced a miscarriage, irregular periods or failing to become pregnant even if trying for one straight yearbody mass index (kg / m)n<18.518.519.92024.92529.93032.432.5p valuedependent variableever experienced miscarriage miscarriage46,3341.06 (1.00, 1.12)1.02 (0.97, 1.07)1.001.11 (1.03, 1.20)1.11 (0.92, 1.35)0.97 (0.80, 1.16)0.06 miscarriage44,9751.04 (0.98, 1.10)1.02 (0.97, 1.07)1.001.07 (0.99, 1.16)1.04 (0.85, 1.27)0.92 (0.76, 1.11)0.46 miscarriage42,9791.04 (0.98, 1.11)1.02 (0.97, 1.08)1.001.05 (0.97, 1.14)1.01 (0.82, 1.24)0.87 (0.72, 1.06)0.40menstrual irregularities menstrual irregularities45,7011.03 (0.95, 1.12)0.98 (0.91, 1.04)1.001.18 (1.07, 1.31)1.89 (1.53, 2.33)1.98 (1.64, 2.39)<0.0001 menstrual irregularities44,4291.03 (0.95, 1.12)0.97 (0.90, 1.04)1.001.16 (1.05, 1.28)1.84 (1.49, 2.28)1.91 (1.58, 2.31)<0.0001 menstrual irregularities42,9791.04 (0.96, 1.13)0.97 (0.90, 1.04)1.001.16 (1.04, 1.29)1.79 (1.44, 2.23)1.87 (1.54, 2.27)<0.0001problems with becoming pregnant problems with becoming pregnant46,5821.16 (1.08, 1.25)1.13 (1.06, 1.20)1.001.11 (1.01, 1.22)1.46 (1.17, 1.82)1.55 (1.26, 1.90)<0.0001 problems with becoming pregnant45,2461.14 (1.06, 1.23)1.12 (1.05, 1.20)1.001.11 (1.00, 1.22)1.49 (1.19, 1.87)1.53 (1.25, 1.89)<0.0001 problems with becoming pregnant42,9791.13 (1.04, 1.22)1.13 (1.06, 1.21)1.001.07 (0.97, 1.19)1.36 (1.08, 1.72)1.36 (1.10, 1.69)<0.0001odds ratio (95% ci). Adjusted for age when filling in the questionnaire and marital statusalso adjusted for ever smoking, ever use of alcohol, ethnic background (blacks vs. other) and level of educationalso adjusted for ever smoking, ever use of alcohol, ethnic background (blacks vs. other), level of education as well as problems with becoming pregnant, menstrual irregularities and age at menarchealso adjusted for ever smoking, ever use of alcohol, ethnic background (blacks vs. other), level of education as well as experienced a miscarriage, problems with becoming pregnant and age at menarchealso adjusted for ever smoking, ever use of alcohol, ethnic background (blacks vs. other), level of education as well as experienced a miscarriage, menstrual irregularities and age at menarche relationships between body mass index at age 20 and the likelihood of having experienced a miscarriage, irregular periods or failing to become pregnant even if trying for one straight year odds ratio (95% ci). Adjusted for age when filling in the questionnaire and marital status also adjusted for ever smoking, ever use of alcohol, ethnic background (blacks vs. other) and level of education also adjusted for ever smoking, ever use of alcohol, ethnic background (blacks vs. other), level of education as well as problems with becoming pregnant, menstrual irregularities and age at menarche also adjusted for ever smoking, ever use of alcohol, ethnic background (blacks vs. other), level of education as well as experienced a miscarriage, problems with becoming pregnant and age at menarche also adjusted for ever smoking, ever use of alcohol, ethnic background (blacks vs. other), level of education as well as experienced a miscarriage, menstrual irregularities and age at menarche no relationship was found for the risk of any miscarriage . Furthermore, we found no linear relationship between body mass index at age 20 and the number of miscarriages (not shown in the table). Women with bmi 32.5 kg / m at age 20 had approximately 2.0 and 1.5 higher odds for irregular menstruation or failing to get pregnant, respectively, than women with bmi in the 2024.9 kg / m bracket . Underweight (bmi <18.5 kg / m) when aged 20 marginally (approximately 15%) increased the risk of failing to get pregnant within a year (p value for quadratic term <0.001). Also for irregular menstruation, a u - formed relationship was statistically significant (p <0.001), but the increased risk associated with underweight was negligible . Adjustments for ever smoking, ever use of alcohol, ethnic background (blacks vs. other) and education in addition to age and marital status had little impact on these relationships (table 2). Further adjustments for age at menarche as well ever experienced one of the two other dependent variables included in our analyses did not explain the statistically significantly increased risk associated with obesity . However, the increased risk of failing to become pregnant associated with both underweight and obesity was attenuated with the last set of adjustments (table 2), but even fully adjusted (line 3), there was a statistically significant (p <0.001) u - formed relationship between body mass index at age 20 and problems of becoming pregnant . It may, however, be debatable whether it is correct to adjust for miscarriages and irregular periods when assessing the relationship between body mass index at age 20 and failing to become pregnant, as these variables may be considered intermediary . Thus, the impact of these two variables on the relationship with failing to become pregnant was assessed in a separate analysis . When adjusted for irregular periods and miscarriages in addition to smoking, alcohol, ethnic background, education and age at menarche (including 42,979 women in both analyses), the odds ratio associated with obesity (bmi 30 some of the results regarding failing to become pregnant are presented in table 3 . As evident, the displayed relationship did not depend on the age when completing the questionnaire (p value for interaction = 0.9), or whether the women had ever been married (p value for interaction = 0.6). The relationship may be somewhat weaker in blacks than in other ethnic groups (p value for interaction = 0.03).table 3stratified analyses of the relationships between body mass index at age 20 and having experienced problems getting pregnantbody mass index (kg / m) at age 20n<18.518.519.92024.92529.93032.432.5p valueall women46,5821.16 (1.08, 1.25)1.13 (1.06, 1.20)1.001.11 (1.01, 1.22)1.46 (1.17, 1.82)1.55 (1.26, 1.90)<0.0001age (years) 405418,5141.21 (1.09, 1.35)1.08 (0.98, 1.19)1.001.23 (1.07, 1.42)1.56 (1.15, 2.12)1.55 (1.18, 2.04)<0.0001 556916,7951.11 (0.98, 1.25)1.10 (1.00, 1.23)1.000.95 (0.79, 1.13)1.42 (0.96, 2.10)1.60 (1.12, 2.28)0.014 70 + 11,2731.15 (0.97, 1.35)1.28 (1.13, 1.46)1.001.10 (0.90, 1.34)1.26 (0.73, 2.17)1.31 (0.72, 2.41)0.008married never2,6071.31 (0.81, 2.12)0.76 (0.45, 1.30)1.001.27 (0.77, 2.09)1.49 (0.58, 3.85)1.64 (0.81, 3.31)0.33 ever43,9751.16 (1.08, 1.25)1.14 (1.07, 1.21)1.001.10 (1.00, 1.21)1.45 (1.16, 1.83)1.53 (1.23, 1.89)<0.0001ethnic group blacks11,5831.12 (0.97, 1.28)0.97 (0.85, 1.10)1.001.22 (1.03, 1.46)1.43 (0.97, 2.11)1.29 (0.90, 1.85)0.03 other34,4891.17 (1.07, 1.27)1.19 (1.11, 1.27)1.001.07 (0.95, 1.20)1.47 (1.12, 1.94)1.69 (1.31, 2.17)<0.0001parity 07,3181.33 (1.13, 1.55)1.11 (0.96, 1.28)1.001.14 (0.93, 1.39)1.52 (0.98, 2.35)1.42 (0.99, 2.04)0.004 1 + 38,1181.10 (1.01, 1.19)1.13 (1.05, 1.21)1.001.08 (0.96, 1.21)1.31 (1.00, 1.72)1.40 (1.08, 1.81)0.0008smoking never38,3771.18 (1.09, 1.28)1.17 (1.10, 1.26)1.001.14 (1.02, 1.27)1.55 (1.19, 2.01)1.38 (1.07, 1.80)<0.0001 ever7,9441.05 (0.89, 1.25)0.93 (0.80, 1.09)1.000.97 (0.78, 1.21)1.26 (0.83, 1.92)1.77 (1.26, 2.48)0.014alcohol never29,0491.17 (1.07, 1.28)1.17 (1.09, 1.27)1.001.14 (1.01, 1.29)1.44 (1.05, 1.98)1.49 (1.10, 2.01)<0.0001 ever17,1941.14 (1.01, 1.28)1.05 (0.94, 1.16)1.001.05 (0.91, 1.23)1.49 (1.10, 2.03)1.60 (1.21, 2.11)0.002never used tobacco or alcohol27,8911.18 (1.08, 1.30)1.19 (1.10, 1.29)1.001.15 (1.02, 1.31)1.44 (1.04, 2.02)1.35 (0.96, 1.88)<0.0001coffee / caffeine containing soft drinks <monthly28,3751.15 (1.05, 1.26)1.16 (1.07, 1.25)1.001.18 (1.04, 1.34)1.59 (1.18, 2.15)1.55 (1.17, 2.06)<0.0001 monthly or more often15,7871.18 (1.05, 1.34)1.09 (0.98, 1.21)1.000.96 (0.81, 1.13)1.49 (1.06, 2.09)1.66 (1.22, 2.26)0.0005odds ratio (95% ci). Adjusted for age when filling in the questionnaire and marital status stratified analyses of the relationships between body mass index at age 20 and having experienced problems getting pregnant odds ratio (95% ci). Adjusted for age when filling in the questionnaire and marital status of particulate interest in this population are associations in never smokers and women who have never used alcohol . Table 3 demonstrates that the relationships displayed in table 2 were found in women who had abstained from smoking for their entire life and in women who had not, although the relationship may be somewhat stronger in the latter group (p value for interaction = 0.04). The association was the same in women who were lifelong abstainers from alcohol and other women (p value for interaction = 0.5) and was also found in the 27,891 women who denied ever having used either stimulant (alcohol and tobacco). There was in addition a seemingly interaction for age at menarche, but the relationships for women with early and late menarche were the same (p - value for interaction = 0.9) if the two categories of obesity were merged . The same stratified analyses were conducted also for irregular periods and miscarriages, and the relationships displayed in table 2 were found consistently in the different strata of the population . For irregular periods, there were no indications of any significant interactions . For miscarriages, we found a weak u - formed (p = 0.003) relationship in parous women which was unaffected by further adjustments for parity . We refer to web appendix (web tables 13) for more detailed presentation of stratified analyses . Obesity at age 20 years was in this large study of women associated with increased risk of irregular periods and failing to become pregnant even if trying for one straight year, but not with the risk of experiencing at least one miscarriage . As we are exploring relationships between body mass index at age 20 and reproductive problems, we have restricted the analytical sample to women at an age (aged 40 and above) when they most likely will have experienced reproductive problems if they will ever do so, particularly if these problems should have clinical consequences . As detailed above, we did not include irregular periods or failing be become pregnant before the age of 20 as an outcome in the study, only problems after the age of 20 . We do not, however, know when the women experienced her (first) miscarriage; this may have happened as a teenager . Due to the mean age of the included women (nearly 60 years), modern treatment for infertility (like in vitro fertilization) has played a minor role for our findings . The relationship between bmi at age 20 and irregular periods and problems of becoming pregnant may be explained by an increased risk of oligo- and anovulation in obese women and a number of other adverse effects of obesity on reproductive physiology in women [13, 25]. Our results support previous findings of a u - formed relationship between body mass index at age 23 and problems of becoming pregnant and menstrual problems before age 33 . Data from a self - administered questionnaire hamper the possibilities for further discussion with regard to etiology . One explanation may be that a relatively high percentage of the obese women may have had polycystic ovary syndrome (pcos). Polycystic ovary syndrome affects 510% of women in a general population, and many (at least one out of three) of the patients with pcos are obese [26, 27]. No relationship was found between obesity and the odds of reporting one or more miscarriages . Previous population - based studies have not given a consistent picture with regard to body mass index as a risk factor for miscarriages; underweight may be just as important as obesity [612]. Traditionally, psos has been thought to play a major role also for the risk of recurrent miscarriages, but this has recently been questioned . The lack of relationship between body mass at age 20 and miscarriages may to some extent be due to misclassification as early miscarriages often are overlooked and any relationship will tend to be attenuated . However, the positive, direct relationship between the number of miscarriages and the odds of reporting failing to become pregnant indicates that information about the miscarriages has some validity and may further suggest that some women have interpreted the question did you ever try for one straight year or more to become pregnant and, during that time, not become pregnant? Less precisely than was the intention, answering that they for one straight year or more were not able to get pregnant or give birth to a live born child . Given that there is no, or only a very weak, relationship between obesity and the risk of a miscarriage and that some women have interpreted the question as suggested above, this points to a possible stronger relationship between obesity (and possible underweight) and failure to become pregnant than the results presented in table 2 may indicate . Whereas irregular periods and failing to become pregnant reflect problems of becoming pregnant, the women who have a miscarriage have conceived and are therefore fertile . Thus, according to our findings, weight may be more important for becoming pregnant than for remaining pregnant . When the relationship with failing to become pregnant was adjusted for irregular periods and miscarriages, the odds ratio associated with obesity was attenuated . However, it is debatable whether it is correct to adjust for these variables which most likely are on the causal pathway . If not, obesity (bmi 30 kg / m) increases the odds of having problems of becoming pregnant with approximately 45%, even after adjustments for other likely confounders . Table 1 shows that women who were obese at age 20 were less likely to ever have been married . We adjusted for marital status in all analyses (table 2) and the stratified analyses by ever married status (table 3) clearly demonstrate that the relationship we found is not due to women who never were married and therefore may not have tried to become pregnant . One might assume that experiencing irregular periods, the variable most strongly related to obesity, was independent of marital status, but the risk was found to be higher in never married women . Thus, the higher odds for reporting problems of becoming pregnant in obese women may be due to obesity in the male partner . It is however unlikely that the positive (and stronger) association (table 2) between obesity and irregular periods is related to male obesity . The information from the women did not make it possible to differentiate between a miscarriage (a spontaneous loss of a fetus before the 20th week of pregnancy) and a stillbirth (a delivery after 20 completed weeks gestation of a fetus showing no signs of life). The former is much more frequent, and our results will pertain largely to miscarriages . . Found that 31% of pregnancies were lost, two out of three before the pregnancy was detected clinically . Currently <1% of all pregnancies in the us end as a stillbirth, but the percentage is higher in blacks than in other ethnic groups . The risk of a stillbirth was higher during the childbearing years of the women included in our analysis, though . Obesity has in most studies been found to increase the risk of stillbirths [32, 33]. We have used this height when computing the body mass earlier in life, at age 20 . However, as the associations we found were basically independent of age at enrollment (aged 4054, 5569, or 70, and thus time since the women were 20 years old), little bias is introduced when applying current height when computing bmi earlier in life . The main weakness of our study is that weight is self - reported and recalled . Underweight women tend to overestimate the self - reported weight whereas obese women underestimate it . Thus, in women with bmi <18.5 kg / m, the reported bmi is probably higher than the true bmi and the opposite is true for obese women . However, the most important in our context is the ability to rank the women according to bmi and measured and self - reported bmi has been found to be highly correlated (rs = 0.94) in this population as in the previous adventist health study (ahs-1). The mean age at enrolment was 59.9 years, and the women were asked to recall their weight nearly 40 years earlier, at age 20 . Data from the nurses health study indicate that women are able to recall their weight at age 18, the correlation coefficient between recalled and measured weight was 0.87 . The women in the nhs cohort (aged 2542) were, however, significantly younger than in our study . Data from women who took part in both this adventist health study (ahs-2) and the former one (ahs-1 in 1976) demonstrate strong correlations (r = 0.82 for women of all ages) between recalled weight in the 1970s and weight stated in the questionnaires in 1976 . Thus, misclassification of recalled bmi at least in terms of relative rank appears to be quite small for recall of 2530 years . We do not find it likely that our results can be explained by differential recall of weight at age 20 as this would imply a strong correlation between reporting problems of becoming pregnant or, in particular, irregular periods and falsely recalled overweight and obesity when aged 20 years old . It is probable that a more relevant measure is the percentage of body fat, a measure that was strongly correlated (r = 0.84) with bmi in us women aged 2039 . Information about adipose tissue distribution, like waist circumference or waist / hip - ratio, may have given additional information, although the correlation between bmi and waist circumference in relatively young women is high (r = 0.93) according to recent nhanes data . One possible source of bias would be that women who complete the lifestyle questionnaire are survivors . Obese, relatively young, women have higher mortality than women with normal weight [40, 41]. However, the mortality in women aged less than 40 is low, particularly in this relatively healthy group of subjects with low smoking prevalence, and the relationships did not depend on the age of the women when completing the questionnaire (table 3 and web tables 13). Thus, it is unlikely that survival bias has impacted on our results to any measurable degree . The prevalence of obesity at age 20 (2.4%) is relatively low, but it is for instance similar to the prevalence of obesity in women included in the swan cohort which was based on women aged 1718 years old in the late 1960s, furthermore, the study population is somewhat selected as all the women were adventists . It could be that obesity is associated with irregular periods and miscarriages differently in this group of women than in the general population . However, we find this unlikely and the stratified analyses did not suggest any interaction with lifestyle . It is large in terms of women included, which has allowed detailed stratified analyses . The main findings were found to be very consistent in the different strata of the population . Another related strength is that this study has been conducted in a rather unique us population with a relatively high proportion of women who have abstained from alcohol and smoking for their entire life . Additionally, 25% of the analytical population are black adventists of us and caribbean origin; approximately 90% of the remaining 75% are white, non - hispanic women . Both underweight and obesity after adjustment for age and marital status and compared to other women, black women were only slightly more likely to report irregular periods (2%) or failing to become pregnant even if trying for least 1 year (6%). However, blacks were more likely to report at least one miscarriage [or = 1.34 (95% ci: 1.271.40)]. However, as detailed in table 3 and the web appendix, stratified analyses demonstrate that there are few indications that ethnicity has influenced our findings significantly . In summary, this large study found that women who were obese when they were 20 years old were at a significantly increased risk of failing to become pregnant even if trying for one straight year . One of the explanations for this seems to be that obese women have difficulties to conceive due to irregular periods, rather than increased risk of miscarriages . Supplementary material 1 (docx 36 kb) supplementary material 1 (docx 36 kb)
The incidence of acute pancreatitis is increasing around the world, where it is associated with morbidity and even the risk of mortality . It may presents with various skin manifestations as rash and rarely, with erythematous tender subcutaneous nodules, known as pancreatic panniculitis . Alcohol and gallstones are the most important causes, while routinely prescribed drugs have been linked to the causation of acute pancreatitis . Metformin, one of the most widely prescribed oral hypoglycemic agents, was linked to pancreatitis, secondary to overdose or in case of impaired renal function . Here, we report a case of metformin induce acute pancreatitis in young healthy man with normal renal function . Nineteen year - old - man, known case of type 2 diabetes mellitus for 4 y on 1 g metformin twice daily since diagnosis of his diabetes . He was in his usual state of health till he presented to the emergency department reporting nausea, vomiting and epigastric pain for 3 d. on physical examination, his height was 170 cm and body weight 99 kg; body mass index (bmi) 34.3 kg / m2, looked mildly dehydrated . Laboratory investigations showed hba1c 7.7%, creatinine 58 mol / l, amylase 462 units / l (normal range <100), lipase 1378 units / l (060), white blood cells 16.8/mm3 (411) 80% of which was neutrophils, crp 258 mg / l (05), mg 0.76 mmol / l (0.71.05), ca 2.17 mmol / l (2.22.6), ast 18 units / l (<39), alt 34 units / l (<41), tg 0.95 mmol / l (<2.3), lactate 1.4 mmol / l (0.51.6). Abdominal ultrasound and ercp were done for the patient, results showed no gallstones and clear biliary tract, respectively . Normalization of amylase and lipase was reached after metformin cessation, and supportive treatment in the form of iv insulin and iv fluids . Few days later, after re - exposure to metformin, he presented with recurrence of his previous symptoms, and elevation of amylase and lipase was documented . As a result, metformin was suspended with improvement of his symptoms and biochemical profile . Metformin is one of the most effective and valuable oral hypoglycemic agents in the biguanide class . It has been selected as the drug of choice in management of majority of patients with type 2 diabetes mellitus, as it works by several mechanisms; decreasing hepatic gluconeogenesis, increasing glucose transport into glucose utilizing cells, decreasing appetite and caloric intake and reducing intestinal glucose absorption . As any other medication, metformin is associated with undesirable side effects and it has been reported in few cases to cause lactic acidosis and pancreatitis . Acute pancreatitis is attributed to many etiological factors; one of them is medication, where a large number of single case reports on drug - induced pancreatitis were published . Diuretics, antimicrobial agents, hiv therapy and neuropsychiatric agents as well as metformin are known medication to induce pancreatitis . The underlying pathogenesis in drug induced pancreatitis in some medications as codeine was due to spasm of sphincter of oddi, in others as azathioprine, immune- mediated mechanism or a hypersensitivity to the drug is the postulated pathogenesis . In metformin, the exact mechanism is not known, but toxicity is probably secondary to acinar cell injury leading to intercellular leakage of digestive enzymes from ductules . Fortunately, drug - induced pancreatitis is an acute edematous pancreatitis of a benign course and good prognosis, but fatal outcome may ensue if proper management is not initiated . Among the published case reports of metformin induced pancreatitis, one was attributed to metformin overdose, other was caused by metformin accumulation, resulting from combination of drug overdose and acute renal failure triggered by vomiting in patient with concealed renal insufficiency . Additionally, two case reports were found to be associated with drug induced (angiotensin converting enzyme inhibitor, angiotensin receptor blocker, non - steroidal anti - inflammatory drug as ibuprofen and celecoxib and diuretics) renal failure, which reduced excretion of therapeutic dose of metformin causing toxicity, leading to sever lactic acidosis and pancreatitis . Lastly, in a case report of patient presented with hyperglycemia after an ingested dose between 64- 85 g of metformin in a suicide attempt, the potential mechanism of hyperglycemia is not clear and nothing among metformin s known mechanisms would logically explain the progressive and severe hyperglycemia, pancreatitis remains a potential mechanism, as the patient s clinical presentation with reportts vomiting and abdominal pain is consistent with pancreatitis . In our case, several signs can guide to and support the diagnosis of metformin induced pancreatitis; first is the clinical presentation of abdominal pain, nausea, vomiting and dehydration . Second, high levels of lipase and amylase, which correlate positively with ct findings . The lack of other known causes of acute pancreatitis such as gall stones, alcohol abuse, hypercalcaemia, hypertriglyceridaemia, and trauma, added more evidence to the diagnosis . Apart from the therapeutic dose of metformin (2 g daily), our patient has never consumed any other medications known to cause pancreatitis . Finally, resolution of his symptoms upon metformin cessation and relapse upon re - exposure would strongly suggest that metformin is deemed responsible of our patient s clinical presentation . Available evidence suggests that acute pancreatitis in our patient was probably precipitated by therapeutic dose of metformin with normal renal function, with unknown exact mechanism . In summary, our case demonstrates the possibility of metformin to induce pancreatitis in a healthy patient without preexisting renal disease . Accordingly, every diabetic patient on metformin should be counseled about symptoms of acute pancreatitis and the urgency to discontinue metformin and visit the emergency department to receive the appropriate treatment . And for us as physicians, metformin should be kept in back of our minds as a cause that would contribute to acute pancreatitis even in a healthy patient.
Alzheimer's disease (ad) is a neurodegenerative disorder which causes a progressive and irreversible loss of cognition and physical function . The number of ad patients will increase sharply in the near future, and it has been estimated that over 80 million individuals will be diagnosed with this condition by the year 2040 . To date, no cure is available to modify the progression of ad, although available pharmacological treatments have been proven to positively control cognitive and functional symptoms of the disease and improve quality of life of patients and their families for the time they are responsive to treatment [3 .] Most often, care for ad patients is provided by informal caregivers including relatives and friends . Caregivers are individuals who are called to assist patients in their activities of daily living, to administer medications to them and to deal with the patients progressive personality change and physical, cognitive and emotional deterioration . Such aspects make providing care to people with ad a burdensome experience which affects the physical and psychological state of caregivers and their quality of life . Managing and administering medications to ad patients may substantially contribute to the caregivers workload and distress . Compliance to treatments and efficacy of medications may depend on how well caregivers are able to manage drug administrations . The caregivers satisfaction with treatments is crucial for a good compliance to treatments, and it has been associated with an increased likelihood of positive clinical outcomes, decreased need of sedatives and anxiolytics, and delayed patient institutionalization . Currently, there are four medications approved for the treatment of ad by regulatory authorities . Three are acetylcholinesterase inhibitors (acheis) including donepezil, rivastigmine and galantamine; the fourth is memantine, an n - methyl - d - aspartic acid receptor antagonist . Since the rivastigmine patch has been marketed, experimental data from randomized clinical trials have suggested that the transdermal application would be preferred by caregivers of ad patients compared to oral medications . To date, no information is available regarding patient compliance to treatment and caregiver satisfaction with respect to different formulations of ad medications in a real clinical setting . The present study aimed at comparing compliance to treatment of community - dwelling patients with mild - to - moderate ad treated with transdermal application compared to oral formulation drugs and caregiver satisfaction in a real clinical setting . The secondary purpose of the study was to provide a full description of sociodemographic and clinical characteristics of mild - to - moderate ad patients and to draw a sociodemographic profile of their caregivers . A sub - study was conducted to perform the linguistic and psychometric validation of the caregiver medication interview (cmi), which is the instrument adopted in the current investigation to assess patient compliance to treatment and caregiver satisfaction . The axept study consisted of a main study, which was conducted to primarily compare patient compliance to treatment and caregiver satisfaction with respect to oral and transdermal formulations of anti - ad medications, and a sub - study, which was conducted to validate the cmi instrument . The main study had an observational, cross - sectional, multicenter design . In the sub - study, a sample of participants was observed longitudinally and assessed with the cmi at the baseline visit and 1 week later (follow - up visit) to measure the psychometric properties of this instrument . The study was conducted in 45 memory clinics uniformly distributed throughout italy between september 8, 2010 and january 31, 2011 . Patients with ad were considered eligible for participation if: (a) they had a mini - mental state examination (mmse) score between 18 and 26 at the screening visit; (b) they were 18 years old or more; (c) they signed an informed consent, and (d) they were treated either with acheis or memantine in monotherapy regimens, and such treatment was initiated 46 months prior the baseline visit as either the first anti - ad treatment ever received or a new drug option after switching from an old treatment . All the above - mentioned inclusion criteria had to be met for patients to be enrolled in the study . Patients were excluded from the study if they were treated with a combination of acheis or with acheis plus memantine . Caregivers had to be present at the baseline visit and had to provide care to the patients at least 4 h per day for at least 4 months prior to the baseline visit . They had to speak and understand the italian language and to be able to answer a questionnaire including a numeric rating scale . Participants in the main study were assessed at one point in time during the baseline visit . Participant assessment was conducted by site physicians who received specific training on the study procedures during a centralized investigator meeting . Information was collected by direct interviews with patients and caregivers and extracted from medical charts . The caregivers were asked about the daily amount of time spent providing care to patients . Information on the patients weight, height, blood pressure, heart rate, disease duration, swallowing difficulties, medical history and concomitant gastrointestinal or dermatological symptoms or diseases was also collected . The activities of daily living scale (adls), scoring from 0 (total dependence) to 6 (total independence), was used to evaluate the patients functional status . Data on the patients current anti - ad pharmacological treatment were recorded, including the drug ingredient, dose, formulation, start date of treatment and eventual reason for switching to a different medication . The patients use of concomitant medications with particular respect to psychotropic drugs was also assessed . The cmi is an ad hoc questionnaire developed for the purpose of the axept study . It was initially developed in the english version by the axept researchers as an instrument which could have been used internationally . It is available in two forms, one designed to be used in case of oral treatment and one designed to be used in case of transdermal applications . It consists of three questions directed to the caregiver and exploring the ease of administration, the global compliance and the satisfaction relative to the treatment the patient is receiving . For questions on compliance and satisfaction, the caregiver has to indicate, on a numeric rating scale, a score between 0 and 10, with 0 being the lowest degree and 10 the highest degree of compliance and satisfaction . Data collected from direct interviews with caregivers and from medical charts during the baseline visit were used to assess the characteristics of mild - to - moderate ad patients and their caregivers . According to the methods proposed by divine et al . For estimating the sample size for the wilcoxon - mann - whitney test, it has been calculated that a sample of 730 participants was required to observe a p(x> y) equal to 0.57 with a power of 0.90 and a 0.05 type i error . Given that x and y are random observations from the two distributions being compared (transdermal and oral formulation), p(x> y) represents the probability that x is greater than y. since previous data were missing, it has been conservatively assumed that p(x> y) equals 0.57, under the null hypothesis that p(x> y) equals 0.50 . A zero variance associated with ties and an allocation ratio of 3 to 4 between the transdermal and the oral formulation according to data on prescription patterns in italy have also been assumed . Characteristics of the study participants according to the type of drug formulation received were assessed using descriptive statistics . Compliance to treatment and caregiver satisfaction were described according to the type of drug formulation (oral or transdermal). Descriptive statistics included mean, standard deviation, median, mode, quantiles and interquartile range . Comparison between patient compliance and caregiver satisfaction according to the type of drug formulation was conducted using the wilcoxon - mann - whitney test . Statistical analysis was performed using sas v 9.1.2 . For the purpose of the current study, the english version of the cmi was translated into the italian language, and a linguistic and psychometric validation of the italian version was performed . The linguistic validation process included the following steps: forward translation of the english cmi into italian: two forward translations were produced by two independent professional translators who were native italian speakers; reconciliation: creation of a preliminary combined version (reconciled version) on which both translators and investigators agreed; backward translation of the italian reconciled version into english: the preliminary italian version was translated back into english by a professional translator who was a native english speaker and had no access to the original version of the instrument; comparison between the backward translated version with the original english version; mistranslations and inaccuracies were detected and the preliminary italian version was revised accordingly in a consensus meeting including the backward translator and the investigators; comprehension test: the italian revised version of the cmi was administered to a sample of 18 caregivers of patients who were potential study participants according to the inclusion criteria; these caregivers were identified from the three expected highest recruiting sites; this was a qualitative examination performed to determine possible problems in the respondents understanding of the instructions; creation of a final italian version by the investigators and implementation of this instrument in the study . The psychometric evaluation of the translated cmi was then conducted in a sub - sample of 188 caregivers participating in the study and selected by the six expected highest recruiting sites . The cmi was administered to the sub - study caregivers during the baseline evaluation and 1 week later . The reliability of the instrument was evaluated by measuring its internal consistency, expressed by cronbach's, and its test - retest reliability, expressed by the intraclass correlation coefficient (icc) and the spearman correlation coefficient (). No gold standard among the instruments which measure compliance to treatment and caregivers satisfaction is available in the literature . Therefore, the convergent validity was evaluated comparing the cmi scores with the responses obtained from the same caregivers who were administered with a visual analogue scale (vas). For the purpose of this study, a vas including a 10-cm line with one extreme indicating the lowest and the other extreme indicating the highest compliance and satisfaction was used . The icc and spearman's were measured to estimate the convergent validity of the italian version of the cmi compared with the vas . The axept study consisted of a main study, which was conducted to primarily compare patient compliance to treatment and caregiver satisfaction with respect to oral and transdermal formulations of anti - ad medications, and a sub - study, which was conducted to validate the cmi instrument . The main study had an observational, cross - sectional, multicenter design . In the sub - study, a sample of participants was observed longitudinally and assessed with the cmi at the baseline visit and 1 week later (follow - up visit) to measure the psychometric properties of this instrument . The study was conducted in 45 memory clinics uniformly distributed throughout italy between september 8, 2010 and january 31, 2011 . Patients with ad were considered eligible for participation if: (a) they had a mini - mental state examination (mmse) score between 18 and 26 at the screening visit; (b) they were 18 years old or more; (c) they signed an informed consent, and (d) they were treated either with acheis or memantine in monotherapy regimens, and such treatment was initiated 46 months prior the baseline visit as either the first anti - ad treatment ever received or a new drug option after switching from an old treatment . All the above - mentioned inclusion criteria had to be met for patients to be enrolled in the study . Patients were excluded from the study if they were treated with a combination of acheis or with acheis plus memantine . Caregivers had to be present at the baseline visit and had to provide care to the patients at least 4 h per day for at least 4 months prior to the baseline visit . They had to speak and understand the italian language and to be able to answer a questionnaire including a numeric rating scale . Participants in the main study were assessed at one point in time during the baseline visit . Participant assessment was conducted by site physicians who received specific training on the study procedures during a centralized investigator meeting . Information was collected by direct interviews with patients and caregivers and extracted from medical charts . The caregivers were asked about the daily amount of time spent providing care to patients . Information on the patients weight, height, blood pressure, heart rate, disease duration, swallowing difficulties, medical history and concomitant gastrointestinal or dermatological symptoms or diseases was also collected . The activities of daily living scale (adls), scoring from 0 (total dependence) to 6 (total independence), was used to evaluate the patients functional status . Data on the patients current anti - ad pharmacological treatment were recorded, including the drug ingredient, dose, formulation, start date of treatment and eventual reason for switching to a different medication . The patients use of concomitant medications with particular respect to psychotropic drugs was also assessed . The cmi is an ad hoc questionnaire developed for the purpose of the axept study . It was initially developed in the english version by the axept researchers as an instrument which could have been used internationally . It is available in two forms, one designed to be used in case of oral treatment and one designed to be used in case of transdermal applications . It consists of three questions directed to the caregiver and exploring the ease of administration, the global compliance and the satisfaction relative to the treatment the patient is receiving . For questions on compliance and satisfaction, the caregiver has to indicate, on a numeric rating scale, a score between 0 and 10, with 0 being the lowest degree and 10 the highest degree of compliance and satisfaction . Data collected from direct interviews with caregivers and from medical charts during the baseline visit were used to assess the characteristics of mild - to - moderate ad patients and their caregivers . According to the methods proposed by divine et al . For estimating the sample size for the wilcoxon - mann - whitney test, it has been calculated that a sample of 730 participants was required to observe a p(x> y) equal to 0.57 with a power of 0.90 and a 0.05 type i error . Given that x and y are random observations from the two distributions being compared (transdermal and oral formulation), p(x> y) represents the probability that x is greater than y. since previous data were missing, it has been conservatively assumed that p(x> y) equals 0.57, under the null hypothesis that p(x> y) equals 0.50 . A zero variance associated with ties and an allocation ratio of 3 to 4 between the transdermal and the oral formulation according to data on prescription patterns in italy have also been assumed . Characteristics of the study participants according to the type of drug formulation received were assessed using descriptive statistics . Compliance to treatment and caregiver satisfaction were described according to the type of drug formulation (oral or transdermal). Descriptive statistics included mean, standard deviation, median, mode, quantiles and interquartile range . Comparison between patient compliance and caregiver satisfaction according to the type of drug formulation was conducted using the wilcoxon - mann - whitney test . For the purpose of the current study, the english version of the cmi was translated into the italian language, and a linguistic and psychometric validation of the italian version was performed . The linguistic validation process included the following steps: forward translation of the english cmi into italian: two forward translations were produced by two independent professional translators who were native italian speakers; reconciliation: creation of a preliminary combined version (reconciled version) on which both translators and investigators agreed; backward translation of the italian reconciled version into english: the preliminary italian version was translated back into english by a professional translator who was a native english speaker and had no access to the original version of the instrument; comparison between the backward translated version with the original english version; mistranslations and inaccuracies were detected and the preliminary italian version was revised accordingly in a consensus meeting including the backward translator and the investigators; comprehension test: the italian revised version of the cmi was administered to a sample of 18 caregivers of patients who were potential study participants according to the inclusion criteria; these caregivers were identified from the three expected highest recruiting sites; this was a qualitative examination performed to determine possible problems in the respondents understanding of the instructions; creation of a final italian version by the investigators and implementation of this instrument in the study . The psychometric evaluation of the translated cmi was then conducted in a sub - sample of 188 caregivers participating in the study and selected by the six expected highest recruiting sites . The cmi was administered to the sub - study caregivers during the baseline evaluation and 1 week later . The reliability of the instrument was evaluated by measuring its internal consistency, expressed by cronbach's, and its test - retest reliability, expressed by the intraclass correlation coefficient (icc) and the spearman correlation coefficient (). No gold standard among the instruments which measure compliance to treatment and caregivers satisfaction is available in the literature . Therefore, the convergent validity was evaluated comparing the cmi scores with the responses obtained from the same caregivers who were administered with a visual analogue scale (vas). For the purpose of this study, a vas including a 10-cm line with one extreme indicating the lowest and the other extreme indicating the highest compliance and satisfaction was used . The icc and spearman's were measured to estimate the convergent validity of the italian version of the cmi compared with the vas . Patients and caregivers consecutively visiting the participating memory clinics since the beginning of the study were screened for enrolment . The duration of the screening period was 5 months, and a competitive enrolment was allowed . A total of 896 patients and their caregivers were screened; 41 of them were excluded because they did not meet the inclusion criteria . Sociodemographic, cognitive, functional and clinical characteristics of the study patients by type of drug formulation received are reported in table 1 . The mean age of patients was 77 years in both groups, and over 60% were female . Both groups showed a mild - to - moderate cognitive impairment (mean mmse score 21.0) and a good level of preserved functional abilities (mean adl score 5.0). Cardiovascular diseases were highly prevalent in both groups, with nearly 50% of patients affected by hypertension and 20% diagnosed with any cardiac disease . Patients on oral medications were more likely to be diagnosed with psychiatric illnesses such as major depression, bipolar disorder and schizophrenia than patients on patch (14.6 vs. 7.9%). However, the patients compliance to treatment and the caregivers satisfaction by type of drug formulation did not change after stratifying the sample on the presence of psychiatric illnesses . Moreover, correlation analyses showed that psychiatric illnesses were not associated with the type of drug formulation and with compliance and satisfaction . The prevalence of gastrointestinal diseases was slightly higher among patients treated with oral medications compared to those treated with the rivastigmine patch . Dermatological conditions were rarely reported, with a slightly increased prevalence in the group receiving oral medications . Other conditions including chronic obstructive pulmonary disease, asthma, cancer, swallowing problems and renal or liver failure were infrequent and equally distributed in the two groups . The estimated mean time from the onset of symptoms to ad diagnosis was nearly 2 years in both groups . Over 17% of patients in both groups had switched from a previous anti - ad medication to the current one received at the study time . The most frequently reported medication changes were from either donepezil or oral rivastigmine to patch (40%) and from either patch or oral rivastigmine or donepezil to memantine (34%). The most frequently reported reason for switching was low tolerability of the previous treatment . With respect to other psychotropic medications, 30% of patients on oral medications and 26% of patients on patch were taking antidepressants mostly belonging to the class of selective serotonin re - uptake inhibitors . Antipsychotic prescription was not frequent in the study population, with more atypical than conventional medications used in both groups . A low prevalence of use was also reported for benzodiazepines and anticonvulsants in both groups . Characteristics of the caregivers in the study according to the type of drug formulation their patients were receiving are reported in table 2 . The mean age of the caregivers was 58 years, and over 70% were female . Over 95% of the caregivers were relatives, and more than half of them were living with the patient . The mean time spent providing care reported by caregivers in both groups was around 12 h per day . The rivastigmine patch was the most frequently prescribed anti - ad drug in the study population . Over 46% (n = 396) of the study patients were on rivastigmine patch, while 54% (n = 461) were receiving any type of oral anti - ad drugs . With respect to oral drugs, 25% of patients were treated with donepezil and 21% were receiving memantine . The median and the mode in addition to the range of the daily drug dose are reported in table 3 . Nearly 80% of caregivers of patients on patch were not concerned about patient adherence to anti - ad medical treatment compared to 64% of caregivers of patients on oral medications (fig . Did not report any difficulties in remembering to administer treatment compared to 73% of caregivers of patients on oral medications (fig . Both compliance to treatment and satisfaction were significantly higher in the group treated with the rivastigmine patch compared to the group on oral medications (table 4). Nearly 90% of caregivers of patients on transdermal application versus 78% of caregivers of patients on oral medications reported a score between 9 and 10 at the cmi question on compliance . Over 60% of caregivers of patients treated with the patch compared to 46% of caregivers of patients on oral medications reported a score between 9 and 10 at the cmi question on satisfaction . The translated version of the cmi showed a high level of internal consistency (cronbach's = 0.74) and a high test - retest reliability (icc = 0.96 and = 0.94 for items assessing compliance; icc = 0.98 and = 0.97 for items assessing satisfaction). Convergent validity of the instrument compared with the vas was also high (icc = 0.79 and = 0.72 for items assessing compliance; icc = 0.92 and = 0.92 for items assessing satisfaction). Findings from the axept study indicate that the use of a transdermal application may improve patient compliance to anti - ad treatment and caregiver satisfaction . These results are in line with previous experimental data which highlighted the benefits of a transdermal application for patients and caregivers . Specifically, more than 70% of the 1,059 caregivers of ad patients included in the ideal trial preferred the patch over capsules for drug delivery . Caregivers reported that their preference for the patch was based on the ease of use and following the schedule, which was eventually related to spare of time and reduced workload . The only previous large observational study investigating caregiver satisfaction with drug treatment in ad suggested that donepezil orally disintegrating tablets may have been preferred by caregivers; however, this study did not include patients on rivastigmine patch . Such results appear to support the current hypothesis that new effective drug formulations may be preferred by caregivers of ad patients due to the ease and convenience of administration . It has been shown that the use of the rivastigmine patch is associated with reduced side effects and access to optimal dosages with possible improved efficacy due to constant drug delivery, steady plasmatic drug levels and prolonged cholinesterase inhibition . In general, the patch represents a user - friendly mode of drug delivery in geriatric patients who are likely to present with multimorbidity and complex polypharmacy . The potential for overdose due to erroneous multiple administrations may be reduced by using a transdermal application . Finally, the patch provides caregivers with a visual reminder that the medication has been correctly administered, thus helping improve patient compliance to treatment . All these advantages may relieve the strain among caregivers in managing drug administration while improving treatment compliance and effectiveness . Recently, the who has recommended studies focusing on the role of the caregiver and particularly investigating the level of caregiver satisfaction in relation to daily workload and drug administration . Also, the fda has stated that effective health outcomes in ad should be identified taking into account the needs of both patients and caregivers . In this respect, the caregivers perspective may provide unique insight into clinical research because of their own experience with this condition . Cognitive and functional impairment, behavioral symptoms and susceptibility to side effects make compliance to drug regimens in ad patients particularly challenging . It has been estimated that over 70% of ad patients require assistance in managing and taking medications . The development and implementation of strategies to help caregivers administrate medications to their patients may indeed reduce the strain related to such tasks and increase their level of satisfaction with the care delivered . A reduced level of caregiver burden and distress has also been associated with a decreased risk of developing behavioral symptoms in ad patients and with an overall improved quality of life for their families . Such positive effects result in a more prolonged in - home care provision for patients, with an overall reduction of costs of institutionalization . The study population included patients with multimorbidity who were relatively preserved in their functional abilities, as it may have been expected in consideration of their mild - to - moderate level of cognitive impairment . Cardiovascular diseases and metabolic conditions such as diabetes and dyslipidemia were highly prevalent in our study patients, and this is in accordance with a growing body of literature which indicates that ad patients are likely to have an even worse cardiovascular and metabolic profile compared to elderly individuals who are not cognitively impaired . The management of comorbidities represents an additional contribution to the overall burden of care, requiring caregivers to monitor medical symptoms and signs and administer complex drug therapy regimens . In our study patients, the time interval between first appearance of symptoms and ad diagnosis was nearly 2 years . This estimate is in line with previous european data indicating that for a clinical diagnosis of ad nearly 3 years are required in the uk, 2 years in france and spain and less than 1 year in germany . The role of families and general practitioners has been recognized as crucial to the process of early identification of ad symptoms, and public health interventions to target possible barriers to an early diagnosis have been proposed . An early diagnosis allows to provide medical treatments which are most effective in the early stages of the disease, to implement adequate multidisciplinary interventions and to offer tailored socioeconomic support and services to patients and their families . The sociodemographic profile of caregivers of community - dwelling patients with mild - to - moderate ad has been described in this study . Caregivers are likely to be women of middle or old age, mostly daughters or spouses, living with the patients . They spend half a day, 7 days per week in providing assistance to the patients . This is in line with previous epidemiological data indicating that family caregivers are those who provide most of the care and social support to the ad patients, thus being daily confronted with the psychological and socioeconomic burden of the disease . The needs of these people are among the most urgent public health issues of our society and need to be addressed by tailored interventions aimed at providing social, financial and psychological support . The cross - sectional study design does not make possible to establish any temporal relationship between the exposure to a particular drug formulation and the level of compliance to treatment and caregiver satisfaction reported . Data on concomitant medications other than psychotropic drugs were not collected . Also, no information was gathered regarding the presence and severity of behavioral and psychological symptoms of dementia . The instrument used to assess the patient compliance and caregiver satisfaction is based on a report of the caregivers perspectives, which may not necessarily correspond to the patients opinions . Although all caregivers in the study reported to administer medications to their patients, it is not possible to exclude that some patients may have self - administered treatments . Finally, results have been derived from a sample of community - dwelling mild - to - moderate ad patients and their caregivers in italy and they may not be extendable to other populations . The current study has provided evidence regarding patient compliance to and caregiver satisfaction with ad pharmacological treatments derived from the observation of a real clinical setting . Caregivers of patients treated with a transdermal application appeared to be more satisfied and reported higher levels of compliance to treatment than caregivers of patients receiving anti - ad oral medications . These findings indicate that new convenient modes of drug administration may be appreciated by caregivers and may contribute to reduce their daily workload, thus producing beneficial effects for the patients and their families . Clinicians should consider such evidence together with efficacy and safety data and the patients individual clinical profile when choosing the best medical treatment option for persons with ad . The following questions ask about your opinion about the capsule medication used by the patient with alzheimer's dementia for whom you provide care . Please answer all of the questions as honestly as you can and without help from anyone . Taking your medication in the dose and at the times prescribed can be very important to the action of the medication . During the past 1 week of therapy with the oral medication for alzheimer's dementia, to the best of your knowledge: the following questions ask about your opinion about the treatment received by the patient with alzheimer's dementia for whom you provide care . Please answer all of the questions as honestly as you can and without help from anyone . Taking your medication in the dose and at the times prescribed can be very important to the action of the medication . During the past 1 week of therapy with the patch medication for alzheimer's dementia, to the best of your knowledge: roberto bernabei from universit cattolica del sacro cuore, roma; daniela gragnaniello from universit sant'anna, ferrara; emilio luda di cortemiglia from ospedale di rivoli, rivoli; paolo maria rossini from universit cattolica del sacro cuore, roma; emanuele cassetta from ospedale fatebenefratelli, roma; alfonso iudice from ospedale santa chiara, pisa; luca cipriani from istituto nazionale riposo e cura per anziani (inrca), roma; alberto pilotto from casa sollievo della sofferenza, san giovanni rotondo; luigi di cioccio from ospedale santa scolastica, cassino; claudia bauco from servizio geriatrico integrato dottore angelico, aquino; maria lia lunardelli from policlinico s. orsola malpighi, bologna; niccol marchionni from universit degli studi di firenze, firenze; vito ferrara from ospedale regionale f. miulli, acquaviva delle fonti; mario barbagallo, from universit di palermo, palermo; maurizio russotto from ospedali riuniti villa sofia cervello, palermo; salvatore ferrara from azienda sanitaria provinciale di siracusa, siracusa; massimo franceschi from multimedica holding, castellanza; giancarlo comi from fondazione san raffele del monte tabor, milano; carlo valente from azienda ospedaliera usl 4, prato; antonio lacetera from ospedale santa croce, fano; evelina bianchi from ospedale civile san bortolo, vicenza; domenico consoli from ospedale g. jazzolino, vibo valentia; flavio devetag from ospedale santa maria del prato, feltre; piero secreto from casa di cura beata vergine della consolata fatebenefratelli, san maurizio canavese; marinella turla from ospedale vallecamonica, esine; gianpaolo ben from ospedale civile san martino, belluno; francesco saverio caserta from assistenza anziani asl napoli 1, napoli; fabio di stefano from asl vco omegna, omegna; massimo zanasi from ospedali riuniti, foggia; ciro mundi from ospedali riuniti, foggia; mauro minervini from opera don uva, bisceglie; sandra fanfoni from ospedale nuovo regina margherita, roma; carlo serrati from ospedale san martino, genova; stefania boschi from policlinico le scotte, siena; cristina paci from ospedale civile madonna del soccorso, san benedetto del tronto; leonardo cocito from universit degli studi di genova, genova; carlo de lena from universit degli studi di roma la sapienza, roma; lucilla parnetti from universit di perugia, perugia; lino pasqui from ospedale civile di monselice, monselice; maria giovanna marrosu from policlinico universitario di monserrato, monserrato; edoardo dal monte from ospedale civile salute degli infermi, faenza; antonio tetto from ospedale san leopoldo mandic, merate; giovanni giannelli from centro esperto asl cesena, cesena; stefano viola from ospedale civile san pio da pietralcina, vasto; carlo sabba from ospedale policlinico consorziale, bari, italy.
Single nucleotide polymorphisms (snps) are the simplest and most frequent type of dna sequence variation among individuals and, with the recent availability of high - throughput methodologies, are considered one of the most powerful tools in the search for e.g. Disease susceptibility genes and drug response - determining genes (1,2). However, complex diseases, for which markers display weak associations, still constitute a challenge . Most probably, advancement in the knowledge of such diseases will come from improved genotyping methods in combination with the proper bioinformatics design strategies (3). It is generally believed that multigenicity reflects disruptions in proteins that participate in a protein complex or in a pathway (4). Typically, snps have been used as markers; that is, the real determinant of the disease was not the snp itself but some other mutation in linkage disequilibrium (ld) with it . Because of this, the use of functional snps could be an important factor in increasing significantly the sensitivity of association tests . In fact, several complex genetic disorders such as alzheimer's disease (5) and crohn' disease (6) have been associated with functional snps, lending weight to strategies giving priority to candidate markers based upon predictable function . Several estimations suggest that, on average, some 20% of snps could directly damage proteins (7). Much attention has been focused on modelling by different methods the possible phenotypic effect of snps that cause amino acid changes (713), and only recently has interest focused on functional snps affecting regulatory regions or the splicing process (14). However, there is increasing evidence that many human disease genes are the result of exonic or non - coding mutations affecting regulatory regions (1517). A recent large - scale screening over a set of 16 chromosomes found snps in the promoter regions of 35% of the genes, and experimental evidence suggested that around a third of promoter variants may alter gene expression to a functionally relevant extent (18). Alternative splicing produced by mutations in intron / exon junctions, or in distinct binding motifs, such as exonic splicing enhancers (eses) (19), has also been related to different diseases (20). In fact, it has been estimated that 15% of point mutations that result in human genetic diseases cause rna splicing defects (21). In addition to functional information, population frequency is another important factor to be taken into account when selecting snps . Also, ld is another interesting factor in selecting snps as markers since, if two snps are in strong ld, only one of them will provide enough information for any association or linkage test . With the idea of selecting optimal sets of snps using as much information as possible on putative phenotypic effect, population frequencies and ld, we have developed pupasview (putative phenotypic alterations caused by snps viewer), a server that can be used alone or in combination with pupasnp (14). Pupasview works not only as a viewer of where snps are located, but also as a selector in which different filters based on combinations of functionality and population frequencies can be interactively applied over the ld parameters in order to obtain an optimal selection of snps for genotyping studies, in such a way that with a minimum number of snps maximum information on the genic region is obtained . There are three important properties for an snp to be considered an optimal candidate for genotyping purposes: functional effect, minor allele frequency and ld with respect to other snps . Finding such optimal snps is not always possible, but the idea behind pupasview is to facilitate the selection process in order to achieve a final collection of snps bearing the maximum amount of information . Different filters can be interactively applied to the ld information available based on distinct functional properties, cross - species conservation and population frequency . This permits a final selection of a minimum number of snps with optimal properties in terms of population frequencies and potential phenotypic effect . Pupasview uses a precompiled database which contains a collection of dbsnp entries mapped to the golden path genome assembly, as implemented in the human section of ensembl (). Regions 10 000 bp upstream of the genes belonging to the promoter region of each gene in the list have been scanned for the presence of possible different regulatory motifs . Promoter regions were scanned for the presence of possible transcription factor binding sites . The program match (22) was used for this purpose, using only high - quality matrices and with a cut - off to minimize false positives from the transfac database (23). Snps located within these motifs are considered to have a putative phenotypic effect in the expression of the gene . Almost four million such motifs were found, with 130 373 snps mapping onto them.intron/exon border consensus sequences . Ensembl apis (24) were used to extract the intron / exon organization of the genes and the corresponding sequences . The two conserved nucleotides on each side of the splicing point, which constitute the splicing signal (21), were then located and all the snps altering these signals were recorded . More than 700 000 intron / exon boundaries could be defined in human genes with 1786 snps mapping onto them.eses . Mutations that inactivate or activate an ese sequence may result in exon skipping, errors in alternative splicing patterns, malformation and so on . Different classes of ese consensus motifs have been described, but they are not always easily identified . Exon sequences were scanned to identify putative eses responsive to the human sr proteins sf2/asf, sc35, srp40 and srp55, using the available weight matrices (20). A score was obtained that is related to the likelihood that the site found is a real ese . Only ese sites with scores over the threshold [see (20) for details] were taken into account in the analysis . More than 11 million eses were found, with 299 106 snps located in them.triplex-forming oligonucleotide target sequences (ttss). It has been found that the population of ttss is much more numerous than expected from simple random models (25). The population of ttss is large in the whole genome, without major differences between chromosomes, but with a large concentration in regulatory regions, especially in promoter zones, which suggests a tremendous potential for triplex strategy in the control of gene expression (25). Although the role of ttss in regulation is still a matter of speculation, the program also reports snps disrupting these structures . Some 5.4 million putative triplex - forming sequences were found, and 364 314 snps mapped onto them.snps in exons that cause an amino acid change . Any snp causing a change of amino acid, independent of any speculation on its possible phenotypic effect, is reported . There are 45 906 such snps.snps in exons that cause an amino acid change with putative pathological effect . The putative pathological effect of an amino acid change can be predicted using neural networks (nns) carefully trained to predict disease - associated amino acidic polymorphism (12,13). The server implements a small nn (1 hidden layer and 20 nodes) and three sequence - derived descriptors (pam40, pssm and variability), which are either retrieved from databases or determined internally from multiple alignments using two - iterations psi - blast (26) run over a non - redundant swissprot / trembl database . The trained method displays a success rate> 80% in cross - validation experiments . According to the algorithm, 19 309 snps displayed a high probability of having pathological effect.humanmouse conserved regions . Untranslated whole genome comparisons by blastz were performed for species pairs which are thought to be similar enough to be able to detect homology directly at the dna level (27). Of particular interest is mouse (or rat) because of its phylogenetic position with respect to humans: distant enough to interpret conservation as important but not so distant as to lose most of the similarity . The phenotypic effect of a change in such regions is quite speculative, but cross - species conservation can be useful in cases in which no other information is available . It is also useful for reinforcing the likelihood of other predictions (e.g. An ese in a conserved region is more likely to be real than one in a non - conserved region). Promoter regions were scanned for the presence of possible transcription factor binding sites . The program match (22) was used for this purpose, using only high - quality matrices and with a cut - off to minimize false positives from the transfac database (23). Snps located within these motifs are considered to have a putative phenotypic effect in the expression of the gene . Almost four million such motifs were found, with 130 373 snps mapping onto them . Ensembl apis (24) were used to extract the intron / exon organization of the genes and the corresponding sequences . The two conserved nucleotides on each side of the splicing point, which constitute the splicing signal (21), were then located and all the snps altering these signals were recorded . More than 700 000 intron / exon boundaries could be defined in human genes with 1786 snps mapping onto them . Mutations that inactivate or activate an ese sequence may result in exon skipping, errors in alternative splicing patterns, malformation and so on . Different classes of ese consensus motifs have been described, but they are not always easily identified . Exon sequences were scanned to identify putative eses responsive to the human sr proteins sf2/asf, sc35, srp40 and srp55, using the available weight matrices (20). A score was obtained that is related to the likelihood that the site found is a real ese . Only ese sites with scores over the threshold [see (20) for details] were taken into account in the analysis . More than 11 million eses were found, with 299 106 snps located in them . It has been found that the population of ttss is much more numerous than expected from simple random models (25). The population of ttss is large in the whole genome, without major differences between chromosomes, but with a large concentration in regulatory regions, especially in promoter zones, which suggests a tremendous potential for triplex strategy in the control of gene expression (25). Although the role of ttss in regulation is still a matter of speculation, the program also reports snps disrupting these structures . Some 5.4 million putative triplex - forming sequences were found, and 364 314 snps mapped onto them . Any snp causing a change of amino acid, independent of any speculation on its possible phenotypic effect, is reported . The putative pathological effect of an amino acid change can be predicted using neural networks (nns) carefully trained to predict disease - associated amino acidic polymorphism (12,13). The server implements a small nn (1 hidden layer and 20 nodes) and three sequence - derived descriptors (pam40, pssm and variability), which are either retrieved from databases or determined internally from multiple alignments using two - iterations psi - blast (26) run over a non - redundant swissprot / trembl database . The trained method displays a success rate> 80% in cross - validation experiments . According to the algorithm untranslated whole genome comparisons by blastz were performed for species pairs which are thought to be similar enough to be able to detect homology directly at the dna level (27). Of particular interest is mouse (or rat) because of its phylogenetic position with respect to humans: distant enough to interpret conservation as important but not so distant as to lose most of the similarity . The phenotypic effect of a change in such regions is quite speculative, but cross - species conservation can be useful in cases in which no other information is available . It is also useful for reinforcing the likelihood of other predictions (e.g. An ese in a conserved region is more likely to be real than one in a non - conserved region). There are> 10 million snps stored in the last build of dbsnp (build 124), and more than half of these have been validated by different means (). Validation status is annotated and is an important field in terms of trusting an snp . But, in addition to being real, an snp must exist in the population at frequencies which make it a suitable marker . The program haploview (28) is used to infer blocks using different procedures . In one of the most common procedures (29), 95% confidence bounds based on the d ld parameter are generated and each comparison is called strong recombination. A block is created if 95% of informative (i.e. Non - inconclusive) comparisons are two other methods are used: the four gamete rule (30) and the solid spine of ld (28). . Also d, r and lod parameters between adjacent snps can be visualized by placing the cursor between them . Only hapmap genotyped snps (31) are used to calculate blocks and ld parameters . The main purpose of pupasview is to provide the user with an optimal set of snps for genotyping experiments by filtering the annotated snps using a series of filters related to their impact in protein functionality and pathology, their population frequency and ld . The input is a gene identifier (ensembl ids or external ids, which include genbank, swissprot / trembl and other gene ids supported by ensembl). The program presents a list of options that can be selected and applied as many times as desired . The options include validation status obtained from dbsnptype of snp (coding, intron, untranslated region, local), according to its position in the genefrequency and population, an option that allows the possibility of filtering by a range of frequencies of the minor allele in one or more populations (europe; europe, multinational; europe, north america; north america; central / south america; north / east africa and middle east; central / south africa; west africa; central asia; east asia; pacific; multinational; unknown; hapmap)functional properties as follows: non - synonymous snps [all or only those predicted as pathological by the pmut algorithm (12,13)]snps disrupting predicted transcription factor binding sites (all or only those that are in regions conserved in the mouse genome)snps disrupting predicted eses (all or only those that are in regions conserved in the mouse genome)snps disrupting potential triplex - forming regions (all or only those that are in regions conserved in the mouse genome)snps disrupting intron / exon boundaries regions conserved in mouseoptions for the way in which blocks are constructed: confidence intervals (29)four gamete rule (30)solid spine of ld (28). Validation status obtained from dbsnp type of snp (coding, intron, untranslated region, local), according to its position in the gene frequency and population, an option that allows the possibility of filtering by a range of frequencies of the minor allele in one or more populations (europe; europe, multinational; europe, north america; north america; central / south america; north / east africa and middle east; central / south africa; west africa; central asia; east asia; pacific; multinational; unknown; hapmap) functional properties as follows: non - synonymous snps [all or only those predicted as pathological by the pmut algorithm (12,13)]snps disrupting predicted transcription factor binding sites (all or only those that are in regions conserved in the mouse genome)snps disrupting predicted eses (all or only those that are in regions conserved in the mouse genome)snps disrupting potential triplex - forming regions (all or only those that are in regions conserved in the mouse genome)snps disrupting intron / exon boundaries regions conserved in mouse non - synonymous snps [all or only those predicted as pathological by the pmut algorithm (12,13)] snps disrupting predicted transcription factor binding sites (all or only those that are in regions conserved in the mouse genome) snps disrupting predicted eses (all or only those that are in regions conserved in the mouse genome) snps disrupting potential triplex - forming regions (all or only those that are in regions conserved in the mouse genome) snps disrupting intron / exon boundaries regions conserved in mouse options for the way in which blocks are constructed: confidence intervals (29)four gamete rule (30)solid spine of ld (28). Confidence intervals (29) four gamete rule (30) solid spine of ld (28). Figure 1a shows the result of running pupasview on the gene tp53 without applying any filter . If the cursor is over an snp, information on it is displayed by means of pop - up text . Figure 1b shows a subselection of these snps obtained after selecting only snps for which population frequency was available . Finally, figure 1c shows the selection obtained if only snps with putative functional effect are chosen . The upper horizontal bar below the figure represents ld parameters (which can be individually obtained by placing the cursor over them). The blocks are displayed graphically with brown rectangles going from the first to the last snp within the block . When the cursor is over the rectangles, a tooltip text pops up in the block showing the snps and the haplotypes (with hapmap frequencies in parentheses). There are three important properties for an snp to be considered an optimal candidate for genotyping purposes: functional effect, minor allele frequency and ld with respect to other snps . Finding such optimal snps is not always possible, but the idea behind pupasview is to facilitate the selection process in order to achieve a final collection of snps bearing the maximum amount of information . Different filters can be interactively applied to the ld information available based on distinct functional properties, cross - species conservation and population frequency . This permits a final selection of a minimum number of snps with optimal properties in terms of population frequencies and potential phenotypic effect . Pupasview uses a precompiled database which contains a collection of dbsnp entries mapped to the golden path genome assembly, as implemented in the human section of ensembl (). Regions 10 000 bp upstream of the genes belonging to the promoter region of each gene in the list have been scanned for the presence of possible different regulatory motifs . Promoter regions were scanned for the presence of possible transcription factor binding sites . The program match (22) was used for this purpose, using only high - quality matrices and with a cut - off to minimize false positives from the transfac database (23). Snps located within these motifs are considered to have a putative phenotypic effect in the expression of the gene . Almost four million such motifs were found, with 130 373 snps mapping onto them.intron/exon border consensus sequences . Ensembl apis (24) were used to extract the intron / exon organization of the genes and the corresponding sequences . The two conserved nucleotides on each side of the splicing point, which constitute the splicing signal (21), were then located and all the snps altering these signals were recorded . More than 700 000 intron / exon boundaries could be defined in human genes with 1786 snps mapping onto them.eses . Mutations that inactivate or activate an ese sequence may result in exon skipping, errors in alternative splicing patterns, malformation and so on . Different classes of ese consensus motifs have been described, but they are not always easily identified . Exon sequences were scanned to identify putative eses responsive to the human sr proteins sf2/asf, sc35, srp40 and srp55, using the available weight matrices (20). A score was obtained that is related to the likelihood that the site found is a real ese . Only ese sites with scores over the threshold [see (20) for details] were taken into account in the analysis . More than 11 million eses were found, with 299 106 snps located in them.triplex-forming oligonucleotide target sequences (ttss). It has been found that the population of ttss is much more numerous than expected from simple random models (25). The population of ttss is large in the whole genome, without major differences between chromosomes, but with a large concentration in regulatory regions, especially in promoter zones, which suggests a tremendous potential for triplex strategy in the control of gene expression (25). Although the role of ttss in regulation is still a matter of speculation, the program also reports snps disrupting these structures . Some 5.4 million putative triplex - forming sequences were found, and 364 314 snps mapped onto them.snps in exons that cause an amino acid change . Any snp causing a change of amino acid, independent of any speculation on its possible phenotypic effect, is reported . There are 45 906 such snps.snps in exons that cause an amino acid change with putative pathological effect . The putative pathological effect of an amino acid change can be predicted using neural networks (nns) carefully trained to predict disease - associated amino acidic polymorphism (12,13). The server implements a small nn (1 hidden layer and 20 nodes) and three sequence - derived descriptors (pam40, pssm and variability), which are either retrieved from databases or determined internally from multiple alignments using two - iterations psi - blast (26) run over a non - redundant swissprot / trembl database . The trained method displays a success rate> 80% in cross - validation experiments . According to the algorithm, 19 309 snps displayed a high probability of having pathological effect.humanmouse conserved regions . Untranslated whole genome comparisons by blastz were performed for species pairs which are thought to be similar enough to be able to detect homology directly at the dna level (27). Of particular interest is mouse (or rat) because of its phylogenetic position with respect to humans: distant enough to interpret conservation as important but not so distant as to lose most of the similarity . The phenotypic effect of a change in such regions is quite speculative, but cross - species conservation can be useful in cases in which no other information is available . It is also useful for reinforcing the likelihood of other predictions (e.g. An ese in a conserved region is more likely to be real than one in a non - conserved region). Promoter regions were scanned for the presence of possible transcription factor binding sites . The program match (22) was used for this purpose, using only high - quality matrices and with a cut - off to minimize false positives from the transfac database (23). Snps located within these motifs are considered to have a putative phenotypic effect in the expression of the gene . Almost four million such motifs were found, with 130 373 snps mapping onto them . Ensembl apis (24) were used to extract the intron / exon organization of the genes and the corresponding sequences . The two conserved nucleotides on each side of the splicing point, which constitute the splicing signal (21), were then located and all the snps altering these signals were recorded . More than 700 000 intron / exon boundaries could be defined in human genes with 1786 snps mapping onto them . Mutations that inactivate or activate an ese sequence may result in exon skipping, errors in alternative splicing patterns, malformation and so on . Different classes of ese consensus motifs have been described, but they are not always easily identified . Exon sequences were scanned to identify putative eses responsive to the human sr proteins sf2/asf, sc35, srp40 and srp55, using the available weight matrices (20). A score was obtained that is related to the likelihood that the site found is a real ese . Only ese sites with scores over the threshold [see (20) for details] were taken into account in the analysis . More than 11 million eses were found, with 299 106 snps located in them . It has been found that the population of ttss is much more numerous than expected from simple random models (25). The population of ttss is large in the whole genome, without major differences between chromosomes, but with a large concentration in regulatory regions, especially in promoter zones, which suggests a tremendous potential for triplex strategy in the control of gene expression (25). Although the role of ttss in regulation is still a matter of speculation, the program also reports snps disrupting these structures . Some 5.4 million putative triplex - forming sequences were found, and 364 314 snps mapped onto them . Any snp causing a change of amino acid, independent of any speculation on its possible phenotypic effect, is reported . The putative pathological effect of an amino acid change can be predicted using neural networks (nns) carefully trained to predict disease - associated amino acidic polymorphism (12,13). The server implements a small nn (1 hidden layer and 20 nodes) and three sequence - derived descriptors (pam40, pssm and variability), which are either retrieved from databases or determined internally from multiple alignments using two - iterations psi - blast (26) run over a non - redundant swissprot / trembl database . The trained method displays a success rate> 80% in cross - validation experiments . According to the algorithm untranslated whole genome comparisons by blastz were performed for species pairs which are thought to be similar enough to be able to detect homology directly at the dna level (27). Of particular interest is mouse (or rat) because of its phylogenetic position with respect to humans: distant enough to interpret conservation as important but not so distant as to lose most of the similarity . The phenotypic effect of a change in such regions is quite speculative, but cross - species conservation can be useful in cases in which no other information is available . It is also useful for reinforcing the likelihood of other predictions (e.g. An ese in a conserved region is more likely to be real than one in a non - conserved region). There are> 10 million snps stored in the last build of dbsnp (build 124), and more than half of these have been validated by different means (). Validation status is annotated and is an important field in terms of trusting an snp . But, in addition to being real, an snp must exist in the population at frequencies which make it a suitable marker . The program haploview (28) is used to infer blocks using different procedures . In one of the most common procedures (29), 95% confidence bounds based on the d ld parameter are generated and each comparison is called strong recombination. A block is created if 95% of informative (i.e. Non - inconclusive) comparisons are two other methods are used: the four gamete rule (30) and the solid spine of ld (28). . Also d, r and lod parameters between adjacent snps can be visualized by placing the cursor between them . Only hapmap genotyped snps (31) are used to calculate blocks and ld parameters . The main purpose of pupasview is to provide the user with an optimal set of snps for genotyping experiments by filtering the annotated snps using a series of filters related to their impact in protein functionality and pathology, their population frequency and ld . The input is a gene identifier (ensembl ids or external ids, which include genbank, swissprot / trembl and other gene ids supported by ensembl). The program presents a list of options that can be selected and applied as many times as desired . The options include validation status obtained from dbsnptype of snp (coding, intron, untranslated region, local), according to its position in the genefrequency and population, an option that allows the possibility of filtering by a range of frequencies of the minor allele in one or more populations (europe; europe, multinational; europe, north america; north america; central / south america; north / east africa and middle east; central / south africa; west africa; central asia; east asia; pacific; multinational; unknown; hapmap)functional properties as follows: non - synonymous snps [all or only those predicted as pathological by the pmut algorithm (12,13)]snps disrupting predicted transcription factor binding sites (all or only those that are in regions conserved in the mouse genome)snps disrupting predicted eses (all or only those that are in regions conserved in the mouse genome)snps disrupting potential triplex - forming regions (all or only those that are in regions conserved in the mouse genome)snps disrupting intron / exon boundaries regions conserved in mouseoptions for the way in which blocks are constructed: confidence intervals (29)four gamete rule (30)solid spine of ld (28). Validation status obtained from dbsnp type of snp (coding, intron, untranslated region, local), according to its position in the gene frequency and population, an option that allows the possibility of filtering by a range of frequencies of the minor allele in one or more populations (europe; europe, multinational; europe, north america; north america; central / south america; north / east africa and middle east; central / south africa; west africa; central asia; east asia; pacific; multinational; unknown; hapmap) functional properties as follows: non - synonymous snps [all or only those predicted as pathological by the pmut algorithm (12,13)]snps disrupting predicted transcription factor binding sites (all or only those that are in regions conserved in the mouse genome)snps disrupting predicted eses (all or only those that are in regions conserved in the mouse genome)snps disrupting potential triplex - forming regions (all or only those that are in regions conserved in the mouse genome)snps disrupting intron / exon boundaries regions conserved in mouse non - synonymous snps [all or only those predicted as pathological by the pmut algorithm (12,13)] snps disrupting predicted transcription factor binding sites (all or only those that are in regions conserved in the mouse genome) snps disrupting predicted eses (all or only those that are in regions conserved in the mouse genome) snps disrupting potential triplex - forming regions (all or only those that are in regions conserved in the mouse genome) snps disrupting intron / exon boundaries regions conserved in mouse options for the way in which blocks are constructed: confidence intervals (29)four gamete rule (30)solid spine of ld (28). Confidence intervals (29) four gamete rule (30) solid spine of ld (28). Figure 1a shows the result of running pupasview on the gene tp53 without applying any filter . If the cursor is over an snp, information on it is displayed by means of pop - up text . Figure 1b shows a subselection of these snps obtained after selecting only snps for which population frequency was available . Finally, figure 1c shows the selection obtained if only snps with putative functional effect are chosen . The upper horizontal bar below the figure represents ld parameters (which can be individually obtained by placing the cursor over them). The blocks are displayed graphically with brown rectangles going from the first to the last snp within the block . When the cursor is over the rectangles, a tooltip text pops up in the block showing the snps and the haplotypes (with hapmap frequencies in parentheses). It is believed that improved genotyping methods in combination with the proper bioinformatics design strategies will offer better opportunities for the study of complex diseases (3). The use of functional snps could be an important factor in increasing the sensitivity of association tests . Different bioinformatics approaches have been focused mainly on the effect of coding snps, but also recently on snps affecting the regulation or the splicing of genes (14). Pupasview is the first tool that integrates both transcriptional and translational phenotypic effects caused by polymorphisms . It provides an interactive environment in which functional information and population frequency data can be used over ld parameters as sequential filters to obtain a final list of snps optimal for genotyping purposes . Pupasview is closely linked to our previous program pupasnp (14), which is a tool for selecting snps with putative phenotypic effects . Pupasnp, designed for high - throughput experiments, has been used to design> 9000 sets of snps, and has a daily average of 50 uses . Pupasview assists in the last refinement step of gene - by - gene selection of snps . Figure 1 illustrates the effect of applying successive filter steps, which are, conceptually, first to select only those snps which are real (with reported population frequencies) and then to select only functional snps . In the last view (figure 1c), ld parameters can be used to help in the final selection . More than 5000 snps have been selected using pupasnp and pupasview in the first step of the pipeline for the study of polymorphisms at the spanish national genotyping centre (cegen). (c) snps with any functional characteristic . Depending on the versions of ensembl and dbsnp, the appearance of
Parrots are among the most endangered group of birds worldwide, and 15 of the 84 brazilian species are classified as being vulnerable or critically endangered . Studies on parrot populations are important to establish a database that can be assessed in the event of outbreaks, which could also be useful for subsequent epidemiological studies and conservation efforts . However, health surveys of free - ranging wild animals are mostly focused on retrospective studies on mortality [3, 4]. Research on diseases affecting free - ranging parrots is still scarce and studies performed often fail to maximize the scientific information that could be gathered [2, 58]. Such data could be of extreme importance in guiding conservation measures ex situ and in situ . The hyacinth macaw and lear's macaw are well - known flagship species that have suffered heavily owing to the destruction of habitat and illegal trade . A number of studies aimed at promoting their recovery have been performed and actions taken; however, they are still classified as endangered species and face severe threats to their long - term survival . On the other hand, the blue - fronted amazon parrot is rated as a species of least concern regarding its conservation status . It is, however, the most illegally traded parrot species in brazil, and therefore, it is possible that decades of successive capturing of nestlings and concomitant ageing of the adult population could cause local extinctions in several areas where it is still common today . Studies have established that the intestinal flora of most species of healthy captive psittacines is composed essentially of gram - positive bacteria [11, 12]. Parrots in captivity are frequently affected by infections caused by gram - negative bacteria, and these microorganisms are considered either pathogenic or opportunistic . One such bacterium, escherichia coli (e. coli), is frequently involved in respiratory, digestive, and septicemic disorders in captive parrots . It is possible to classify e. coli into pathotypes by using genes responsible for the expression of virulence factors . Commonly described pathotypes include epec (enteropathogenic e. coli), apec (avian pathogenic e. coli), and upec (uropathogenic e. coli) [14, 15]. Epec is an important category of diarrheagenic e. coli and a major cause of infant diarrhea in developing countries, while apec is recognized for significant economic losses to the poultry industry, resulting in respiratory diseases and septicemia [14, 15]. Upec is a serious cause of urinary diseases in humans, causing cystitis that may progress to pyelonephritis . Although, certain e. coli classification studies performed on wild birds [1722] and those involving captive psittacines have found a certain degree of correlation between disease and specific pathotypes [2325], current serological methods to determine the pathogenicity of e. coli strains do not accurately predict which strains will be pathogenic in which birds . The purpose of this study was to test cloacal samples from asymptomatic free - living nestlings (blue - fronted amazon parrots as well as hyacinth and lear's macaws) to determine if they could be carriers of recognized e. coli pathotypes . In addition, we discuss the role that these strains could play in both free - living and captive parrots . The samples of this study were collected during field surveys of the nestlings of hyacinth macaws and blue - fronted amazon parrots in the pantanal (wetlands) region of the refgio ecolgico caiman and neighboring farms (1958s, 5624w) in mato grosso do sul state, and of lear's macaws at the estao ecolgica de canudos (095348s, 390135w) in the caatinga (semi - arid) of the bahia state . Cloacal swabs (cultureswab sterile, difco becton dickenson and company, sparks, maryland, usa) were moistened with care using a sterile saline solution so as not to contaminate the swab during insertion in the cloaca . All chicks had no evident signs of disease (soiled vent, emaciation, prostration, or delays in development according to their estimated age). In total, 44 samples were obtained, of which 10 were from hyacinths, 13 from lear's, and 21 from blue - fronted amazon parrots . The swabs were refrigerated up to processing at which point they were aerobically incubated in bhi broth (brain heart infusion, difco) for 24 hours at 37c . They were then streaked onto macconkey (difco) agar plates and incubated for another 24 hours at 37c . Bacteria were identified using a specific enterobacteria identification kit (newprov, pinhais, paran, brazil) and stored at 20c . Isolates were tested using polymerase chain reaction (pcr) (table 2 . ), for the presence of e. coli attaching and effacing (eae) gene and bundle - forming pili structural (bfpa) gene of epec . For apec, the aerobactin (iucd), cytotoxic necrotizing factor (cnf1), s fimbrial adhesin (sfa), and p fimbrial adhesin (papef) genes were amplified . Upec utilized alpha hemolysin (hlya) in addition to the genes used for apec . Additional apec genes for serum resistance (iss) and temperature - sensitive hemagglutinin (tsh) were also tested . E. coli was obtained from all 44 samples . Details on the positive samples are given in table 1 . One sample was positive solely for eae and not for bfp and was characterized as atypical epec . A large number of samples were positive for virulence factors commonly found in apec (14 samples), with most originating from blue - fronted amazon chicks . This gene was also found associated with other virulence factors in 4 samples, all from blue - fronted amazon parrots . There was also an association between other virulence factors among some isolates (table 1). Epec have the ability to cause lesions on the intestinal mucosa, leading to severe diarrhea . This process is initiated by adherence to the epithelial cell membrane and is mediated by the adhesin intimin (encoded by the eae gene). Typical epec strains possess both intimin and bundle - forming pili (encoded by the bfp gene), which are responsible for the initial contact between the bacteria and the host cell; atypical isolates, on the other hand, lack the bfp gene . Humans are considered the primary reservoir for the typical pathotype although it has also been found in dogs and cats [29, 30]. There are few reports of the presence of atypical epec in birds, especially the isolates obtained from poultry [3032]. There are also reports of atypical epec causing fatal outbreaks in backyard passerine species, and carriers have been found among feral pigeons and rehabilitated seagulls [20, 22]. A study using 103 samples from captive psittacines detected 4 samples characterized as typical epec and 3 others as atypical isolates, all of which originated from clinical cases of diarrhea, enteritis, or septicemia . Another survey conducted in brazil regarding necropsy cases of symptomatic parrots also showed the presence of the eae gene in 2 atypical samples of e. coli isolated from the livers of 2 individual pet amazon parrots . The fact that we found an atypical isolate in an asymptomatic bird, especially a free - ranging individual, suggests that this pathotype is not pathogenic in all parrots depending on the situation to which the birds are subjected . Apec and upec share several genes that encode virulence factors such as p fimbrial adhesin (pap gene) and s fimbrial adhesin (sfa gene). Apec has been extensively studied in poultry where it has been observed that the p fimbrial adhesion enables binding of bacteria to internal organs and protects against heterophilic inflammation, while the s fimbrial adhesin is associated with omphalitis, salpingitis, chronic respiratory diseases, and sepsis . Although several virulence factors have been associated with clinical cases of apec in poultry, no specific factor has been confirmed to be responsible for contributing to the pathogenicity observed, which makes it difficult to interpret results among lesser - studied groups such as wild birds . Siderophores, such as aerobactin (iuc gene), enable e. coli to obtain iron stores from the host, and strains with this gene are quite frequently associated with clinical cases of poultry . Toxins, such as alpha hemolysin (hlya gene characteristic of upec) and cytotoxic necrotizing factor 1 (cnf1 gene), provide the ability to cause tissue damage, contributing to dissemination and release of host nutrients while impairing the immune defenses . In this investigation, 2 samples were positive for the sfa and 1 was positive for the cnf1 gene . These genes have been reported in pathogenic strains of e. coli isolated from septicemic poultry, as well as from human and domestic animals with extraintestinal pathogenic e. coli infections [16, 34]. A survey dealing with healthy feral pigeons detected a number of positive cloacal - swab specimens for the cytotoxic necrotizing factor 1, showing the potential for disease spread by carriers of this species . Other genes such as the tsh (temperature - sensitive hemagglutinin) and the iss (increased serum survival) are reported to be present in apec strains . The increased serum survival causes sepsis by conferring resistance to the bacteria against the host immune bactericidal defenses . The exact function of tsh is largely unknown, but it has been shown to be involved in mechanisms of adherence to the respiratory tract of poultry . In this study, however, it has been shown in poultry that the presence of the iss gene alone may not be sufficient to identify apec isolates because this gene can also be found in the intestinal microbiota of healthy individuals, and an association with the iuc gene was reported to be necessary for achieving higher levels of virulence . Interestingly, a sample from an asymptomatic blue - fronted amazon chick in this study showed an association between the iss and iuc genes . This research also found 1 positive sample for the iss / tsh association . In domestic turkeys, a relationship between clinical cases of colibacillosis and the presence of iss and tsh has been described . The occurrence of gene associations such as iss / pap, iss / iuc / tsh, and pap / iuc / tsh has previously been reported in e. coli isolated from fecal, liver, and blood samples collected during necropsies performed in symptomatic psittacines in brazil . These results in captive birds demonstrate that there is a connection between the presence of these genes and some clinical cases of colibacillosis as a contributing cause of death whether as primary or opportunistic pathogens . Even though the sample numbers were too small to reach definite conclusions, we observed differences in the presence of virulence factors among the different species . Apec / upec genes were found mostly in species that usually nest on trees and live in a tropical climate (at least in this studied area, for the species a. hyacinthinus and a. aestiva). Epec was found in species that nest on limestone cliffs and inhabit semi - arid regions (a. leari). Differences among feeding habits, direct or indirect contact with other wild animals, and human activities (interference due to human settlements and domestic animals) could possibly have influenced these results, and thus, these results should be further investigated . Although previous studies showed that some virulence factors are indeed involved in clinical cases of colibacillosis in psittacines [2325], this investigation found a number of carriers for virulence factors . These unusual findings focus the attention on the fact that, at least at the time of sampling, there was a stable host / parasite relationship . Unlike wild birds, parrots maintained in captivity are frequently exposed to a number of factors that cause immunosuppression and increase their susceptibility to disease . These include deficient diets, inadequate hygiene, and lack of mental and physical stimuli; all of these are factors that may determine the course of the disease when the animal is exposed to a microorganism . The concept of disease is considered the result of an interactive relationship among the causative agent, the animal, and environmental factors, and a multitude of factors act together in order to initiate the disease process . If a factor is not present, it is probable that the organism will be capable of fighting the pathogen without showing overt clinical signs . The nestlings in our study successfully fledged, indicating that although the potential for disease was present, birds living in their natural environment, without the factors induced by captivity, are more likely to remain disease free . The results presented here are also important for the future conservation of the 2 endangered species (a. hyacinthinus and a. leari) as well as the heavily trafficked a. aestiva because they could better guide ex situ husbandry practices involved in captive breeding and rehabilitation / relocation programs, besides assisting monitoring of the overall health of the wild population . In conclusion, to our knowledge, this is the first study that tested e. coli virulence factors in wild psittacines . It is also the first to describe e. coli carriers in free - ranging parrots, and the results indicate that although the potential to develop disease was present, several factors that are most likely to be found in captivity needed to be involved in triggering disease development . Other studies involving different species as well as a higher number of samples are important to further define the role and risks involved with specific e. coli pathotypes in the case of both wild and captive psittacines.
Methods are described in detail in the supplementary material online, below is a brief summary . We used publicly available chip - seq data produced from the livers of human, macaque, mouse, rat, and dog for four liver - enriched tfs: cebpa, foxa1, hnf4a, onecut1 (schmidt et al . Reads were mapped to each reference genome with bowtie2 (langmead and salzberg 2012) with default parameters . Numbers of mapped reads and quality metrics are shown in supplementary table s2, supplementary material online, for the liver - enriched tfs . Tf peaks were called as reproducible in both replicates if they overlapped by 75% reciprocally (supplementary table s3, supplementary material online). (2010) and were screened by eye for whether they have liver expression in mouse, rat, and dog (based upon previously published rna - seq data, see supplementary material online), as well as whether they have clear 1:1 orthologs based on ensembl annotations and/or based on sequence similarity . The set of 1,373 liver expressed genes that are one - to - one orthologs across the 5 mammals was constructed based on a cut - off of 5 read fragments per kilobase of exon per million reads within the livers of mouse, rat, and dog . The binding level for orthologs across species 2015). Before running these analyses, for each tf and species, read alignment files were downsampled to the number of reads mapped in the replicate with the fewest number of reads . This approach combines the number of binding events, their binding intensity, and their distance from the tss into a single measure . Specifically, binding level (ais), where i is an orthologous gene in species s, is given by ais = log (jkgijksdijks + 0.1) where k is a peak within 100 kb of the tss of gene i, gijks is the intensity of peak k for tf j in species s and dijks is the distance (in bp) from the tss to the summit of peak k. the region size used is 100 kb and is discussed further elsewhere (wong et al . A pseudocount of 0.1 is added to the denominator to ensure this value is never zero . The mean and variance in binding level was calculated for all tfs and species was determined for all liver expressed genes . Because the signal - to - noise ratio differs greatly across chip - seq experiments, binding levels were converted into standard scores (for each species and tf) so that they could be better compared across species . The standard scores of binding levels for each tf were then summed to obtain a single measure of binding level per gene . Maximum - likelihood ancestral binding levels were reconstructed under a bm model in r using the ace function of the ape package (paradis et al . Enrichment levels for the histone modification markers were calculated using the same steps as for binding level . All sequence analyses reported were performed on peak and flank coordinates sliced from the 38 eutherian epo alignment (release 74; cunningham et al . 2015). Peak sequences were taken as 100 bp from peak summits, while flanking regions were taken as 2 kb from peak ends that did not intersect other peaks or exons . These alignments were realigned with mafft (l - ins - i method; v6.882b; katoh and toh 2008). Shared peaks across species were called based upon overlapping summits (taken as the peak center) within 150 bp of each other within the alignment . Ancestral sequences at each node in the primate phylogeny were reconstructed using the prequel program of phast (v1.3; hubisz et al . Lrts were performed with the phylop program of phast to test for accelerated evolution within the human supplementary tables s1s3, figures s1s9, and text are available at molecular biology and evolution online (http://www.mbe.oxfordjournals.org/).
Obsessive compulsive disorder (ocd) can manifest with a wide range of clinical pictures . On the other hand, there has been a long - standing observation that patients with various types of epilepsy / seizures have a higher incidence of many psychiatric disorders including ocd . Though the equation of seizures leading to ocd is a known phenomenon, but to our best knowledge, we could not find literature showing ocd to cause seizures directly or indirectly . Here we present a case of undetected ocd presenting as repeated seizures, which is an indirect relationship might be first of its kind in literature . A 45-year - old female from rural kashmir (india), married with four children, with previous three to four admissions for generalized tonic - clonic seizures from last 3 years was presently again admitted with two episodes of generalized tonic clonic seizures . As in previous instances she again had a low serum sodium level of 115 meq / l . Rest of the investigations viz ., hemogram, liver function tests, kidney function tests, blood sugar, urine examination, calcium, potassium, chloride, protein levels and lipid levels were in normal range . Every time her seizure was ascribed to the only abnormal finding of hyponatremia of 110 meq / l, 114 meq / l, 121 meq / l, in previous admissions and the 115 meq / l at the present . There was no apparent cause for this low sodium except for high intake of thiazides at the first admission . In spite of changing thiazides, she again had seizures with hyponatremia every time . She was evaluated for other possible causes of seizures and hyponatremia, but no concrete cause was found . One out of three electroencephalograms showed nonspecific epileptic discharges . Considering her repetitive enquiring behavior and restlessness a psychiatric evaluation was sought . On detailed psychiatric evaluation she verbalized pathological doubts, excessive cleanliness, excessive worries, repetition of acts . Upon further interview, she said that whenever she took water she felt as if she did not take and did not get satisfied, and she took more and more water for the same . Many a times she knew that she has taken a lot of water, but she felt compelled to take more . She further said that her idea of repeatedly drinking water was useless, but she could not resist it and had taken about 6 - 8 l of water on the day of seizure . She also described the similar repeated intake of antihypertensive tablets (thiazides) prior to her first seizure around 3 years back . Her husband further described her habits of taking medications over the counter, from her local health workers, changing and ill formed pain and ache complaints and corroborated her behaviors of intrusiveness, excessive washing, cleaning, checking and perfectionism . She described the intrusiveness of these thoughts and the disturbance in other psychosocial spheres for more than 15 years as was also reported by the family members . She had been put on tablet phenytoin sodium 300 mg daily since her first seizure, without any significant benefit . On psychiatric evaluation she was diagnosed as ocd (international classification of diseases-10 criteria) and put on fluvoxamine and cognitive behavioral therapy (cbt), however other causes of hyponatremia and polydipsia were still being ruled out . Tablet fluvoxamine was slowly titrated from 50 to 200 mg / day over 5 weeks, and cbt sessions were also given twice weekly . The patient showed a response to therapy . In the meantime, no apparent cause for his low serum sodium / seizure / excessive water intake could be found . After a consensus with the neurologist, phenytoin sodium was slowly tapered from 6 month of the start of flouvoxamine, repeated electroencephalograms and serum sodium levels were followed and she was off phenytoin in 9 month . At present, after 28 months of follow - up she is significantly improved in ocd features and had no further seizures since then . Her yale - brown obsessive compulsive scale score at start and at 28 months is 29 and 10 respectively . She scored 5 (markedly ill) for clinical global impressions severity (cgi - s) at start and at 28 months she scores 1 (very much improved) for cgi improvement and cgi - s also scores 1 (normal - not at all ill, symptoms of disorder not present past 7 days). Presently, she is only on fluvoxamine 200 mg / day, without any antiepileptic . As our findings and investigations, we excluded our differential diagnosis for seizures such as central nervous system infections, space occupying lesions, drug intake, metabolic causes, and other possible causes for seizures . As has been reported, seizures could be the only obvious neurologic manifestation of more moderate levels of hyponatremia . Hyponatremia seems to be most possible cause in this case, as a correction, and future avoidance of hyponatremia prevented further seizures . In light of normal renal functions and normal metabolic profile; a subtle, undiagnosed renal / metabolic defect can explain the development of hyponatremia in an otherwise healthy female, although development of hyponatremia with excessive water intake is known . In this case our hypothesis of ocd leading to excessive water intake and subsequent development of hyponatremia and seizure is more tangible because treatment of ocd led to the overall improvement . More importantly our patient is off antiepileptics, has improved oral contraceptive features and had an overall improvement and functionality . Since epilepsy affects more than 50 million people worldwide, 80% of them live in developing world and a substantial number has no attributable cause, our finding is important in finding an indirect etiology (ocd) of seizures . It also points to the unawareness and the load of the psychiatric presentations in the society . We suggest more involvement of psychiatrists in emergency units, and team approach with other specialties . It will help in avoiding unnecessary continuous antiepileptic and more importantly it shows a different presentation of ocd (close to psychogenic polydipsia) for which both emergency residents and the psychiatrists should scratch into.
Focal cervical dystonia cases were recruited from 2 movement disorders clinics in uk: (1) national hospital for neurology and neurosurgery, london; and (2) salford royal foundation trust, manchester . Only cases that remained focal involvement were recruited and most of them were followed up in botulinum toxin clinic . Cases were reviewed by movement disorder specialists, and only presumptive primary cases were recruited, with secondary causes like wilson s disease or other neurodegenerative disease ruled out where clinically appropriate . Because of small sample size, subclassification according to family history of movement disorders and age of onset was not attempted all patients gave informed consent to the study, approved by the respective local ethics committee . Dna was extracted locally from blood and genotyped in the university college london genomics microarray centre, using illumina human 610-quad beadchip (illumina, san diego, ca, usa). Control data of european descent was drawn from the wellcome trust case control consortium (wtccc) data set as previously reported . A total of 2930 samples from 1958 birth cohort and 2737 samples from national blood services were genotyped in illumina 1.2 m duo array by the wellcome trust sanger institute . Quality control measure was performed both before and after merging with the cases as detailed in the supporting materials . Genotyped data from cervical dystonia cases was assembled in genomestudio (v2011.1) per the manufacturer s suggestion (illumina). Postassembly quality control was performed in genomestudio and plink (version 1.07). In brief, genotyping quality of the sample and single - nucleotide polymorphisms (snps) were controlled for in genomestudio (illumina). Further checking for gender mismatch, sample relatedness (identity - by - descent, excluding pihat> 0.125) (pi - hat a parameter in plink using estimates of pairwise ibd to find pairs of individuals who are possibly related . ), hardy - weinberg equilibrium (excluding p <13 10), allele frequency (excluding minor allele frequency [maf] <0.01), nonrandom missingness (missingness - by - haplotype, excluding p <1 10; missingness - by - genotype, excluding p <1 10) and population substructure (excluding 6 sd from combined mean of northern and western european ancestry (ceu) and toscani in italia (tsi) in multidimensional scaling [mds] component 1 or 2) were performed in plink (details in supporting materials). The dystonia sample data was merged with the wellcome trust case control consortium (wtccc), and association analysis was performed using logistic regression in the plink package . Three covariates were used in the regression: gender and the first 2 components of plink mds analysis to adjust for gender and genetic variation . Because of our relatively small sample size, we did not perform separate analysis that looked specifically at known loci . Imputation for autosomal chromosome was performed with mach and minimac, in chunks of 10 megabases with 1 megabase overlapping at both ends . Postimputation association was done in mach2dat excluding imputed snps with r squared (rsqr) <0.3 as suggested . A higher cutoff was chosen as the small sample size in this study contributed to false - positive association in snps with low maf . Reference for imputation was taken from the 1000 genomes project 2010 august release and composed of 283 individuals from the european continental group . A total of 233 cases were genotyped and 212 cases (66 m, 146 f, mean age 60.6 years, sd 10.9 years) remained after quality control and 494 k snps had maf> 0.01 (exclusion breakdown in supporting tables 1 and 2). The cases were clustered around the ceu - tsi controls in the mds plot confirming european descent (supporting fig . These were compared with 5173 controls (2609 m, 2564 f) from wtccc . Quantile - quantile (qq) plot did not deviate from the expected (supporting fig . No single snp had reached a genomewide significant association (defined as p <5 10). The best signal was rs9416795 (p = 2.00 10), located in an intergenic region on chromosome 10 . This was followed by rs1338041 on chromosome 13, intron region of nalcn, coding for sodium leak channel, nonselective (fig . 1a, supporting table 3). After imputation and quality controls (rsqr> 0.3 and maf 0.03) these had satisfactory genomewide coverage, except a few regions, mainly telomeric and centromeric regions (manhattan plot, fig . The regions not well covered with gwas and imputation are listed in supporting table 7 . There were no snps with genomewide significance (defined as p <5 10). A few clusters of possible associations (defined as p <5 10) were found and shown in the manhattan plot (fig . 1b, table 1). With imputation, the best signals found clustered around nalcn with best p value of 9.8 10 in rs61973742 and 5 more snps in the same gene just short of the best p value . The majority of the associated snps were found in the first intron between the first and second exon . The remaining were within a few kilobases of the 5 region of exon 1 and 5 untranslated region (utr) (fig . The second cluster with peak p at 3.1 10 was found in rs67863238, chromosome 11 base position 48,267,856 (hg19). This cluster codes for a number of olfactory receptors (or4x1, or4x2, or4s1, and or4b1) (fig . 2b, supporting table 5). Remaining imputed snps that passed the possible associations were found on chromosome (chr) 1 rgl1, chr 2 intergenic 3 of kiaa1715, chr 10 intergenic, and chr 11 col4a1 . The snps and local plots of these regions are shown in supporting table 4 and supporting figures 4 and 5 . In this cervical dystonia gwas, no loci reached a statistically significant association with dystonia . The most crucial limitation was the small sample size, which was underpowered to detect loci with a smaller disease effect . Assuming maf of 0.4, odds ratio 1.3, additive model, and prevalence of 430 per million in dystonia, it would need more than 1800 cases to achieve an 80% power . If the odds ratio was as high as with the complement factor h in macular degeneration, the cohort of 212 cases with same assumptions would have a power of almost 100% to detect the snp . Hence, we conclude that a common snp with large effect - size is unlikely to be present in idiopathic cervical dystonia, at least in chr 1 to 22 as shown in manhattan plot (fig . 1) and within the regions that were usually genotyped by microarray (supporting table 7 for uncovered regions). Our group has also recently identified mutations in anoctamin (ano3-dyt23), a calcium - gated chloride channel gene, leading to autosomal dominant craniocervical dystonia . Nalcn protein is a member of 4 6 transmembrane voltage - independent, nonselective, noninactivating ion channel found in all animals studied and universally expressed in mouse brain and spinal cord . Our uk brain expression consortium data shows nalcn is universal expressed in brain (supporting fig . 3). In schizophrenia, the associated snp in nalcn was rs2044117, located at the last intron . The c - terminal end of the protein is the important site in coupling with unc79 and unc80 proteins . This complex senses calcium level and results in alteration of leaking current and neuronal excitability, comparable to ano3 as a calcium - gated chloride channel . Mice with exon 1 knocked out die from disrupted respiratory neuronal firing in the brainstem, suggesting regions other than the c - terminal are also critical . Our snps were clustered around the exon 1 both at 5 and within the first intron . There are a few synonymous and non - synonymous snps found in the exon 1 (rs144447052, rs145910377, rs74707055, rs76774740, rs75606652, rs77203309, rs188237867, rs79047578, and rs9557636). These snps are very rare, with maf well below 0.01 and not included in the imputation and analysis . These may be pathogenic but unidentified rare pathogenic variants tagged to our snps are also possible . The best snp from imputation (rs61973742) has a low r with the best snp rs1338041 in gwas . There is a great discrepancy of the maf, 0.062 in rs61973742 and 0.34 in rs1338041 . A discrepancy in maf can lead to low r (vanliere and rosenberg and wray). These 2 snps have a \|d\| 5 0.88 despite its low r (0.05) in 1000 genomes 378 european population . It was not found in the initial gwas, but multiple snps with borderline p value were identified from imputation (supporting table 5). If replicated, this represents the power of finding new associations that are not well tagged by common snps and are identified through haplotypes inferred during imputation . This group of associations, namely or4x1, or4x2, or4s1, and or4b1, is interesting (fig . They belong to the olfactory receptor, family 4, a type of g - protein - coupled receptors (gpcrs). Olfactory function may seem unrelated to dystonia . Recently fuchs et al . Reported the association of gnal mutation with primary torsion dystonia, and the predominant clinical feature in these patients is cervical dystonia . Gnal, coding an olfactory g protein [g(olf)] is found highly expressed in striatum and coupled with the expression of dopamine d1 receptor (drd1). The 4 olfactory genes (or4x1, or4x2, or4s1, and or4b1) are universally expressed in brain and orb1 is highly expressed in striatum as well (allen brain atlas). This may lead to slightly different p values in gwas and imputation for the same snp . The 3 other hits found in gwas were located at intergenic at chr 6 92 mb, chr 10 28 mb, and chr 19 29 mb . The local plots of postimputation snps in these regions are shown in supporting figure 4 . The sparse imputed snps around chr 10 suggested that might be a false - positive result, as were the imputation finding at chr 1 183 mb and chr 2 176 mb (supporting figure 4). The extra snps found with imputation at chr 6 92 mb and chr 19 29 mb suggested potential association . The p value was lower than the best 2 clusters in nalcn and or4x1 and the functional role within an intergenic region is difficult to predict . Given the recent findings from encode, intergenic region may still play a significant function in transcription . In summary, we found a plausible association, though not statistically confirmed, of cervical dystonia with snps in the nalcn region . Replication on another cohort of cervical dystonia cases would be essential to confirm the association . As dystonia gwas is relatively understudied, we make all these data publicly available to encourage further analyses of the problem.

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