Datasets:

giuliadc

/

pubmed-filtered

like 0

pubmed-601

the 62-year-old male patient in this case report had a smoking history of more than 30 pack-years and a medical history of hypertension and diabetes. he had had a percutaneous coronary intervention 6 years previously due to angina and had noticed a cold and numbing sensation in the extremities of both hands for the previous 3 years. two years previously, the patient's fourth finger on the left hand had experienced blue discoloration, with extreme pain of greater than 90/100 mm on the visual analogue scale (vas), followed by ulceration. after a few tests at the orthopedic clinic, he had been referred to the pain clinic for conservative treatment one year previously. on the patient's upper extremity angiography, greater than 80% stenosis was observed in the first and third finger artery in the area of radial artery and palmer arch, and greater than 90% stenosis was also observed in distal ulnar artery. at the time of referral, the patient had been taking oxycodone 40 mg twice a day, as well as limaprost 5 g and gabapentin 300 mg 3 times day. even with an increase in opiate dose and several chest sympathetic block and stellate ganglion blocks, the analgesic effect was temporal. the pain and ulcers on the fingers worsened, and a finger amputation was planned as arterial bypass surgery was not a valid method for this patient. although a number of treatments were tried, the patient complained about extreme pain, 90/100 mm on vas, and there were severe gangrenous ulcers progressing on the fourth finger of the left hand and the index finger of the right hand. the patient was very much against the amputation even though he was under extreme pain and had been referred to the pain clinic several times. the blood pressure, heart rate, oxygen saturation, and electrocardiography were monitored, and, with the patient in the prone position, local anesthesia was performed at t2-3 intervertebral space. a 15-gauge tuohy needle was used for the paramedian approach with a c-arm fluoroscopic image. after ensuring the needle was positioned in the epidural space, the guidewire was inserted for easy insertion of the electrode, and the electrode was positioned in the posterior epidural space using the c-arm fluoroscopic image. the guidewire was removed, and the octrode electrode lead (advanced neuromodulation system inc, plano, texas, usa) was placed 2 mm left of the radiological center. the electrode was connected to the test stimulator, and the stimulation was profound in the areas with pain. after the test stimulation, the pain was reduced by 50%. during 1 week of test stimulation, the patient's pain was maintained to within 20-30/100 mm on vas, and the use of opiate analgesics was decreased by 50%. the genesis ipg (advanced neuromodulation system inc, plano, texas, usa) was buried under the lower left-side clavicle, and stimulation was well controlled with 3-4+electrode combination, 4.0 v amplitude, 240 msec pulse width, and 34 hz frequency. after the spinal cord stimulation insertion, the ulcers on both hands began gradual recovery and were completely recovered after 6 months. the pain was maintained within 30/100 mm on vas, and the use of opioid analgesics was decreased more than 50% compared with the pre-procedure stage (fig. the cause of buerger's disease is unknown, but smoking is considered to be a major factor in the initiation and progression of the disease. the progression of the disease is relatively fast, with 2-5 years typically passing between the onset of the disease and loss of tissues. therefore, the foundation of treatment is smoking cessation to stop the disease progression and prevent amputation of the lesions. other than smoking cessation, systemic treatments such as thrombolytic agents, anticoagulants and prostaglandin agents can be used, as well as direct arterial bypass surgery, sympathectic block, and amputation [1-4]. a prostaglandin agent has been used in occlusive arterial disease due to its vasodilation and antiplatelet effects, but it has the disadvantage of 80-90% loss of efficacy through lung circulation when it is administered intravenously. bypass surgery or percutaneous angioplasty tend to limit the treatment, as the saphenous vein for the graft can also accompany phlebitis, which often continues to progress even after the bypass surgery, and the lesions generally spread widely. sympathectic nerve block is known to be effective for the ischemic ulcer but does not decrease the rate of the most severe limb amputations. according to a number of studies, spinal cord stimulation in patients with buerger's disease is not only a safe treatment method but also can prevent limb amputation, reduce pain, and improve blood flow and ulceration. spinal cord stimulation in buerger's disease reduces ischemic pain and the rate of amputation by treating the ulceration. hence, it is recommended for people who fail conservative or surgical treatment and for whom arterial bypass surgery is not feasible, as well as for people who complain about severe ischemic pain after failure of medication. performed spinal cord stimulation in 29 patients with buerger's disease who did not respond to medications or arterial bypass surgery and followed up for up to 4 years. most of the patients (limb salvage rate 93.1%) presented with a significant increase in microcirculation of the blood vessels. lower limb amputation, one of the most serious problems in buerger's disease treatment, was performed in only 2 patients, a considerably lower rate than that seen in other studies that conducted sympathectic block or aterial bypass surgery. a number of other studies have suggested spinal cord stimulation in buerger's disease as a safe treatment method that can reduce the pain and improve blood flow and the treatment of ulcers. therefore, it has been argued that spinal cord stimulation should be considered as an important alternative treatment choice. the reduction in the rate of limb amputation due to spinal cord stimulation is hugely beneficial in socioeconomic terms. the cross-sectional problem of the increase in treatment costs due to spinal cord stimulation can easily be offset by decreases in the length of hospital stay after the procedure, prevention of amputation, and the increase in quality of life due to pain reduction. therefore, spinal cord stimulation should be considered in patients who do not find a sympathectic block to be effective in ischemic pain relief or for whom bypass surgery is not indicated. however, spinal cord stimulation is a high-cost treatment and only should be considered in patients with a high severity of disease, consistent symptoms, lack of success with medication, and no indication for arterial bypass surgery. in 1976, cook et al. reported using electronic stimulation of spinal nerves and the dorsal root in vascular disease of limbs, and since then spinal cord stimulation has been successfully carried out in the treatment of atherosclerotic and vasospastic peripheral arterial disease and is mainly used in vascular diseases of lower limbs. there is no reported case of spinal cord stimulation in a patient with buerger's disease in korea. a case has been reported in which spinal cord stimulation was carried out in a 60-year-old male patient with raynaud's syndrome, and improvements in pain and ulceration were seen although the ulceration was not completely cured. spinal cord stimulation improves microcirculation by inhibiting sympathetic vasoconstriction and stimulates the secretion of a number of inhibitory neurotransmitters, leading to a reduction in pain in patients with buerger's disease. spinal cord stimulation reduces pain in buerger's disease by promoting the secretion of gamma-aminobutyric acid (gaba), serotonin, and substance p at the spinal nerve dorsal horn. in addition, it inhibits nociceptive neurotransmission, and intramedullary oxidative nitric oxide and gaba act as an important mediator for the induction of pain relief after spinal cord stimulation [11-13]. spinal cord stimulation in peripheral obstructive arterial disease can treat chronic ischemic pain and ulcers and also prevent amputation. unlike the pain reduction, the mechanism of action for the improvement in microcirculation is unknown, and the improvement in ulceration is considered to be the outcome of the reduction in vasoconstriction due to the inhibition of peripheral sympathetic nerves. the improvement in microcirculation after spinal cord stimulation in patients with peripheral obstructive arterial disease can be quantified by the ankle-brachial index, transcutaneous oxygen pressure, and regional perfusion index. it is simple and non-invasive and can effectively measure the degree of blood circulation of cutaneous tissue. in this case, the progression of ulcers was only observed through gross examination before and after the procedure, and objective measures were not carried out to determine the degree of microcirculation. also, angiography was not carried out after spinal cord stimulation, and thus the change in peripheral vessels could not be confirmed. however, there was a reduction in redness on the patient's palms immediately after the spinal cord stimulation, and significant improvement was seen a week after the procedure. the ulceration on both hands seemed to improve slightly one week after the procedure, and the ulceration gradually improved to achieve complete recovery 6 months after the procedure. although the patient fully recovered from the ulceration within 6 months of spinal cord stimulation, the pain still was present. however, the pain decreased by about 50% from 90/100 mm on vas immediately after the procedure and substantially improved after 6 months to 20-30/100 mm on vas. this pain reduction led to more than a 50% reduction in the use of opiate analgesics and the total amount of medication. after spinal cord stimulation, the patient completely recovered from ulcers on fingers of both hands and achieved a significant reduction in pain, although the cold sensation in his hands remained. therefore, the authors suggest that spinal cord stimulation be considered in patients with buerger's disease who do not response to medications or sympathectic block and for whom arterial bypass surgery is not feasible. spinal cord stimulation would be an excellent treatment choice in patients with buerger's disease when an amputation is planned due to the lack of successful response to various treatments.

buerger's disease (thromboangiitis obliterans) is known as a segmental inflammatory vasculitis that involves the small-sized and medium-sized arteries, veins, and nerves. most effective treatment for buerger's disease is smoking cessation. except for the cessation of tobacco use, surgical revascularization is available in severe ischemia and a distal target vessel. amputation has been used as the last treatment option of the disease up to the present. increasing limb survival and decreasing amputation rate is important. this case describes the use of spinal cord stimulation (scs) in patient with buerger's disease and its effect is not only the complete healing of ulcers but also amputation is not performed.

PMC3766785

pubmed-602

pari passu with the progress of science in medical education, simulation in medical education is also progressing in leaps and bounds. something which was hardly ever talked about even a couple of years ago has now become the new buzz word in medical education circles, and now india is also getting on the bandwagon. simulation has been variably defined as the imitation of the operation of a real-world process or system over time; an imitation of some real thing, state of affairs, or process for the practice of skills, problem solving, and judgment. the aim of medical simulation training is to imitate reality, or rather, to mimic reality to the closest extent possible so that the learner is in a state of suspended disbelief and he believes himself to be undergoing a real experience. simulation has been used at its greatest effect in pilot training-indeed, it is mandatory for all pilots of commercial aircraft to undergo refresher courses on flight simulators, regardless of their real flying experience. medical simulation has unfortunately not kept up with the development of simulation in engineering systems, flight training, etc. however, this situation is now rapidly changing. this change has been brought about by three main reasons: firstly, a big increase in student population in medical education, both at the undergraduate and post-graduate level. this unprecedented growth has occurred in the past two decades (mci vision 2015). approximately 33,528 graduates pass out every year from these colleges (mci annual report 20092010).. there has also been legislation which limits the number of hours residents can now work, reducing further the opportunity for bedside learning. the american medical association's accreditation council for graduate medical education has limited the time that residents can be required to work to 80 h a week. although this action was intended to improve patient safety, it also reduced the number of patient-contact hours needed to train residents. secondly, there is a growing awareness of patients of their rights and reluctance to be practiced upon and the growing incidence of medical litigation leading to a reduction in the number of available patients. finally, there has been a huge technological improvement in the quality and variety of simulators. now, high fidelity computerised human patient simulators have been developed by various companies (e.g. simman by laerdal, the meti family of human patient simulators by cae health care), which mimic very realistically the response of human beings to physiological insults, whether it be an acute cardiac event, or a trauma event; indeed, any disease process that affects the cardiovascular or respiratory system can be accurately modelled. surgical simulators have been developed to practice laparoscopic surgery using a virtual reality environment (e.g. the lapvr laparoscopic simulator by cae health care), which even provide haptic feedback (a sense of tissue resistance)., many countries are now making certification on simulators mandatory before performing procedures on actual patients. even in india, the mci 2015 vision document says that a mandatory and desirable comprehensive list of skills has been planned and would be recommended for bachelor of medicine and bachelor of surgery (mbbs) graduate. the educational technology section of the 2004 aem consensus conference for informatics and technology in emergency department health care concluded that there should be increasing use of web-based, virtual reality and human patient simulation in educating emergency medicine trainees. the authors postulate the philosophy of see one, simulate many, do one competently, and teach everyone. there are now many articles on how simulation can be used in medical training. a key challenge for surgical training is to provide conditions for effective learning without putting patients health at risk. simulation presents an attractive solution, offering a safe, simulated clinical environment where the training agenda can be determined by the needs of the learner, not the patient. evidence is now available to suggest that simulation training actually improves clinical performance. in a review of simulation articles, it was found that four (3%) journal articles provided evidence for the direct correlation of simulation validity with effective learning. another study showed that the use of virtual reality surgical simulation to reach specific target criteria significantly improved the operating room (or) performance of residents during laparoscopic cholecystectomy. simulation in wound management is still relatively in its infancy when compared to the use of simulation in other areas of medical education. it is however reasonable to believe that the use of simulation in wound management will result in improvement in wound care. simulation in relation to wounds can be divided into use of simulation to study the effect of projectiles on tissue (wound modelling) and the use of simulation in the management of wounds. the purpose of this article is to outline the present status of simulation related to wounds, in terms of their creation and management. moulage is the art of creating mock injuries to simulate wounds. theatrical makeup and common everyday materials moulage is commonly used in mass casualty and disaster drills to add realism to the simulation. it is reasonable to believe, though not proven, that the use of moulage would create a more realistic environment to enhance learning in mass casualty and other trauma situations. as far back as in 1964, omissions and deficiencies in disaster ability were dramatically and conclusively revealed by use of what appeared to be a live disaster setting with smoke, fire, explosions; adverse weather and light conditions; realistically simulated casualties especially prepared not only to look but also to act the part. moulage by author (drp) showing bleeding thigh wound and evisceration on metiman (cae health care) mannequin another area where simulation is used in wounds is to simulate the effect of injuring agents on the body so as to understand the various forces that are at work on the tissues during the causation of an injury. this would help in accurate wound assessment and anticipation of likely tissue damage if the causative agent is known. work has mainly been done on cavitating injuries produced by high-velocity projectiles. because gelatin and glycerin soap have approximately the same density as human tissue, the gunshot effects in them are comparable with those in human tissue. the experiments with these substitutes make it possible to understand what happens to the human body as the result of, for example, a penetrating gunshot. some other authors have used transparent gel candles [15% kraton (polymers) in 85% white paraffin oil] for the same purpose. a synthetic skin skull brain model has been developed to simulate gunshot wounds to the head in such a realistic manner as to reconstruct the specific physical characteristics of a given trauma, including its progressive formation. this model uses a silicon cap containing synthetic fibres for artificial skin, a layered polyurethane sphere for the skull (containing the outer and inner tables as well as the diploe), a periosteum of latex and the brain itself is simulated with ordnance gelatin. high fidelity simulators are now available which can mimic the physiological effects of wounds on the body. the aim of management of a wounded individual in the emergency department is mainly to correct and prevent the life-threatening physiological derangements associated with the wound like airway obstruction, hypovolemia, respiratory distress, etc. as such these physiological derangements can be mimicked extremely realistically by these high fidelity systems, examples of which are the meti family of mannequins from cae health care or the simman family from laerdal. these simulators are used the world over to train emergency medical care providers, medical students, post-graduates and practicing physicians and surgeons in emergency trauma management [figure 2]. military specific simulators (e.g. caesar from cae healthcare) are now in advanced stages of development to train soldiers with non-medical backgrounds at the point of injury. john's ambulance team (malaysia) demonstration of trauma scenario on ecs (meti) simulator there are a few articles on the emergence of simulation in wound management, which deal essentially with teaching learners local wound management. this includes wound dressing techniques, performance of local surgery like escharotomy or wound debridement. wound management simulators can be divided into basic part task trainers which use low-technology solutions that are cheap and high-technology virtual reality simulators which, of course, will be much more expensive. the low-technology solutions are discussed first, followed by the high-technology virtual reality solutions. simulated wounds have been created on animal models like pig trotters to teach the technique of wound debridement. a simulated wound covered with thick slough can be created on a pig trotter by excising an area of skin and then applying a layer of hydrocolloid paste (e.g. adapttm, hollister) on the denuded area. a layer of toothpaste may then be applied and covered with a piece of glad wrap to simulate the application of emla cream. a small curette is then used to debride the wound as in a human patient, after washing away the layer of toothpaste (= topical anaesthetic). another study has created a simulated abscess in chicken breast by injecting mock purulent material under the chicken breast skin. the author felt that this new simulation model may be an effective tool to teach skin abscess management. physicians who evaluated the simulated abscess found that it replicates the classic palpable fluctuance and ultrasound findings of an actual abscess, and it can be surgically incised and drained in a similar fashion. yet another researcher has used an orange to simulate debridement-the student is required to remove the peel and the white fibres (representing slough) off the orange without puncturing the inner orange itself. burn wounds offer a challenge to learners in terms of local wound care, and as a result this helps train burn care trainees in the management of such wounds, specifically to perform escharotomy. a major technological development has been that of a thigh wound virtual reality model by delp et al. they have used the data from the visible human project as the basis of reconstruction of the thigh anatomy, on which they superimposed wound models of bullet wounds gleaned from data available in the literature. functional consequences of the injuries were also modelled, which provided both short-term consequences as well as long-term disability of the simulated patient. the user could interact with the virtual thigh model using virtual surgical instruments from a virtual tray to stop the bleeding, assess muscle bleeding and contractility, perform debridement, align bone, etc. the overall effect on the patient's physiology was also calculated and displayed in terms of vital signs readouts, which changed depending upon appropriate treatment, e.g. control of bleeding, etc. the virtual reality medical center (vrmc) conceptualised and developed a unique injury simulator as an adjunct to current combat medic training. this initial technology, called injury creation science (ics), very realistically simulated a number of battlefield injuries such as amputations, eviscerations, blast injuries, punctures and burns. since the initial prototypes, vrmc has developed this technology into wearable part-task trainers that simulate injuries as well as allow combat medics to practice actual medical procedures common to the battlefield. the procedures currently available or under development include treatment of pneumothorax, hemoperitonium and gunshot wounds to an artery. by integrating medical science with cutting-edge simulation and training technologies, realistic prosthetic tissue, wounds and part-task trainers one of the major issues with wounds is the risk of hospital-acquired wound infections, which are usually preventable by simple personal hygiene techniques. unfortunately, many hospital workers are either unaware of them or unwilling to follow these steps. training of all hospital staff to prevent cross infection is therefore high on the agenda for most hospital administrators, impinging as it does on patient safety. if the movement of bacteria that cause wound infection, and their removal from hands and other inanimate hospital objects could be seen, then it would be a very useful training tool and could markedly increase staff compliance with hygiene measures. this is exactly what was done in one study wherein ultraviolet polyethylene microspheres were able to simulate the spread of bacteria through direct and indirect contact on different surfaces and had the ability to be washed off under specific hand washing guidelines. the authors felt that due to the positive results, the use of microspheres as a simulator of bacterial presence and spread should be explored further in order to improve hand washing techniques and compliance. a simulation-based training system for surgical wound debridement has been developed and comprises a multimedia introduction, a surgical simulator (tutorial component) and an assessment component. the multimedia training component describes varieties of wound categories (e.g. burns, lacerations, etc.), methods of debridement (e.g. sharp, mechanical, etc.) and equipment/materials used in the procedure. this module provides the pedagogical context for skills training and assessment, linking together all of the required elements for mastering the procedure. a software virtual agent is included and performs the role of an instructor by providing verbal guidance and assessment feedback. designed to be comprehensive in order to reduce the need for a more senior instructor, the software addresses the patient's condition, initial description of the injury, and provides instruction on operation of the simulator. specific instructions cover the removal of foreign objects with forceps, scrubbing with a brush, rinsing with saline solution and the maintenance of sterility. trainees are instructed to remove debris with forceps, scrub with a brush and rinse with saline solution to maintain sterility. in conclusion, increasing sophistication in simulation technology has been also used in simulation of wounds, both in wound modelling and wound management. the available simulators include low-technology solutions and high-technology virtual reality simulators, all of which train learners in the art of wound management. however, this technology is yet to be validated in wound management to see if simulator training can result in better management of wounds in actual patients. future developments would include research on application of simulator training to real patient care, and the development of better technology to make virtual reality simulators even more realistic by the improvement of design, graphics and haptic feedback.

simulation in medical education is progressing in leaps and bounds. the need for simulation in medical education and training is increasing because of a) overall increase in the number of medical students vis--vis the availability of patients; b) increasing awareness among patients of their rights and consequent increase in litigations and c) tremendous improvement in simulation technology which makes simulation more and more realistic. simulation in wound care can be divided into use of simulation in wound modelling (to test the effect of projectiles on the body) and simulation for training in wound management. though this science is still in its infancy, more and more researchers are now devising both low-technology and high-technology (virtual reality) simulators in this field. it is believed that simulator training will eventually translate into better wound care in real patients, though this will be the subject of further research.

PMC3495369

pubmed-603

asthma is a chronic inflammatory disorder of the airways characterized by bronchial hyperresponsiveness, reversible airflow limitation, and respiratory symptoms (1, 2). internationally, the prevalence of asthma has increased over the last 3 decades (3-5), and in the asia-pacific region asthma causes considerable morbidity, with 15% of teenagers troubled by exercise-induced symptoms during the past 12 months (6). furthermore, asthma mortality rates in more affluent areas, such as hong kong and japan, are similar to those reported in western countries (6). the reported prevalence of asthma in korea ranges from 2 to 13% (9-18). however, its prevalence in the elderly has been reported to be very high, at 12.7% in those aged 65 or more (15). indeed, with a population estimated at more than 48 million, and a life expectancy of 72.0 yr for males and 79.5 yr for females (19), korea faces an important public health challenge in terms of dealing with chronic diseases such as asthma. in addition to its increasing prevalence, the economic impact of asthma is also substantial and continues to grow. the total cost of the disease in the united states was estimated to be u$4.5 billion in the mid-1980s, whereas in the first half of the 1990s, this estimate had increased to between u$6.2 and u$10.7 billion (20-22). in korea, where health insurance is mandatory and the cost of medical care is only covered in part by health insurance, the importance of economic outcomes also continues to grow although little data is currently available. the purpose of this review is to present an overview of the current disease status with respect to the prevalence, mortality rate, socioeconomic burden, quality of life and the treatment patterns of asthma in korea, to identify barriers to improvements in asthma care and to provide recommendations for action at the national, organizational and individual levels. for this, all relevant english and korean language articles were retrieved using pubmed (www.ncbi.nlm.nih.gov/entrez/query.fcgi) and koreamed (www.koreamed.com) from november 2004 to january 2005. recently, several large-scale studies of different population in korea (9-17) have reported asthma prevalence ranging from 2 to 13% (table 1). these differences are probably ascribable to different case definitions, methodologies, and a tendency to survey children rather than adults due to the relative ease of implementing studies in a school environment. according to the latest summary issued by the global burden of asthma by the global initiative for asthma (gina) program, the prevalence of clinical asthma in korea is estimated to be 3.9% (23). the prevalence of asthma tended to be higher in children than in adults and was found to depend significantly on their place of residence, for example, it was found to be higher in seoul than in provincial cities (7). according to 1998 data (11), childhood asthma was less prevalent in korea than in other developed countries. however, among the elderly (aged 65 yr or older), its prevalence was found to be high at 12.7% in 2001 (15), which is about three times higher than among english or us elderly (24, 25). although estimations of asthma prevalence in older age groups differ greatly between countries because of overlapping diagnoses, and poor patient perception of symptoms (24), this rate is unexpectedly high. in the elderly, asthma is an important problem because it is usually underdiagnosed and hence inadequately treated (24-26). interestingly, korean adults and children appear to have a later average age of asthma onset than other asian populations (12) (fig. this is likely to be due to the underdiagnosis of early stage asthma by general physicians. some investigators have reported that an underdiagnosis rate by general physicians was 21% (27). another contributable factor may be that a high proportion of korean people, at least initially, turn to traditional medicine for medical help (28-30), which delays the diagnosis until they are referred to hospitals or emergency department with severe symptoms. age, sex, affluence, genetic predisposition, climate, outdoor air quality and cigarette smoking have all been found to be associated with asthma (7, 12, 15, 31-33). studies suggest a higher incidence of asthma in children than in adults (9-15, 17, 34), and male asthmatic patients were found to be more often hospitalized or treated at a tertiary medical centers than their female counterparts (34, 35). in a regression analysis of the asthma insights and reality in asia-pacific (airiap) data for the eight areas of asia-pacific countries including korea, it was found that a low household income is significantly associated with the likelihood of having moderate or severe persistent asthma symptoms (p=0.002 compared with a high income status) (12). it has been reported that the genetic variations of the chemokine (c-x-c motif) receptor 3 (cxcr3) (36), the signal transducer and activator of transcription 4 (stat4) (37), a disintegrin and metalloprotease 33 (adam33) (38), and tumor necrosis factor-alpha (tnf-) (39) are associated with the susceptibilities to the development of asthma and its intermediate phenotypes in the korean population. the il-4 cytokine gene cluster and/or the t cell receptor/gene complex are believed to be associated with the expression of bronchial responsiveness to methacholone in nuclear families (40), and a polymorphism in exon 7 of chromosome 11q13 was found to be significantly associated with histamine release from basophile to anti-ige stimuli in 80 randomly recruited asthmatic children (41). spider mites (citrus red mite) (42, 43), the european red mite (panonychus ulmi), (44) and the two-spotted spider mite (tetranychus urtcae) (44), have been identified as important allergens in the development of work-related asthma and rhinitis symptoms and toluene diisocyanate has been most commonly implicated allergen in occupational asthma in korea (45). a number of studies have documented an association between air pollution and asthma-related morbidity in korean cities and have linked active smoking with asthma prevalence and severity (15, 28, 29, 46-48). the accurate determination of the asthma mortality in korea is difficult. because large-scale medical databases tend to group asthma data along with those of other disease states, or to group it with less specific group diagnoses such as ' chronic lower respiratory diseases ' (49). according to the korea national statistical office data, the death rate from chronic lower respiratory diseases (including asthma) between 1992 and 2002 increased from 12.9 to 22.6 deaths per 100,000 of the population (50). in 1992, chronic lower respiratory diseases were ranked as the eighth leading cause of death in korea, and in 2002, this had risen to fifth place proceeded only by diabetes mellitus, heart disease, cerebrovascular disease, and cancer (50) (fig. 2). according to the latest gina estimates, there are 4.9 asthma related deaths per 100,000 asthmatics each year in korea (23). globally, respiratory diseases are responsible for 6.3% of all death and asthma for 0.4% (51). these include cigarette smoking, chronic asthma severity, frequent hospitalization, the duration of recent asthma exacerbations, age and a female sex (28, 52, 53). at present, little data is available on the economic burden posed by asthma in korea. however, indirect estimates may be made based on the market shares of asthma medications in korea. according to the data from the pharmaceutical industry (54), from 2001 to 2004, the annual gross value of drugs sold in korea increased by nearly 50% (from u$3.48 to u$5.21 billion) and the proportion shared by drugs for asthma treatment in respiratory drug market increased by approximately 36% (from u$51.8 to u$70.6 million). research in this area is underway, with a large-scale analysis being performed using data from health insurance review agency (hira), the 1998 national survey on health and nutrition in korea, and an ongoing patient survey. to adequately account for cultural and behavioral factors, a quality of life questionnaire for adult korean asthmatics (qlqaka) was developed (55), and in a multicenter study 5 using qlqaks, it was found that dyspnea (87%), difficulty in sputum discharge or throat clearing (87%), strenuous activity limitation (84%), and coughing (82.4%) were most frequently complained about by adult asthmatics (55). the airiap study revealed that approximately one-third of all korean asthmatic patients felt restricted in terms of their abilities to perform the routine activities of daily living such as, exercise, social activities, or sleeping (12). this survey also reported that 52% of asthmatic patients had experienced daytime symptoms during 4 weeks prior to completing the questionnaires, and that 41% of patients with asthma had been woken at night by their symptoms (12). another study conducted to assess the quality of life of 189 patients showed that running, walking, and hurried movements appeared to be the most impaired daily activities (56). the characteristics that significantly impaired an individual's quality of life score were disease severity, level of asthma control and symptom attacks during the previous 3 months (all p<0.001) (56). interestingly, a discrepancy was identified between perceived and actual asthma control in a substantial number of asthma patients. according to airiap data, about one-fifth of korean asthmatic patients with severe persistent symptoms considered that their asthma was adequately controlled, despite their current symptoms suggesting otherwise (12) (fig. this is due in part to the fact that some asthmatics have poor symptom perception (57-59) and partly because asthmatics in korea are not adequately educated about the practical and theoretical aspects of asthma management (60, 61). no national guidelines concerning best management practice of asthma have been issued in korea, although three academic associations have published separate guidelines (62-64). however, almost half of the physicians (43%) in 325 korean clinics surveyed by the hira either did not use or were unfamiliar with these guidelines (65). therefore, few asthma patients used or were prescribed inhaled corticosteroid by their physicians to prevent symptoms (65) (fig. 4). the reasons for this reluctance to prescribing inhaled corticosteroid as cited by physicians in the hira survey were, in decreasing order; lack of patient education concerning inhaler use, high cost, patient refusal to use, and side effects (65). in addition, according to the airiap survey, 65% of patients with asthma in korea have never undergone a lung function test, and only 32% of patients reported that they had undergone a lung function test during the previous year (12). moreover, although 6% of patients owned a peak flow meter for self-monitoring, only 20% of them used it daily, whereas another 20% used it on a symptomatic basis (12). another survey found that patients who used a nebulizer at home were not properly instructed as to how to use it, and did not follow equipment maintenance procedures (66). however, paradoxically, most korean asthmatics were satisfied with their treatment, although many expressed a need for more information on the disease (12). this ' disconnect ' from the guidelines by physicians has had a major impact on the management of asthma patients. to reduce this gap, an ' easy asthma management ' study is currently underway in korea. the purpose of this study may provide a simple stereotyped algorithm using a computer based program to allow clinicians to easily diagnose asthma and implement its correct management. in december 2004, it was estimated that 11.9 million koreans had access to the world wide web (www) and the number of allocated ip addresses allocated in korea ranked eighth worldwide (67). nowadays, computer-based educational programs available on the www are believed to be an effective alternative as they overcome the cost and time barriers that prevent implementation of education programs for asthmatics (68). however studies have shown that asthma educational material on the web sites is highly variable in quality and content, lacks detail of the core concepts essential for asthma education, and fails to meet patients needs (69, 70). this review showed that the prevalence of asthma in korea ranges from 2 to 13%, and that physicians and patients often underestimate its severity. mortality from chronic lower respiratory diseases including asthma increased from 12.9 to 22.6 deaths per 100,000 of the population between 1992 and 2002. disease severity, level of control, and symptom state were all found to negatively impact the quality of life of asthmatics. moreover, although international and korean asthma management guidelines are available, primary care physicians are largely unfamiliar with or fail to implement them.

a systematic review of english and korean articles published between 1990 and 2004 and a search of database and various online resources was conducted to determine the prevalences, mortality rates, socioeconomic burden, quality of life, and treatment pattern of asthma in korean adults and children. asthma morbidity and mortality in korea are steadily increasing. the prevalence of asthma in korea is estimated to be 3.9% and its severity is often underestimated by both physicians and patients. mortality resulting from chronic lower respiratory diseases including asthma increased from 12.9 to 22.6 deaths per 100,000 of the population between 1992 and 2002. disease severity, level of control, and symptom state were all found to negatively impact the quality of life of asthmatics. although international and korean asthma management guidelines are available, familiarity with and implementation of these guidelines by primary care physicians remain poor.

PMC2733988

pubmed-604

asthma in adults is a heterogeneous disease usually characterized by chronic airway inflammation with hyperresponsiveness of airways to various stimuli. the prevalence estimates of asthma, in general, and the prevalence of severe asthma, in particular, are well known and sufficiently illustrative. according to the global asthma report 2014,1 asthma affects 334 million people with projections of 400 million in 2025. in addition, asthma still accounts for one of every 250 deaths worldwide, and almost all of these deaths are avoidable.2 in spain, prevalence rates of 5% and 10% have been estimated in adults and children, respectively, with more than three million people with asthma.35 in addition, there is a large geographical variability is asthma prevalence, with a trend toward stabilization following an increase in the prevalence of the disease observed in the past years.3,6 this complex disease affects patients of all ages. although its diagnosis is usually easily established and most patients respond to therapy, approximately between 3% and 10% of adult asthma patients have disease that is difficult to control despite taking maximal doses of inhaled medications.7 patients with therapy-resistant or difficult-to-control asthma require a rigorous and systematic approach to their diagnosis and treatment. despite rigorous, optimized follow-up treatment, 75% of severe asthma patients did not achieve adequate symptom control and presented with impaired quality of life.8 although much progress has been made in the understanding of difficult-to-control asthma, with consensus documents and clinical practice guidelines focused on severe uncontrolled asthma with proposals for a stepwise diagnostic procedure and phenotype-targeted treatment,9,10 improvements in clinical practice are still limited. in addition, involvement of pulmonology services in the specialized approach for difficult-to-control asthma is insufficient. in a survey carried out in spain, 47 (68.1%) of a total of 69 pulmonology services met criteria for an important level of health care activity in asthma, but only 29 (42%) had a monographic consultation for difficult-to-control asthma and 37 (53.6%) had implemented an education program. as for post-graduate education, only 31 (44.9%) provided their resident physicians with specific asthma training.11 in 2015, the asthma area of the spanish society of pneumology and thoracic surgery (separ) addressed the task of establishing the necessary requirements for the provision of official accreditation standards of the different levels of care for asthma units already existing in hospitals of the spanish national healthcare system. accreditation levels included basic units, specialized units, or specialized units of high complexity, with or without the distinctive of excellence, according to the fulfillment of a series of criteria established by the society. the development and implementation of this nationwide strategic plan had several objectives, including improving the care and quality of life of asthma patients, particularly patients with severe difficult-to-control asthma in order to achieve a decrease and prevention of acute exacerbations, with a subsequent reduction in the number of visits to the emergency department, need of in-patient care, and inadequate use of asthma medications. all these actions have been complemented by the development and implementation of education and self-management strategies (where nursing plays a key and indispensable role) and follow-up of patients in monographic consultations for asthma and in the framework of multidisciplinary involvement of health care professionals. in a recent scientific meeting of health care professionals of the different asthma units, which underwent specialized accreditation, it was agreed that a perspective article should be drafted to achieve greater dissemination of the details regarding the characteristics, service portfolio, and resources available in these units. the present document written and approved by all attendees of this event and members of units provides information and describes the initial global experience of asthma units accredited in spain. it also aims to contribute to raising awareness among pulmonologists and other professionals about the problem of severe refractory asthma and the response offered by asthma units to improve patient care and to minimize the complications and burden of this complex pathology. in this respect, we believe that the spanish experience may be useful and potentially applicable to other settings, in particular with similar health care systems. however, workup details and assessment of outcomes were outside the scope of this report. significant progress and advances in the understanding of asthma and in the care of asthma patients with an increase in the number of medications and development of new protocolized therapeutic strategies have been associated with a substantial reduction in asthma-specific mortality and hospitalization rates for asthma. however, despite all these improvements and the development of clinical practice guidelines with specific recommendations for the control of asthma, it is well known that in clinical practice, adequate asthma control is achieved in only one-third of patients.1214 uncontrolled severe asthma accounts for ~15% of all asthma patients but is associated with a higher morbidity and mortality as well as an important impact on health care costs and the consumption of resources.15 the asmacost study16 based on data of a prospective cohort of 627 patients throughout spain with asthma diagnosed according to the guidelines of the global initiative for asthma (gina)17 and the adapted spanish criteria (gua espaola de manejo del asma [gema])9 and followed up for 12 months revealed that the total societal cost for asthma was 1,726 per patient annually (1,533 to the national health service), with higher costs for patients older than 65 years and for those with a more severe disease (2,635 for severe asthma). based on these findings, the total annual cost of asthma in spain was estimated to be 1,480 million euros. on the other hand, 70% of total costs were attributed to the poor control of the disease, which further reinforces the need to achieve clinical stability of the patients as it has been repeatedly emphasized in clinical practice guidelines.9,17 from another perspective, poor asthma control is a determining factor of the high indirect costs related to absenteeism and reduced productivity. in a naturalistic and observational study (tenor study) of 4,756 asthma patients recruited in 283 study sites from diverse geographical areas in the usa and followed up for 24 months, the mean annual costs for uncontrolled patients with difficult-to-treat asthma were more than double of those for controlled patients throughout the study.18 different studies carried out in other countries have obtained similar results.1921 moreover, chronic comorbidities contribute to the burden and costs of persistent asthma,22 and poor adherence to asthma medication regimens, including patients with difficult-to-control asthma, is a key problem contributing to poor disease control.23 adherence to asthma treatment in patients with uncontrolled difficult asthma is highly relevant, since treatments currently considered for step-up therapy (biologics, bronchial thermoplasty)9,17 are very expensive and, in many cases, are prescribed without adequate adherence to drug regimens in the lower steps.24 a further interesting aspect concerns to deficiencies in the development and implementation of effective education programs for asthma patients, promotion of health, and social support. it has been demonstrated that education in self-management of asthma with symptom or peak flow monitoring, combined with regular medical visits and written action plans, is effective in improving health outcomes in adults with asthma.25,26 in a 1-year cluster randomized controlled multicenter study with the participation of 230 adults with mild-to-moderate persistent uncontrolled asthma, an asthma educational program based on a repeated short intervention, given in four face-to-face sessions at 3-month intervals was effective in improving asthma symptom control, future risk, and quality of life.27 in this study, the education program included administration of a written personalized action plan and training on inhaler technique.27 other experiences reported similar results. in a controlled clinical trial in which a comprehensive asthma intervention program was evaluated in a population of medicaid-insured asthmatic children, a significant improvement in health outcomes (emergency department visits, hospitalizations) and asthma health care costs was observed in the intervention group in the year after enrollment.28 in a randomized patient selection study with crossover, a vigorous medical regimen and intensive educational program were able to decrease hospital use among a group of adult asthmatics who had previously required repeated readmissions for acute asthma exacerbations.29 in a large teaching hospital (glasgow royal infirmary) where asthma management was audited prospectively for 1 year, treatment of asthma patients in wards with a specialist interest in respiratory medicine was associated with a reduction in the rate of readmissions compared to patients admitted to general wards without this special interest (2% vs. 20%).30 given the complexity and multiple factors involved in the control of asthma, there is a need for establishing asthma units involved in the care of asthma patients, especially those patients with severe difficult-to-control disease. however, up to the present time, a few studies have demonstrated that assessment and management of patients in specific units for severe asthma are associated with substantial benefits in terms of health (asthma control, quality of life) and reduction in economic burden. in a study of 346 patients with severe asthma referred to specialist centers across the uk and followed up for a median of 286 days, significant reductions in health-care use (primary care or emergency department visits), hospital admissions, and steroid dose were observed, which were accompanied by significant improvements in quality of life and asthma control.31 in a crossover study carried out in spain, treatment of patients in asthma clinics was cost-effective and beneficial in asthma management in comparison with standard outpatient services.32 the national asthma program in finland, probably one of the most outstanding health care networks for asthma patients, has shown that integration of different health care levels involved in the management of asthma (pneumologists, primary care physicians, and pharmacists) improves control of the disease and reduces the morbidity of asthma.33 the separ has promoted the task of accrediting the levels of the different asthma units already existing in our country, with the following objectives: 1) to improve the level of care of asthma patients, ensuring a framework of quality of care; 2) to establish resources and facilitating their management; 3) to promote the development of training plans in asthma and to advance in the concept of accreditation of knowledge; 4) to favor collaboration with professionals from other clinical disciplines in a cooperative environment; and 5) to promote asthma research. within the separ training framework, a manual on severe asthma and difficult-to-control asthma has been published, which includes a chapter on the provision and organization of a severe asthma unit.34 briefly, three levels of specialized asthma care have accredited based on available resources: specialized unit for highly complex asthma, specialized asthma unit, and basic asthma unit. regardless of the level of accreditation obtained, the distinction of excellence could be granted when more requirements were met at each level. in order to obtain accreditation at each of the levels, there were indispensable requirements (irs) that had to be met; also, there were two other criteria, which included evaluable criteria (ec) and recommended criteria (rc). the certification of each level was achieved if at least 80% of the ec corresponding to each category were fulfilled. the quality of each level was excellent if the result of the formula (ec+rc) 100/(total number of ec+rc items at the level) was 80%. the integral care of the patient with severe asthma in a specialized multidisciplinary and high-quality setting presents many advantages, particularly related to the diagnosis of asthma, including identification of patients according to phenotypes, treatment of comorbidities, protocolized followup, optimization of the therapeutic arsenal, indication of specific treatments, and emphasis on health education for both patients and health care professionals. for instance, to have available a specialized nurse well trained in asthma education and use of inhaler devices will result in a better understanding of the disease, adherence to treatment, and control of the disease. in addition, there is a high prevalence of psycho-comorbidity in asthma, and acting at this level can improve the quality of life of the patients, control of anxiety, low consumption of resources, etc. in this respect, likewise, the possibility of implementing complementary programs contributes to better control of the disease. this approach is associated with cost savings as a result of a better and rational use of the resources. in addition, accredited asthma units could help patients with asthma through identifying misdiagnosed cases of asthma and providing more appropriate treatment/referral. misdiagnosis of non-asthmatic conditions as uncontrolled asthma has been reported to be as high as 12%30%.35,36 the implementation of databases with relevant and updated clinical information is an essential tool for the independent analysis of the data of each unit or aggregated data from different units. this is effectively a proposal for the control and improvement of care provided to our patients, the results of which are pending to be collected and evaluated. we believe that diagnosis and treatment by highly specialized and experienced personnel in asthma will result in the achievement of a better control of disease. a recent study carried out in an asthma clinic of a university-affiliated hospital in lugo in 2012 showed that all cost variables except drugs and diagnostic tests were significantly reduced in comparison with standard outpatient services, giving an annual saving per patient of 338.32 the assessment and management of patients with severe difficult-to-control asthma in accredited asthma units aims to improve the quality of care and control of the disease. in addition, specialized asthma units can improve the cost-effectiveness of pharmaceutical expenditure, especially regarding the new and costly therapies. health care professionals involved in the management of asthma should continue pursuing for unifying the quality of care of patients with asthma in a multidisciplinary collaborative setting.

this paper, developed by consensus of staff physicians of accredited asthma units for the management of severe asthma, presents information on the process and requirements for already-existing asthma units to achieve official accreditation by the spanish society of pneumology and thoracic surgery (separ). three levels of specialized asthma care have been established based on available resources, which include specialized units for highly complex asthma, specialized asthma units, and basic asthma units. regardless of the level of accreditation obtained, the distinction of excellence could be granted when more requirements in the areas of provision of care, technical and human resources, training in asthma, and teaching and research activities were met at each level. the spanish experience in the process of accreditation of specialized asthma units, particularly for the care of patients with difficult-to-control asthma, may be applicable to other health care settings.

PMC5431694

pubmed-605

acetone is the simplest ketone compound. in general, acetone is not considered harmful, and the world health organization has not classified acetone as carcinogenic. however, its prolonged inhalation can not only cause irritation of the mucous membranes, headaches, confusion, and narcotic effects, but lead to coma as well [15]. for etiological reasons, acetonaemia is classified to be of endogenous and exogenous origin [6, 7]. multiple toxicities and physiopathological conditions result in ketosis (acetonaemia particularly), including diabetes mellitus (dm), starvation coupled with physiologic stress, prolonged exercise, during pregnancy, and ethanol toxicity, other alcohol ingestions, drug toxicities, inborn errors of ketone metabolism, alcoholic ketoacidosis, delirium tremens, and hypothermia [69]. the main symptom of dm is a high blood glucose concentration depending on insulin deficiency. in this case the body can not fully use glucose but could use fatty metabolism instead of glucose for energy. dm, especially diabetes and autoimmune associated diseases, thyroid disease, and diseases that can accompany diabetes (hypertension, cardiovascular disease, cerebrovascular disease, and renal insufficiency) can cause pathological changes in most of the tissues, organs, and biological fluids depending on lipotoxicity and glucotoxicity. a lot of medical, chemical, and medicolegal investigations have been carried out with the determination of acetone in blood and other biological fluids [2, 6, 7, 10]. over the decades, several methods have been used for its determination in biological samples. in the beginning, colorimetric methods were developed and used for the determination of acetone in plasma [1113]. these methods have common disadvantages such as the lack of specificity and detection limit. in recent decades, gas chromatographs equipped with flame ionization detectors or mass spectrometric detectors have been developed for determination of acetone concentrations in body fluids and in expired air [1418]. enzymatic methods are more specific but more complex and have long assay times and gas chromatographic methods, although widely used, are applied with difficulty as routine tests. the determination of acetone in the blood is most important in clinical diagnostic laboratory studies. there are three ketone bodies, while the two main ketone bodies are acetoacetate (acac) and 3-b-hydroxybutyrate (3hb), the third ketone body; acetone (ac) is found minimum level. ketone bodies are produced by the liver and used peripherally as an energy source when glucose is not readily available [9, 19]. ketone bodies are three water-soluble compounds that are produced as by-products when fatty acids are broken down for energy in the liver and kidney. ketone bodies are produced from acetyl-coa mainly in the mitochondrial matrix of hepatocytes when carbohydrates are so scarce that energy must be obtained from breaking down fatty acids [9, 20]. acetone can not be converted back to acetyl-coa, so it is excreted in the urine or exhaled. recently, blood or urine testing kits have been used to test for the presence of acetone in clinical biochemistry laboratories. acetone can also be quantified by sampling the human blood and testing by gas chromatography. described a rapid and simple hplc procedure that can be used for the routine measurement of acetone in biological fluids, such as plasma and urine. according to fujii et al., it is proposed that liquid chromatography with fluorescence (lc-fl) seems to be useful for the determination of acetone in the saliva. in this paper, we present a rapid and simple hplc technique using 2,4-dnph as a derivatizing reagent for quantitative determination and metabolomic research of acetone in biological fluid such as human blood. 2,4-dnph (sigma-aldrich, 97%), acetone (merck, 99.8%), acetonitrile (sigma-aldrich, 99.8%), and methanol (merck, 99.8%) were used in this study. for the chromatographic analysis, thermo scientific dionex ultimate 3000 hplc with a thermoacclaim-c18 (15 cm 4.6 mm 3 m) column and uv-vis dad were used. the deionized water was 18.2 mcm (millipore direct-q3 uv) and was used throughout the experiments. for the biochemical analyses, cobas 6000 (roche, germany) autoanalyzer was used for blood glucose levels. qualitative analysis of total ketones in urine was evaluated by iris iricel 2000 analyzer (icem velocity). in the first stage of our study, following a 12-hour fasting venous blood samples were taken from patients admitted to hospital of the faculty of medicine, canakkale onsekiz mart university. the human blood and urine samples were directly collected into a tube (without a collection device) between 08:30 and 11:00 am. clinical biochemistry laboratory blood and urine glucose tests were studied for routine biochemistry using a urine autoanalyzer. the patients were divided into high blood glucose and urine ketone positive subjects (group 1) and high blood glucose and urine ketone negative example subjects (group 2). the patients with hyperglycemia were 8 females and 7 males (age: 2187; n=15), while 5 female and 2 male patients had positive urine ketones (age: 2168, n=7) and 5 male and 3 female patients had negative urine ketones (age: 5587, n=8). the blood glucose levels varied between 110 and 320 mg/dl in our patients (table 1). in the second stage of the study, to determine the probable positive acetone, its quantitative analysis was carried out in biological fluids using the hplc technique. the blood samples were immediately prepared for hplc analysis carried out within 8 hours after sample collection. plasma specimens were deproteinized with acetonitrile (1: 1, v/v); 2,4-dnph is added to the supernatant (filtered blood samples) and treated with acetonitrile (2: 1, v/v) to prevent crystallization of the synthesized phenylhydrazone. an aliquot (20 microliters) of the reaction mixture was subjected to hplc at ambient temperature using thermoacclaim-c18 (15 cm 4.6 mm 3 m) column and uv-vis dad with (methanol/acetonitrile) water as eluent at a flowrate of 1 ml min and detection at 365 nm [2, 3]. the experimental procedures were conducted in accordance with the ethical standards of the helsinki declaration and approved by the canakkale onsekiz mart university human research ethics committee. written informed consent was obtained from all the subjects. the 2,4-dinitrophenylhydrazone standards were prepared by mixing a and b solutions: (a): 0.40 g of 2,4-dnph dissolved in 2.00 ml of h2so4+3.00 ml of h2o+10.0 ml ethanol; (b): 0.50 g or 1.00 ml of the acetone standard dissolved in 20.0 ml ethanol. after this mixing, a precipitate was formed in each case, isolated through filtration, and dried in vacuum [3, 24, 25]. acetone was added into its 2,4-dnph derivatives by mixing 1.00 ml of a solution containing 200 mg/100 ml of 2,4-dnph with 1.0 ml of h3po4, and 4.00 ml of the human serum. after 2 h, a 40.0 l aliquot was withdrawn and analyzed by the hplc technique [3, 2427]. chromatographic separation was achieved in a thermoacclaim-c18 (15 cm 4.6 mm 3 m) column at uv-vis dad detector (max 365 nm). the injection volume was 20.0 l and the detection was performed at 365 nm. the following gradient was used: (methanol/acetonitrile) (8: 2) water 60: 40 (v/v). elution was achieved at retention time (tr) 12.10 and flow-rate of 1 ml min. standard calibration curve was prepared with acetone (0.5, 2.5, 5.0, 10, and 20 mmol l) and 40 l 2,4-dnph in acetonitrile. human samples were prepared by adding 40 l 2,4-dnph and 500 l acetonitrile to 200 l human serum. the calibration curve was constructed by plotting the peak area of the 2,4-dnph derivative of acetone (y) versus the concentration of acetone (x, mmol l) by linear regression (n=4). the equation was found as y=0.7361x+0.0877 with a 0.9967, correlation coefficient (r). the method proposed was validated as described in ich guidelines in parameters of linearity, limit of detection and quantification, accuracy, and precision. the linearity of the method was determined at four concentration levels ranging from 0.5 to 20 mmol l. the calibration curves were constructed by plotting the peak area of the 2,4-dnph derivative of acetone (y) versus concentration of acetone (x). the slope, y-intercept, and correlation coefficient were calculated. to check the linearity, the lod was estimated using signal-to-noise ratio of 3: 1 or (3 s/m) and loq as 10: 1 or (10 s/m), at which accuracy and standard deviation were within 20% as per ich [28, 32, 34, 35]. intraday accuracy and precision were performed for acetone at 5.0 mmol l in replicate (n=3). accuracy (expressed as recovery) and precision (expressed as relative standard deviation) should not deviate by 15% of the nominal concentration. as can be seen from figure 1, the retention time (tr) was obtained as 3.80 min. the most efficient separation of acetone as its 2,4-dnph was obtained with a thermoacclaim c18 column (15 cm 4.6 mm 3 m) at retention time (tr) 12.10 min and flowrate of 1 ml min using a (methanol/acetonitrile) water elution gradient. no elution problem for acetone as its 2,4-dinitrophenylhydrazone derivative was observed. typical hplc chromatogram of 0.681 mmol l acetone as its 2,4-dnph derivative is given in figure 2. the hplc chromatogram of 15 mmol l acetone in human blood in the first patient of group 1 is given in figure 3. in our study, the patients were determined as high blood glucose and urine ketone positive subjects (group 1) and high blood glucose and urine ketone negative example subjects (group 2). the blood glucose levels, given in table 1, vary between 110 and 320 mg/dl in our patients, except for sample 7. many patients in our study have dm disease, except samples numbered 4, 5, 11, 13, and 15 (table 1). the calibration plot of peak area against concentration was obtained linear in the range 0.5 to 20 mmol l. the regression equation and correlation coefficient were obtained as y=0.7361x+0.0877 with a 0.9967, correlation coefficient (r). so this value is smaller than 2.2, and calibration curve is linear. for the human plasma samples, fcritical value for calibration curve at 3 degrees of freedom (p=0.05) is 3.18. so, this value is smaller than fcritical value, and obtained values are appropriate. the lod and loq were found as 0.041 and 0.136 mmol l, respectively. the accuracy for acetone in human plasma samples expressed as recovery was found as 98%. for interday assay, the accuracy for acetone in human plasma samples expressed as recovery was found as 96%. 2,4-dnph can be used to qualitatively detect the carbonyl functionality of a ketone such as acetone or aldehyde functional group. a positive test is signaled by a yellow, orange, or red precipitate known as a dinitrophenylhydrazone. if the carbonyl compound is aromatic, then the precipitate will be red; if aliphatic, then the precipitate will have a more yellow color. the reaction between 2,4-dnph and a ketone such as acetone is shown below:(1)rrco+c6h3no22nhnh2c6h3no22nhncrr+h2othis reaction can be described as a condensation reaction, with two molecules joining together with loss of water. it is also considered an addition-elimination reaction: nucleophilic addition of the -nh2 group to the c=o carbonyl group, followed by the removal of an h2o molecule. 2,4-dnph does not react with other carbonyl-containing functional groups such as carboxylic acids, amides, and esters. for carboxylic acids, amides, and esters, there is resonance associated stability as a lone pair of electrons interacts with the p-orbital of the carbonyl carbon resulting in increased delocalization in the molecule. also with carboxylic acids there is the effect of the compound acting as a base, leaving the resulting carboxylate negatively charged and hence unable to be attacked by this nucleophile [36, 37]. in this study, an analytical method was applied for the quantitative determination of acetone in human blood. the determination was carried out using a uv-vis dad detector with hplc. in most of our patients ' blood samples, higher levels of acetone have been measured in the patients who have high level of blood glucose and positive urine ketone (group 1). the hplc method based on the labeling of acetone with 2,4-dnph seems to offer a rapid, low cost, sensitive, selective, and reproducible methodology for quantification of the acetone level in clinical samples such as human blood. the hplc method described here overcomes many of the problems in the determination of acetone in biological fluids and the preanalytical errors. the volatile ketone is promptly stabilized by conversion into its dnph derivative and rapidly determined without recourse to a solvent extraction step. the method uses very inexpensive reagents. as can be stated by brega et al., the proposed hplc method can therefore be used to great advantage over current gas chromatographic methods; it can be used in experiments requiring multiple samples and specific activity determination for the routine measurement of acetone in diabetic patients and in biological monitoring of exposed workers. we also presented acetone as a useful tool for the hplc-based metabolomics investigation of endogenous metabolism and quantitative clinical diagnostic analysis. in the medical and medicolegal practice however, the detection and early identification of acetone could be used as an initial indicator of detection of all these physiopathological conditions and the biological monitoring test and to determine further diagnostic management and timely treatment. determination of acetone levels in blood may be a valid clinical approach in the symptomatic or/and nonsymptomatic cases in the literature for determining treatment strategy and controlling glycemic levels of patients. consequently, the advanced studies which evaluate blood and urine ketone bodies level together should be performed in different physiopathological conditions including clinical and forensic toxicological studies.

using high-performance liquid chromatography (hplc) and 2,4-dinitrophenylhydrazine (2,4-dnph) as a derivatizing reagent, an analytical method was developed for the quantitative determination of acetone in human blood. the determination was carried out at 365 nm using an ultraviolet-visible (uv-vis) diode array detector (dad). for acetone as its 2,4-dinitrophenylhydrazone derivative, a good separation was achieved with a thermoacclaim c18 column (15 cm 4.6 mm 3 m) at retention time (tr) 12.10 min and flowrate of 1 ml min1 using a (methanol/acetonitrile) water elution gradient. the methodology is simple, rapid, sensitive, and of low cost, exhibits good reproducibility, and allows the analysis of acetone in biological fluids. a calibration curve was obtained for acetone using its standard solutions in acetonitrile. quantitative analysis of acetone in human blood was successfully carried out using this calibration graph. the applied method was validated in parameters of linearity, limit of detection and quantification, accuracy, and precision. we also present acetone as a useful tool for the hplc-based metabolomic investigation of endogenous metabolism and quantitative clinical diagnostic analysis.

PMC4889849

pubmed-606

in addition to being a crucial underlying circumstance in the primary cardiovascular risk factors (cvrfs), obesity is an independent risk factor for cardiovascular illness and mortality [1, 2]. increased body mass index (bmi) alters the behavior of adipose tissue, which provides insulin resistance as well as resistance to type-2 diabetes, arterial hypertension, dyslipidemia, and proinflammatory and prothrombotic states, thereby favoring the onset of ischemic cardiopathy. an estimated 42.3% of coronary episodes in the spanish population may be attributable to excess weight after adjusting for age, sex, and other risk factors. it would therefore be logical to expect obesity to have a lethal effect on patients who have suffered a coronary event. however, some studies have reported better short- and medium-term prognoses in overweight coronary patients [5, 6]. this situation, in which obesity seems to protect patients with acute coronary syndrome (acs), has been called the obesity paradox and has been described in other facilities. other authors have obtained conflicting results and question the existence of this protective effect [7, 8]. in light of this controversy, the goal of this study is to determine the relationship between bmi and intrahospital mortality in patients admitted consecutively for acs. all patients admitted consecutively between 2009 and 2010 for acs were included in the renaci database of the working group on ischemic heart disease and coronary care units of the spanish cardiology society. a total of 853 patients were initially admitted during the period studied with a discharge diagnosis of unstable angina or myocardial infarction. therefore, the final sample consisted of 824 patients. the demographic characteristics and base anthropometry were recorded for all the patients studied. the patients were divided into three groups: normal (bmi<25 kg/m), overweight (bmi=2530 kg/m), and obese (bmi>30 we analyzed various clinical variables including the classic cvrfs (smoking, diabetes, hypertension, and dyslipidemia), cardiovascular background and treatment prior to the acute event, hemodynamic variables at admission, risk scores according to the timi and grace scales, electrocardiographic data, analytical parameters and noninvasive cardiologic examination parameters, treatment administered at admission, coronarography examination, and results, as well as percutaneous and surgical revascularization. all data were analyzed with the one-sample kolmogorov-smirnov test to evaluate the normality of their distribution. continuous variables were expressed as the average and standard deviation and were compared using an unpaired student's t-test. the one-way anova test was used to compare more than two groups, and the bonferroni test was used to detect differences between groups. percentages were compared using chi-squared statistics and fisher's exact test when any of the expected values in any of the cells were below 10. univariate and multivariate logistic regression analyses were used to determine the variables associated with hospital mortality. the average age of the sample was 65.84+0.4 years (ranging from 2595), and the sample was predominantly male (73.5%). bmi had an inverse relation to mortality, that is, to say, higher bmis were associated with a reduced mortality rate. in the multivariate logistic regression analysis (table 1), bmi along with other clinical variables (age, diabetes mellitus, grace score, and cardiogenic shock) were the independent predictors of death in our sample. table 2 shows the hospital admission data for the sample, and table 3 shows not only the distribution of subjects in the normal weight, overweight, and obese categories but also the main clinical variables (risk factors and relevant background factors). the incidence of risk factors is high in this population (95.8%). in the obese group, the prevalence of diabetes mellitus and chronic obstructive pulmonary disease is significantly higher than in the other two groups. a larger proportion of the obese group had previously undergone treatment with angiotensin ii receptor antagonists and diuretics although there were no other differences in prior treatments. at admission, overweight patients presented higher blood pressure levels, and lower killip classes and timi scores, as well as higher initial glycemia and cholesterol levels. a coronariography was conducted in 76% of the patients in our sample, and no differences were found between obese, overweight, or normal weight subjects. no differences were found among the three groups in the percentage of angioplasties conducted or in the medical treatment received during admission. the prevalence of complications such as angina pectoris, reinfarction, hemorrhage, and mechanical complications was similar across groups with the exception of stroke, which was lower in obese patients. thirty-five patients died (4.2%) with no statistically significant differences among the different bmi groups. most deaths occurred within 48 hours of having been admitted (19 patients), followed by 7 patients between day 2 and day 7, and a further 9 cases after day 7. no statistical differences were found in the combined endpoint (mortality, reinfarction, bleeding, and cerebrovascular accident). our study shows that bmi is an independent predictor of hospital mortality in that a higher bmi is associated with a lower mortality rate. this finding is correlated with a lower mortality rate in obese and overweight patients compared to patients in the normal range although this result is not statistically significant. unlike other authors [5, 9], we did not find pronounced differences among these groups in terms of clinical data, hemodynamics, electrocardiographs, analyses, echocardiographs, angiographs, treatments administered, or resulting complications except that obese subjects have a greater incidence of diabetes, cardiovascular history, chronic obstructive pulmonary disease, and lower killip classes and timi scores. this data suggests that evolution would be less favorable and the mortality rate higher during hospital stays. although bmi is an inverse predictor of mortality in our series, indicating that overweight patients with acs have better survival rates than normal weight subjects, when subjects are classified into bmi subgroups (normal weight, overweight, and obese), we were unable to demonstrate with statistical significance that patients classified as obese have lower hospital mortality although this tendency does exist. among other reasons, this may be due to the sample size. our data is consistent with other data published, among which are the results of the synergy, merlin-timi 36, and crusade studies, which also describe lower mortality rates in obese patients suffering from acs. in a meta-analysis of 40 studies with more than 250,000 patients, romero-corral et al. observed that overweight patients with coronary disease have a lower risk of cardiovascular and total mortality than patients in the low and normal weight groups. however, this tendency disappears in the bmi 35 kg/m patient group (morbid obesity), which runs a greater risk of cardiovascular mortality. analyzed the prognostic value of bmi in medium-term hospital mortality in a cohort of 1,063 consecutive patients with first infarction in 15 hospitals in spain and found no association between bmi and medium-term hospital mortality. furthermore, in patients with acute st-elevated myocardial infarction, das et al. described a less-favorable prognosis for very obese patients in a sample of 501,489 patients. several hypotheses have been proposed to explain the inverse relationship between obesity and the prognosis of patients with ischemic cardiopathy. the higher mortality rate in the normal weight group may be due to a higher mortality rate in underweight patients. some studies [1, 2] show that the optimal bmi for the general population is 22.525 kg/m in smokers, and that people in the lower range of normal bmi (18.522.5 kg/m) have a higher mortality rate than individuals in the upper range of overweight bmi (27.530 kg/m). so the high-risk, underweight group is subsumed within the group of patients that is often considered normal. in contrast, most studies have found that a higher mortality rate is associated with extreme obesity [8, 9, 11]. another aspect that is often not given appropriate consideration is the role of pharmacological treatment, especially the adverse effects of multiple medicines used in acs (fibrinolytic agents, antiplatelet drugs, anticoagulants, inotropes, antiarrhythmic agents, diuretics, nitrates, beta blockers, etc.), which could explain a higher mortality rate. given the difficulty of anthropometric measurement in the first hours of cardiologic emergency treatment, errors in medicinal dosage may arise, primarily in those patients with lower body weight. nevertheless, unlike other authors, in our series we found that bmi group had no effect on either treatments administered or hemorrhages. since bmi can not differentiate between muscle and fat mass, overweight and obese subjects with coronary disease may have more muscle mass. when bmi is very high and better reflects body adiposity, the obesity paradox disappears. the main approaches used to measure obesity are bmi, waist-hip ratio, and waist circumference. there is considerable disagreement on which of them is best [3, 1315]. most authors recommend the simultaneous use of all of these parameters to better assess patient risk. although the worst scenario is the obese individual with a high waist circumference, patients categorized as normal with a high waist circumference also face an elevated risk. other authors suggest that obesity should be expressed as the percentage of body fat (> 25 for men and>35 for women), but this is difficult to quantify clinically. correctly identifying the different compartments of body fat, and specifically visceral fat, using more sophisticated techniques [16, 17] may help to clarify the role of obesity in acs patient mortality. studies have also been conducted which measure the degree of activity or physical condition and classify the subgroups normal weight-sedentary, overweight active, obese active, overweight sedentary, and obese sedentary from least to greatest risk, with obese-sedentary being the group at greatest risk [18, 19]. although the sample consisted of more than 800 patients, classifying the patients into three groups may prevent hospital mortality from being appropriately assessed because its incidence is relatively low. the clinical appraisal of obesity has many limitations, and an alternative might be the combined appraisal of other anthropometric measurements such as waist circumference, which was an unknown variable in our sample. better anthropometric appraisal using data on abdominal obesity and an understanding of the physical condition of the patients so that they can be more accurately classified into groups could play a critical role in clarifying this paradox. the relationship between higher bmi and a greater incidence of coronary disease in the general population is well documented, and excess weight should clearly be avoided. however, once coronary heart disease arises, the association between bmi and the prognosis becomes more complex, even paradoxical according to some authors. so the controversy over the predictive value of bmi in patients with acs remains latent. our study confirms that bmi is an independent predictor of hospital mortality; a higher bmi is associated with a lower mortality rate. our overweight and obese patients had a higher incidence of diabetes, cardiovascular history, chronic obstructive pulmonary disease, and lower killip classes and timi scores. however, they did not show an increased mortality rate, which is apparently paradoxical.

although obesity is a well-established cardiovascular risk factor, some controversy has arisen with regard to its effect on hospital mortality in patients admitted for acute coronary syndrome. methods. clinical and anthropometric variables were analyzed in patients consecutively admitted for acute coronary syndrome to a university hospital between 2009 and 2010, and the correlation of those variables with hospital mortality was examined. results. a total of 824 patients with a diagnosis of myocardial infarction or unstable angina were analyzed. body mass index was an independent factor in hospital mortality (odds ratio 0.739 (ic 95%: 0.597 0.916), p=0.006). mortality in normal weight (n=218), overweight (n=399), and obese (n=172) subjects was 6.1%, 3.1%, and 4.1%, respectively, with no statistically significant differences between the groups. conclusions. there is something of a paradox in the relationship between body mass index and hospital mortality in patients with acute coronary syndrome in that the mortality rate decreases as body mass index increases. however, no statistically significant differences have been found in normal weight, overweight, or obese subjects.

PMC3414064

pubmed-607

this has been attributed to procedure time, cost, and the known risks, which include arterial puncture, pneumothorax, and infection. numerous studies done in the last few years have paid attention to peripheral venous pressure (pvp) and more specifically its pressure waveform. although controversy still exists concerning the role of peripheral veins and their contribution to the central volume in face of blood loss, many studies in the late 1990s and early 2000s have shown a consistent correlation between central venous pressure (cvp) and pvp. this implies that in emergency conditions and situations where anatomical sites are inaccessible for central venous catheterization as seen in burns patients, the estimation of cvp is possible via measurement of peripheral intravenous (iv) catheter. hemodynamic monitoring has been shown to provide valuable additional information if burn resuscitation is not proceeding as planned or volume therapy guided by the typical vital signs is not attaining the desired effect. the goal of this study was to determine a reliable association between changes in cvp and pvp in patients with burns and to assess the long-term correlation in varied hemodynamic status during the first 10 h. after obtaining institutional ethical committee approval and informed consent from each patient, 30 consecutive patients admitted to our burns intensive care unit (bicu) from june to august 2014 were included in our study. the exclusion criteria were patients on the mechanical ventilator and untimely death during the study period. the sample size was decided after doing a pilot study for a month in our bicu and based on power of analysis. central venous pressure access was obtained using a 7 french double-lumen, arrow international catheter placed via the left or right internal jugular or subclavian vein. tip of central venous catheter was inserted at the junction of the superior vena cava and right atrium confirmed by chest x-ray. the peripheral measurement of cvp was obtained from a peripheral iv site (dorsum of the hand, forearm or antecubital region) using a standard iv catheter (18, 20 or 22 gauges). cvp was measured from both the central venous catheter and the peripheral iv catheters using philips and spacelab monitors equipped with invasive blood pressure monitoring transducers, which were zeroed at the phlebostatic axis. simultaneous measurements of cvp from central and peripheral venous catheters were made hourly for 10 consecutive hours. age, weight, height, site of cvp and pvp and peripheral iv catheter size for each patient were recorded. the predictability of cvp by pvp was examined using linear regression analysis at a p 0.05. among the 30 patients in this study, there were 20 females and 10 males. the age range was from 18 to 65 years, and their weight ranged from 45 to 60 kg. the predictability of cvp by pvp was tested by applying the linear regression which is shown in figure 1. this regression formula shows a reliable and significant association between cvp and pvp (p<0.001). the overall mean difference between cvp and pvp was 1.628 0.84 mmhg. the mean difference between cvp and pvp in each hour we used the bland-altman diagram for estimation of agreement between cvp and pvp during the 10 h period. this showed a perfect agreement (difference of 1.2 with an sd of+1.96) as seen in figure 3. the predictability of central venous pressure by measuring peripheral venous pressure tested by applying linear regression the hourly mean difference between central venous pressure and peripheral venous pressure agreement between central venous pressure and peripheral venous pressure during the 10 h period using bland the present study demonstrated reliable agreement between cvp and pvp over a 10 h period, suggesting that pvp monitoring can be used as a simple, cost-effective and less invasive substitute for cvp monitoring in patients admitted to the bicu. previous studies comparing cvp measured from central and peripheral access have shown a consistent correlation, but most of these studies were done in surgical patients, and we could not find any similar study in burns patients despite extensive literature search. cvp monitoring in the critically ill patients is usually performed by catheterization of either the subclavian or internal jugular veins. in burns patients, the site for catheterization of the central veins maybe inaccessible and in such patients monitoring the cvp via a peripheral vein is definitely an attractive option. the results of our study show that we can estimate cvp through simultaneous measurement of pvp and the difference between cvp and pvp measurements remain almost in a constant range over a period of time. hence, evaluation of hemodynamic changes occurring with dehydration or volume overload can be made by measuring pvp., showed that pvp measured from a peripheral iv catheter in infants and children with congenital heart disease was an accurate estimation of cvp and its changes had good concordance with cvp over a long period of time., however, reported that pvp measurement in critically ill patients did not give an accurate estimate of absolute value of cvp. tugrul et al., also reported that pvp showed strong correlation with cvp and mean difference between pvp and cvp was 2+1.8 mmhg, but the upper limit of agreement of pvp-cvp (5.6 mmhg) indicated the difference between two pressures might reach a clinically unacceptable value. however, in our study, the overall mean difference between cvp and pvp was 1.628 0.84 mmhg and the upper limit of agreement correlated to the clinically acceptable limits and were comparable with data described in other studies., found the mean difference between pvp and cvp to be 1.6 mmhg in all intraoperative patients and 2.2 mmhg in the postoperative period. hoftman et al., showed that pvp correlated with cvp even under adverse hemodynamic conditions in patients undergoing liver transplantation. studies have also shown that neither the peripheral iv catheter size nor the site of catheter placement interfered with the agreement of pvp and cvp. in our study, we used three different sites in the upper limb and three sizes of the peripheral catheter but did not find any statistically significant variations in the agreement of pvp and cvp values. although cvp waveforms characteristically showed a-waves, c-waves, and v-waves, pvp waveforms appeared as a more dampened sinusoidal pattern. we also noticed that the pvp tracing had more typical cvp waveforms when the peripheral catheter diameter was of a larger gauge and when the site was antecubital but since our subgroup sample size were small, we could not stratify this. although by placing the peripheral iv catheter more distally and decreasing its diameter increased the pvp-cvp gradient, these failed to reach statistical significance in the study done by tugrul et al. several studies have also demonstrated that the difference between cvp and pvp varies with the value of cvp. in nine patients undergoing orthotopic liver transplantation, hoftman et al., reported a weaker correlation between pvp and cvp at lower cvp values. another study by cave and harvey showed that when cvp increased, the difference between pvp and cvp tended to decrease. the number of patients was too small for subgroup analysis with regard to the percentage of burns and to the site and size of peripheral catheters affecting the pvp-cvp gradient. we also did not have a protocol for fluid management and its effect on our data. hence, the trends in pvp may be useful as an alternative for hemodynamic monitoring in the bicu during emergencies, in situations where central venous site is inaccessible and also to avoid the complications of central venous catheterization in critically ill burns patients. further studies are needed to determine the clinical usefulness of pvp as a trend monitor for evaluating the intravascular volume in this patient population.

background: optimizing cardiovascular function to ensure adequate tissue oxygen delivery is a key objective in the care of critically ill patients with burns. hemodynamic monitoring may be necessary to optimize resuscitation in serious burn patients with reasonable safety. invasive central venous pressure (cvp) monitoring has become the corner stone of hemodynamic monitoring in patients with burns but is associated with inherent risks and technical difficulties. previous studies on perioperative patients have shown that measurement of peripheral venous pressure (pvp) is a less invasive and cost-effective procedure and can reliably predict cvp. objective:the aim of the present prospective clinical study was to determine whether a reliable association exists between changes in cvp and pvp over a long period in patients admitted to the burns intensive care unit (bicu).subjects and methods: the cvp and pvp were measured simultaneously hourly in 30 burns patients in the bicu up to 10 consecutive hours. the predictability of cvp by monitoring pvp was tested by applying the linear regression formula and also using the bland altman plots of repeated measures to evaluate the agreement between cvp and pvp. results:the regression formula revealed a reliable and significant association between cvp and pvp. the overall mean difference between cvp and pvp was 1.628 0.84 mmhg (p<0.001). the bland altman diagram also showed a perfect agreement between the two pressures throughout the 10 h period. conclusion:peripheral venous pressure measured from a peripheral intravenous catheter in burns patients is a reliable estimation of cvp, and its changes have good concordance with cvp over a long period of time.

PMC4397625

pubmed-608

iodinated contrast is frequently given to enhance the diagnostic utility of computed tomography (ct). the contrast material results in a large increase in body iodine stores, which is progressively cleared over several weeks to months. a typical ct study uses about 100 cc of intravenous contrast material, which translates to about 30 g of iodine. there is concern that this transient increase in iodine can compete with i and interfere with subsequent management, such as whole-body scans and treatment with radioactive iodine in thyroid cancer patients. in addition, many groups recommend iodine depletion diets to optimize the therapeutic response to radioactive iodine in these patients [2, 3]. thus, avoidance of iodinated contrast material is particularly important as it can lessen the effectiveness of radioactive therapy [4, 5]. the duration of time required for body iodine stores to return to normal following iodinated contrast administration has not been well studied in thyroid cancer patients. recommendations for avoidance of radioiodine administration after iodinated contrast vary from 4 weeks to 1 year, with most suggesting about 3 months. iodinated contrast should be avoided if radioactive iodine therapy is planned within the subsequent few months. the european consensus guidelines also state that radioactive iodine administration should be postponed for two to three months after the event of iodine contamination. the normal thyroid has a considerable storage pool of organified iodine and since the body recirculates this iodine, thyroidectomised patients may possibly have a quicker elimination time than euthyroid individuals. if confirmed, then a briefer interval between iodinated contrast administration and subsequent radioiodine therapy could be considered and would expedite therapy. the objective of this study is to compare iodine clearance following iodinated contrast administration in thyroidectomised thyroid cancer patients and euthyroid individuals. a convenience study population was drawn from patients referred to the saint boniface general hospital diagnostic imaging department for iodinated contrast-enhanced ct studies. thyroidectomised thyroid cancer patients were notified of the study by members of the cancercare manitoba thyroid cancer disease site group who identified eligible patients requiring contrast-enhanced ct for clinical reasons. eligible thyroidectomised patients had previously undergone total or near-total thyroidectomy for thyroid cancer and were on a stable dose of suppressive l-thyroxine therapy. control euthyroid subjects were recruited from routine diagnostic ct referrals coded to receive iodinated contrast. eligible control individuals included those without documented thyroid disease and who were not taking thyroid hormone replacement. the university health research ethics board approved the study for the university of manitoba and signed and informed consent was obtained. exclusion criteria included pregnancy, other major sources of iodine including any history of amiodarone use ever or iodinated contrast in the preceding 12 months, treatment with lithium, abnormal renal function (serum creatinine>170 umol/l), inability or unwillingness to participate, and control subjects with known thyroid disease or those who were found to have abnormal thyroid function or anticipated survival of less than one year. routine ct protocols used the same dye load (100 cc of omnipaque 350, ge healthcare) for all adult patients undergoing scanning of the neck, chest, abdomen, or pelvis. we recorded routine demographics and clinical characteristics including sex, age, height, weight, and any iodine-containing medications, vitamin supplements, or herbal products. prior to the patient undergoing the iodinated contrast enhanced ct, a urine sample subjects were asked to collect a urine specimen every 2 weeks for the first 8 weeks and then monthly up to 6 months for 9 collections in total. urinary iodine concentration is currently the most practical biomarker for determining the iodine nutritional status [8, 9]. approximately 90% of all iodine ingested in the diet will be excreted in the urine. urine samples were stored in a 60 c freezer until analysis at the end of the study. all samples were batch-run for assessment of urinary iodine in conjunction with creatinine at health canada. urinary iodine concentrations were determined using ammonium persulfate digestion followed by colorimetric analysis based on the sandell-kolthoff reaction according to a modified microplate method. urinary creatinine concentrations were determined by enzymatic idms-standardized two-point rate on an ortho-clinical diagnostics vitros 5, 1fs analyser. the urinary iodine to creatinine ratio (microg/g) was calculated as the measure of iodine clearance as used by the third national health and nutrition examination survey (nhanes iii) (19841994). a review concluded that spot urinary iodine concentration is a reliable measure of the iodine intake in the population when expressed as a function of urinary creatinine to correct for the influence of fluid intake. demographic and clinical characteristics of the thyroidectomised thyroid cancer patients and of the euthyroid controls are described using medians, means, and standard deviations (sd) for continuous variables and frequencies and percentages for categorical variables. group comparisons for continuous data were conducted with the student's t-test for specific time points. models based upon generalised estimating equations (gee) were used to analyse combined data for all time points from 4 weeks onwards assuming an exchangeable correlation structure. separate gee models were then constructed to look for an effect of time since contrast administration or a time group interaction. the dependent variable used for the primary analysis was the urinary iodine creatinine ratio. in a secondary analysis we enrolled 6 thyroidectomised thyroid cancer patients and 7 control subjects. in total 50 samples from the thyroidectomised thyroid cancer group there were no missing collections from the control group; there were 4 missing collections from the thyroidectomised thyroid cancer group. the mean urinary iodine creatinine ratio was 248.2 microg/g at baseline for the thyroidectomised thyroid cancer patients and 138.3 microg/g at baseline for the control patients. as anticipated, the two-week mean values were still elevated above baseline (thyroidectomised thyroid cancer 770.3 microg/g versus 670.3 microg/g control groups). figure 1 shows the mean urinary iodine creatinine ratios for the two groups, demonstrating a return to baseline values 4 weeks following iodine contrast administration. pairwise testing showed no significant group differences in urinary iodine creatinine ratios at baseline or any subsequent time point. when expressed as the change from baseline, only the 2-week sample showed significantly increased urinary iodine clearance (table 2). gee was used to examine for differences in urinary iodine creatinine ratios between the two groups from week 4 onwards and no significant group effect was identified (p=0.53). in a separate gee model we did not identify any significant time effect (p=0.19) or time group interaction (p=0.84). a gee model with change from baseline from week 4 onwards showed a statistically significant difference between the two groups, lower in the thyroidectomised thyroid cancer group (parameter 113.1 microg/g [95% confidence interval 12.8 to 213.3 microg/g], p=0.027). following exposure to a large iodine load, the plasma concentrations of free iodine remain elevated as the body's iodine stores are expanded in the interstitial fluids and in the thyroid colloid. current literature suggests that, in euthyroid individuals, the body's iodine stores are increased for at least 3 months following iodinated contrast. our study finds that the urinary iodine creatinine ratio returns to baseline by 4 weeks following iodinated contrast in both euthyroid and thyroidectomised thyroid cancer subjects. in addition, there was no significant difference between the thyroidectomised and euthyroid patients at 4 weeks, nor throughout the rest of the study period up to 6 months. in which urinary iodine levels returned to baseline at one month in thyroidectomised thyroid cancer patients after receiving iodinated contrast. a prospective study by nimmons et al. showed that the median time for urinary iodine levels to normalize in euthyroid subjects was 43 days, with 75% of subjects returning to baseline by 60 days. our study enrolled only 13 subjects in total and may have been underpowered to detect significant differences between the two groups. the small numbers of cases and controls are compensated for by the multiple time points assessed, which allowed for all urinary collections from 4 weeks onwards to contribute to the statistical analysis using gee. in addition, thyroid cancer patients need to be on a low iodine diet prior to radioiodine treatment. while this study demonstrates that urinary iodine creatinine ratio returns to baseline by 4 weeks in both thyroidectomised thyroid cancer and euthyroid subjects, it does not look at the effect of a low iodine diet on the urinary iodine creatinine ratio post iodinated contrast. we did not collect data on noncontrast sources of exogenous iodine (e.g., diet, vitamin supplements), but our analysis of the change in iodine excretion from baseline partially compensates for interindividual differences in dietary iodine intake. increasingly, ct is being compared to ultrasound, magnetic resonance imaging, or positron emission tomography techniques. as more information is gained about the sensitivity, specificity, and accuracy of these techniques, ct may be used less frequently which would ameliorate the concern of iodinated contrast and therapeutic radioactive iodine. in conclusion, we found that 4 weeks may be sufficient for urinary iodine creatinine ratios to return to baseline value following iodinated contrast in both thyroidectomised thyroid cancer and euthyroid subjects. this suggests that clearance time may be more rapid than previously thought and that a shorter time interval between iodinated contrast and radioiodine therapy might be considered but requires confirmation in larger studies that include a broad case mix in terms of age and dietary iodine intake.

purpose. to compare iodine clearance following iodinated contrast administration in thyroidectomised thyroid cancer patients and euthyroid individuals. methods. a convenience population (6 thyroidectomised thyroid cancer patients and 7 euthyroid controls) was drawn from patients referred for iodinated contrast-enhanced computed tomography (ct) studies. subjects had sequential urine samples collected up to 6 months (50 samples from the thyroidectomised and 63 samples from the euthyroid groups). t-tests and generalised estimating equations (gee) were used to test for group differences in urinary iodine creatinine ratios. results. groups had similar urinary iodine creatinine ratios at baseline, with a large increase 2 weeks following iodinated contrast (p=0.005). both groups had a return of urinary iodine creatinine ratios to baseline by 4 weeks, with no significant group differences overall or at any time point. conclusions. thyroidectomised patients did not have a significantly different urinary iodine clearance than euthyroid individuals following administration of iodinated contrast. both had a return of urinary iodine creatinine ratios to baseline within 4 weeks.

PMC4247980

pubmed-609

cervical cancer (cc) is listed among the top five cancers that affect women globally. according to the international agency for research on cancer (iarc), in 2008 there were 530,000 new cases of cervical cancer worldwide, with 85% of the disease burden occurring in developing countries. caribbean women are at an increased risk of dying from cc, which is the second most frequent cancer among women and also ranks as the third most frequently diagnosed cancer in both sexes. in the united states (us), cc has decreased in incidence and mortality since the mid 19th century, primarily because of screening. even with the introduction and widespread use of the pap test, cc still ranks among the top ten cancers diagnosed in the us among minority populations, which includes blacks, american indians, and hispanics. persistent high-risk (hr) human papillomavirus (hpv) infection, specifically hpv types 16 and 18, has been linked to the development of cc, anogenital cancers, and oropharyngeal cancers [4, 5]. in 2006, the fda approved the hpv vaccine gardasil (against hr hpv types 16 and 18 as well as low-risk types 6 and 11) for all females aged 9 through 26. in 2009, the fda also approved the use of this vaccine in males aged 9 through 26. cervarix, which aims to protect against hr hpv types 16 and 18 only, was approved by the fda in the fall of 2009 for females aged 10 through 25. the centers for disease control and prevention (cdc) advisory committee on immunization practices (acip) recommended routine hpv vaccination for adolescent boys and girls as young as 9 years and men and women as old as 26. however, even with the availability of these vaccines, there is still a disparity in their utilization between races. minorities are not receiving and/or completing the vaccine process at the same rate that their white counterparts are in the us. a study conducted among 363 african-american college females showed that only about 25% of young women report uptake of the hpv vaccine. vaccine uptake was associated with significantly higher levels of hpv knowledge, lower perceived barriers to vaccination, and younger age. with respect to vaccine completion, niccolai et al. reported that completing the vaccination series was associated with being white and having an annual household income>$75,000. reasons as to why disparity exists for hpv vaccine uptake and completion remain unclear in the literature. some studies attribute racial disparity to income level differences, while others believe it is due to lack of knowledge [1012]. the poor response and completion rate of hpv vaccination amongst minorities still need to be fully assessed. in our earlier study of hpv knowledge amongst the general population, we reported that the general population was aware of hpv as well as the hpv vaccine; however, the benefits of the vaccine to the study population were not evident. when looking at knowledge according to race, people of african descent were less informed than whites (black 89% versus white 97%, p>0.1). cultural beliefs or perceptions and lifestyle are distinct between persons born in the us and those born in other countries [1416]. while there is a similar concern for high cc incidence among black women in the caribbean and the us, interventions that promote hpv vaccine uptake and completion may need to be tailored differently for each group. prior to the development of needed interventions, information on knowledge and attitudes toward hpv and its vaccine is needed. yet, no previous publication has assessed hpv knowledge and/or attitude towards hpv vaccination in the caribbean. therefore we have expanded our previously published study to include an example of a caribbean nation (the bahamas) where cc incidence (17.6 per 100,000) is similar to the incidence of cc for the caribbean region (20.8 per 100,000). we sought to determine knowledge and attitudes of hpv and the vaccine in the bahamas and determine whether differences exist between different cultures of african descent. the original study population consisted of 202 volunteer participants from the university of pittsburgh and hampton university. the recruitment was expanded and involved two additional locations: the new york city (nyc) metropolitan area and the bahamas (bhm), west indies. therefore, this study includes a diverse study population recruited from the general population between 2008 and 2010 from three urban cities in the north eastern us differing in size and demographics, (small (hampton, va, population 136,401) medium (pittsburgh, pa, population 307,484) and large (nyc, ny, population 8.245 million). both hampton, va and pittsburgh, pa, comprise more of an african-american demographic compared to nyc where a higher proportion of the population consists of immigrant families, including the caribbean. we believe that this mix is diverse and may be more representative of the us population. institutional review board approval was obtained from the university of pittsburgh, suny downstate medical center, and princess margaret hospital in the bahamas. for all study locations, study participants were recruited face-to-face or using posted flyers in various settings including hospitals, private and other clinics, and community locations (e.g., parks, restaurants, barber shops and laundromats, etc.). the original english questionnaire was formally translated into haitian creole by the haitian embassy in the bahamas in order to accommodate creole-speaking participants. those who were not able to read or write well had assistance in completing the questionnaire. a total of 793 surveys were administered and returned; of these, 231 were excluded from the present analysis because they were completed by people who identified themselves as some race other than black. the study population was then divided into two groups based on place of current residence us and west indies. the questionnaire used in this study consisted of demographic questions pertaining to age, race, sex, place of birth, religion, household income, level of education, health insurance status, parental status, and number of children. the study instrument included measures of hpv knowledge and the safety, efficacy, and impact of the hpv vaccine. a content assessment of the survey was conducted prior to the launch of the study. descriptive statistics were generated for demographic and knowledge variables according to the place of residence for study participants. fisher's exact test was used to determine statistical significant differences between proportions; differences in continuous variables were assessed using two-sample t-tests. adjusted prevalence of correct answers to hpv and hpv vaccine knowledge questions was calculated and stratified by geographic location after adjusting for age, level of education, marital status, parental status, health insurance status, and income level. ordered logistic regression was performed to assess differences in response to knowledge questions pertaining to hpv and hpv vaccination between the two groups, adjusting for demographic variables and the covariates previously mentioned. statistical analysis was performed using stata version 10.1 software (stata lp, college station, tx, usa). a total of 555 black participants residing in the us and the bahamas (bhm) completed the survey (41% from the us and 59% from the bhm (table 1)). between both populations, 4% of the subjects (n=23) were immigrants and not born in their current country of residence. twenty-five percent of the participants were male, and 75% were female; there was no difference in gender between the two geographic locations. in both locations, the majority of the participants were between the ages of 18 and 35, with a higher proportion of this age group in the us sample compared to the bhm (80.5% versus 49%, p<0.0001). eighty-four percent of the us population was single compared to only 42% of the bhm population. parenthood also demonstrated differences, where more bahamians tended to be parents (bhm: parent=65.2% versus us: parent=23.9%, p<0.0001). the correlation between parental status and marital status also differed between the us and bahamas. the majority of bahamian parents were married (45%), while the majority of us parents were single (41%). us participants were more likely to have attended or completed college (us: some college=55.3%, college graduate=27.4% versus bhm: some college=17.9%, college graduate=22.0%, p<0.001). income levels varied modestly between groups up to the us $50,000 mark; 40.7% of us respondents fell into this bracket compared to 11.7% of bahamian respondents reporting this as their income. general knowledge about hpv and the hpv vaccine differed between the two countries significantly (table 2). when asked if they ever heard of hpv, bahamian participants were less aware of the virus than participants in the us (us 89.5% versus bhm 61.5%, p<0.001). this difference in knowledge remained consistent for 6 out of the 10 questions related to hpv knowledge, while there was no difference in knowledge for four specific questions. although the majority of respondents from both locations knew that only women could develop cervical cancer, lower proportions of respondents knew that hpv causes genital warts (28.2% of us and 33.2% of bahamians) and that genital warts are not caused by the same hpv types that cause cervical cancer (13.8% of americans and 7.5% of bahamians). for the questions related to hpv vaccination (table 3), the majority of both populations heard of the vaccine, however, the proportion was higher among american respondents (us 66.9% versus bhm 50.6%, p=0.021). for two of the three questions assessing knowledge about the hpv vaccine (table 3), bahamian study participants were more likely to answer questions incorrectly when compared to american blacks. at the time the questionnaire was administered, the hpv vaccine had not yet been approved for males; when asked who was eligible to receive vaccination, approximately 51% of americans answered correctly versus only 35% of bahamian participants (p=0.013). more than half of both populations knew that, in spite of vaccination, annual pap smears were still needed to screen for cervical cancer according to the recommendation at that time. even so, a significantly lower proportion of bahamians answered this question correctly (us 75.3% versus bhm 51.5%, p<0.0001). there was no difference between the two groups when asked what age group was eligible to receive the vaccine; less than half of each group answered with the correct age group, 926 (us 47.6% versus bhm 40.4%, p=0.264). for the participants that had previous knowledge of the hpv vaccine, the source of this information was similar between the two groups (figure 1). for the us, hpv vaccine awareness was first provided through an advertisement followed by a health care professional, the news, and school, although, in the bahamas, the primary mechanisms for promoting awareness were advertisement followed by a health care professional and the news with minimal contribution from school. table 4 shows cumulatively the number of answers that respondents from each geographic location answered correctly. after adjusting for covariates (table 5), bahamian respondents were less likely to have higher numbers of correct knowledge answers when compared to americans (adjusted odds ratio [adj. or] 0.47, 95% confidence interval [ci] 0.300.75). older age, regardless of location, was also associated with answering fewer questions correctly (adj. or 0.61, 95% ci 0.400.92). having health insurance and higher income levels, regardless of location, was associated with answering higher numbers of questions correctly (adj. or 1.76, 95% ci 1.072.92; adj. or 1.21, 95% ci 1.081.35, resp.). when asked whether or not the hpv vaccine should be given to both boys and girls, both us and bhm participants had mixed views; however, the majority of both groups agreed that both genders should receive the vaccine (figure 2). for both us and bahamian parents, the majority indicated that they were willing to vaccinate their daughters (us=21/51, 41% and bahamas=96/187, 51%). for the us parents unwilling to vaccinate their daughters, however, it was not possible to make comparisons between the two populations since 66% of the bahamian parents who were unwilling to vaccinate their daughters did not indicate the reason. for both groups, 90% or more respondents agreed that assurances were still needed for vaccine safety and efficacy. overall, the majority of participants from both populations did not feel that administration of the vaccine would encourage risky sexual behavior and felt that discussing issues of sexuality before vaccination was necessary. when asked if an informed child should be able to request vaccination at sexual health clinics without parental consent, nearly 80% of bahamian respondents felt that children should not be able to receive the vaccine without parental consent compared to 57% of american respondents. a number of international studies have focused on knowledge and attitudes related to hpv and the hpv vaccine. however, a majority of these studies have been conducted in europe, asia, and africa [1723], with little work done in latin america and no studies conducted in the caribbean. this study is the first to report on overall hpv and hpv vaccine knowledge and perception in a sample of black volunteers containing a large number of subjects from the us and caribbean, and we have compared our findings by geographic residence. our previous study showed a lack of knowledge among people of african descent in the us, which remained consistent with our current findings and other published literature [24, 25]. however, the present study indicates that overall knowledge was significantly lower in bahamian participants when compared to american blacks, even after adjusting for possible confounders. more than half of participants from each location were aware that a vaccine for hpv is available; however bahamian participants were less aware of this. studies that focused on populations of african descent, outside of the us, report a similar lack of knowledge in regard to vaccine availability [21, 26], and educational attainment has been shown to be associated with familiarity of hpv and its vaccine [23, 25, 27]. in our study population, us participants had higher levels of education compared to bahamian participants, which could explain the lesser familiarity with hpv and its vaccine in the bahamas compared to the us. for persons who were aware of the hpv vaccine, there were slight differences between the us and bahamas in the type of resource from which they received this information. while advertisements were the major resource for obtaining information about hpv vaccines for both the us and bahamian populations, health professionals were also an important resource for the us population but not as much for the bahamas. further investigations that help clarify the reasons why health professionals are not currently important resources for educating the bahamian population about hpv vaccines are warranted and might help provide insight for improving hpv vaccine awareness in this geographic region. published literature suggests that in most cases, after parents were well informed about the risks and benefits of the vaccine, they were willing to vaccinate their children [11, 19, 28], and we have noted that in our study the majority of parents were willing to vaccinate their daughters. for the us, parents who were unwilling to vaccinate their daughters identified their reasons as safety concerns. however, in this study we were not able to compare the reasons for unwillingness of parents to vaccinate their children between the two countries, because of low response rates to those questions for the bahamian parents. it was not clear why the response rate for this question was low; however, it is possible that the participants may not have been comfortable with disclosing their reasons. in general, attitudes related to hpv vaccination were similar between the us and bhm but differed when the two populations were asked if a well-informed child should be able to request vaccination at sexual health clinics without parental consent. us participants were more likely to agree with this statement (p<0.001). this difference may suggest that americans have a higher self-worth in regard to their health, which is not the case when it comes to children. hughes reports that children considered themselves to be passive participants when determining their course of medical care. in our study population, the majority of parents from bhm were married in contrast to the parents from the us. in the west indies, the importance of family has been distinguished and deemed quite atypical to that of the western world. although speculative, the rationale behind the majority of bahamian respondents disagreeing with children being able to receive the vaccine without parental consent might be explained by differences in overall family structure and child rearing practices, which have been shown to differ when compared to the us. still, with the spread of american culture globally, family values have begun to change within the caribbean, which would explain why younger respondents from bhm tended to agree with the statement. respondents who agreed with the statement about children being able to decide about their own vaccination tended to be younger people (< 35 years), regardless of geographic location (p<0.001). lack of knowledge in regard to hpv and the hpv vaccine is not a problem that is limited to peoples of african descent but extends to ethnicities within developing countries all over the world. regardless of geographic location, whites have consistently displayed higher levels of knowledge concerning hpv and the vaccine when compared to other ethnic groups [13, 26]. effective methods in disseminating necessary information to these populations with lower health education need to be assessed and put into action to minimize future cases of hpv-related cc. given that the majority of study participants first heard about the hpv vaccine via advertisements or their health care providers, design of an intervention that combines the two may be effective in reaching the public. in cases where access to health care providers is limited, the use of community-based educators may prove to be more effective than gaining information from a physician. moreover study findings suggest that interventions to increase the uptake of hpv vaccine in the caribbean must include parental involvement. in summary, the loss of productive lives and quality of life caused by morbidity and early death from mortality due to cervical cancer in the caribbean and latin american region is significant. determining the best strategies to educate and increase hpv prevention practices in this population is crucial and needs to be treated with a sense of exigency in order to decrease the burden of cc in this region.

objective. to compare knowledge and attitudes of human papillomavirus (hpv) and the vaccine between different cultures of african descent. methods. a cross-sectional survey of 555 african-americans and afro-caribbeans residing in the us and the bahamas (bhm) was conducted. results. general knowledge about hpv and the hpv vaccine differed between the two countries significantly. bahamian respondents were less likely to have higher numbers of correct knowledge answers when compared to americans (adjusted odds ratio [adj. or] 0.47, 95% confidence interval [ci] 0.300.75). older age, regardless of location, was also associated with answering fewer questions correctly (adj. or 0.61, 95% ci 0.400.92). attitudes related to hpv vaccination were similar between the us and bhm, but nearly 80% of bhm respondents felt that children should not be able to receive the vaccine without parental consent compared to 57% of american respondents. conclusions. grave lack of knowledge, safety and cost concerns, and influence of parental restrictions may negatively impact vaccine uptake among african-american and afro-caribbean persons. interventions to increase the vaccine uptake in the caribbean must include medical provider and parental involvement. effective strategies for education and increasing vaccine uptake in bhm are crucial for decreasing cervical cancer burden in the caribbean.

PMC3730153

pubmed-610

when biologists teach contemporary or historical discoveries in the life sciences, we often aim not only to impart the facts but also to explore how we know what we know focusing on the scientific process that led to the revelation of such new scientific information. many scientist-educators would also like to include in their courses the examination of the future implications of such discoveries, encompassing both the future basic and applied scientific potential as well as the accompanying bioethical dilemmas. both involve exploring the scientific reaches of new discoveries, but examining bioethical future concerns additionally requires some familiarity with ethical theory and ethical reasoning. in effect, looking forward biologically, we ask what can we do? while looking forward ethically we ask what should we do and why? to educate our students responsibly, and to introduce bioethics effectively, we should not simply allude to future bioethical challenges thereby raising awareness but should also help students to begin developing a framework for reasoning through those challenges to some resolution about what ought to be done ethically. since its emergence as a discipline in the 1970s, bioethics has taken shape as an important academic discipline that intersects with the life sciences in myriad ways, and not only with genetic, biomedical, or biotechnological advances. the unesco universal declaration on bioethics and human rights (15) established in 2005 has identified 15 principles that range from human dignity, social responsibility, equality, and justice, to protecting the environment, the biosphere, biodiversity, and future generations. however, the integration of clearly reasoned bioethical thinking into undergraduate biology and microbiology curricula has not kept pace with this wide-ranging international awareness and is often limited to research or professional ethics. during this emergence of bioethics, the traditions of research and professional ethics have evolved with their respective mandates, and have been the default for integrating ethics into undergraduate or graduate courses in the life sciences. research ethics prepares students to perform research ethically, in part by understanding the need to propose research with animals to institutional animal care and use committees (iacucs) or submit research proposed with humans to institutional review boards (irbs). professional ethics teaches students the importance of abiding by professional codes of conduct such as the code of ethics for the american society for microbiology (http://www.asm.org/cclibraryfiles/filename/000000001596/asmcodeofethics05.pdf). although research and professional ethics are necessary, they do not adequately address the range of bioethical questions that continue to emerge as the fields of biology and microbiology advance. these questions relate to scientific course content more directly than do policies, regulations, and codes. indeed, there is some evidence that students appreciate the need for professional ethics but do not recognize the personal effects of morals and behavior (6), which is in itself a reason for folding ethics into biology courses. when women s studies began in the 1970s a favored pedagogical tactic advised instructors to add women and stirtake a traditional course and append a unit that would address women s issues. though initially useful, the measure was eventually seen to marginalize the most crucial discussions about women (10). since those early days, infusing women s issues into the general curriculum has proven an effective tactic, especially when viewed in relation to the original add women and stir approach, for infusion tactics require integration of material into the stream of the courses in question, discussing both together, contextualizing and enriching both standard and gender analyses. in this perspective, we heed that lesson and apply it to the teaching of bioethics in undergraduate science curricula: instead of adding ethics and stirring, that is, instead of inserting ethical issues at several points in each course necessarily very briefly, and without requiring much depth of thinking we recommend introducing one ethical topic allied to the professor s area of expertise or interest and taking the time to work this topic into the science material by means of ethical reasoning. questions generated by such topics are many and varied. for instance, should we try to clone animals that are on the verge of going extinct even though that extinction is driven by habitat destruction? how do we balance the need to improve agricultural yields with stewardship of the environment? is feeding fast food/junk food to children in school lunches ethical when we know it can not only negatively affect their health but also potentially the health of subsequent generations? should there be any limits placed on the genetic information commercially available to individuals? with advances in neuroscience, if pharmaceuticals that enhanced cognition were developed, who could/should take them? should people, particularly those who are terminally ill, be able to choose the time and means of their own death? what responsibility do we have to maintain the increasingly large pool of embryos developed in vitro but which have not been chosen for implantation? is it ethical to bypass the safety standards for developing safe drugs and vaccines when an epidemic such as ebola is rapidly expanding? this vertical infusion approach is also consistent with the reforms currently taking place in undergraduate biology education with more active, student-centered learning, greater interdisciplinary focus and problem-based learning, as detailed in the aaas report vision and change in undergraduate biology education: a call to action (1). in this report of a 2009 national conference designed to develop a blueprint for real change in biology education, the ability to understand the relationship between science and society is identified as a core competency in the practice of science with one example being evaluating ethical implications of biological research many excellent paths have been proposed to invite students of science disciplines to stretch their reasoning abilities toward the analysis of ethical issues. in 1990, when forming the national human genome research institute (nhgri) the national institutes of health (nih) included the ethical, legal, and social implications (elsi) of the human genome as a significant component of its funded research programs (8). biology textbooks then began to incorporate elsi into their auxiliary information, enclosed in textboxes or simply as questions in the margins for those faculty and students who wished to explore these implications further. however, though most often these textbooks dutifully ensure that students get the science right, they do not ask students to reflect more deeply, reasoning with the aid of ethical principles, in order to reach a just resolution of the dilemmas presented. another approach is the establishment of science-based bioethics courses within undergraduate biology curricula, as seen at columbia university in its crossroads in bioethics and bioethics for biomedical engineers courses. in this approach, the scientific issues, ethical challenges and ethical theories appropriate for resolving bioethical challenges are melded into one course (5). columbia s integrated bioethics courses have the advantage of giving the students a chance to reason challenging dilemmas through to an ethical conclusion based on both the science and the ethical principles. one, entitled gynebioethics: reproduction and beyond, melded reproductive biology and bioethics, treating, for instance, the genetics of sex determination with the ethics of sex selection. another course, the bioethics of food, explored the biological features of the western diet along with its consequent bioethical entanglements, such as, for example, the duty of a state to outlaw certain substances, such as trans fats, or genetically modified crops. the advantage of such courses is that the science content is more focused, so less competition exists for coverage of that content, leaving more time to actually explore and analyze bioethical issues in depth. however, not all biology programs can afford the faculty resources to develop such integrated approaches. still another strategy for including bioethics in the undergraduate biology curriculum could be requiring life science majors to complete an allied bioethics course, as is often the practice for other foundational courses such as chemistry, physics, or math. although we are not aware of many programs that do so (outside of pre-health studies, such as nursing) one example is the microbiology undergraduate program at rutgers (http://dbm.rutgers.edu/microbio.php) which requires a professional ethics course. we have mentioned the limits of such courses: they canvas professional codes and policies but not, as a rule, the types of ethical issues that scientists and citizens together may encounter as science advances, such as the possibility of three-parent embryos, the ownership of water rights as sources become scarce, the ethics of transgender fertility, the prospect of cloning beloved pets, or the question of what responsibilities biomedical research institutes have to patients whose tissues are harvested. yet, even if expanded beyond professional ethical material, such stand-alone bioethics courses are most often taught by philosophers who are not necessarily prepared to integrate the science needed. in cases where this allied course requirement is the approach taken, it would be best to offer bioethics courses team-taught by a scientist and a philosopher to be able to incorporate effectively the science with the ethics. we have offered such team-taught bioethics courses and find students from many backgrounds biology, philosophy, anthropology, history, psychology, exercise science, and business fully engaged in the interfaces between science and ethics. such courses are not required in our biology curriculum at transylvania university but remain very popular electives. both of these kinds of stand-alone courses the science-based with constant bioethical integration and the team-taught courses with both philosopher and biologist have definite merits, but they do not address the concerns of biology professors who are committed to challenging their students directly, in the heat of a standard biology or microbiology course. chamany and colleagues put it this way: as biology instructors we may choose to teach biology devoid of social context, believing that students can make these connections on their own. but students model their instructors behaviors, and follow their lead (4). given the ever-increasing bioethical concerns attached to practical biological applications, it is not surprising to find many of us wanting to integrate bioethical questions into our own courses choosing to be the role models chamany and colleagues discuss. confident voices of professionally-trained biologists will be needed more urgently as new biotechnologies and other bioscience discoveries emerge, especially in the span of our current students careers. if the question is, what can i do in my own courses to help stimulate mature and considered thinking about ethical aspects of what i teach? biology professors should not apologize for being concerned primarily with scientific content and student ability to understand, apply, and integrate biology principles appropriately delivered to challenge student problem-solving and analytical thinking skills. when asked what impedes their treatment of ethical issues in their courses, most point to a lack of time (3). whatever ethical content we examine, it should not interfere significantly with the science being taught but rather complement it and encourage students to see how important good science is to sound ethical reasoning and practice. even though textbooks use elsi textboxes and thought-provoking questions to encourage discussion of bioethical issues, instructors may avoid doing so because these novel issues are thought to deflect student concentration at crucial stages of biology instruction. even if the bioethical prompts are used to raise a number of ethical questions during a term (without going beyond merely showing that an ethical dimension exists), first, such bioethics instruction can be distracting and time-consuming; second, and just as important, since faculty may not want to spend much time on each insertion of bioethical thinking, students could easily get the impression that ethical issues are either too vague and indefinable to contemplate or too unimportant to squander cognitive resources on (11). complaints like these, especially the latter, contributed to the diminishment of the add women and stir approach in women s studies (10). the horizontal approach seems most appropriate for specialized, integrated science-based bioethics courses discussed above, where one is not limited to brief analysis of each bioethical issue, rather than for introductory or advanced biology or microbiology courses in a standard curriculum. vertical infusion would involve a single in-depth analysis of a relevant bioethical issue in each or several courses in a biology/microbiology program. horizontal infusion would involve introduction of multiple bioethical issues in less depth across a single or a few courses in a biology/microbiology program. so what do we suggest for professors who responsibly commit to coverage and understanding of the science but also realize the importance of encouraging students to think seriously about ethical issues? we advise what we call a vertical approach to teaching the ethical dimension. instead of trying to invest ethical import wherever it might fit, or, at several convenient points along the progress of the course, the vertical approach focuses on only one issue during the term and only for one occurrence but that issue is explored in more detail than would be possible if several had to be envisioned. further, the topic in question could be selected in light of the professor s research interests, thus making preparation less onerous and more enticing for professor and students alike. if, for instance, one is studying the establishment of the human gut microbiome using 16s rdna analysis, and the course in question is an introduction to microbiology, then one could expand the normal flora section by discussing biobanking microbes from the human intestinal tract and the subsequent bioethical questions of consent, confidentiality, and privacy (see, for instance, the treatment of related ethical issues in ref. in this fashion, the question could be addressed more deeply, even to the point of encouraging students to think with the aid of ethical principles, such as the principlist ones now common in suggested bioethics curricula, both globally and nationally (5, 9, 12, 14). (note: we are not advocating for an exclusive focus on principlist ethical theory but suggest that it is a comfortable place to begin.) these basic ethical principles include autonomy/respect for persons, beneficence, non-maleficence, and justice. their use became more common in 1979 after the release of the belmont report from the national commission for the protection of human subjects of biomedical and behavioral research (7) because they are acceptable principles in most cultural and religious traditions. hence, instead of merely pointing out current policies guiding the just operation of biobanks (professional practice approach), one could ask why these practices are ethical, what principles justify them, and how these principles might radiate out to other similar biomedical practices. undergraduate biology and microbiology professors have considerable content to cover, whether in an introductory or advanced course, as we have mentioned. we believe that the horizontal means of infusion of ethics (many examples in one course that are not covered in much depth) can distort the focus of the course, and diminish the importance and urgency of bioethical challenges. conversely, a vertical approach, where each faculty member might develop a single ethical issue out of their own area of interest, would encourage deeper, more responsible thinking on the part of students given the parameters of a standard, single-term biology course. with the intense focus comes a relatively smaller portion of time one needs to allot to integrating ethical concerns. a lab period, or a couple of class periods, especially when devoted to and motivated by one s own passion, could suffice for deep engagement and productive conversation. using the example of rights to a person s microbiome, a professor could explore how students have come to their conclusions about the issue, what ethical principle might lie behind that conclusion, and whether that ethical reasoning might itself be hiding a clash of principles. imagine also if a number of faculty members in a department began vertically infusing a single ethical issue in each of their courses. throughout a student s major pattern, they would have encountered several ethical issues and been encouraged to give them respectable consideration. on the best construal, each student would be integrating these issues from each course into a coherent style of thinking and critiquing. any individual student would then have thought about the ethics of gmo foods, stem cells, antibiotic use, preservation of biodiversity, dna fingerprinting and biobanking, for example, finding similarities and seeking a consistent application of ethical principles. in such a program, students would grow to see their discipline in an even richer dimension and would not be hesitant to engage in the common social discourse about ethical issues related to scientific discoveries. with the vertical approach, instructors choose their own occasions for infusing bioethical questions, according to their own scientific strengths. how then to help students think clearly about ethical implications that attend those strengths? as we have mentioned, many excellent resources abound, attached to stand-alone courses and large programs, which might prove valuable, but by and large they are not targeted to aid undergraduate biology faculty who want to infuse a single issue. they are often too narrowly focused on medical ethics or human rights, or they target high school or professional school audiences. the nsf-sponsored national center for case study teaching in science (http://sciencecases.lib.buffalo.edu/cs/collection/) holds in its peer-reviewed collection more than 20 cases related to bioethics that are appropriate for introductory biology courses as well as upper-level ones. however, while well conceived, up to date, accurate with respect to science and technology, and framed with innovative pedagogical suggestions, they nonetheless do not offer help when it comes to actual reasoning about ethical principles. for biology professors using the vertical approach, we would, ideally, suggest that they consult a major classical formulation of principlism found in the principles of biomedical ethics by thomas beauchamp and james childress (2). also, reliable but briefer introductions to those principles are widely available (for instance, thomas mccormick s at https://depts.washington.edu/bioethx/tools/princpl.html). the best way to learn on one s own how to infuse such bioethical principles into one s pedagogy is, as we have suggested, to begin where one is most comfortable, in one s own field, select an ethical issue, and work the principles into one s teaching with that single issue. the best assistance for busy faculty would be for asm through jmbe or microbelibrary to add a new area to their curriculum resources that focused on resources to support infusing ethics into biology and microbiology curricula. ideally this would include a variety of resources on different ethical theories that could be applied in science courses, how they could be applied, and possibly additional case studies that demonstrate how to utilize various ethical theories to address bioethical issues. an online course designed by philosophers and biologists together or a summer team-taught workshop (possibly at the asm conference for undergraduate educators) on infusing bioethics into biology and microbiology courses would be most helpful. these would not be strictly human- or human rights-focused but would include the broad range of ethical issues appropriate for undergraduate biology and microbiology courses from responsible use of antibiotics, biotechnology, and neuroscience to agronomy, physiology, and climate change.

with the rise of biomedicine and biotechnology, there has been a corresponding growth in the need for better understanding of consequent ethical questions. increasingly, biologists are being asked not only to offer technical clarifications but also to venture ethical opinions, for which most feel poorly equipped. this expectation puts pressure on biology instructors at the university level to provide biology majors the skills and experience to discuss with some confidence and competence bioethical issues which may arise in either the workplace or through public discourse in everyday contexts. many fine curricular resources about bioethics are available for varied pedagogical purposes, but few target undergraduate biology or microbiology student audiences. when it occurs in the context of a course, bioethics instruction often is taught by non-biologists outside standard biology curricula. we propose that biologists should strive to infuse bioethical thinking into their courses and major curricula but not in such a way as merely to point at ethical problems, treating them at a surface level. we suggest what we call vertical infusion: taking one bioethical issue per course and integrating this issue within the context of a relevant biological topic, challenging students to push their thinking beyond their initial intuitions toward underlying scientific and ethical principles. while the vertical approach lacks widespread coverage of ethical issues throughout a single course, it has the advantage of taking the bioethical dimension seriously and in intimate relation to contemporary discoveries in biology and to the biological principles, processes, or procedures that occasioned the ethical quandaries in the first place.

PMC4278480

pubmed-611

discoveries of restriction enzymes by hamilton smith and daniel nathans, and reverse transcriptase by howard temin and david baltimore in the 1970s set the stage for an explosion in molecular and clinical genetics. armed with a sufficient amount of the mrna product of a gene such as the beta globin gene, investigators could now produce a radioactive dna copy (cdna) of the mrna and use it as a valuable probe of gene expression. cloned into an expression plasmid and transduced into an appropriate cell, the cdna would produce mature beta globin mrna, and cdnas could be manipulated at will in what became the era of recombinant dna technology. the new discoveries were greeted with enthusiasm by most biologists and clinical investigators but with mounting horror and suspicion by many members of the public and their elected officials, as well as some academics. frankensteins were thought to be loose in biomedical laboratories; monsters would be created; plagues of vicious e. coli would be loosed on an innocent population; mad scientists would forever contaminate the food supply; the new genetics would lead to a resurgence of social darwinism. the specter of nazi medicine roiled some faculty meetings and communities in which the science was rapidly advancing. rules and restrictions were demanded that would deliberately inhibit the work. to their credit, leaders of this new genetics revolution met in asilomar california, where they established laboratory standards intended to reassure themselves, their colleagues, and the general population that gene manipulation could be rendered safe and useful. while eager clinical investigators hoped to apply the new genetics in the treatment of inherited diseases, cooler heads recognized that the technology was not sufficiently powerful or predictable. the more cautious advised the national institutes of health to be very wary of human application because the biological ' rules of the game ' had not yet been established. that did not stop an american clinical entrepreneur from injecting beta globin cdna into the bone marrows of thalassemic patients in italy and israel. his reward was failure and opprobrium, and academic medicine encountered a congress increasingly determined to surround clinical research with an ever-growing net of regulation. how could a cdna be introduced efficiently into a rare, quiescent, mammalian cell, such as a bone marrow stem cell, remain potentially active, and be sufficiently expressed when that stem cell developed into a hematopoietic precursor and subsequently fully differentiated functional blood cell? furthermore, could a defective gene be actually replaced in human cells in a targeted fashion by a normal counterpart and still maintain high transcription efficiency, or would such ' plug and socket ' technology be so inefficient that correction would be impossible? instead, could cdnas such as a beta globin cdna be carried into the target cell chromosomes on the back of a virus, such as a retrovirus, and could the transduced sequence express its mature mrna regardless of its genomic location? the idea of a retrovirus as a gene-transfer agent was first seen as dangerously oncogenic, until 1983 and 1984 when mann, mulligan and baltimore devised cell lines that would produce replication-defective retroviruses that still exploited the capacity of the viruses to incorporate themselves efficiently in the dna of dividing cells. for the most part, the modified retroviral vectors infected human cells at comfortingly low multiplicities of infection. shortly after this, williams and mulligan and dick and bernstein, and their colleagues, showed that murine bone marrow cells could be transduced with defective retroviruses carrying cdnas, and that mature nucleated blood cells would carry the foreign cdna for weeks, proving that the murine hematopoietic stem cell, despite its very low rate of division, could be so transduced. but the percentage of infected cells was very low and expression of the transferred gene was vestigial. the results suggested that successful gene transfer with cdnas borne on replication-defective retroviruses would require high recombinant viral titer, cell culture systems that would encourage stem cell division without differentiation, and a setting in which target cells would have a selective advantage following gene transfer. the entire field of gene therapy was energized by the findings of williams and dick and their colleagues, but the barriers to translation and clinical application were soon found to be almost insurmountable. the slow pace prompted orkin and motulsky to lower their expectations for immediate clinical application, and focus instead on solving the basic technical and biological problems. in an attempt to gain some clinical traction, blaese and his colleagues introduced retroviral cdna ex vivo into the mature t cells of patients with immunodeficiency due to mutations in adenosine deaminase. the treatment provided little if any clinical benefit, and the risk of malignancy was obviously high because the t cells were influenced to divide in culture in order to enhance transduction. concerns about unwieldy and potentially unsafe clinical research protocols began to mount in the united states. four levels of review, the recombinant dna advisory committee, individual institutional review boards, individual institutional biosafety committees, and the food and drug administration all established barriers that slowed the pace of gene therapy clinical research to a crawl. this necessary regulatory environment was onerous enough, but it became even more obstructive when investigators at the university of pennsylvania performed a study of gene replacement in a rare metabolic disorder using adenovirus as a vector in order to infect non-dividing liver cells. one young adult with the disease died after a high titer of virus was administered. an investigation revealed that the gene therapists had a financial stake in the company that produced the vector. that revelation initiated an even higher burden of regulation and added massively to a growing concern about conflict of interest in clinical research-a conflict that continues to roil academic waters to this day. meanwhile, after four decades of development, the clinical application of allogeneic hematopoietic stem cells in transplantation for the treatment of congenital bone marrow diseases was moving ahead reasonably briskly (sans gigantic regulatory hurdles). this form of cell-based therapy, though initially applicable in only the 25-30% of patients with histocompatible donors, was associated with success in several patients with severe immunodeficiencies, congenital bone marrow failures of several types and even the inherited hemoglobin disorders. adenovirus vectors were thought to be too immunogenic to be useful but they and their cousin, the tiny adeno-associated virus, were considered to be worthy of evaluation in the transduction of non-dividing cells such as liver cells or endothelial cells. indeed, high and her co-workers have made quiet progress in the correction of canine hemophilia with adeno-associated virus. most groups interested in hematopoietic targets or cancer vaccine protocols continue to focus on defective murine leukemia viruses or lentiviruses. the latter are thought to have a higher capacity than murine leukemia viruses to integrate within the dna of non-dividing cells. both the science and the regulatory apparatus seemed to be daunting, and the funding was fragmented and difficult to obtain. but in 2002 cavazzana-calvo and his colleagues blew new life into the field when they made the startling announcement that they had successfully treated nine of ten patients with x-linked severe combined immunodeficiency (scid), utilizing a fairly standard murine leukemia viral vector that carried the common gamma chain of the interleukin-2 receptor into autologous bone marrow cells. shortly thereafter, however, cynicism returned when the authors reported that several of the patients had developed t cell leukemia. careful work by the investigators revealed that the long terminal repeat (ltr) of the vector may have a predilection for the lmo2 proto-oncogene on chromosome 11. but even random integration of the ltr at the lmo2 site would favor selection of such cells. the lmo2 proto-oncogene is often activated by translocation (11:14) in t cell leukemia. clearly, the gene-corrected immunocytes had a survival advantage, but the malignant t cells had an even greater survival advantage as well as a growth advantage. what was once a promising new start for gene therapy became an enormous set-back. vector safety had always been a pressing issue-now it had become a yawning chasm: of 20 patients with x-linked scid treated by gene transfer in these two trials, 18 are currently alive and with good immune reconstitution, but five have experienced a serious side-effect. of these five children, one died of therapy-related leukemia, and one died of complications of a subsequent stem cell transplant that was performed as a result of the failure of gene therapy. have been the results of retroviral correction of oxidase deficiency in chronic granulomatous disease. in two well-described cases, correction of granulocyte oxidase deficiency has been achieved but at the cost of clonal proliferation of cells activated at the sites of the mds-1-evi1, prdm16 or setbp1 proto-oncogenes. furthermore, other in vitro studies have demonstrated similar insertions by lentiviruses bearing beta globin genes. finally, williams and his co-workers have temporarily discontinued their pioneering work on the correction of the deficiency of dna repair pathways in hematopoietic stem cells of patients with fanconi anemia, because they are concerned that current vectors that permit rescue of those cells may induce insertional mutagenesis and leukemia in the treated patients, and that focus should be on developing methods of expanding deficient hematopoietic stem cells in this disease. despite these serious setbacks there have been some recent bright lights. in 2008, maguire and bainbridge and their colleagues reported progress on the treatment of leber's optic atrophy with an adeno-associated viral vector applied to the retina. and retrovirally transfected epidermal stem cells have been grown into keratinocytes to correct the lesions of epidermolysis bullosum. more follow-up is needed but the initial results hold promise for local applications of gene transfer. an encouraging report emerged at a recent annual meeting of the european society of gene and cell therapy. lentivirus vectors have been used to transduce hematopoietic stem cells in x-linked adrenoleukodystrophy, a progressive demyelinating disease that causes severe debilitation by early teenage years. long-term and stable gene modification has been observed in 20% of myeloid cells, well within the range to reverse phenotypes in some red cell and myeloid disorders. finally, auiti and his colleagues have recently reported highly encouraging results in the treatment of adenosine deaminase deficiency. thus, gene therapy of hematopoietic diseases with retroviruses lumbers in choppy straits twixt the scylla of insufficient gene transfer and the charybdis of leukemia, while the therapy of metabolic and coagulation disorders with adeno- and adeno-associated viruses is blunted by immune reponses to the vectors. to committed gene therapists, these are simply the challenges that they have always faced, while those who are engaged in stem cell transplantation, or in finding better halfway measures that support afflicted patients, work as best they can, all the while hoping that the holy grail of gene replacement will one day become a safe reality. the future could lie in the promising field of site-specific gene correction using modified nucleases, and the conversion of corrected somatic cells such as fibroblasts to functioning hematopoietic stem cells. cdna: copy dna; ltr: long terminal repeat; scid: severe combined immunodeficiency. the authors are particularly grateful to our colleague david a williams, md, for his contributions to this field and his invaluable assistance during the development of this review.

though the field has moved with glacial speed, gene therapies have been carried out successfully in patients with bone marrow disorders including immune deficiencies. the field may be poised to move forward more rapidly, but many barriers have yet to be surmounted.

PMC2684659

pubmed-612

a recent institute of medicine (iom) report on this subject acknowledges that the rates and impact of medication errors are huge but are poorly understood. the president of the institute of safe medication practices (ismp), michael cohen, in his testimony to a committee of the us congress estimated that the dollar cost of adverse drug events was about $200 billion across all settings. in ambulatory settings, medication errors and adverse drug events (ades) are one of the most important safety issues. a study based on the national ambulatory medical care survey (namcs) found that office-based physicians prescribed at least 1 inappropriate medication to nearly 8% of the elderly who received prescriptions. another study of ambulatory elderly patients with polymorbidity and associated polypharmacy documented that 35% reported experiencing at least one ade within the previous year. gurwitz and colleagues have estimated (by extrapolation) that medicare enrollees alone suffer approximately 500,000 preventable ades per year. a 2003 report of a multidisciplinary group (composed of the ahrq-supported medical group management association center for research, the centers for medicare and medicaid services, and the partnership for patient safety) draws attention to the fact that while safety risks are widespread in ambulatory settings, there has been insufficient attention paid to them. some estimates suggest that ambulatory settings are at least equally important as inpatient settings, with up to 200,000 avoidable deaths annually in the united states of america (usa) alone [7, 8]. lack of awareness of the type, the incidence, and consequences of errors in any setting is one of the most important barriers to reducing these errors and improving quality of care. the most commonly used method for estimating vulnerabilities in healthcare is to retrospectively collect and count errors through voluntary reporting systems (often referred to as these are fraught with difficulty due to underreporting (according to iom's 1999 report, only 5% of known errors are typically reported and then there are unknown errors) and abuse (e.g., reports filed and counterfiled as a means of retaliation against colleagues). error reporting often does not promote understanding of the organizational structure and processes of care. instead it tends to be associated with blame and shame, and frequently results in antagonism between team members undermining mutual respect, trust, and cooperation. the patient safety and quality improvement act (2005) was introduced in large part to stimulate increased error reporting through the creation of patient safety organizations (psos). bates and colleagues have described difficulties involved in defining and quantifying errors; they report that even direct observational studies, which are highly labor intensive, often miss errors. an alternative approach that is prospective, rather than retrospective, and encourages involvement of all teammembers for identifying and prioritizing safety and quality problems invokes failure modes and effects analysis (fmea). this has been widely used in other high-risk industries and has been advocated by the iom as a means of analyzing a system to identify its weaknesses (failure modes), possible consequences of failure (effects), and to prioritize areas for improvement. we have adapted and tailored this methodology to allow for the levels of resources and expertise available in ambulatory settings, and developed an instrument that has been shown to be effective in a variety of these settings. the details of the rationale and processes behind this instrument termed safety enhancement and monitoring instrument that is patient centered have done, that a vital step toward creating the medical home is to close the physician staff divide so as to maintain communication and coordination. they draw attention to the fact that currently practice meetings are universally unpopular despite their indispensability. our trm methodology, invoking the paradigm of complex adaptive systems, is designed to aid formation of central attractors in the form of self-empowered effective learning teams with a common vision to help their complex microsystems to adapt and thrive [1721]; thriving systems are endowed with simple rules, shared vision, and opportunities for team members to innovate. an outcome-oriented team has to be enabled to (a) own and identify vulnerabilities in their settings, (b) design and implement interventions tailored for its settings, (c) monitor the efficacy of these interventions, and (d) continue the never ending journey in pursuit of safe care, that is, continuing quality improvement. interestingly, a recent study by quinn et al. found that physicians from practices that were involved in evaluation of quality improvement activities had significantly less isolation, stress, and dissatisfaction. objectivethe primary objective of this study was to evaluate the impact of the prospective team resource management methodology (trm), based on the semi-p instrument, (with and without the use of a practice enhancement associate (pea)) onthe number of preventable ades in a vulnerable population aged 65 and above;the severity of these preventable ades. the primary objective of this study was to evaluate the impact of the prospective team resource management methodology (trm), based on the semi-p instrument, (with and without the use of a practice enhancement associate (pea)) onthe number of preventable ades in a vulnerable population aged 65 and above;the severity of these preventable ades. the number of preventable ades in a vulnerable population aged 65 and above; the severity of these preventable ades. this was a cluster randomized trial in which 12 practices in the upstate new york practice-based research network were each randomized to one of 3 states (4 practices each): (1) team resource management intervention; (2) team resource management intervention with pea; (3) no intervention (comparison group). these can be repeated to make a cycle, as shown in figure 1, for continuous quality and safety improvement. this anonymous survey is an opportunity for all staff to freely express opinions about the care processes in their setting. this survey asks about each of the steps in the medication use process. to help orient staff to the overall process, the survey uses a diagram (figure 2) that shows who and what is involved in the processes and how they work (or are supposed to work) together. each page of the survey is about a different area and consists of a list of errors or causes of error that can occur in that area. the lists are based on review of the literature and consultation with practicing physicians and nursing leaders. the survey asks staff to think about each of the errors in turn and, for each, to indicate their opinion about how often it occurs and, when it does happen, how severe the consequences usually are. the diagram of the testing process is included on each page, with red highlighting to show which part of the process is being asked about. if willing, they can mark their job category (provider, nursing, or administrative). scores (called hazard scores obtained by multiplying frequency of each error with its respective severity of consequence) are calculated for each error in the survey, based on respondents ' answers to the frequency and severity questions. the items are then listed in rank order (highest to lowest) and presented to staff in a graphical format in a group meeting for their discussion. this helps the team to form a common vision and consensus regarding which problems need to be addressed first. in further group meetings, staff discuss the prioritized problems and work together to design solutions, keeping in mind the resources available and the capabilities of their unique setting. teams are formed to implement the solutions, with clear allocation of responsibilities and an agreed time schedule. she had completed a master's degree in nutrition but with no prior experience working with practices. she participated in all study-related team meetings at each practice and made herself available to each practice for up to half a day a week throughout the study period to support safety and quality improvement activities (not limited to study-related activities). the main contribution of the pea was to follow through on plans developed by the practice team, that is to support stages 3 and 4 in the trm cycle outlined above. examples of pea activities include developing patient education materials, preparing draft protocols, and collecting resources for patient prescription assistance. the rate and severity of ades and preventable ades were measured by chart review for the two 12-month periods before and after the start of the intervention. eligible patients were those aged 65 years and above, who had at least one visit for cardiovascular disease (icd-9-cm codes 390459) during the measurement period and at least one visit for any diagnosis in the prior year. if there were 200 or fewer eligible charts at a site, all were screened, otherwise a random sample of 200 was taken. the chart review was performed using a trigger tool method that involves 2 steps, namely a screening step and a review step [5, 2332]. the screening step involves identifying the presence of certain chart findings, known as triggers, that are known to be possible evidence of an adverse event. in this study, the triggers of interest are those that might represent an adverse drug event (ade). examples include an elevated inr (often associated with adverse effects of warfarin) and abrupt discontinuation of a medication (that sometimes occurs because of an ade). in our study research assistants performed the screening step, using a previously published ade trigger tool that we adapted from the work of others [5, 25, 26]. in the second step, known as the review step, a physician and pharmacist reviewed the identified triggers to determine for each trigger: (1) whether an ade took place and if so, (2) whether the event was preventable, (3) the stage of the medication use process where the ade originated (prescribing, dispensing, administration, and monitoring), and (4) the effect on the patient (none/minimal, mild, moderate, or severe). examples of preventable errors include missed allergy, wrong dosage, errors of dispensing, administration errors, and failure to order or complete laboratory monitoring. the physician and pharmacist worked together, reviewing, discussing, and recording their consensus opinion of each trigger. when they were unable to reach consensus a final determination was made by a third reviewer. if they identified any potential or actual harm that had not been previously recognized and addressed, they notified the primary care physician or site medical director. the study protocol was approved by the social and behavioral sciences institutional review board of the state university of new york at buffalo. part way into the study, one of the comparison group practices withdrew from the study, citing concerns over the administrative burden of the chart reviews (the practice was undergoing administrative changes). table 1 shows the characteristics of the 11 sites that completed the study. at baseline, of these, 1066 (54.1%) had triggers, yielding a total of 2898 triggers. this far exceeded our expectation and therefore posed a practical problem due to the effort (and therefore cost) associated with reviewing this number of triggers. therefore, we elected to reduce the sample by randomly eliminating a proportion of patients at each site, sufficient to reduce the number to approximately 100 patients per site. this yielded a total of 1125 patients, of whom 598 patients had one or more triggers, all of which underwent review. a similar process was used to make the endpoint data manageable, yielding 1050 patients, of whom 564 had triggers that were reviewed. table 2 summarizes the rates of preventable ades (normalized per 100 patient-years) for each arm of the study. for each arm, we compared the pade rate (per 100 patient-years) at the two time points (after versus before) by means of a paired t test with sites as the unit of analysis. as shown in table 2, in the intervention with pea group there was a statistically significant decrease in the overall rate of pades after the intervention compared to before (7.4 per 100 patient-years versus 12.6, p=0.018) and in the rate of moderate or severe (combined) pades(1.6 versus 6.4, p=0.035). in the comparison arm and the unaided intervention arms, analysis of variance (anova) with study arm and time as the factors and total pades as the outcome variable showed no significant interaction between arm and time. for the outcome of moderate or severe pades, the interaction term was significant (p=0.023) supporting the notion that the intervention with pea practices had a greater reduction in moderate/severe pades over time than the comparison group. the trm approach, when aided by a pea, demonstrated a significant reduction in pades, especially those with the highest severity. the reduction in pades represents a significant improvement in patient safety through the collective efforts of practice staff guided by a structured trm process. it appears that the additional resource offered in the form of a pea was important, as the non-pea practices did not achieve the same statistically significant improvement that the pea practices did. while the mechanism of action of the pea is not clearly understood, it is reasonable to surmise that the extra human resource represented by the pea enabled practices to more effectively implement their planned interventions. the addition of a pea may have helped to minimize the incremental burden on teams that are already overburdened. while we did not formally evaluate this, it is the authors ' observation that the humanistic self-empowerment approach used in this study helped to energize front-line workers to maintain and continually improve quality. staff commented that seeing other people's perspectives (as reflected in semi-p results) helped to improve mutual understanding, leading to consensus. further, we believe that by closing the physician staff divide, and encouraging closer communication and coordination between team members in addressing practice issues, the trm approach can achieve progress toward the creation of the medical home. firstly, the outcome measure is based on a trigger tool methodology that has limited sensitivity. in fact, the sensitivity of the trigger tool is unknown because there is no gold standard against which to compare it. this means that the rates of pades reported should not be seen as estimates of true rates. instead, they represent only a subset of pades, that is, those that are identifiable by the trigger tool. however there is no reason to expect that the sensitivity of the trigger tool would vary between study groups or over time so the validity of the study findings is not jeopardized. another significant weakness is the small size of the study. having only 3-4 practices in each arm limited the power. in addition, further exploration of the role of the pea is required to establish the most important active ingredients so that these can be deployed in an efficient way.

background. most safety issues in primary care arise from adverse drug events. team resource management intervention was developed to identify systemic safety issues to design and implement interventions to address prioritized issues. objectives. evaluate impact of intervention on rates of events and preventable events in a vulnerable population. design. cluster randomized trial. 12 practices randomly assigned to either: (1) intervention; (2) intervention with practice enhancement assistants; (3) no intervention. the intervention took 12 months. main outcome measure. rate and severity of events and preventable events measured using a trigger tool chart review method for the 12-month periods before and after the start of the intervention. results. in the intervention with assistants group there was a statistically significant decrease in the overall rate of events and in the rate of moderate/severe events. analysis of variance with study arm and time as the factors and moderate/severe events as the outcome showed a significant interaction between arm and time supporting the notion that the intervention with assistants practices had a greater reduction in moderate/severe preventable events. conclusions. the intervention had a significant effect on medication safety as estimated using a trigger tool. further exploration of role of assistants and trigger tool is warranted.

PMC3296195

pubmed-613

in pregnant women with mechanical heart valves, the frequency of valve thrombosis increases due to pregnancy-related hypercoagulability. therefore, effective anticoagulation is critical in pregnant patients with mechanical heart valves but remains problematic because both oral anticoagulation and heparins have been associated with important fetal and maternal side effects (1). coumarin derivatives are anticoagulants of choice for mechanical heart valves, but they cross the placenta and are associated with coumarin-induced fetal loss or embryopathy (1-3). unfractionated heparin (ufh) provides an alternative therapy that avoids fetal side effects, however, the use of ufh is associated with increased maternal thromboembolic and bleeding complications (1, 4, 5). low molecular weight heparin (lmwh) may be more advantageous than ufh (6), and appears a good alternative. however, little clinical information and no reliable data are available regarding its efficacy and safety. no consensus has been reached about optimal antithrombotic therapy in pregnant patients with a mechanical heart valve. coumarins are contraindicated in pregnancy in north america due to fetal concerns, but european experts have recommended low dose coumarins (less than 5 mg daily) throughout pregnancy given a very low frequency of fetal anomalies (2, 3). reports of lmwh use began to appear, and many physicians now use lmwh because of its good safety profile for both mother and baby (7-10). however, treatment failures have been reported (11, 12), and no lmwh has been licensed for use in pregnant patients with mechanical heart valves. available published data regarding the efficacy and safety of lmwh in this clinical setting have been derived from small case series, and usually enoxaparin has been used. here, we report our experience of nadroparin treatment, and its associated pregnancy outcomes and maternal complications. between 1997 and 2005, 31 pregnancies were analyzed retrospectively in 25 women with mechanical heart valves. basic characteristics and previous operative data are listed in table 1. in 23 of these 31 pregnancies, nadroparin was used as an anticoagulant during the first trimester with given informed consent, concerning the risks and benefits of lmwh. others were anticoagulated with coumarin derivatives (cmd) with or without aspirin due to unawareness of pregnancy until the first trimester, or because of refusal or poor compliance on self-injected lmwh. in the lmwh-treated group, when pregnancy was confirmed, coumarins were stopped and changed to subcutaneous nadroparin (7,500 u, twice daily), from 6 weeks to 12 weeks of gestation. aspirin, at 100 mg/day, was also administrated throughout the pregnancy. at gestation week 38, women were scheduled for labor induction and changed to nadroparin to avoid the delivery of an anticoagulated fetus. after establishing labor, we carefully checked for hemorrhages and other complications, and babies were examined for congenital anomalies and weight. in the cmd-treated group, coumarins and aspirin were continued throughout the pregnancy and the target international normalized ratio (inr) was maintained between 2.5 to 3.5. coumarins were changed to nadroparin before 2 weeks prior to the expected delivery date to avoid fetal bleeding complications during delivery. pregnancy outcomes, namely, numbers of healthy babies, fetal anomalies, fetal losses, and maternal complications, including thromboembolism or bleeding, were analyzed. to evaluate the efficacy and safety of lmwh, eight pregnancies, maintained using coumarins throughout pregnancy, were compared. data were analyzed using spss for windows version 10.0 software and compared using the student's t-test, at a level of significance of p<0.05. no maternal death or bleeding complication occurred in either the lmwh-treated group or the cmd-treated group. frequencies of maternal thromboembolism were not different between the two groups (table 2). a maternal transient ischemic attack (tia) occurred in one case in each group, and both patients had previously undergone mitral valve replacement. prosthetic mitral valve thrombosis occurred in three pregnancies, two in the lmwh group and one in the cmd group (table 3). the two of these three patients underwent redo surgery, and other patient was managed on thrombolytic therapy. numbers of live born and healthy babies were higher in the lmwh group (table 4). in both groups, two babies had low birth weights of 2.1 kg and 2.4 kg, but were otherwise healthy. in the cmd group, one baby had hydrocephalus. however, the frequency of fetal loss including therapeutic abortion and stillbirth were significantly higher in the cmd group. two fetal losses occurred in the lmwh group, both occurred in cases of maternal valve thrombosis. four fetal losses occurred in the cmd group, and one of these involved maternal valve thrombosis. this study demonstrates that lmwh-based therapy is superior to coumarin therapy in pregnant women with a prosthetic heart valve, and that the use of nadroparin during the first trimester with 100 mg of aspirin throughout pregnancy could be a safe and effective protocol for thromboprophylaxis in these women. recent recommendations, published in 2004 as part of the 7th american college of chest physicians (accp) consensus on antithrombotic therapy (12), included the following three regimens: 1) aggressive adjusted-dose lmwh throughout pregnancy; 2) adjusted-dose ufh, throughout pregnancy; or 3) either lmwh or ufh between 6 and 12 weeks and close-to-term only and the use of cmd at other times. our protocol was similar to the third regimen, but we also administered aspirin (100 mg daily) throughout pregnancy to reduce coumarin dosages and the risk of thromboembolism. the overall frequencies of maternal thromboembolism, including valve thrombosis, were similar in both groups, but the frequencies of live and healthy baby births were higher in the lmwh group. these results demonstrate that lmwh-based therapy is a good alternative to coumarins, because it has similar anticoagulation effects with lower fetal side effects. exposure to coumarins during the second part of the first trimester is associated with fetal loss, primarily due to spontaneous abortion or coumarin-induced embryopathy. the reported frequencies of coumarins-related embryopathy vary for debatable reasons (14, 15), a recent study suggested that coumarin risk is dose related and that adverse effects occur mainly in women taking>5 mg daily., the target inr was maintained with less than 5 mg of coumarins in all patients in the cmd group; however, a half of these lost their babies due to abortion or stillbirth. our results represent only observational data, and the effect of dose-related embryopathy remains uncertain. lmwh does not cross the placental barrier and offers potential advantages compared with ufh in terms of better safety profile with less thrombocytopenia, less bleeding, less osteoporosis with prolonged treatment, a more predictable and rapidly reached anticoagulant effect, and the possibility of self-administration of anticoagulant therapy without laboratory monitoring. however, treatment failures have been reported, and the use of lmwh for pregnant women with mechanical heart valves has become controversial due to small numbers of patients and a lack of accurate postmarketing data (16). a recent review of 81 pregnancies in 75 women treated with lmwh reported an 8.6% rate of valve thrombosis (17), and found that appropriate dose adjustments could reduce the frequency of thromboembolism. the 7th accp recommendations call for the use of lmwh at levels that achieve peak anti-factor xa values of around 1.0 u/ml (12). a recent prospective study with deltaparin reported that dosages based on body weight were inadequate to maintain a therapeutic level of lmwh in pregnancy (18). our data demonstrate that valve thrombosis occurred in 2 patients treated with nadroparin; a prevalence of 8.7%. unfortunately, we did not monitor anti-xa levels during nadroparin administration, and thus, we can not conclude that valve thrombosis is associated with an inadequate nadroparin dose. further studies, with sufficient statistical power, are required to clarify the clinical significance of anti-xa levels. in conclusion, despite the retrospective design of the present study, it might be worth to mention that lmwh appears a safe and effective substitute for any other anticoagulants in pregnant women with mechanical heart valves. we have experienced that pregnancy outcomes are acceptable with lmwh, but that its efficacy for preventing valve thrombosis remains uncertain. further studies are needed in order to establish appropriate management protocols for pregnant women with mechanical heart valves.

no definitive recommendation is available concerning optimal antithrombotic therapy in pregnant women with a mechanical heart valve. the purpose of the current study was to evaluate the clinical results of nadroparin treatment with respect to pregnancy outcome and maternal complications. from 1997 to 2005, 31 pregnancies were reviewed in 25 women. nadroparin (7,500 u, twice daily) was used in 23 pregnancies between 6 and 12 weeks of gestation and close-to-term only, and coumarin derivatives were used with aspirin at other times. eight pregnant women treated with coumarin derivatives throughout pregnancy were compared to evaluate the safety and efficacy of nadroparin. no maternal death or bleeding complication occurred in either of the two groups, and frequencies of maternal thromboembolism including valve thrombosis (8.7% vs. 12.5%, p>0.05) were similar. however, the frequencies of live born (91.3% vs. 50%, p=0.01) and healthy babies (90.4% vs. 25%, p<0.01) were significantly higher, and the fetal loss rate was significantly lower (8.7% vs. 50%, p=0.01) in the nadroparin-treated group. regarding the efficacy and safety of antithrombotic treatment in pregnant women with prosthetic heart valves, nadroparin treatment during the first trimester is an acceptable regimen and produces better results than coumarin derivatives.

PMC2693592

pubmed-614

pulmonary hypertension is an increasingly recognized complication of sickle-cell anaemia and a risk factor for early death [14]. recurrent episodes of acute and subacute pulmonary crises reflecting in situ sickling within the lung has been postulated to be the primary event leading to pulmonary hypertension. haemolysis may participate in its pathogenesis by limiting nitric oxide (no) bioavailability and producing vasculopathy [5, 6]. an initial study in howard university, usa, using echocardiographic assessment of tricuspid valve regurgitant jet velocity 2.5 m/sec as diagnostic criteria, demonstrated pulmonary hypertension in 32% of adult sickle-cell patients, and the prevalence appeared to increase with age of the patients. in patients between 40 and 49 years old, the prevalence was 40% and increased to 5560% by age 50 and above. other studies have documented prevalence rates between 20 and 40% [4, 810]. sickle-cell anaemia patients with pulmonary hypertension have a significantly increased mortality rate compared with patients without pulmonary hypertension. sutton and colleagues reported 40% mortality rate in sickle-cell patients with pulmonary hypertension at 22 months after diagnosis (odd ratio 7.86; 95% confidence interval=2.6323.4) compared with sickle-cell patients without pulmonary hypertension. castro et al. in a study of 34 adult sickle-cell patients who underwent right heart catheterization for evaluation of pulmonary hypertension found increased pulmonary artery pressure in 20 (58.8%) on initial catheterization. during 2345 months of follow up, 11 of these 20 (55.0%) died compared with 3 of the 14 without pulmonary hypertension (21.0%). every 10 mmhg increase in mean pulmonary artery pressure was associated with 1.7 increase in mortality (95% confidence interval=1.12.7; cox proportional hazard model, p=0.028). the study was aimed at comparing the clinical and electrocardiographic findings in sickle-cell anaemia patients with raised pulmonary artery pressure with those of patients without pulmonary hypertension. the study was carried out in the adult outpatient sickle-cell clinic and the cardiac centre of the university of nigeria teaching hospital (unth), enugu, nigeria. the study subjects were drawn from adult patients (age 18 years), attending the adult sickle-cell clinic of the hospital, who had haemoglobin genotype ss on haemoglobin electrophoresis, were in steady state, and consented to participate in the study. steady state is defined as absence of any crisis in the preceding four weeks, absence of any symptoms, or signs attributable to acute illness. a total of sixty two sickle-cell anaemia patients, and sixty two age- and sex-matched normal controls were studied. resting 12-lead electrocardiography was performed on all subjects using cardioline ar-600 model electrocardiography machine at a paper speed of 25 mm/s and standardized at 0.1 mv/mm. a single observer analyzed the electrocardiogram. measurements of the heart rate, cardiac axis, pr- interval, qrs duration, and qtc interval were done in the standard fashion. heart rate correction of the qt interval was performed using bazett's formula (qtc=qt/ rr). the dispersion of p-wave, qrs, and qtc intervals was measured manually under magnifying glass by the same observer and was taken as the difference between the maximum and minimum values of each parameter on standard 12-lead electrocardiogram. left ventricular hypertrophy on electrocardiogram was based on sokolow and lyon voltage criteria, while right ventricular hypertrophy was based on the criteria described by allenstein and mori (dominant or tall r-waves or rs pattern in a vr, v1, and v2 with deep s-wave in i, avl, v5, and v6). echocardiography was done using hewlett packard sonos 2500 echocardiography machine with 3.7 mhz transducer. pulmonary artery flow acceleration time was obtained from a doppler signal of the pulmonary flow in the parasternal short-axis view at the aortic valve level and was described as the time from onset to peak flow velocity. the mean pulmonary artery pressure (mean pap) was calculated from the formula: mean pap (m m/h g)=90 (0.62 a t), where at is the acceleration time of the pulmonary artery flow. pulmonary hypertension was defined as calculated mean pulmonary artery pressure 30 mm/hg [4, 7]. ethical clearance for the study was obtained from the ethical committee of the university of nigeria teaching hospital, enugu, nigeria. the declaration of helsinki's recommendations for guiding physicians in biomedical research involving human subjects were followed. data were presented as means standard deviation for continuous variables and as proportions for categorical variables. comparisons of continuous variables between groups were made with independent student's t-test. for discrete variables, distribution between groups was compared with chi-square test and fishers exact test as appropriate (where an expected cell is less than 5). multivariate pearson's correlation coefficient was used to determine the relations between clinical, electrocardiographic data and mean pulmonary artery pressure. all statistical analyses were carried out using the statistical packages for social sciences (spss inc., chicago, illinois) software version 11.0 and epi-info version 3.4. statistical tests with 2-tailed probability values less than 0.05 were considered statistically significant. the age, gender, and anthropometric parameters of the patients and controls are shown in tables 1 and 2. values for pulmonary artery pressure in the patients and controls are shown in table 3. elevated pulmonary artery pressures (pap) as defined by pap 30 mmhg was demonstrated in (26) 41.9% of patients with sickle-cell anaemia and in (2) 3.2% of the controls; =26.571, df=1, p<0.001 (figure 1). p-wave duration, qtc interval, and qtc dispersion were increased in patients with pulmonary hypertension (table 5). significant correlation was found between mean pap and (1) frequency of crisis (spearman correlation=0.320; p=0.011), (2) body mass index (pearson's correlation=0.297; p=0.019), and (3) qtc interval (pearson's correlation 0.261; p=0.040), (table 6). the prevalence of pulmonary hypertension in adult nigerian sickle-cell anaemia patients in this study was 41.9%. this value is slightly higher than previous report of 25% by aliyu et al. in northern nigeria. however, in that study, neither the mean pap nor the mean age of the subjects was stated. previous studies have relied on tricuspid regurgitant jet velocity as an indirect estimate of pap. this has resulted in an underestimation of the prevalence of pulmonary hypertension in sickle-cell anaemia patients. results of right heart catheterization for evaluation of pulmonary artery pressures in sickle-cell patients have shown prevalence rate of 58.8%. autopsy studies suggest that up to 75% of sickle-cell anaemia patients have histological evidence of pulmonary arterial hypertension at the time of death. mean pap derived from the echocardiographic estimation of pulmonary artery flow acceleration time in steady-state patients as was used in this study has been shown to correlate significantly with values from cardiac catheterization. several studies have corroborated the role of vaso-occlusive crisis and acute chest syndrome in initiating nitric oxide depletion resulting in the pathological changes in pulmonary hypertension [6, 19]. this study identified a positive correlation between pulmonary artery pressure and frequency of vaso-occlusive crisis. patients with pulmonary hypertension were found to have reduced body mass index when compared with patients without pulmonary hypertension. this difference could be due to increased disease severity in patients with pulmonary hypertension. recurrent insitu vaso-occlusive crisis in the pulmonary vascular bed with resultant pulmonary hypertension and right ventricular hypertrophy has been demonstrated in sickle-cell disease patients. this is in keeping with the finding by this study of a significantly increased prevalence of right ventricular hypertrophy in patients with pulmonary hypertension. right ventricular hypertrophy in these patients explains the significant positive correlation between pulmonary artery pressure and qtc interval. the significance of increased p-wave duration and the spatial dispersion of qtc-interval noted in this study in patients with pulmonary hypertension are unclear. qtc dispersion is a measure of the disparity among qtc intervals in various electrocardiographic leads and reflects the variability of myocardial repolarization. this corroborates the finding in a study by akgul et al. in turkey which recorded higher qtc dispersion in sickle-cell patients with pulmonary hypertension. pulmonary hypertension in adult sickle-cell anaemia patients is significantly associated with electrocardiographic evidence of right ventricular hypertrophy, increased p-wave duration, qtc interval, and qtc dispersion and correlates significantly with frequency of vaso-occlusive crisis and qtc interval. the observations by this study tend to suggest that these parameters could be useful for early detection and prevention of pulmonary hypertension in patients with sickle-cell anaemia.

pulmonary hypertension is an emerging complication of sickle cell anaemia with associated increased risk of mortality. in order to evaluate the clinical and electrocardiographic findings in adult sickle-cell patients with pulmonary hypertension, a cross sectional study was conducted on sixty two sickle cell anaemia patients and sixty two age and sex matched normal controls. elevated pulmonary artery pressures (pap), defined by pap 30 mm hg on echocardiography, was demonstrated in 41.9% of patients with sickle cell anaemia and in 3.2% of the controls; 2=26.571, p<0.001. right ventricular hypertrophy, increased p-wave duration, qtc interval, and qtc dispersion were significantly associated with pulmonary hypertension. significant correlation was found between mean pap and (1) frequency of crisis (spearman correlation=0.320; p=0.011), (2) body mass index (pearson's correlation=0.297; p=0.019), and (3) qtc interval (pearson's correlation 0.261; p=0.040). pulmonary hypertension in adult sickle anaemia patients is associated with electrocardiographic evidence of right ventricular hypertrophy, and correlates significantly with frequency of vaso-occlusive crisis, and qtc interval. the observations by this study tend to suggest that these parameters could be useful for early detection and prevention of pulmonary hypertension in patients with sickle cell anaemia.

PMC3320006

pubmed-615

retinoblastoma (rb) is the most common intraocular malignant tumor in childhood, with an incidence of 1 in 15000 live births. it may affect one eye (unilateral rb) or both (bilateral rb) during the first five years of life. although extensive epidemiologic studies have been done to study this tumor, the results have been more often misinterpreted at the expense of mutation theory which has prevailed until recently in spite of the outstanding evidence against it. in the present review the authors analyze the most relevant epidemiological issues concerning retinoblastoma, in the light of recent developments highlighting the role of aneuploidy and genetic instability in the pathogenesis of this eye cancer. the most important studies to investigate the pathogenesis of retinoblastoma began with a paper published by knudson in 1971, when the author, after investigating the age distribution and laterality of a cohort of 48 rb patients, concluded that the disease could be inherited and formulated the so-called two-hit theory in order to explain its pathogenesis. in reality, no clues about the inheritance of retinoblastoma could be deducted from such a small sample. in fact, in his first report on this matter, knudson referred to earlier, smaller series showing that, in retinoblastoma survivors with bilateral disease, the proportion of affected offspring closely approximated 50%, as in dominant (mendelian) inheritance. from an original, mathematical analysis of the above data, knudson inferred that retinoblastoma is caused by two sequential (two hit) mutational events. according to this hypothesis, in the dominantly inherited form of the disease, one mutation is inherited via the germinal cells and the second spontaneously occurs in somatic cells of the retina and other tissues of the body. on the contrary, in the nonhereditary form the different timing and cell type involved by the two mutations determines the different clinical phenotype, with all bilateral and a minority of the unilateral cases being classified as hereditary, and the remaining unilateral cases being included in the sporadic group (table 1). during the following forty years of epidemiological, clinical, genetic, and biological research in this field, with the discovery of the rb1 as the prototype tumor suppressor gene, the medical establishment agreed on the pathogenetic two-hit theory which was further expanded by knudson, in many other scientific articles and review papers [624]. this generated the widespread conviction that rb is caused by two mutational events leading to the loss or inactivation of both alleles of the rb1 gene, as still believed by some authors. as mentioned above, the original input into the possible genetic derivation of retinoblastoma was based on limited evidence showing an apparently dominant mendelian distribution of the disease in the offspring of bilaterally affected individuals, thus allowing knudson to conclude that bilateral rb is inherited through the germ cells. minimum or no disagreement had been appeared on this account in the literature during the last four decades. in an attempt to elucidate this issue, we have performed an analysis of the distribution of the disease in the offspring of unilaterally affected rb survivors, referred to the department of ophthalmology we discovered that in a total of 16 children born to 12 unilaterally affected patients, 8 (50%) were healthy and 8 (50%) affected with rb (table 2). using the reasoning of knudson, it would be easily concluded that the unilateral disease phenotype is inherited and not sporadic, and this would be in sharp contrast with the current knowledge according to which bilateral rb is always hereditary and unilateral rb is almost always sporadic. it was reported by knudson and confirmed by others that about 10% of all rb cases do have a positive family history. in other words, the family history of the index case offers at least one other affected member, either a parent or another close relative. in this case, it is assumed that the events leading to the inactivation of the rb1 gene run in the family, and therefore the first hit is transmitted through the germline, exactly what happens in bilateral rb, but with one difference; bilateral rbs, which after knudson are all to be considered hereditary, are also assumed to have inherited the first hit through a mutation in one of the parent's germ cells, but they are the only affected members in their families. we should therefore be reasoning that, since familial rbs share the same pathogenetic mechanism with bilateral (hereditary) rb, the vast majority of these cases should show the bilateral phenotype. to be more accurate, we could make a calculation of the percentage of familial rbs carrying the unilateral phenotype. as a matter of fact, table 1 shows that the unilateral phenotype accounts for about 1/3 of all hereditary cases (or about 30%), and since familial rb represents the 10% of all rbs, it comes out that rbs carrying the unilateral phenotype, within the familial group, should not be more than 3% (i.e., the 30% of the 10%). as a matter of fact, a meta-analysis of a cohort of 3584 patients (table 3), reported by us elsewhere [4, 5], reveals that on a total of 344 (9.5%) familial cases, 83 (24%) show the unilateral phenotype, instead of the predicted 3%, a rather unexplainable figure, in the light of the predictions made by the in 1986, potluri and coworkers observed that the association of rb with the constitutional chromosome 13q deletion syndrome and the finding of 13q deletions or monosomy 13 in rb cells in individuals with normal constitutional karyotypes seemed to suggest that chromosome 13q could contain a gene responsible for tumor development in retinoblastoma. although the authors themselves acknowledged that other chromosome abnormalities, in addition to those involving chromosome 13, are evident in retinoblastoma (additional copies of 1q material in 44% of cases, isochromosome 6p, in 45% of cases, monosomy 16, in 18% of cases, marker 1p+, in 13% of cases, and homogeneously staining regions and double minutes, in 9% of cases), further investigations on this matter stressed the role of 13q deletions in the genesis of rb, thus reinforcing the belief that the loss or inactivation of the rb1 was the only responsible for rb to develop. but it is well known that retinoblastoma is only one among many different tumors associated with deletions of chromosome 13. cancers linked with these deletions include chronic lympocytic leukaemia (cll), chronic myeloproliferative disorders, multiple myeloma, hepatocellular carcinoma (hcc), nasopharyngeal carcinoma, benign and low-grade malignant lipomatous tumors, bladder cancer, malignant mesothelioma, and prostate cancer. the same pleiomorphism in the phenotypic expression of cancers associated with rb1 gene mutations is therefore evident in 13q deletion syndrome. but the most important consideration to be made about the association of 13q deletion syndrome and rb concerns the evident discrepancy existing between the expected and the real number of bilateral tumors among the patients affected by this genetic disorder. as a matter of fact, the 13q deletion syndrome must be confirmed by the cytogenetic analysis of peripheral blood lymphocytes, and it is due to a deletion of the long arm of chromosome 13 which involves, by definition, the rb1 gene locus. since the constitutional deletion of the rb1 gene can only be present if transmitted through the germ cells of one parent, it follows that all patients affected by 13q deletion syndrome and retinoblastoma belong to the hereditary group of knudson's and must, therefore, express the bilateral disease phenotype. it happens, however, that this assumption does not fit the clinical reality. in table 4 a series of 13 cases of 13q deletion syndrome and retinoblastoma referred to us over the last four decades is reported. of these patients, only 4 had the bilateral disease phenotype, while the remaining 9 were unilaterally affected (table 4). the mean age at diagnosis in this group, which is about 10 months, further reinforces the assumption that they must belong to the hereditary group of knudson's, but the unilateral disease phenotype is unexplainably high. in the light of the two hit theory, the data summarized above do not have any rationalization, and the only plausible conclusion is that the assumptions made in regard to the role of the rb1 gene in retinoblastoma are incorrect. clinical epidemiology is a leading discipline in the understanding of disease pathogenesis and etiology, but, as any other scientific endeavor, it relies on the correct interpretation of the available data. the proper analysis of data, in turn, relies not only on the individual researcher's skill but also on social, economic, and political environment in which the data are analyzed. the presumed genetic origin of rb and its relationship with the rb1 gene represent a clear example of how an entire body of prominent researchers may fail to question a flawed pathogenetic hypothesis (i.e., the two-hit theory), for the sake of personal, academic, or other interests. it was not by chance that we had to approach many different scientific journals to have access to the medical community about the role of aneuploidy and genomic instability in the genesis of rb [4, 5, 39]. we are still optimistic, however, because our alternative pathogenetic explanation has finally appeared in recent ophthalmologic literature.

retinoblastoma (rb) is considered to represent the prototype of cancer linked to the sequential loss or inactivation of both alleles of a so-called tumor suppressor gene, the rb1 gene. the pathogenetic mechanism behind this tumor was first hypothesized by knudson in 1971 and further confirmed by others who identified the rb1 gene whose loss or inactivation was claimed to be responsible for the disease. however, after about four decades of continuous research in the field of molecular biology, the evidence behind the role of the rb1 gene in rb appears to be seriously flawed in the light of epidemiological, biological, and clinical evidences. this editorial summarizes the inconsistencies on this subject. nevertheless, the molecular biology establishment still adheres to the biased view of the genetic origin of rb and other cancers, and hardly any alternative explanations are taken into account.

PMC2859046

pubmed-616

the etiology underlying temporomandibular joint osteoarthritis (tmj-oa) is not fully understood; however, some contributing factors are known, including internal derangement, macrotrauma, and parafunctional habits.1-3 in addition, functional overloading appears to be an important step in the cascade of events leading to osteoarthritis of the tmj,1,4,5 which can lead to physical disruption of cells, impaired cellular function, transient ischemia of certain cell populations and the production of neurogenic irritants.4 as a result, the joint tissues collapse. if the joint collapse occurs in both tmjs, condylar resorption causes morphologic collapse of the tmjs and a subsequent decrease in ramus height, which results in progressive mandibular retrusion with anterior open bite.4 this is called acquired open bite associated with tmj-oa. the malocclusion and retrognathic facial profile associated with acquired open bite can be improved by orthognathic surgery, including maxillomandibular advancement with counterclockwise rotation. however, patients with active tmj disease and concomitant or resultant maxillofacial skeletal discrepancies who are treated with orthognathic surgery alone often have poor outcomes and significant relapse.6-8 this implies that patients with presurgical tmj symptoms requiring mandibular advancement appear to have an increased risk of condylar resorption.9 in addition, orthognathic surgery requires surgical invasion, which carries risks and can result in significant postoperative discomfort.10 however, no significant alternative treatment for these patients has yet been reported. therefore, we suggest orthodontic treatment for patients with skeletal mandibular retrusion and anterior open bite due to tmj-oa, including molar intrusion, which has a beneficial effect on both esthetic appearance and occlusion. as a result of the counterclockwise rotation of the mandible caused by molar intrusion, the condyle is repositioned, and a functional adaptation in circumoral musculature can be achieved. here, we present an adult case of acquired open bite associated with tmj-oa treated using miniscrew anchorage. a 46-year 9-month-old woman presented with mandibular retrusion and circumoral musculature strain on lip closure (figure 1). she had a vertical and horizontal open bite and severe crowding of the upper anterior teeth. the only occlusal contacts were at the bilateral molars; however, significant abrasion was detected on the anterior teeth and premolars. although the mandibular midline was shifted 2.4 mm to the left, the maxillary midline was nearly aligned with the facial midline. the molar relationship was angle class iii on the left side due to a missing lower left second premolar, and angle class i on the right side. during mandibular excursive movement, molar guidance with balancing side contacts was detected. in her history, she had been conscious of her retropositioned mandible and chin; but during the past several years, the retrognathia increased. model analysis showed an arch length discrepancy of -5.8 mm in the upper arch and -3.5 mm in the lower arch. panoramic radiography showed the absence of the upper and lower third molars, and that the lower left second premolar had been extracted (figure 2). the lower left second molar had little or no alveolar bone support and was a floating tooth. lateral transcranial radiography of the tmj showed that both condyles were located anterior to the glenoid fossa, and no translations of the condyles were induced during mouth opening. cephalometric analysis revealed a skeletal class ii malocclusion with a severe retropositioned mandible (figures 2 and 3). the mandibular plane and gonial angles were significantly larger than the japanese norms (frankfort-mandibular angle, 52.7; gonial angle, 134.1).11 the mandible exhibited a backward and downward rotation with a short ramus. although the inclination of the maxillary incisors was within the normal range, the lower incisors were labially inclined. the patient had experienced frequent tmj pain during mastication and at maximum mouth opening for at least 5 years. maximum mouth opening without pain was 28.0 mm, and tmj crepitus was detected on both sides. the diagnosis was skeletal open bite with a short mandibular ramus associated with tmj-oa. the treatment objectives were to correct the anterior open bite, establish an ideal interincisal relationship, and to achieve an acceptable occlusion with a good functional class i occlusion. this case was treated according to the following plan: extraction of both maxillary first premolars and the mandibular right second premolar.placement of multi-bracket appliances on both dentitions to align the teeth.placement of titanium miniscrews in both sides of the posterior maxilla to intrude the molars, since molar intrusion should lead to subsequent counterclockwise mandibular rotation.use of a transpalatal arch to prevent buccal tipping of the maxillary molars during intrusion. placement of titanium miniscrews in both sides of the posterior maxilla to intrude the molars, since molar intrusion should lead to subsequent counterclockwise mandibular rotation. use of a transpalatal arch to prevent buccal tipping of the maxillary molars during intrusion. although mandibular advancement with orthognathic surgery is considered an effective treatment method, surgical treatment is not strongly recommended for patients with progressive mandibular retrusion associated with tmj-oa. in addition, surgical treatment requires prolonged hospitalization and higher medical costs, and is the most invasive option. we did not want to close the anterior open bite by extruding the anterior teeth, because the vertical relationship between the incisors and jaws was considered acceptable prior to orthodontic treatment, and tooth extrusion is generally considered an unstable movement. therefore, intrusion of the maxillary molars and subsequent counterclockwise rotation of the mandible were considered good options to treat the anterior open bite in this patient. a transpalatal arch was placed between the maxillary first molars, and both the upper first and the lower right second premolars were extracted. after the extraction, standard edgewise appliances with 0.018 0.025-inch slots were placed on both dentitions, except for the upper incisors (figure 4a). after leveling and alignment, titanium miniscrews (dual top anchor; jeil medical co., seoul, korea) were placed at the buccal sites of the posterior maxilla, and molar intrusion and canine retraction were initiated using elastic chains from the anchor screws. at this time, the brackets were bonded onto the upper incisors. at 1 year, 0.016 0.022-inch co-cr alloy wires were placed on both arches (figure 4b). as a result of molar intrusion, her anterior open bite was nearly corrected without the aid of vertical intermaxillary elastics. after intrusion, the canine and molar relationships were changed to almost class i. at 1 year and 6 months, the space closing process and anterior retraction continued with the use of elastic chains from the miniscrews (figure 4c). after 2 years and 7 months of orthodontic treatment, an acceptable occlusion was achieved without any recurrence of tmj symptoms, and the multi-bracket appliances were removed. immediately after removal, lingual bonded retainers were fixed on both dentitions, and a wraparound retainer was added to the upper arch. an acceptable occlusion was achieved, and overjet and overbite were increased to 2.2 mm and -0.7 mm, respectively. the canine and molar anteroposterior relationships were improved to class i on both sides (figure 5). lateral transcranial radiography of the tmj showed that both condyles were still anterior to the glenoid fossa, and that condylar movements during mouth opening were still poor. cephalometric analysis revealed approximately 1.5-mm maxillary molar intrusion and subsequent counterclockwise rotation of the mandible (figure 7). reduction of the excessive overjet was due to lingual inclination of the upper incisors. throughout the treatment period, maximum mouth opening without pain was 38.0 mm; however, tmj crepitus was still detected on both sides. magnetic resonance imaging taken after treatment showed anterior disc displacement without reduction in both tmjs, and the condylar head was only black in color, which indicated cortical bone without cancellous bone and bone marrow, suggesting an avascular necrosis-like structure (figure 8). two years after retention, the mandibular position was nearly unchanged (figure 7). the circumoral musculature strain upon lip closure disappeared, and an acceptable occlusion was maintained without recurrence of tmj symptoms (figure 9). the canine and molar relationships remained class i, and no relapse of the anterior open bite was found. panoramic radiographs showed little or no change in condylar structure, with condylar resorption and deformity (figure 10). lateral transcranial radiography of the tmj showed that the condylar movements during mouth opening were still poor. although successful outcomes have been reported for orthognathic surgical management of maxillofacial skeletal discrepancies with signs and symptoms of tmj disease,12 the outcomes depend on the pretreatment tmj condition. orthognathic surgeries in patients with active tmj disease and concomitant or resultant maxillofacial skeletal discrepancies often have poor results and significant relapse.6-8,13,14 this implies that patients with tmj symptoms have an increased risk of condylar resorption. symptomatic and asymptomatic pre-existing tmj pathologies that can lead to unfavorable outcomes include the following; internal derangements, progressive condylar resorption, condylar hyperplasia, osteochondroma, and congenital deformities.14 since the tmjs are the foundation of orthognathic surgery, the resultant pathology of tmj conditions with gross erosive changes in the articulating components of the fossa and condyle resulting in vertical height loss offers a poor base for maxillofacial skeletal and functional reconstruction. furthermore, the degenerative and osteolytic changes in the joint components due to these conditions make these tmj components highly susceptible to failure under the new functional loading resulting from orthognathic surgical repositioning of the maxillofacial skeleton. maxillomandibular advancement with counterclockwise rotation of the occlusal plane is an established procedure for patients with healthy tmjs. however, patients who had presurgical tmj symptoms and underwent orthognathic surgery alone had a statistically significant rate of skeletal relapse related to condylar remodeling and resorption. in addition, orthognathic surgery requires surgical invasion, which carries risks and can cause postoperative discomfort. therefore, orthognathic surgery is not recommended for patients with progressive mandibular retrusion associated with tmj-oa. in the present case, the patient had an anterior open bite with a retropositioned mandible caused by severe condylar resorption and deformity, indicative of tmj-oa. morphologic collapse of the joint component by tmj-oa induces a decrease in ramus height, leading to clockwise rotation of the mandible and an anterior open bite. the results of finite element model analysis showed that an open bite can induce greater tmj stress than normal occlusion.15 furthermore, clockwise rotation of the mandible, which is a major character of skeletal anterior open bite, leads to a synergistic increase in tmj stress during clenching.15 this suggests that improvement of mandibular clockwise rotation, which results in the reduction of tmj overloading, may be indispensable for treatment of acquired open bite associated with tmj-oa. however, it is nearly impossible to provide skeletal improvement in patients with anterior open bites using traditional orthodontics. several recent studies have demonstrated effective treatment of anterior open bite patients with class i or ii jaw relationships using temporary anchorage devices (tads), which has now been established as a new treatment strategy.10,16-19 absolute molar intrusion, which was previously impossible with traditional orthodontic mechanics, using tads results in counterclockwise rotation of the mandible, and the reduced overbite is increased without incisal elongation. kuroda et al.10,17,18 reported that although the mandibular plane was rotated more than 5 by molar intrusion, the patients had no functional problems after treatment. in addition, the procedure is definitely less invasive than a le fort i osteotomy for maxillary impaction with a mandibular repositioning osteotomy, and provides superior morphologic improvement over orthognathic surgery.18 therefore, absolute anchorage with tads was used in the present case. superimposition of the cephalometric tracings before and after treatment showed counterclockwise rotation of the mandible due to upper molar intrusion. because of this mandibular rotation, the mandibular plane angle was decreased by 2.3. the mandibular condyles also exhibited rotational repositioning in the inferior direction. consequently, due to increases in the anterior and superior areas, the joint space became nearly uniform. it would be reasonable to assume that these changes in condylar position, if accomplished with optimal occlusal support, may lead to biomechanical equilibrium in the tmj. when tmj-oa is progressing, the condyle deformity is more prominent with a shorter ramus height. although tmj-oa often has an unpredictable course, optimal condylar position and stable occlusion can achieve biomechanical equilibrium in the tmj, and this may inhibit progression of tmj-oa, and occasionally lead to functional and adaptive remodeling of the condyles through resorption repair.20 in the current case, there was no recurrence of tmj symptoms during the orthodontic treatment. although anterior disc displacement without reduction and condylar resorption and deformity persisted after treatment, all symptoms of tmj disease disappeared, and long-term stability of both the occlusal and symptomatic states was obtained. growing evidence suggests that in tmj-oa, similar to oa in other joints, overloading may initiate a series of degenerative changes, such as condylar resorption, decreased mandibular ramus height, mandibular clockwise rotation, progressive mandibular retrusion, and an anterior open bite. to date, many treatment modalities for tmj-oa have been reported; however, the treatment outcomes depend on the preoperative tmj conditions. therefore, understanding the pathogenesis of tmj-oa and its current clinical treatment is essential for developing a " good as new " treatment for tmj-oa, including the orthodontic approach. we showed here that orthodontic correction through molar intrusion using titanium miniscrews was effective for the management of an open bite and clockwise-rotated mandible associated with tmj-oa and jaw deformity.

this article reports the orthodontic treatment of a patient with skeletal mandibular retrusion and an anterior open bite due to temporomandibular joint osteoarthritis (tmj-oa) using miniscrew anchorage. a 46-year-old woman had a class ii malocclusion with a retropositioned mandible. her overjet and overbite were 7.0 mm and -1.6 mm, respectively. she had limited mouth opening, tmj sounds, and pain. condylar resorption was observed in both tmjs. her tmj pain was reduced by splint therapy, and then orthodontic treatment was initiated. titanium miniscrews were placed at the posterior maxilla to intrude the molars. after 2 years and 7 months of orthodontic treatment, an acceptable occlusion was achieved without any recurrence of tmj symptoms. the retropositioned mandible was considerably improved, and the lips showed less tension upon lip closure. the maxillary molars were intruded by 1.5 mm, and the mandible was subsequently rotated counterclockwise. magnetic resonance imaging of both condyles after treatment showed avascular necrosis-like structures. during a 2-year retention period, an acceptable occlusion was maintained without recurrence of the open bite. in conclusion, correction of open bite and clockwise-rotated mandible through molar intrusion using titanium miniscrews is effective for the management of tmj-oa with jaw deformity.

PMC3481975

pubmed-617

fisher used the expression crash migraines to describe acute, high-intensity headaches similar to those caused by ruptured saccular aneurysms, but with normal lumbar puncture and angiography. day and raskin coined the term thunderclap headache to refer to a similar type of headache occurring in a patient who had three severe, acute episodes of headache within 1 week. the brain ct scan and the cerebrospinal fluid (csf) examination were normal in this patient, while cerebral angiography showed diffuse, multifocal, and segmental cerebral vasospasm with a saccular aneurysm at the origin of the right posterior cerebral artery. the authors concluded that unruptured intracranial aneurysms can present with thunderclap headache and that angiography is necessary in patients presenting headache episodes with the thunderclap profile thunderclap headache (tch) is a severe and headache of the explosive type that appears suddenly, like a clap of thunder, with peak intensity of the pain occurring at onset (usually within 30 s). the international classification of headache disorders, second edition (ichd-ii, 2004) categorizes headaches into primary and secondary forms based on the absence or presence of intracranial lesions. this classification has included tch in group iv other primary headaches (code 4.6) (table 1) and has proposed precise diagnostic criteria . table 1 4. other primary headaches 4.1 primary stabbing headache 4.2 primary cough headache 4.3 primary exertional headache 4.4 primary headache associated with sexual activity 4.4.1 preorgasmic headache 4.4.2 orgasmic headache 4.5 hypnic headache 4.6 primary thunderclap headache 4.7 hemicrania continua 4.8 new daily-persistent headache (ndph) the nosography of most headaches included in group iv is not immediate. although some of them are simply identified according to their trigger it is mandatory to accurately rule out any possible organic cause (that is to exclude a possible secondary origin) when a patient with one of these provoked headaches is seen for the first time. indeed a recent publication shows that 18 of 30 patients with sexual headaches with an abrupt and severe headache which was similar to tch in most cases had reversible cerebral vasoconstriction syndrome (rcvs) (code 6.7.3 in ichd-ii). thunderclap headache is a rare type of headache with an incidence of 43 cases per 100,000 adults per year. primary tch closely mimics secondary forms of tch and therefore appropriate instrumental investigations are absolutely mandatory to rule out possible organic causes. the close similarity between primary and secondary tch has led to the hypothesis that the primary form may, in some cases, actually be a wrong diagnosis resulting from the incapacity of instrumental diagnostics to identify the organic cause; for this reason, the alternative term thunderclap headache of undefined origin has also been proposed. some authors showed that patients with recurrent tch can be divided in two groups based on the result of mra: those with diffuse vasospasm and those without any (visible) vasospasm. however, both groups had similar clinical features and both showed the same rate of ischaemic complications [67% of posterior reversible encephalopathy syndromes (pres) and of ischaemic stroke]. these authors therefore suggested that primary tch and rcvs may be part of the same spectrum.. showed that 21% of 67 patients with a proven rcvs had an initial normal mra and they would have been classified as primary tch in the absence of a second mra repeated after a few days later or of a conventional angiogram. furthermore, other authors showed that the arterial abnormalities of rcvs, as assessed by non invasive tools, increase several days after tch onset. indeed mean flow velocities of middle cerebral artery (mca) measured by transcranial doppler are maximal at 1825 days after tch onset, and mra vasoconstriction scores are maximal 16 10 days after headache onset, close to headache resolution at 16 9 days. therefore, it is possible that some of primary tchs are actually forms of rcvs whose arterial abnormalities went undetected by neuroimaging. the clinical and diagnostic overlapping is also reflected in the pathophysiological field where the proposed mechanisms for the so-called primary tch are vasospasm and autonomic dysfunction, which are also thought to underline rcvs. a certain degree of overlapping between tch and rcvs also exists as regards treatment because nimodipine shows beneficial effect both in the presence and in the absence of vasospasm. rcvs is a vascular disorder that is frequently, but by no means always, benign. stroke occurs in 515% of the prospective series [4, 9] and death has also been reported. thus, the clinical spectrum of rcvs is large and it ranges from isolated recurrent and self-limiting tch to life-threatening forms. severe forms of rcvs were previously misdiagnosed as benign forms of primary angiitis of the central nervous system (pacns), with good evolution and prognosis, and normalization of arterial irregularities after a short course of steroids. calabrese et al. first proposed that these patients had benign angiitis of the central nervous system (bacns) and later on recognized that bacns was equivalent to rcvs. although headache can recur within the first week after onset, it generally does not recur regularly over subsequent weeks or months. as regards the pathophysiology of primary tch the proximal portions of the large intracranial arteries are indeed innervated by neuropeptide y and noradrenaline-containing sympathetic afferents, which modulate vascular tone [13, 14]. it has been suggested that the head pain in tch may be due to acute vasoconstriction or alterations in vascular tone secondary to heightened sympathetic tone, and indeed there exist experimental and clinical data supporting a pivotal role of the sympathetic nervous system (e.g. tch attacks associated with hypertension or preceded by events associated with elevated sympathetic tone, such as episodes of anger, sexual intercourse, and exertion) [15, 16]. vasoconstriction per se does not seem to be the cause of pain, since in the cases of tch with rcvs it is a long-lasting phenomenon, which may persist for hours up or weeks, even when headache has already disappeared. it seems more likely that pain in tch results from a combination of vasoconstriction with systemic or local autonomic changes. in patients with sexual headache resembling tch associated with rcvs, additional factors that may contribute to pain induction are possibly represented by the psychological stress associated with sexual arousal and by the activation of systemic autonomic reflexes, which result in increased blood pressure and heart rate. as regards the pathophysiology of the arterial spasm, several factors encompassing mechanical stimulation, biochemical mediators, and neurogenic events vasoactive substances, i.e. ergotamine, amphetamine, cocaine may trigger it. alternatively, the formation of circulating metabolites (i.e. during eclampsia or pheochromocytoma) or the exposure to toxins (i.e. angiographic contrast material) are all possible causes for vasospasm. it is essential to remember that a diagnosis of primary or secondary tch can be made only when exhaustive instrumental investigations have been performed. to manage a patient with tch correctly, the clinician must be aware of all the organic disorders that can act as a causative factors. these are, first of all, subarachnoid haemorrhage (sah), but also cerebral venous sinus thrombosis, carotid artery dissection, hypertensive encephalopathy, spontaneous retroclival haematoma, sentinel headache, ischaemic stroke, pituitary apoplexy, spontaneous intracranial hypotension, colloid cyst of the third ventricle, intracranial infection, pacns. it is also worth noting that a headache with clinical features of tch has been described in conditions not associated with a structural abnormality, such as bathing headache, primary cough, sexual and exertional headaches, and rcvs. recently, some authors have proposed diagnostic criteria for tch attributed to idiopathic reversible cerebral vasoconstriction (tharcv) syndrome based on the clinical and radiological features. table 2 shows the diagnostic work up and the main criteria for the differential diagnosis of primary tch versus other disorders that can cause/be associated with tchs [1288]. table 2differential diagnosis of tchdiseaseneurological symptoms/signsprecipitating factorscommentssubarachnoid haemorrhage (sah) [1932]headachephysical exertionabout 70% of pts with sah present with headache aloneloss of consciousnesssexual intercoursefocal neurological symptomscerebral venous sinus thrombosis (cvst) [3342]headachepuerperium1530% of pts present with isolated headache that can worsen in the recumbent positionaltered consciousnessdehydrationfocal neurological symptoms/signscancercervical artery dissection (cad) [3, 4345]headachehead and neck injurygenerally headache is ipsilateral to the cadamaurosis fugaxhorner s syndromefocal neurological symptoms/signsacute hypertensive crisis (ahc) [4651]headachehypertensive crisisheadache occurs in about 20% of pts with ahcaltered mental statusseizuresfocal neurological symptoms/signsspontaneous retroclival hematoma (srh) [52, 53]headachenonesrh is very raremild nuchal rigidityoculomotor nerve palsyupper limb paresissentinel headache (sh) headachephysical exertionsh is present in 1040% of pts with sahfocal neurological symptoms/signs generally absentsexual intercourseischaemic stroke (is) [5558]headachenoneheadache is more common with large isfocal neurological symptoms/signspituitary apoplexy (pa) [22, 5860]headachepregnancypa commonly occurs in pts with no known pituitary tumour historyvisual disturbancesspontaneous intracranial hypotension (sih) [6164]orthostatic headachevalsalva manoeuvretch is present, at onset, in about 15% of pts with sihhearing disturbancesmild nuchal rigiditycolloid cysts of third ventricle [7375]headachenoneheadache can be relieved by recumbencyloss of consciousnessseizurescomareversible cerebral vasoconstriction syndrome (rcvs) [8, 6973]headachepostpartumrcvs is spontaneous in about 30% of casesfocal neurological symptoms/signssexual intercoursedrugs exposure (see bottom of table)prognosis is uncertain, but most pts do wellblood products (see bottom of table)head traumaneurosurgical proceduresbenign hot bath-related headache [7478]headachehot bathheadache disappears spontaneously after a period of 2 weeks to 3 monthsnormal neurological examinationhot showerprimary cough, headachecoughthese headache forms aresexual and exertional headaches [3, 79]normal neurological examinationphysical exertionan exclusion diagnosissexual activityprimary angiitis of the central nervous system (pacns) [72, 8083]headachenoneheadache is the most common presenting symptomseizuresbehavioural disturbancesfocal neurological symptoms/signsprimary thunderclap headache (tch) [3, 1215, 24, 26, 8489]headachenonetch is an exclusion diagnosis and has a relatively benign prognosisnormal neurological examinationdrugs exposure phenylpropanolamine, ergotamine tartrate, methergine, bromocryptine, lisuride, tricyclic antidepressants, selective serotonin reuptake inhibitors, sumatriptan, isometheptine, cocaine, ecstasy, amphetamine derivatives, marijuana, lysergic acid diethylamide, tacrolimus (fk-506), cyclophosphamideblood products erythropoietin, intravenous immune globulin, and red blood cell transfusions differential diagnosis of tch drugs exposure phenylpropanolamine, ergotamine tartrate, methergine, bromocryptine, lisuride, tricyclic antidepressants, selective serotonin reuptake inhibitors, sumatriptan, isometheptine, cocaine, ecstasy, amphetamine derivatives, marijuana, lysergic acid diethylamide, tacrolimus (fk-506), cyclophosphamide blood products erythropoietin, intravenous immune globulin, and red blood cell transfusions while defining the diagnosis, tch must always be managed as a medical emergency in order to avoid potentially catastrophic consequences that can occur with secondary tch. non-contrast brain ct is the first examination in this assessment, to be performed within the first 12 h after the onset of the headache, preferably using third-generation ct scanners that have a specificity of 98% and a sensitivity close to 100%. the sensitivity of ct for the detection of sah declines with increasing time from haemorrhage onset, falling to 86% on day one, 76% after 2 days, and 58% after 5 days. therefore, although the sensitivity of ct scan in detecting sah is very high in the early phase, if we consider also the possibility of human error, which, in the case of sah, may be fatal, we recommend to perform lumbar puncture even in the presence of a normal brain ct. csf assessment is definitely mandatory in patients who come to clinical attention 12 h after tch onset and have normal or non-diagnostic brain ct scans. blood and csf work up (routine cell counts, measurement of protein, glucose, opening pressure, and inspection for xanthochromia) should be performed. because visual inspection for xanthochromia is associated with a high rate of false-negative interpretations, spectrophotometry should be performed when available. spectrophotometry also helps to overcome the problem of false-positives; analysis for bilirubin by spectrophotometry has a sensitivity close to 100% when lumbar puncture is performed between 12 h and 2 weeks after sah. magnetic resonance imaging (mri), which can detect many of the possible causes of secondary tch, should be performed in all tch patients with normal or non-diagnostic ct scans and csf analysis. in most cases, magnetic resonance studies should include cerebral mri, magnetic resonance angiography, magnetic resonance venography and, if necessary, mri of the cervical arteries using the fat saturation technique. ct angiography can be used instead of magnetic resonance angiography for the diagnosis of an intracranial aneurysm. evidence suggests that conventional cerebral angiography is not necessary in the assessment of patients with tch, normal neurological examinations, and normal ct and lumbar puncture. some authors believe that conventional cerebral angiography should be avoided, since the contrast medium could enhance the vasospasm and this, in turn, would increase the chance of a stroke or even cause further deterioration of a critical neurological condition. however, since there is no adequate evidence to conclude that conventional angiography can result in worsening vasoconstriction, in highly selected cases, when the clinical suspicion of intracranial aneurysm remains high despite normal or non-diagnostic ct, lumbar puncture, and mri studies, conventional angiography has to be considered. the following indications have been derived from the information collected from a systematic analysis of the international literature. we conducted a literature search covering the period 19322010, employing available electronic databases (national library of medicine, national institute of health, embase) with the following medical search terms: thunderclap, cough, exertional, exercise, orgasmic, sex, or abrupt in association with unfortunately, the literature contains very few reports on the treatment of primary tch and those that are available are mostly represented by single case reports., a woman with an apparent primary tch had frequent recurrences of the headache until she reached (by day 14) a therapeutic dosage of gabapentin 600 mg three times a day. the exact mechanism of action through which gabapentin decreases headache pain is not known, although multiple mechanisms might be involved. gabapentin enhances gaba-mediated inhibition, inhibits gaba metabolism and modulates l-type calcium channels by binding to their 2 subunit. iv nimodipine and magnesium were administered to a 63-year-old woman with tch and vasospasm; a posterior ischaemic infarct had occurred before infusion. the patient s headaches resolved within hours, and transcranial doppler sonography revealed normalized mean cerebral blood flow velocity, suggesting relief of the vasospasm. the mean cerebral blood flow velocity increased again when oral nifedipine, another calcium channel blocker, was used in place of nimodipine. nimodipine infusion effectively stopped tch in another woman, aged 58 years, who had vasospasm and posterior ischaemic infarct. described 11 patients with primary tch (nine without vasospasm and two with vasospasm) treated with nimodipine. in eight of the nine patients without vasospasm, tch stopped within 24 h of oral nimodipine administration; the other patient had one further attack 2 days later. the highest dosage of nimodipine was 30 mg every 4 h in four patients, 45 mg every 4 h in one, and 60 mg every 4 h in four. in the two patients with arterial vasospasm, oral nimodipine was only temporarily effective and tch recurred. when iv nimodipine was given instead, tch subsided in 6 h without recurrence. in one of the patients, the dose infused was 2 mg/h; in the other, it was lower (0.51 mg/h) because of the appearance of nimodipine-induced hypotension. the iv route was switched to oral when mr angiography or transcranial doppler sonography no longer showed evidence of vasospasm. the duration of iv nimodipine treatment was variable, ranging from 5 to 10 days. no patient reported a relapse of tch during a mean 6-month follow-up (range 119 months) after nimodipine discontinuation. these reports suggest that iv nimodipine is the drug of choice for primary tch with cerebral vasospasm, whereas oral nimodipine can be used in patients without vasospasm, although the optimal dose and time window remain to be determined. it is noteworthy that the patients with tch associated with vasospasm described in the above reports [8789], the correct diagnosis should be rcvs rather than tch, while only the nine without vasospasm described by lu et al. can technically be classified as having primary tch, although with a certain degree of approximation since one can not exclude a rcvs without visible vasoconstriction at the time of angiogram. therefore, these nine subjects can be regarded either as probable rcvs or probable primary tch. following this line of reasoning, if we consider that, in some cases, rvcs may be missed by mra because of timing issues, it seems wise to avoid tricyclic antidepressants (e.g. amitriptyline) and propranolol because they may facilitate the development of vasoconstriction as suggested by valenca et al.. in analogy, vasoconstrictor medications, such as ergots and triptans, should be contraindicate during the treatment of headache of patients with potential cerebral vasoconstriction syndromes, at least in the acute phase and until ongoing or impending rvcs has been ruled out. all patients with tch profiles must be assessed urgently and thoroughly in order to consider and rule out all the possible organic causes. after the exclusion of all secondary causes, including rcvs, there are very few patients left with true primary tch. the data gathered in recent years suggest that, in order to avoid false primary tchs, it is appropriate to perform neuroimaging studies (brain mri angiography or ct angiography) 34 weeks after the onset of all cases of tch with negative instrumental findings during the acute phase in order to exclude the presence of delayed vasospasm [10, 11] and therefore identify the true primary tchs. once the diagnosis has been defined with certainty, secondary forms of tch must be managed through treatment of the underlying brain disorder. for primary tch, as well as for forms associated with rcvs, the therapeutic options are restricted to nimodipine, intravenously or orally administered, although gabapentin was reported effective in one case of primary tch .

thunderclap headache (tch) is an excruciating headache characterized by a very sudden onset. recognition and accurate diagnosis of tch are important in order to rule out the various, serious underlying brain disorders that, in a high percentage of cases, are the real cause of the headache. primary tch, which may recur intermittently and generally has a spontaneous, benign evolution, can thus be diagnosed only when all other potential underlying causes have been excluded through accurate diagnostic work up. in this review, we focus on the management of tch, paying particular attention to the diagnostic work up and treatment of the condition.

PMC3072477

pubmed-618

electronic health records (ehrs) are increasingly used by medical providers and offer a wide-reaching source of information on utilization of preventive services. numerous measures used for quality assessment and public reporting are estimated based on ehr data. however, sources of error and misclassification can lead to over- or underestimation of true utilization rates. ehr-derived measures of screening test use are subject to error due to misclassification of screening and diagnostic tests. the implications of this misclassification for ehr-based screening utilization estimates have not been well explored. we calculated the bias in estimates of screening test utilization associated with several published ehr-based algorithms for identifying screening colonoscopies and propose two simple methods to correct this bias. we apply these corrections to obtain adjusted estimates of screening colonoscopy utilization using ehr data from group health, an integrated health care system in washington state. the bias in screening colonoscopy utilization estimates ranged from an underestimation of 3 percentage points to an overestimation of 12 percentage points across classification methods. if the operating characteristics of the classification method are known or if a statistical model that returns predicted probabilities of screening indication is applied in the population of interest, this information can be used to obtain unbiased estimates through simple corrections to the utilization rates with little loss of precision. when applied to data on colonoscopies received at group health, we found that an unadjusted estimate was 4 percentage points higher than our adjusted estimate. error in classification of tests as screening when using ehr data to study screening utilization should be accounted for in order to eliminate bias and prevent spurious findings. electronic health records (ehr) data, including administrative data used for billing and clinical medical records collected as part of patient care, are potential sources of information on a wide range of quality measures. these data include information on a broad patient population and facilitate research and quality assessment that is representative of care in community medical practice. ehr data will become increasingly available and valuable as adoption of electronic systems expands, a process that has been accelerated by incentives for health it provided under the affordable care act. rates of breast, cervical, and colorectal cancer screening are examples of measures of preventive services utilization that are commonly estimated using ehr data. for instance, cancer screening measures estimated via ehr data are included among the national committee on quality assurance s healthcare effectiveness data and information set (hedis) measures,1 and have also been tied to reimbursement through incentives mandated by the affordable care act and the centers for medicare and medicaid services (cms) hospital outpatient quality reporting program.2 estimating these measures using ehr data is preferable to self-report of screening test utilization due to the possibility of bias in estimates based on self-report.3,4 use of ehr data for evaluating screening utilization is also far more efficient than manual chart review. despite the potential value of ehr data for estimating screening test utilization, obtaining accurate estimates is challenging for several reasons. for instance, the national quality forum (nqf) colorectal cancer screening measure is an estimate of the proportion of individuals ages 5075 years who have been screened with colonoscopy within the past 10 years, flexible sigmoidoscopy within the last 5 years, or fecal occult blood test within the past year.5 an accurate assessment of this outcome requires reliable measures of the number of individuals in the screening-eligible population and the number of individuals receiving screening with each of the tests of interest. identifying individuals in the screening-eligible population is challenging because individuals may have recently entered the health system population, making it difficult to determine if they have pre-existing diagnoses that should result in their exclusion from the eligible population. similarly, identifying screened individuals is challenging because new enrollees may have been screened prior to enrollment in the health system. additionally, there are no unique codes for screening as distinguished from diagnostic colonoscopy. including diagnostic colonoscopies in estimates of screening utilization this may obscure important information about access to and utilization of cancer screening services and may produce biased or spurious findings regarding disparities in screening if there is differential misclassification across patient subgroups. the extent of the error in estimates of cancer screening utilization based on ehr data attributable to misclassification of screening indication depends on the operating characteristics of methods used to classify cancer screening tests. several previous studies have estimated the operating characteristics of approaches to distinguishing screening and diagnostic tests using administrative ehr data.68 while these studies have focused on the use of administrative ehr data, similar considerations apply to studies using data from clinical encounters, if the classification approaches in question produce measures of screening indication that have errors. in the context of breast cancer screening, several algorithms have been developed for classifying mammograms as screening or diagnostics using diagnosis and procedure codes from claims data.6,9,10 a number of algorithms have also been developed for identifying screening colonoscopies based on ehr data with varying operating characteristics.7,1113 the sensitivity of these approaches ranges from 70 to 90 percent and specificity from 60 to 90 percent. a natural question arises: how might estimates of screening test utilization based on ehr data be influenced by misclassification of screening and diagnostic examinations? in this paper we explore the implications of using ehr data with imperfect information on screening indication to estimate rates of screening test utilization. we use numerical examples and simulations focusing on screening colonoscopy to demonstrate the magnitude of bias that can be induced by using an imperfect measure of screening indication. we then demonstrate via simulation studies how to correct this bias using information on the operating characteristics of the algorithm used to assign screening indication or a predicted probability that the test was performed for screening purposes. finally, we demonstrate the application of unadjusted and adjusted measures of screening colonoscopy utilization using ehr data from group health, an integrated health plan and health care delivery system in washington state. to illustrate the bias arising from imperfect ascertainment of screening-test utilization we introduce the following notation. let y represent the true classification of the test with y=1 indicating a screening test and y=0 indicating a diagnostic test. we assume that y is unobserved in ehr data but that a proxy is available, denoted y. for instance, in the case of colonoscopy, procedure codes do not distinguish colonoscopy performed for screening versus colonoscopy performed to evaluate signs or symptoms (i.e., a diagnostic exam). however, by applying existing algorithms to administrative data it is possible to obtain a predicted probability of screening indication that can then be dichotomized to yield an indicator that the examination was a screening test. in this example the operating characteristics of y are the sensitivity, se=p(y=1|y=1), and specificity, sp= p(y=0|y=0). in studying utilization of cancer screening tests, our objective is to estimate the proportion of the population receiving a screening test, p(y=1). if we use instead p(y=1) as a measure of screening utilization, the difference, p(y=1) we can express this bias as a function of the sensitivity and specificity of y, p(y=1) p(y=1)=se it can be easily seen that for a test with perfect operating characteristics (sensitivity and specificity equal to 1), p(y=1) will be unbiased. the relative impact of sensitivity and specificity on bias will depend on p(y=1), which is unknown in practical applications. in table 1, we illustrate the bias in estimates of screening-colonoscopy utilization when using three different algorithms for identifying screening examinations. previously, self-report data have been used to estimate screening-colonoscopy utilization.14,15 however, ehr data offer the opportunity to evaluate utilization in a broad population without the bias inherent in self-report. motivated by estimates of lower endoscopy utilization,15 we assume that the true proportion of individuals who have been screened with colonoscopy in the past 10 years is 58.5 percent. in this setting, bias in screening-colonoscopy utilization ranges from underestimation by 5.7 percentage points to overestimation by 11.6 percentage points. in this section we present two methods for correcting the bias in estimates of screening utilization based on imperfect ehr-based algorithms for assigning screening indication. the first relies on the existence of validation studies that have established the operating characteristics of the classification algorithm in the population of interest. we can use the known sensitivity and specificity of the algorithm for classifying tests as screening to correct estimates through direct algebraic manipulation of the formula for bias, providing a simple approach for obtaining unbiased utilization estimates. specifically, a corrected formula for screening-test utilization is given by (p(y=1)+sp1)/(se+sp1). utilization is first estimated using algorithm-assigned screening indication, providing an estimate of p(y=1). this estimate and the estimated sensitivity and specificity of the algorithm are then substituted into the expression above to produce a bias-corrected estimate of p(y=1). the second approach to correcting estimates of screening utilization requires an algorithm that assigns individual-level probabilities of screening indication. some methods for identifying screening tests return a predicted probability that a test was used for screening purposes. for instance, in the context of colorectal cancer screening, a lasso algorithm has been proposed that provides the probability that a given colonoscopy was a screening test.8 lasso is a statistical prediction model that combines variable selection and parameter estimation under a constraint on the size of the regression coefficients.16 assuming that these probabilities are well calibrated, summing the predicted probabilities of screening indication in the patient population of interest provides an unbiased estimate of the number of screening tests performed in the population. a well-calibrated probability implies that the proportion of tests with screening indication is equal to the predicted probability that a test has screening indication. for instance, among all tests with predicted probability of screening indication equal to 0.2, 20 percent will truly be screening tests if the prediction model is well calibrated. if the algorithm was developed in a different population, it may not be well calibrated and might over- or underestimate the probability of screening indication in a new population. given a well-calibrated predicted probability, the utilization rate can be estimated without bias by taking the mean of the predicted probabilities in the population of interest. to demonstrate the bias and precision resulting from using uncorrected classifications of screening indication to estimate utilization as well as performance of the two proposed approaches to bias correction, we conducted a simulation study motivated by the context of screening with colonoscopy. in this study we assumed a population of size 10,000 and that 59 percent of this population were screened at least once over a 10-year period. we assumed that predicted probabilities of screening indication in this population arose from a beta(0.25, 0.17) distribution. this distribution implies that the cutpoint maximizing the average of sensitivity and specificity has a sensitivity of 0.89 and a specificity of 0.90. these parameter values were selected to create a scenario similar to the adams algorithm for classifying screening colonos-copies.8 for each simulated individual, both the true classification of the test as screening or diagnostic and the algorithm-assigned predicted probability were known. we first computed an uncorrected utilization estimate by dichotomizing the predicted probability at the value maximizing the average of sensitivity and specificity and then computing the proportion of individuals classified as having received a screening examination. we then computed the two bias-corrected estimates, first by applying the correction factor presented above and then by computing the mean of the predicted probabilities. we repeated this process across 10,000 simulated populations and plotted the distribution of screening-test utilization estimates provided by each approach in figure 1. in these simulated populations, the approach directly using the algorithm-assigned classification without adjustment underestimated screening utilization by 2.5 percentage points. both of the bias-corrected approaches had a bias of less than 0.01 percentage points. the uncorrected method had a standard error of 0.5, the direct adjustment method of 0.6, and the mean probability method of 0.4 percentage points. to demonstrate the application of an adjusted utilization approach in a real data set derived from an ehr, we used data on receipt of colonoscopy from group health, an integrated health plan and health care delivery system in washington state. because sensitivity and specificity in this population are unknown we used the mean probability method to obtain adjusted estimates. we constructed a sample consisting of members ages 50 years or older who were continuously enrolled in group health for at least 5 years between 2002 and 2012 with no prior diagnosis of colorectal cancer. in this population, we estimated the proportion of members receiving a screening colonoscopy at least once during a 5-year period. we used current procedural terminology (cpt) codes (45355, 4537845387, 45391, 45392), healthcare common procedure coding system (hcpcs) codes (g0105, g0121) and international classification of diseases ninth revision, clinical modification (icd-9-cm) codes (45.2145.24, 45.25, 45.42, 45.43) to identify each colonoscopy received by a member of the denominator population. we then applied the algorithm of adams8 to each colonoscopy to obtain a predicted probability that the colonoscopy was a screening examination. if an individual received more than one colonoscopy during this time frame we retained the colonoscopy with the highest probability of screening indication we first dichotomized the predicted probabilities at a threshold of 0.261, the threshold maximizing the average of sensitivity and specificity in a validation study,8 to obtain a binary indicator of receipt of screening colonoscopy. we then computed an unadjusted measure corresponding to the proportion of individuals with a screening colonoscopy based on this binary indicator. finally, we computed an adjusted measure by taking the mean of the predicted probabilities. this approach is appropriate for accounting for misclassification in identification of screening tests when the sensitivity and specificity of the algorithm are unknown in the target population. in this population of 139,163 individuals, we compare this to an estimate of 7.8 percent based on averaging the predicted probabilities. thus, the unadjusted approach appears to substantially overestimate screening-colonoscopy utilization in this population. estimates of screening-test utilization based on algorithms for assigning screening indication with imperfect operating characteristics will be biased. this bias leads to under- or overestimation of the proportion of the population making use of a screening test. in the case of underestimation this could lead to unnecessary efforts to improve utilization, and in the case of overestimation this could lead to failure to address the needs of underserved populations. the magnitude and direction of the bias depend on the sensitivity and specificity of the ehr-based algorithm as well as the prevalence of screening utilization in the population of interest. we have demonstrated two simple approaches to correcting bias, which can easily be applied to ehr data, and implemented an adjusted approach using ehr data from group health. these methods require that either the operating characteristics of the algorithm are known based on a prior validation study or that the algorithm returns predicted probabilities of screening indication and is well calibrated in the population under study. we recommend that one of these two approaches be used to correct utilization estimates whenever possible. use of these approaches is particularly well suited to ehr data because the depth of information available via data from clinical encounters facilitates validation studies for evaluation of the discrimination and calibration of the measure in a small sample of the target population. the measure and appropriate methods for correcting for misclassification can then be rapidly applied to a broad population using administrative ehr data. in this study, we focused on estimation of screening-test utilization when screening indication is imperfectly ascertained. however, there are a variety of other uses for ehr-derived data on cancer screening that rely on accurate ascertainment of test indications. for instance, estimates of screening-test effectiveness including the cancer stage at diagnosis and the mortality among screened and unscreened individuals may be of interest. misclassification of screening and diagnostic tests will bias these measures of screening-test effectiveness. several previous studies have discussed considerations for using algorithm-assigned outcomes and exposures in studies based on ehr data.17,18 these provide guidance on the relationship between algorithm operating characteristics and bias in various measures of effectiveness and can be used in conjunction with the current study to guide the choice of algorithm and analysis strategy when using data with known error in screening indication. calibration and discrimination of the ehr-based measure are key to obtaining accurate estimates. if the measure is not well calibrated all three of the approaches discussed in this paper will return biased estimates. we recommend using a validation sample, in which both clinical and administrative data are available, to evaluate the calibration of any screening indication algorithm before it is applied broadly to ehr data. additionally, an algorithm with poor discrimination (sensitivity and specificity) will lead to biased estimates and may inflate the standard errors of the estimates. the direct adjustment method, in particular, will have inflated standard errors if its denominator, se+sp 1, is close to 0. this will occur in the case of a measure where the sum of sensitivity and specificity is close to 1, which would occur, for instance, if both cases and controls are correctly classified only about 50 percent of the time. if each site uses a different approach to classifying screening indication or if a common approach is used that has differing operating characteristics across study sites, then differential bias may lead to the spurious appearance of variability in screening utilization across sites. if patient characteristics differ across sites this could also lead to the appearance of associations between patient characteristics and utilization that are entirely attributable to bias. in the case of a multisite study the classification approach should be validated at each study site allowing for bias correction at the study site level. similarly, if operating characteristics vary across patient subgroups this could result in apparent variation in utilization across subgroups where none truly exists or, conversely, might obscure true variation in utilization. to protect against this bias, validation studies within patient subgroups of interest must be conducted to provide estimates of operating characteristics within each population or to demonstrate that algorithm-assigned probabilities are well calibrated in each group. as use of electronic medical records expands, ehr data will become increasingly valuable as a source of information on screening test utilization. the considerations provided here allow for evaluation of the bias that will occur if algorithms for screening indication are used without correction for potential misclassification of screening tests as diagnostic tests. we have provided simple approaches to correct this bias that can be readily implemented and have demonstrated their potential for eliminating bias without inflating standard errors. the need to classify tests as screening or diagnostic is not unique to the setting of cancer screening using ehr data but exists across a broad range of prevention measures including use of tests for depression, high cholesterol, and osteoporosis. moreover, the considerations described here do not apply solely to misclassification of diagnostic and screening tests but are relevant broadly to measures that rely on imperfect ascertainment of the service of interest. error in ascertainment of utilization due to misclassification should be considered and appropriate adjustment methods such as those proposed in this paper should be applied to avoid bias.

background: electronic health records (ehrs) are increasingly used by medical providers and offer a wide-reaching source of information on utilization of preventive services. numerous measures used for quality assessment and public reporting are estimated based on ehr data. however, sources of error and misclassification can lead to over- or underestimation of true utilization rates. ehr-derived measures of screening test use are subject to error due to misclassification of screening and diagnostic tests. the implications of this misclassification for ehr-based screening utilization estimates have not been well explored. objectives:we calculated the bias in estimates of screening test utilization associated with several published ehr-based algorithms for identifying screening colonoscopies and propose two simple methods to correct this bias. we apply these corrections to obtain adjusted estimates of screening colonoscopy utilization using ehr data from group health, an integrated health care system in washington state. findings:the bias in screening colonoscopy utilization estimates ranged from an underestimation of 3 percentage points to an overestimation of 12 percentage points across classification methods. if the operating characteristics of the classification method are known or if a statistical model that returns predicted probabilities of screening indication is applied in the population of interest, this information can be used to obtain unbiased estimates through simple corrections to the utilization rates with little loss of precision. when applied to data on colonoscopies received at group health, we found that an unadjusted estimate was 4 percentage points higher than our adjusted estimate.discussion:error in classification of tests as screening when using ehr data to study screening utilization should be accounted for in order to eliminate bias and prevent spurious findings.

PMC4346157

pubmed-619

laparoscopic surgery (ls) for both benign and neoplastic colonic disease has become a standard procedure worldwide [18], although its distribution is currently limited. many authors reported adequacy and short-term benefits also for laparoscopic rectal procedures [1013]; nevertheless, large randomized studies and oncologic results are still lacking. in recent years, innovative endoscopic procedures such as single-port laparoscopic surgery (sils), natural orifices transluminal endoscopic surgery (notes), and needlescopic surgery (ns) have been introduced to further reduce surgical invasiveness and abdominal wall trauma. this goal has been achieved by reducing the number of ports (sils), avoiding transabdominal incisions (notes), or reducing port size (ns). this should possibly reduce postoperative pain and lower the incidence of wound infections and port site hernias, besides improving cosmetic results. notes has been performed mainly on experimental models [17, 18], and its application in clinical environment is very limited [19, 20]. several attempts with single-port technique have been made for various procedures, including appendectomy, cholecystectomy, splenectomy, inguinal hernia repair, and in paediatric, gynaecologic, and urologic surgery; few preliminary experiences are available also for colorectal surgery [2846]. likewise, ns has been gradually introduced in the aforementioned surgical fields, with some preliminary results also in colorectal surgery [4755]. the main drawback of sils is the loss of triangulation of surgical instruments in the operative field, which despite recent development of curved instruments and flexible endoscopes enhances technical difficulty and requires a long learning curve. needlescopic technique keeps port positioning unchanged compared to standard laparoscopic procedures and therefore has minimal impact on the surgeon. since reports are limited in this field, we aim to review technical points such as instrumentation and its use in the different steps of the operation. in our practice, laparoscopic colorectal resections are currently performed with a 3- to 5-port (512 mm size) technique, intracorporeal anastomosis whenever possible, and specimen extraction through a suprapubic transverse incision. laparoscopic instrumentation consists of 30 scope, atraumatic graspers, coagulating hook, bipolar grasper, clip applier, ultrasonic dissector (optional), suction device, retractor, needle holder, and linear stapler. apart from the clip applier, the ultrasonic dissector, and the stapler, all instruments are available in 3 mm size (figure 1) still keeping a high standard of quality and performance. only 3 mm laparoscopes, although providing a good vision, are still less performant than 5 mm hd scopes which may be preferable in advanced laparoscopic procedures. since a minilaparotomy is always planned, open access with a hasson port may be performed at the suprapubic site allowing introduction of 1012 mm devices. further trocars ranging from 3 to 5 mm size are placed after insufflation under direct vision. port positioning for minilaparoscopic left colectomy is shown in figure 2. after placement of the 12 mm hasson port at the site of the planned minilaparotomy, one 5 mm port is inserted through the umbilicus for the scope, and two 3 mm ports are placed in the right hypochondrium on the midclavicular line and in the right lower quadrant. such position allows good triangulation in order to work between the left hypochondrium and the pelvis. an additional 3 mm port may be placed in the left lower quadrant for the surgeon to switch hands and improve triangulation during mobilization of the splenic flexure or dissection of the lower rectum. when in place, this port may be used by the assistant for additional grasping or to expose the operative field with a retractor when working in the pelvis. a standard medial to lateral approach clips for vascular ligation are inserted through the 12 mm suprapubic port (figure 3). mobilization of the splenic flexure may be performed indifferently as a first step or before bowel section. dissection is performed with the 3 mm coagulating hook; should the ultrasonic dissector be used, the 3 mm port in the right and/or left lower quadrant is to be replaced with a 5 mm port. three mm instruments allow fine grasping of elements such as vessels and peritoneum, but care must be taken during lifting of the mesocolon as the small contact surface may result in the tearing of the vessels which need to be preserved; it is therefore advisable to interpone a sponge (inserted through the 12 mm port) between the grasper and the tissue to be handled. similarly, since mesorectal integrity is of utmost importance during total mesorectal excision in rectal cancer surgery, grasping of the mesorectal fascia with small instruments is to be avoided, and a wad of gauze held by the grasper should be used to expose the holy plane (figure 4). if a stronger retraction is needed to achieve dissection of the lower rectum or in case of bulky tumours in obese patients, a 10 mm retractor may be introduced through the 12 mm suprapubic port. the same port is used to place the linear stapler and transect the rectum at any level down to the pelvic floor (figure 5). after specimen retrieval, the suprapubic minilaparotomy is closed leaving in place the 12 mm port which may be useful for extraction of the staple trocar, anterior retraction during confection of low colorectal anastomosis, and introduction of sutures if the peritoneum is to be closed. alternatively, the suprapubic minilaparotomy may be performed as a first step of the operation and sealed temporarily with a device which allows air-tight placement of a 12 mm port. at the end of the procedure, the ports are removed under vision to check eventual bleeding, and the 12 mm port is extracted at last. the 12 mm hasson port is inserted above the pubis using the open technique, and two additional ports are placed under vision: one 5 mm port is placed in the left lower quadrant for the introduction of the scope and one 3 mm port in the left hypochondrium on the midclavicular line. such position allows good triangulation when working in the right abdomen and on the middle transverse colon. the use of the ultrasonic dissector requires a 5 mm port in the left upper quadrant. an optional 3 mm port may be placed in the right hypochondrium to allow grasping and retraction by the assistant (figures 6 and 7). the clip applier and linear stapler are introduced through the 12 mm port. after completing the mobilization and the bowel transaction, a double enterotomy is performed in the distal ileum and transverse colon, and a stapled side-to-side isoperistaltic anastomosis is performed. due to the direction of the linear stapler introduced through the suprapubic port, the visceral stumps must be correctly oriented using one or two traction sutures held by graspers. the anastomosis is completed with a running suture, and the ileal mesentery and transverse mesocolon are approximated. five and 3 mm ports are retrieved under vision, and the specimen is extracted via a suprapubic incision. laparoscopy has been widely proven to be a feasible, safe, and effective technique to perform colorectal resections [1, 2, 5661] leading to clinically relevant advantages in selected patients such as reduction of postoperative pain [1, 62] and complications, shortening hospital stay and improving recovery [1, 58, 63], wound healing [1, 64], and cosmesis [65, 66]. moreover, minimally invasive surgery has facilitated the application of enhanced recovery programs in colorectal surgery [6769]. long-term outcome of laparoscopic colonic resection for cancer is not different from what has been achieved by open surgery procedures. therefore, some authors suggest that laparoscopy should be the preferred technique to perform colectomy in patients suitable for this approach. new trends have been developed in order to further reduce the impact of surgical procedure in patients undergoing colorectal resections. three main directions have been undertaken in specialized centres: sils, which aims to the reduction of port number, notes, in which surgical instruments are inserted in hollow organs trough natural openings, and minilaparoscopic colorectal surgery, based on reduction of port size. sils was first described by piskun and rajpal for cholecystectomy as early as 1999; this term currently identifies surgical procedures that provide the placement of one port having three or more working channels within the umbilicus. surgeons who perform single-port colorectal surgery seem to agree that this technique, though should be suitable for the resection of colon cancer with respect to oncologic principles, is demanding because of the difficulties of exposure of the operative field and because of the risk of crowding while maneuvering laparoscopic instruments, although specially designed for this purpose.: this term currently identifies surgical procedures that provide the placement of flexible endoscopic systems through natural orifices (per-oral, transvaginal, transanal, transumbilical, or transvesical routes) entering the peritoneal cavity through an incision of hollow organs and approaching target organs to perform intra-abdominal procedures. many procedures ranging in complexity from cholecystectomy to colorectal resections may be theoretically performed entirely endoscopically without the need for abdominal incisions [70, 71]. the advantages of such an approach include absence of incisional pain and wound complications (including infection and hernias), improved cosmetic results, and faster recovery. although studies have shown the feasibility of an notes approach, significant constraints have been identified with the use of a flexible endoscopy platform, including a relative inability to apply off-axis forces, mechanical stability, inadequate triangulation, and limits in passing multiple instruments simultaneously into the peritoneal cavity. concerns have also been expressed about the risk of postoperative leak and infections: with the intestinal closure systems currently adopted for notes access sites, it is doubtful that 100% safety can be achieved. at present, the need for improved technology remains a major limitation for sils and notes. the use of smaller ports to perform laparoscopic procedures is defined with different terms such as minilaparoscopy, microlaparoscopy, miniendoscopic or microendoscopic surgery, and microinvasive surgery. in general, ns is the term used to describe ls with instruments with an external diameter of 2-3 mm, as defined by gagner and garcia-ruiz. santoro et al. have defined miniendoscopic surgery as any procedure that uses endoscopic instruments and optics 5 mm in diameter or smaller. needlescopic colorectal surgery is feasible, effective, and easy to perform since no specific training is required. surgeons who experienced ns in the aforementioned surgical fields [4755] report several advantages over standard ls. in general, reduction of laparoscopic port size is associated with limited trauma on the abdominal wall. smaller incisions result in decreased incisional pain and reduced risk of complications such as port-site bleeding, infection, and herniation. moreover, minimal scarring allows better cosmetic results. on the other hand, narrow operative field, lower image quality due to lack of definition and reduced light transmission [16, 74], and blurred vision with the use of electrocautery achilles ' heel of this technique and cause more stress for the surgeon especially when using 3 mm scopes. the use of modern 5 mm optics with high-definition cameras and powerful light sources is much more comfortable in performing advanced laparoscopic procedures, though a 3 mm optic inserted through an ancillary port may be useful if the 5 mm port is to be used for a larger instrument such as the clip applier. as for smaller instruments, they may show a weaker grasping capability and a lack of tensile strength due to increased flexibility, particularly in the presence of fibrosis or inflammation. manipulation of tiny laparoscopic instruments may result in an increased risk of tissue damage during dissection [16, 74, 7679]. apart from these precautions, moving from standard laparoscopic technique to needlescopic colorectal resections is not to be considered as approaching a new technique but simply an adaptation of a well-established practice and does not require a long learning curve. none of the steps of the operation has shown difficulties resulting from the use of miniaturized instruments. a good exposition of the surgical field has been always achieved during vessel ligation and viscera dissection, transection, and anastomosis. building on the experience gained from needlescopic procedures such as cholecystectomy and appendectomy, we decided not to give up the greater definition provided by 5 mm scopes, since the 3 mm optics are still less performant for more advanced and complex procedures. the 3 mm grasper has been shown to provide good traction, also during gentle dissection. we used a simple trick to overcome its aforementioned limits: a wad of gauze held within the jaws of the instrument itself was used for lifting and retracting viscera in order to increase its strength and decrease the risk of injury of other organs. one aspect that has been reconsidered performing needlescopic colorectal surgery is the position of trocars: we thought it would be logical to incorporate the only 12 mm port that must necessarily be placed for the introduction of the stapler in the minilaparotomy which is generally a transverse suprapubic incision; we therefore started introducing the stapler from a suprapubic port not only for low rectal resection but also to transect the upper rectum and transverse colon. the use of the stapler from the suprapubic port did not result in substantial differences in bowel transection. nevertheless, performing an intracorporeal side-to-side mechanical ileocolic anastomosis from the suprapubic port requires wider mobilization of the transverse colon in order to place it parallel to the stapler. approximation and orientation of the ileal and colonic stumps is best achieved by pulling on two stitches placed at each end of the anastomosis, the proximal one being held by the 3 mm grasper in the right hypochondrium and the distal one passing through the 12 mm suprapubic port. the 3 mm grasper in the right hypochondrium is also useful during hand suturing of the enterotomies. finally, attention must be paid when maneuvering 3 mm instruments, which must be done under direct vision throughout the operation. our experience suggests that in well-trained hands and for properly selected patients, ports can be reduced in size safely without a negative impact on the surgeon's ability to perform laparoscopic colorectal resections. these findings should promote a larger prospective randomized comparison with conventional laparoscopy to determine whether this refinement of laparoscopic colorectal surgery confers concrete and incontrovertible benefits to the patients.

laparoscopic colorectal resections have been shown to provide short-term advantages in terms of postoperative pain, general morbidity, recovery, and quality of life. to date, long-term results have been proved to be comparable to open surgery irrefutably only for colon cancer. recently, new trends keep arising in the direction of minimal invasiveness to reduce surgical trauma after colorectal surgery in order to improve morbidity and cosmetic results. the few reports available in the literature on single-port technique show promising results. natural orifices endoscopic techniques still have very limited application. we focused our efforts in standardising a minilaparoscopic technique (using 3 to 5 mm instruments) for colorectal resections since it can provide excellent cosmetic results without changing the laparoscopic approach significantly. thus, there is no need for a new learning curve as minilaparoscopy maintains the principle of instrument triangulation. this determines an undoubted advantage in terms of feasibility and reproducibility of the procedure without increasing operative time. some preliminary experiences confirm that minilaparoscopic colorectal surgery provides acceptable results, comparable to those reported for laparoscopic surgery with regard to operative time, morbidity, and hospital stay. randomized controlled studies should be conducted to confirm these early encouraging results.

PMC3323854

pubmed-620

asthma affects an estimated 8.9% of usa children under 17 years and continues to be one of the most common childhood chronic illnesses. uncontrolled asthma is associated with more school days missed among children, more work days missed among caregivers, and poorer quality of life among both [2, 3]. a special case of poor asthma control, nighttime awakenings from asthma, have been linked to school absences, lower school performance, and parents ' lost workdays. the national heart, lung, and blood institute (nhlbi) guidelines provide several recommendations for proper asthma management to minimize uncontrolled asthma. these guidelines include: use of pharmacologic therapy, patient education, reduce environmental triggers, and assess and monitor asthma control. the guidelines emphasize the importance of using a collaborative approach between providers, parents, and children to develop an appropriate asthma management plan for the child. however, recent studies have found that these guidelines are not being met, with less than half of families ever receiving any education about their child's asthma [7, 8]. in another study, most hospitalizations for asthma attacks were found to be preventable and had medications been taken regularly. prior research indicates that asthma patients who report poor communication with their physicians are less adherent with inhaled steroids [10, 11]. few studies have examined how physicians communicate about medications during medical visits using actual communication data and to our knowledge, no prior study has investigated how physicians communication about control medications during pediatric asthma visits [1215]. findings from these prior adult studies suggest that communication about medications can be improved. in a sample of 40 veterans who were on continued or newly prescribed antidepressants, providers asked 6% of patients about adverse events and 15% of patients how well the antidepressants were working. moreover, providers only gave 10% of patients ' information about adverse events and 5% information on how well the medication works. young et al. used standardized patients (n=131) and found that physicians provided information about side effects to 85% of patients but only gave information about how well the drug works to 38% of patients. to our knowledge, there are no studies that have examined how providers communicate about control medications during pediatric asthma visits. it is important to better understand how providers communicate about control medications during medical visits because the clinical practice guidelines of the national asthma education and prevention program of nhlbi encourage physicians to discuss medications with patients at every follow-up asthma visit. therefore, the purpose of this study was to: (a) describe the extent to which providers discuss, educate, and ask children and their caregivers questions about control medications and (b) examine how child, caregiver, and provider characteristics are associated with provider communication about control medications during pediatric asthma visits.. providers were recruited at five pediatric practices in nonurban areas of north carolina, and consent was obtained. children and their caregivers of these participating providers were recruited. children were eligible if they: (a) were ages 8 through 16 years, (b) were able to speak english, (c) could read the assent form, (d) had been seen at the clinic at least once before, (e) were present at the visit with an adult caregiver (parent or legal guardian) who could read and speak english and who was at least 18 years of age, and (f) had mild, moderate, or severe persistent asthma. persistent asthma was defined as experiencing asthma-related daytime symptoms more than twice a week, asthma-related nighttime symptoms more than twice a month, or receiving one or more long-term control therapies for asthma [16, 17]. clinic staff referred potentially eligible and interested patients to a research assistant who explained the study, obtained caregiver consent and child assent, and administered the eligibility screener. providers and families were told that the study was examining communication during pediatric visits. all of the medical visit audio-tapes were transcribed verbatim, and a detailed coding tool was developed to assess provider communication behaviors. all of the transcripts were coded using the coding tool and more detail about the types of communication behaviors coded is provided below. the research assistants showed caregivers a list of asthma medications and asked them to indicate which one(s) the child was taking. responses were dichotomized based on whether the caregiver reported that the child was taking a control medication versus not taking a control medication. asthma severity was classified as mild versus moderate/severe by a research assistant based on recent symptoms and medication use reported by the caregivers when research assistants administered the eligibility screening instrument for the study [16, 17]. our eligibility screening instrument utilized the primary asthma severity classification system that was being used when the study was designed and conducted [16, 17]. all child study information was then reviewed by a pediatric pulmonologist or a clinical pharmacist with expertise in asthma to verify the severity classification as mild or moderate/severe persistent asthma. the first method was medication use; any child receiving a single long-term control agent was considered to have mild persistent asthma. any child receiving two or more long-term control agents was categorized as having moderate to severe persistent asthma. a long-term control medicine included inhaled corticosteroids, leukotriene modifiers, cromolyn, nedocromil, or a long-acting beta agonist as defined by the national heart lung and blood insititute's guidelines [16, 17]. subjects who reported the occurrence of any one of eight symptoms as occurring two or more times per week or who reported awakening with asthma symptoms two or more times per month was classified as mild persistent. the eight daytime symptoms included: wheezing with a cold, wheezing without a cold, attack of wheezing that made it hard to breath or catch breath that lasted longer than a day or more, had a cough that would not go away, complained that chest felt tight or heavy, used rescue inhaler for symptoms, wheezed with exercise or running or playing hard, and coughed with exercise or running or playing hard. the nighttime symptoms asked about how often the child's sleep has been disturbed because of wheezing, coughing, chest tightness, or shortness of breath. reports of daily symptom occurrence or awakening 5 times a month resulted in a classification as moderate or severe persistent. in situations where the two methods (medication use and symptom frequency) resulted in discordant classification, the more severe category was used. child and caregiver age, caregiver education, and years the child had asthma were measured as continuous variables. child race was recoded into four categories: white, african american, native american/american indian, or other (includes categories of: hispanic, asian american, other). however, for the bivariate analyses, child race was recoded into a dichotomous variable (white versus non-white). the child's insurance status was measured using the following categories: none, private insurance, medicaid, the state children's health insurance program (schip), and others. how well the child thinks the provider knows them as a person was measured with the following categories: hardly at all, slightly, moderately well, and very well. reason for visit was measured as asthma-related versus other (e.g. physical). length of visit was measured in minutes, and whether the child was taking a control medication was measured as a dichotomous variable. the categories used in the coding tool for communication about asthma medications were adapted from the categories used in prior studies of provider-patient communication about medications [1215]. the transcripts were reviewed by two research assistants who met twice a month with the investigators to develop and refine the coding rules until themes were saturated. using the coding tool for transcribed medical visits, coders recorded the following: was there any discussion of control medications, did the provider give any education about control medications, and how many questions did the provider ask the child and caregiver about control medications. the research assistants then coded whether discussion, education, and question-asking occurred in each of the following areas: adherence, fears/concerns, frequency/timing, generic/brand, how well it works, purpose, side effects, strength/dose, supply, and others. two research assistants coded 20 of the same transcripts throughout the study period to assess intercoder reliability which was calculated using interrater correlations. inter-rater reliability was 1.0 for whether control medications were discussed, 0.87 for the number of areas discussed, 0.91 for whether the provider educated about control medications, 0.80 for number of areas the provider educated about, and 0.95 for the number of questions the provider asked about control medications. all children were included in the analyses because even if children were not on a control medication, control medications could have been discussed during the visit as a possible treatment. first, we present descriptive statistics for the demographic, clinical, and provider communication variables. second, we examine bivariate relationships between the demographic variables and provider communication variables using correlation coefficients, t-tests, or pearson chi-square statistics, as appropriate. next, we used generalized estimating equations (gee) to examine how demographic and clinical characteristics of the child and caregiver were associated with: (a) whether the provider discusses control medications, (b) whether the provider educates about control medications, and (c) how many questions the provider asks about control medications. a poisson gee was used to examine provider question-asking because the variable was skewed. forty-one providers agreed to participate in the study; two providers refused to participate for a participation rate of 95.3%. three hundred and thirty-three of the 377 families (88%) that approached the research assistant to learn more about the study agreed to participate in the study. two-hundred and ninety six patients of the 333 participating patients (89%) had useable audio-tape data, and these patients were seen by 35 of the 41 providers who agreed to participate in the study. four of the 35 providers were nurse practitioners or physician assistants, and they saw seventeen of the participating children. twenty-seven of the providers were white, two were american indian, three were african american, one was asian, and two classified their race as other. providers ranged in age from 30 to 70 years (mean=44.8 years, standard deviation=9.4). approximately 62% of the children were white, 30% were african american, and 10% were native american/american indian. in terms of the child's asthma providers discussed control medications during 87.4% of visits where children were taking control medications and during 63.8% of visits where children were not taking control medications. the average number of topic areas discussed was 2.92 (standard deviation=2.0; range 0 to 8 areas). table 2 shows the control medication areas that providers discussed most often during the medical visits: (a) frequency/timing of use (63%), (b) supply of medication (50%), (c) strength/dose of medication (48%), (d) adherence (47%), and (e) purpose of the controller medication (34%). side effects were only discussed during approximately 11% of encounters and fears/concerns were only discussed during 4.4% of encounters. in the bivariate results, control mediations were significantly more likely to be discussed if a child was on a control medication (pearson chi-square=16.1, p<0.001), if the child had moderate/severe persistent asthma (pearson chi-square=12.8, p<0.001), and if the child was present for an asthma-related visit (pearson chi-square=14.48, p<0.001). providers were significantly more likely to discuss control medications if a child was on a control medication and during visits with children who had moderate to severe persistent asthma compared to children with mild persistent asthma. providers were also more likely to discuss control medications when the reason for visit was asthma-related versus not asthma-asthma-related. the average number of areas educated about was 1.54 (standard deviation=1.6; range 0 to 7 areas). as presented in table 2, providers educated about control medications most often in the following areas: (a) frequency/timing of use (37%), (b) strength/dose (32%), and (c) purpose (30%). providers were significantly more likely to provide education about control medication side effects if a child was not on a control medication (pearson chi-square=5.1, p=0.02). in the bivariate results, providers were more likely to educate about control medications if the child had moderate/severe persistent asthma (pearson chi-square=13.3, p<0.001), if the child was younger (t-test=2.71, p=0.007), and if the child was present for an asthma-related visit (pearson chi-square=12.9, p<0.001). table 3 demonstrates which child and caregiver characteristics were associated with whether providers educated families about control medications. providers were significantly more likely to educate about control medications during visits with children who had moderate to severe persistent asthma, younger children, and during visits that were primarily asthma-related. the average number of questions asked was 2.68 (standard deviation=3.1; range 0 to 16 questions). providers were most likely to ask children and their caregivers questions about control medications in the following areas: (a) frequency/timing of use (38%), (b) adherence (37%), and (c) supply (29%). providers only asked about side effects during 2% of encounters, fears/concerns during 1% of encounters, and how well the control medications were working during approximately 12% of encounters (table 2). in the bivariate results, providers were more likely to ask control medication questions if the child was on a control medication (t-test=3.5, p=0.001) and if the child was younger (pearson correlation coefficient=0.12, p=0.04). table 4 presents the poisson gee results predicting the number of questions providers asked about control medications. providers asked more questions about control medications if a child was currently treated with a control medication. providers also were more likely to ask younger children more questions about control medications than older children. providers discussed the frequency of use, supply of medication, and strength/dose of medication with families most often, but they only discussed the purpose of the control medication during about one third of all visits and how well the medication works during about a quarter of all visits. providers rarely discussed side effects and fears/concerns about control medications. according to clinical practice guidelines of the national asthma education and prevention program of nhlbi the national clinical practice guidelines instruct providers to teach and reinforce the roles of control medications at every opportunity, yet providers in this study only educated children and their caregivers about control medications during about two-thirds of the visits. it is especially critical for families to understand the purpose of asthma control medications so that they understand the difference between control and rescue medications. providers rarely educated about side effects and how well the medications work even though according to the practice guidelines of the national asthma education and prevention program of nhlbi, providers should ask about specific side effects from control medications during routine asthma visits. our results provide evidence that discussion of asthma controller medication does not occur at every follow-up visit. given recent evidence that poorly controlled asthma (56%) is common among children receiving asthma care from community pediatricians, this study points to provider discussion and education as a key area for improvement. if children and their caregivers better understand what to expect when taking the medications, they might be more adherent. better adherence could lead to improved asthma control and fewer school days missed for children and reduced health care costs [2, 3]. future work needs to assess the relationship between provider education about control medications and medication adherence and asthma control. specifically, providers were more likely to educate younger children about control medications and they were more likely to ask younger children more questions about control medications. perhaps this is because younger children are less likely to volunteer information about their use and experiences in using control medications. it is important to make sure that children of all ages understand how to use their control medications. providers also were more likely to engage children with more severe asthma in discussions about controller medications. it is important for providers to ask about child adherence to control medications so they can work with families to improve adherence and asthma control. providers asked few questions about side effects and how well the control medications were working, which is similar to findings of medication communication in adults with other medical conditions [12, 13]. if providers want to detect and prevent problems with asthma control medication use and adherence, they should consider asking at least one open-ended question about how the medications are working and a second question about any side effects or barriers to use (e.g., how are your asthma medications working? and what types of problems have you had with your medications?). the study's generalizability is limited in that it was conducted in five pediatric clinics in nonurban areas of north carolina. another limitation is that clinic staff referred potentially eligible patients to the research assistant; thus, we do not know how many referred patients chose not to talk with the research assistant. however, we could not ask the clinic staff to track these numbers because of the busyness of the clinic and our promise not to interrupt clinic flow. providers and caregivers knew they were being recorded and may have changed their communication, but they did not know the study hypotheses. it is also important to note that we did not assess the level of control in this study based on the current nhlbi guidelines because this study was initiated prior to the release of the new guidelines. another limitation is that we did not include children ages 27 years and adolescents ages 17-18 years. future research should examine provider discussion, education, and question-asking about control medications in these age groups. despite its limitations, this study presents information on the extent to which providers discuss and educate children about control medications during pediatric medical visits.

background. few studies have explored how providers communicate about control medications during pediatric asthma visits. objectives. the purpose of this study was to: (a) describe the extent to which providers discuss, educate, and ask children and their caregivers questions about control medications and (b) examine how child, caregiver, and provider characteristics are associated with provider communication about control medications during pediatric asthma visits. methods. children ages 8 through 16 with mild, moderate, or severe persistent asthma and their caregivers were recruited at five pediatric practices in nonurban areas of north carolina. after audio-tape recording medical visits, caregivers completed questionnaires and children were interviewed. generalized estimating equations were used to analyze the data. results. providers educated families about control medications during 61% of the visits, and they asked questions about control medications during 67% of visits. providers were significantly more likely to discuss control medications if a child was taking a control medication, if the child had moderate to severe persistent asthma, and if the child was present for an asthma-related visit. conclusion. providers need to educate and ask more questions of families about side effects and how well control medications are working.

PMC3155790

pubmed-621

most dysentery cases in the tropics are caused by shigella, whereas dysentery in the developed countries is usually caused by salmonella. death rates as high as 6.2% have been reported during epidemics of shigella dysenteriae type 1. the provision of effective anti-microbial therapy is important especially for reducing the prevalence of shigella and other organisms causing dysentery in children. decreasing the bacterial load excreted by a child with dysentery also reduces the probability of fecal oral transmission to close contacts, such as neighbours, friends or members of the child s household. anti-microbial therapy is particularly important in developing countries, where prolonged diarrhoea episodes, including dysentery, can significantly decrease the growth and nutritional status in the affected children., the world health organization (who) recommends that all episodes of diarrhoea with blood in the stool be treated with antibiotics. the who currently recommends treatment with ciprofloxacin (a quinolone) or one of the three second-line antibiotics, pivmecillinam, azithromycin and ceftriaxone (a third-generation cephalosporin). here, we review the scientific evidence supporting the who-recommended antibiotics ciprofloxacin, ceftriaxone and pivmecillinam for the effective treatment of dysentery. we systematically reviewed all literature published between 1 january 1990 and 31 january 2009 to identify the studies describing the efficacy of ciprofloxacin, ceftriaxone and pivmecillinam for the treatment of dysentery in children aged 5 years. methods paper), we searched pubmed, cochrane libraries and all who regional databases, including literature published in other languages. shigella and salmonella. studies were included if they reported the effect of the antibiotics on severe morbidity as observed by decreased blood in the stool or the effect of the antibiotics on shigella and/or salmonella bacteraemia, in the stool of paediatric dysentery cases. we abstracted data describing study identifiers and context, study design and limitations, intervention specifics and outcome effects, into a standardized abstraction form from any publications that met final inclusion and exclusion criteria (ref. bacteriologic failure and bacteriologic relapse. clinical failure was defined as an absence of marked improvement in, or worsening of, illness with the presence of bloody mucoid stools, more than a trace of blood in stool, abdominal pain, tenesmus and/or fever. bacteriological failure was defined as failure to clear an enteropathogen isolated from an individual on admission to the study, by the end of the treatment period. bacteriological relapse was defined as the reappearance of an enteropathogen in stool after that enteropathogen was cleared by treatment. each study was assessed and graded according to the cherg adaptation of the grade technique. one- to two-point grade increases were allotted to studies with statistically significant strong levels of association (> 80% reduction). any study with a very low final grade laird pooled relative risk and corresponding 95% confidence interval because there was heterogeneity in the study design. we also ran, but did not report, the mantel haenszel pooled relative risk and corresponding 95% ci. we summarized the evidence by outcome, including qualitative assessments of the study quality and quantitative measures, according to the standard guidelines for each outcome. we applied the cherg rules for evidence review to the collective diarrhoea morbidity outcomes to estimate the effects of ciprofloxacin, ceftriaxone and pivmecillinam on eliminating severe morbidity due to diarrhoea in children with dysentery. we identified 586 titles from searches conducted in all databases (figure 1). after screening titles and abstracts because very few studies reported data exclusively for children aged 5 years, we expanded our study population to include children aged up to 16 years. eight papers were included in the final dataset with some papers contributing data for multiple antibiotics or more than one outcome measure (supplementary table 1). we found eight studies that reported on clinical failure (12 unique data points),, with most studies evaluating clinical failure status 3 days after treatment was initiated (range 36 days). four studies reported on bacteriological failure (six unique data points),, and five reported on bacteriological relapse (seven unique data points),, (table 1). figure 1synthesis of study identification to review effect of ciprofloxacin, ceftriaxone and pivmecillinam on diarrhoea treatment failure, bacteriological failure and bacteriological relapse table 1quality assessment of trials of antibiotics for the treatment of diarrhoeaquality assessmentsummary of findingsdirectnessno. of eventsno. of studies (ref)designlimitationsconsistency (based on the heterogeneity of the meta-analysis)generalizability to population of interestgeneralizability to intervention of interestinterventioncontrolpooled failure rate (95% ci)clinical failure: moderate outcome-specific quality eight, rctrct but only able to use treatment failure rates (0.5)0.55/12 data points from israel7/12 hospital based; 2/12 multicentre820.1% (0.2 to 0.5%)bacteriological failure: moderate outcome-specific quality four, rctrct but only able to use treatment failure rates (0.5)borderline heterogeneity based on the meta- analysis (p=0.077) (0.5)3/6 data points from bangladeshgeneralizable90% (0.1 to 0.1%)bacteriological relapse: moderate outcome-specific quality five,,rctrct but only able to use treatment failure rates (0.5)consistent based on meta-analysis3/7 data points from israelgeneralizable70% (0.1 to 0.1%)random effects meta-analysis.rct, randomized controlled trial. synthesis of study identification to review effect of ciprofloxacin, ceftriaxone and pivmecillinam on diarrhoea treatment failure, bacteriological failure and bacteriological relapse quality assessment of trials of antibiotics for the treatment of diarrhoea random effects meta-analysis. rct, randomized controlled trial. in table 1, we report the quality assessment of trials by study outcome as well as results from corresponding meta-analyses. based on 12 data points from eight studies, treatment with one of the three antibiotics resulted in a clinical failure rate of 0.1% (95% ci 0.2 to 0.5%). based on six datasets abstracted from four studies evaluated in this review, the effect size of antibiotic therapy on a child s relative risk of bacteriological failure is 0% (0.1 to 0.1%). seven datasets from five studies indicate that the effect size of antibiotic therapy on a child s relative risk of bacteriological relapse is 0.0% (0.1 to 0.1%). assuming treatment failure rate to be an extremely conservative proxy for dysentery deaths not preventable with prompt antibiotic treatment, it can be estimated that treatment of dysentery with ciprofloxacin, ceftriaxone or pivmecillinam will reduce diarrhoea mortality attributable to dysentery by 99% (figure 2). figure 2application of the cherg guidelines for the effect of antibiotics on dystenteric morbidity and mortality application of the cherg guidelines for the effect of antibiotics on dystenteric morbidity and mortality diarrhoeal disease, including dysentery, is a major cause of morbidity and mortality among children in developing countries. this systematic review of the literature summarizes the evidence supporting the use of the antibiotics recommended by who: ciprofloxacin, ceftriaxone and pivmecillinam. it also suggests that the bacteria isolated from a stool sample of a child with dysentery rarely relapses if the child has received full-course treatment with one of these antibiotics, and the disease-causing bacteria is sensitive to the antibiotic. reducing a child s risk of bacteriological relapse is beneficial, because the likelihood of subsequent episodes of dysentery occurring in that child, and of transmission occuring to others, are reduced as a result. the studies contibuting data in this review were conducted in middle- and low-income countries increasing their generalizability to paediatric populations in countries with the highest diarrhoea mortality rates. extrapolating clinical failure to mortality, our meta-analyses indicate that>99% of dysentery deaths can be prevented with ciprofloxacin, ceftriaxone or pivmecillinam treatment. for application in the lives saved tool, it is essential to extrapolate severe morbidity to mortality, although this leap has many limitations. children with functioning immune systems do not always progress to death as a result of dysentery. it is possible for some children to successfully fight the infection without antibiotics and make a full recovery. in addition, many children who present for medical care and are prescribed one antibiotic are put on a second-line treatment if the first choice fails, thus further reducing the treatment failure rate. nearly, all studies were conducted in a clinic or hospital, where staff could monitor treatment. in a community or outpatient setting, the therapeutic effect of the antibiotics reviewed here may not be as great as our analyses indicate, because caregivers may not comply with the dosage and duration specifications of the treatment. caregivers may also fail to manage the dehydration that often accompanies diarrhoea, thereby increasing a child s risk of death. the 99% reduction in diarrhoea mortality that we estimate is attributable to the treatment of dysentery with ciprofloxacin, ceftriaxone or pivmecillinam and assumes antibiotic susceptibility. the variability in the types of dysentery-causing organisms that occur worldwide and their sensitivity to the antibiotics recommended for treatment by the who may decrease the generalizability of the findings presented in this review. because bacteria that cause dysentery can acquire resistance to antibiotics, drugs used for treatment should be selected based on resistance patterns prevalent in the community. future research with regard to site-specific antibiotic resistance may provide additional data and help refine recommendations for national or local planning. there is strong evidence in favour of the continued use of the antibiotics recommended by who ciprofloxacin, ceftriaxone and pivmecillinam to reduce morbidity and mortality in children with dysentery. us fund for unicef from the bill&melinda gates foundation (grant 43386 to promote evidence-based decision making in designing maternal, neonatal and child health interventions in low- and middle-income countries). national institutes of health (grant t32hd046405 for international maternal and child health).

background ciprofloxacin, ceftriaxone and pivmecillinam are the antibiotics currently recommended by the world health organization (who) for the treatment of dysentery in children; yet there have been no reviews of the clinical effectiveness of these antibiotics in recent years. methods we reviewed all literature reporting the effect of ciprofloxacin, ceftriaxone and pivmecillinam for the treatment of dysentery in children in the developing countries. we used a standardized abstraction and grading format and performed meta-analyses to determine the effect of treatment with these antibiotics on rates of treatment failure, bacteriological failure and bacteriological relapse. the cherg standard rules were applied to determine the final effect of treatment with these antibiotics on diarrhoea mortality. results eight papers were selected for abstraction. treatment with ciprofloxacin, ceftriaxone or pivmecillinam resulted in a cure rate of>99% while assessing clinical failure, bacteriological failure and bacteriological relapse. conclusions the antibiotics recommended by the who ciprofloxacin, ceftriaxone and pivmecillinam are effective in reducing the clinical and bacteriological signs and symptoms of dysentery and thus can be expected to decrease diarrhoea mortality attributable to dysentery.

PMC2845863

pubmed-622

oral drug delivery systems are essential to optimize both the residence time of the system within the gastrointestinal tract and the release rate of the drug from the system. various attempts have been made to prolong the residence time of the dosage forms within the stomach. rapid gastrointestinal transit could result in incomplete drug release from the drug delivery system to absorption window leading to diminished efficacy of the administered dose. prolonged gastric retention is important in achieving control over the gastric residence time because it helps to maintain the controlled release system in the stomach for a longer time in an expected manner. floating systems (hydrodynamically controlled systems) are low-density systems which means they are less dense than gastric fluid. these systems have sufficient buoyancy to float over the gastric contents and remain buoyant in the stomach for a prolonged period of time without disturbing the gastric emptying rate. while the formulation is floating on the gastric contents, drug is released slowly from it at a desired rate [3, 4]. for oral sustained release, multiple unit dosage forms (i.e., microballoons) are more beneficial than single unit dosage forms. because single unit dosage forms have the disadvantage of a release all or nothing emptying process and multiple unit dosage forms have advantages of disperse widely and release drug uniformly along the gastrointestinal tract, which results in more reproducible drug absorption, less dose dumping, and reduced risk of local irritation than the use of single unit dosage form [57]. these microballoons are characteristically free flowing powders consisting of proteins or synthetic polymers, ideally having a size less than 200 m. pentoxifylline (ptx), trisubstituted xanthine derivative (3,7-dimethyl-1-(5-oxohexyl)-3,7-dihydro-1h-purine-2,6-dione), is a hemorheologic agent used for the treatment of peripheral arterial disease and intermittent claudication. the apparent plasma half-life of the drug and its metabolite is 2-3 hours. on the basis of using ptx as drug of choice in chronic occlusive aterial diseases, it is of a wise candidate drug to be formulated in sustained release oral dosage form. thus, the aim of the present research work was to design, develope, and characterization of pentoxifylline loaded floating microballoons. mumbai, india. ethyl cellulose, hydroxypropyl methylcellulose (hpmc k4 m), and tween 80 were purchased from central drug house (cdh), new delhi, india. all other solvents and chemicals were of analytical grade. pentoxifylline, hpmc k4 m, and ethyl cellulose were dissolved in a mixture of ethanol and dichloromethane at room temperature. these were poured into 250 ml of water containing 0.01% tween 80 maintained at a temperature of 3040c and consequently stirred at ranging agitation speed to allow the volatile solvent to evaporate. the formulated microballoons were filtered, washed with distilled water, and dried at 40c. the size of microballoons of each formulation was determined using a microscope fitted with an ocular micrometer, and stage micrometer and average particle size was determined. the surface morphology of microballoons was examined by scanning electron microscopy (jeol jsm-1600, tokyo, japan) operated at 15 kv on samples gold sputtered at 10 ma, under argon at low pressure. the weight of microballoons was divided by the total weight of all the nonvolatile components that were used for the preparation of the microballoons and multiplied by 100 gives the% yield of microballoons as follows: (1)% yield=(weight of microballoons collected) (weight of all nonvolatile components used for the preparation)1100. percentage loading efficiency. to determine loading efficiency, microballoons were taken, thoroughly triturated, and suspended in a minimal amount of alcohol. the estimation of drug was carried out using uv spectrophotometer (uv-vis double beam spectrophotometer 2201, systronics) at 272 nm max. the percentage loading efficiency was calculated as follows: (2)loading efficiency (%) =amount of drug actually presenttheoretical drug load expected100. the buoyancy test of the microballoons was carried out using usp ii (paddle type) dissolution apparatus (ds 8000, labindia). dissolution test solution simulated gastric fluid (sgf) containing tween 80 (0.02% v/v) was used as a dispersion medium to simulate gastric fluid. the microballoons were spread over the surface of the sgf, ph 1.2 (900 ml, 37 0.5c), which was agitated by a paddle rotated at 100 rpm for 12 h. after agitation for a previously determined interval, the microballoons that were floating and the ones that settled to the bottom of the flask were recovered separately. after drying, the fraction of the microballoons was weighed. the% buoyancy of the microballoons was calculated by the following formula: (3)% buoyancy=weight of floating microballoons after dryingweight of floating+settled microballoons after drying 100. in vitro drug release studies. the dissolution test was performed using 0.1 n hcl (ph 1.2) as dissolution fluid (900 ml) maintained at 37 0.5c at 100 rpm. the samples (5 ml) of the solution were withdrawn from the dissolution apparatus for 12 h, and the samples were replaced with fresh dissolution medium each time to maintain the sink condition. withdrawn samples were analyzed using uv-vis double beam spectrophotometer at 272 nm against suitably constructed calibration curve. all measurements were carried out in triplicate, and average values were plotted. statistical analysis. two-way analysis of variance (anova) was applied to check significant differences in drug release from different formulations. the kinetic models used were zero order (cumulative percentage of drug release versus time), first order (log cumulative percentage of drug remaining versus time), the higuchi model (cumulative percentage of drug release versus square root of time), and korsmeyer-peppas (log cumulative percent drug release versus log of time). regression (r) values were calculated for the linear curves obtained by regression analysis. preparation of pentoxifylline loaded floating microballoons was done by using hpmc k4 m and ethyl cellulose as sustained release polymers by the solvent evaporation technique. ethanol and dichloromethane were used as solvents to keep both polymers and drug in solution. the solution was poured with the help of syringe into 250 ml water containing 0.01% tween 80 maintained at a temperature of 3040c and subsequently stirred at ranging agitation speed to allow the volatile solvent to evaporate. the formulations f1, f2, and f3 were formulated by varying the concentration of ethyl cellulose, and formulations f4, f5, and f6 formulated by varying the concentration of hpmc k4 m. microballoons with higher concentration of ethyl cellulose gave much retarded drug release than higher concentration of hpmc k4 m. from the result of this study, the average particle size of microballoons were found to be 74.63 1.04, 85.18 3.12, and 104.0 2.87 for f1, f2, and f3 formulations and 82.96 2.13, 99.32 1.45, and 110.4 2.94 for f4, f5, and f6 formulations, respectively. this is due to the increase in viscosity of the solution and the decrease in stirring efficiency. also with increasing polymer concentration, therefore, a shorter time was provided for the breakup of droplets, and larger microballoons were formed. the scanning electron microphotograph showed that the developed floating microballoons were spherical with porous surface which facilitate diffusion of drug as shown in figure 1. production yields were found to be 75.76 1.54, 78.13 1.21, 80.89 2.24, 76.79 1.38, 74.66 2.61, and 72.57 1.85 for f1, f2, f3, f4, f5, and f6 formulations, respectively, as shown in table 2. the percentage loading efficiencies were found to be 75.5 1.82, 76.36 1.27, 77.85 0.61, 76.22 0.82, 77.29 0.12, and 77.66 1.35% for f1, f2, f3, f4, f5, and f6 formulations, respectively. the buoyancy percentage for all batches was almost above 70%, which was studied for 12 h. the highest percentage was obtained with formulation f6. average buoyancies in percentage were found to be in the range of 72.43 0.21% to 78.19 0.63% for f1 to f6 formulations. in general, with the increase in the amount of polymers, there was an increase in the buoyancy percentage. the increase in the buoyancy percentage may be attributed to air and gel-forming polymer hpmc k4 m which caused swelling because of increased amount of the polymers present. the in vitro drug release of formulations f1, f2, f3, f4, f5, and f6 was found to be 96.81 0.16, 88.84 0.46, 82.21 1.29, 93.13 1.48, 90.16 0.98, and 87.09 1.73 in 12 h, respectively. results indicate that proportion of polymers in formulation was the key factor governing the release of drug from microballoons. as the concentration of polymer increased, there was an increase in diffusional path length. formulations comprised of ethyl cellulose in higher proportion exhibited much retarded drug release as compared to formulations comprised of hpmc k4 m in higher proportion. the release profile of pentoxifylline from microballoons for all formulations was shown in figure 2. the release profile of pentoxifylline from microballoons containing varying concentrations of ethyl cellulose and hpmc two-way analysis of variance (anova) was applied to check significant differences in drug release from different formulations containing different concentrations of hpmc k4 m and ethyl cellulose. on increasing the amount of polymers, a significant decrease (p<0.05) was obtained in the cumulative drug release. the kinetics and mechanism of drug release were determined using zero order, and first order, higuchi's model, and further analysis was performed using korsmeyer-peppas equation. all formulations were found to be following higuchi's model as the plot showed high linearity (r=0.985 to 0.991) as shown in table 3. this equation indicates that the cumulative amount of drug release is proportional to the square root of time for diffusional release of drug from the formulation. n values from the power law equation (korsmeyer-peppas equation) for drug release profiles were between 0.776 and 0.842, suggesting that drug release mechanism from formulations followed the non-fickian (anomalous) transport mechanism, which may indicate that diffusion was predominant mechanism of drug release. the release kinetic data obtained from different plots of models for all formulations are given in table 3. floating microballoons of pentoxifylline were prepared by the solvent evaporation technique using different concentrations of polymers like hpmc k4 m and ethyl cellulose (ec) dispersed in ethyl alcohol and dichloromethane as a solvent system. prepared floating microballoons showed significant floating ability, good buoyancy, and sustained drug release. in vitro drug release of microballoons was influenced by polymers concentration. from the percentage loading efficiency and in vitro drug release studies, it was observed that f3 formulation exhibits greater drug loading efficiency and sustained release behavior. on fixing the in vitro drug release data of optimized formulation to various kinetic models, it was found that it exhibits the higuchi order of kinetics followed by zero order and first order. the formulation undergoes anomalous (non-fickian) diffusion, which indicates that the drug release rate was controlled by swelling, erosion, and diffusion from microballoons. thus, pentoxifylline loaded floating microballoons can prove to be potential pharmaceutical dosage form for prolonging the gastric retention time of dosage form.

the floating microballoons have been utilized to obtain prolonged and uniform release in the stomach. the objective of the present study involves design, development, and characterization of pentoxifylline loaded floating microballoons to prolong their gastric residence time. pentoxifylline (trisubstituted xanthine derivative) loaded microballoons were prepared by the solvent evaporation technique using different concentrations of polymers like hpmc k4 m and ethyl cellulose (ec) in ethyl alcohol and dichloromethane organic solvent system. microballoons were characterized for their particle size, surface morphology, production yield, loading efficiency, buoyancy percentage, and in vitro drug release studies. from the characterization it was observed that increases in amount of polymers (hpmc k4 m and ec) led to increased particle size, loading efficiency, and buoyancy percentage, and retarded drug release. the particle size, particle yield, loading efficiency, buoyancy percentage and in vitro drug release for optimized formulation (f3) were found to be 104.0 2.87 m, 80.89 2.24%, 77.85 0.61%, 77.52 2.04%, and 82.21 1.29%, respectively. the data was fitted to different kinetic models to illustrate its anomalous (non-fickian) diffusion. the in vitro result showed that formulations comprised of varying concentrations of ethyl cellulose in higher proportion exhibited much retarded drug release as compared to formulations comprised of higher proportion of varying concentrations of hpmc k4 m.

PMC4590787

pubmed-623

musicians often experience musculoskeletal pain as a result of lengthy and long-term instrument performance. more students enrolled in music schools note upper limb pain compared with students enrolled in ordinary schools1. it has also been reported that the incidence of upper limb pain is higher in those who have longer practice times. therefore, it can be said that upper limb pain in musicians is a symptom of overuse, with lengthy and long-term instrument performance being contributing factors. musculoskeletal pain in musicians is common not only in the upper limbs but also in the lumbar and cervical regions, as well as in the shoulder girdle2. for example, it is conceivable that when playing a heavy instrument such as the tuba, a large load is applied to the trunk to maintain the performance posture. furthermore, as instruments in a marching band are played while marching, a large load is probably also applied to the lower limbs. however, the performance postures of various types of instruments have not been sufficiently investigated. thus, the present study clarified the characteristics of the standing performance posture for the trumpet and the marching euphonium and investigated the effect of the performance postures of these instruments on the lower limb musculoskeletal system. the subjects were 10 female university students in japan. their (mean sd) age was 20.5 1.3 years, their height was 159.6 6.2 cm, and their weight was 55.1 7.2 kg. because the subjects were not professional trumpet or marching euphonium performers, they all received sufficient instruction from the instructor regarding the trumpet and marching euphonium performance posture prior to measurements. the purpose and contents of the study were sufficiently explained to the subjects, whose consent to participate in the study was obtained. additionally, approval was obtained from the shijonawate gakuen university ethics committee (approval number 23-2). the subjects adopted a closed-leg resting standing position, a trumpet performance posture, and a marching euphonium performance posture. all instrumental performance postures were adopted in the standing, and the subjects focused directly ahead on a mark positioned at eye level. subjects maintained performance posture without actually playing the instrument. the angle and muscle activity of the trunk and lower limbs were measured while the postures were maintained. the measurement was performed in the sequence of the resting standing position, the trumpet performance posture, and the marching euphonium performance posture. the trunk and lower limb angles were measured using a three-dimensional motion analysis device (cms-hs, zebris medical gmbh, isny, germany). markers were attached to the lateral malleolus, lateral epicondyle of the femur, greater trochanter, and acromion on the left side of the subjects. the sampling frequency was 100 hz, and the measurement time was 10 s. measurements were performed as soon as the subjects could maintain a stable performance posture. the collected data were processed by image analysis software (win-date, zebris medical gmbh), and the angles of knee flexion, hip flexion, and anterior tilt of the trunk were calculated. the angle of knee flexion was considered as the angle formed between the line linking the lateral malleolus and the lateral epicondyle of the femur and the line linking the lateral epicondyle of the femur and the greater trochanter. the angle of hip flexion was considered as the angle formed between the line linking the lateral epicondyle of the femur and the greater trochanter and a vertical line. the anterior tilt angle of the trunk was considered as the angle formed between the line linking the acromion and the greater trochanter and a vertical line. a surface myograph (myosystem 1200, noraxon inc., the muscle activities measured were those of the cervical paraspinal muscles, upper fibers of the trapezius, lumbar paraspinal muscles, gluteus maximus, rectus femoris, biceps femoris, and lateral head of the gastrocnemius on the left side. after treating the skin sufficiently to create a skin resistance of no greater than 10, electrodes (blue sensor p-00-s, ambu a/s, ballerup, denmark) were attached parallel to the muscle fibers at the center of each muscle. spacing between the electrodes was 25 mm, the sampling frequency was 1,000 hz, and the sampling time was 10 s. measurements were performed as soon as subjects could maintain a stable performance posture. the collected data were subjected to full-wave rectification using electromyogram analysis software (myoresearch, noraxon inc.), and the average amplitude of the central three seconds was determined. the average amplitude of each muscle was normalized as 100% according to the average amplitude of the 3-s maximum voluntary contraction. statistical analysis entailed a one-way analysis of variance and multiple comparison using tukey's method. mean sd. a: significant difference between the standing position and the trumpet performance posture (p<0.01). b: significant difference between the standing position and the marching euphonium performance posture (p<0.01). c: significant difference between the trumpet performance posture and the marching euphonium performance posture (p<0.05) mean sd. a: significant difference between the standing position and the marching euphonium performance posture (p<0.01). b: significant difference between the standing position and the marching euphonium performance posture (p<0.05). c: significant difference between the trumpet performance posture and the marching euphonium performance posture (p<0.01) the anterior tilt angle of the trunk decreased significantly in the trumpet and marching euphonium performance postures compared with the resting standing position, as well as in the marching euphonium performance posture compared with the trumpet performance posture (table 1). the muscle activity of the cervical paraspinal muscles, upper fibers of the trapezius, and lumbar paraspinal muscles increased significantly in the marching euphonium performance posture compared with the resting standing position, as well as in the marching euphonium performance posture compared with the trumpet performance posture (table 2). however, there were no significant differences in other measurement items between the postures. the anterior tilt angle of the trunk can be rephrased as an increase in the posterior tilt angle of the trunk. this is likely a means for maintaining the combined center of gravity of the instrument and the body in the most stable position possible. an increase in the posterior tilt angle of the trunk increases the lumbar lordosis angle. this increase results in tension being applied to the anterior tissues of the lumbar spine, such as the anterior longitudinal ligament, and compressive force being applied to the zygapophyseal joints4. christie et al. reported that an increase in the lumbar lordosis angle while standing is associated with chronic low back pain5. consequently, maintaining the performance posture for the trumpet and the marching euphonium for a long period can contribute to low back pain. no significant difference was found in the muscle activities of the lumbar paraspinal muscles between the resting standing position and the trumpet performance posture, but a significant increase was noted in the marching euphonium performance posture compared with the resting standing position. the lumbar paraspinal muscles are antigravity muscles which are contracted continuously while standing, thus easily incurring mechanical stress. it is conceivable that, since the muscle activity of the lumbar paraspinal muscles increases during the marching euphonium performance posture, holding the performance posture for a long time increases the load on the lumbar paraspinal muscles and is therefore a factor leading to lumbar fatigue and low back pain. the muscle activity of the upper fibers of the trapezius increases in line with instrument weight in the trumpet to marching euphonium postures. when holding these instruments, the shoulder joint flexes and abducts, while the scapula rotates up and elevates. the muscle activity of the upper fibers of the trapezius is likely to increase to maintain such a posture. we also found that muscle activity of the cervical paraspinal muscles increased significantly in the euphonium performance posture compared with the resting standing position. since the posterior tilt angle of the trunk increases in the marching euphonium performance posture, it is necessary to tuck in the chin to face forward chin-in posture and is considered a posture in which the muscle activity in the cervical area including the cervical paraspinal muscles increases6. sustained contraction of these muscles is a likely factor of muscle fatigue. furthermore, obstruction of blood flow by sustained muscle contraction will result in myalgia7. therefore, holding the trumpet and marching euphonium performance postures for a long time would be factors of myalgia. the present findings suggest that the trumpet and marching euphonium performance postures increase the load on the cervical and trunk musculoskeletal system. moreover, the load on the musculoskeletal system increases in line with increased instrument weight. however, the trumpet and marching euphonium performance postures do not affect the musculoskeletal system of the lower limbs. therefore, we consider it important to evaluate the cervical and trunk musculoskeletal systems and to conduct conditioning coaching appropriate for trumpet and marching euphonium performers. because these instruments are supported by the left hand and played with the right hand, the trunk and lower limb musculoskeletal system on the left side, that is, the supporting side, was measured in the present study. we plan to measure the trunk and lower limb musculoskeletal system on the right side to elucidate the effect of performance postures on the whole body. in addition, it is possible that the trunk and lower limb musculoskeletal system is affected differently by the technique used to hold the instrument with the left hand during trumpet and the marching euphonium performance. because the angles and muscle activities of the upper arm were not measured in the present study, we plan to perform these measurements in a future study to elucidate the influence of different instrument holding techniques on the trunk and lower limb musculoskeletal systems.

[purpose] the purpose of the present study was to investigate the effect of trumpet and marching euphonium performance posture on the trunk and lower limb musculoskeletal system. [subjects] the subjects were 10 female university students. [methods] subjects maintained a resting position, a trumpet performance posture, and a marching euphonium performance posture. the angles and muscle activities of the trunk and lower limbs were then measured. [results] the anterior tilt angle of the trunk decreased significantly in the trumpet and marching euphonium performance postures compared with the resting standing position, as well as in the marching euphonium performance posture compared with the trumpet performance posture. the muscle activity of the cervical paraspinal muscles, upper fibers of the trapezius, and lumbar paraspinal muscles increased significantly in the marching euphonium performance posture compared with the resting standing position, as well as in the marching euphonium performance posture compared with the trumpet performance posture. [conclusion] the results suggest that the performance position for trumpet and the marching euphonium performance increases the load on the cervical and thoracic musculoskeletal system, which increases with greater instrument weight. however, the same instrument performance postures had no affect on the musculoskeletal system of the lower limbs.

PMC3818765

pubmed-624

protein glycosylation is a phenomenon shared by all domains of life. over 70% of the eukaryotic proteome although it is too early to predict the full extent of prokaryotic glycosylation, it is clear from the diversity of prokaryotic glycoproteins discovered in recent years that glycosylation in these organisms is the norm rather than the exception. a great deal of progress has been made in understanding prokaryotic glycosylation since the seminal review of szymanski and wren in 2005 which focused on the discovery, five years earlier, of a general n-glycosylation system in campylobacter jejuni. the best understood prokaryotic glycoproteins are s-layers, pilins, and flagellins plus a selection of cell surface and secreted proteins which are known to be involved in adhesion and/or biofilm formation. notably, novel general o-glycosylation systems have recently been uncovered in both pathogenic and symbiotic bacteria. in this paper, our primary aim is to articulate commonalities and differences in eukaryotic and prokaryotic glycosylation rather than provide full coverage of specific areas. there are many excellent specialist reviews referred to throughout our paper which the reader should consult for in depth coverage of particular topics. until recently it was widely believed that n-glycosylation of proteins is a eukaryotic phenomenon. nevertheless, it was as long ago as 1976 that mescher and strominger reported that the s-layer protein from an archaeal prokaryote, halobacterium salinarum, contained glycans covalently linked to asparagine residues. over the ensuing three decades sporadic evidence emerged suggesting that n-glycosylation was likely to be common in the s-layers of archaea. then, in the early years of the 21st century, groundbreaking research on the bacterial pathogen campylobacter jejuni, showed that this prokaryote has a general n-glycosylation system [3, 4]. it soon became clear that all three domains of life (eukarya, bacteria, and archaea) perform n-glycosylation in a similar manner. thus, all engage in stepwise assembly of sugars in the cytoplasm, donated by soluble nucleotide-activated sugars, to form an oligosaccharide precursor attached via pyrophosphate (all domains) or phosphate (archaea) to a lipid carrier (the so-called lipid-linked oligosaccharide or llo). after assembly of the oligosaccharide, the llo is flipped from the cytoplasm to face the lumen of the endoplasmic reticulum (er), or the periplasmic face of the inner membrane, in eukaryotes and gram-negative bacteria, respectively (figures 1(b) and 1(c)). thus far n-glycosylation has not been observed in gram-positive bacteria. in the case of archaea, which do not have a compartment equivalent to the er or periplasm, flipping across the cytoplasmic membrane will position the llo on the exterior surface of the cell where the subsequent transfer to proteins is believed to occur (figure 1(a)). in all three cases, the oligosaccharide is subsequently transferred en bloc from the lipid carrier onto the acceptor protein in a step catalysed by the ubiquitous oligosaccharyltransferase enzyme (n-ost). shared and unique aspects of the three hallmark events of n-glycosylation (cytoplasmic assembly of the llo, flipping across the er/periplasmic/cytoplasmic membranes, and en bloc transfer of the oligosaccharide to the protein acceptor) within the three domains are examined in more detail in the next sections. interestingly these hallmark processes are mirrored in the biosynthesis of bacterial lipo-oligosaccharides (los) and lipo-polysaccharides (lps) (compare figures 1 and 2). in eukarya and archaea, the lipid constituent of the llos is dolichol, which is a polymer of isoprene units (ch3c(ch3)=ch ch2) numbering about 12 in archaea, 14 in yeast, and up to 19 in mammals. bacteria also have a polyisoprene as their llo lipid but, instead of dolichol, they use undecaprenol (11 isoprene units) which has one more double bond than the same length dolichol. this double bond is located between carbons 2 and 3 with respect to the alcohol group (see figure 3). the absence of this specific double bond will confer greater rotational mobility to the oligosaccharidic chain in the dolichol llos, compared with the undecaprenol llos, which might facilitate chain extension after flipping. the llo biosynthetic pathway has been exhaustively characterized in eukaryotes and is very well understood [7, 8]. thus the cytoplasmic llo carries a unique heptasaccharide (man5glcnac2; figure 1(b)) which is further elaborated in all higher eukaryotes, after flipping to the lumen of the er, by the stepwise addition of 4 additional mannoses plus 3 glucoses, donated by dolichol-phosphate-linked sugars, to form glc3man9glcnac2-p-p-dol (figure 1(b)). the glucoses play a pivotal role in lectin-mediated quality control of glycoprotein folding in the er and are removed by glucosidases during the folding process. in protozoa, however, there is some divergence from the conserved 14 sugar llo [9, 10]. it has been discovered that these primitive eukaryotes are characterized by llos that lack glucose and some are further deficient in the four er-derived mannoses. this lack of llo processing in the protist's er is reminiscent of periplasmic events in bacteria which do not appear to involve the addition of further sugars to their translocated llos (see below). although the n-biosynthetic pathways of the three domains have much in common, the archaeal and bacterial llo processes differ from eukaryotes in two key respects. firstly, there is no evidence for oligosaccharide sequences being conserved amongst the archaea and bacteria, in contrast to the conserved glc3man9glcnac2 sequence of all higher eukaryotes. indeed, as shown in figure 4, a great diversity of glycans are known to be transferred by n-osts to bacterial and archaeal proteins. despite this diversity, there is some commonality with respect to the type of linking sugar utilized in the three domains. secondly, the bacterial and archaeal llos do not appear to be further elaborated after flipping. however, it should be borne in mind that knowledge of bacterial and archaeal n-linked glycosylation is only just emerging, and very few biosynthetic pathways have been investigated thus far. therefore, it remains an open question as to whether llos can be extended by stepwise addition of extra sugars in the periplasmic and cell surface compartments. although there exists a very substantial body of evidence, assembled over more than three decades, demonstrating unequivocally that in eukaryotes the llo precursor is assembled in the cytoplasm and then flipped across the er membrane to the lumen, remarkably no er flippase has yet been biochemically identified. hence comparisons of flippase structures and mechanisms between the three domains are not yet possible. fortunately, genetic tools have enabled considerable progress to be made in uncovering likely candidates for flippases. thus, genetic experiments in yeast have provided very good evidence that the rft1 protein is involved in transfer of the man5glcnac2-llo across the er membrane. in accordance with the conclusion that they play a role in translocation, rft1 proteins are conserved in eukaryotic organisms, although it is still not clear whether they are actually the elusive flippases. the elucidation of archaeal n-biosynthetic pathways is still in its infancy (see calo et al. for an in-depth review of recent discoveries) and in contrast, bacterial flippases are quite well understood, not the least because llo translocation is integral to lps biosynthetic pathways which have been intensively studied for many years. it is known, for example, that the product of the wzx gene, a non-abc-type transporter, mediates transport of undecaprenol-linked o-antigen subunits across the plasma membrane in lps biosynthesis. with respect to the bacterial n-glycosylation pathway, which has been rigorously studied in the paradigm organism, c. jejuni, aebi, and coworkers have shown that pglk (previously called wlab), which is an abc-type transporter, is responsible for flipping the llo. interestingly, these workers found that pglk has a relaxed substrate specificity exemplified by its ability to complement a wzx deficiency in o-antigen biosynthesis in e. coli. notably, all bacterial n-glycans identified to date have seven or fewer sugar residues, with many archaeal structures being of a similar size (figure 4). as described earlier, the eukaryal cytoplasmic llo contains seven sugars (see figure 1(b)). these observations suggest that a maximum of seven sugars might be optimal for the flipping mechanism, though it has also been suggested that the flipping process might be affected by monosaccharide composition at the reducing end of the glycan. in this context, it could be significant that the large archaeal n-linked polysaccharide shown in figure 4 is composed of tandem repeats of a short oligosaccharide. this type of structure is reminiscent of bacterial lps and could therefore be assembled from short llo precursors, after flipping across the cytoplasmic membrane, in a similar way to wzx/wzy-dependent o-antigen polymerization in the periplasm of bacteria. alternatively, it possible that this n-linked polysaccharide might be flipped across the membrane in an atp-binding cassette (abc) transporter-dependent manor. the transfer of oligosaccharides from the llos to asparagine acceptors in n-linked glycoproteins is catalysed by homologous oligosaccharyltransferase enzymes (n-osts) in the three domains of life. in eukaryotes and archaea; n-osts are ubiquitous. consequently, n-linked glycoproteins are found in abundance throughout both domains. on the other hand, bacteria have probably not evolved n-osts of their own (see below) and n-glycosylation is restricted to a limited number of species. this is the pglb gene of campylobacter jejuni which was found to be highly homologous to the catalytic subunit (called stt3) of eukaryotic n-osts. a similar degree of homology was found in archaeal n-ost genes (which are called aglb) when their identity was confirmed a few years later [16, 17]. when the general n-glycosylation system was first discovered in c. jejuni, it was thought to be unique, and it was postulated that this organism might have acquired the pglb gene by lateral gene transfer from either the archaeal or the eukaryal domains. it is now considered most likely that pglb originated from archaea rather than eukarya (brendan wren, london school of hygiene and tropical medicine, personal communication). this conclusion is based on knowledge emerging from searches of bacterial genomes for pglb orthologues. thus far, bacterial n-ost candidates have been found exclusively in a subset of species belonging to the phylogenetic grouping known as the epsilon subdivision of the proteobacteria, which include campylobacter, helicobacter, and wolinella genera. amongst these, n-glycosylation has been rigorously confirmed by mass spectrometry for c. jejuni, w. succinogenes, and h. pullorum (figure 4) [1, 18, 19]. note, however, that although h. pullorum has the machinery for n-glycosylation, the pglb gene is absent in related mammalian pathogens such as h. pylori and h. hepaticus. it may be significant that in primordial deep sea vents, which are the homes for many archaea, the majority of bacteria are epsilon proteobacteria. so it is tempting to speculate that these extreme environments have provided the conditions for n-ost gene transfer between the prokaryotic domains (brendan wren, personal communication). we now overview current understanding of the biochemistry of n-osts across the three domains of life. n-osts in archaea, bacteria, and primitive eukaryotes (protozoa) are comprised of a single subunit (the catalytic subunit) which is the product of the aforementioned aglb, pglb, and stt3 genes, respectively. in contrast, all n-osts of higher eukaryotes are multi-subunit complexes in which the catalytic subunit (stt3) is accompanied by a total of seven additional proteins whose roles remain poorly understood [14, 2022]. suggested functions of these accessory proteins include regulating substrate specificity, possibly by expanding the range of acceptor sequences, and assisting in protein translocation and/or folding. why primitive eukaryotes do not require a multiprotein complex remains enigmatic, but even more enigmatic is the observation that a single stt3 from leishmania major can substitute for the whole n-ost complex in yeast [23, 24]. for example, l. major expresses four stt3 paralogs, whilst trypanosoma brucei has three. yeast, however, has only a single stt3 gene (called stt3p), whilst vertebrates, insects, and plants have two, encoding for stt3a and stt3b, respectively. it has been shown, via sirna knockdown experiments in mammalian cells, that stt3a glycosylates cotranslationally, whilst stt3b, which is normally coexpressed, acts posttranslationally, although the protein must not be folded. also, stt3b is required for efficient glycosylation adjacent to the n-terminal signal sequence. thus the two isoforms appear to function in concert to ensure maximal efficiency of n-glycosylation. information is only just beginning to emerge concerning the number of n-ost genes in prokaryotes. campylobacter has only a single pglb gene but h. pullorum has two unrelated genes, denoted pglb1 and pglb2, the first of which has been proven to mediate glycosylation. bioinformatic searches for aglb genes in archaea have suggested multiple candidates in individual organisms but confirmation of expression and activity has not yet been determined experimentally. n-osts from all domains have been found to exhibit quite relaxed specificity with respect to the oligosaccharide donor. thus each is capable of transferring short glycans from biosynthetic llo intermediates in addition to the full length glycans of the mature llos. eukaryotic and bacterial n-osts transfer glycans whose reducing sugar carries at least one acetamido (nac) group. thus the eukaryotic n-glycan has a chitobiose core (glcnacb1-4glcnac) and the linking sugar in characterized bacterial n-glycans is either 2,4-diacetamido-2,4,6-trideoxyglucopyranose (bacillosamine; campylobacter and wolinella) or hexnac (h. pullorum). interestingly, the pglb protein of c. jejuni is capable of transferring a variety of o-antigen oligosaccharides onto protein acceptors in engineered e. coli cells, provided their reducing sugar has an nac moiety. this discovery has important implications for the development of o-antigen containing neoglycoprotein vaccines. archaea appear to have a greater diversity of linking sugars, including glc as well as glcnac and galnac [5, 17]. interestingly, a sulfolobus archaeal species, which is very close phylogenetically to primitive eukaryotes, has mannose rich chitobiose-linked n-glycans reminiscent of the eukaryotic core sequence. when comparing mechanisms of prokaryotic and eukaryotic n-glycosylation, it is important to remember that the folding status of their proteins is very different at the time of glycosylation. thus, eukaryotic oligosaccharides are transferred to nascent proteins before they are folded, whilst in prokaryotes the proteins are presumably fully folded, having already been transported from the cytoplasm, where translation occurs, into the periplasm or onto the surface, where glycosylation takes place. in all three domains, the asparagine acceptor must normally be located in a consensus sequence (asn-x-ser/thr or, rarely, asn-x-cys, where x can not be proline); however, not all consensus sequences are glycosylated. sequence motifs contributing to specificity of site occupancy are not yet fully understood, but it is already clear that bacterial glycosylation is much more restricted than eukaryotic glycosylation. for example, consensus sites in c. jejuni require an upstream glu or asp residue in the extended consensus sequence d/eznxs/t, where neither z nor x can be proline. high throughput glycoproteomic efforts are beginning to provide comprehensive site-occupancy data in eukaryotic systems. it is hoped that these and similar experiments will facilitate the development of algorithms that will be capable of accurately predicting which consensus sequences in eukaryotic proteomes are likely to be occupied. in contrast to eukaryotes, very few prokaryotic glycoproteins have had their glycosylation sites determined. based on a limited body of data, some predictions have been made for sequences favouring archaeal glycosylation but emerging data from studies of sulfolobus s-layers suggest that these rules probably will not be universally applicable (see and unpublished work from our laboratory). bearing in mind that glycosylation in prokaryotes occurs posttranslationally, it is conceivable that general rules for site occupancy may not prevail in these organisms, because of the unique nature of individual proteins with respect to accessible consensus sequences. this could be especially relevant in s-layer glycosylation, because these proteins self-assemble into crystalline monolayers and all their consensus sequences are therefore likely to be in exposed locations [30, 31]. in concluding this section on n-osts, we draw attention to the fact that crystal structures are now available for the c-terminal domains, that include the wwdyg motif implicated in the catalytic mechanism, of both an archaeal aglb and the pglb of c. jejuni, although not so far for the eukaryotic stt3, despite many valiant efforts. mechanistic and evolutionary understanding provided by the crystal structures has been reviewed very recently so will not be covered here. all eukaryotic glycoproteins are subjected to extensive remodelling in the golgi apparatus after they exit the er, resulting in heterogeneous mixtures of glycoforms exhibiting a great variety of peripheral structures, many of which are rich in functionally important sugars such as fucose and sialic acid [7, 8]. prokaryotes have no counterpart to the golgi apparatus, and there is no evidence so far that they remodel their n-linked glycoproteins. about seven years ago a study of the hmw1 adhesin of h. influenzae uncovered a potentially novel n-glycosylation pathway occurring in the cytoplasm of this bacterium. this intriguing discovery has now been confirmed by rigorous structure analyses which, remarkably, have identified 31 glycosylated asn residues within the hmw1 protein [33, 34]. all sites carry either hex or hex-hex, where hex can be gal or glc, and all but one of the glycosylation sites has the normal n-glycosylation consensus sequence (asn-x-ser/thr, see section 2). interestingly it transfers glucose to all glycosylated asparagines but only transfers galactose to a subset of these sites. moreover, the same enzyme appears to be responsible for the hex-hex glycoforms as well as those carrying a single glc or gal. homology analysis suggests that a variety of other bacteria possess hmw1c-like proteins, so it is likely that this type of cytoplasmic n-glycosylation will be found elsewhere. whether similar glycosylation occurs in archaea is not known. as shown in figure 4, glucose has been observed as a linking sugar in some archaeal n-glycans, but it is likely that the n-ost pathway is employed in their biosynthesis. the presence of a glc-asn moiety was reported in eukaryotic laminin in 1994 but this observation has not been independently confirmed. during the past five years, intensive research on neisseria and pseudomonas pilin glycosylation has uncovered a general o-glycosylation pathway that, remarkably, has all the hallmarks of n-linked glycosylation (figure 5). moreover, this general pathway does not appear to be restricted to a few pilin proteins in a handful of pathogens. thus, very interesting data are emerging from research on bacteroides species that suggests that these bacteria are capable of glycosylating a great number of proteins in this way. so far oligosaccharyltransferase-mediated o-glycosylation has only been found in gram-negative bacteria, which is perhaps not surprising, bearing in mind that it mirrors lps biosynthesis. much of our knowledge of bacterial o-linked glycosylation pathways has been elucidated from studies in neisseria species. o-linked glycosylation was first characterised in neisseria meningitidis, where the pilin protein was shown to be modified by a trisaccharide, with a similar glycan being found on n. gonorrhoeae. subsequent bioinformatics and directed mutagenesis led to the identification of an o-ost, called pgll, in n gonorrhoeae [38, 39]. pgll o-osts belong to a family of bacterial osts responsible for o-linked glycosylation of type iv pilins. this family appears to be widespread amongst pathogenic bacteria, including some strains of pseudomonas aeruginosa, where it is called pilo [40, 41]. moreover, it has recently been demonstrated that neisseria are able to decorate a diverse set of proteins via the o-ost pathway [42, 43]. research using neisseria and pseudomonas glycosylation systems in engineered e. coli cells has demonstrated that the biosynthesis of the o-linked glycan has a number of similarities to its n-linked counterpart (compare figures 1 and 5). the o-linked glycosylation pathway involves llos, and the glycans are transferred en bloc by the o-osts from the llos carrier onto the protein. the translocation of the llo substrate into the periplasm is required for activity and it has been shown that pglf, a protein with homology to o-antigen flippase, is required for pilin glycosylation which is thought to occur in an analogous manner to the wzy-dependent addition of o-antigen to the core-lps. in a similar fashion to the n-osts in archaea, bacteria, and lower eukaryotes, the o-ost's catalytic subunit is sufficient for glycosylation. as with o-glycosylation in eukaryotes, there appears to be no consensus sequence for defining sites of o-glycan attachment. interestingly, the neisseria o-osts display a pronounced substrate promiscuity when compared to n-osts, as demonstrated by their ability to transfer virtually any glycan from an llo carrier onto pilin in engineered e. coli cells. for example, it was shown that the neisseria pgll could transfer peptidoglycan subunits onto pilin, highlighting the potential for exploitation of such pathways for biotechnological purposes. as such it appears that the substrate specificity of the o-osts is found in the lipid carrier, a hypothesis nicely demonstrated using an in vitro glycosylation system that utilised purified neisseria pgll, pilin, and the lipid farnesyl pyrophosphate carrying a synthetic pentasaccharide that was successfully transferred onto the pilin protein. bacteroides comprise one of the most abundant genera of commensals in the human colon. exciting recent research suggests that these bacteria are not only capable of o-glycosylating many of their proteins but, unusually, they exploit a host-like pathway to add fucose (apparently acquired from their host glycans and/or from plant polysaccharides present in the gut) onto their glycoproteins and polysaccharides. a combination of cell biology and molecular biology experiments has provided convincing evidence for the existence of a general o-glycosylation system in these symbiotic bacteria which has all the hallmarks of the pilin o-ost-mediated pathogen pathway described earlier. notably, o-glycosylation appears to be central to the physiology of b. fragilis as well as its ability to colonise its ecological niche. although the structures of the b. fragilis o-glycans remain to be defined, many elements of the biosynthetic pathway are beginning to be unraveled. thus five glycosyltransferases, plus an unrelated fucosyltransferase, have been proposed to be involved in assembly of the llo on the cytoplasmic face of the inner membrane. translocation to the periplasm is thought to be mediated by the o-antigen flippase (wzx, see figure 2). however, there is no candidate gene as yet for the putative o-ost. interestingly, very recently it has been reported that fucosylated o-glycans are present on the fimbriae of porphyromonas gingivalis. like b. fragilis, it is conceivable, therefore, that p. gingivalis glycosylates its proteins via a similar pathway to b. fragilis. monosaccharide compositional analysis has shown that the p. gingivalis glycans are likely to be complex (fuc, xyl, man, gal, glc, galnac and glcnac have all been detected) but so far no sequences are available for this glycoprotein. all eukaryotic o-glycosylation is processive that is, it is a stepwise process which begins with the attachment of the linking monosaccharide to the acceptor serine or threonine. many eukaryotic o-glycans are of the mucin-type which are linked via galnac, but other classes exist which are attached to proteins via a variety of sugars including fuc, man, glc, gal, ara, xyl, and glcnac. most eukaryotic o-biosynthetic events take place in the golgi, although some classes of o-glycans, for example, o-man linked glycans (see later), are initiated in the er. an enormous variety of sequences can be attached to these linking sugars. thus there is a great diversity of o-glycosylation in the eukaryotic domain [6, 7]. the only substantive study was about twenty years ago when the s-layers of halobacterium salinarum and haloferax volcanii were shown to carry several o-linked disaccharides of sequence glc1-2gal. in contrast, there is a large body of evidence pointing to a rich diversity of o-glycans in the bacterial domain. the most complex structures have been found on bacterial s-layers which have been investigated in many species of bacteria over the last thirty years. their structures and biosynthesis have been comprehensively reviewed on several occasions [5, 31, 49, 50] and the reader is referred to these articles for further information. below we discuss emerging understanding of other families of bacterial cell surface and secreted o-linked glycoproteins, many of which, in contrast to the s-layers, have structural and/or functional counterparts in the eukaryotic domain. mucins are high molecular weight eukaryotic glycoproteins, produced in abundance by epithelial and goblet cells, whose polypeptide chains are coded by the muc genes. mucins are characterized by the presence of tandem repeats of serine/threonine/proline-rich sequences which are extensively o-glycosylated. mucins readily form gels and are a key component of most gel-like secretions in eukaryotes where they have functions ranging from lubrication to serving as receptors for microbes. in recent years, it has become evident that many bacterial biofilms contain glycoproteins whose compositions indicate that they are mucin-like molecules [5153]. the best characterized are the serine-rich repeat (srr) glycoproteins belonging to the fap1 family, which are conserved in streptococci, staphylococci, and lactobacilli, and are required for bacterial biofilm formation and pathogenesis. the polypeptides of srr family members are comprised of a long signal peptide followed in turn by a short serine-rich domain, an acidic or basic region, a long serine-rich domain and a c-terminal anchoring motif. the srr sequences are reminiscent of the eukaryotic mucins in that the majority of the polypeptide is comprised of tandem repeats of short motifs which have related sequences (see figure 6 for comparison of a portion of an srr domain compared with part of the human muc-1 sequence). the serine-rich domains have the key hallmark of the mucins, namely, variable repeated sequences rich in potential o-glycosylation sites (see figure 6), but in contrast to the mammalian mucins, proline is absent from the srr domains. thus their proline residues are important for ensuring that exposed, accessible sites are available for glycosylation. currently little is known about the process of glycan attachment to the bacterial srrs, other than the fact that attachment of the linking sugar appears to occur very rapidly in the cytoplasm, before transport to the cell surface via the accessory sec transporter. it has been proposed that the glycosylation mechanism is a two-step process, with the second step requiring several accessory secretion components and thus is probably coupled with secretion. recent electron microscopy structural studies have indicated that the serine dipeptide repeat domains have a super-helical extended structure with exposed serine side-chains, which are expected to be readily accessible to o-glycosylation. the glycan content of the srrs has been explored in five species, streptococcus parasanguinis, s gordonii, s. agalactiae, s. pneumoniae, and staphylococcal aureus. this has been largely done using lectins such as wheat germ agglutinin (wga) which recognize terminal glcnac, and by sugar compositional analyses. lectin blotting, supplemented by sugar composition data, has indicated that the linking sugar in the srrs is probably glcnac. interestingly two glycosyltransferases, called gtf1 and gtf2, are required for this initial glycosylation step by s. parasanguinis. gtf1 and gtf2 homologs from s. pneumoniae also form an enzyme complex that catalyzes the transfer of glcnac to serine-rich sites of psrp. a similar requirement for two glycosyltransferases adding a single sugar occurs in the o-mannosyl glycans of higher eukaryotes (see later) whose biosynthesis is initiated by a heterodimer enzyme complex composed of protein o-mannosyltransferases (pomt) 1 and 2. it should be noted that although o-linked glcnac is found on eukaryotic cytoplasmic and nuclear proteins, it is probably not analogous to srr o-glycosylation, because eukaryotic o-linked glcnac residues are not further extended. moreover, this ubiquitous eukaryotic glycosylation is unusual because it is dynamic and involves cross-talk with phosphorylation. a glucosyltransferase has recently been identified in s. parasanguinis that transfers glucose to the glcnac-modified fap1. although the structure of the product of glycosylation remains to be determined, it is tempting to speculate that these bacterial proteins might carry glycans whose core sequences are glucosyl analogues of the core type 1 sequence, gal1-3galnac, that is ubiquitous in mammalian mucins. sugars additional to glc and glcnac, including galnac and rha, have been observed at low levels in sugar analyses of the fap1 glycoproteins. it remains to be established whether the putative glc-glcnac moieties are further elongated or whether other glycans account for the compositional data. the title of a recent review protein o-mannosylation: conserved from bacteria to humans o-mannosyl glycans are abundant in yeast and fungi, whilst in mammals they occur on a restricted number of proteins, such as -dystroglycan where their impairment is a cause of congenital muscular dystrophy [63, 64]. yeast and fungi express short mannosyl oligomers, with galactose being present on terminal sites in some species. in contrast, mammalian o-mannosyl glycans carry sialylated and fucosylated n-acetyllactosamine sequences similar to those found in mucins. eukaryotic o-mannosylation is initiated in the er by the concerted action of two protein o-mannosyltransferases (pomt1 and 2) which employ dol-p-man as the mannose donor. chain extension subsequently takes place in the golgi. o-mannosyl glycans analogous to those found in yeast and fungi have been found on glycoproteins from members of the actinomycetes class of gram-positive bacteria which include the mycobacteria and streptomyces genera [62, 65]. the first to be characterised were the surface glycoproteins of mycobacterium tuberculosis [66, 67]. subsequently m. bovis, corynebacterium glutamicum, and streptomyces coelicolor were shown to be similarly glycosylated. all contain o-glycans whose sequences are restricted to short stretches of mannose (usually three residues or less). like in eukaryotes, the mannosyl donor is a polyprenol phosphate, and their protein o-mannosyltransferases (pomts) are membrane associated. the activities of the products of candidate pomt genes in m. tuberculosis, c. glutamicum, and s. coelicolor have been genetically and biochemically confirmed. the fact that bioinformatic screening has uncovered a plethora of pomt homologs in other species, indicates that o-mannosylation constitutes a general o-glycosylation pathway in actinomycetes. steps equivalent to the eukaryotic golgi processes of o-mannose extension have not been determined thus far in bacteria. there are, nevertheless, candidate mannosyltransferases, for example, those involved in the biosynthesis of the mannan core of cell wall lipomannan/lipoarabinomannan in mycobacteria. it has been suggested that o-mannose extension in mycobacteria is coupled with sec-dependent secretion in a manner akin to that proposed for the serine-rich proteins in streptococci and staphylococci described earlier. aida-i, tiba, and ag43 are three autotransporter proteins in pathogenic e. coli which are associated with virulence phenotypes, such as the formation of biofilms and aggregates. all three are extensively glycosylated with o-linked heptose on their so-called passenger domains [7174]. glycosylation occurs in the cytoplasm and the heptoses are derived from adp-glycero-manno-heptopyranose which is recruited from the lps biosynthetic pathway. the passenger domains are secreted to the extracellular environment where their glycosylation appears to enhance bacterial attachment to human cells. the heptoses are attached at multiple sites in ser/thr rich domains (figure 7) that are reminiscent of eukaryotic mucin sequences (see earlier) although they lack the hallmark tandem repeats of the latter. flagellin o-linked glycosylation has been widely reported in a number of bacteria, where it appears to be restricted, with the archaeal counterparts being n-linked. current knowledge of the o-linked sugars involved in flagellin glycosylation has been covered in recent reviews [75, 76]. probably the best studied flagellin glycans are found to be glycosylated with a family of sialic acid-like monosaccharides, based around the sugars pseudaminic acid and legionaminic acid, with a diversity being generated by variation in their decorating appendages. these sugars have been found in several species including campylobacter jejuni, helicobacter pylori, and aeromonas caviae. in c. jejuni, the o-linked glycosylation gene cluster has been identified and the function of a number of gene products involved in the pseudaminic acid biosynthetic pathway has been elucidated. it appears to have evolved to share some of the same biosynthetic machinery as the n-linked glycosylation pathway, allowing the organism to maintain a compact genome and avoid redundancy. flagellin glycosylation in p aeruginosa has also been shown to share biosynthetic machinery, in this case with the o-antigen pathway. the most complex flagellin o-glycans identified thus far have been found in hypervirulent strains of clostridium difficile [75, 78]. the c. difficile flagellins carry hexnac-linked oligosaccharides up to at least five sugars in length. in contrast to the en bloc transfer in the n-linked pathway, it is apparent that flagellin o-linked glycosylation is likely to proceed in a sequential fashion. given that a single sugar residue is often added to sites of attachment, specific glycosyltransferases are thought to be involved in the glycosylation process, but our present understanding of the glycosyltransferases involved in the glycosylation process remains limited. the current proposed model for o-linked flagellin glycosylation occurs at the cytoplasmic face of the inner membrane in the vicinity of the type iii secretion complex. nucleotide activated sugars are utilised by specific glycosyltransferases in the glycosylation machinery and are added to exposed serine and threonine residues. the glycosylated flagellin monomers are then secreted to the tip of the growing flagellin filament. however, it is well known that certain sequence motifs are preferred in mucin type o-glycosylation. indeed the netoglyc open access tool, which can be very helpful for predicting possible sites of eukaryotic mucin o-glycosylation, was developed using knowledge of preferred sequence motifs. the rapid progress that is being made in defining o-glycosylation sites in diverse prokaryotic glycoproteins, coupled with the fact that some researchers are beginning to employ the netoglyc tool to guide them in the choice of targets for mutation in searches for prokaryotic glycosylation [67, 80], have made it timely to assess the applicability of the netoglyc tool to prokaryotic glycoprotein research. in a preliminary unpublished study, we have ascertained netoglyc predictions for selected members of each of the families of prokaryotic o-glycoproteins described in the previous sections, for which there is published experimental data on site occupancy. the outputs send the very clear message that netoglyc does not, in fact, correctly predict o-glycosylation in most families of prokaryotic glycoproteins. thus we found that no sites were correctly predicted in the pilins, flagellins, serine-rich proteins, the autotransporters, or the bf2494 glycoprotein of b. fragilis. experimental data suggests that the latter has an essential three-residue glycosylation site motif (d)(s/t)(a/i/l/v), which is not typical of eukaryotes, so it is therefore not surprising that netoglyc appears to be not suitable for predicting this type of o-glycosylation. in conclusion, netoglyc is likely to only be useful for predicting o-glycosylation in prokaryotic glycoproteins like mycobacterial lipoproteins whose pro/ser/thr-rich glycosylation domains have sequence characteristics which they share with the mammalian mucins. prediction in other families will require the development of new algorithms which take account of their specific glycosylation domain characteristics. although much remains to be uncovered concerning protein glycosylation in prokaryotes, several themes are emerging from the discoveries of the past decade. firstly, like in eukaryotes, n-glycosylation is largely restricted to asn-x-ser/thr consensus sequences, even when the canonical oligosaccharyltransferase pathway is not involved. secondly o-glycosylation is far more abundant in bacteria than in archaea whilst the reverse is true for n-glycosylation. thirdly, oligosaccharyltransferase-mediated o-glycosylation is likely to be widespread in gram-negative bacteria. in contrast this type of o-glycosylation has not thus far been proven to occur experimentally in gram-positive bacteria or archaea. finally cytoplasmic o-glycosylation appears to be both more common and more diverse in gram-positive compared with gram-negative bacteria, possibly because of the existence of the alternative periplasmic o-ost pathway of the latter.

recent years have witnessed a rapid growth in the number and diversity of prokaryotic proteins shown to carry n- and/or o-glycans, with protein glycosylation now considered as fundamental to the biology of these organisms as it is in eukaryotic systems. this article overviews the major glycosylation pathways that are known to exist in eukarya, bacteria and archaea. these are (i) oligosaccharyltransferase (ost)-mediated n-glycosylation which is abundant in eukarya and archaea, but is restricted to a limited range of bacteria; (ii) stepwise cytoplasmic n-glycosylation that has so far only been confirmed in the bacterial domain; (iii) ost-mediated o-glycosylation which appears to be characteristic of bacteria; and (iv) stepwise o-glycosylation which is common in eukarya and bacteria. a key aim of the review is to integrate information from the three domains of life in order to highlight commonalities in glycosylation processes. we show how the ost-mediated n- and o-glycosylation pathways share cytoplasmic assembly of lipid-linked oligosaccharides, flipping across the er/periplasmic/cytoplasmic membranes, and transferring en bloc to the protein acceptor. moreover these hallmarks are mirrored in lipopolysaccharide biosynthesis. like in eukaryotes, stepwise o-glycosylation occurs on diverse bacterial proteins including flagellins, adhesins, autotransporters and lipoproteins, with o-glycosylation chain extension often coupled with secretory mechanisms.

PMC3068309

pubmed-625

all reactions were carried out under a nitrogen atmosphere in oven- or flame-dried glassware. all catalysts and reagents were obtained from commercial sources and were used without further purification with the exception of bf3oet2, which was purified by distillation from cah2. n-allyl-4-methylaniline, 2-allyl-4-methylaniline, 2-allylaniline, 2-bromocyclohex-1-ene-1-carbaldehyde, and 2-(but-3-en-2-yl)aniline were prepared according to published procedures. structural and stereochemical assignments were made on the basis of 2-d cosy, hsqc, and noesy experiments. ratios of diastereomers were determined by h nmr analysis. high resolution esi mass spectra were acquired using an instrument equipped with at tof mass analyzer, and high resolution ei mass spectra were acquired using an instrument equipped with a magnetic sector mass analyzer. yields refer to isolated yields of compounds estimated to be 95% pure as determined by h nmr analysis unless otherwise noted. the yields reported in the supporting information describe the result of a single experiment, whereas the yields reported in scheme 2 and table 1 are average yields of two or more experiments. thus, the yields reported in the supporting information may differ from those shown in scheme 2 and table 1. a thick-walled glass pressure tube equipped with a stir bar was sealed with a septum. the pressure tube was flame-dried and cooled under a vacuum and then purged with nitrogen. the flask was charged with n-allyl-4-methoxyaniline (2.5 g, 15.2 mmol) and xylenes (30.5 ml). the solution was cooled to 0 c, bf3oet2 was added dropwise, and the resulting solution was then allowed to warm to room temperature. the septum was removed, the pressure tube was replaced with a teflon screw cap, the tube was immersed in a 170 c oil bath, and the reaction mixture was allowed to stir for 2 h. the mixture was then cooled to room temperature, and 1 m naoh (15 ml) was added. the resulting mixture was transferred to a separatory funnel, the layers were separated, and the aqueous layer was extracted with dichloromethane (3 15 ml). the combined organic layers were dried over anhydrous na2so4, filtered, and then concentrated in vacuo. the crude product was purified via flash chromatography on silica gel using 5% ethyl acetate: hexanes as the eluent to afford 870 mg (35%) of the title compound as an orange oil: h nmr (700 mhz, cdcl3) 6.666.62 (m, 3 h), 5.975.91 (m, 1 h), 5.135.08 (m, 2 h), 3.74 (s, 3 h), 3.40 (s, br, 2 h), 3.28 (d, j=5.6 hz, 2 h); c nmr (175 mhz, cdcl3) 152.9, 138.3, 135.7, 125.7, 116.9, 116.2, 116.0, 112.7, 55.7, 36.6; ir (film) 3357, 1500 cm; ms (esi+) 164.1071 (164.1070 calcd for c10h13no, m+h). a flame-dried flask equipped a stir bar was cooled under a vacuum and then purged with nitrogen. this flask was charged with 2-iodonitrobenzene (3.6 g, 14.5 mmol) and thf (58 ml). the resulting solution was cooled to 40 c in a mecn/co2(s) bath, and then a solution of phmgbr (16 ml, 16.00 mmol, 1 m in thf) was added dropwise. the resulting mixture was stirred at 40 c for 5 min, and then a solution of cucn2licl (29 ml, 29 mmol, 1 m in thf) was added dropwise. the reaction mixture was stirred at 40 c for 10 min, and then 2-methylallyl bromide (2.4 g, 17.5 mmol) was added dropwise. the mixture was stirred at 40 c for 1 h, and then the reaction was quenched with nh4cl (50 ml) and transferred to a separatory funnel. water (50 ml) was added, the layers were separated, and the aqueous layer was extracted with etoac (2 50 ml). the organic layers were then combined, washed with brine, dried over anhydrous na2so4, filtered, and concentrated in vacuo. the crude product was purified via flash chromatography on silica gel using 5% ethyl acetate: hexanes as the eluent to yield slightly impure 2-(2-methylallyl)nitrobenzene (1.8 g, 70% yield), which was carried on to the next step without further purification. a flame-dried flask equipped with a stir bar was cooled under a vacuum, purged with nitrogen, and charged with 2-(2-methylallyl)nitrobenzene (1.8 g, 10 mmol) and etoh (67 ml). the resulting mixture was stirred at rt until the 2-(2-methylallyl)nitrobenzene was completely dissolved, then powdered zinc (9.9 g, 151 mmol) and acoh (8.6 ml, 151 mmol) were added. the resulting mixture solution was stirred at rt for 1 h and then was filtered through celite, and the celite was washed with et2o. the crude product was purified via flash chromatography on silica gel using 25% ethyl acetate: hexanes as the eluent to afford 1.16 g (54%) of the title compound as a yellow oil that contained ca. data are for the major product: h nmr (400 mhz, cdcl3) 7.097.02 (m, 2 h), 6.74 (t, j=7.6 hz, 1 h), 6.67 (d, j=7.6 hz, 1 h), 4.87 (s, 1 h), 4.74 (s, 1 h), 3.68 (s, br, 2 h), 3.28 (s, 2 h), 1.73 (s, 3 h); c nmr (100 mhz, cdcl3) 145.2, 143.6, 131.0, 127.5, 123.9, 118.7, 115.9, 111.6, 41.1, 22.3; ir (film) 3394, 1480 cm; ms (esi+) 148.1125 (148.1121 calcd for c10h13n, m+h). a flame-dried flask equipped with a stir bar and a reflux condenser the flask was charged with the appropriate 2-allylaniline derivative (1.25 equiv), the appropriate 2-bromobenzaldehyde derivative (1.0 equiv), 4 molecular sieves (1 g/mmol), and toluene (0.2 m). the resulting solution was heated to reflux with stirring overnight. the mixture was then cooled to rt and filtered, and the solids were washed with etoac. the combined organic solutions were then concentrated in vacuo, and the resulting crude imine was immediately used without further purification. a flame-dried flask with a stir bar was cooled under a vacuum and then purged with nitrogen. the flask was charged with the crude imine (1 equiv) and et2o (0.5 m) and was cooled to 0 c. a solution of lialh4 (2 equiv, 4 m in et2o) was added dropwise, and the resulting solution was allowed to warm to rt and stir overnight. the mixture was diluted with et2o, and then water (0.1 ml/mmol of lah) was carefully added dropwise to the solution followed by dropwise addition of 1 m naoh (0.1 ml/mmol of lah). additional water was then added dropwise until a white solid precipitated and clung to the walls of the flask. the solution was filtered, the flask was washed with et2o, and the resulting solution was also filtered. the combined organic solutions were then concentrated in vacuo, and the crude product was then purified via flash chromatography on silica gel using 25% ethyl acetate: hexanes as the eluent. the coupling of 2-allylaniline (894 mg, 6.72 mmol) with 2-bromobenzaldehyde (994 mg, 5.37 mmol) was accomplished according to general procedure a. this procedure afforded 1.01 g (62%) of the title compound as a white solid: mp 4244 c; h nmr (400 mhz, cdcl3) 7.62 (d, j=8 hz, 1 h), 7.39 (d, j=7.6 hz, 1 h), 7.267.22 (m, 1 h), 7.147.06 (m, 3 h), 6.71 (t, j= 7.2 hz, 1 h), 6.53 (d, j=8 hz, 1 h), 6.035.93 (m, 1 h), 5.165.09 (m, 2 h), 4.43 (s, 2 h), 4.27 (s, br, 1 h), 3.35 (d, j=6 hz, 2 h); c nmr (175 mhz, cdcl3) 145.7, 138.2, 136.0, 132.8, 129.9, 129.0, 128.6, 127.7, 127.5, 123.6, 123.2, 117.6, 116.4, 110.9, 48.2, 36.6; ir (film) 3440, 1512 cm; ms (esi+) 302.0544 (302.0539 calcd for c16h16brn, m+h). the coupling of 2-allyl-4-methoxyaniline (1.22 g, 7.47 mmol) with 2-bromobenzaldehyde (1.10 g, 5.97 mmol) was conducted according to general procedure a. this procedure afforded 1.09 g (55%) of the title compound as an orange oil: h nmr (400 mhz, cdcl3) 7.56 (d, j=8 hz, 1 h), 7.34 (d, j=7.2 hz, 1 h), 7.267.23 (m, 1 h), 7.12 (t, j=8 hz, 1 h), 6.726.63 (m, 2 h), 6.49 (d, j=8.8 hz, 1 h), 6.025.92 (m, 1 h), 5.155.09 (m, 2 h), 4.38 (s, 2 h), 3.97 (s, br, 1 h), 3.74 (s, 3 h), 3.33 (d, j=6 hz, 2 h); c nmr (175 mhz, cdcl3) 152.1, 139.8, 138.5, 135.7, 132.7, 129.1, 128.5, 127.5, 125.7, 123.3, 116.6, 116.6, 112.2, 112.1, 55.7, 49.0, 36.5; ir (film) 3428, 1508 cm; ms (esi+) 332.0645 (332.0645 calcd for c17h18brno, m+h). the coupling of 2-allyl-4-methylaniline (2.20 g, 15.0 mmol) with 2-bromobenzaldehyde (2.21 g, 12.0 mmol) was conducted according to general procedure a. this procedure afforded 2.07 g (55%) of the title compound as an orange oil: h nmr (400 mhz, cdcl3) 7.56 (d, j=8 hz, 1 h), 7.34 (d, j=7.6 hz, 1 h), 7.257.21 (m, 1 h), 7.11 (t, j=7.2 hz, 1 h), 6.916.89 (m, 2 h), 6.44 (d, j=8.8 hz, 1 h), 6.035.93 (m, 1 h), 5.145.09 (m, 2 h), 4.40 (s, 2 h), 4.14 (s, br, 1 h), 3.32 (d, j=6.4 hz, 2 h), 2.23 (s, 3 h); c nmr (175 mhz, cdcl3) 143.4, 138.4, 136.1, 132.7, 130.7, 129.0, 128.5, 128.0, 127.5, 126.7, 123.8, 123.3, 116.3, 111.1, 48.5, 36.6, 20.4; ir (film) 3437, 1513 cm; ms (esi+) 316.0695 (316.0695 calcd for c17h18brn, m+h). the coupling of 2-allylaniline (832 mg, 6.25 mmol) with 2-bromocyclohex-1-ene-1-carbaldehyde (945 mg, 5.00 mmol) was conducted according to general procedure a. this procedure afforded 989 mg (65%) of the title compound as a yellow oil that contained ca. data are for the major product: h nmr (400 mhz, cdcl3) 7.15 (t, j=8 hz, 1 h), 7.04 (d, j=6.8 hz, 1 h), 6.70 (t, j=8 hz, 1 h), 6.64 (d, j=8 hz, 1 h), 6.005.90 (m, 1 h), 5.145.08 (m, 2 h), 4.123.91 (m, 3h), 3.29 (d, j=6.4 hz, 2 h), 2.602.50 (m, 2 h), 2.182.09 (m, 2 h), 1.711.60 (m, 4 h); c nmr (175 mhz, cdcl3) 146.2, 136.1, 133.9, 129.8, 127.8, 123.6, 121.3, 117.3, 116.3, 110.8, 49.4, 36.8, 36.6, 29.3, 24.7, 22.4; ir (film) 3438, 1508 cm; ms (esi+) 306.0852 (306.0852 calcd for c16h20brn, m+ h). the coupling of 2-allylaniline (1.66 g, 12.5 mmol) with 2-bromo-5-fluorobenzaldehyde (2.02 g, 10.0 mmol) was conducted according to general procedure a. this procedure afforded 2.28 g (72%) of the title compound as a pale yellow solid: mp 4647 c; h nmr (700 mhz, cdcl3) 7.49 (dd, j=4.9, 8.4 hz, 1 h), 7.107.06 (m, 3 h), 6.83 (t, j=7.7 hz, 1 h), 6.72 (t, j=7 hz, 1 h), 6.43 (d, j=7.7 hz, 1 h), 6.025.96 (m, 1 h), 5.175.12 (m, 2 h), 4.38 (s, 2 h), 4.30 (s, br, 1 h), 3.37 (d, j=4.9, 2 h); c nmr (175 mhz, cdcl3) 162.4 (d, j=245.3 hz), 145.3, 140.8 (d, j=6.8 hz), 136.0, 133.9 (d, j=7.3 hz), 130.1, 127.8, 123.7, 117.9, 116.8 (d, j=2.6 hz), 116.5, 115.9 (d, j=23.8 hz), 115.6 (d, j=22.9 hz), 110.8, 48.1, 36.6; ir (film) 3440, 1463 cm; ms (esi+) 320.0450 (320.0445 calcd for c16h15brfn, m+h). the coupling of 2-allylaniline (1.66 g, 12.5 mmol) with 6-bromo-1,3-benzodioxole-5-carboxaldehyde (2.29 g, 10.0 mmol) was conducted according to general procedure a. this procedure afforded 2.51 g (73%) of the title compound as a white solid: mp 5355 c; h nmr (400 mhz, cdcl3) 7.137.06 (m, 2 h), 7.02 (s, 1 h), 6.85 (s, 1 h), 6.71 (t, j=7.6 hz, 1 h), 6.51 (d, j=8.4 hz, 1 h), 6.035.94 (m, 3 h), 5.165.09 (m, 2 h), 4.31 (d, j=5.6 hz, 2 h), 4.22 (s, br, 1 h), 3.34 (d, j=6 hz, 2 h); c nmr (175 mhz, cdcl3) 147.6, 147.4, 145.6, 136.0, 131.6, 129.9, 127.7, 123.7, 117.6, 116.4, 113.3, 112.8, 110.9, 109.0, 101.6, 48.1, 36.5; ir (film) 3439, 1478 cm; ms (esi+) 346.0440 (346.0437 calcd for c17h16brno2, m+h). the coupling of 2-allylaniline (1.7 g, 12.5 mmol) with 1-bromo-2-naphthaldehyde (2.35 g, 10.00 mmol) was conducted according to general procedure a except after addition of lialh4 the reaction mixture was heated to reflux overnight. this procedure afforded 2.77 g (79%) of the title compound as an orange solid: mp 9597 c; h nmr (400 mhz, cdcl3) 8.34 (d, j=8.4 hz, 1 h), 7.80 (d, j=8 hz, 1 h), 7.74 (d, j=8.8 hz, 1 h), 7.60 (t, j=7.2 hz, 1 h), 7.527.48 (m, 2 h), 7.107.06 (m, 2 h), 6.71 (t, j=7.2 hz, 1 h), 6.55 (d, j=8.8 hz, 1 h), 6.055.95 (m, 1 h), 5.165.11 (m, 2 h), 4.66 (s, 2 h), 4.39 (s, br, 1 h), 3.37 (d, j=6.0 hz, 2 h); c nmr (175 mhz, cdcl3) 145.8, 136.6, 136.0, 133.9, 132.4, 130.0, 128.1, 127.8, 127.8, 127.4, 126.9, 126.3, 126.1, 123.7, 123.0, 117.6, 116.5, 111.0, 49.2, 36.6; ir (film) 3442, 1541 cm; ms (esi+) 352.0700 (352.0695 calcd for c20h18brn, m+h). the coupling of 2-(2-methylallyl)aniline (1.16 g, 7.84 mmol) with 2-bromobenzaldehyde (1.16 g, 6.28 mmol) was conducted according to general procedure a. this procedure afforded 767 mg (39%) of the title compound as a white solid: mp 4346 c; h nmr (400 mhz, cdcl3) 7.56 (d, j=8 hz, 1 h), 7.32 (d, j=6 hz, 1 h), 7.257.21 (m, 1 h), 7.147.04 (m, 3 h), 6.70 (t, j=7.2 hz, 1 h), 6.51 (d, j=8 hz, 1 h), 4.88 (s, 1 h), 4.76 (s, 1 h), 4.424.39 (m, 3 h), 3.33 (s, 2 h), 1.73 (s, 3 h); c nmr (175 mhz, cdcl3) 146.1, 143.7, 138.3, 132.7, 130.8, 128.8, 128.5, 127.7, 127.5, 123.5, 123.2, 117.4, 112.0, 110.9, 48.1, 41.4, 22.3; ir (film) 3430, 1510 cm; ms (esi+) 316.0692 (316.0695 calcd for c17h18brn, m+h). the coupling of 2-(but-3-en-2-yl)aniline (298 mg, 2.02 mmol) with 2-bromobenzaldehyde (300 mg, 1.62 mmol) was conducted according to general procedure a. this reaction afforded 451 mg (88%) of the title compound as a yellow oil that contained ca. data are for the major product: h nmr (400 mhz, cdcl3) 7.57 (d, j=8 hz, 1 h), 7.34 (d, j=7.6 hz, 1 h), 7.247.22 (m, 1 h), 7.157.07 (m, 3 h), 6.75 (t, j=8 hz, 1 h), 6.54 (d, j=8 hz, 1 h), 6.035.94 (m, 1 h), 5.125.07 (m, 2 h), 4.424.37 (m, 3 h), 3.52 (quint, j=6.4 hz, 1 h), 1.43 (d, j=7.2 hz, 3 h); c nmr (175 mhz, cdcl3) 145.2, 142.3, 138.3, 132.8, 129.0, 128.6, 128.5, 127.5, 127.4, 126.9, 123.3, 117.7, 114.2, 111.1, 48.3, 38.1, 18.9; ir (film) 3434, 1506 cm; ms (esi+) 316.0693 (316.0695 calcd for c17h18brn, m+h). the coupling of 2-allylaniline (1.66 g, 12.5 mmol) with 2-bromoacetophenone (2.00 g, 10.0 mmol) was conducted according to general procedure a except after addition of lialh4 the reaction mixture was heated to reflux overnight. this procedure afforded 1.75 g (55%) of the title compound as a yellow oil that contained ca. 6% of an unknown impurity: h nmr (400 mhz, cdcl3) 7.54 (d, j=8 hz, 1 h), 7.37 (d, j=7.6 hz, 1 h), 7.19 (t, j=7.2 hz, 1 h), 7.087.02 (m, 2 h), 6.97 (t, j=8 hz, 1 h), 6.63 (t, j=7.6 hz, 1 h), 6.18 (d, j=8 hz, 1 h), 6.075.97 (m, 1 h), 5.235.18 (m, 2 h), 4.84 (quint, j=6.4 hz, 1 h), 4.25 (s, br, 1 h), 3.40 (d, j=6 hz, 2 h), 1.48 (d, j=6.4 hz, 3 h); c nmr (175 mhz, cdcl3) 144.7, 143.6, 136.3, 132.9, 129.7, 128.3, 128.0, 127.6, 126.7, 122.9, 122.6, 117.2, 116.3, 111.6, 52.4, 36.9, 23.1; ir (film) 3746, 1506 cm; ms (esi+) 316.0692 (316.0695 calcd for c17h18brn, m+h). a flame-dried schlenk tube equipped with a stir bar was cooled under a vacuum and then purged with nitrogen. the tube was charged with naobu (96 mg, 1 mmol), pd2(dba)3 (9 mg, 0.01 mmol), and pcy3hbf4 (15 mg, 0.040 mmol). the substrate (0.5 mmol) was dissolved in toluene (5 ml) and added to the flask. the resulting solution was heated 105 c with stirring for 18 h, at which time the substrate was judged to be completely consumed by gc, tlc, or h nmr analysis of an aliquot removed from the reaction mixture. the mixture was then cooled to rt, and saturated aqueous nh4cl (10 ml) was added. the mixture was transferred to a separatory funnel, the layers were separated, and the aqueous layer was extracted with etoac (3 15 ml). the combined organic layers were then dried over anhydrous na2so4, filtered, and concentrated in vacuo. the crude product was then purified via flash chromatography on silica gel using 2% ethyl acetate: hexanes as the eluent to afford a product that still contained small amounts of impurities. the product was then dissolved in 30% et2o/hexanes (5 ml, 0.1 m) and 2 m hcl in et2o (1.5 ml, 3 equiv) was added dropwise. the resulting solution was stirred at rt for 1 min, and a white solid (the product hcl salt) precipitated from the solution. the salt was then dissolved in dicholoromethane (10 ml) and transferred to a separatory funnel. a solution of 4 m naoh was added, and the mixture was shaken vigorously for 1 min. the layers were then separated, and the aqueous layer was extracted with dichloromethane (3 15 ml). the combined organic layers were dried over anhydrous na2so4, filtered, and concentrated in vacuo to afford the desired product. the cyclization of 3a (151 mg, 0.500 mmol) was conducted according to general procedure b to afford 94 mg (85%) of the title compound as a yellow solid: mp 125127 c; h nmr (400 mhz, cdcl3) 7.207.10 (m, 6 h), 6.72 (t, j=7.2 hz, 1 h), 6.55 (d, j=8 hz, 1 h), 4.58 (d, j=15.6 hz, 1 h), 4.02 (d, j=15.2 hz, 1 h), 3.583.50 (m, 1 h), 3.15 (dd, j=7.6, 15.2 hz, 1 h), 3.053.00 (m, 2 h), 2.75 (dd, j=11.2, 15.0 hz, 1 h); c nmr (175 mhz, cdcl3) 151.8, 134.7, 133.5, 129.5, 129.3, 127.5, 126.9, 126.3, 126.1, 124.4, 118.5, 107.0, 61.6, 48.5, 35.7, 35.6; ir (film) 2778, 1456 cm; ms (ei) 221.1201 (221.1204 calcd for c16h15n, m). the cyclization of 3b (166 mg, 0.500 mmol) was conducted according to general procedure b except only 1 equiv of 2 m hcl in et2o was used. this procedure afforded 86 mg (68%) of the title compound as a yellow solid: mp 134 c (dec); h nmr (700 mhz, cdcl3) 7.197.14 (m, 4 h), 6.80 (s, 1 h), 6.68 (d, j=7 hz, 1 h), 6.50 (d, j=7 hz, 1 h), 4.54 (d, j=14.7 hz, 1 h), 3.93 (d, j=14.7 hz, 1 h), 3.74 (s, 3 h), 3.453.40 (m, 1 h), 3.11 (dd, j=7.7, 14.7 hz, 1 h), 3.053.02 (m, 2 h), 2.73 (dd. j=11.9, 14.7 hz, 1 h); c nmr (175 mhz, cdcl3) 153.4, 146.2, 134.6, 133.6, 131.2, 129.3, 126.9, 126.3, 126.0, 112.2, 111.6, 107.3, 62.6, 56.0, 49.6, 36.0, 35.4; ir (film) 2781, 1486 cm; ms (ei) 251.1314 (251.1310 calcd for c17h17no, was conducted according to general procedure b to afford 98 mg (81%) of the title compound as a brown solid: mp 6870 c; h nmr (400 mhz, cdcl3) 7.217.15 (m, 4 h), 6.976.92 (m, 2 h), 6.49 (d, j=8 hz, 1 h), 4.58 (d, j= 15.2 hz, 1 h), 3.98 (d, j=15.2 hz, 1 h), 3.523.44 (m, 1 h), 3.11 (dd, j=14.8, 7.6 hz, 1 h), 3.043.02 (d, j=6.8 hz, 2 h), 2.74 (dd, j=14.8, 11.2 hz, 1 h), 2.27 (s, 3 h); c nmr (175 mhz, cdcl3) 149.8, 134.8, 133.7, 129.9, 129.3, 128.0, 127.6, 127.0, 126.4, 126.1, 125.4, 107.0, 62.2, 49.2, 35.7, 35.6, 20.9; ir (film) 2918, 1491 cm; ms (ei) 235.1358 (235.1361 calcd for c17h17n, m). the cyclization of 3d (153 mg, 0.500 mmol) was conducted according to general procedure b. in order to remove a small amount of unreacted starting material from the product, the crude product was dissolved in dichloromethane (2.5 ml), and dmap (6.1 mg, 0.050 mmol) and et3n (104 l, 0.750 mmol) were added to the solution. acetic anhydride (61 l, 0.650 mmol) was added, and the solution was allowed to stir at rt for 3 h and was then concentrated in vacuo. the crude product was purified via flash chromatography to afford 62 mg (55%) of the title compound as a brown solid: mp 8890 c; h nmr (700 mhz, cdcl3) 7.097.06 (m, 2 h), 6.67 (t, j=7.0 hz, 1 h), 6.45 (d, j=7.7 hz, 1 h), 3.68 (d, j=15.4 hz, 1 h), 3.343.29 (m, 1 h), 3.18 (d, j=15.4 hz, 1 h), 3.03 (dd, j=7.0, 14.7 hz, 1 h), 2.61 (t, j=12.6 hz, 1 h), 2.21 (t, j=14.7 hz, 1 h), 2.12 (d, j=16.1 hz, 1 h), 2.021.91 (m, 4 h), 1.771.71 (m, 2 h), 1.571.52 (m, 2 h); c nmr (175 mhz, cdcl3) 151.9, 129.6, 127.5, 127.3, 125.7, 124.2, 118.1, 106.8, 61.5, 49.6, 36.5, 35.8, 30.2, 27.7, 22.9, 22.8; ir (film) 2916, 1607 cm; ms (ei) 225.1518 (225.1518 calcd for c16h19n, m). the cyclization of 3e (161 mg, 0.505 mmol) was conducted according to general procedure b to afford 105 mg (87%) of the title compound as a yellow solid: mp 9293 c; h nmr (400 mhz, cdcl3) 7.137.07 (m, 3 h), 6.896.85 (m, 2 h), 6.73 (t, j=7.2 hz, 1 h), 6.54 (d, j=8 hz, 1 h), 4.54 (d, j=15.6 hz, 1 h), 3.98 (d, j=15.6 hz, 1 h), 3.553.47 (m, 1 h), 3.15 (dd, j=4.6, 14.8 hz, 1 h), 3.022.90 (m, 2 h), 2.74 (dd, j=11.2, 14.8 hz, 1 h); c nmr (175 mhz, cdcl3) 161.3 (d, j=242.6 hz), 151.6, 135.4 (d, j=7 hz), 130.7 (d, j=7.5 hz), 130.3 (d, j=2.6 hz), 129.5, 127.6, 124.5, 118.8, 113.6 (d, j=21 hz), 113.3 (d, j=21.7 hz), 107.1, 61.8, 48.6 (d, j=1.9 hz), 35.6, 34.9; ir (film) 2789, 1480 cm; ms (ei) 239.1116 (239.1110 calcd for c16h14fn, m). the cyclization of 3f (173 mg, 0.500 mmol) was conducted according to general procedure b to afford 95 mg (72%) of the title compound as a white solid: mp 9092 c; h nmr (400 mhz, cdcl3) 7.127.09 (m, 2 h), 6.72 (t, j=7.2 hz, 1 h), 6.60 (d, j=6.8 hz, 2 h), 6.53 (d, j=8 hz, 1 h), 5.88 (s, 2 h), 4.46 (d, j=14.8 hz, 1 h), 3.92 (d, j=14.8 hz, 1 h), 3.523.43 (m, 1 h), 3.13 (dd, j=7.6, 15.0 hz, 1 h), 2.90 (d, j=7.2 hz, 2 h), 2.74 (dd, j=10.8, 15.0 hz, 1 h); c nmr (175 mhz, cdcl3) 151.6, 146.1, 146.1, 129.4, 127.6, 127.4, 126.2, 124.3, 118.5, 108.8, 106.9, 106.5, 100.8, 61.5, 48.5, 35.5, 35.5; ir (film) 2891, 1484 cm; ms (ei) 265.1103 (265.1103 calcd for c17h15no2, m). the cyclization of 3 g (173 mg, 0.491 mmol) was conducted according to general procedure b to afford 106 mg (80%) of the title compound as a red solid: mp 9597 c; h nmr (400 mhz, cdcl3) 7.94 (d, j=8.4 hz, 1 h), 7.79 (d, j=8 hz, 1 h), 7.66 (d, j= 8.4 hz, 1 h), 7.50 (t, j=7.2 hz, 1 h), 7.44 (t, j=6.8 hz, 1 h), 7.22 (d, j=8.4 hz, 1 h), 7.167.12 (m, 2 h), 6.75 (t, j=7.2 hz, 1 h), 6.60 (d, j=8 hz, 1 h), 4.64 (d, j=15.6 hz, 1 h), 4.12 (d, j=15.6 hz, 1 h), 3.603.51 (m, 2 h), 3.22 (dd, j=7.2, 14.8 hz, 1 h), 3.09 (t, j=12.8 hz, 1 h), 2.85 (dd, j=11.6, 14.2 hz, 1 h); c nmr (175 mhz, cdcl3) 151.9, 132.5, 132.4, 130.9, 129.9, 129.8, 128.6, 127.6, 126.6, 126.3, 125.4, 125.4, 124.6, 122.9, 118.8, 107.3, 61.8, 49.3, 36.1, 32.0; ir (film) 2924, 1482 cm; ms (ei) 271.1351 (271.1361 calcd for c20h17n, m). the cyclization of 3h (163 mg, 0.517 mmol) was conducted according to general procedure b to afford 97 mg (80%) of the title compound as a yellow solid: mp 4750 c; h nmr (700 mhz, cdcl3) 7.197.14 (m, 3 h), 7.06 (m, 3 h), 6.63 (t, j=7.0 hz, 1 h), 6.41 (d, j=7.7 hz, 1 h), 4.50 (d, j=16.1 hz, 1 h), 4.25 (d, j=16.1 hz, 1 h), 3.10 (d, j=15.4 hz, 1 h), 2.94 (s, 2 h), 2.70 (d, j=15.4 hz, 1 h), 1.15 (s, 3 h); c nmr (175 mhz, cdcl3) 150.5, 134.6, 132.9, 129.8, 128.3, 127.6, 126.5, 126.4, 126.0, 124.8, 117.2, 106.2, 62.3, 43.7, 43.7, 40.0, 20.9; ir (film) 2924, 1482 cm; ms (ei) 235.1364 (235.1361 calcd for c17h17n, the cyclization of 3i (64 mg, 0.202 mmol) was conducted according to general procedure b to afford 36 mg (75%) of the title compound as a yellow oil. this product was judged to have been formed with 18:1 dr by h nmr analysis. data are for the major isomer: h nmr (700 mhz, cd3od) 7.197.15 (m, 4 h), 7.097.06 (m, 2 h), 6.76 (t, j=7.7 hz, 1 h), 6.62 (d, j=7.7 hz, 1 h), 4.58 (d, j=15.4 hz, 1 h), 3.83 (d, j=14.7 hz, 1 h), 3.06 (d, j=14.7 hz, 1 h), 2.922.84 (m, 3 h), 1.37 (d, j=6.3 hz, 3 h); c nmr (175 mhz, cd3od) 152.7, 136.1, 135.6, 134.6, 130.3, 128.4, 127.9, 127.4, 127.1, 123.5, 120.3, 108.7, 71.5, 50.3, 43.3, 35.0, 16.1; ir (film) 3676, 1506 cm; ms (ei) 235.1363 (235.1361 calcd for c17h17n, m). the cyclization of 3j (165 mg, 0.500 mmol) was conducted according to the general procedure b to afford 64 mg (52%) of the title compound as a yellow oil. 2:1:1 mixture of the two possible diastereomers and a side product tentatively assigned as 6-methyl-11-methylene-5,6,11,12-tetrahydrodibenzo[b, f]azocine, which results from competing intramolecular heck reaction. data are for the mixture: h nmr (700 mhz, cdcl3) 7.267.24 (m, 1 h), 7.227.20 (m, 1.5 h), 7.197.16 (m, 2.5 h), 7.167.14 (m, 2.5 h), 7.127.07 (m, 4.5 h), 7.02 (d, j=7.7 hz, 1 h), 6.97 (t, j=10.5 hz, 1 h), 6.686.63 (m, 2 h), 6.60 (d, j=7.7 hz, 0.5 h), 6.48 (d, j=8.4 hz, 1 h), 6.45 (d, j=7.7 hz, 0.5 hz), 6.29 (ddd, j=3.5, 10.5, 17.3 hz, 0.5 h), 5.235.13 (m, 1 h), 4.844.78 (m, 1.5 h), 4.534.50 (m, 1 h), 3.973.92 (m, 1 h), 3.763.60 (m, 1 h), 3.23 (dd, j=8.4, 14.7 hz, 1 h), 3.062.92 (m, 4 h), 2.772.72 (m, 1.5 h), 1.55 (d, j=6.3 hz, 1.5 h), 1.53 (d, j=7.0 hz, 1.5 h), 1.36 (d, j=7.0 hz, 3 h); c nmr (175 mhz, cdcl3) 151.3, 149.8, 146.0, 143.5, 140.9, 140.4, 139.8, 139.2, 134.5, 134.0, 132.6, 130.2, 129.6, 129.3, 129.0, 128.8, 127.7, 127.7, 127.5, 127.4, 127.4, 127.2, 127.1, 127.0, 126.5, 126.3, 126.0, 124.5, 124.2, 123.5, 122.3, 117.9, 117.8, 117.5, 117.3, 115.3, 106.9, 106.3, 62.7, 56.4, 54.6, 53.9, 50.6, 48.0, 36.3, 36.1, 35.8, 35.3, 24.9, 19.2, 17.2; ir (film) 3362, 1617 cm; ms (esi+) 236.1442 (236.1434 calcd for c17h17n, m+h).

intramolecular pd-catalyzed alkene carboamination reactions of substituted 2-allyl-n-(2-bromobenzyl)anilines are described. the substrates for these reactions are generated in two steps from readily available 2-allylanilines and 2-bromobenzaldehyde derivatives. the transformations afford substituted tetrahydroindoloisoquinolines, an uncommon class of fused bicyclic heterocycles, in good yield. the mechanism of these transformations is described, and a model that accounts for the observed product stereochemistry is proposed.

PMC4011572

pubmed-626

one of the shortcomings of the available treatments for major depressive disorder (mdd) is the time delay between commencing the treatment and achieving an antidepressant response. most of the approved drugs for such treatment need at least 2 to 4 weeks for the onset of their effects, and sometimes the peak of drug efficacy is achieved after 6-8 weeks (1). the fast effectiveness and the short period between the onset of action and the relief of symptoms are the major advantages of electroconvulsive therapy (ect) compared with common antidepressant drugs; however, similar to drugs, the time delay between the start of ect and its effect is still a weak point of this treatment modality (2, 3). despite the relative superiority of ect to drugs in the treatment of mdd, the patients suffering caused by the symptoms and the possibility of harmful behaviors in the period between the start of treatment and the response to it still remains one of the major challenges in the treatment of depression. ketamine is a non-competitive inhibitor of the n-methyl-d-aspartic acid (nmda) receptors. it is sometimes used for induction of anesthesia in ect because it neither raises the convulsive threshold nor impairs cognition (4). furthermore, there are some evidences showing the elevated mood in patients receiving ketamine (5-7). hence it seems that the induction of anesthesia by ketamine, which has mood enhancing effects, during ect, may expedite the process of mdd recovery. recent studies suggest the probability of the synergic effects of ect and ketamine in accelerating the recovery of patients, but controversial results make performing more research in this field mandatory (8). some reports show that ketamine during ect can have neuroprotective effects and reduce its cognitive side effects to some extent (7, 9), while other studies report effects to the contrary. additionally, according to the results of some other investigations, ketamine itself may cause cognitive impairments (10). it, therefore, seems that more studies are needed to illuminate the effect of ketamine on patients cognitive conditions after ect. the present report evaluates the role of ketamine in accelerating antidepressant effects and its cognitive effect after ect. this double-blind randomized clinical trial study recruited mdd patients who received ect in qods hospital, sanandaj, west of iran, between july 2010 and june 2012. the inclusion criteria were comprised of age between 18 and 65 years, normal iq, suffering from mdd according to the diagnostic and statistical manual of mental disorders, 4th edition, text revision (dsm-iv-tr) criteria, and having a score of 20 or higher in the hamilton rating scale for depression (hrsd). the hrsd scores, vital signs, and duration of reorientation were collected by an author who was blind to group assignment. the patients were also blind to the received medication and only the anesthesiologist responsible for the induction of anesthesia knew which patient received which drug. the exclusion criteria consisted of substance or drug dependence for up to at least 3 months before the study, any contraindication for receiving ect, cognitive impairment, epilepsy, receiving ect in the previous 3 months, and anesthesiology class>ii according to the american society of anesthesiologists (asa) physical status classification system. this study was approved by the ethics committee of kurdistan university of medical sciences and was registered in the iranian registry of clinical trials (irct.ir#irct138811022935n2). as the study was done under close observation in the hospital, no changes were made in the type and doses of the already prescribed drugs. considering =0.05, =0.1, =3.5, and d=2.5, the sample size was calculated as 22 patients in each intervention and control group. the patients in the intervention group received ketamine with propofol and those in the control group received normal saline and propofol. the hrsd scores were obtained one day before ect onset, the day after the third session, the day after the last session, and finally 2 weeks after the last session. the minimum score needed to participate in the study was considered 20 and response to treatment was considered as at least a 50% reduction in the score after the last session. at time 0, the intervention group received 0.3 mg/kg of ketamine hydrochloride (manufactured by rotexmedica, germany) intravenously (iv) which was diluted with 5 ml of saline; and in the control group, ketamine was replaced with 5 ml of normal saline. thirty seconds later, after the loss of eyelid reflex, anesthesia was induced with 1 mg/kg of propofol 1% iv (manufactured by fresenius kabi, austria gmbh). muscle relaxation was achieved by the administration of 0.5 mg/kg of succinylcholine iv. before succinylcholine administration, a manometer cuff was fastened to one of the legs and blown up to 30 ml higher than systolic pressure and was retained until the end of seizure. propofol complementary doses were administered if needed. then, in all the patients, lung ventilation with 100% oxygen was performed by mask. the anesthesiologist was the only person aware of the administered drugs. after complete relaxation, electric stimulation was performed using an ect apparatus (iec 601-1 type bf class 1, iran) with bifrontotemporal electrode placement. for the first session, the patients received 30% to 50% of maximum output stimulus. the blood pressure and heart rate of the patients were also evaluated 5 minutes after the motor seizures were ended. to assess the patients cognitive performance recovery time, 10 questions relating to orientation were asked at 5-minute intervals, after terminating the motor seizures (box 1). the minimum cognitive performance recovery time was considered when the patient was able to answer all the 10 questions correctly. similar assessments have been performed to measure the patients cognitive performance recovery time in previous studies (7, 11). repeated measurement was used to compare the trend of the hrsd, blood pressure, heart rate, seizure duration, and cognitive performance recovery time at different time points. the within-subject contrasts test was employed to assess the interaction effects between group and time. the average of the p values after adjusting the degree of freedom through the greenhouse-geisser and huynh-feldt was reported for some of the variables for which sphericity assumption was rejected. in the control group, one patient was excluded because of consent withdrawal for ect and one more patient was excluded because of early discharge from the hospital. finally, of the 42 patients whose data were analyzed at the end of the study, 22 patients were in the intervention group (27.3% men and 72.7% women) and 20 patients (35% men and 65% women) were in the control group (figure 1). the two groups had no significant difference in sex distribution (p= 0.74, =0.29, df=1), education level (p=0.72, =1.5, df=3), and mean age (p=0.82, t=0.23, df=40) (table 1). there was no significant difference in the motor seizure duration between the two groups (p=0.395, df=5, f=1.04) (table 2). the mean cognitive performance recovery time in the intervention group was lower than control group (p=0.042, df=5, f=2.36) (figure 2 and table 2). there were no significant differences between the groups in terms of the rate and speed of the recovery process (p=0.82, df=3, f=0.31) (figure 3, table 2). t1, one day before ect onset; t2, a day after the third session; t3, a day after the last session; t4, two weeks after the last session. the results of this study did not show any significant difference in cardiac side effects between the patients in both groups before and after ect (table 2). other plausible side effects of ketamine such as postictal agitation, hallucination, or derealization were not seen in the participants of either group. in this study, we assessed the efficacy of ketamine on the rate of recovery from mdd by adding it to one of the routine methods of anesthesia induction in ect. we could not find significant differences in the symptom recovery, seizure duration, and blood pressure alterations between the two groups. however, the cognitive performance recovery time in the ketamine group was better than in the control group. in the current study, the evaluation of cognitive performance recovery time in both groups revealed that the patients who received ketamine were able to answer the cognitive ability questions faster than the control group. in mc daniel and colleagues study, the ketamine-administered patients for ect showed less memory impairment than the etomidate-administered patients.the authors suggested the antagonistic effect of ketamine on the nmda receptors as a protective agent against the side effects of ect on the cognitive abilities of patients (9). krystal and colleagues also reported a reduction in cognitive disabilities caused by ect after the ketamine usage (7). in contrast, in a report by loo and colleagues, administering ketamine with thiopentone did not affect the neuropsychological side effects of ect in the treated patients. the ketamine dose was 0.5 mg/kg in that study, and the patients underwent ultrabrief pulse-width right unilateral ect (8). as unipolar ect has normally fewer side effects, one of the probable reasons for the lack of efficacy of ketamine on cognition in their study could be the different ect modality. in addition, evidence has also shown that the ketamine effect on glutamate transportation via the nmda receptors is dose-dependent and is shaped like an inverted u (14-16), which can be a possible explanation for the variations in different reports. in our study, although in 5 out of 6 sessions of ect, the motor seizure in the ketamine-administered patients was more than that in the control patients, the difference was not significant. in both groups, our finding is in line with that reported by kayhan and colleagues, who stated that their groups had no difference in motor seizure duration, but the quality of seizure was better in the ketamine group. in that study, the ketamine dose was 0.5 mg/kg and the authors also succeeded in recording the seizure time by electroencephalography (eeg) (13). on the other hand, krystal and colleagues showed that increasing the ketamine dose (0.7 to 2.8 mg/kg) could promote the seizure duration significantly compared with the administration of methohexitone only (7). it seems that the difference in results stems from the ketamine dose or mixing it with other drugs and the methods of seizure recording. as we could not record the seizures using eeg, we can make no claims regarding the quality of seizure. the present study did not show any significant differences in terms of the cardiac side effects of ketamine between the two groups. although ketamine may cause cardiac side effects due to the systemic release of catecholamines (17), the small dose of ketamine used in this study may be the reason for the absence of cardiac side effects. it is deserving of note that some studies have shown that the combination of ketamine and propofol may provide better hemodynamic stability (18, 19). in our study, the decreasing trend of the scores in the hrsd during the treatment for both groups showed the efficacy of both anesthesia induction methods. however, there were no significant differences in the rate and quality of recovery between the two groups (p=0.92). in machado-vieira and colleagues study, which used ketamine as an iv single dose, the patients showed recovery in the first 24 hours (20). the rapid antidepressant effects of ketamine were also shown in a report by pheilps and colleagues, who studied 26 treatment-resistant patients with major depression (24). similar effects have been reported about using ketamine on patients with major depression and chronic pain syndrome (22), treatment-resistant patients with depression at risk of suicide (23), and also during ect (24). in wang s study, the patients who received ketamine or a ketamine-propofol combination during ect experienced less depression on days 2, 3, and 7 after ect. it is important to note that the patients in that study received only one ect session and underwent further ect if necessary (12). in a study done by goforth and colleagues, the depression symptoms of a patient were relieved 8 hours after administering 100 mg of ketamine and one ect session (24). loo showed that adding ketamine led to better treatment efficacy in the first week but this effect had no continuity (8). it is noteworthy that there are limited studies on the combined effect of ketamine and ect and that they were retrospective (25) or case reports (26) or limited to one ect session (12). moreover, a few other similar studies did not assess the continuity of the effect (21, 22). consequently, it seems that although our study could not show the effect of ketamine on the mdd recovery in a 2-week follow-up period, the fact that there are conflicting reports on the effects of ketamine means that such effects on depression can not be ruled out and that they may be caused by the ketamine dose, the onset of administering the drug before ect, and the time of evaluating the changes in depression intensity. first and foremost among the limitations of the present study is that we could not record the seizure duration by eeg. another salient drawback to this study is its small sample size due to the unavailability of patients for follow-up in iran, which may have affected the results. additionally, ect discontinuation because of patient noncompliance and incomplete hrsd questionnaires because of low literacy are also the weak points of our study. another shortcoming is the small ketamine dose in our study by comparison with other similar studies. indeed, achieving more reliable results requires the use of higher doses of ketamine. finally, some degree of misinterpretation by our patients in answering the hrsd questionnaire may be a possible source of bias in this study.

background: one of the shortcomings of the available treatments for major depressive disorder (mdd) is the time delay between starting the treatment and achieving an antidepressant response. objectives:we aimed to determine the effect of ketamine as a synergistic antidepressant and anesthetic agent on mdd in electroconvulsive therapy (ect). patients and methods: twenty-two patients with mdd received ketamine and propofol as anesthetic agents compared with 20 patients as the control group who received propofol in a double-blind randomized clinical trial. the hamilton rating scale for depression was used to determine the changes in symptoms severity during ect and a 2-week follow-up. results:both groups showed a reduction in depression severity, but there was no significant difference between the groups in the recovery process (p=0.92). however, the cognitive performance recovery time in the ketamine group was lower than that in the control group (p=0.042). conclusions: this study could not show the effect of ketamine on depression recovery in a 2-week follow-up period. nevertheless, ketamine may provide a better cognitive performance in patients under ect.

PMC4644613

pubmed-627

an estimated 207 million cases of human schistosomiasis have been reported worldwide and about 90% of these live in sub-saharan africa, with nigeria having the highest prevalence. schistosoma infections cause significant morbidity and mortality with peak prevalence and intensity of infection occurring between the ages 10 and 20 years and subsequent decline by age 65 years. chronic human circulatory system infection by schistosoma haematobium is reported to affect the urinary bladder and is a possible risk factor in the aetiology of cancers of the bladder and the urinary tract system. s. haematobium infection has been linked with the development of squamous cell carcinoma of the bladder [4, 5]. s. haematobium associated bladder damage has been closely linked to the immune reaction elicited against the parasite egg deposited in the bladder which eventually induces chronic inflammation related granulomatous injury. schistosomiasis and bladder cancer share common symptoms such as haematuria, dysuria, and pain with micturition. this may prevent early diagnosis of bladder cancer and the resultant severe bladder damage particularly in people living in s. haematobium endemic areas. in nigeria, most studies have focused on the epidemiology of s. haematobium infection [3, 7, 8] particularly in school-age children, with limited information about the morbidity resulting from urinary schistosomiasis in adults. this study was therefore aimed at determining the prevalence of schistosomiasis and associated bladder pathology in adults living in eggua, yewa, north local government area, ogun state, nigeria. the study was carried out in eggua, a rural agrarian community, between august 2012 and may 2013. it is one of the wards that make up yewa north local government area as previously described. it consists of settlements at sagbon, imoto, tata, agbon-ojodu, and igan alade. two major rivers (yewa and iju) flowing through the area serve as the main water source, resulting in high water contact by the inhabitants. these rivers are used for religious, domestic, and entertainment activities which enhance the transmission of schistosomiasis. participants aged 30 to more than 60 years old from the community were enrolled for the study. children were excluded from the study in line with the objective of the study to determine the effect of chronic urinary schistosomiasis on adult members of the community. informed consent was obtained from each participant under a protocol approved by the local government and local health officials. ethical approval for the study was also obtained from the ogun state ministry of health. blood (5 ml) and urine specimens were collected from each study participant. the urine samples were collected between 10:00 and 14:00 hours to ensure maximum egg yield. the urine samples (10 ml) were processed for microscopic examination and egg count [3, 10]. the eggs were quantified by counting under the microscope and classified as light infection if there were 50 (149) eggs/10 ml urine and heavy infection if there were>50 eggs/10 ml urine. a blind ultrasound examination was carried out on each participant approximately 1 h after drinking potable water (0.11.5 litre depending on the age of the participant) to distend the bladder. the classification of bladder pathology or damage was based on the definition of the who [11, 12] and shiff et al.; the abnormalities assessed included abnormal bladder shape, bladder wall irregularities, bladder masses, presence of polyps, calcification, and presence of hydronephrosis in the kidneys. bladder lesions were considered severely abnormal when four of the above conditions or three conditions as well as hydronephrosis were present in a single individual. lesions were considered moderate if fewer conditions were seen and negative when no specific lesions were observed. a structured, pretested questionnaire was used to obtain information about participants ' habits regarding smoking and alcohol consumption, which are determinants of bladder cancer. statistical analysis of data obtained was done using spss version 20.0 (p<0.05). a total of 257 (79 males and 178 females) participants aged 3090 years were screened for s. haematobium infection and associated bladder pathologies. the overall prevalence of s. haematobium in the sampled population was 25.68% (66/257), 21 (31.8%) in males and 45 (68.2%) in females. the highest prevalence of infection was observed in participants over 60 years old (table 1). the majority (56/66) (84.8%) of those positive for s. haematobium had a light intensity of infection with the egg mean intensity of 16.7 eggs/10 ml urine. the yewa river was the main source of water for most (49/62) (79.0%) of the participants infected with s. haematobium (table 4). bladder pathologies were observed in 33.9% (87/257) of the sample population and included abnormal bladder wall thickness (39/66) (59%), abnormal bladder shape (10/66) (15.2%), bladder wall irregularities (15.2%), bladder masses (1.5%), and bladder calcification (1.5%) (table 2). bladder wall thickness, the most common abnormality, was recorded in 46/79 (58.2%) males and 90/178 (50.6%) females (table 3). among the participants, 56 (84.8%) with bladder pathologies also had an existing schistosomiasis infection, 48 (87.3%) of which were light intensity and 8 (72.7%) of which were heavy intensity: =267.5, p=0.001 (table 5). thus, there was an association between urinary tract pathology and the intensity of s. haematobium infection (= 375.4, p=0.001, table 2). among the participants with light and heavy intensity of s. haematobium infections, bladder wall thickness was the most common bladder structural pathology identified in 33/56 (58.9%) and 6 (60.0%) participants with light and heavy s. haematobium infections, respectively (table 5). abnormal bladder shape and bladder wall irregularity were seen in 8/56 (14.3%) and 2 (20%) participants with light and heavy infections, respectively (figures 13). hydronephrosis was present in only one participant with light infection, while calcification was identified in only one participant with heavy infection. mild bladder pathology was more common than severe bladder pathology in this study and was found in 48 of the participants (table 5). there was a higher incidence of bladder pathologies among female participants (table 3); bladder mass and hydronephrosis were also seen only in female participants. there was no significant relationship between cigarette smoking and bladder pathology in the study (table 6). among participants with bladder pathology, 29 (33.3%) admitted consuming alcohol while 58 (66.7%) said that they had never consumed alcohol (table 7). the overall prevalence rate (25.98%) of adults with s. haematobium infection recorded in this study was slightly higher than 20.8% and 20.0% reported in yewa north local government, ogun state, and owan east local government, respectively, in nigeria [3, 13]. most (81.3%) of the participants depended solely on the s. haematobium contaminated river water, which could account for the higher s. haematobium prevalence; and little or no schistosomiasis control (drug) intervention targeted to adults has been recorded in this area. the higher frequency of light intensity s. haematobium infection observed in this study could be explained by some level of acquired protected immunity by adults in that community due to chronic exposure to schistosomiasis.. found that the proportion of egg-positive individuals falls progressively with age and is a feature in populations with lifelong exposure to the parasite. therefore, chronicity of infections in older people will more likely be difficult to ascertain using egg count method. the higher frequency of mild bladder pathology observed in this study was also similar to another study which observed a higher incidence of mild bladder than severe bladder pathology. this result could be explained by the low number of participants who smoked cigarettes and consumed alcohol; these conditions may serve as promoting factors either in progression of bladder pathology to cancer or in making the bladder pathology more severe (table 4). in addition, this lifestyle could buttress the possibility of s. haematobium being the principal cause of the reported bladder structural pathology in the study population. the close relationship between the intensity of s. haematobium infection and the presence of bladder abnormalities was similar to previous reports [3, 1416]. the presence of hydronephrosis in participants with light infection is however at variance with the report of nmorsi et al. although hydrocalycosis (a condition mostly mistaken for hydronephrosis) was observed in some patients with heavy infection, indicating the likely contribution of this infection to kidney pathology. females (64.7%) had more structural bladder pathology compared to males (35.3%). this may be due to higher water contact by females and also to the higher number of female study participants than an indication of a female predilection to bladder pathology. however, since hydronephrosis and bladder mass or bladder calculi were found together in a female participant, female predilection to bladder pathology may not completely be ruled out. the structural changes to the bladder recorded in this study were in consonance with observations in west madagascar and nigeria [3, 15] where bladder irregularities and bladder wall thickness were identified as the most common pathologies in individuals infected with s. haematobium. in conclusion, there is evidence that s. haematobium infections may be associated with bladder pathology, on ultrasound examination. individuals with bladder pathologies could have heavy or light intensity of schistosomiasis infection or have no existing infection at all. however, a long term exposure to schistosomiasis is necessary for the development of bladder cancer. further research on the determinants and progress of the bladder pathologies seen in this study population is needed.

screening for schistosoma haematobium infection and its possible morbidity was carried out in 257 adult participants in eggua community, ogun state, nigeria. parasitological assessment for the presence of ova of s. haematobium in urine and abdominopelvic ultrasonographic examination for bladder and secondary kidney pathology were carried out. s. haematobium prevalence of 25.68% (66/257) was recorded among the participants. there was a significantly higher prevalence of 69.2% of urinary schistosomiasis in the females than the prevalence of 31.8% in males (p=0.902). the intensity of infections was mostly light (55) (21.8%) compared to heavy (10) (3.9%) with the mean intensity of 16.7 eggs/10 ml urine. structural bladder pathology prevalence among participants was 33.9%. the bladder and kidney pathologies observed by ultrasound in subjects with s. haematobium infections included abnormal bladder wall thickness (59%), abnormal bladder shape (15.2%), bladder wall irregularities (15.2%), bladder masses (1.5%), bladder calcification (1.5%), and hydronephrosis (3%). infection with s. haematobium was associated with bladder pathology. higher frequencies of bladder abnormalities were observed more in the participants with light intensity of s. haematobium infection than in those with heavy infection. more bladder pathology was also seen in women than in men, although this was not statistically significant. in conclusion, there is evidence that the development of bladder pathology may be associated with s. haematobium infection.

PMC5011230

pubmed-628

therapeutic high intensity focused ultrasound (hifu) or focused ultrasound (fus) is a noninvasive medical treatment that allows the deposition of energy inside the human body. the energy levels carried in the ultrasound beam are several orders of magnitude greater than those of a standard diagnostic ultrasound beam. in the case of focused ultrasound, the high energy levels carried in a hifu beam can therefore be magnified further and delivered with precision to a small volume, while sparing surrounding tissues. fus energy can be deposited in small areas providing a substantial advantage for drug targeting. the volume of energy deposition following a single hifu exposure is small and will vary according to transducer characteristics but is typically cigar shaped with dimensions in the order of 13 mm (transverse) 815 mm (along beam axis). hifu transducers deliver ultrasound with intensities in the range of 10010,000 w/cm to the focal region, with peak compression pressures of up to 30 mpa peak and rarefaction pressures up to 10 mpa. the ultrasound wave propagates through tissues, causing alternating cycles of increased and reduced pressure (compression and rarefaction, resp.). in the case of tissue ablation during hifu treatments, the temperature at the focus can rise rapidly (up to 80c) which can cause cell damage. ultrasound affects the molecular structure of the tissues during the alternating cycles of compression and rarefaction. during rarefaction, gas can be drawn out of the solution to form bubbles, which can collapse rapidly. in this case injury is induced through a combination of mechanical stresses and thermal effects at a microscopic level. when ultrasound is applied in biological systems it can induce local tissue heating, cavitation, and radiation force, which can be used to initiate local (focal) drug delivery, increase permeation through membranes, and enhance diffusivity of drugs, respectively, only at the site of sonication therefore allowing control of local drug release. the ability of fus to induce thermal or mechanical effects at very defined (focal) locations in living tissue has been first described in 1942, when lynn et al. they used a sonication system in combination with x-rays to determine the target location relative to skull and to focus the ultrasound beam through a craniotomy into deep brain for effective functional neurosurgery. later on, in the 1980s the first fda-approved fus system, sonocare cst-100, was developed to treat ocular disorders such as glaucoma and many patients were successfully treated with this system. more recently substantial technological developments have led to new fus equipment for a number of different applications. current research and development aims to explore transducer technology and array design to achieve faster delivery of focal sonications, to improve transducer accessibility (smaller devices) or fit them to certain parts of the body such as a helmet of arrays for brain focal treatment. these devices can operate under image guidance to provide real-time monitoring of the treatment. guidance and monitoring of acoustic therapy controls the treatment region and minimizes damage to adjacent structures. monitoring using real-time imaging, such as with sonography (diagnostic ultrasound), ensures that the targeting of the fus beam is maintained on the correct area throughout the procedure. mri and sonography are the two imaging modalities currently being used for guidance and monitoring fus therapy. mri has the advantage of providing temperature data during fus treatment. however, mri guidance is expensive, labor-intensive, and of lower spatial resolution in some cases. sonographic (ultrasound) guidance provides the benefit of imaging using the same form of energy that is being used for therapy. therefore, if the target can not be well visualised with sonography, then it is unlikely that fus therapy will be effective. insightec manufactures the exablate2000 which uses mri for extracorporeal treatment of uterine fibroids (fda-approved) with significant success, and extensive current research focuses on investigating its application in other parts of the body [7, 8]. exablate technologies are used for prostate cancer or bone metastasis (exablate 2100 conformal bone system); these applications are currently under development by insightec. the ablatherm hifu/us consists of a transrectal probe for prostate treatment and has ce mark approval. the sonablate 500, an ultrasound guided system uses a transrectal probe to carry out prostate cancer focal ablation. the sonalleve hifu/mr is an mr compatible device developed to examine a series of applications as fibroids and other body sites., tirat carmel, israel) with each element driven separately, providing individual correction of skull distortion as well as electronic steering. the device has been used for the treatment of neuropathic pain essential tremor and there is also evidence of possible application for brain tumours [12, 13]. essential tremor noninterventional functional neurosurgery treatment has shown an immense potential of transcranial mrgfus application to induce lesions focally and treat patients nonsurgically. ultrasound propagates as mechanical vibrations that induce molecules within the medium to oscillate around their positions in the direction of the wave propagation. the rate of energy flow through a unit area, normal to the direction of the wave propagation, is called acoustic intensity. at 1 mhz the ultrasound wave is attenuated about 50% while it propagates through 7 cm of tissue. the attenuated energy is transformed into temperature elevation in the tissue [15, 16]. ultrasound is transmitted from one soft tissue layer to another. usually in soft tissues a small amount of wave is reflected back except at the soft tissue-bone interface where approximately one-third of the incident energy is reflected back. in addition, the amplitude attenuation coefficient of ultrasound is about 1020 times higher in bone than in soft tissues. each particle moves small distances from its rest position but the vibrational energy is propagated as a wave traveling from particle to particle through the material. ultrasound is attenuated as it travels through a tissue due to beam divergence, absorption, and deflection of the acoustic energy. the energy required for a sound wave to travel through a tissue must overcome the internal friction intrinsic to any material. as a sound wave travels through tissue, it continually loses a proportion of its energy to the tissue (attenuation). divergence of the sound beam spreads the acoustic energy over a larger beam area and reduces the intensity along the beam axis. the greatest cause of attenuation in the body is absorption, in which energy is transferred from the sound beam to the tissue and ultimately is degraded to heat. the amount of absorption depends on the frequency of the ultrasound beam. whenever a sound beam encounters a boundary between two materials, the direction of the reflected wave, or the echo, depends on the orientation of the boundary surface to the sound wave. the major physical effects of ultrasound are heat, mechanical effects, cavitation, and chemical effects. acoustic impedance is a measure of the resistance that a material offers to the passage of an ultrasound wave and is expressed in units of rayls (kg/m/sec). acoustic impedance of water is 1.5 10 mrayls whereas that of bone is 8 10 mrayls. the greater the difference in acoustic impedance between two materials, the stronger the echo (reflected wave) arising from their interface. when the rate of heat generation is higher than the rate of heat dissipation in the body, the body temperature will rise significantly. mechanical effects, such as the breaking of bonds, can occur if the amplitude of the ultrasound wave is significantly large. cavitation occurs when an ultrasound beam of sufficient intensity travels through a liquid in which gas bubbles have been generated. the alternating high- and low-pressure periods of the ultrasound wave forces the bubbles to contract and expand. the amplitude of the bubble oscillation increases with increasing ultrasonic intensity. during the bubble contraction, the internal pressure can increase and the temperature can reach 10,000c. a sonic explosion can occur, releasing a large amount of energy, although for very short (m) distances. chemical effects, such as the acceleration of chemical reactions, can occur due to an increase in the temperature and pressure. when ultrasound beams are focused a focal diameter of 1 mm can be achieved at 1.5 mhz. if the ultrasound beam is transmitted from an applicator 23 cm in diameter, the ultrasound intensity at the millimeter-sized focal spot can be several hundred times higher than in the overlying tissues. typical diagnostic ultrasound transducers deliver ultrasound with time-averaged intensities of approximately 0.1100 mw/cm or compression and rarefaction pressures of 0.0010.003 mpa, depending on the mode of imaging. in contrast, hifu transducers deliver ultrasound with intensities in the range of 10010,000 w/cm to the focal region, with peak compression pressures of up to 30 mpa and peak rarefaction pressures up to 10 mpa. the ultrasound exposure drops off rapidly across the area within the sonication path and therefore focusing provides a method to overcome attenuation losses and to concentrate energy deep in the body while avoiding the surrounding tissues. focusing is dramatically improved with the use of transducer arrays that are driven with signals having the necessary phase difference to obtain a common focal point. the main advantage of these phased arrays is that the focal spot can be controlled. in addition, the electronically focussed beam allows multiple focal points to be induced simultaneously or fast electronic scanning of the focal spot which increases the size of the focal region. the combination of high-intensity focused ultrasound together with high-resolution mr guidance has created a system that can produce tissue destruction deep within solid organs without any invasion. accurate targeting and detailed accurate thermal mapping are provided by mri. in recent years imaging has been combined with fus to provide real-time manipulation of drug guidance within the targeted area. ultrasound and magnetic resonance (mr) imaging are widely used clinical imaging modalities that can be combined with fus for image guided fus treatments. in the area of drug delivery ultrasound microbubbles or nanocarriers providing contrast enhancement can be used. when using nanocarriers sensitive to mechanical forces (the oscillating ultrasound pressure waves) and/or sensitive to temperature, the content of the nanocarriers can be released locally. thermosensitive liposomes have been suggested for local drug release in combination with local hyperthermia more than 25 years ago. real-time imaging methods, such as magnetic resonance, optical and ultrasound imaging, have led to novel insights and methods for ultrasound triggered drug delivery. image guidance of ultrasound can be used for: (1) target identification and characterization; (2) spatiotemporal guidance of actions to release or activate the drugs and/or permeabilize membranes; (3) evaluation of biodistribution, pharmacokinetics and pharmacodynamics; and (4) physiological read-outs to evaluate the therapeutic efficacy. the enhanced permeability and retention effect has served as a basic rationale for using liposomes and other nanoparticles to treat solid tumors. however, it has been recently noticed that the enhanced permeation and retention effect does not guarantee a uniform delivery. this heterogeneous distribution of therapeutics is a result of physiological barriers presented by the abnormal tumor vasculature and interstitial matrix. in a recent review by jain and stylianopoulos first, the abnormal structure of tumor vessels results in heterogeneous tumor perfusion and extravasation, and a hostile tumor microenvironment that supports drug resistance and tumor progression. second, in highly fibrotic tumors, the extracellular matrix blocks penetration of large nanoparticles leaving them concentrated in perivascular region. to overcome these barriers the authors suggest normalization of the vascular network and the extracellular matrix as well as development of nanoparticles that release therapeutic agents in response to the tumor microenvironment or an external stimulus (such as heat light and hifu). however, development of thermosensitive liposomal carriers for cancer was only introduced as recently as 1999 when needham's group evaluated phase transition enhanced liposomal permeability. in vivo data using cancer models were presented one year later when the authors described a new lipid formulation containing doxorubicin optimized for mild hyperthermic temperatures (39c to 40c) that are readily achievable in the clinic leading to very rapid release times of the drugs. this new liposome, in combination with mild hyperthermia, was found to be significantly more effective than free drug or current liposome formulations at reducing tumour growth in a human squamous cell carcinoma xenograft. these low temperature-sensitive liposomes (ltsl) were further developed in dogs having canine tumours to show a superior efficiency [27, 28]. a formulation based on these thermosensitive liposomes took the brand name thermodox and was further developed by celsion corporation. thermodox liposomes can be triggered to release their payload by any heat-based treatment such as radiofrequency thermal ablation (rfa), microwave hyperthermia, and high intensity focused ultrasound (hifu). results from a phase i study that used thermodox was recently published. in a phase i study researchers used escalating dose of thermodox with radiofrequency (rf) ablation and concluded that thermodox can be safely administered at 50 mg/m in combination with rf ablation. currently thermodox in combination with rf ablation is being tested in a large phase i study to treat hepatocellular carcinoma. the concept of using liposomes and hifu was introduced recently, in 2006 when frenkel et al. used liposomal doxorubicin (doxil) in combination with pulsed high-intensity focused ultrasound (hifu) exposures in a murine breast cancer tumor model. doxil is a stable liposomal preparation that has no response to increased temperature and was developed to minimise doxorubicin's cardiotoxicity, by encapsulating doxorubicin within stealth liposomes. although doxil achieves long circulation of doxorubicin with minimum cardiotoxicity it does not rapidly release the drug within the tumour. pulsed-hifu exposures were not found to enhance the therapeutic delivery of doxorubicin and did not induce tumour regression. however, a fluorescent dextran showed blood vessels to be dilated as a result of the exposures. experiments with polystyrene nanoparticles of similar size to the liposomes showed a greater abundance to be present in the treated tumours. although this study did not achieve or prove a therapeutic advantage of the use of hifu with temperature stable liposomes it showed clearly that pulsed hifu induces a substantial increase of permeation of macromolecules and nanoparticles in tumours. presented the first study on thermosensitive liposomes (low temperature sensitive liposomes (ltsl)) and hifu. the authors investigated pulsed-high intensity focused ultrasound as a source of hyperthermia with thermosensitive liposomes to enhance delivery and efficacy of doxorubicin in murine adenocarcinoma tumours. in vitro treatments simulating the pulsed-hifu thermal dose (42c for 2 min) triggered release of 50% of doxorubicin from the thermosensitive liposomes; however, no detectable release from the nontemperature sensitive liposomes (similar to doxil) was observed. similarly, in vivo experiments showed that pulsed-hifu exposures combined with the ltsl resulted in more rapid delivery of doxorubicin as well as significantly higher concentration within the tumour when compared with ltsls alone or nonthermosensitive liposomes, with or without exposures. in a later study the same team developed mr imageable thermosensitive liposomes (iltsl), with the objective to characterise drug release in phantoms and in vivo. an mri contrast agent (prohance gd-hp-do3a) and doxorubicin were loaded and drug release was quantified by spectroscopic and fluorescence techniques, respectively. release with hifu under mr guidance was examined in tissue-mimicking phantoms containing iltsl and in a vx2 rabbit tumour model. release of doxorubicin and prohance from iltsl was minimal at 37c but fast when heated to 41.3c. relaxivity of iltsl increased significantly from 1.95 0.05 to 4.01 0.1 mms when liposomes were heated above the phase transition temperature indicating the release of prohance from liposomes and its exposure to the aqueous surroundings. importantly, the signal increase corresponded spatially and temporally to mr-hifu-heated locations in phantoms. in vivo, iltsl injection and after each 10-min heating, with greatest increase in the heated tumour region. the authors concluded that mr-hifu combined with iltsl may enable real-time monitoring and spatial control of drug release from liposomes. in a follow-up study the authors investigated the effect of iltsl in rabbits bearing vx2 tumours. in that study image-guided noninvasive hyperthermia was applied for a total of 30 min, completed within 1 h after ltsl infusion and quantified doxorubicin release in tumours with hplc and fluorescence microscopy. sonication of vx2 tumours resulted in accurate and spatially homogenous temperature control in the target region. ltsl+mr-hifu resulted in significantly higher tumour doxorubicin concentrations (3.4-fold greater compared ltsl resp.). the authors observed that free doxorubicin and ltsl treatments appeared to deliver more drug in the tumour periphery as compared to the tumour core indicating that hifu induced hyperthermia and ltsl increases doxorubicin's permeability as doxorubicin was found in both the tumour periphery and core. the group further developed a heating algorithm using the same rabbit tumour model proving that the use of the binary feedback algorithm results in accurate and homogenous heating within the targeted area. a computational model that simulated the tissue heating with hifu treatment and the resulting hyperthermia that leads to drug release was developed by haemmerich. in this model a spatiotemporal multicompartmental pharmacokinetic model simulated the drug release in the blood vessels and its transport into the interstitium as well as cell uptake. two heating schedules were simulated each lasting 30 min, the first corresponding to hyperthermia, (ht; 43c) and the second corresponding to hyperthermia followed by a high temperature (50c) for 20s pulse, (ht+). using the computational model (validated in rabbit vx2 tumours) the study indicates the importance of simulations in the application of drug delivery mechanisms to tumours. in addition to the progress in the understanding of the physical mechanism of drug delivery from well validated thermosensitive liposomes carrying doxorubicin, researchers further investigated the chemical composition of such liposomes in response to hifu induced hyperthermia. de smet et al. two temperature-sensitive systems composed of the following lipids dppc: mppc: dppe-peg2000 (low temperature-sensitive liposomes; ltsl) and dppc: hspc: cholesterol: dppe-peg2000 (traditional temperature-sensitive liposomes; ttsl) were investigated for their stability and release profile at 37c and 42c in phantoms using mri 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (dppc), 1-palmitoyl-sn-glycero-3-phosphocholine (mppc), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-n[methoxy(polyethyleneglycol)-2000] (dppe-peg2000), hydrogenated-l--phosphatidylcholine (hspc). the ltsl system showed a higher leakage of doxorubicin at 37c, but a faster release of doxorubicin at 42c compared to the ttsl system indicating that lipid composition plays an important role on stability and release profile. the authors further investigated the more stable traditional temperature sensitive liposomes carrying doxorubicin and prohance in vivo in rats bearing 9l gliosarcoma tumours. a clinical mri-hifu system was applied in a proof-of-concept study to induce local hyperthermia for 30 min. the local temperature-triggered release of prohance was monitored with interleaved t1 mapping of the tumour. a good correlation between the r1 (change in longitudinal relaxation rate r1=(1/t1)) and the intratumour doxorubicin and gadolinium concentration was found, implying that the in vivo release of doxorubicin from the thermosensitive liposomes can be probed in situ with the longitudinal relaxation time of the coreleased mri contrast agent (dose painting). temperature sensitive liposomes release their encapsulated drugs at the melting phase transition temperature (tm) of the lipid bilayer. at this tm the lipid membrane changes its structure as it transfers from a gel to the liquid crystalline phase. when the liposomal membranes are in the gel phase they show less permeability to molecules and water compared to the liquid crystalline phase. the liposomes ' transition to the liquid crystalline phase can be achieved with the incorporation of a lyso-phospholipid such as mspc (r=c17h35).. a potential disadvantage of mspc containing liposomal formulations is their rapid doxorubicin leakage at 37c. prepared temperature sensitive liposomes using nonionic surfactants brij which are peg-ylated lysolipids resembling the chemical structures of mspc and dspe-peg(2000). results indicated that the optimal acyl chain length of the surfactant was between c(16) and c(18) with a saturated carbon chain and a peg repeating unit ranging between 10 and 100 with a molecule weight above 600 da. in the panel of surfactants tested, brij78 was optimal and could be incorporated into the liposomes by the thin film hydration or the postinsertion method with an optimal range of 1 to 8 mol%. the authors continued with in vivo experiments in mice bearing mammary carcinoma cells emt-6, investigating gddtpa (diethylene triamine pentaacetic acid) release with relaxometry. the authors observed a good correlation between relaxation enhancement in the heated tumour and the inhibition of tumour growth at day 21 after treatment. kono et al. investigated the effect of poly [2-ethoxy(ethoxyethyl)vinyl ether] chains (having a lower critical solution temperatures) and polyamidoamine g3 dendron-based lipids having gd chelate residues into pegylated liposomes. these designed liposomes exhibited excellent ability to shorten the longitudinal proton relaxation time. when administered intravenously into tumour-bearing mice, accumulated liposomes in tumours increased with time, reaching a constant level 8 h after administration by following t1-weighted mri signal intensity in tumours. liposome size affected the liposome accumulation efficiency in tumours: liposomes of about 100 nm diameter were accumulated more efficiently than those with about 50 nm diameter. tumour growth was strongly suppressed when liposomes loaded with doxorubicin were administered intravenously into tumour-bearing mice and the tumour was heated mildly at 44c for 10 min at 8 h after administration. in our group we have investigated the potential of an mri labelled phospholipid/lysolipid containing liposome to accumulate in tumours and release the drug under conditions of mild hyperthermia induced by fus. we label the liposome nanoparticles with a lipid that consists of a dota [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid] headgroup (figure 1) [44, 45]. introducing the imaging lipid in the lipid bilayer provides a better and clearer monitoring of liposomal particle kinetics and a better knowledge of the time required for maximum nanoparticles accumulation in tumours (monitored by mri). although most research studies have focused mainly in thermoresponsive liposomes and fus activation of drug release, there is limited work on the use of polymers (thermoresponsive or not) and their application in fus triggered drug delivery. the effect of ultrasound on drug release from polymers was studied in 1989 by kost et al. and indeed the authors found that ultrasound can increase the polymer degradation rate leading to 20 times higher release rate. interestingly the authors observed that the release rate increased in proportion to the intensity of ultrasound proposing that cavitation appeared to play a significant role. triggered drug delivery using an external physical force provides the required control of drug deposition in certain tissues avoiding exposure of healthy tissues to high (toxic) concentrations. the trigger induced delivery should be acute and the effect induced on nontargeted tissues nondamaging and reversible. hyperthermia induced by a means like ultrasound can be exploited as an external trigger in drug delivery [3, 47]. mild hyperthermia can be induced by pulsed fus that can reduce extreme tissue heating by allowing the tissue to cool down between us exposures. the increase in temperature can be 35c (hyperthermia) despite the high energy deposited in the tissue. studies with canine soft tissue sarcoma and human tumour clinical studies have also demonstrated that hyperthermia improves tumour oxygenation and enhances response of such tumours to radiotherapy or chemoradiotherapy. the increased blood flow and vascular permeability caused by temperatures such as 42c may also improve the delivery of chemotherapy drugs, immunotherapeutic agents and genes to tumour cells. fus exposures in pulsed mode lower the rates of energy deposition and generate primarily mechanical effects for enhancing tissue permeability to improve local drug delivery. these pulsed exposures can be modified for low-level hyperthermia as an enhancement of drug delivery that would lead to better drug deposition and better therapeutic effect. mild hyperthermia of 42c can improve the degree of nanocarrier extravasation as shown by kong et al.. the reason that this leads to increased extravasation maybe due to downregulation of ve-cadherin that contributes to vascular integrity as it was shown in huvec endothelial cells. it is clear that hyperthermia can provide a boost to extravasation and drug deposition in tumours. this should provide an adjuvant effect when nanocarriers are used and accumulate in tumours due to enhanced permeation and retention effect. it would be interesting to investigate the effect of hyperthermia on tumour/tissue drug clearance. acoustic cavitation can be defined as the growth, oscillation, and collapse of gas containing bubbles under the influence of the varying pressure field of sound waves in a fluid and can have an effect on the permeability of a biological tissue [5355]. the noninertial (stable) cavitation occurs when bubbles persist for a number of acoustic cycles. in this case the bubble's radius increases and decreases (expands and contracts) according to the applied us frequency. inertial (transient cavitation) occurs when bubbles grow faster expanding 2- or 3-fold their resonant size, oscillate unstably, and collapse in a single compression half cycle. it has been considered that the primary mechanism to affect the structure of intact cells is inertial cavitation that can induce irreversible damage as well as increase cell membrane permeability [56, 57]. an important application of hifu and microbubbles lies in the area of altering the permeability of the blood brain barrier (bbb). in a study in 2002, hifu induced reversible, nondestructive, bbb disruption in a targeted area and this opening reversed after 72 h. the authors showed with microscopy that hifu either entirely preserved brain architecture while opening the bbb, or generated tissue damage in a small volume within the region of bbb opening. further electron microscopy suggested that hifu disrupted the bbb by opening capillary endothelial cell tight junctions, a mechanism that was not observed in other methods used to open bbb. the effect of fus on tight junctions ' integrity was later confirmed in a study investigating rat brain microvessels after this bbb disruption. the authors used immunoelectron microscopy to identify tight junctional proteins such as occludin, claudin-1, claudin-5, and submembranous zo-1 after sonication. monitoring the leakage of horseradish peroxidase (mw 40 kda) the authors observed that the bbb disruption appears to last up to 4 h after sonication. in a later study the role of caveolin in the mechanism of fus-bbb enhanced permeation was suggested. in a study investigating caveolae density it was found that caveolae and caveolin-1 were primarily localized in the brain microvascular endothelial cells of all the animals tested (rats) regardless of treatment, and that caveolin-1 expression was the highest in the rats treated with both fus and microbubbles. the authors concluded that caveolin-1-mediated transcellular transport pathway may cooperate with other transport pathways (e.g., tight junctional disruption) to induce opening of the bbb. the bbb opening was measured by an mri contrast agent evaluating the local enhancement in the brain. the authors found that low ultrasound powers and pressure amplitudes were found to cause focal enhancement of bbb permeability. trypan blue injected before animals were sacrificed indicated blue spots in the areas of the sonicated locations. the authors concluded that hifu disruption of bbb could be used enhancing drug delivery to the brain. tested the safety of this method by searching for ischemia and apoptosis in areas with bbb disruption induced by pulsed ultrasound in the presence of gas bubbles and by looking for posttreatment effects up to one month after sonication. pulsed ultrasound exposures (sonications) were performed in the brains of rabbits under monitoring by mri. whole brain histologic examination was performed using staining for ischemic neurons and tunel staining for apoptosis. tiny regions of extravasated red blood cells scattered around the sonicated locations, indicated capillaries. despite these vasculature effects, only a few cells in some of the sonicated areas showed evidence of apoptosis or ischemia. the authors found that ultrasound-induced bbb disruption is possible without inducing substantial vascular damage that would result in ischemic or apoptotic death to neurons. the method could find application in the delivery of large therapeutic molecules that do not normally permeate the bbb. herceptin (trastuzumab), a humanized anti-human epidermal growth factor receptor 2 (her2/c-erbb2) monoclonal antibody, was delivered locally and noninvasively into the mouse central nervous system through the blood-brain barrier under image guidance by using an mri-guided focused ultrasound. the amount of herceptin delivered to the target tissue was correlated with the extent of the mri-monitored barrier opening, making it possible to estimate indirectly the amount of herceptin delivered. it was further shown that dopamine d(4) receptor-targeting antibody could also be delivered using the same technique in the brain [65, 66]. delivery of small molecules can also be enhanced with the use of hifu cavitation disruption of the bbb. demonstrated relatively high concentrations of doxorubicin in the brain with minimal healthy tissue damage effects. mri signal enhancement in the sonicated region correlated strongly with tissue doxorubicin concentration, suggesting that contrast-enhanced mri could perhaps indicate drug penetration during image-guided interventions. konofagou and coworkers assessed the spatial permeability of the bbb-opened region using dynamic contrast-enhanced mri (dce-mri) in mice. the authors processed dce-mr images using the general kinetic model and the reference region model. permeability maps were generated and the ktrans (the transfer rate constant from the intravascular system to the extracellular extravascular space) values were calculated for a predefined volume of interest in the sonicated and the control area for each mouse. the results demonstrated that ktrans in the bbb-opened region was at least two orders of magnitude higher when compared to the contralateral (control) side. there are several parameters to affect the level of bbb enhanced permeability and the endothelial tight junctions disruption; the pulse sequence comprising short bursts, the spacing between bursts or the rate of infusion of the microbubbles, and the size of microbubbles were found to affect the effect on bbb disruption [69, 70]. the brain-derived neurotrophic factor (bdnf) was delivered to the left hippocampus in mice through the noninvasively disrupted blood-brain barrier (bbb) using focused ultrasound. the bdnf bioactivity was found to be preserved following delivery as assessed quantitatively by immunohistochemical detection of the ptrkb receptor and activated pakt, pmapk, and pcreb in the hippocampal neurons. it was shown that bdnf delivered this way induced signalling effects in a highly localized region in the brain. however it is the area of targeting brain tumours that have attracted most interest in the fus disrupted bbb. mei and colleagues investigated the effects of targeted and reversible disruption of the blood-brain barrier by mri-guided focused ultrasound and delivery of methotrexate to the rabbit brain. the authors recorded that the methotrexate concentration in the sonicated group was notably higher than that in both the control group (intravenous administration) and the internal carotid artery administered group. they observed a greater than 10-fold increase in the drug level compared to internal carotid administration without fus. liu et al. investigated the delivery of 1,3-bis(2-chloroethyl)-1-nitrosourea (bcnu) to glioblastomas in rats with induced tumours with the help of fus. the authors found that fus significantly enhanced the penetration of bcnu through the bbb in normal and tumour-implanted brains without causing bleeding. surprisingly, treatment of tumour-implanted rats with focused ultrasound alone had no beneficial effect on tumour progression. however, treatment with focused ultrasound before bcnu administration controlled tumour progression and improved animal survival relative to untreated controls. liu and colleagues recently assessed fus-mediated delivery of an iron oxide magnetic nanoparticle (mnps) conjugated to an antineoplastic agent, epirubicin. they used mnps because of the favourable mr imaging characteristics, which could facilitate imaging. they demonstrated a substantial accumulation of mnps, as well as epirubicin, up to 15 times the therapeutic range in the brain when delivered with fus. they further showed decreased tumour progression in animals with brain tumours that received mnp with epirubicin via fus. receptors targeting liposomal nanocarriers have been combined with mrgfus to treat brain tumours. in a recently presented study it was shown that pulsed hifu and human atherosclerotic plaque-specific peptide-1- (ap-1-) conjugated liposomes containing doxorubicin (ap-1 lipo-dox) acted synergistically in an experimental brain tumour model. prior to each sonication, ap-1 lipo-dox or unconjugated lipo-dox were administered intravenously, and the concentration in the brain was quantified. drug injection with sonication increased the tumour-to-normal brain doxorubicin ratio of the target tumours by about twofold compared with the control tumours. moreover, the tumour-to-normal brain ratio was the highest after the injection of ap-1 lipo-dox with sonication. the results of this study indicate that combining targeting strategies can substantially enhance delivery of chemotherapy in the brain. in a separate study the authors investigated the pharmacokinetics of i-labeled ap1-lipo-dox using microspect. the authors confirmed that sonication increased liposomal doxorubicin concentrations in tumour areas (murine glioblastoma) and that molecular targeting acts synergistically with fus. targeted gene transfer into central nervous system was investigated using mri-guided focused ultrasound-induced blood-brain barrier disruption. the results of this study showed that mri-guided fus achieved plasmid dna transfer across the opened bbb furthermore plasmid ware internalized into the neurons presenting heterogeneous distribution and numerous transparent vesicles were observed in the cytoplasm of the neurons in the sonicated region, suggesting vesicle-mediated endocytosis. bdnf (and bdnf-egfp) expressions were markedly enhanced by the combination of ultrasound and pbdnf-egfp-loaded microbubbles about 20-fold than that of the control group. the method by using mri-guided fus to induce the local bbb disruption could accomplish effective targeted exogenous gene transfer in the cns. in this the investigators conjugated plasmid onto the surface of microbubbles and they coated these carriers using polymers in a layer by layer technique. an exciting application is the delivery of therapeutic stem cells to the brain using fus to potentially treat neurodegenerative diseases, traumatic brain injury, and stroke. mri guidance was used to target the ultrasound beam thereby delivering iron-labeled, green fluorescent protein (gfp) expressing neural stem cells specifically to the striatum and the hippocampus of the rat brain. immunohistochemical analysis confirmed the presence of gfp-positive cells in the targeted brain regions suggesting that mrigfus may be an effective alternative to invasive intracranial surgery for stem cell transplantation. although a very efficient approach, the use of microbubbles to enhance drug permeation through tissues, it may require significant safety consideration. in a key study in 2005 prentice et al. presented clearly in a well-designed experimental setup that there are important interactions between individual cells and violently cavitating microbubbles leading to large pores in the cell membrane (sonoporation). these effects on cell membrane will need to be thoroughly investigated at microscopical and molecular level to design efficient and safe fus regimes. during the last few years the main dosage forms tested in mrgfus drug delivery strategy are the thermosensitive liposomes and the lipid based microbubbles that can be conjugated with drugs or other liposomes on their surface [78, 81]. rapoport discussed recently the potential of using micelles and fus for enhanced tissue permeation. micelles are nanosized carriers able to carry hydrophobic drugs; their combination with fus could substantially enhance their delivery in tissues. kostarelos and colleagues suggested the incorporation of thermosensitive peptides onto liposome bilayers to enhance thermoresponsiveness, and the group of lammers designed polymer-based microbubbles for ultrasound drug release. it is clear that already established delivery systems such as different structurally nanocarriers have not been investigated in combination with image guided fus. it would be interesting to see the effect of fus on the enhanced permeability of micelles, polymers (dendrimers cyclodextrins), or metal nanoparticles (gold-iron) to tissues. polymers or proteins that respond to small change of temperature could form suitable image guided fus triggered platforms. the effects of fus in biological tissues with or without carriers will require a more thorough investigation to understand the short- and long-term effects of ultrasound in the body and the complex environments such as tumours, blood vessels, and bone. the mechanism of fus induced hyperthermia and/or the fus tissue permeability increase is not well understood at cellular and molecular levels. there is limited knowledge on the effects of fus on genomic dna and if certain proteins are overexpressed after fus treatment. in addition to the above, the frequency of fus drug delivery treatments (or dosing) and the long-term effects in the body will have to be investigated in preclinical studies in order to design a fus drug treatment regime. an imaging modality will have to be used for accurate image guided fus therapy. in the case of mri clinically approved contrast enhancing agents will have to be added to the delivery system to monitor carriers ' distribution in the treatment area as well as efficient and rapid release. considering the approval in clinical applications, such treatments will require the control of several factors such as drug and drug carrier, mri contrast enhancing agents, and mrgfus parameters, and this could mean several regulatory hurdles. however, the fact that most of the components (fus, liposomes) have been tested in clinical trials is encouraging for such approach to move forward. most of the current strategies to increase tumour specificity of nanocarriers include the use of tumour biomarkers for either targeting (receptors) or for triggered release (internal stimuli; ph proteases) and/or the use of external stimuli such as light and ultrasound. biomarkers and internal stimuli may vary in different tumours indicating that such nanocarriers for cancer treatments should be individualised. external stimuli can be used independent the tumours characteristics and therefore guarantee a more uniform effect. it also shows the significant advantages of being noninvasive as well as controlled and focused. overall mrgfus drug delivery is a novel and valuable tool to increase drug targeting and tissue specific drug delivery. it is expected that future studies will prove the clinical efficacy of mrgfus drug delivery applications.

ultrasound-mediated drug delivery under the guidance of an imaging modality can improve drug disposition and achieve site-specific drug delivery. the term focal drug delivery has been introduced to describe the focal targeting of drugs in tissues with the help of imaging and focused ultrasound. focal drug delivery aims to improve the therapeutic profile of drugs by improving their specificity and their permeation in defined areas. focused-ultrasound- (fus-) mediated drug delivery has been applied with various molecules to improve their local distribution in tissues. fus is applied with the aid of microbubbles to enhance the permeability of bioactive molecules across bbb and improve drug distribution in the brain. recently, fus has been utilised in combination with mri-labelled liposomes that respond to temperature increase. this strategy aims to activate nanoparticles to release their cargo locally when triggered by hyperthermia induced by fus. mri-guided fus drug delivery provides the opportunity to improve drug bioavailability locally and therefore improve the therapeutic profiles of drugs. this drug delivery strategy can be directly translated to clinic as mrg fus is a promising clinically therapeutic approach. however, more basic research is required to understand the physiological mechanism of fus-enhanced drug delivery.

PMC3666208

pubmed-629

a 33-year-old male (height 172.3 cm and weight 61.5 kg) visited our pain clinic due to neck pain radiating to both shoulders, which started 3 years ago. he complained of tingling sensations of the bilateral hand along with shoulder pain, and difficulty in neck flexion. upon physical examination, the results of the jackson compression test and the spurling's test did not appear positive. on the cervical mri, c5/6 and c6/7 intervertebral disc protrusions were found and chronic right c6 radiculopathy was found on the electromyography. this patient had already received an epidural steroid injection and decompressive neuroplasty using a racz's catheter at a local hospital. therefore, we decided to perform cervical nucleoplasty at the c5/6 and c6/7 intervertebral discs. on the pre-procedural laboratory tests, there were no abnormalities in the cbc, esr, and blood chemistry test (table 1). nucleoplasty was performed on the c5/6 and c6/7 intervertebral discs without any perioperative events. under a supine position, skin preparation was performed with betadine soap and 2% chlorhexidine-70% isoprophyl alcohol solution, and a sterile surgical drape was applied. after the internal carotid was laterally displaced, a 19 gauge 3 inch introducer needle was introduced till the anterolateral annulus fibrosus. after the needle was advanced deeper, the position of the needle was adjusted to reach the target site of the herniated disc under a c-arm guide. when the needle reached the proper target site, the stylet of the introducer needle was withdrawn, and the perc dc spine wand (arthrocare co., sunnyvale, ca, usa) was replaced and fastened to the needle hub. next, the perc dc spine wand was connected to the arthrocare system 2,000. after confirming that there was no cervical root stimulation, coblation was then carried out by rotating the flange 180 for 20 seconds. after this, the wand was retracted 1-2 mm under c-arm guidance and the same procedure was repeated 3 times. stay, the body temperature was 36.7-36.9 and there were no signs or symptoms of acute complications. an additional amount of intravenous cefazolin was administered three times a day and a routine prophylaxis for 2 more days during the hospital stay. additional oral cefradine was prescribed 1,000 mg/day for 1 week after the patient's discharge. after 8 days, the patient revisited our outpatient clinic for routine postoperative checkup, but there were no improvements in the patient's symptoms. in addition, there were no symptoms or signs of infection including fever or chills, and the laboratory findings, such as the cbc and esr, were in the normal range. after 2 months, during his second postoperative visit to our clinic, the patient complained of right shoulder pain and worsening neck pain during flexion. he also complained of feelings of weakness in both arms, but his motor functions were intact when a physical examination was performed. in addition, new physical signs developed; the jackson compression test and spurling's test were positive, with radiating pain in both shoulders. there was tenderness on the right cervical facet joint, but no tenderness was observed on the left side. the body temperature was normal, and there was no sensation of chills. in order to evaluate the focus of the pain, a cervical mri was performed, and the findings were compatible with spondylodiscitis at the c6/7 intervertebral disc and the vertebral body (fig. the patient was referred to the neurosurgery department immediately, and intravenous antibiotics therapy was planned. however, with the patient's will, he was transferred to another hospital for long-term antibiotics therapy. early diagnosis and treatment of spondylodiscitis is important for the prevention of catastrophic sequelae and huge additional expenses. therefore, it is essential to understand the natural course of postoperative spondylodiscitis. usually, short-term relief of the symptoms comes first after surgery, and then back pain recurs within 6 weeks. according to bavinzski et al. in addition, fever was a typical symptom associated with this infection. in our case, neck pain was aggravated and new positive signs in the physical exam appeared 8 weeks after the procedure. moreover, our patient was afebrile, which can be explained by the lack of elevation of an inflammatory marker. the esr and crp are known as the earliest and most sensitive screening tools for the infection. meyer et al. suggested the sensitivity and specificity of crp as 100% and 95.8%, respectively, when predicting postoperative infections. in addition, mustard et al. conducted a study on 108 patients, and a positive crp response was defined as meeting two criteria: on days 3 and 4, the crp level is>80% of day 2 (positive diagnosis by day 4); and after day 4, the crp rises on 2 consecutive days with a level greater than 15 mg/l for each day (positive diagnosis by day 6). the above criteria had a sensitivity of 63%, a specificity of 82%, a positive predictive value of 68% and a negative predictive value of 78%. recommended that an esr of more than 50 mm/hr for more than 2 weeks after surgery be used as an early diagnostic marker for spondylodiscitis, indicating a need to perform more tests. however, during the early postoperative period, the crp and esr may rise as a consequence of tissue damage caused by the surgery itself. kwon et al. found that the crp and white blood cell (wbc) rose starting on the first day of surgery and decreased after the third postoperative day, and the esr elevation lasted up to 11 days after spine surgery. to screen postoperative infections with laboratory tests, adequate follow up periods and tests should be considered, with further evaluations being needed to confirm spondylodiscitis. based on the previous studies, crp should be checked 4 days after the surgery and compared with a baseline value (preoperative value), while the esr should be checked 12 days after the surgery. there has been a reported case of spondylodiscitis following cervical nucleoplasty: in theat case, the complication was detected 3 weeks after the procedure. in our case, outpatient follow-ups were performed 9 days and 8 weeks after the operation. during the first visit, in our case, neither the infection nor the procedure itself increased the esr and crp. further research is needed to clarify the changes of the inflammatory marker after nucleoplasty. at the second visit, the crp was checked and was identified to be within the normal range. during the 8-week postoperative follow-up, we could not find any abnormalities in the laboratory tests even when the mri showed apparent infection. bavinzski et al. reported a series of 13 patients with postoperative disc space infection. all patients suffered from severe local lumbar pain with muscle cramps and radiating pain to the hips, abdomen, legs, scrotum, groin, or perineum. many patients were unable to stand, with the pain exacerbated by motions of the spine segments. in a recent review article, the typical course of spondylodiscitis is the return of low back pain approximately six weeks later. similar to the previous studies, our patient also complained about pain being aggravated by motion and local tenderness around the infection site about 8 weeks after the spinal procedure. therefore, clinicians should be careful if patients complain of aggravated pain about 6 weeks post-operation, while additional studies are required to diagnose the infection. if there is no neurologic or structural instability, it is recommended that antimicrobial therapy should be withheld until positive identification of the organism is achieved unless back pain is accompanied by systemic symptoms. however, in our case, the infection was identified at the site of nucleoplasty 2 months later, and the patient's symptoms were aggravated. therefore, the infection was considered to be a procedure-related problem and empirical antibiotics therapy was initiated. surgical intervention is recommended in cases of impending pathologic fractures, functionally significant neurologic deficits, paravertebral or epidural abscess formations, persistent septicemia despite antibiotic treatment, intractable pain, and unacceptable saggital or coronal plane deformity. we postulate that the pathogen may be sequestrated from the blood stream completely, therefore, systemic inflammatory changes were not triggered, or the immunity of this patient (33 year-old man) was strong enough to overcome the dispersion of the pathogen. for detecting postoperative discitis, mri is the imaging method of choice, with its high sensitivity and specificity. mri is considered to be the most sensitive and specific method, 93% and 97%, as reported. furthermore, mri is the gold standard for diagnosing spondylodiscitis. in conclusion, therefore, an mri should be taken when there is clinical suspicion of infection in order to not miss the complications after interventional procedures, even if the laboratory findings were normal.

infective spondylodiscitis is a rare complication that can occur after interventional spinal procedures, of which symptoms are usually back pain and fever. early diagnosis of infective spondylodiscitis is critical to start antibiotics and to improve prognosis. laboratory examinations including complet blood cell count (cbc), erythrocyte sedimentation rate (esr), and c-reactive protein (crp) are conventional tools for the early detection of infectious spondylitis. however, we experienced infective spondylodiscitis after cervical nucleoplasty which did not display any laboratory abnormalities, but was diagnosed through an mri. a patient with cervical disc herniation received nucleoplasty at c5/6 and c6/7. one month later, the patient complained of aggravated pain. there were neither signs of chill nor fever, and the laboratory results appeared normal. however, the mri findings were compatible with infectious spondylodiscitis at the nucleoplasty site. in conclusion, infectious spondylodiscitis can develop after cervical nucleoplasty without any laboratory abnormalities. therefore, an mri should be taken when there is a clinical suspicion for infection in order to not miss complications after interventional procedures, even if the laboratory findings are normal.

PMC3629348

pubmed-630

methylmercury is efficiently accumulated through estuarine food webs, such that concentrations in fish are controlled by initial ch3hg assimilation at the base of the food web. sources and pathways of ch3hg and hg accumulation in lower trophic levels are therefore fundamental to understanding hg cycling in estuaries and controlling hg levels in top predator fish. estuarine sediments are a sink for hg and also a dominant site for methylation of hg to the more bioavailable species, ch3hg. benthic organisms, which dwell at the sediment water interface, are exposed to hg from both sediments and the water column and are also a food source to larger invertebrates and fish. as such, they present an important link between hg bioaccumulation in benthic and pelagic food webs, yet dominant sources and pathways of hg uptake in benthic fauna are not well understood. benthic species are exposed to dissolved metals from porewater and overlying water and also take up metals from dietary sources, which can originate in both the sediment and water column. identifying the uptake and trophic transfer between sediment and water food sources and primary consumers is important to linking water and sediment quality data to bioaccumulation. an important benthic species along the east coast of north america is the estuarine amphipod, leptocheirus plumulosus, which is abundant over a wide range of sediment types and salinity and temperature conditions.l. plumulosus is also used as a test organism to assess both acute and chronic toxicity of estuarine sediments. l. plumulosus are facultative feeders; they live in u-shaped burrows through which they filter water and ingest suspended particles and also roam the surface of the sediment and deposit feed, thus deriving their nutrition and exposure to contaminants from both the water column and sediments. microcosm and modeling studies have shown dietary ingestion of organic matter, rather than uptake from the dissolved phase, to be the dominant process for ch3hg and hg accumulation in l. plumulosus(6,18) over a range of assimilation efficiencies (ae) and ingestion rates (ir), the relative contribution of food sources to body burden was modeled to be>80% for ch3hg and 1060% for hg, although accumulation was determined in water with no appreciable dissolved organic carbon (doc), and uptake of both species from natural water is inversely correlated to doc concentrations. ingestion pathways are ignored in toxicity testing, which assumes metal exposure is predominantly from sediment porewater. biokinetic models of metal accumulation currently lack estimates of uptake from deposit vs suspension feeding. feeding behavior in l. plumulosus is a critical piece of information to understanding hg accumulation and trophic transfer. in this study, we used enriched isotopic ch3hg and hg tracers in microcosm experiments to directly determine hg uptake pathways in l. plumulosus. two experiments were conducted to study feeding pathways of ch3hg (experiment 1) and hg (experiment 2). this approach had several advantages: (1) using multiple enriched isotopes of hg allowed us to simultaneously decipher uptake from different pathways. (2) we could expose amphipods to ch3hg and hg at environmentally relevant levels. (3) unlike radiometric tracer studies, this technique enabled hg species transformation to be traced throughout the experiment. because ae of ch3hg is much higher than that of hg, methylation of hg will increase availability to the biota. methylation occurs in anoxic zones in sediment and has also been observed in algal cultures. if hg speciation is not monitored during incubation in uptake experiments, bioaccumulation of hg species may be misinterpreted. enriched stable isotopes of hg species have been used as a powerful tool to study hg cycling and uptake into freshwater foodwebs. in this study, we applied the technique to identify trophic transfer of hg species from live phytoplankton in water and sediment compartments in estuarine benthic infauna. all solutions were made up using ultrapure water (> 18 m cm) produced by a purelabpluswater purifier (us filter, ma, usa). enriched stable isotopes of hg (as hgo, hgo, and hgo) single isotope spikes of hg and hg were prepared by dissolution of hgo in hcl, followed by dilution to 400 mg/l in 5% hcl (fisher optima grade, pittsburgh, pa, usa). solutions of ch3hgcl and ch3hgcl were synthesized by conversion of hgo to hgcl2 followed by reaction with methylcobalamin. stock solutions were diluted to 50 mg/l of ch3hg and 5 mg/l of ch3hg in 0.5% acetic acid and 0.2% hcl (fisher optima). isotopic abundances of natural hg and the enriched isotope spikes are given in the supporting information. the marine phytoplankton species, i. galbana, was used as the food source for the ch3hg and hg exposures of l. plumulosus. cells of i. galbana were grown in a 19 l carboy of 20 ppt artificial seawater (instant ocean prepared in ultrapure water) to a density of 1.3 10 cells per milliliter and then reduced by centrifugation to 800 ml. the concentrated culture was then divided into four 200 ml solutions, and each algal solution was spiked with one isotopically enriched hg species. in experiment 1 (ch3hg uptake), the suspensions were tagged with 2.5 g ch3hg or ch3hg and in experiment 2 (hg uptake), with 34 g of hg or hg. the isotopically labeled cultures were allowed to equilibrate for 24 h and then spun down into pellets and the supernatant discarded. aliquots (1 ml) of each cell suspension were removed and then centrifuged, and the resulting algal pellets were freeze-dried and weighed. the pellet and supernatant solutions (both n=4) were analyzed for ch3hg and thg, and the fraction of algal-bound hg was determined (94 1% for ch3hg, and 95 2% for ch3hg; 98.1 0.1% for hg and hg). concentrations of hg spikes in the algal slurries are included in tables 1 and 2. leptocheirus plumulosus was purchased from aquatic research organisms (hampton, nh) and cultured according to epa method 1994. similarly sized (> 1 mm) amphipods were retrieved from culturing tanks prior to the experiment and depurated for 4 h in salt water. microcosm experiments were conducted in 100 ml (5 cm diameter, 10 cm height) glass jars (qorpak, bridgeville, pa, usa). the organic carbon content was determined to be 3.2 0.1% by loi (dry sediment was heated at 550 c for 4 h). for each experiment, 16 identical microcosms were assembled. in each microcosm, 1 ml of algal suspension containing enriched isotope ch3hg (experiment 1) or hg (experiment 2) was mixed with 16 g of wet sediment, and the other enriched isotope-tagged algae was mixed with 60 ml of 20 ppt artificial seawater (table 2; henceforth isotopically enriched hg species are denoted by a subscript (hgsed or hgwater) indicating the compartment to which the spiked algae was added). the sediment was added to a jar (1 cm depth), along with 5 amphipods, and overlying water was added. microcosms were kept in an environmental chamber (20 c, 10:14 (l/d) h photoperiod), covered loosely with plastic to minimize evaporation, and slowly agitated on an orbital shaker (100 rpm) to maintain aeration and suspension of algae. a water sample was taken from four microcosms immediately after assembly (t=0); then four microcosms were dismantled for analysis after 12, 24, 36, and 48 h. this two-day time series was chosen to monitor relative accumulation of hg from different sources in amphipods over time, and to minimize the effects of algal death and decay on bioavailability of ch3hg and hg associated with longer incubation periods. changes in hg concentration and speciation were monitored in whole and filtered water and sediment throughout the time series. a second set of microcosms was assembled under the same conditions but using algae suspensions not spiked with hg species, to monitor dissolved oxygen (do), ph, and oxidation reduction potential (eh) throughout the experiment. results are reported in the supporting information (si). to dismantle microcosms after each time point, a 15 ml aliquot was filtered through a 0.45 m poresize 25 mm syringe tip filter (fisherbrand), and a second 15 ml sample was collected unfiltered. both filtered and whole water samples were acidified to 0.5% hcl (fisher optima). amphipods from each microcosm were removed from the sediment by pasteur pipet and thoroughly rinsed in two sequential water baths and placed in a preweighed 15 ml glass vial (i-chem). sediments were removed from the microcosm jars and placed in 60 ml pfa vials (sarstedt, nmbrecht, germany). amphipod and sediment samples were freeze-dried; all concentrations are reported on a dry weight basis. all sample containers were double bagged and stored refrigerated in the dark until analysis. freeze-dried amphipod samples were weighed and extracted in glass vials. to each vial, 1.5 ml 4 m hno3 was weighed, and samples were heated overnight at 60 c. a 50 l aliquot of each extract was transferred to a 40 ml amber glass vial with teflon-lined septa (brooks rand laboratories, seattle, usa). samples (8 ml) of filtered and whole overlying water were weighed into 40 ml vials. portions (0.5 g) of freeze-dried, homogenized sediment samples were leached with kbr/h2so4/cuso4, extracted into ch2cl2, then back extraction into 10 ml water; then 2 ml of the extract was weighed into 40 ml glass vials. samples were buffered and derivatized, then analyzed using a merx-m methylmercury analysis system (brooks rand laboratories, seattle, usa) coupled with a 7700x agilent icp-ms (agilent technologies, santa clara, usa); the details of this technique are given in taylor et al. isotope deconvolution was used to separate hg species derived from three sources: the ambient hg in the sample (determined from the hg signal) and hg inputs from two enriched hg-tagged algal spikes added to the water and sediment compartments. external calibration was performed using six standards (0.025 to 25 ng/l), and a secondary source calibration check was analyzed every 10 samples. method detection limits (mdl) were 0.05 ng/g (sediment), 5 ng/g (amphipods), and 0.13 ng/l water. recoveries for ch3hg were 85 13% (n=2) in bcr-580 estuarine sediment (irmm, geel, belgium) and 106 10% (n=3) in nist 2976 mussel; spike recoveries in water were 99 5% (n=5). quantification of total hg from biological tissues was determined from 4 m hno3 extracts. aliquots (0.5 ml) of amphipod extracts were diluted 10 times by weight and analyzed directly by icp-ms (agilent 7700x), with a mdl of 20 ng/g. portions of sediment samples (0.25 g) were weighed into 60 ml pfa tubes (sarstedt) and 5 ml of 45% hno3: 5% hcl v/v was added to each tube. samples were digested in an open vessel microwave (mars xpress, cem corps, matthews, nc, usa) at 95 c for 1 h. digested samples were diluted to 50 ml with ultrapure (18 m) water, and weighed, then analyzed by icp-ms, with a mdl of 0.1 ng/g. signal intensities were deconvoluted (si) prior to conversion to concentration by external calibration. recovery of standard reference materials nist 2711a (n=2) and nist 2976 (n=3) were 80 1% and 115% 11%, respectively. filtered and whole overlying water and supernatant samples from algal suspensions were analyzed by cold vapor-icp-ms, using an automated merx t purge and trap system (brooks rand) coupled with icp-ms based on epa method 1631. samples (8 ml overlying water or 0.4 ml supernatant) were weighed into 40 ml clear glass vials with teflon-lined septa (brooks rand) and diluted to 25 ml with 1% hcl. samples were digested overnight in capped vials, by the addition of 0.1 ml freshly prepared brcl (0.16 g kbr was dissolved in 15 ml hcl, then 0.38 g kbro3 added and stirred for 1 h). to each sample, 0.1 ml sncl2 and 0.1 ml hydroxylamine (brooks rand) were added, then vials were recapped for analysis. bioaccumulation factors (baf) were calculated for each uptake pathway to normalize body burden to different exposure concentrations. body burdens of isotopically enriched hg from 36 and 48 h time points were divided by sediment concentrations in each microcosm; e.g., in experiment 1, baf for ch3hgsed was calculated as the concentration of ch3hgsed (ng/g) in amphipods relative to its concentration in sediment. metal accumulation from ingestion has been described by the model in eq 1(40,41)1where the body burden of a metal species taken up from food, css, is calculated from the metal concentration in food (cf), the assimilation efficiency of the metal species (ae, unitless), the ingestion rate, ir (mg food/g body wt/d), the metal efflux rate constant following uptake from food, kef (1/d), and the growth rate constant, g (1/d). the equation was rearranged to calculate ir. reported values of ae (ch3hg=80%, hg=6%), kef (ch3hg=0.052 d, hg= 0.089 d), and g (0.07 d) for l. plumulosus were applied, and concentrations of isotopic tracers in algae cf and amphipods css were used to distinguish ingestion from benthic and pelagic sources. nonparametric analyses were chosen because distribution could not be ascertained due to small sample sizes. significant trends in spike concentrations in sediment, water, and amphipods with time were assessed by kruskal wallis rank tests; specific differences in concentration between time points were determined using the mann whitney rank sum statistic. using the two enriched isotopes, ch3hg concentrations from spiked algae added simultaneously to the water column and sediment were tracked in the sediment, water column, and in amphipods over time (table 1). for sediment, differences in concentrations of both enriched isotope hg species were not significant between time points (kruskal wallis: d.f. =3, p>0.05), so data for all time points were pooled for each experiment. only 0.2% of the recovered ch3hgwater was suspended in the water column after 12 h, whereas concentrations of ch3hgwater and ch3hgsed in the homogenized sediment samples were similar. variation in ch3hg tracer levels occurred in the water column between time points (table 1). levels of ch3hgwater in the initial whole water sample (t=0) were elevated and variable between microcosms. concentrations of ch3hgwater decreased from 0 to 24 h and then reached a steady state between 24 and 48 h (mann whitney u, p>0.05). in the filtered water fraction, low levels of the ch3hgsed tracer were also present in the water compartment. enriched isotope ch3hg tracers were present in amphipods after 12 h (figure 1), and concentrations of both tracers in amphipods did not vary significantly across time series (kruskal wallis, d.f.=3, p>0.05), although variance between replicates at each time point was large (1646%). body burdens of isotopically enriched ch3hg tracers in l. plumulosus over time (experiment 1). the two enriched isotopes of hg were tracked over time in microcosms (table 2). as with experiment 1, both tracers were present in the bulk sediment, including 99.9% hg-tagged algae added to the water column. concentrations of hg tracers in sediment did not vary with time (kruskal wallis d.f.= 3, p>0.05), whereas tracer concentrations in the whole water fraction were variable. whole water samples were elevated in hgwater at t=0 (mann whitney u, z=2.3, p=0.02) immediately following the addition of spiked algae to the surface water but reached an apparent steady state from 24 to 48 h (p>0.05). concentrations of hgwater in the filtered water were also highly variable at t=0 but did not change significantly from 0 to 48 h (kruskal wallis, d.f. methylation of the hg spikes was evident during incubation of the i. galbana suspensions (table 2; 0.70.8% ch3hg). the% ch3hg of tracers in sediment increased slightly relative to the starting algal suspension (mann whitney u,% ch3hgsed: z=2.5, p=0.01;% ch3hgwater: z= 2.0, p=0.04). the% ch3hg in the hgsed tracer, mixed with the sediment, was slightly higher than in the hgwater tracer initially added to the water column (mann whitney u, z=2.2 p=0.03), although variability in% ch3hg in tracers among microcosms was high (rsd=5863%). in the whole and filtered water samples,% ch3hgsed was higher than in the algal suspensions (table 2) prior to addition to the sediment (mann whitney u, z=2.5, p=0.01). in the overlying water,% ch3hgsed was not significantly different than in the sediment (p> 0.05). there was no difference between% ch3hgwater in the water column and in the algal suspensions or sediments (p>0.05). neutral ph (8) and oxic conditions (do>6 mg/l) in the sediment and water column were maintained over the time course under these experimental conditions (si). body burdens of isotopic tracers in amphipods, as thg, ch3hg, and% ch3hg are shown throughout the time series in figure 2. ambient thg in amphipods (from hg) was 15 ng/g with 39 12% ch3hg (all time points; n=16). at t=12 h, body burdens of hgwater were higher than in later time points, even when one high value (1812 ng/g) was removed (mann whitney u, z=2.3, p=0.02). conversely, increases in hgsed body burden were significant with time (figure 2a). (a) body burdens of isotopically enriched hg tracers, as thg concentrations. (b) ch3hg concentrations and% ch3hg in l. plumulosus over time (experiment 2). letters (a, b, c for hgsed; or y, z for hgwater) signify statistical differences between time points by mann amphipods accumulated methylated forms of both tracers, comprising up to 18% of thg (figure 2b and c). uptake of ch3hgwater was evident, but there was no relationship between accumulation and time (kruskal wallis, d.f.=3, p>0.05), whereas ch3hgsed body burden was significantly related with time. the trend was also significant when normalized to total hgsed (as% ch3hgsed). because 99.8% ch3hgwater and 99.9% hgwater added to the water column were recovered in the bulk sediment, bafs for both enriched isotopes were calculated as body burdens normalized to sediment concentrations. the baf for ch3hgwater was 682 442 (unitless), whereas for ch3hgsed, the baf was 30 19, such that uptake of ch3hg from organic material of pelagic origin was 23 higher than from algae mixed with the sediment. for experiment 2, bafs for hg were 0.9 0.2 for hgsed and 5.8 2.0 for hgwater, and bioaccumulation from settling algae was only 6 higher than for sediment sources. for the ch3hg experiment, ir was determined to be 65 mg/g/day from ingestion of algae added to the water column and 3.1 mg/g/day for ingestion of algae mixed in with the sediment. for the hg experiment, ir was 30 mg/g/day for algae originating in the water column and 5.2 mg/g/day for sediment feeding. the ir is a measure of the milligrams of food consumed (per body wt/day) and should not vary with metal species, although its calculation does depend on the accuracy of the applied constants (ae, kef, g) as well as the experimentally derived food concentration and body burden. microcosm studies using enriched stable isotopes showed trophic transfer from sedimenting algae was a major source of hg to benthic-dwelling amphipods. this suggests that newly deposited phytoplankton is an important and potentially overlooked source of hg to benthic infauna in ecosystem studies and toxicity testing. enriched stable isotopes are a powerful tool for studying accumulation of hg in aquatic food webs; we applied this technique to a microcosm study to compare trophic transfer of hg by two ingestion pathways. microcosms were scaled down from recommended conditions for culturing l. plumulosus, to minimize the quantity of enriched isotopes needed, but maintaining recommended amphipod densities (< 1.5 cm), sediment depth (1 cm), and water conditions (20 ppt, 20 c, do>4.4 mg concentrations of ch3hg and thg in sediments and whole surface waters in microcosms were within ranges found in new england and estuaries worldwide, although dissolved water concentrations were slightly higher. body burdens of hg from tracers were up to 6 higher than those found in new england estuaries (chen, unpublished: 17105 ng/g). in estuaries, sediment characteristics and food quality have been shown to affect ae and kef in amphipods, as well as survival and reproduction, but specific effects on amphipod feeding behavior have not been reported. because amphipods feed selectively, behavior may change with habitat and food availability. accumulation of hg species from sediment mixed with algae was shown to be higher than from sediment alone, suggesting the abundance of high quality food spiked with hg in this experiment may lead to higher accumulation in amphipods than in a natural environment. homogenized sediment and standardized conditions used in this experiment allow for direct comparison of metal accumulation from different feeding modes under optimal conditions. algae added to the water column was rapidly deposited to the sediment surface, such that>99.8% of ch3hg and hg were recovered from the bulk sediment within 12 h. concentrations of hg species added to the water column varied over time, due to settling of algae added to the water, and diffusion/suspension of tracers added to the sediment. variability between samples was high at initial time points due to the capture of large suspended particles during sampling. while sediment concentrations were similar for both tracers added to the microcosm, spiked algae originating in the water column was deposited to the sediment surface, whereas the spikes originating in the sediment were thoroughly mixed in the bulk sediment. tracking speciation throughout the experiment proved important as methylation of hg spikes was observed. in radiometric methods, hg species can not be distinguished, and species transformation alters the bioavailability of hg, which could lead to inaccuracies in uptake modeling. methylation of tracers was observed in spiked algal cultures (0.70.8%) and has previously been observed in cultures of periphyton. further methylation was also evident during sediment incubation (23% ch3hg), despite maintaining oxic conditions throughout the experiment. this may also occur during toxicity testing, where sediment incubation conditions are similar to those used here, altering the bioavailability of hg to test organisms relative to natural environments. because the experiment focused on uptake, a short exposure duration was used to minimize artifacts from algal decay, water quality changes, and amphipod death/reproduction. the body burden of ch3hg reaches an apparent steady state after 12 h in this experiment, although variability at each time point was high, possibly due to feeding behavior, and to variable metal distribution in the algal food source. lawrence and mason reported an apparent steady state in amphipods sampled at three and six days, but calculated much longer exposures (50 day) are needed to achieve equilibrium. the apparent steady state in body burden may be due to the long time intervals (12 h) used in this study, relative to reported gut passage times of 35228 min for l. plumulosus fed i. galbana. longer exposure to hg sources may lead to higher accumulation if a true steady state was not reached in this experiment, but body burdens represent relative accumulations from different feeding pathways. because tracers were ingested simultaneously and prepared from the same algal source, ae and kef will be the same for both ingestion pathways. amphipods in this study were not depurated, although for ch3hg, this is likely to have little effect on the determined body burden due to its high ae where rate of loss from excretion is negligible relative to growth dilution. variability in body burden of hg tracers was high, likely due to spatial variability of tracers in both food sources. the body burden of hgsed increased significantly between time points, but hgwater did not. the pattern may be explained by preferential uptake of hgwater from settling algae during the first time interval (t=0 to 12 h), then increased consumption of hgsed as amphipods stir up sediment while they burrow. this may be explained by the higher ae of ch3hg (80%) than hg (6%), causing hg to be excreted while ch3hg is mostly retained. this difference in assimilation should be consistent for both isotopic tracers, however, but was not significant for ch3hgwater. uptake of hg tracers initially added to the water column and deposited on the sediment surface was significantly higher than for tracers mixed in the sediment, suggesting ingestion from the sediment surface is the major feeding mode in amphipods. while bafs are useful to normalize body burden to different exposure concentrations, they can be difficult to evaluate between different food sources and habitats. in this study, because the same algal food source was added to both compartments, bafs were used to compare relative uptake from different sources. for both spikes, bafs were determined using concentrations in amphipods relative to the sediment; this assumes that ingestion from the sediment surface or the bulk sediment were distinct uptake pathways and ignores accumulation from the water column. conversely, if both sediment sources of ch3hg were assumed to have the same baf, then bafch3201hg+ (for bulk sediment) could be applied to the concentration of ch3hgwater in the sediment compartment, to predict an amphipod body burden of 45 ng/g ch3hgwater. to accumulate another 625 ng/g of ch3hgwater from the fraction of algae suspended in the water, a 5 mg amphipod would have to ingest 3.9 ng of ch3hgwater (assuming ae=80%). whole water ch3hgwater concentrations were 2.7 to 16.5 ng/l (12 to 48 h), meaning that only 0.15 to 1 ng of ch3hgwater was available for uptake from the entire in 60 ml water compartments. similar calculations suggest amphipods would need to acquire 40 ng of hgwater from suspended algae to achieve determined body burdens, whereas only 1.54 ng of hgwater were present in the microcosms. only a small portion of hg spikes was present in the dissolved water fraction. lawrence and mason found an inverse effect of doc-binding on hg uptake and derived a laboratory-based relationship between doc concentration (mg/l) and water bioaccumulation factor (wbaf): log wbafch3hg=1.740.173[doc], where wbafch3hg is the concentration of ch3hg in amphipods (ng/g wet wt) divided by the dissolved concentration of ch3hg in the water column (ng/l). using this equation, wbafch3200hg was estimated to be 7.5 for a doc concentration of 5 mg/l measured in this study. for a dissolved ch3hg concentration of 2 ng/l (experiment 1; time points 12 to 48 h), amphipod concentrations are calculated to increase 100 ng/g (dry wt, assuming 15% dry/wet weight ratio), which is less than 15% of the determined ch3hgbody burden. wbafhg2+is reportedly 10 times lower than wbafch3hg, under the same conditions. ng/l (experiment 2; time points 12 to 48 h), amphipod concentrations are calculated to increase 80 ng/g (dry wt), which is also 15% of the determined body burden. uptake from both dissolved and suspended algae sources was therefore considered minor in this study, and while newly deposited algae may be considered part of the sediment, this study suggests accumulation was much higher from this new surface layer than from the bulk sediment. as expected, bafs for ch3hg were significantly higher than for hg. in phytoplankton, ch3hg accumulates predominantly in the cytoplasm of algal cells, whereas hg adheres to the cell membrane; the fraction of metal in the cytoplasm is attributed with higher ae during trophic transfer. comparing the bioaccumulation of the two hg species, bafch3hg was relatively higher than bafhg for tracers originating in the water column than for sediment sources. uptake of ch3hg into the cytoplasm of phytoplankton only occurs in live cells, whereas both hg species are bound to the cell membrane in dead cells, causing lower assimilation of ch3hg during trophic transfer. accumulation of ch3hg in some invertebrates (copepods) was shown to decrease with decay of the marine algae, thalassiosira weissflogii; however, ch3hg assimilation in l. plumulosis was not affected, which is thought to be due to this species being well adapted to detrital feeding. release of trace metals from decaying algae decreases exponentially with time and varies between elements. in this study, death and decay may have occurred more rapidly in the phytoplantkon mixed in the sediment, which would explain this relative difference, but the microcosm incubation time was relatively short (48 h), and algal decay has been shown not to affect ch3hg assimilation in amphipods. although amphipods were rinsed, residual particle binding may lead to adsorption of hgsed to the amphipod exoskeleton elevating apparent body burdens from the sediment. given that hg is strongly particle reactive, adsorption of hgsed to the exoskeleton may have been higher than for ch3hgsed, causing the relatively higher baf from sediment sources. to advance biokinetic modeling studies, ir for both feeding pathways of l. plumulosus were calculated. values of ir for surface feeding determined here are in reasonable agreement with values for suspension feeding determined from five different metal species using radiometric techniques (50 to 151 mg/g/day). in this study and in williams et al., ir was calculated using values of ae determined by radiometric techniques, which may have led to overestimation of ae for hg if methylation of hg spikes occurred during incubation. this would lead to underestimation of ir, which was lower for hg than for ch3hg in both studies. no other studies report ir from deposit feeding in amphipods, although shlekat et al. reported a single measurement of 3 g/g/day, which is much higher than was observed here. this may reflect variation in feeding behaviors and effects of food substrate on test organisms. phytoplankton rapidly accumulate ch3hg, but rapid algal growth has been shown to dilute hg in algal food sources and therefore decrease uptake into higher trophic levels. deposition of phytoplankton blooms is a major source of organic carbon flux to sediment surfaces, and this study suggests deposited algae are a source of hg species to primary benthic infaunal consumers. in estuaries, increases in growth and reproduction of l. plumulosis have been correlated to chl a production associated with sedimenting algae. in a study of c-labeled tracers, however, phytodeposition was found to be a less prominent food source than microphytobenthos production for benthic fauna, including amphipods. however, the study also demonstrated that pelagic organic carbon sources were consumed during pulses which mimicked deposition of spring phytoplankton blooms.. turbulence and resuspension of sediment can increase transfer of sediment ch3hg into organisms, and sedimenting algal blooms have also been shown to increase ch3hg production in surface sediments. previously, bioavailability of hg species in estuaries has been inversely correlated with sediment organic matter. this study suggests hg in suspended and freshly deposited algal cells are an important uptake source, and that newly deposited algal particles should be distinguished from bulk sediment organic carbon as compartments controlling hg accumulation. in field collection, bulk sediments are typically collected from the top 4 to 15 cm. field collection of amphipods is reported within the top 57 cm of the sediment, but imaging of amphipod burrows in sediment incubations show the highest burrow volume within the top 1 cm of sediment. our findings suggest that amphipods feed primarily from the sediment surface, suggesting much smaller sampling depths (< 1 cm) are appropriate for assessing hg sources to benthic infauna. l. plumulosus are routinely used in both acute and chronic toxicity tests for estuarine sediments. in acute (10 d) tests, sediments are the only food source, whereas during 28 day chronic toxicity tests, l. plumulosus are fed by an external uncontaminated food source 23 times per week. if in natural systems, algae originating in the water column are a preferred food source, these tests do not accurately assess exposure to contamination, as both tests overlook the influence of contamination originating in the water column, and acute toxicity tests ignore ingestion pathways completely. bioaccumulation varies with feeding behavior, suggesting uptake pathways in test organisms need careful investigation to relate to sediment and water quality criteria.

mercury is a widespread contaminant in marine food webs, and identifying uptake pathways of mercury species, ch3hg+ and hg2 +, into low trophic level organisms is important to understanding its entry into marine food webs. enriched stable isotope tracers were used to study benthic vs. pelagic pathways of ch3hg+ and hg2+uptake via food to the infaunal estuarine amphipod, leptocheirus plumulosus. algal cells differentially labeled with isotopically enriched ch3hg+ or hg2+were added simultaneously to the sediment and water column of microcosms, and hg species were monitored in amphipods and in sediment and water compartments. methylation of hg2+occurred during the course of the experiment, enhancing the uptake of hg2+spikes. trophic transfer of hg from algae added to the water column was determined to be the major uptake route for amphipods, suggesting inputs of contaminated organic matter from the pelagic zone are important to mercury bioaccumulation even in organisms living in sediments.

PMC4014141

pubmed-631

spinal epidural abscess (sea) is a relatively rare disease but frequently, emergent surgery is needed2,9,12,18). their incidence is on the rise because of the various factors such as the increase of the elderly population, intravenous drug use, epidural steroid injection for pain control, epidural anesthesia and development of imaging methods such as computed tomography (ct) and magnetic resonance imaging (mri). with the development of diagnostic radiological evaluations such as ct or mri, rapid and accurate diagnosis has been available, leading to the significant improvement of mortality and morbidity. however, neurological complete recovery rate and mortality were reported to be still 16% and 41-47%, respectively despite appropriate treatments4,15). the aim of this study is to describe the clinical characteristics of 35 patients with sea and to disclose the risk factors, treatments and neurologic outcomes. this study was conducted on 35 patients who had been diagnosed with sea at the department of neurosurgery in our hospital for 24 years from april 1987 to april 2011. we excluded osteomyelitis or discitis without true epidural collection and sea due to tuberculous spondylitis. we retrospectively reviewed the following data from the medical records including age, gender, clinical manifestation, laboratory data, imaging finding such as ct and mri and neurological outcome data at the time of discharge. the patients who underwent either emergent operation within 24 hours or delayed surgery after 24 hours, or conservative management were dealt case by case. emergency surgeries within 24 hours were conducted when patients had neurological deficit or neurological deterioration and 16 patients received antibiotics treatment alone without surgery. neurological outcome and clinical outcome were classified by comparing the mrc scale at the time of diagnosis and at the time of discharge. to elucidate improved to worsened outcomes of patients by treatment methods on admission to our department, there were 22 males (62.9%) and 13 females (37.1%). the presented symptoms were neck pain or back pain in 12 cases (34.3%), radiating pain in 22 cases (62.9%), and neurologic deficits such as motor weakness and sensory change in 28 cases (80%). fever upon hospitalization was shown in 10 cases (28.6%), and the mean number of white blood cell (wbc) was shown to be 9,8863,776 cells/ml. the mean c-reactive protein (crp) was shown to be 4.944.05 mg/l, and the increased crp was shown in 19 cases (54.3%). non-spinal infections were the most common cause of sea. among them, paravertebral abscess was the utmost cause, and skin and soft tissue abscess was followed by it. in addition, risk factors were found in spinal procedure such as epidural injection for pain control, acupuncture and previous spine surgery after spinal trauma. risk factors such as diabetes mellitus, liver disease, and cancer were also found. the location of epidural abscess collection was in the cervical spine in 4 patients, thoracic in 8, thoracolumbar in 3, lumbar in 19, and lumbosacral in 1 patient. the extents of spinal epidural abscesses ranged from 1 to 6 segments, and the mean was 2.7 segments. the spinal epidural collection was dorsal segment to the thecal sac in 4 cases, ventral segment to the thecal sac in 24 cases, and circumference to the thecal sac in 7 cases (table 2). gadolinium enhanced mri was performed in 34 cases and ct was conducted in 1 case when mri was unavailable. mri showed iso or low signal intensity in t1 weighted image (t1wi), whereas it exhibited high signal intensity in t2 weighted image (t2wi). after gadolinium administration, rim enhancement was found in 22 cases, whereas heterogeneous enhancement was found in 12 cases (fig. the initial mrc scale of patients was 1/5 in 5 patients, 2/5 in 1 patient, 3/5 in 3 patients, 4/5 in 18 patients, and 5/5 in 8 patients. and the post treatment mrc scale was 1/5 in 1 patient, 2/5 in 4 patients, 3/5 in 6 patients, 4/5 in 6 patients, and 5/5 in 18 patients (table 3). once sea was diagnosed in the patients, an immediate treatment was initiated to all sea patients. laminectomy, decompressive debridement, abscess drainage, or spinal fusion was performed in the patients with minor or severe neurologic deficit, and spinal fusion was performed in the patients with instability or spinal cord compression in the imaging study even if there were no neurologic deficit. laminectomy was performed in 15 cases and laminectomy with spinal fusion was performed in 4 cases. antibiotics alone therapy was performed in patients with no neurologic deficit and minimal spinal cord compression in the imaging study. empirical antibiotics covering staphylococcus aureus was initially used and was changed with sensitive antibiotics according to the culture of abscess. causative pathogens were identified in 13 cases (37.1%) in the abscess culture, of which staphylococcus aureus was found in 7 cases (53.8%). streptococcus species was found in 2cases (15.3%), klebsiella, and burholderia cepacia were found in one case (7.6%) each. a positive result in the blood culture was shown in 8 cases (22.9%), and staphylococcus aureus was most frequently found. for the period of antibiotics use, an intravenous antibiotic was used for 4-8 weeks while monitoring wbc count, erythrocyte sedimentation rate (esr) and crp, and an oral antibiotic was additionally used in some patients for 2-3 weeks more. 14 cases (40%) showed improvement of mrc scale and 13 cases (37.1%) were unchanged. nevertheless, mrc scale was worsened in 8 cases (22.9%). of the 14 cases showing improvement, 10 patients underwent the emergent surgery within 24 hours and 3 patients had the delayed surgery over 24 hours and 1 patient received conservative treatment with antibiotics alone. among 13 patients who showed no change in mrc scale, 1 patient underwent the emergency surgery, 2 patients experienced the delayed surgery and 10 patients underwent conservative treatment with antibiotics alone. of the 8 patients who showed worsened neurologic deficit, no patient received emergency surgical treatment, 3 patients underwent delayed surgical treatment and 5 patients experienced antibiotic treatment (table 4). between operative and conservative management, operation, regardless of emergency or delay, was more effective than conservative management to improve neurological deficit measured by mrc scale (table 5). initially, patients with decreased mrc scale and rapidly progressing neurological deficit underwent emergency surgery within 24 hours. patients with intact mrc scale and minor neurological deficit got delayed surgery over 24 hours or conservative management with antibiotics (table 6). among 19 patients those who underwent emergent operation within 24 hours showed better prognoses than those who received delayed operation after 24 hours. 13 patients who had positive culture revealed more improvement of mrc scale than the patients with negative culture (p=0.009) (table 8). sea mainly occurs in patients aged from 30 to 70 years old and a very rare disease with a prevalence of 1-2 persons/10,000 patients before 1990s. however, it has been recently increasing with a prevalence of 10-12 persons/10,000 patients due to increased elderly population, intravenous drug abuse, patient controlled analgesia, and spinal anesthesia for pain treatment as well as decreased immune response caused by various reasons1,10,22). in our series, a specific co-morbid condition with spinal epidural abscess was non-spinal infection in 19 cases (55%), spinal procedure in 6 cases (17%), diabetes mellitus in 1 case (3%), spinal trauma in 4 cases (11%), liver disease in 3 cases (8%), cancer in 1 case (3%) and no risk in 1 case (3%). three main mechanisms of sea are as follows: first, results from hematogenous dissemination of distant infectious foci such as urinary tract infection, pneumonia, and laryngitis, account for the highest proportion. second, infections are directly transmitted from adjacent structures such as vertebral osteomyelitis, spondylodiscitis, and perispinal mass abscess. third, infections are transmitted via the hands such as spinal surgery, epidural injection therapy, and spinal anesthesia7,21). staphylococcus aureus has been known to be the most common causative pathogen accounting for 42-84%, and streptococcal strains and gram-negative bacteria are followed by staphylococcus aureus4). in this study, staphylococcus aureus was shown to be the most frequently found causative pathogen. however, due to the early use of antibiotics prior to bacteria culture and insufficient tissue for bacteria culture, the unidentified causative pathogen and the negative result in the blood culture has been known to account for 24.4% and 50%, respectively6,8,19). in this study, the successful bacteria culture in the abscess and in the blood was shown in 13 cases (37.1%) and in 8 cases (22.9%), respectively. sea most frequently occurs in the thoracic region, and then in the lumbar region, whereas it has a lowest prevalence in the cervical region. the higher prevalence of sea in the thoracic region is attributable to larger epidural fat tissue and space. the lower prevalence of sea in the cervical region is attributable to the limited epidural fat tissue and space7,11). in this study, sea was shown to occur in the lumbar regions in 19 cases, almost half of all the presented cases (54.2%). mri with gadolinium enhance is the most commonly used radiologic diagnostic method in these days. ct has an advantage of identifying small bone destruction or paraspinal abscess, but has disadvantages of difficulties in identifying the small amount of abscess and accurately assessing the spreading level of abscess into the adjacent area due to low contrast among the tissues11). in addition, the distribution of epidural abscess can be accurately identified via mri10,13,20). typical findings include iso or low signal intensity in t1wi and heterogeneous high signal intensity in t2wi3). as mri has a disadvantage of a difficulty in assessing the change of the concurrent adjacent bone structure, simple x-ray or ct is conducted with mri to accurately assess the state of the lesions14). treatment of sea is comprised of immediate decompression and surgical drainage, and appropriate administration of antibiotics before and after the surgery3,10,14). through immediate decompression and surgical drainage, the neurologic deficit can be repaired as well as pathological diagnosis, and the identification of causative pathogens can be achieved15,20). operation strategy should be determined according to abscess location, concurrent osteomyelitis, and vertebral body destruction. laminectomy is the most commonly used in the case of abscess located in the posterior spine without lesions in the vertebral body. in addition, in the cases of compression rate of the vertebral body more than 50%, defected posterior spinal structure and the kyphotic angle of more than 20, internal fixation with autologous bone graft is required16,17). because most infections are caused by staphylococcus aureus, empirical antibiotics covering staphylococcus aureus was initially used and was changed with sensitive antibiotics according to the sensitivity. the 4-week period of antibiotics use is generally recommended in the case of abscess without pyogenic spondylitis. the 8-week period of antibiotics use is recommended in the case of abscess with pyogenic spondylitis. furthermore, an oral antibiotics is additionally used for extra 2-3 weeks while monitoring esr and crp4,8). in the past, a mortality of 100% was reported in the case of no surgical treatment, and a mortality of 32% was reported in the case of surgical treatment, which showed high morbidity and mortality11). at present, a neurologically complete recovery rate of 41-47% and a mortality of 16% are still reported even in the case of appropriate treatments4,15). in general, there were no statistical differences between groups in age, gender, location of abscess, number of involved vertebral levels or risk factors. complete recovery can be expected in the cases of no paraplegia before operation or paraparesis period of 36 hours or less. however, neurological recovery can not be achieved in the case of paraplegia of more than 48 hours as neurological paraplegia causes not only mechanical decompression, but also circulatory disturbance due to thrombosis caused by poor inflammatory response, thereby causing spinal neurologic deficit5,11,20). our study suggests that neurological improvement in spinal epidural abscess is more easily achieved via decompressive laminectomy or abscess drainage than conservative treatment after neurological deterioration. poor prognoses are expected in the cases of intraspinal high signal intensity in t1wi, thrombocytopenia, increased esr, increased crp or pancytopenia in hematologic test, pure abscess with no granulation tissue, and antibiotics treatment alone without surgical treatment4,6,8). in particular, prognoses are poorer in the case of thoracic region than in the cases of cervical or lumbar region. this is likely to be attributable to rapid neurologic deficit due to narrow spinal subarachnoid space and poor circulation14). in this study, better recovery of neurologic deficit was shown in the patients with surgical treatment than in the patients with antibiotics treatment alone. patients who had decreased mrc scale at diagnosis, had more incidence to receive surgery than who had intact mrc scale. we had decompressive surgery and abscess drainage to patients with decreased mrc scale at diagnosis if condition of patient was not excessively bad for operation. in the patients with surgery, better prognoses were shown in the patients with emergent surgery within 24 hours than in the patients with delayed operation after 24 hours. each patient had its own reasons for treatment and as our study is retrospective, we could not compare with the other treatments. thus, it is insufficient to conclude early surgery is better than the other treatments and it is the limitation of our study. however, good outcomes can be expected if decompression and surgical drainage are conducted immediately after diagnosing sea. and the improvement of neurological symptoms can be expected through emergency surgery particularly in the patients with neurological deficit with decreased mrc scale. sea is a rare disorder that can cause neurological deterioration or death if it is not recognized or treated early. therefore, sea should be considered in patients with back or radiating pain, motor weakness or sensory change. and if sea is diagnosed, although the patients have minor neurological deficit or no motor weakness, early surgical removal of sea should be considered. we can expect desirable outcomes through immediate surgical drainage and decompression when sea is diagnosed. in particular, satisfactory outcomes can be achieved from emergent surgery within 24 hours from diagnosis of sea even if neurological deficit is observed at the time of diagnosis.

objectivethe aim of this study is to elucidate the clinical characteristics of patients with spinal epidural abscess (sea) and demonstrate the risk factors, treatments and neurologic outcomes. methodswe retrospectively reviewed the medical records and radiologic images of 35 patients admitted to our department with sea between march 1987 and april 2011. while we performed decompressive laminectomy and abscess drainage on 19 patients (54.3%), and 16 patients (45.7%) initially received conservative therapy with antibiotics alone. medical research council (mrc) scale was applied to estimate results objectively. resultsthe neurological outcome data showed improved mrc scale from 14 (40%) patients. 13 (37.1%) patients showed unchanged mrc scale and 8 (22.9%) patients revealed worsened mrc scale at the time of discharge. the patients with surgical treatment showed more improved mrc scale than the patients with conservative treatment and this was statistically significant (p=0.001) on univariate analysis. initially, patients with decreased mrc scale and rapidly progressing neurological deficit underwent emergency surgery within 24 hours. patients with intact mrc scale and minor neurological deficit received delayed surgery or conservative management with antibiotics. among 19 patients those who experienced emergent operations within 24 hours showed better prognosis than those who underwent delayed operations after 24 hours. conclusionsurgical treatment is the modality of choice in patients with sea and urgent surgery especially is indicated in patients with neurological deficits. and early surgery is more effective in neurological improvements than delayed surgery and conservative management.

PMC4432386

pubmed-632

with an annual death toll of more than one million people, the tropical disease malaria is considered one of the most significant infectious diseases worldwide. malaria is caused by protozoan parasites of the genus plasmodium and transmitted by blood-feeding anopheline mosquitoes. during their life cycle, plasmodia alternate between the human host and the insect vector, and thus the transmission stages of the parasite had to develop mechanisms for rapid adaptation to the new environment in order to coexist with the respective host. like most apicomplexan parasites, plasmodia further switch between tissue-specific multiplication cycles and a phase of sexual reproduction, which mediates the transition from the human to the mosquito and thus plays a crucial part in the spread of the disease. the malaria sexual phase begins with the differentiation of gametocytes in human erythrocytes, followed by their uptake during the blood meal of the mosquito and the formation of gametes within the insect midgut. the transformation of the fertilized zygote into the infective ookinete subsequently marks the end of the malaria sexual phase (reviewed in). historically, scant research has been devoted to the malaria sexual stages, namely, gametocytes, gametes, and zygotes, since they neither contribute to the clinical picture of patients nor do they play a role for vector control. however, within the last two decades the dramatic increase of drug resistance in malaria parasites has forced researchers to broaden their consideration of tactics to combat the disease, including transmission blocking strategies aimed at the sexual stages. such transmission blocking strategies, on the level of either drugs or vaccines, are designed to disrupt parasite reproduction and further development in the mosquito midgut, thus breaking the life cycle of the parasite. research on the malaria transmission stages, however, was formerly hampered by cost- and time-consuming cultivation, as well as by the technically challenging infections of mosquitoes with parasites. this was particularly true for work on p. falciparum, the causative agent of malaria tropica. in recent years knowledge on the malaria sexual phase has benefited from a dramatic resurgence provided by proteomic, microarray, and annotation projects that arose out of the genome sequence projects for multiple malaria species (e.g., [27]). as a result, a number of new sexual stage antigens have been identified, and progress has been made in the identification and functional characterization of enzymes and regulatory proteins that are involved in gametocyte differentiation and fertilization (reviewed in). nowadays, three main questions regarding the malaria sexual phase are in the focus of interest. (1) which are the mechanisms that cause a subset of erythrocytic parasites to enter the sexual stage pathway and to differentiate to gametocytes ?. (2) how do gametocytes become activated within the mosquito midgut and how do they transform into gametes ?. (3) in which way do the sexual stages interact with factors of the mosquito midgut? this review addresses the role of gametocytes during malaria transmission and particularly discusses the recent findings on gametocyte activation following entry of the mosquito midgut, as well as their egress from the host erythrocyte and transformation into gametes. additional aspects of gametocytogenesis, sexual stage proteins, and malaria transmission can be found in other recent reviews [1, 811]. the gametocytes are the only stages within the life cycle of malaria parasites that are able to mediate the transition from the human host to the insect host. the development of asexual blood stage parasites to intraerythrocytic gametocytes, which is referred to as gametocytogenesis, starts approximately 715 days after the appearance of parasites in the human blood. it is not known to which degree gametocytes develop stochastically, with a small proportion of committed parasites leaving the asexual cycle and entering the sexual pathway, versus gametocytogenesis as a response to complex environmental signals during infection. gametocytogenesis was previously shown to be influenced by different kinds of stress, including parasite density, anemia, host immune response or drug treatment (e.g., [1220]; reviewed in [8, 10]). up to date, little is known about parasite genes that regulate gametocytogenesis, but it was observed that reduced levels of gametocytes in parasite cultures are often associated with the loss of genetic information following subtelomeric deletion in the right arm of chromosome 9. while in most plasmodium species the sexual stages mature within less than two days, a time period of about 10 days is required for gametocyte development in the human malaria pathogen p. falciparum. gametocyte maturation can be classified into five morphological stages (stages i v), and mature p. falciparum gametocytes show an eponymous falcipare form. in these stages, the host erythrocyte has conformed to the crescent shape of the parasite and is reduced to a small cytoplasmic hem. the intraerythrocytic gametocyte lies within the parasitophorous vacuole (pv) and is shielded from the erythrocyte cytoplasm by the pv membrane (pvm), which is located adjacent to the parasite plasma membrane (ppm) (figure 1(a)). underneath the ppm is a typical gametocyte feature, the pellicular complex, which consists of a subpellicular membrane (spm) vacuole subtended by an array of longitudinally oriented microtubules. these structures probably give the gametocyte stability, and the electron-dense spm disappears during gametogenesis (figures 1(a), 1(b), 1(c), and 1(d)). besides morphological changes, maturation of gametocytes also includes alterations on the molecular level in order to prepare the parasites for the rapid adaptation to the mosquito midgut. for instance, a large amount of mrna is transcribed and stored in the cytoplasm of female gametocytes, as shown for the transcripts of the sexual stage surface proteins pbs25 and pbs28 in the rodent malaria model p. berghei, which will be transcribed only in the mosquito vector (see below). gender specificity becomes established in the schizont committed to gametocytogenesis, and the gender ratio is typically female-biased with one male for about five female gametocytes, depending on the respective parasite clone. this difference might be explained by the fact that one male gametocyte forms approximately eight microgametes, thus establishing a roughly 1: 1 ratio of micro- and macrogametes in the mosquito midgut, thereby leading to most efficient fertilization in a monoclonal infection [8, 28]. recently, reece et al. demonstrated in the mouse model that parasites were able to adjust the sex ratio according to parasite density and the number of parasite clones coinfecting the mammalian host. for instance, a less female-biased sex ratio would increase the probability of a successful fertilization of females of the respective clone, when competing with others [2932]. in contrast, in vitro studies on p. falciparum did not show an adjustment of sex ratio to gametocyte density. however, an impact of the sex ratio on the infection rate, depending on gametocyte density, was observed. the fact that single, haploid asexually replicating malaria parasites are able to develop into gametocytes of both sexes in the absence of sex chromosomes indicates that gametocyte gender determination is governed by differential gene expression. gametocyte stages i to iv were reported to sequester in the bone marrow and spleen, while terminally differentiated stage v gametocytes are then released in the peripheral blood system [35, 36] and only become infectious to mosquitoes after a further two or three days of circulation [37, 38]. bloodstream gametocytes might not be distributed homogeneously, as evidenced by a significant aggregation pattern observed in midgut smears of p. falciparum-fed mosquitoes, and it is an intriguing hypothesis that parasites increase the likelihood of fertilization in the mosquito midgut by promoting uptake in preformed complexes of gametocytes. while feeding on an infected human, the female mosquito takes up malaria gametocytes together with the blood meal. by entering the midgut, the parasites receive environmental signals, which indicate the switch from warm-blooded host to insect vector and which initiate the development of gametes. such signals include a drop of temperature by approximately 5c, and the presence of the mosquito-derived molecule xanthurenic acid (xa), a byproduct of eye pigment synthesis [41, 42]. an additional signal reported to induce gametocyte activation is an increase of ph from 7.2 to about 8 [40, 43], but such a ph shift was later discussed to be an artificial inductor of exflagellation. while xa appears to initiate a number of signaling events in the parasite (see below), the quest for a receptor that binds xa was hitherto unsuccessful. gametocyte activation is routinely measured by the formation of exflagellation centers, although a time period of almost 15 minutes lies between these two events. this is due to the fact that exflagellation can easily be observed under the light microscope and quantified by counting of exflagellation centers. exflagellation is the process when the activated male microgametocyte forms motile flagellar microgametes, which detach from the residual body by binding to erythrocytes (see below). two previous studies showed that induction of exflagellation involves a fast increase in intracellular calcium and cgmp [45, 46]. an initial benchmark in elucidating sexual stage signaling was the identification of two guanylyl cyclases (gc and gc) as integral membrane proteins in p. falciparum, which are activated by addition of xa (figure 2). noteworthy, the subsequent disruption of the gc ortholog in p. berghei resulted in normal exflagellation, but motility-impaired ookinetes, indicating that the role of gc is not essential for gametocyte activation. the increase of cgmp triggers the activation of a cgmp-dependent protein kinase, pkg. activation of pkg leads to rounding up of the gametocyte, a process that appears to be independent from calcium increase. gametocyte exflagellation further involves the presence of the second messengers diacylglycerol and inositol triphosphate (ip3), hydrolysis products of phospholipase c activity. the latter eventually mediates the release of intracellular calcium from the endoplasmic reticulum (er) (figure 2). it is not yet known how the signaling pathway involving ip3 and calcium release and the pathway involving cgmp and pkg activation are linked together, and whether pkg has an additional effect on calcium release from the er (figure 2). current data suggest that at least three effector pathways exist (discussed in ): (1) a pkg-dependent, calcium-independent pathway that mediates rounding up of the activated gametocytes, (2) a calcium-dependent pathway that initiates microgamete formation, and (3) a calcium-dependent pathway that regulates emergence of activated gametocytes of both genders. following uptake by the mosquito, both male and female gametocytes round up and then escape from the enveloping erythrocytes within about 10 minutes postactivation (figures 1(b), 1(c), and 1(d)). in this period the microgametocyte replicates its genome three times in order to produce eight motile microgametes (reviewed in [1, 9]). egress of the activated gametocyte from the host erythrocyte has been linked to the presence of osmiophilic bodies, gametocyte-specific secretory organelles that were first identified by electron microscopy due to their electron-dense features (figure 1(b)) [24, 52]. they appear first in stage iv gametocytes and are particularly present in the female sexual stages. the osmiophilic bodies migrate to the ppm during activation and disappear within a few minutes post-activation, coevally with the rupture of the pvm (figures 1(b), 1(c), and 1(d)) (g. pradel, unpublished observations). osmiophilic bodies contain a gametocyte-specific and highly hydrophilic protein, pfg377, which is considered a marker for these organelles [53, 54]. gene-disruption studies showed that female p. falciparum gametocytes lacking this protein reveal a reduced number of osmiophilic bodies and fail to egress from the host erythrocyte, pointing to a pivotal role of this protein in gametocyte emergence. another protein, which was only recently identified, mdv-1/peg3, has also been implicated with gametocyte egress. in p. falciparum, expression was reported to be initiated in stage i gametocytes in association with all membranous structures of the pvm and to persist until gametocyte maturation [5658]. first gene disruption studies on p. falciparum described a reduced formation of particularly male gametocytes. two subsequent studies, however, indicated a role of the protein post-activation of gametocytes.lal et al. reported the presence of mdv-1/peg3 in p. berghei gametocytes of both sexes and a subsequent focal localization at the anterior pole of the developing ookinete. studies on parasites in which the respective gene was knocked out resulted in reduced ookinete formation. a study by ponzi et al., on the other hand, showed that p. berghei mdv-1/peg3 was associated with the gametocyte osmiophilic bodies. gametocytes lacking this protein failed to egress from the host erythrocyte, thus resulting in reduced fertilization and ookinete formation. therefore suggested that mdv-1/peg3 plays a major role in disrupting the pvm and the erythrocyte membrane (em). independent from the life-cycle stage, host cell egress of malaria parasites involves rupture of two membranes, pvm and em. the time line of rupture, however, was recently object to several brisk discussions. particularly two models are currently under investigation, the inside-out model, in which the pvm ruptures prior to the em, and the outside-in model, in which the em is degraded first [62, 63] (reviewed in). the timeline of parasite egress was hitherto mainly investigated in the asexual blood and liver stages, and no data are available for the egress of gametocytes. in the above suggested that mdv-1/peg3 is involved in pvm destabilization and that em rupture depends on the absence of the pvm. in accord with this hypothesis, new studies from our laboratory indicated that the pvm disappears within a few minutes after gametocyte activation, and that the rupture of the em follows several minutes later (g. pradel, unpublished observations), thus supporting the inside-out model of egress. the coming-out of malaria parasites from the host cell requires protease activity. a number of new studies engaged with the identification of proteases that mediate emergence of asexual blood stage merozoites. data point to the involvement of the cytoskeleton-degrading malaria proteases falcipain-2 and plasmepsin ii. particularly sera (serine-rich antigen) proteins, which were identified in the pv of blood stage schizonts (e.g., [65, 66]), are supposed to mediate pvm rupture. it was shown for sera-5 of p. falciparum that the protease is proteolytically activated by the serine-like subtilisin protease pfsub1. while no detailed studies were yet performed on malaria gametocytes, it is worth mentioning that transcripts of select representatives of the above mentioned protease families are expressed in these stages, including falcipain-1, plasmepsin vi, sera-6, sera-7, and pfsub3 [68, 69]. a popular strategy in investigating protease activity during rupture is the treatment of parasites with protease type-specific inhibitors. again, these studies were mostly performed on blood stage parasites, particularly using cysteine protease inhibitors like e64. treatment with this inhibitor, however, resulted to date in contradictory results, and it was reported that the inhibitor blocked either degradation of the pvm [62, 63] or rupture of the em. a similar egress study using protease inhibitors was recently performed on activated p. berghei gametocytes and showed that exflagellation can be blocked by the cysteine/serine protease inhibitors tpck and tlck. treatment of activated gametocytes with tpck, tlck, pmsf, or two novel falcipain-targeting cysteine protease inhibitors during activation reduced the formation of microgametes. furthermore, the aspartic protease inhibitor epnp appeared to interfere with rounding up of gametocytes. the exact modes of action for these proteases during gametocyte egress from its host cell remain to be investigated. exflagellation is the process in which the newly formed microgametes adhere to neighboring erythrocytes, thus forming rosettes called exflagellation centers, and then detach from the residual body of the activated male microgametocyte. the exflagellating microgamete adheres to sialic acids and glycophorin a of the erythrocyte surface and this binding is probably mediated by pfs230, an abundantly expressed adhesion protein that is associated with the gamete surface. interestingly, pfs230 is proteolytically processed during gametocyte activation, and this processing can be inhibited by the metalloprotease inhibitor 1,10-phenanthroline [75, 76]. the same inhibitor blocks exflagellation by leaving the microgamete amotile, and it is tempting to speculate that processing of pfs230 increases the adhesive properties of this protein, which are needed for the binding of the exflagellating microgamete to erythrocytes. this might be explained by the fact that sexual stage proteins can easily be disrupted because of their nonessentiality for parasite proliferation, and thus a functional characterization can be obtained by phenotype analysis of parasites, in which the respective genes have been knocked out. genome annotation has revealed an extensive catalog of parasite-encoded kinases, the malaria kinome, with at least 86 hypothetical kinases identified in p. falciparum [77, 78]. an initial elegant study showed that the calcium-dependent protein kinase pbcdpk4 is involved in sexual stage signaling and regulation (figure 2). the kinase becomes activated by calcium increase following xa activation, resulting in genome replication in microgametocytes. in p. falciparum, pfcdpk4 was reported to be gametocyte-specific and activated by phospholipase c. in a subsequent step, the mitogen-activated protein kinase pbmap-2 controls formation of male gametes at the stage of cytokinesis [8183]. downstream of these events, the protein kinases pbnek-2 and pbnek-4 trigger genome replication to the tetraploid level in the zygote stage [81, 84, 85]. furthermore, pbcdpk3 is required for ookinete motility and engagement with the mosquito midgut epithelium [86, 87]. when highlighting these novel signaling pathways during gametogenesis, it has to be taken under consideration, however, that in some cases the results obtained for p. berghei and p. falciparum might differ. for example, a recent reverse genetics approach on the p. falciparum ortholog pfmap-2 pointed to an essential function of this kinase for the parasite asexual blood cycle, contradictory to the abovementioned results on pbmap-2. this indicates that insights gained by studying the rodent malaria model p. berghei can not be as easily applied to human malaria pathogens as has so far been assumed. ingestion by the blood-feeding mosquito triggers molecular changes in the sexual stages of p. falciparum, with approximately 20% of stage-specific genes being activated during sexual stage development and parasite transmission [3, 5, 6]. this molecular switchover adjusts the gametocytes to the invertebrate host and on one hand initiates reproduction but on the other hand prepares the emerging gametes for the hostile environment of the mosquito midgut. gametocyte development and gamete formation are particularly accompanied by the coordinated expression of numerous surface-associated proteins, including the egf domain-containing proteins pfs25 and pfs28, the cysteine motif-rich proteins pfs230 and pfs48/45, as well as the multiadhesion domain pfccp proteins. these proteins and their potential as transmission blocking targets were discussed previously and will therefore not be focus of this review. noteworthy is that the majority of these surface proteins have adhesive properties and can be divided in two classes. one class of sexual stage proteins, including pfs230, pfs48/45, and the six pfccp proteins, is expressed within the pv of the developing gametocyte and subsequently present on the gamete surface, but expression of these proteins usually ceases during fertilization (figure 3). the expression of the second class of surface proteins starts at the time point of fertilization, as was shown for pfs25 and pfs28, and expression often persists until the ookinete has formed. the reason for this sudden onset of protein expression during fertilization is the translational repression of messenger rna encoding for these proteins. this was interalia shown for the repression of pbs25 and pbs28 by the p. berghei rna helicase dozi (development of zygote inhibited) as part of a ribonucleoprotein complex. the factor, however, is only translated in the ookinete stage, where it then activates a set of genes encoding for adhesion proteins important for midgut invasion. the reason for such a high number of adhesive proteins in the malaria parasite sexual stages remains elusive, but a new study from our laboratory might provide a first step towards answering this question. we showed that the six pfccp proteins, which are characterized by a high number of adhesion modules, assemble to form multiprotein complexes during their expression in the pv, and these complexes are subsequently present on the surface of the newly emerged macrogametes. preliminary data point to an additional involvement of other surface-associated adhesion proteins in these complexes, like the transmembrane protein pfs48/45, which might link the complex to the gamete surface (s. scholz, a. kuehn, n. simon, and g. pradel, unpublished observations). we hypothesize that these protein complexes cover the macrogamete in the form of a sticky coat and that they are involved in important adhesive processes during malaria transmission to the mosquito. the complexes might play a role in promoting contact between the emerging gametes within the blood meal or in protecting the gametes from the aggressive environment of the mosquito midgut. noteworthy, the gametes and zygotes are the only stages within the parasite's life cycle that, for more than one day, have to persevere outside a host cell. here they are exposed to factors of the blood meal, including midgut bacteria and digestive enzymes, as well as components of the human immune system. this exposure results in an approximate 300-fold loss of parasite abundance during transmission to the mosquito, and the malaria transmission stages are therefore considered bottleneck stages of the parasite's life cycle. during exflagellation, the microgamete detaches from the residual body and is freely motile, moving via sinusoidal or helical waves. it is not known whether the microgamete meets the macrogamete by coincidence, whether it actively scans the blood meal, or whether it migrates along a gradient of an attractant that is released by the macrogamete. interestingly, we recently identified filamentous protrusions of the p. falciparum gamete surface, which form immediately upon activation and which appear to establish long-distance contacts between parasites in the mosquito midgut (g. pradel, unpublished observations). these filaments fit the typical characteristics of socalled nanotubes, novel organelles that were recently described for a number of animal cells (reviewed in [95, 96]). it has been proposed that nanotube-like filaments can be formed by almost all cells serving as a medium for exploring the extracellular environment and therefore are likely to represent ancient features of unicellular eukaryotes. nanotubes were reported to have a function in communication between cells, including calcium signaling and organelle transfer [95, 96]. we therefore hypothesize that the nanotubes of malaria gametes might be tools to facilitate association within the midgut in order to increase the chance of parasite mating. once the microgamete adheres to a macrogamete, fertilization begins by fusion of the plasma membranes. two recent studies on p. berghei described the identification of the microgamete protein gcs1 (generative cell specific 1), also termed hap2, which enables gamete fusion, and disruption of the respective gene results in male sterility and blocked fertilization [97, 98]. gcs1/hap2 is a conserved protein of algae and plants, where it is involved in pollen tube guidance and seed formation [99, 100], and was also identified in protozoan parasites [97, 98, 101]. importantly, gcs1/hap2 does not mediate the initial binding between the two mating partners, which appears to involve other adhesion proteins. cell fusion is followed by nuclear fusion, and over the next 3 hours, meiosis occurs and the zygote becomes tetraploid. during the following 24 hours, the zygote transforms into the infective ookinete stage, thus marking the end of the malaria sexual phase. the ookinete is motile and possesses an apical complex which enables it to disrupt and traverse the midgut epithelium, before settling down between epithelium and basal lamina. parasite tetraploidy persists throughout the ookinete stage until sporozoite budding in the oocyst restores the haploid state. despite intense work on the sexual stages of malaria parasites, they represent the least understood stages of the parasite's life cycle. gametocyte differentiation and gametogenesis have mostly been studied in the human malaria pathogen p. falciparum, and for these sexual stages, a variety of proteins have been identified and characterized. on the other hand, the implication of malaria sexual stage proteins for malaria transmission was preferentially investigated in the murine p. berghei model, which is more easily accessible for genetic manipulations and transmission studies. up to date it is challenging to combine information gained by both systems to receive the big picture on the malaria sexual phase. we expect that in the near future research on the sexual stages of malaria parasites will be dominated by two major tasks: (i) the big hunt for the gene, which enables the blood stage parasite to enter the sexual pathway and (ii) the analysis of the molecular mechanisms and signaling events of sexual stage parasites during fertilization in the mosquito midgut.

the tropical disease malaria, which results in more than one million deaths annually, is caused by protozoan parasites of the genus plasmodium and transmitted by blood-feeding anopheline mosquitoes. parasite transition from the human host to the mosquito vector is mediated by gametocytes, sexual stages that are formed in human erythrocytes, which therefore play a crucial part in the spread of the tropical disease. the uptake by the blood-feeding mosquito triggers important molecular and cellular changes in the gametocytes, thus mediating the rapid adjustment of the parasite from the warm-blooded host to the insect host and subsequently initiating reproduction. the contact with midgut factors triggers gametocyte activation and results in their egress from the enveloping erythrocyte, which then leads to gamete formation and fertilization. this review summarizes recent findings on the role of gametocytes during transmission to the mosquito and particularly focuses on the molecular mechanisms underlying gametocyte activation and emergence from the host erythrocyte during gametogenesis.

PMC2810480

pubmed-633

protein solvation determines to a large extent the physicochemical behavior of proteins, and it is therefore an essential part of their exerted function. in particular, complex formation, which is involved in almost all protein functions, depends on the detailed balance between solvent solute and solute solute forces. too strong solvent solute forces would impair protein function, while too strong solute solute forces would lead to unspecific protein aggregation. protein folds are generally stable under physiological conditions, but changes in the environment can induce unfolding and denaturation. urea is among the most frequently used denaturants in studies of protein folding and stability. the effect of cosolvents on proteins has been studied experimentally and theoretically (for reviews, see refs (13)), but to date the molecular mechanisms and particularly the driving forces of urea-induced protein denaturation are not yet fully understood. several theoretical studies using molecular dynamics (md) on single amino acids, peptides, and proteins have been performed to elucidate the atomic details of the interactions between urea and amino acids, peptides, or proteins. most simulation studies focused on the energetic components governing the interaction between the proteic solute and the water urea solvent mixture. different interaction mechanisms have been suggested, debating whether urea-induced denaturation is driven by polar interactions or by hydrophobic interactions and if these are induced by direct or indirect interactions with the solute atoms, but in the meantime there is sufficient experimental evidence to exclude a urea-induced structural change of water. a recent experimental study expands thermodynamic data to the spectrum of atoms occurring in proteins with the conclusion that urea interacts favorably, compared to water, with most atom types. a general consensus attributes a dominant role to van der waals interactions between urea and protein atoms over pure electrostatic contributions. the hydration shell model explained the solvation terms of small aliphatic hydrocarbons in urea water mixtures. an essential term of this model is an enthalpic contribution arising from the van der waals forces between the solute and the cosolvent urea. however, the model neglected the cavity formation, an integral element of solvent transfer models. urea does not reduce the free energy of cavity formation; on the contrary, the strong interaction between urea and water increases the surface tension. therefore, the replacement of water from the solvation shell around the hydrophobic solute seems not to contribute to the transfer free energy. also, the hydrogen bond reorganization in the first solvation shell has only a compensatory energetic effect; i.e., enthalpic interaction of water with the protein surface is replaced by a gain of entropy in the bulk solvent. an alternative thermodynamic approach is the solvent exchange model that considers the solute surface as a collection of small interaction sites. this model is closer in spirit to molecular simulations, because (i) system trajectories provide direct information about the urea protein interactions and (ii) the model allows for the existence of heterogeneous sites; i.e., the heterogeneity of the protein surface may be accounted for by the formalism. a strong interest in protein unfolding and the principles underlying conformational transitions comes from observations that denatured cellular proteins tend to aggregate, a phenomenon that is often associated with a diseased cellular state. a prominent example of a potentially fatal condition caused by protein misfolding and aggregation is the prion disease that is associated with the formation of aggregates of the prion protein (prp). while the native structure of the globular c-terminus of the cellular prion protein (prp) is mostly helical, its amyloid fibrillar aggregates contain -rich conformers (prp). the details of the conformational transition as well as the structure of the fibrillar aggregates are still elusive. in a quest to determine a minimal set of structural elements that retain the aggregation propensity of the prion protein, the 36-residue fragment h2h3, comprising the helices h2 and h3, has been designed. as demonstrated by our previous studies, h2h3 shows fibrillation, gpi anchoring, and insoluble pk-resistant aggregate formation analogous to prp. we evaluate here the forces acting at the initial stage of urea-induced denaturation on three prp constructs, a stable structure and two h2h3 intermediates isolated from misfolding simulations of h2h3 in water. these two intermediates of h2h3 represent an -rich state h2h3 as an analogue of prp and a -rich state h2h3 as an analogue of prp. these two conformers have been previously extensively characterized by md simulations, and their role in the assembly of misfolded states has been evaluated. besides being very different in their secondary structure content, they differ remarkably in their solvent exposure, with the h2h3 state exposing considerably more hydrophobic surface. h2h3 and h2h3 are therefore ideal systems to study the solvation effects. the solvent forces on h2h3 and h2h3 in pure water and in 4 m aqueous urea solution were analyzed. we have previously introduced shannon entropy maps and analyzed the hydration properties of water at the surface of prp, identifying particular loci with different entropies. by studying the effect of urea on folded and intermediate structures, we (i) isolate the urea effect on defined conformers from the unfolding process itself and by restraining the protein structure (ii) observe the competition of urea and water for protein interaction in the absence of protein dynamics. it is clear that urea denaturation is induced spontaneously, because protein surface atoms interact stronger with urea than with water. to our knowledge, no study has yet been directed to the role of solvent forces in the initiation of urea-induced unfolding. here, the preference of the proteic solute to interact with either urea or water was computed on the basis of atomic force distributions. shannon entropies of the solvent were computed to illustrate the replacement of water from the solvation shell. we compare our results of atomic solvation forces to thermodynamic data and observe good correlations, as well as general trends in hydrophobicity scales extracted from molecular dynamics simulations and based on energetical considerations. three starting structures were used in this study: -rich h2h3 (h2h3), -rich h2h3 (h2h3), and the crystal structure of the c-terminal globular domain of prp (pdb: 1uw3). h2h3 is a truncated form of the prion protein comprising mainly helices h2 and h3 (residues 183218). h2h3 has been shown previously to undergo a conformational transition from an -rich (h2h3) to a -rich (h2h3) conformation. these two conformations were used here to compare forces on the same sequence in two different folds. the c-terminal globular domain of prp is used as a native reference protein for the analyses. the urea model of smith et al. was used in its implementation as molecular building block ' urea ' in this force field. the temperature was set to 300 k and controlled by weak coupling to a temperature bath with a coupling constant t= 0.1 ps. the non-bonded pair list was updated every time step for pairs within 0.8 nm and every fifth time step for the range 0.81.4 nm. twin-range cutoff radii of 0.8/1.4 nm were used to compute non-bonded interactions. long-range electrostatic interactions were approximated by a reaction-field force, using a dielectric constant of 54. simulations were kept at 0.061020 kj mol nm (1 atm) with a coupling time of p=0.5 ps and an isothermal compressibility of 5.575 10 (kj mol nm). initial protein structures h2h3, h2h3, and 1uw3 were energy minimized using 100 steps of steepest descent. energy minimized protein conformations were solvated in a periodic box of 5.2 edge length. the minimum solute systems were electrostatically neutralized by replacing water molecules with sodium ions to compensate the net charge (h2h3, 1; 1uw3, 2) of the protein at a neutral ph value. the neutralized systems were energy minimized using 100 steps of steepest descent, while the protein was harmonically positionally restrained using a force constant of 2.5 10 kj mol nm. the systems were run for 5 10 steps (1000 ps) of md while keeping the solute positionally constrained. configurations, energies and forces were saved at intervals of 250 steps (0.5 ps), yielding 2000 conformations per trajectory. the final urea concentrations were curea(h2h3)=3.8 m, curea(h2h3)=4.5 m, and curea(1uw3)=4.2 m. forces between the following groups of atoms were recorded: protein, ion, water, and urea. two sets of simulations were performed, (i) in pure water (ii) and in a urea/water mixture. since water equilibration around solutes occurs on the time scale of 1020 ps, explicit solvent simulations of 1000 ps are sufficiently long for the pure water simulations to sample representative force distributions. the urea/water systems equilibrated after about 5 ns, as shown by the radial distribution functions of urea and water in supporting information figure s1. simulations of these systems were run for 10 ns, and the last 3 ns were used for the analysis. given that we estimate the equilibration to occur at approximately 5 ns, we considered 10 ns as sufficiently long to obtain accurate equilibrium solvent properties. the first 300 ps of the trajectories were excluded from analysis to remove potential equilibration effects. the contact coefficient is the contact ratio, the fraction of protein urea contacts, normalized by the mole ratio of urea:1where n is the number of contacts, m is the number of molecules, p: u are protein urea interactions, and p: s are protein(co)solvent (urea and water) interactions. normalized force differences f were computed for protein atoms with at least one contact to urea via2where f denotes the mean force over a specified interval of the trajectory. rescaled forces were computed for protein atoms with at least one contact to urea via3 statistical values were computed over the last 700 ps of the trajectories using the statistics functions of the gnu scientific library and plotted using the r-project software. the ability of prp to form fibrils is well recognized, but only recently has the neurotoxic activity been correlated with the formation of soluble oligomeric species. we have shown that oligomers from the h2h3 subdomain have very similar physicochemical charateristics to those of the entire ovine prp protein. the formation of these oligomeric species is accompanied by an increase in -sheet (h2h3) content. we have studied in detail this conformational transition in water by molecular dynamics simulations and have characterized the process of interconversion from an all -helical to a -rich conformer in water. after only 90 ns, a -sheet seed forms close to the disulfide bridge (nucleating at residues v183n184); later the elongation of this sheet resulted in the refolding of the entire structure to what we call a double--hairpin (first hairpin, v183t191/i208m216; second hairpin, v192t196/q199q203) (figure 1a, b). this conformer was very stable in solution and has been repetitively observed in simulations of oligomer-forming mutants. we therefore suspect that this is one of the -rich species in solution leading to oligomer formation. transition from (a) the -rich h2h3 to (b) the -rich h2h3 conformation. (c) c-terminal globular domain of ovine prp (1uw3). the model systems illustrated in figure 1 were selected, because we assume that they are among the species present in the oligomerization pathway. the comparison of the organization of the urea mixture around these structures can reveal the tendency of denaturants to favor the species that is more prone to forming oligomers. by computing the solvent accessible surface area (sasa) calculated by pops and divided into hydrophilic and hydrophobic contributions (figure 2), one observes the exposure of backbone atoms in the transition from the -rich to the -rich conformer observed in pure water solution (hydrophobic sasa in yellow, hydrophilic sasa in red). this is intriguing for the study of solvation forces, because one of the postulated effects of urea-induced denaturation is strong interactions with backbone atoms that become exposed. the only region in which the -rich conformer exposes more hydrophobic surface is observed in the stretch 198204, which corresponds to the loop connecting the two helices. this loop becomes quite buried in the -rich conformer, forming a minimal core between the two aforementioned hairpins. solvent-accessible surface areas (in) of the prion protein conformers h2h3 (blue) and h2h3 (red). the bars are divided into hydrophobic (light colors) and hydrophilic (dark colors) contributions. in denaturant-induced unfolding experiments, the number of denaturant molecules bound to the protein can be derived from the depression of the transition temperature, and thermodynamic parameters can broken down into increments per contact. in computational studies, this type of information is directly accessible if one defines a contact distance threshold (here 3.5) between protein atoms and (co)solvent atoms. the relative preference of protein surface atoms to bind to either the cosolvent (urea) or the solvent (water) can be expressed by the contact coefficient, which is the fraction of contacts to a given cosolvent normalized by the mole fraction of the cosolvent in the mixture. a contact coefficient above 1 indicates a favored contact to the cosolvent and below 1, a disfavored contact. in figure 3, the normalized force difference between the protein hydrophobic c atoms show the strongest preference for urea, and their force difference is positive; i.e., protein urea forces are stronger than protein water forces for hydrophobic atoms. the polar amide atoms n and o have a preference for urea contacts, but the interaction forces with urea are on average lower than those with water. polar oh and charged o and n atoms show the highest preference and force differences for water. the normalized force differences between protein urea and protein water interactions in figure 3 follow a hydrophobicity scale. urea and protein water interactions per atom type as a function of the contact coefficient. the linear fit through all data points (solid line) has a regression coefficient of r=0.6, exclusion of the outlier solvent forces on individual protein atoms are cumulative; i.e., the recorded forces originate from interactions with one to several (co)solvent atoms and molecules. the effect of denaturants is concentration dependent, because the number of interactions and therefore the cumulative solvation forces increase with the mole ratio. the concentration effect can be compensated computationally by a scaling of the solvation forces with the contact ratio (eq 3). this creates a useful theoretical scenario, in which solvent and cosolvent form an equal number of contacts with the protein, and therefore the forces are represented on a symmetric basis. a detailed picture of this scenario is obtained from a plot of the force distributions. the quantile quantile (q q) plots in figure 4 provide a graphical summary of the differences between the force distributions. q plots, while increased frequencies in one distribution bend the curve toward that dimension. the axes show the scaled forces fc of protein urea versus protein it is apparent that almost all atom types favor urea interactions compared to water interactions, with the exception of hydroxyl o and partially carboxylate o and amide n establish particularly strong forces with urea, while forces on amide o are moderate. the increased backbone exposure of h2h3 compared to h2h3 is notable in the relatively large forces on the backbone atoms amide n and amide o. the surface interaction potentials (in units 10 m) determined by guinn et al. however, the potentials aromatic c 8.9 and amide o 8.7 were not reproduced by our simulations, possibly because the exposure of these atoms was significantly higher in the model compounds of the thermodynamic study than in the protein folds used here. the mean forces (not scaled), contacts, and contact ratios are tabulated in table 1. interaction forces normalized by the contact ratio between h2h3 and urea (fcp: u) compared to h2h3 and water (fcp: w). shown are the force distributions of selected types of atoms as q q plot. (a) h2h3, (b) h2h3, and (c) 1uw3. data points above the diagonal line indicate a preferred interaction with urea, below a preferred interaction with water. fc: mean force per contact standard deviation in units kj mol nm. entropic solvation effects are as important as the enthalpic force contributions discussed in the previous section. as urea replaces water in the first solvation shell, water gains entropy, but the reorganization of hydrogen bonds is an endothermic process. therefore, the water replacement has been refuted as a driving force (gibbs energy change) of denaturation. the solvent entropy in simulated systems can be approximated by the shannon entropy h of the solvent. the atom occupancy within cells of a virtual grid were recorded along the simulation and transformed into an entropy hper grid cell (for details, see the methods). the orange distributions show the values of grid cells close to the protein surface (00.25 distance); the blue distributions show the values of the second solvation shell (0.50.75 distance). the differences between the orange and blue distributions illustrate that the h values are a sensitive measure of local atom fluctuations, although the resolution of the grid cells is limited (here 2.0) to provide a sufficiently diverse cell occupancy, because each additional atom contributes to the shannon term. water mixtures (hugc) and pure water (hwgc) are given as q q plots in the right column of figure 5, the color code representing the same distance ranges as in the left column. the entropy distributions of the bulk are about the same for protein urea mixtures and pure water, with the exception of the lower entropy range of the h2h3 system. h2h3 shows a reduction, compared to the pure water system, of the number of water molecules in the urea/water mixture in the entropy range of 0.40.7, which indicates a loss of low entropy water molecules in the solvation shell of the protein. h2h3 exposes the largest fraction of hydrophobic surface area among the three prion conformers, and this entropic effect is most likely due to the replacement of water by urea at the protein surface. this effect is not detectable in the urea/water mixture systems of the helical proteins h2h3 and 1uw3. however, all urea/water systems show an increase, compared to the pure water system, in the number of water molecules in the entropy range 0.71.2, which is caused by the presence of urea in the solvation shell and the concurring reduction of solvent mobility. shannon entropy distributions of the solvent around prion conformers h2h3 (a, b), h2h3 (c, d), and 1uw3 (e, f). distributions are divided into grid cells close to (00.25, orange) and far from (0.50.75, blue) the protein surface. right: q q plots of the shannon entropy distributions of the solvent environment of water (hwgc) and urea (hcgc) atoms. urea does not replace water molecules that are tightly bound to the protein surface and therefore create a low entropy environment. urea replaces water molecules at protein surface locations where the bound solvent water fluctuates. the water urea shannon entropy maps are shown as isosurfaces in figure 6 for both h2h3 and h2h3. these maps highlight the structural and dynamical properties of the solvent mixture around the molecules. high entropy solvents close to the protein surface are visible as locations where the contour map touches the protein. these are sites that are prone to (water) desolvation by exchange with cosolvents like urea that form stronger interactions with these sites. for the h2h3 -rich conformer, the loop between the two helices shows a high entropy environment, and urea molecules interact closely with the backbone. for both conformers, one can observe clustering of urea molecules at exposed amide n atoms (blue space-filling spheres), in agreement with observations in the atomic force analysis, where amide n atoms gave rise to the largest scaled forces in water urea mixtures. shannon entropy of the solvent surrounding (a) h2h3 and (b) h2h3 illustrated as isosurface in wire mesh rendering. solvation forces are a sensitive measure to gauge the solution properties of protein atoms in urea water mixtures. the individual preferences to interact with urea or water follow a hydrophobicity scale in a similar trend as force field energies of amino acid analogues. the comparison of the scaled forces showed largely agreement with data of surface interaction potentials. particularly strong force differences were observed for carboxylate o and amide n atoms. the direct comparison of urea and water forces on the protein was performed on rescaled forces to compensate for the dependence of the solvation forces on the urea concentration. solvent species appear in two forms: occupying a site and as a solution component. this has implications for the relative weights of the contributions to the solvation force of the (co)solvent components. in the direct comparison of the force distributions of the protein urea and protein water interactions (figure 4), the forces were scaled by the inverse of the contact ratio, but not additionally by the mole fraction as in the contact coefficient. therefore the interacting solvent components were viewed as occupying a site, which reflects the fact that the relative concentrations of urea and water are different close to the protein surface compared to in solution, as has been shown by radial distribution functions here and in previous studies. the urea molecules close to the protein surface remained largely outside the first water solvation shell, except at locations where the solvent entropy is relatively high. it was noted earlier for the 1uw3 molecule in pure water that surface sites surrounded by high entropy water molecules are likely to be structural defects or interaction sites, because of the low energetic cost of desolvation. one of these sites, the loop between the two helices, is also an apparent interaction site in the h2h3 -rich conformer: it is surrounded by high entropy solvent, and urea molecules bind to the backbone amide n atoms. the use of other cosolvents like fluorinated derivatives in the stabilization of folded and unfolded species of prpc has been explored in experimental and theoretical approaches. generally, a stabilization of helical structure is observed in these solvents. hexafluoro-2-propanol has been shown to affect the ultrastructure of prp amyloid and to decrease the -sheet content as well as prion infectivity. in contrast, 1,1,1-trifluoro-2-propanol does not inactivate prion infectivity but alters the morphology of the rods and abolishes congo red binding. protein simulations in urea water mixtures provide a rich source of information about solvation effects that can be used to detect potential structural defects and interaction sites. the solvent interaction forces of individual atom types could be converted to solvation parameters and embedded in an implicit solvation model for urea water mixtures. another, more challenging, perspective is the combination of the observed surface site interactions in md simulations with a thermodynamic site model.

solvation forces are crucial determinants in the equilibrium between the folded and unfolded state of proteins. particularly interesting are the solvent forces of denaturing solvent mixtures on folded and misfolded states of proteins involved in neurodegeneration. the c-terminal globular domain of the ovine prion protein (1uw3) and its analogue h2h3 in the -rich and -rich conformation were used as model structures to study the solvation forces in 4 m aqueous urea using molecular dynamics. the model structures display very different secondary structures and solvent exposures. most protein atoms favor interactions with urea over interactions with water. the force difference between protein urea and protein water interactions correlates with hydrophobicity; i.e., urea interacts preferentially with hydrophobic atoms, in agreement with results from solvent transfer experiments. solvent shannon entropy maps illustrate the mobility gradient of the urea water mixture from the first solvation shell to the bulk. single urea molecules replace water in the first solvation shell preferably at locations of relatively high solvent entropy.

PMC3466777

pubmed-634

melting point determinations were performed by the open capillary method and are reported uncorrected. h and c nmr chemical shifts () are reported in parts per million (ppm) relative to tms, and coupling constants j are reported to the nearest 0.1 hz. c, ch, ch2, or ch3c signals are assigned from dept-90 and 135 spectra. in a number of cases, carbon atoms attached to boron gave very broad peaks in c nmr spectra, and these could not always be distinguished. hydrogen atoms attached to these carbons were not always observed in h nmr spectra. low- and high-resolution mass spectra were recorded on a time-of-fight mass spectrometer using electron impact (ei). ir spectra were recorded on a ft-ir spectrometer as a thin film (liquid samples) or applied as a solution in chloroform, and the chloroform was allowed to evaporate (solid samples). gc determinations were performed using a gas chromatograph fitted with a zb-5 column (30 m, 0.32 mm inner diameter, 1.0 m film thickness). the carrier gas was he at 69.3 kpa, and a split injection mode was used. the oven temperature was increased from 70 to 260 c at 6 c min and then held for 4 min. authentic samples of products were used to determine response factors relative to tetradecane, a known amount of which was added to reaction mixtures to allow quantification of product yields. a mixture of propiolic acid (8.79 ml, 0.143 mol) and aq hbr (48%, 40 ml) was heated at 95 c for 2.5 h, then left to cool. the crude product was filtered, washed with cold water (3 25 ml), and dried in air to give (e)-3-bromoacrylic acid as colorless needles (13.49 g, 63%); mp 120121.5 c (lit. a two-necked round-bottomed flask equipped with a magnetic stirrer bar, septum, and septum-capped dropping funnel was assembled hot and flushed with n2. (e)-3-bromoacrylic acid (8.00 g, 53 mmol) was dissolved in dry diethyl ether (40 ml) and transferred via syringe to the dropping funnel. powdered lialh4 (4.02 g, 106 mmol) was transferred quickly to the reaction flask, and dry diethyl ether (40 ml) was added. the reaction mixture was cooled in an ice bath, and the solution of 3-bromoacrylic acid was added dropwise via the dropping funnel with vigorous stirring. the mixture was stirred for 2 h, then water (2.5 ml) was added dropwise, followed by 15% naoh (5.25 ml) and more water (7.4 ml). the resulting salts were washed with diethyl ether (3 50 ml), and the combined extracts were dried over sodium sulfate and concentrated under reduced pressure to give fairly pure (e)-3-bromoprop-2-en-1-ol as a yellow liquid (4.16 g, 57%). a 50 ml round-bottomed flask equipped with a magnetic stirrer bar was charged with (e)-3-bromoprop-2-en-1-ol (3.52 g, 25.7 mmol) and cooled using an ice bath. hexachloroacetone (7.79 ml, 51.4 mmol) was added and the solution stirred for 5 min. powdered triphenylphosphine (6.74 g, 25.7 mmol) was added portionwise over a period of 25 min. the mixture was stirred for 2 h at 0 c, then fractionally distilled under reduced pressure to give 90% pure (e)-1-bromo-3-chloroprop-1-ene, which was fractionally distilled a second time to give pure (e)-1-bromo-3-chloroprop-1-ene (5) as a colorless liquid (0.52 g, 13%): h (500 mhz, cdcl3) 6.48 (1h, dt, j=13.5, 1.1 hz), 6.34 (1h, dt, j=13.5, 7.1 hz), 4.02 (2h, dd, j=7.1, 1.1 hz); c (125 mhz, cdcl3) 133.1 (ch), 110.8 (ch), 43.6 (ch2). boronate esters 3 where r=bn and 4-meobn were purchased from sigma-aldrich and tci, respectively, and used without further purification. boronate esters 3 where r=bu, i-pr, and cyclohexyl were prepared from the reaction of their corresponding boronic acids with pinacol in pentane in 73, 36, and 32% yields, respectively. boronate ester 3 where r=tert-bu was prepared by the literature method from pinacolborane and tert-bumgcl in 40% yield. boronate ester 3 where r=thexyl was prepared by the method previously described from the monohydroboration of 2,3-dimethyl-2-butene and subsequent reaction with pinacol, while boronate esters 11 and 12 were also prepared as described previously. the procedure for preparation of 13 (r=c-hex, r=phch=ch) is typical. characterization details of the other boronate esters 13 can be found in our previous publication. dicyclohexylborane was prepared by the dropwise addition of cyclohexene (2.12 ml, 20.9 mmol) to borane dimethyl sulfide complex (10 m, 1.00 ml, 10.0 mmol). the mixture was stirred at room temperature for 2 h, dry thf (10 ml) was added, then the mixture was kept at 0 c during the dropwise addition of phenylacetylene (1.15 ml, 10.5 mmol). the mixture was stirred for 1 h at 0 c and 1 h at room temperature then recooled to 0 c. dichloromethyl methyl ether (1.36 ml, 15.0 mmol) was added dropwise, followed by a freshly prepared solution of lithium triethylcarboxide (dropwise addition of n-buli (1.6 m in hexanes, 9.38 ml, 15.0 mmol) to a solution of 3-ethyl-3-pentanol (2.12 ml, 15.0 mmol) in thf (10 ml)) dropwise via a cannula over a 20 min period. the ice bath was removed and the mixture left to stir for a further 1 h. 2,2-dimethyl-1,3-propanediol (1.57 g, 15.0 mmol) in dry thf (5 ml) was added and the reaction mixture left to stir overnight at room temperature. the crude product following workup was separated by column chromatography on silica (petroleum ether) to give pure (e)-2-(1,1-dicyclohexyl-3-phenylallyl)-5,5-dimethyl-1,3,2-dioxaborinane (1.19 g, 20%) as cubic crystals: mp 188189 c; h (400 mhz, cdcl3) 7.40 (2h, d, j=7.2 hz), 7.29 (2h, app t, j=7.6 hz), 7.16 (1h, t, j=7.3 hz), 6.36 (1h, d, j= 16.7 hz), 6.25 (1h, d, j=16.7 hz), 3.65 (4h, s), 0.911.82 (22h, m), 1.01 (6h, s); c (125 mhz, cdcl3) (quat c next to boron not seen) 139.4 (quat c), 135.9 (ch), 129.0 (ch), 128.4 (ch), 126.2 (ch), 126.1 (ch), 71.9 (ch2), 41.1 (ch), 31.4 (quat c), 30.5 (ch2), 29.2 (ch2), 27.7 (ch2), 27.4 (ch2), 27.3 (ch2), 22.6 (ch3); b{h} (96.2 mhz, cdcl3) 29.3; hr-ei ms m/z (%) calcd for c26h39bo2 394.3043, found 394.3049 (m, 39%). a mixture of the appropriate boronic ester 3, 11, 12, or 13 (1 equiv), 1-bromo-3-chloroprop-1-ene (5, 1.1 equiv), and dry thf in a dry 50 ml round-bottomed flask equipped with a magnetic stirrer bar and septum was cooled to 78 c, and t-buli in hexanes (1.1 equiv) was added dropwise with vigorous stirring, and the mixture was stirred for an additional 30 min. the cooling bath was removed and the reaction mixture left to warm to 20 c over 3 h. an accurately weighed amount of tetradecane (0.2 ml) was added as a standard to enable gc determination of the amount of desired product formed following workup. the reaction mixture was cooled to 0 c, then oxidized by dropwise addition of excess naoh (0.6 g in 5 ml of h2o), followed by hydrogen peroxide (30% by weight, 3 ml). once the initial exothermic reaction subsided, the cooling bath was removed and the mixture left to stir overnight at room temperature. diethyl ether (10 ml) was added, the aqueous layer saturated with sodium chloride, and a small aliquot taken from the organic phase for gc analysis. the extract was washed with water (10 ml) and brine (10 ml), then dried over mgso4 to give a crude product containing the desired 3-alkylprop-1-en-3-ol 9 or 14, which was estimated by gc. in some cases (see below), mixtures were separated by column chromatography on silica (prewashed with 3% triethylamine in petroleum ether, petroleum ether/ethyl acetate 95:5 through 85:15) to provide the pure products. from 3 (r=thexyl) (0.564 g, 2.66 mmol), 5 (0.416 g, 2.68 mmol), thf (15 ml), and t-buli in hexanes (1.9 m, 1.41 ml, 2.68 mmol) (note that the excess of 5 and organolithium reagent was smaller in this example); colorless oil (0.105 g, 28%); h (400 mhz, cdcl3) 5.94 (1h, ddd, j= 17.2, 10.5, 6.7 hz), 5.23 (1h, d, j=17.2 hz), 5.18 (1h, d, j=10.5 hz), 4.01 (1h, d, j=6.7 hz, choh), 1.73 (1h, app sept, j=7.0 hz), 1.47 (1h, br s), 0.87 (3h, d, j=6.9 hz), 0.850.82 (6h, m), 0.73 (3h, s); c (125 mhz, cdcl3) 138.5 (ch), 116.5 (ch2), 78.1 (ch), 39.6 (quat c), 32.9 (ch), 19.0 (ch3), 18.6 (ch3), 17.5 (ch3), 17.4 (ch3); hrms (ei) m/z calcd for c9h18o 124.1252, found 124.1252 (m h2o). from 13 (r=r=et) (0.335 g, 1.58 mmol), 5 (0.274 g, 1.76 mmol), dry thf (10 ml), and t-buli in hexanes (1.6 m, 1.09 ml, 1.74 mmol); colorless liquid (0.042 g, 17%); h (500 mhz, cdcl3) 6.02 (1h, ddd, j=17.2, 10.5, 6.6 hz), 5.24 (1h, app dt, j=17.2, 1.6 hz), 5.16 (1h, app dt, j=10.5, 1.6 hz), 4.003.96 (1h, m), 1.411.28 (6h, m), 0.84 (9h, t, j=7.6 hz); c (125 mhz, cdcl3) 138.8 (ch), 115.9 (ch), 78.4 (ch), 41.7 (quat c), 26.0 (ch2), 8.5 (ch3); hrms (ei) m/z calcd for c10h20o 156.1514, found 156.1509 (m). from 3 (r=bn) (0.280 g, 1.28 mmol), 5 (0.226 g, 1.45 mmol), dry thf (10 ml), and t-buli in hexanes (1.5 m, 0.97 ml, 1.46 mmol); colorless oil (0.097 g, 52%); h (400 mhz, cdcl3) 7.24 (2h, app t, j=6.9 hz), 7.197.15 (3h, m), 5.86 (1h, ddd, j=17.2, 10.5, 5.8 hz), 5.18 (1h, app dt, j=17.2, 1.4 hz), 5.06 (1h, app dt, j=10.5, 1.3 hz), 4.314.24 (1h, m), 2.82 (1h, dd, j=13.6, 5.1 hz), 2.72 (1h, dd, j=13.6, 8.0 hz), 1.64 (1h, br s); c (125 mhz, cdcl3) 140.2 (ch), 137.8 (quat c), 129.7 (ch), 128.6 (ch), 126.7 (ch), 115.1 (ch2), 73.8 (ch), 43.9 (ch2); lrms (ei) m/z 148 (m, 4%). from 4-methoxybenzylboronic acid pinacol ester (0.292 g, 1.18 mmol), 5 (0.210 g, 1.35 mmol), dry thf (10 ml), and t-buli in hexanes (1.5 m, 0.84 ml, 1.26 mmol); colorless oil (0.185 g, 52%); h (400 mhz, cdcl3) 7.15 (2h, d, j=8.7 hz), 6.86 (2h, d, j=8.7 hz), 5.93 (1h, ddd, j=17.2, 10.5, 5.8 hz), 5.24 (1h, app dt, j=17.2, 1.4 hz), 5.13 (1h, app dt, j=10.5, 1.4 hz), 4.344.27 (1h, m), 3.79 (3h, s), 2.83 (1h, dd, j=13.7, 5.1 hz), 2.73 (1h, dd, j=13.7, 7.9 hz), 1.65 (1h, br s); c (125 mhz, cdcl3) 158.4 (quat c), 140.3 (ch), 130.5 (ch), 129.7 (quat c), 114.8 (ch2), 114.0 (ch), 73.7 (ch), 55.3 (ch3), 42.9 (ch2); lrms (ei) m/z 178 (m, 45%). an oven-dried 50 ml round-bottomed flask equipped with a magnetic stirrer bar and septum was flushed with n2. vinylmagnesium bromide solution in thf (0.7 m, 1.2 equiv) was added, and the mixture was cooled to 0 c. the appropriate aldehyde (1 equiv) was added dropwise, and the mixture was stirred for 15 min. distilled water (10 ml) was added and the product extracted with diethyl ether (3 20 ml). the combined organic extracts were washed with water (2 10 ml), dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the product. from vinylmgbr (0.7 m, 20 ml, 14 mmol) and valeraldehyde (1.24 ml, 11.7 mmol); light yellow liquid (0.73 g, 55%); h (400 mhz, cdcl3) 5.86 (1h, ddd, j=17.2, 10.5, 6.3 hz), 5.21 (1h, app dt, j=17.2, 1.4 hz), 5.09 (1h, app dt, j= 10.5, 1.4 hz), 4.08 (1h, app q, j=6.7 hz), 1.77 (1h, br s), 1.601.44 (2h, m), 1.411.23 (4h, m), 0.89 (3h, app t, j=7.2 hz); c (125 mhz, cdcl3) 141.5 (ch), 114.6 (ch2), 73.4 (ch), 36.9 (ch2), 27.6 (ch2), 22.8 (ch2), 14.1 (ch3). from vinylmgbr (0.7 m, 30 ml, 21 mmol) and isobutyraldehyde (1.60 ml, 17.5 mmol); colorless liquid (1.49 g, 85%); h (500 mhz, cdcl3) 5.86 (1h, ddd, j=17.2, 10.5, 6.4 hz), 5.22 (1h, app dt, j=17.2, 1.5 hz), 5.15 (1h, app dt, j=10.5, 1.5 hz), 3.86 (1h, app t, j=6.1 hz), 1.781.67 (1h, m), 1.58 (1h, br s), 0.93 (3h, d, j=6.8 hz), 0.90 (3h, d, j= 6.8 hz); c (125 mhz, cdcl3) 139.7 (ch), 115.7 (ch2), 78.4 (ch), 33.8 (ch), 18.3 (ch3), 17.9 (ch3). from vinylmgbr (0.7 m, 10 ml, 7 mmol) and cyclohexanecarboxaldehyde (0.71 ml, 5.9 mmol); colorless liquid (0.50 g, 61%); h (500 mhz, cdcl3) 5.86 (1h, ddd, j= 17.2, 10.4, 6.6 hz), 5.20 (1h, app dt, j=17.2, 1.6 hz), 5.14 (1h, app dt, j=10.4, 1.6 hz), 3.84 (1h, app t, j=6.4 hz), 1.881.81 (1h, m), 1.791.70 (2h, m), 1.701.62 (2h, m), 1.55 (1h, br s), 1.440.93 (6h, m); c (125 mhz, cdcl3) 140.0 (ch), 115.6 (ch2), 77.9 (ch), 43.7 (ch), 28.9 (ch2), 28.5 (ch2), 26.7 (ch2), 26.3 (ch2), 26.2 (ch2). borane dimethyl sulfide (10 m, 1.94 ml, 19.4 mmol) was added to a 50 ml round-bottomed flask equipped with a stirrer bar and under n2. the flask was cooled in an ice bath, and a solution of pinacol (2.29 g 19.4 mmol) in dry dichloromethane (10 ml) was added dropwise over a period of 10 min. the reaction mixture was stirred for an hour at 0 c and a further hour at room temperature before being transferred dropwise via cannula to a 100 ml round-bottomed flask equipped with a stirrer bar and a suspension of wilkinson s catalyst (0.16 g, 0.17 mmol, 1 mol %) in dry dichloromethane (5 ml). the reaction mixture was stirred for 5 min, then tert-butyl but-3-enoate (2.50 g, 17.6 mmol) was added dropwise over a period of 10 min. hexane (20 ml) was added, the mixture was filtered, and the filtrate was concentrated to give the crude product (4.30 g, 91%), which consisted of boronic esters 15 and 16 (85:15 by relative integrations by h nmr). this was separated by column chromatography on silica (98:2 petroleum ether/ethyl acetate, followed by 95:5, 90:10, and finally 85:15) to give boronate ester 15 as a colorless oil (1.80 g, 50%): h (400 mhz, cdcl3) 2.21 (2h, t, j=7.6 hz), 1.69 (2h, app pent, j=7.7 hz), 1.43 (9h, s), 1.23 (12h, s), 0.79 (2h, t, j=7.8 hz); c (125 mhz, cdcl3) 173.1 (quat c), 83.1 (quat c), 80.0 (quat c), 38.0 (ch2), 28.3 (ch3), 24.9 (ch3), 19.9 (ch2), 10.7 (br, ch2b); b{h} (96.2 mhz, cdcl3) 32.8; hr-ei ms m/z (%) calcd for c10h18bo3 197.1349, found 197.1353 (m oc(ch3)3, 41%). tert-butyl-5-hydroxyhept-6-enoate (17, 96% yield by gc) was prepared under the standard conditions (see section on reactions of alkylboronic esters with 5 and tert-buli; general procedure) from boronate ester 15 (0.631 g, 2.34 mmol), 5 (0.423 g, 2.72 mmol), dry thf (10 ml), and t-buli in hexanes (1.5 m, 1.81 ml, 2.72 mmol), except that after oxidation of the organoboron compound with alkaline hydrogen peroxide the mixture was made acidic (ph 3) by addition of hcl, prior to saturation with sodium chloride and extraction. the crude product was separated by column chromatography on silica gel (90:10 petroleum ether/ethyl acetate, followed by 85:15) to give 17 as a colorless oil (0.183 g, 39%): h (400 mhz, cdcl3) 5.84 (1h, ddd, j=17.2, 10.4, 6.2 hz), 5.21 (1h, dt, j=17.2, 1.4 hz), 5.09 (1h, dt, j=10.4, 1.4 hz), 4.09 (1h, app q, j=6.6 hz), 2.24 (2h, app t, j=7.5 hz), 2.01 (1h, br), 1.721.57 (2h, m), 1.591.48 (2h, m), 1.42 (12h, s); c (125 mhz, cdcl3) 173.1 (quat c), 140.9 (ch), 114.8 (ch2), 80.3 (quat c), 72.7 (ch), 36.3 (ch2), 35.2 (ch2), 28.1 (ch3), 20.8 (ch2); hr-ei ms m/z (%) calcd for c7h11o2 127.0759, found 127.0759 (m tert-butyl 5-hydroxyhept-6-enoate (17, 0.069 g, 0.345 mmol), dry benzene (10 ml), and p-toluenesulfonic acid monohydrate (0.018 g, 0.093 mmol) were added to a 25 ml round-bottomed flask equipped with a stirrer bar and a condenser. the reaction mixture was heated to reflux (bath temperature =100 c) for 4 h. the reaction mixture was concentrated under reduced pressure, hexane (20 ml) was added, and the mixture was filtered. the filtrate was concentrated under reduced pressure to give lactone 18 as a light yellow oil (0.044 g, 100%): h (500 mhz, cdcl3) 5.86 (1h, ddd, j= 17.2, 10.6, 5.5 hz), 5.34 (1h, dt, j=17.2, 1.4 hz), 5.23 (1h, dt, j=10.6, 1.4 hz), 4.844.79 (1h, m), 2.622.55 (1h, m), 2.522.44 (1h, m), 2.021.81 (3h, m), 1.701.61 (1h, m); c (125 mhz, cdcl3) 171.2 (quat c), 136.1 (ch), 117.0 (ch2), 80.4 (ch), 29.7 (ch2), 28.0 (ch2), 18.2 (ch2); hr-ei ms m/z (%) calcd for c7h10o2 126.0681, found 126.0678 (m, 20%).

the reagent 3-chloro-1-lithiopropene (4) can be generated by treating 1-bromo-3-chloropropene with t-buli. it is unstable but if generated at low temperature in the presence of alkylboronic esters, such as 3, is trapped in situ to give rearrangement products 2, which on oxidation give 3-alkylprop-1-en-3-ols in good yields. the reaction works for primary, secondary, benzylic, and even tertiary alkylboronic esters, providing allylic alcohols bearing almost any alkyl group available using organoborane chemistry and incorporating all features of such groups.

PMC3806150

pubmed-635

survival rates continue to improve with the advent of more refined treatments and better supportive care. today over 80% of children with cancer are alive for at least five years, and the majority of these are cured. as a consequence, many sufferers of childhood cancer are now living into adulthood and having families of their own. it has been estimated that about 1 in 900 adults aged 18 to 44 years is a cancer survivor. among this group, those with an inherited susceptibility to cancer will transmit their genetic fault to a proportion of their children. they are also at risk of developing a second cancer during their adult life. while the causes of the majority of childhood cancers are largely unknown, there are a number of clinical syndromes for which the evidence for an excess cancer risk in children is most persuasive. these include li-fraumeni syndrome, neurofibromatosis type i, inherited retinoblastoma mutations, familial wilms tumor, and some disorders of dna repair. these syndromes collectively account for less than 10% of all childhood cancers, however. beyond this, it is not clear if cancer in childhood increases the risk of cancer in relatives. a number of recent studies have described an association between childhood cancer and cancer in first-degree relatives [57], particularly siblings [5, 7] and mothers. in this study, we describe the distribution of cancers in australian children and estimate the risks of cancer in their relatives. it was established from the clinical services at the royal children's hospital (rch) and the monash medical centre (mmc), between which over 95% of children under 10 years of age and 83% of children aged 1015 years with cancer within the state of victoria, australia, are treated. children were eligible for inclusion if they had a diagnosis of any cancer before age 15 and when initial treatment was given at one of the participating centres between october 1998 and october 2002. the human research ethics committee at each participating institution reviewed and approved the protocol before enrolment commenced. a total of 486 (422 at rch and 64 at mmc) incident cases of childhood cancer were diagnosed during the study period. of these, 21 families were found to be ineligible (seven non-english speaking parents, seven where the proband was aged over 15 years, six where the proband did not have their initial treatment at either service, and one where the proband was adopted). of the remaining 465 families, 21 were not contactable, 53 refused to participate, 6 were lost to followup, and 6 later withdrew consent. this left 379 (82%) families participating (340 at rch and 39 at mmc), from which we interviewed 351 (92.6%) fathers and 371 (97.9%) mothers. in 343 (90.5%) families, both the mother and father were interviewed, while in 28 (7.4%) families only the mother was interviewed and in 8 (2.1%) families only the father was interviewed. at the time of enrolment, both parents of the affected child were asked a baseline questionnaire that included information on lifestyle and environmental risk factors and a detailed family history of cancer. where half siblings were identified, the appropriate, biologically relevant family history was obtained. the family history of cancer questionnaire asked about all of the affected child's siblings, parents, aunts, uncles, and grandparents (i.e., all first-degree and second-degree relatives). parents were asked to identify each first-degree and second-degree relative, any known cancer diagnoses with the age (in years) at diagnosis, and type of malignancy, together with their current age or age at the time of death. the information collected was reviewed and classified by the authors with expertise in cancer (jah; es). the world health organization's international classification of diseases for oncology, 3rd edition (id-o-3) scheme, was used for classification of site of malignancy. nonmelanoma skin cancers, nonmalignant tumours, and in situ cancerswere not included in the analyses. to address the potential contribution of known familial cancer syndromes, we examined the medical records of the 26 affected children who had a first-degree relative with cancer. in particular, we sought clinical evidence for li-fraumeni syndrome, neurofibromatosis type 1, familial retinoblastoma, and familial wilm's tumour syndrome. person-years at risk for the cohort of relatives were calculated as the time to date of diagnosis of cancer, date of death, or date of interview completion, whichever occurred earliest. australian population-based cancer incidence data, specific for gender, age, and year of birth, were obtained from the australian institute of health and welfare. standardised incidence ratios were used to compare the number of observed cancers in relatives with the number expected in the australian population. robust estimates for confidence intervals were calculated to account for potential clustering within a family. all statistical tests were two-sided and a p value<0.05 was considered statistically significant. the distribution of childhood cancers diagnosed in the cohort of 379 children is summarized in table 1. a family history for cancer was identified in 4,736 (1,337 first-degree and 3,399 second-degree) relatives with a total of 211,394 person-years of followup. fourteen of the 379 (3.7%) families reported a positive history for childhood cancer in any relative, with none having more than one case identified. twenty-six children with cancer (6.9%) had a first-degree relative (parent or sibling) with a history of cancer. the results for the family history of cancer in all childhood cancer patients are summarized in table 2. there was a higher than expected, though not statistically significant, incidence of childhood cancer among first-degree relatives (sir 1.43; 95% ci 0.545.08). there was also a higher than expected, though not statistically significant, incidence of cancer among first-degree relatives (sir 1.45; 95% ci 0.932.1). there was a statistically significant increase in cancer among female first-degree relatives (sir 1.82; 95% ci 1.263.39). the increased family cancer history in first-degree females was largely attributable to an effect in mothers (sir 1.78; 95% ci 1.273.33) but was also observed in sisters (sir 2.15; n=1). elevated cancer incidence rates were also observed in aunts (sir 1.48; 95% ci 1.112.00). the gender-specific association was reflected in higher than expected incidence rates of breast cancer in mothers (sir 1.92; 95% ci 0.726.83), aunts (sir 1.64; 95% ci 0.982.94) and to a lesser extent grandmothers (sir 1.04; 95% ci 0.781.40). although numbers were small, an increased cancer incidence in relatives appeared to be most apparent in the children with sarcomas (sir 2.58; 95% ci 0.998.52), embryonal tumors (sir 2.12; 95% ci 0.719.34), and brain tumors (sir 1.52; 95% ci 0.703.92). of the 26 families with a history of cancer within a first-degree relative, only one met the criteria for a clinically recognizable familial cancer syndrome (li fraumeni syndrome). the increased rates of cancer in female first-degree relatives (sir 1.68; 95% ci 1.032.93), including mothers (sir 1.78; 95% ci 1.093.09) and aunts (sir 1.49; 95% ci 1.122.02), remained when this child and family were removed from the analyses. our small, population-based study suggests an increased risk of childhood cancer and some adult cancers among relatives of childhood cancer patients that are not accounted for by clinically identifiable familial cancer syndromes. these findings were seen in both first- and second-degree relatives. although some of our findings did not reach statistical significance, they are largely consistent with previously published unselected childhood cancer cohorts [57, 12] and add to a growing body of evidence that unidentified genetic risk exists in some families. in addition to the small size of the study, other limitations include the use of family history in isolation and the absence of some parental input to family history of cancer. while we were not able to cross-reference reported history of cancer with cancer registry data, a similar study in sweden confirmed underreporting of cancer in relatives by history. furthermore, in the small minority of cases where both parents were not available to provide the relevant family history of cancer, underreporting of the family history of cancer is also likely. allowing for these two study biases towards the null hypothesis is therefore likely to strengthen the positive findings we have reported. this underreporting may also explain why a large number of apparent protective effects occurred in second-degree relatives, where direct, personal access to a family history of cancer is not always possible. overall, first-degree relatives (siblings and parents) had an increased incidence of childhood tumors. this is consistent with the higher occurrence of childhood cancers among siblings demonstrated in a study of 51,000 children who developed cancer under the age of 15 in the uk, even after those with a genetic etiology were excluded. it was also reported in a cohort of 13,703 childhood cancer survivors in north america (sir 1.5; 95% ci 1.351.7). the apparent association of childhood cancer with adult cancers in female relatives is fascinating and supports a recently published population-based study from utah, usa, where a higher risk of adult cancer was restricted to mothers and siblings (sir 1.31; 95% ci 1.111.56) but was not observed in fathers. it is interesting to note that the effect was greatest for children younger than 5 years at diagnosis (sir 1.48; 95% ci 1.131.95), suggesting a strong ante- or perinatal effect. factors which may be related to maternal effects on childhood cancer risk include maternal age of pregnancies, altered in utero exposures, and birth weight. our finding of an increased prevalence of breast cancer in female relatives also supports recently published swedish data. a number of related studies have also observed higher rates of breast cancer in mothers and sisters [1416]. the potential link between breast cancer and childhood cancer is of great interest, given past reports of increased rates of childhood cancer in families carrying a brca1 or brca2 mutation [17, 18]. our study was not designed nor powered to examine the relationship between specific childhood cancer types and family history of cancer, so it must be acknowledged that potential associations may have gone undetected. certainly, others have postulated a link between family history of cancer and the more common subtypes, childhood leukaemia, childhood lymphomas, and childhood brain tumours. the epidemiology of childhood cancer described here is the starting point for further explorations into identifying previously unrecognized genetic predisposition to childhood cancer. we are currently undertaking whole genome sequencing of these children with cancer and their affected first-degree relatives ' germline dna in order to identify the role of previously described and novel genetic mutations to account for our findings.

we determined the extent and distribution of cancers in relatives of 379 children newly diagnosed with cancer. family history was collected from 1,337 first-degree and 3,399 second-degree relatives and incidence compared with national age- and gender-specific rates. overall, 14 children (3.7%) had a relative with a history of childhood cancer and 26 children (6.9%) had a first-degree relative with a history of cancer, with only one of these having an identifiable familial cancer syndrome. there was a higher than expected incidence of childhood cancer among first-degree relatives (parents and siblings) (standardized incidence ratio (sir) 1.43; 95% ci 0.545.08). there was also a higher than expected incidence of adult cancers among first-degree relatives (sir 1.45; 95% ci 0.932.21), particularly in females (sir 1.82; 95% ci 1.263.39). the increased family cancer history in first-degree females was largely attributable to an effect in mothers (sir 1.78; 95% ci 1.273.33). the gender-specific association was reflected in higher than expected incidence rates of breast cancer in both mothers (sir 1.92; 95% ci 0.726.83) and aunts (sir 1.64; 95% ci 0.982.94). these findings support the hypothesis that previously undetected familial cancer syndromes contribute to childhood cancer.

PMC3985329

pubmed-636

women of childbearing age appear to be particularly susceptible to the exacerbation of existing mental illness and the development of new mental illness [15]. indeed, it has been estimated that over 500,000 pregnancies annually are complicated by psychiatric illness that either precedes pregnancy or arises during pregnancy. untreated psychiatric disease during pregnancy is associated with increased risks for the mother (including self-harm/suicide, self-neglect, and reduced compliance with prenatal and postnatal care) and risks for the child (including impaired fetal development, infanticide, and impaired mother-child bonding) (reviewed in [7, 8]). historically, antipsychotics have been extensively and effectively used for the treatment of schizophrenia and bipolar disorder, and more recently they are becoming part of the treatment of depression [912]. conventional antipsychotics (also known as typical or first generation antipsychotics) which were more commonly used to treat these conditions caused a significant decrease in fertility. however, the newer atypical antipsychotics do not have this side-effect. as a consequence, the number of women taking antipsychotics, who are becoming pregnant, is on the rise. indeed, appointments at a motherisk program clinic related to the use of antipsychotic medications increased 170% between 1989 and 2001; a rise which was for the most part attributable to an increased use of second-generation or atypical antipsychotics. the vast majority of women who use antipsychotics during pregnancy do so because of ongoing illness. in fact, only in cases of where the first schizophrenic episodes are reported in pregnancy or there is a risk of puerperal psychosis would the exposure of antipsychotic exposure be restricted to the pregnancy period. functionally, typical antipsychotics exhibit high affinities for d2 receptors [15, 17] in the mesolimbic, mesocortical, and nigrostriatal dopamine pathways. this, rather nonspecific targeting of dopaminergic pathways, can result in a range of undesirable motor disorders. atypical antipsychotics, however, are more selective for the d2 receptors in mesolimbic pathway as compared to those in the mesocortical and nigrostriatal pathways. in addition, they also target the serotonin receptor subtype 2a (5ht2a) which may help to reduce the negative side-effects associated with typical antipsychotics [19, 20]. it is important to note that the proper correlation between the ratio of the clinical dosage and d2 receptor affinity necessary to treat the symptoms of conditions such as schizophrenia has not been completely established. it is well understood that the suitability of antipsychotic medications during pregnancy is a balance between the risks of adverse obstetrical and neonatal outcomes and the risks associated with untreated or inadequately treated psychiatric illness. the most common atypical antipsychotics administered during pregnancy are olanzapine, clozapine, risperidone, quetiapine, and aripiprazole. complicating the matter further is that almost 4057% of women taking atypical antipsychotics during are prescribed a combination of these drugs (polytherapy) [2325]. in general, current practice guidelines discourage changing medications during pregnancy as this may leave the patient on nontherapeutic doses during a period of time; a situation which is not in the best interests of the mother. therefore, the usual standard of care dictates that dosages be increased or polytherapy be implemented. although general clinical practice guidelines have been established (acog, 2008), there remains significant uncertainty regarding effects of these drugs on the fetus. to date, the teratogenic effects of antipsychotic exposure have received significant attention; however, the effects of these medications on long-term health outcomes of the offspring have not been well studied. in adults, one of the major side-effects of antipsychotic use is the dysregulation of body weight homeostasis (reviewed in [26, 27]). similarly, maternal use of antipsychotics has been reported to result in aberrant fetal growth. indeed maternal antipsychotic use has been reported to result in an increased incidence of both low and high birth weight relative to the general population [23, 24, 28, 29]. since being either too small or too large at birth is a risk factor for the development of metabolic syndrome in postnatal life [3032], children exposed to antipsychotic medications in utero may be at increased risk of developing obesity in postnatal life. however, there are no human studies which have tested this hypothesis and the mechanism(s) by which atypical antipsychotics may affect fetal growth have yet to be elucidated. it is very difficult to decipher, based on the available literature, why atypical antipsychotics can cause both small for gestational age (sga) and large gestational age (lga) fetuses. for example, mckenna et al. report an increased risk of sga in women taking the atypical antipsychotics olanzapine, risperidone, quetiapine, and clozapine. report an increased risk of lga for women taking the atypical antipsychotics amisulpride, clozapine, olanzapine, quetiapine, and risperidone. since the sample sizes in these studies are small for women taking each of the individual drugs, the risks associated with each drug can not be accurately reported. furthermore, the paucity of data evaluating the action of antipsychotics in pregnancy using animal models makes the discussion of mechanisms for these drugs difficult. it is well accepted that atypical antipsychotic use can alter body weight homeostasis in humans and nonpregnant rodent models, resulting in significant drug-induced weight gain and visceral fat accumulation [26, 27, 33], an effect which is more pronounced in females. the weight gain varies among the atypical antipsychotics, ranging from 2 to in excess of 25 kg [27, 35], and can affect up to 60% of patients after 312 months of use. furthermore, there is now considerable evidence from animal experiments and clinical studies that the use of atypical antipsychotics is a major risk factor for impaired glucose homeostasis and type 2 diabetes in the nonpregnant population [26, 27, 3640]. however, the effects of atypical antipsychotics on gestational weight gain, postpartum weight retention, and gestational diabetes have not been systematically addressed; although, one study has reported that the use of antipsychotics during pregnancy significantly (or 1.78, 95% ci 1.043.01) increases the risk of developing gestational diabetes. in addition to metabolic consequences of antipsychotic medications, there are a number of other risk factors which may explain the increased incidence of diabetes in mentally ill patients including hereditary factors, poverty and poor access to good nutrition, physical inactivity, and antipsychotic medication use (reviewed in holt et al., 2004). these changes in glucose homeostasis may explain, in part, the increased incidence of large for gestational age (lga) babies born to women taking atypical antipsychotics during pregnancy as lga is regarded as the most common fetal complication when women have gdm or preexisting diabetes [4346]. the mechanisms by which atypical antipsychotics can cause maternal obesity and dysglycemia are varied, and the relative contribution of each pathway to an aberrant metabolic state is not completely understood. atypical antipsychotic-induced weight gain and fat accumulation may be the result of altered food intake (i.e., hyperphagia and decreased satiety) [4750], direct effects of the drug on adipocytes to alter lipogenesis and lipolysis in favour of lipid accumulation [5153], or effects on peripheral tissues to induce insulin resistance [51, 54, 55]. this insulin resistance in combination with increased gluconeogenesis [51, 54, 56] and impaired insulin secretion from the pancreatic beta cell [55, 57, 58] may in turn be responsible for the observed increase in type 2 diabetes with the use of atypical antipsychotics. however, there is a great deal of heterogeneity amongst the responses evoked by different antipsychotics. indeed, while some evoke insulin release, others such as risperidone, ziprasidone, and quetiapine do not. the paucity of data using individual antipsychotics makes it difficult to conclude how these drugs induce such heterogeneous responses. regardless of the mechanism, aberrant maternal glucose control and maternal obesity can have significant implications for the long-term health of both the mother and her child. there is compelling evidence that obesity in women, whether as a result of prepregnancy obesity, excessive gestational weight gain, and/or postpartum weight retention, is associated with an increased risk for many pregnancy-related health complications such as gestational diabetes and hypertensive disorders of pregnancy [60, 61]. in addition, maternal obesity also considerably increases the risk of fetal complications such as spontaneous abortion, stillbirth, congenital anomalies, neonatal death, and altered fetal growth. indeed, in humans, obesity during pregnancy is associated with a significantly increased risk of macrosomia (commonly defined as birth weight 4500 g (8 lb 13 oz to 9 lb 15 oz)) as well as an increased risk of delivering a low birth weight baby [43, 60, 6266]. similarly, animal studies have also shown that maternal overweight and obesity results in altered fetal growth with reports of both increased and decreased birth weight [30, 6769]. therefore, based on the well-documented relationships between maternal obesity/diabetes and altered fetal growth, it is plausible that the altered fetal growth in women taking atypical antipsychotics may be a reflection of antipsychotic-induced changes in maternal metabolic or nutritional status. in addition to the impact of maternal metabolic status on the pregnancy, atypical antipsychotics may also be transferred across the placenta and directly impact fetal growth. while there is limited evidence quantifying the extent to which these drugs are transferred across the placenta, the work of schenker et al. suggests that somewhere in the range of 514% of a labeled olanzapine can be transferred from maternal to fetal system in 4 h. however, the effects of atypical antipsychotics on fetal growth may be also mediated via altered placental development and/or function (figure 1). prospective and retrospective analyses of maternal antipsychotic use and fetal outcomes have provided evidence that the use of atypical antipsychotics during pregnancy can result in dysregulated fetal growth. the results from these studies are conflicting; with some studies reporting that maternal use of atypical antipsychotics results in an increased incidence of high birth weight babies relative to the normal population and some studies reporting an increased incidence of low birth weight babies [23, 24, 28, 29]. such observations suggest the actions of atypical antipsychotics on fetal growth may be described as a u shaped growth curve. understanding the mechanisms driving such complex interrelationships may prove to be difficult, but the most likely place to initiate this search may be placental development and function. mechanistically, it is known that atypical antipsychotics can act via dopamine d2 or serotonin 5-ht2a receptors. in addition to their well documented, but rather heterogeneous effects on various regions of the brain, the atypical antipsychotics also affect a number of the other tissues. recent work from viau and colleagues reports the identification of the 5-ht2a serotonin receptor in human trophoblasts, a lineage of cells which are of central importance to placental development and function. in other cell types, signaling via the 5-ht2a receptor has been shown to affect cellular differentiation, proliferation, and migration, all of which are central to the function of placental trophoblast cells in the establishment of proper placentation [75, 76]. such a relationship becomes more relevant because serotonin is synthesized de novo in human trophoblast cells where it likely serves an important endocrine, paracrine, and autocrine role in regulating placental function. indeed, treatment of cultured trophoblasts, bewo and jeg-3 cells, with serotonin results in increased aromatase activity; an effect which may result in altered estrogen biosynthesis. control of estrogen biosynthesis is important not only for successful implantation of the blastocyst but also for the regulation of leptin expression (review in). furthermore, the levels of leptin in both maternal and fetal plasma have been associated with the regulation of fetal growth [81, 82]. taken together, this evidence raises the possibility that atypical antipsychotics may affect fetal growth through the regulation of estrogen biosynthesis and leptin expression. dopamine receptors, another target of atypical antipsychotics, are also known to be present in the human placenta, in trophoblast cells. two subtypes, d1 and d2 receptors, have been localized to the spongiotrophoblasts and giant cells of the junctional zone of rat placenta at gestational days 1216 (term 2123 days). these cell lineages play important roles in the establishment, development, and maintenance of pregnancy in rodents [85, 86]. like the serotonin receptor, d2 receptors have also been linked to important hormone regulatory processes such as the inhibition of basal and hormone stimulated release of human placental lactogen (hpl) from trophoblast cells. furthermore, the observation that the pattern of d2 receptor expression varies during the course of a normal pregnancy argues for an important role for dopamine signaling during placental/fetal development [87, 88]. while the function of these receptors have not been directly related to altered fetal growth, their presence in the placenta suggests that drugs which target these receptors may have potential regulatory consequences for placental function and fetal development. while compounds such as clozapine also have a relatively high affinity for the dopamine d4 receptor subtype, which has been detected in the placenta, the functional role(s) of these receptors in placenta there is currently a paucity of data supporting the direct action of atypical antipsychotics on placental function. however, it is biologically plausible that this class of drugs might impact placental function given the presence of the putative receptors for these drugs on trophoblast cells as discussed above. moreover, activation of dopamine receptors by other drugs, namely, bromocriptine, has been shown to alter the expression of pit-1; a pituitary-specific transcription factor which is synthesized in the rat placenta and is involved in the regulation of rat placental lactogen (rpl) gene expression, a hormone known to impact fetal development. alternatively, atypical antipsychotics may affect placental development and/or function via alterations in oxidative stress in this tissue. oxidative stress is a term used to describe the imbalance between the production of reactive oxygen species and the ability of the cell to limit their damage. there is some evidence that olanzapine and quetiapine may act as antioxidants in cultured peripheral neurons by decreasing oxidative stress associated with an increased accumulation of -amyloid protein [92, 93] and can even work to reverse the effects of compounds known to increase oxidative stress. however, the majority of evidence suggests that atypical antipsychotics are associated with increased oxidative stress [95, 96]. these, apparently, divergent effects of atypical antipsychotics may once again reflect the paucity of data in this area of research. systematic investigation of these drugs on both neuronal and uterine systems will be important understanding the effects of these drugs on cellular stress response pathways. clozapine, olanzapine, and aripiprazole have been found to differentially evoke oxidative stress in different regions of the brain. for example, chronic treatment (28 days) of rats with clozapine increased oxidative damage in the hippocampus. olanzapine and aripiprazole actually decreased oxidative damage in the prefrontal cortex, despite the observation that the aripiprazole increased mitochondrial superoxide formation, a radical species known to cause both mitochondrial and cytosolic oxidative damage [98, 99], in the same tissue. atypical antipsychotics have also been linked to increased superoxide formation and apoptosis in nave neutrophils. furthermore, clozapine treatment of isolated lymphoblasts increased oxidative damage of a limited group of proteins. the increased damage of proteins associated with cellular metabolism suggests that the oxidative stress may arise from the mitochondria; known to be a primary producer of cellular superoxide. production of free radicals from mitochondrial sources tends to preferentially damage mitochondrial proteins, the majority of which are associated with energy metabolism. evidence that mitochondria are a target of atypical antipsychotics arises from the observation that the cerebral cortex and hippocampus of rats exposed to clozapine exhibited altered mrna expression for 14 mitochondrial proteins. six of these proteins comprise various subunits of the electron transport chain, including complex i as well as complex v (atp synthase). this is particularly pertinent to oxidative stress since inhibition of the mitochondrial respiratory chain inhibition is known to be associated with increased oxidative stress [101, 103]. while typical and atypical antipsychotics have both been shown to inhibit mitochondrial complex i activity [104, 105], the inhibitory effects of atypical antipsychotics generally require higher concentrations. there are only minimal inhibitory effects of antipsychotics reported on complex ii of the mitochondria [104, 106, 107]. in addition, activation of the 5-ht2a receptor has also been associated with mitochondrial biogenesis. taken together, it is feasible to propose that some of the actions of the atypical antipsychotics may occur via mitochondrially generated oxidative stress in the placenta especially since the placenta is characterized by particularly high levels of mitochondria. increased oxidative stress along with poor mitochondrial function in the placenta has been linked to growth restriction and premature fetal demise. mechanistically, oxidative stress may impact the ability of trophoblasts to transport nutrients between maternal and fetal circulation or carry out their endocrine functions. however, the exact linkage between placental oxidative stress and altered fetal growth and development remains undiscovered (figure 2). oxidative stress and endoplasmic reticulum (er) stress have been linked and may jointly influence a number of cellular processes [19, 113, 114]. however, there has been very little work done to determine whether antipsychotics impact cellular er stress. research by kurosawa et al. suggests that the atypical antipsychotic olanzapine alleviates chemically induced er stress in cultured neurons. in addition to their effects on neuronal function, these drugs also impact reproductive functions such as fertility and may also effect placental function. the mechanisms by which these drugs directly, as well as indirectly (via modulation of metabolic homeostasis), influence fetal programming need to be more thoroughly investigated. there is currently considerable uncertainty regarding prescribing practices for pregnant women with severe and persistent psychiatric disorders. the physician and the mother have to balance the risks of untreated psychiatric illness, during pregnancy, against the potential fetal toxicity of atypical antipsychotic medications. atypical antipsychotics are suspected to be teratogenic, although there are no randomized controlled studies for obvious ethical reasons. the adverse metabolic side-effects of these medications compound the risks associated with teratogenicity. given that the number of women exposed to atypical antipsychotic drugs during pregnancy is increasing, it is important to more clearly delineate the risks associated with the administration of these drugs. there is limited information regarding their impact of these compounds on placental development and/or function. the use of appropriate animal models may be crucial in understanding the effects of these drugs on fetal growth and development which have profound consequences for the long-term health of the offspring.

there is currently considerable uncertainty regarding prescribing practices for pregnant women with severe and persistent psychiatric disorders. the physician and the mother have to balance the risks of untreated psychiatric illness against the potential fetal toxicity associated with pharmacological exposure. this is especially true for women taking atypical antipsychotics. although these drugs have limited evidence for teratological risk, there are reports of altered fetal growth, both increased and decreased, with maternal atypical antipsychotic use. these effects may be mediated through changes in the maternal metabolism which in turn impacts placental function. however, the presence of receptors targeted by atypical antipsychotics in cell lineages present in the placenta suggests that these drugs can also have direct effects on placental function and development. the signaling pathways involved in linking the effects of atypical antipsychotics to placental dysfunction, ultimately resulting in altered fetal growth, remain elusive. this paper focuses on some possible pathways which may link atypical antipsychotics to placental dysfunction.

PMC3401548

pubmed-637

the prognosis of the newly diagnosed breast cancer patient depends on a number of factors, among the most important of which is the extent of spread of disease to the axillary lymph nodes [1, 2]. because treatment is influenced by the presence and number of axillary lymph nodes involved, evaluation of the axillary nodes has been performed in every patient that could tolerate it after a diagnosis of invasive carcinoma. in the past, a complete surgical dissection of the axilla was performed, resulting in significant morbidity, including a 30% incidence of lymphedema. the development of sentinel node mapping resulted in a notable reduction in morbidity; however, if a sentinel node was positive, often not discovered until final pathologic processing done postoperatively, complete axillary dissection would be performed at a later date to assess the total number of lymph nodes involved, thus requiring a second surgical procedure and anesthesia [57]. a preoperative diagnosis of axillary metastasis by ultrasound guided node biopsy would streamline patient care and reduce operating room time and expense by allowing definitive breast surgery and complete axillary dissection in the same operative setting, eliminating an unnecessary sentinel node procedure. while the need for preoperative node sampling in patients with nonpalpable nodes and t1 and t2 cancers has been challenged by the acosog z 11 study, there are subgroups of women for whom initial ultrasound guided node sampling is desirable, including those planning mastectomy or neoadjuvant chemotherapy. using current high frequency transducers, studies have been performed attempting to identify malignant lymph nodes by their morphology but there is overlap both in the appearance of benign and malignant lymphadenopathy and in the appearance of normal and abnormal lymph nodes, with benign causes of axillary adenopathy being relatively common [911]. therefore, tissue diagnosis of axillary node status remains important when it will affect patient management. tissue sampling of suspicious axillary lymph nodes with ultrasound guidance has been performed for many years with a fine needle to obtain aspirates for cytologic evaluation. reports in the literature have shown that fna is useful for evaluation of metastatic disease, with sensitivities ranging from 44 to 100%, the variability being likely due at least in part to patient selection [1218]. studies comparing fna to large needle core biopsy of breast masses have shown core biopsy to be more accurate and easier to interpret than fna, leading breast care specialists to wonder if that is the case for sampling of axillary lymph nodes. while it is reasonable to expect the use of a core biopsy device to obtain a larger and architecturally intact piece of tissue to increase diagnostic accuracy as compared to obtaining an aspirate of cells with a fine needle, if the only question to be answered in sampling the axillary node is whether or not metastatic carcinoma cells are present along with lymphocytes, a larger architecturally intact piece of tissue may not be required. although core biopsy is more invasive than fna due to the larger needle size, reports have shown that core biopsy of axillary nodes is a safe and effective procedure [20, 21]. retrospective comparisons of fna and core accuracy in the axilla have been performed [22, 23], but to our knowledge, only one other prospective study has been published directly comparing fine needle aspiration to core biopsy of an axillary lymph node for the diagnosis of metastatic breast cancer. the purpose of this study was to determine if there is a difference between ultrasound guided core biopsy and fna in their ability to detect metastatic disease in the axillary lymph nodes of patients with a current diagnosis of ipsilateral breast cancer. from december 2008 through december 2010, women with suspected or recently diagnosed breast carcinoma and at least one lymph node in the ipsilateral axilla judged to be abnormal in sonographic appearance were approached for participation in this irb-approved, hipiaa-compliant prospective study. 105 women gave written informed consent to undergo ultrasound guided fna, immediately followed by core biopsy of the same node, followed by clip placement. patients unable to consent due to language or comprehension difficulties and patients deemed emotionally too fragile to discuss the subject of metastatic disease required in the consent form were excluded. other patients excluded themselves, not wishing to undergo a second needle procedure for research purposes. one patient was excluded due to difficulty accessing the node with a core biopsy due to its location. the outcome of percutaneous node sampling was correlated with surgical pathology from sentinel node excision or axillary dissection. prior to node sampling, the cortical thickness, presence or absence of a hilus, presence or absence of cortical flow, node shape, and number of nodes thought to be abnormal were recorded. a phillips iu22 ultrasound unit (phillips healthcare, andover, ma) with 12 or 17 mhz transducers was used for imaging. criteria used in determining appropriateness for node sampling were loss of the normal hilus, abnormal shape including focal bulging of the cortex, presence of cortical flow, and cortical thickening. a strict threshold for cortical thickness was not used; a node with a cortex between 2 and 3 mm was considered suspicious if the other nodes had cortices less than 2 mm. the patients were asked to rate their pain during each procedure on a scale of 1 to 10 and were informed of which procedure was being performed. the degree of bleeding (minimal or moderate) or hematoma formation, if any, was documented. of the 105 patients, 7 patients ' percutaneous breast biopsies were unexpectedly negative for malignancy (their node biopsies were all negative also). of the remaining 98 with breast malignancy, 3 patients with both fna and core negative nodes were excluded due to lack of histopathologic axillary surgical correlation. we included 4 patients who did not have axillary surgery but were assumed to be true positives as they were both core and fna positive. tumor size ranged from 6 mm to 10.7 cm, with 12 patients having tumor size greater than 5 cm. cortical thickness of the sampled node ranged from 2 mm to over 6 mm. table 1 shows features of the study population. to mimic the actual range of clinical practice, variability in sampling devices fna was performed using a 21 or 25 g, 2-inch needle, with one (90 cases), 2 (11 cases), or 3 (4 cases) needle entries of multiple needle excursions through the cortex of the node. the number of excursions was not recorded, but radiologists preferring more than one entry typically made fewer excursions per entry, estimated at 10 versus 20 to 30. the aspirated material was placed both on slides in 95% alcohol and in buffered formalin for cell block preparation; a pathologist was not present at the time of the procedure to evaluate the adequacy of the samples. core biopsy was performed immediately following the fna with either a 14 g (86 cases), 12 g (16 cases), or 18 g (3 cases) biopsy device. the 12 g device used was celero (hologic, bedford, ma). the 14 g devices were bard monopty, bard maxcore, finesse (bard, tempe, az), and achieve (cardinal health, dublin, oh), and the 18 g devices were achieve. one to four cores were obtained (one core in 23 cases, 2 cores in 54 cases, 3 cores in 20 cases, and 4 cores in 8 cases). with 14 g devices, only one core was obtained in 11 of 86 cases. with 12 g devices, one core only was obtained in 12 of 16 cases. with 18 g devices, 2, 3, or 4 the procedures were performed by 12 different academic radiologists experienced (range 321 years, with over half performed by those with over 16 years of experience) in breast imaging and biopsy (figure 1). however, for a given patient, the same radiologist performed both the fna and the core biopsy. the cytologic material obtained was evaluated by one of three pathologists experienced in breast pathology and cytology, without the knowledge of the core biopsy result. the fna result was categorized as negative if reported as containing suspicious cells but not actually stating that metastatic cancer cells were present. suboptimal specimens were categorized as negative for both core and fna, because the course of action in our institution in most cases would be to proceed with sentinel node biopsy rather than to repeat the percutaneous biopsy. the sensitivities of the fna and core biopsy procedures were compared using mcnemar's exact test for correlated proportions. the trends in sensitivities with changing numbers of passes, entries, or needle sizes were assessed with the exact cochran-armitage test. patients ' subjective perception of pain levels was compared using the exact wilcoxon sign test. of the 95 patients in the study cohort, 70 patients (74%) had metastatic adenopathy. this group included 5 patients that were both core and fna positive at percutaneous biopsy but were node negative after chemotherapy, 2 discordant (fna negative, core positive or fna positive, core negative) cases with complete pathologic response to chemotherapy resulting in negative nodes at surgery, and 4 core and fna positive patients without axillary surgical correlation. fna was positive in 55/70 (78.6%) and core was positive in 61/70 (87.1%) (p=0.18 (95% ci 0.0320.166)). 65 of the 70 (92.9%) patients had a positive axillary node by tissue sampling: 51 by both fna and core, 4 only by fna, and 10 only by core biopsy. thus, in this group of 70, there were 14 cases (20%) where fna and core results were discordant (k=0.3 (95% ci 0.03 to 0.58)). the sensitivity for single pass core biopsy was 78.6% (11/14) and for multipass cores was 89.3% (50/56), which was not a statistically significant difference (p=0.37). the sensitivity for the 12 g device was 89.9% (8/9), for 14 g devices was 86.7% (52/60), and for 18 g devices was 1/1; the differences in sensitivity using the 12 g vacuum assisted device versus other devices together were not statistically significant (p>0.99). the sensitivity for 21 g single entry fna was 76.1% (35/46) not different from 25 g single entry fna at 78.6% (11/14) (p=0.85). the sensitivity for single entry fna was 76.7% (46/60) not statistically significantly different from multientry fna at 90% (9/10) (p>0.41) (table 2). the sensitivities of fna and core biopsy were compared in table 2 for numbers of suspicious nodes, for node hilus, cortex, and shape, and also for tumor size. fna sensitivity was inferior to core sensitivity (p=0.04) when the node hilus was visible but improved with hilar absence. both fna and core sensitivities improved with cortical thickness increasing beyond 2 mm, and when 3 or more abnormal appearing nodes were noted. of the 10 fna negative/core positive patients, all but one had positive nodes at surgery; that patient had a complete pathologic response to chemotherapy and 20 negative nodes. one cytology specimen was reported as less than optimal, and 2 mentioned suspicious cells but were not diagnostic for malignancy. of the 4 fna positive/core negative cases, each performed by a different radiologist, all of the core specimens were reported as suboptimal, 2 with scant lymphoid tissue, and two with absent lymphoid tissue. in 2 of the 4 (one scant and one absent lymphoid tissue), only one core was obtained. three of the 4 had positive nodes at surgery, and one patient had negative nodes but had a complete pathologic response to chemotherapy with neoadjuvant related changes in the axilla. of the 5 core and fna negative patients with positive nodes at surgery, the clip was noted to be in a negative node in 2 cases indicating that the wrong node was chosen for sampling. the presence of a clip or evidence of biopsy was commented on in 59% (54/91) axillary surgical reports. there was no difference in bleeding between the 2 procedures, which was minimal for all but one case that was moderate for both fna and core. the mean pain score for fna was 2.0 and for core was 2.4 while the range was from 1 to 8 for fna and from 1 to 10 for core. reported pain levels were similar during fna and core in 63 patients (60%), greater with core in 31 patients (29.5%), and greater with fna in 11 patients (10.5%). the higher pain level was reported significantly more frequently for core than for fna (p<0.01). our results show that although core biopsy had greater sensitivity than fna in detecting metastasis, it did not approach statistical significance, probably primarily due to the small number of patients. these results are in agreement with the meta-analysis by houssami et al. who reported a sensitivity for fna (24 studies) of 72.2% and sensitivity for core biopsy (4 studies) of 83.3%, which were not statistically significantly different. the only other prospective comparison study reported a significantly greater sensitivity for core biopsy (88.2%) than for fna (72.5%) but had a small sample size of 51 patients undergoing percutaneous node biopsy with axillary metastasis. our study included several experienced radiologists and allowed a variety of sampling devices to simulate actual clinical practice. while axillary node fna is technically easy to perform for one experienced in image-guided procedures, the radiologist must obtain an aspirate that is both sufficient in the amount of material and at the same time not overly bloody, to enable an optimal interpretation. it is not clear why there were fewer false negative results when multiple fna entries were performed, as the total number of needle excursions likely did not differ greatly. perhaps the chance of obtaining a better sample was increased by using different entry sites or obtaining less blood mixed with cells from the node. the number of slides used, actual number of excursions, and length of procedure were not recorded, which could have affected the results. in some institutions, a pathologist is present when cytologic samples are obtained and can request additional sampling if the specimen is deemed suboptimal; the presence of a pathologist at the time of sampling could have improved the yield from fna. in our institution, immunostains may be used to aid in interpretation when fna alone is performed. our pathologists have extensive experience in cytopathology but in this study there were no immunostains used in the cytologic evaluation; because the pathologists knew that additional tissue would be examined by core biopsy, a factor that may have decreased the sensitivity of fna. as demonstrated by the core negative/fna positive cases, care must be taken to be certain that the core specimen is being taken from the node; the node may be more difficult to visualize due to its depth and is frequently very mobile, making the core biopsy procedure quite challenging. obtaining more than one core sample should insure a greater chance of obtaining an adequate specimen, as shown by the trend (albeit statistically not significant) for increased sensitivity with a greater number of core passes. obtaining one core with a 14 g device was the least sensitive technique in this study and would not be recommended. if it is not certain that adequate cores were obtained, the radiologist should perform fna of the node or take additional cores. in either case, samples should be taken from the node's cortex, where the metastatic cells would lodge, avoiding the hilus where the vascular supply to the node is located. both fna and core biopsy (excluding the insufficient cores of ill-defined nodes) were least sensitive when the node appearance was least abnormal. this can be due to difficulty in choosing the appropriate node for sampling or due to smaller metastatic deposits in the sampled node. in 4 of the 5 cases that were both core and fna negative, the nodes had a normal shape, visible hilus, and cortical thickness of 2.1 to 4 mm. in our study core biopsy had no more morbidity than fna, even with the largest gauge device. use of a biopsy device with a nonthrow option should diminish the chance of vascular injury. however, patients whose suspect node was immediately adjacent to a vessel or very deep and difficult to access were not asked to participate in the study and hence were not subjected to core biopsy. despite the statistically significant difference we observed in the number of patients reporting pain being greater during core than fna, the majority of patients tolerated the pain equally well during both procedures, and we do not believe this should be a factor in deciding which procedure to perform. limitations of our study included its small size, in particular, the small size of subgroups of needle types and number of samples obtained. although there may have been some selection bias due to excluding patients with nodes not suited to core biopsy, the aim of the study was to compare the two methods when both were possible. in all cases, the core biopsy was performed after the fna, with additional lidocaine, which may have minimized the pain associated with core biopsy. fna was always performed first because of concern that core biopsy might cause sufficient bleeding to have to abort the second sampling procedure, but bleeding was not a significant problem. a large fraction of patients underwent neoadjuvant chemotherapy, which was not predicted at the time of initiation of the study. this could have rendered some patients node negative that were initially node positive, but there were only 7 that were node negative after chemotherapy and node negative by both core and fna. if fna and core were both falsely negative, there would be a similar reduction in sensitivity for each method. however, the 5 patients which were both fna and core negative and with positive nodes at surgery did not have neoadjuvant chemotherapy. table 2 shows the sensitivities of core biopsy and fna in patients chosen to receive chemotherapy to be better than in those going directly to surgery, which is likely a reflection of the fact that the neoadjuvant group had more abnormal appearing nodes with thicker cortices. our study began before the acosog z0011 trial that reported in 2010 no statistically significant differences in local or regional recurrence after median follow-up of 6.3 years between those randomized to sentinel node dissection alone versus completion axillary dissection in patients with clinically negative axillae and t1 or t2 invasive breast cancers treated with lumpectomy and radiation and 1 or 2 positive sentinel nodes. as a result of this trial, surgeons indicated that they would perform sentinel node biopsy even after a positive percutaneous node biopsy to determine if only one or two nodes were positive rather than perform axillary dissection in patients with tumors less than 5 cm. consequently some surgeons have requested that radiologists not biopsy suspicious nodes in these patients. however, the z0011 patient population had a low tumor burden with median tumor sizes of 1.6 and 1.8 cm, and a high percentage of micrometastases and solitary positive nodes, suggesting that their outcome may be different than those with positive percutaneous node biopsies. although the z0011 trial results have called into question the value of preoperative tissue sampling of axillary nodes in a selected population, the preoperative detection of metastatic adenopathy at the time of breast cancer diagnosis will continue to be helpful in management of patients with a larger tumor burden and allow many women to have axillary dissection at the time of definitive breast surgery, sparing them an unnecessary sentinel node procedure. for patients who will undergo neoadjuvant chemotherapy, the z0011 results do not apply; percutaneous axillary node sampling will aid in proper staging prior to treatment. the decision to perform core biopsy versus fna should be based on the pathologist's experience in interpreting cytology and the accessibility of the lymph node. fine needle aspiration is a good alternative to core biopsy when a smaller needle is desired due to node location or other patient related factors.

rationale and objectives. to compare the sensitivities of ultrasound guided core biopsy and fine needle aspiration (fna) for detection of axillary lymph node metastases in patients with a current diagnosis of ipsilateral breast cancer. materials and methods. from december 2008 to december 2010, 105 patients with breast cancer and abnormal appearing lymph nodes in the ipsilateral axilla consented to undergo fna of an axillary node immediately followed by core biopsy of the same node, both with ultrasound guidance. experienced pathologists evaluated the aspirate cytology without knowledge of the core histology. cytology and core biopsy results were compared to sentinel node excision or axillary dissection pathology. sensitivities were compared using mcnemar's test. results. of 70 patients with axillary node metastases, fna was positive in 55/70 (78.6%) and core was positive in 61/70 (87.1%) (p=0.18). the fna and core results were discordant in 14/70 (20%) patients. ten cases were fna negative/core positive. four cases were fna positive/core negative. conclusion. core biopsy detected six (8.6%) more cases of metastatic lymphadenopathy than fna but the difference in sensitivities was not statistically significant. core biopsy should be considered if the node is clearly imaged and readily accessible. fna is a good alternative when a smaller needle is desired due to node location or other patient factors. this trial is registered with nct01920139.

PMC3932200

pubmed-638

the ageing process is characterised by progressive loss in cellular homeostasis and a decline in physiological function (balcombe and sinclair, 2001), resulting in an overall decline in fecundity and increased risk of mortality over time. while ageing was historically believed to be an inevitable and intractable process (for discussion see (kirkwood and holliday, 1979; kirkwood, 2005, 2008; munch et al., 2008)), we now know that ageing can be modulated through various environmental, genetic and pharmacological interventions such as dietary restriction (dr) (kenyon, 2005; mair and dillin, 2008; rikke et al., 2010; swindell, 2012; gems and partridge, 2013). dr is defined as the reduction in overall energy intake, or in specific components of the diet, relative to that consumed normally by individuals with free ad libitum (al) access to food. dr has been conclusively shown to extend lifespan in a wide range of taxonomically diverse organisms (mair and dillin, 2008; selman, 2014). in addition, studies have demonstrated that particular genetic pathways modulate ageing through the observation that deletion of single key genes can extend lifespan and healthspan in model organisms. in particular, reduced signalling via the insulin/insulin like growth factor-1 (igf1) signalling (iis) pathway or the target of rapamycin (tor) pathway shows highly conserved effects on lifespan across wide evolutionary distances (kenyon, 2005; piper et al., 2008; broughton and partridge, 2009; indeed, differential expression of several iis and tor genes is associated with longevity in humans (deelen et al., 2013; more recently pharmacological interventions, including rapamycin and metformin, have been shown to extend lifespan in model organisms. this has led to renewed hope in identifying realistic, efficacious and safe pharmacological interventions that will increase the period of our life free from age-related diseases (selman, 2014). excitingly, evidence exists that some molecular processes affected by these different interventions may overlap, thereby suggesting some level of commonality (selman et al., 2009; yan et al., 2012), although other studies suggest that specific interventions may function through distinct mechanisms (masternak et al., 2004; bhattacharya et al., 2012; fok et al., furthermore, recent research has suggested that the effectiveness of each intervention on ageing is highly sensitive to the effects of genetic background. therefore, the use of such interventions in genetically diverse human beings becomes questionable unless we fully understand the precise role that genetic background plays. in this review, we will discuss the evidence that genetic background plays a role in lifespan extension in response to dietary, genetic and pharmacological interventions in mice. the first studies on lifespan extension in animals in response to dr were published almost 100 years ago (osborne et al., 1917; mccay et al., 1935). consequently, dr is the most reproducible and widely used intervention to modulate lifespan (masoro, 1995; speakman and mitchell, 2011; selman, 2014). in addition, dr has been shown to induce significant health benefits, with mouse studies showing dr protects animals against a range of age-related and non-age-related pathologies, including various cancers, glaucoma, glucose intolerance and sarcopaenia (weindruch and walford, 1982; sheldon et al., 1995a, 1995b; mckiernan et al., 2004; hempenstall et al., furthermore, dr decreases pathogenesis and increases survival in a range of mouse models of disease including parkinson's disease (duan and mattson, 1999), alzheimer's disease (halagappa et al., 2007), viral myocarditis (kanda et al., 2007) and pancreatic cancer (lanza-jacoby et al., 2013). the positive effects of dr on health also extend to rhesus monkeys, where it has been shown that the incidence of cancer, cardiovascular disease, type-ii diabetes mellitus and brain atrophy with advancing age are reduced relative to al controls (colman et al., 2009, 2014; similarly, dr has been shown to induce significant beneficial effects on metabolism (weiss et al., 2006) and cardiac function (stein et al., 2012) in humans, and lowers several risk factors associated with coronary heart disease (fontana et al., 2007). despite the large number of studies that have used dr to extend lifespan we still do not understand the precise mechanism(s) through which dr acts to modulate lifespan. in addition, studies using an increasing number of different model systems have implicated that the effects of dr on lifespan extension are not universal and question whether dr is indeed a public modulator of longevity. several studies in organisms ranging from yeast to non-human primates have reported no effect of dr on lifespan (harrison and archer, 1987; kirk, 2001; carey et al., 2002 ;, 2010; rikke et al., 2010; mattison et al., 2012; another caveat has recently emerged from research on mice which indicates that specific genotypes can affect the extent and direction of the lifespan response to dr. as an example, the effect of dr on lifespan in dba/2 mice has ranged from lifespan extension to lifespan shortening (fernandes et al., 1976; turturro et al. clear differences in key variables including terms of husbandry conditions, extent of restriction, diet, gender and age of dr initiation will undoubtedly vary across different studies which may help explain the differences in the findings reported. however, it is also well established that dba/2 mice show distinct differences in a range of metabolic parameters (e.g., insulin sensitivity, glucose tolerance, metabolic rate) under both al and dr feeding when compared to strains such as c57bl/6 mice (funkat et al., 2004; goren et al., 2004; berglund et al., 2008; hempenstall et al., it is also clear that extended lifespan, when observed, in dba/2 mice in response to dr is more moderate than the extension reported in c57bl/6 mice (turturro et al., 1999). recently, lifespan was assayed in heterogeneous ilsxiss recombinant inbred (ri) mice derived from eight distinct mouse strains (liao et al., 2010; in two separate studies undertaken by the universities of texas and colorado, clear lifespan differences existed between distinct ilsxiss lines under 40% dr. the earlier study examined 39 female lines and 41 male lines under dr and reported that only 21% of female and 5% of male lines showed a significant lifespan extension under dr (liao et al., 2010). in this study, a higher number of lines (26% and 27% for males and females, respectively) showed a significant shortening of lifespan under dr. similarly, the second study examined 42 female lines again and reported a significant difference in terms of the response to dr across different lines, with 21% of females showing lifespan extension and 19% showing significant truncation of lifespan under dr (rikke et al., 2010). similarly, the differential effect of dr on lifespan reported in rhesus monkeys by the wisconsin national primate centre (colman et al., 2009, 2014) and the national institute of aging (mattison et al., 2012) may be partly explained by inter-study differences in geographical origin and genetic background of the experimental animals (mattison et al., 2012; partridge, 2012; currently, we do not understand how genetic background may impact on how dr acts to extend life. in ilsxiss mice at least it may be that the optimal dr regime differs between lines and consequently the 40% dr regime employed (liao et al., 2010; rikke et al., 2010) was too extreme to maximise lifespan in all lines (swindell, 2012; selman, 2014). furthermore, it is not known if specific lines are more prone to particular pathologies under both al and dr conditions. however, comparative-type approaches by taking advantage of the differential responses to dr across different mouse strains or ri lines may be a powerful approach to understand precisely how dr acts to slow ageing (selman, 2014). consequently, a larger subset of more genetically diverse rodent models should be studied under dr, along with the greater use of non-model organisms to increase our understanding of how dr acts. significant research efforts over the last couple of decades, initially using invertebrate model organisms, have demonstrated that decreased iis and tor signalling extends lifespan in a highly conserved fashion across model organisms (kenyon, 2005; piper et al., 2008; gems and partridge, 2013). in addition, in mice a large number of studies have also shown that specific disruptions in somatotrophic function also extends lifespan significantly relative to wild type controls (bartke, 2011). furthermore, it is evident that long-lived genetically mutant mice tend to display a much greater proportion of their life free from age-related pathologies (selman and withers, 2011). as genetic interventions modulating lifespan is a more recent finding compared to studies on dr, it is unsurprising that few studies, so far, have investigated how genetic background can impact on the life-extending effects of specific mutations. the most widely used genetically modified mice in ageing research are those which harbour mutations that result in growth hormone (gh) deficiency or gh resistance (for review see bartke, 2011; bartke et al., 2013); ames mice (prop1), snell mice (pit1) including growth hormone receptor knockout (ghrko) and the little mouse (ghrhr). in addition to altered somatotrophic function, these animals appear to have secondary suppression of iis (bartke, 2011; bartke et al., 2013). these models show reproducible lifespan extension across different studies and appear to be protected against a range of age-related pathologies (brown-borg et al., 1996; flurkey et al., 2001; kinney et al., 2001 ;, 2002; coschigano et al., 2003; ikeno et al., the impact of genetic background on lifespan has been studied in snell mice, where it has been shown that the lifespan extension in these animals is consistent across different genetic backgrounds (flurkey et al., 2001; flurkey et al., however, it should be noted that the additive effects on lifespan with dr reported on a mixed genetic background (bartke et al., 2001) were lost with a greater penetrance of the c57bl/6j background (garcia et al., 2008). in addition, ames mice backcrossed on to a c57bl/6 or 129s1/svlmj background led to a significant increase in perinatal mortality (nasonkin et al., 2004). in 2003, two separate studies were the first to demonstrate that reduced iis extended lifespan was also conserved in mammals. these studies reported that the loss of the insulin receptor specifically in white adipose tissue (bluher et al., 2003) or haploinsufficiency in the gene encoding the insulin like growth factor 1-receptor (igf1r) (holzenberger et al., 2003 (2003) showed that female igf1r mice maintained on the 129sv background were 33% longer lived than wild type mice, although the effect in male igf1r mice was non-significant (16%). in addition, they showed that female but not male mice were resistant to the effects of the oxidant stressor paraquat. this study has since been criticised due to the short lifespan of the control animals and the small sample size used (liang et al., 2003). lifespan and end-of-life pathology were examined under housing conditions optimised to maximise the lifespan of the control mice (bokov et al., 2011). in addition, the igf1r mice were re-derived on the c57bl/6 background for at least 10 generations before lifespan was assayed. this second study reported a more modest increase in female lifespan (5%; significant using the log-rank test) relative to controls and a slight, but non-significant, reduction in male lifespan. while genetic background and the targeting event used may help explain some of the differences reported between the two studies, the authors in the second study (bokov et al., 2011) tend to discount this as significant overlap in terms of the metabolic and oxidative stress resistant phenotype which was seen between each study. (2011) also examined lifespan in igf1r female mice on a f1 hybrid c57bl/6 129sv background and showed no effect of haploinsufficiency on lifespan. consequently, the authors of the second study (bokov et al., 2011) proposed that sub-optimal housing conditions may have led to increased stress exposure of all mice in the earlier study (holzenberger et al., 2003). this was hypothesized to lead to a survival advantage of the stress resistant igf1r females over wild type females, which was not enjoyed by igf1r males. while evidence suggests dr may not have the ubiquitous effect on lifespan as originally proposed, animals under dr tend to retain a longer period of life free from ill health, including the onset and impact of age-related pathologies such as type-2 diabetes, cardiovascular disease and cancer. consequently, much current research aims to identify drugs which mimic the effects of dr, i.e., extend vitality in old age. several compounds have been identified, including rapamycin, metformin and resveratrol, which appear to have significant potential in the development of efficacious and safe dr mimetics (selman, 2014). however, the precise mechanism through which these interventions act is still unclear and research from murine studies has demonstrated that genotype appears to be important in how particular dr mimetics impact on an individual's phenotype. the tor pathway appears to be a highly conserved lifespan determinant (kapahi et al. 2010; lamming et al., 2012; wu et al., 2013), which plays a key role in growth and metabolism (bjedov and partridge, 2011) by responding to a range of stimuli including various growth factors, nutrients and energy status (fig. 1). tor kinase, the central component of the tor pathway, forms two functionally different complexes: tor complex 1 (torc1), which plays a central role in regulating cellular processes associated with growth and differentiation, and torc2 which has a regulatory role in the insulin signalling cascade (lamming and sabatini, 2011; lamming et al., 2012, 2013; selman and partridge, 2012). rapamycin, a macrolide compound, has been shown to be a highly potent inhibitor of mtorc1, and more recently also of mtorc2 (lamming and sabatini, 2011; lamming et al., 2012, 2013a, 2013b; selman and partridge, 2012). in model organisms,, 2009; anisimov et al., 2010b; bjedov et al., 2010; neff et al., 2013) and in mice can attenuate some, but not all, ageing-related phenotypes (wilkinson et al., 2012; however, mouse studies using rapamycin to modulate lifespan have shown that its effects on metabolism appear to be highly sensitive to genetic background. as an example, rapamycin treatment induces overt insulin resistance in c57bl/6 mice (lamming et al., 2012) but insulin resistance was not reported in young or old het3 mice (lamming et al., 2013a). in addition, rapamycin appears to have temporal effects on metabolism in mice, leading to hyperinsulinaemia, insulin resistance and glucose tolerance after 2 weeks of rapamycin treatment but hypoinsulinaemia and insulin sensitivity after 20 weeks of treatment (fang et al., 2013). recent findings have shown that rapamycin induces sex-dependent effects on lifespan (miller et al., 2014) and that there appears to be less overlap with dr, in terms of metabolism and transcription, than was originally believed (fok et al., 2014; miller et al., 2014). similarly, the effect of the biguanide metformin on lifespan and health in mice appears to be, at least partly, dependent on genotype, but also dependent on sex and age at which metformin treatment is initiated (anisimov et al. it is clear that the dramatic rise in the proportion of elderly individuals making up our population is going to have significant ramifications. ageing is associated with a decrease in the quality of life linked to an increase in the risk of developing a range of pathologies, including various dementias, type-2 diabetes, osteoporosis, many cancers and cardiovascular disease. consequently understanding the fundamental processes that drive ageing and increase the susceptibility to develop disease is undoubtedly one of the greatest current challenges in biomedical research. to add to this challenge, it is becoming increasingly prevalent in the literature that genetic heterogeneity is likely to play a critical role in the response to interventions which modulate lifespan and healthspan. another potential confounding issue is the focussed use of model organisms in such studies, for example, the small number of mouse strains used in ageing research has been highlighted as a potential limitation to the identification of longevity-associated genes (yuan et al., 2013). future research efforts should perhaps complement model organism studies with those examining ageing in non-model organisms (selman et al., 2012), for example, employing comparative-type approaches to study ageing in fast-ageing and slow-ageing species (austad, 2010a, 2010b). additionally, we should also take greater advantage of the inherent differences in longevity and pathology at death seen across different mouse strains (storer, 1966) to further understand how specific interventions, including dr, genetic inactivation of iis/mtor and rapamycin treatment, act to modulate lifespan and health in the background of increasing genetic heterogeneity.

we are currently in the midst of a revolution in ageing research, with several dietary, genetic and pharmacological interventions now known to modulate ageing in model organisms. excitingly, these interventions also appear to have beneficial effects on late-life health. for example, dietary restriction (dr) has been shown to slow the incidence of age-associated cardiovascular disease, metabolic disease, cancer and brain ageing in non-human primates and has been shown to improve a range of health indices in humans. while the idea that dr's ability to extend lifespan is often thought of as being universal, studies in a range of organisms, including yeast, mice and monkeys, suggest that this may not actually be the case. the precise reasons underlying these differential effects of dr on lifespan are currently unclear, but genetic background may be an important factor in how an individual responds to dr. similarly, recent findings also suggest that the responsiveness of mice to specific genetic or pharmacological interventions that modulate ageing may again be influenced by genetic background. consequently, while there is a clear driver to develop interventions to improve late-life health and vitality, understanding precisely how these act in response to particular genotypes is critical if we are to translate these findings to humans. we will consider of the role of genetic background in the efficacy of various lifespan interventions and discuss potential routes of utilising genetic heterogeneity to further understand how particular interventions modulate lifespan and healthspan.

PMC4257991

pubmed-639

adenomyosis is a common gynecological condition that is characterized by ingrowths of the endometrial cells into the myometrium. adenomyosis and leiomyoma are benign conditions that are often responsible for uterine enlargement, menorrhagia, anemia, and infertility. adenomyosis or internal endometriosis may occur as a result of increased overgrowth of the endometrium with invasion of the underlying myometrium or the displacement of the endometrium during pregnancy, delivery, endometrial curettage, cesarean section, myomectomy, or metroplasty. because of its similarities to leiomyoma, it can be difficult in some cases to accurately diagnose adenomyosis by ultrasound. most gynecologists will consider conservative medical treatment more often for adenomyosis only, while surgical options will be chosen frequently for leiomyoma or combined adenomyosis and leiomyoma, especially in large uteri. the three most common methods of clinical diagnosis of adenomyosis are magnetic resonance imaging (mri), transabdominal ultrasonography (tas), and transvaginal ultrasonography (tvs). there is limited diagnostic capacity with tas, while tvs is a more feasible option. moreover, tvs is also much more cost effective than mri and is generally more readily available in the office to most practicing gynecologists. the aim of this retrospective study was to determine the accuracy, sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) of tvs in the diagnosis of adenomyosis and leiomyoma confirmed by postsurgical histopathological findings. the importance of an accurate noninvasive diagnostic method like ultrasound for adenomyosis, leiomyoma, or combined is an ongoing need in the gynecological community. this was an institutional review board (irb) approved retrospective consecutive case series study. from november 2006 to may 2012, patients underwent surgery for the treatment of adenomyosis, leiomyoma, or combined adenomyosis and leiomyoma. diagnostic criteria of uterine adenomyosis include two of the five sonographic features on tvs: (1) no distinction of the endometrial-myometrial junction; (2) asymmetry of the anterior and posterior myometrium; (3) subendometrial myometrial striations; (4) myometrial cysts and fibrosis; and (5) heterogeneous myometrial echotexture. diagnostic criteria of uterine leiomyoma include two of the five sonographic features on tvs: (1) clear demarcation of the tumor margin; (2) whorly appearance of the tumor content; (3) the presence of blood vessels (by color doppler) surrounding the tumor; (4) irregularities of the uterine surface (subserous and intramural tumors); and (5) irregularities of the endometrial surface (submucous tumors type 1 and 2). symptomatic patients diagnosed with adenomyosis and leiomyoma via tvs underwent myomectomy with excision of the surrounding myometrium which presumably contained adenomyosis. following surgery, a histopathological examination was performed by the hospital pathologists. student's t-tests were used for parametric continuous variables; and the chi-square or fisher's exact test, where suitable, was used for categorical variables. sensitivity, specificity, npv, ppv, positive and negative likelihood ratios, and accuracy were determined for ultrasound findings as they corresponded to the final histopathological diagnosis. from november 2006 to may 2012, 163 patients underwent surgery for the treatment of adenomyosis, leiomyoma, or combined adenomyosis and leiomyoma. one hundred and twenty-three patients were diagnosed with adenomyosis, and 134 patients were diagnosed with leiomyoma. twenty-nine patients were diagnosed with adenomyosis only, 40 patients were diagnosed with leiomyoma only, and 94 patients were diagnosed with combined adenomyosis and leiomyoma, as illustrated in figure 1. patient diagnosis groups one hundred and thirty patients diagnosed with adenomyosis or combined adenomyosis and leiomyoma via tvs underwent hysterectomy. the mean age was 43.7 years with a standard deviation (sd) of 6.7 years and median of 43 years. there was no significant difference in the mean age, weight, height, gravidity, and parity of the patient diagnosis groups. patient characteristics for 163 females who underwent hysterectomy or myomectomy between 2006 and 2012 of the 123 patients (75.46%) who were positively diagnosed with adenomyosis, 93 of the patients (75.61% ppv) diagnoses were confirmed by the histopathological findings. histopathological reports found 23 (57.50% npv) confirmed negative diagnoses and 17 (42.50%) positive findings. the sensitivity of tvs in the diagnosis of adenomyosis was 84.55% (95% ci 76.4-90.7, p<0.0001) and the specificity was 43.40% (95% ci 29.8-57.7, p=0.41). table 2 shows the sensitivities, specificities, ppvs, npvs, and accuracy for each of the initial tvs diagnoses. this could be explained by the difficulty to diagnose adenomyosis in patients with other intrauterine abnormalities or conditions. patients positively diagnosed with adenomyosis via tvs are 1.49 (95% ci 1.16-1.92, p<0.002) times more likely to have the condition. conversely, patients negatively diagnosed with adenomyosis via tvs are 2.81 (95% ci 1.65-4.79, p=0.0002) times less likely to have the condition. number of patients, positive and negative predictive values, sensitivity, specificity, and accuracy of transvaginal ultrasound for initial diagnosis with histopathological correlation (n=107) one hundred and thirty-four patients (82.21%) were diagnosed with leiomyoma and 29 patients (17.79%) were negative for leiomyoma upon the initial tvs. of the 134 patients diagnosed with leiomyoma, 133 (99.25% ppv) had a confirmed diagnosis represented in their postsurgical pathology report and one patient's (0.75%) pathology report did not confirm the diagnosis. of the 29 patients whose tvs diagnosis was negative for leiomyoma, 24 (82.76%) patients histopathological findings were also negative, and five patients (17.24%) had a positive histopathological diagnosis. the corresponding sensitivity and specificity of tvs as a diagnostic test for leiomyoma was 96.38% (95% ci 91.75-98.81) and 96.00% (95% ci 79.65-99.90), respectively. patients with a positive diagnosis of leiomyoma via tvs are 24.09 times (95% ci 35.30-164.45, p=0.001) more likely to have leiomyoma. patients with a negative diagnosis of leiomyoma via tvs are 26.53 times (95% ci 11.16-62.89, p<0.0001) less likely to have leiomyoma. for the statistical calculations of adenomyosis and leiomyoma as coexisting conditions, analyses were performed excluding the patients who were diagnosed with a singular condition. these patients were classified as negative solely for the purpose of calculations regarding the combined condition. of the 94 patients (57.67%) that were positively diagnosed with combined adenomyosis and leiomyoma, 70 (74.47% ppv) had histopathological confirmation of both conditions and 24 patients (25.53%) did not have evidence of both conditions in their post-surgical pathology report. sixty-nine (42.33%) patients had a negative tvs diagnosis for combined adenomyosis and leiomyoma, and 49 (71.01% npv) had histopathological confirmation of the negative diagnosis. however, 20 patients (28.99%) had a positive histopathological diagnosis for both adenomyosis and leiomyoma. the sensitivity and specificity of tvs in the diagnosis of combined adenomyosis and leiomyoma was 77.78 (95% ci 67.79-85.87) and 67.12% (95% ci 55.13-77.67), respectively. patients who obtained a positive tvs diagnosis are 2.37 times (95% ci 1.67-3.34, p<0.0001) more likely to be diagnosed with combined adenomyosis and leiomyoma. alternatively, patients who are negatively diagnosed with adenomyosis and leiomyoma via tvs are 3.02 times (95% ci 1.99-4.59, p<0.0001) less likely to be diagnosed with combined adenomyosis and leiomyoma. adenomyosis is a gynecological disorder that is characterized by the overgrowth of the endometrium into the underlying myometrium. the difficulty in diagnosing adenomyosis clinically is due to the lack of strong positive pathognomonic signs and/or clinical findings. the explanation for this wide range of values, as described by azziz, is the result of differences in the histological criteria for the diagnosis of adenomyosis, the care of which the pathologic specimens are handled, and the number of blocks of sampling specimens taken. various measures of accuracy were calculated for the diagnosis of adenomyosis, leiomyoma, and combined adenomyosis and leiomyoma. table 3 shows a comparison of sensitivity, specificity, ppv, and npv of this study with several previous studies that investigated the diagnosis of adenomyosis. it is important to note that the mean number of patients included in the 13 previous studies in table 3 is 76.8 patients; however, this study utilized 163 patients in the analyses. along with differences in inclusion criteria, ultrasound equipment, and/or the differences in the criteria to diagnose adenomyosis, the larger sample size could contribute to the difference in results. the sensitivity and specificity of tvs for the diagnosis of adenomyosis in this study was 84.55 and 43.40%, respectively. the associated p value for the specificity was not significant, suggesting tvs as a diagnostic tool is sensitive, but not specific in the diagnosis of adenomyosis. the sensitivity in this study is similar to those previously reported in table 3, but the specificity is the lowest of those reported. this could be due to the difficulty in diagnosing adenomyosis in the presence of other uterine abnormalities and conditions, especially uterine leiomyomata that may distort the uterus. patients that were diagnosed with multiple intrauterine conditions were not excluded from this study, since the principle inclusion criteria was patients who underwent hysterectomy or myomectomy and a preoperative tvs. sensitivity, specificity, positive and negative predictive values of tvs for the diagnosis of adenomyosis from previous series compared with this series the sensitivity of tvs for the diagnosis of leiomyoma was 96.38% and the specificity was 96.00%, which is similar to that of a previous study. the sensitivity for the diagnosis of combined adenomyosis and leiomyoma was 77.78%, and the specificity was 67.12%. it was accurate, sensitive, and specific in the diagnosis of leiomyoma and combined adenomyosis and leiomyoma. tvs was both accurate and sensitive in the diagnosis of adenomyosis, but not specific. moreover, tvs is cost effective and readily available in the office to the majority of practicing gynecologists. this study demonstrated that tvs is a valuable noninvasive method that should be utilized in the diagnosis of leiomyoma and combined adenomyosis and leiomyoma.

objective: to evaluate the accuracy, sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) of the diagnosis of adenomyosis, leiomyoma, or combined adenomyosis and leiomyoma by the use of transvaginal ultrasonography (tvs) compared to the histopathological findings.subjects and methods: this is a retrospective study of patients with a preoperative tvs diagnosis of adenomyosis, leiomyoma, or combined. patients diagnosed with adenomyosis or combined adenomyosis and leiomyoma via tvs underwent hysterectomy. symptomatic patients diagnosed with adenomyosis and leiomyoma via tvs underwent myomectomy with excision of the surrounding myometrium which contained possible adenomyosis. following surgery, a histopathological examination was performed by the hospital pathologists. the microscopic diagnosis of the specimen was recorded. results:tvs diagnosis of adenomyosis was sensitive but not specific. tvs was sensitive, specific, and accurate in the diagnosis of leiomyoma and combined adenomyosis and leiomyoma. conclusion:this study demonstrated that tvs is a valuable noninvasive method that should be utilized in the diagnosis of leiomyoma and combined adenomyosis and leiomyoma. tvs is sensitive, but is not specific in the diagnosis of adenomyosis only.

PMC3853875

pubmed-640

the human gastrointestinal mucosa constitutes the largest mucosal surface area in the human body interfacing the external environment. a network of complementary regulatory interactions between different types of immune and nonimmune cells maintains mucosal homeostasis in the gut. these regulatory interactions occur in the midst of a complex mixture of proteins, known as extracellular matrix (ecm) or stroma [1, 2], which together with soluble mediators such as cytokines and growth factors from mesenchymal cells, immune cells, and epithelial cells regulate cell activation and differentiation. mesenchymal cells are actively involved in the inflammatory process in the gut and can perpetuate chronic gut inflammatory conditions like inflammatory bowel disease (ibd) [35]. transforming growth factor- (tgf-) is one of the potential soluble mediators in homeostatic mechanisms in the gut that downregulates effector t-cell responses in the mucosa by reducing proliferation and interferon- (ifn-) production [4, 6]. however, tgf- together with il-6 and il-1 from the inflamed mucosa induces proinflammatory th17 cells, suggesting an innate regulatory function of the gut mucosal microenvironment [4, 7, 8]. research is ongoing to elucidate the role of soluble factors and ecm in immunopathological mechanisms in ibd, but no such studies have so far been performed on microscopic colitis (mc) where the inflammation is subtler. mc comprises two entities, collagenous colitis (cc) and lymphocytic colitis (lc). both conditions are characterized by chronic nonbloody, watery diarrhoea, often associated with abdominal pain and weight loss [911]. the colonic mucosa is macroscopically normal or almost normal and the diagnosis relies on microscopic assessment of mucosal biopsies. cc is presented with increased densities of lymphocytes and a thickened subepithelial collagen band (10 m) adjacent to the basal membrane. the pathophysiological data of cc are still limited, but it is postulated to be at least partially caused by disturbed immune responses to various luminal antigen(s), such as drugs, gluten, or infectious agents, in predisposed individuals. nonsteroidal anti-inflammatory drugs (nsaids), proton pump inhibitors, aspirin, and selective serotonin reuptake inhibitors have been associated with cc. in the majority of patients, however, no precipitating factor is found. dysregulated myofibroblast function has also been implicated for collagen deposition in cc patients [10, 13]. recently, we reported on increased local activation of both cd4 and cd8 t cells in the lamina propria and epithelium of cc patients, demonstrated as increased expression of cd45ro and the proliferation marker ki67, using flow cytometric analysis of freshly isolated lymphocytes from colonic biopsies. in addition, mucosal transcript levels of ifn-, il-12, il-1, il-6, il-17a, il-21, il-22, and il-23 are enhanced in the inflamed mucosa of cc patients compared to normal mucosa, together with elevated protein levels of il-6, il-21, and tnf. although the above findings suggest that the mucosal microenvironment is involved in cc immunopathology, the interplay between these factors and the proinflammatory activity of local mucosal t cells in cc patients has not been elucidated. we therefore investigated the role of soluble factors from the intestinal mucosa of cc patients in the regulation of t cells using a novel in vitro model system with the aim of mimicking the in vivo exposure of newly recruited peripheral blood t cells to the soluble factors in the colonic milieu of inflamed cc and normal mucosa. cc diagnosis was confirmed by clinical symptoms: 3 loose stools/day and/or abdominal pain and a macroscopically normal colonic mucosa with characteristic histopathological findings: increased numbers of lymphocytes in the epithelium and lamina propria with deposition of a 10 m thick subepithelial collagen layer. patients with enteric infection, ischemic colitis, colonic cancer, or a previous history of crohn's disease or ulcerative colitis were excluded. we investigated colonic biopsies from 7 cc patients (female; n=6) and 20 noninflamed controls (female; n=11) without diarrhoeal symptoms, recruited among patients undergoing colonoscopy for examination of gastrointestinal bleeding or of abnormal radiological findings. twelve biopsies from the hepatic flexure from each individual were obtained using standard biopsy forceps, placed in phosphate buffered saline (pbs), and processed within 1 hour. the colonoscopies were performed at the division of gastroenterology, rebro university hospital, sweden, between november 2012 and november 2013. peripheral blood from healthy donors (n=6) was collected in heparin tubes for cd4 lymphocyte isolation as described below. the study was approved by the regional ethical committee of rebro-uppsala county, sweden (i d no. we investigated the influence of two different preparations of the mucosa: one where the epithelium and intraepithelial cells were removed enzymatically, the denuded biopsies (dnb), and one where collagenase was used to digest the lamina propria after the removal of the epithelium. the dnb and the isolated mononuclear cells were cultured overnight, and the latter were termed lamina propria mononuclear cells (lpmcs). the cell populations are intact in the dnb fraction, whereas the lamina propria mononuclear cells (lpmcs) fraction is composed of a free leukocyte population as well as tissue, collagen, and cell debris. twelve biopsies were thoroughly washed with pbs and incubated with prewarmed hank's balanced salt solution (hbss) (sigma aldrich, st. louis, mo, usa) containing 1 mm edta, 20 mm hepes, and 5% heat inactivated fetal bovine serum (fbs) at 37c, with constant stirring 4 times (15 min), to remove the epithelial layer. six denuded biopsies were kept in serum-free rpmi-1640 containing 20 mm hepes, 100 g/ml streptomycin, 10 g/ml gentamycin, and 100 u/ml penicillin (hereafter referred to as culture medium) on ice until further use, and the remaining six biopsies were further digested with collagenase type viii and dnase i type iv (sigma aldrich) for 11.5 hrs to digest the collagen. isolated lamina propria mononuclear cells were washed twice in pbs and resuspended in culture medium. dnbs and lpmcs were cultured in culture medium overnight at 37c under 5% co2-95% air, to generate conditioned medium (cm). cm from the dnb and lpmc fractions from cc patients and noninflamed controls, respectively, were pooled. endotoxin levels were quantified using the pierce lal chromogenic endotoxin quantitation kit (pierce, rockford, il, usa) according to the manufacturer's protocol. the maximum endotoxin level for dnb-cm and lpmc-cm was 4 eu/ml and 4.2 eu/ml, respectively. total protein concentrations were determined using the bio-rad dc protein assay kit (bio-rad, hercules, ca, usa). dnb-cm was used at the total protein concentrations of 250 and 62.5 g/ml, whereas lpmc-cm was used at the total protein concentrations 125 and 62.5 g/ml, the highest possible concentrations of the respective conditioned media. cm from the intestinal mucosa of noninflamed controls is referred to as ctrl-dnb-cm and ctrl-lpmc-cm, whereas cm derived from cc patients is referred to as cc-dnb-cm and cc-lpmc-cm. cd4 peripheral blood lymphocytes were isolated from healthy donors using a human cd4 t-cell enrichment cocktail kit (stemcell technologies, grenoble, france) according to the manufacturer's protocol. the purity of cd4 t cells was 9095% as determined by flow cytometric analysis (epics altra, beckman coulter, fullerton, ca, usa). purified cd4 pbls were cultured in 96-well flat bottom assay plates (sarstedt, newton, nc, usa) precoated with 50 l of 5 g/ml anti-cd3 (ucht1, bd biosciences, san diego, ca, usa) for 2 hrs at 37c, followed by two washes with pbs. 1 10 cd4 t cells were added to the washed wells together with 1 g/ml soluble anti-cd28 (bd biosciences) and were incubated in culture medium with the addition of 2 mm l-glutamine and 5% ab serum, with or without dnb-cm or lpmc-cm from noninflamed controls or cc patients in a total volume of 200 l. as controls, ctrl/cc-dnb-cm and ctrl/cc-lpmc-cm were cultured without cd4 t cells, and cd4 t cells alone were incubated without anti-cd3/anti-cd28. the assay was performed in duplicate wells for cytokine analysis and triplicate wells for the proliferation assay. the cells were cultured at 37c under 5% co2-95% air for three days; thereafter, supernatants were harvested and stored at 80c until determination of cytokine content, and t-cell proliferation was measured using the celltiter 96 aqueous one solution cell proliferation assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium, promega, madison, wi, usa). forty microliters of aqueous one solution reagent was added into each well and incubated at 37c for 4 hrs in a humidified, 5% co2 incubator followed by recording of the absorbance at 490 nm. the pooled conditioned medium from inflamed cc mucosa and controls as well as supernatants from peripheral cd4 t cells were analyzed for il-1, il-4, il-6, il-10, il-17a, ifn-, and tnf using the xmap technology developed by luminex (austin, tx, usa), using two milliplex map kits (cat. number hcyp2mag-62k), according to the manufacturer's instructions (millipore, ma, usa). the assays were performed in duplicate and the levels of different cytokines were expressed as pg/ml, according to a standard curve with known amounts of each analyte (millipore). tgf- levels were determined by elisa, according to the manufacturer's instructions (bd biosciences, san diego, ca, usa). cytokine amounts were calculated as follows: cytokine amounts released by peripheral t cells incubated with cm minus cytokine amounts in cm alone. for each blood donor, the cytokine amounts released by cd4 t cells incubated with cm from cc patients were compared with the cytokine amounts released by cd4 t cells incubated with cm from controls. as the data obtained were not normally distributed, wilcoxon's signed rank nonparametrical test was used for statistical comparison between groups. to mimic in vitro the exposure of peripheral t lymphocytes that have newly arrived into the colonic mucosa and to determine whether the local intestinal milieu affects t-cell activation and differentiation, we investigated t-cell cytokine production by -cd3 plus -cd28 stimulated cd4 peripheral blood t cells in the presence of soluble factors from the intestinal mucosa. there was significantly increased production of the proinflammatory cytokines ifn-, il-17a, il-6, and il-1 in the presence of cm generated by culture of denuded biopsies (dnb-cm) from the colon of collagenous colitis patients, compared to dnb-cm from noninflamed controls (figure 1). this was evident with the lower protein concentration of cm tested for il-17a, il-6, and il-1 and with the higher concentration of cm tested for ifn- production. we also noted a significantly increased production of the anti-inflammatory cytokines il-4 and il-10 in the presence of dnb-cm from cc patients compared to noninflamed controls, with the lower protein concentration of cm (figure 1). in contrast, no significant differences were noted for tgf- production by peripheral cd4 t cells in the presence of dnb-cm from cc patients compared to noninflamed controls (data not shown). in general, lpmc-cm had less impact on peripheral cd4 t-cell activation and differentiation. a trend towards increased production of il-17a and il-10 (both p=0.06) was noted in the presence of lpmc-cm from cc patients compared to noninflamed controls in the lower protein concentration (figure 2). we next investigated the ability of soluble factors from the colonic mucosa to inhibit t-cell proliferation, as this has previously been demonstrated in vitro [4, 16, 17]. a tendency towards reduced proliferation inhibition of peripheral cd4 t cells was noted in the presence of cm from culture of denuded biopsies (dnb-cm) (p=0.06) from inflamed cc patients compared to noninflamed controls (figure 3). this was evident with both total protein concentrations of dnb-cm tested for peripheral t-cell proliferation. in contrast, no differences in proliferation inhibition were observed in the presence of lpmc-cm from noninflamed controls compared to cc patients (figure 3). as t-cell differentiation and function are regulated by different cytokines, we next analysed eight cytokines in pooled conditioned medium from dnb and lpmc fractions derived from inflamed cc mucosa compared to controls. we found more than twofold and eightfold increased levels of il-6 and il-1, respectively, in dnb-cm from cc patients compared to controls, whereas no alterations were found in the levels of ifn-, il-17a, tnf, il-4, and il-10 (table 1) or tgf- (data not shown). similar trends of increased levels of il-6 and il-1 were noted in lpmc-cm from cc patients compared to controls, though it was investigated in pooled cm from only two cc patients (data not shown). we here report on a novel in vitro model for analysis of the impact of the soluble factors from the colonic mucosa of cc patients on peripheral t lymphocyte activation and differentiation. we found that despite the subtle inflammation in the mucosa of collagenous colitis patients, not visible by the naked eye upon colonoscopy, soluble factors in the mucosa are sufficient to significantly enhance the production of ifn-, il-17a, il-6, il-1, il-4, and il-10 by peripheral cd4 t cells exposed to them in vitro. this is the first study to investigate the role of soluble factors from the intestinal mucosa of cc patients in the regulation of t cells, where this novel system reflects the impact of in vivo exposure of newly recruited peripheral blood t cells to soluble factors in the colonic milieu of inflamed mucosa from cc patients compared to normal mucosa. cm from denuded biopsies from inflamed cc mucosa induced increased production of both pro- and anti-inflammatory cytokines by peripheral t cells. as microscopic colitis is subtler compared to ulcerative colitis and crohn's disease, this may suggest that the colonic microenvironment in cc promotes production of anti-inflammatory cytokines to counterbalance inflammatory responses. a study on the effects of stroma conditioned medium from crohn's patients mucosa on cytokine production by t cells demonstrated increased ifn- and il-17 production but provided no data on the effect of anti-inflammatory cytokine production. no differences were noted in tgf- production by peripheral cd4 t cells in the presence of cm from cc patients compared to controls. whereas tgf- in the normal mucosa likely suppresses t-cell function, the significantly increased amounts of il-6 and il-1 in the inflamed cc mucosa instead likely promote differentiation of proinflammatory th17 cells [7, 18] producing large amounts of il-17a. the colonic milieu from cc patients might also promote differentiation of peripheral cd4 t cells into il-17/ifn- double producing th17/th1 cells that have been suggested to mediate gut inflammatory processes [1921], corroborating our findings of enhanced levels of both il-17a and ifn-. we found a trend towards reduced inhibition of t-cell proliferation by soluble factors from denuded biopsies from the colonic mucosa of cc patients compared to controls. older studies have demonstrated that the mucosal microenvironment reduces the proliferative responses of lamina propria lymphocytes to antigen receptor stimulation [17, 22] but they are still active in their helper and cytolytic functions [22, 23]. the present study together with the study by huff et al. indicates that these effects are at least partly imprinted in the t lymphocytes by the local milieu, rather than an intrinsic characteristic. in contrast to cm from denuded biopsies, lamina propria mononuclear cell- (lpmc-) cm from cc patients did not affect t-cell proliferation compared to lpmc-cm from controls. these different effects of the two types of cm are unclear and further experiments need to be performed to elucidate the differences in the composition of the cms. despite our observed enhanced production of il-10 by peripheral t cells in the presence of cm from cc patients, known to inhibit both t-cell proliferation and cytokine production, we found neither reduced proliferation of peripheral t cells nor production of proinflammatory cytokines. this indicates that other immunoregulatory molecules drive synthesis of these proinflammatory cytokines in collagenous colitis. the production of cytokines did not increase with higher total protein concentrations in the cm. one explanation could be the presence of inhibiting and/or toxic factors in the cm limiting the t-cell responses. in addition, various molecules have different optimal concentrations for their function and high concentrations can limit their activity. to further elucidate this and explain the differences observed on cd4 t-cell differentiation we want to investigate a larger panel of cytokines and compare the protein profile between cm from cc patients and that from noninflamed controls and between dnb and lpmc fractions by proteomics. in conclusion, we have set up an in vitro model for analysis of the impact of the soluble factors from the colonic mucosa of cc patients on peripheral t lymphocyte activation and cytokine production. despite the subtle inflammation in cc, our data demonstrate significant alterations in cytokine production by peripheral cd4 t cells in the presence of mucosa-derived soluble factors from cc patients compared to controls. one of our future goals is to test this in vitro model on differentiation of cd8 t cells, as we have previously reported on their increased numbers in the colonic mucosa of cc patients. we also want to evaluate its use in evaluating the effect of drugs, including those in present use, on the colonic mucosal milieu and the lymphocytes there within, thereby facilitating the decision on optimum molecules as well as doses required for suppression of t-cell inflammatory responses.

soluble factors from intestinal mucosal cells contribute to immune homeostasis in the gut. we have established an in vitro model to investigate the regulatory role of soluble factors from inflamed intestinal mucosa of collagenous colitis (cc) patients in the differentiation of t cells. peripheral blood cd4+t cells from healthy donors were polyclonally activated in the presence of conditioned medium (cm) generated from denuded biopsies (dnb) or isolated lamina propria mononuclear cells (lpmcs) from mucosal biopsies from cc patients compared to noninflamed controls, to determine proliferation and secretion of cytokines involved in t-cell differentiation. compared to controls, we observed significantly increased production of the proinflammatory cytokines ifn-, il-17a, il-6, and il-1 and the anti-inflammatory cytokines il-4 and il-10 in the presence of cc-dnb-cm. the most pronounced effect of cc-lpmc-cm on peripheral cd4+t cells was a trend towards increased production of il-17a and il-10. a trend towards reduced inhibition of t-cell proliferation was noted in the presence of cc-dnb-cm. in conclusion, our in vitro model reveals implications of soluble factors from cc colonic mucosa on peripheral t cells, enhancing their production of both pro- and anti-inflammatory cytokines.

PMC4190132

pubmed-641

patients with schizophrenia present major impairment in social functioning, independent living and work status, compared to healthy subjects [1, 2]. besides the well-established positive and negative symptoms, recently it has been largely recognized that a generalized cognitive deficit is at the core of schizophrenia, and it significantly affects patient's social functioning [2-5]. cognitive impairment in schizophrenia ranges from sensory and perceptual dysfunctions to higher order cognitive dysfunctions like working and episodic memory, attention, problem solving, processing speed [6-8]. the course of cognitive impairment in schizophrenia is characterized by deterioration close to and during the first psychotic episode and a relative stability afterwards. nevertheless, patients with schizophrenia tend to present lower cognitive performance than the general population even in the premorbid phase [9, 10]. antipsychotic medications have shown modest positive effects on different cognitive domains, without a preferential effect over a specific function [11-14]. better treatment approaches to cognition could be achieved with a greater understanding on the pathogenesis of cognitive impairment in schizophrenia, which remains poorly understood. evidence from studies with other neuropsychiatric disorders indicates that immuno-inflammatory processes may play a central role in cognitive deficits. for instance, it has been shown that inflammation may be an important neuropathological mechanism underlying cognitive decline and dementia in elderly population. executive dysfunction was associated to inflammatory parameters in bipolar patients, with inhibitory control being positively correlated to tnf- levels. levels of inflammatory markers have also been linked to cognitive function in major depressive disorder. for instance, high il-6 levels were associated to low performance in immediate and delayed verbal recall tests in recurrent depressed women. in schizophrenia, there is considerable evidence of changes in the immune system. previous studies have shown an imbalance between type-1 and type-2 immune responses with a predominant type-2 response. however, a recent meta-analysis showed dominant pro-inflammatory changes in schizophrenia, but not type-2 or type-1 predominant immune response. after controlling for antipsychotic use as a confounding factor, only il-1ra and il-6 levels were elevated in schizophrenia. another meta-analysis, that considered clinical status and antipsychotic effects, did not find an increase in type-2 cytokines either. instead, the authors suggested that some cytokines are state markers of acute exacerbations (il-1, il-6 and tgf-), while others may be trait markers (il-12, ifn-, tnf-, sil-2r). th1 derived cytokines il-12 and ifn- were elevated only in acute relapses and first episode psychotic patients, as well as macrophage derived cytokines il-6, tnf- and il-1. prenatal and perinatal infections could disrupt fetal neurodevelopmental processes, leading to brain long-lasting changes and increasing the risk of psychotic disturbances in early adulthood. prenatal and perinatal infections could also act on the priming of immune activation in this early period of life. in this scenario, altered levels of il-1 and il-6 could influence the release of hormones by the hypothalamic-pituitary-adrenal axis, contributing to changes in monoamine neurotransmission. therefore, infection and inflammation may trigger pathological mechanisms, resulting in proneness to psychosis and, possibly, cognitive dysfunction. given the relevance of the cognitive impairment and the immune changes in schizophrenia as well as the putative role of the immune system in cognitive dysfunction, it turns relevant to investigate the association between cognitive and immune variables in schizophrenia. therefore, the aim of the present study was to systematically review all the papers published concerning schizophrenia, cognition and immunity. we focused on studies that assessed cognitive and immune variables directly on the patients. by excluding studies that used other populations (e.g. alzheimer, bipolar disorder) or basic research we tried to reduce complexity, homogenize results and to emphasize on studies with direct clinical implications nevertheless, articles not included in our selection, but whose content is relevant to our results, were included in the discussion. a search was conducted in the medline database, up to april 2013, comprising studies written in english, with the following terms in the abstract and/or title: schizophrenia or psychosis or psychotic and inflamm*or immun*or cytokine or il-*or tnf-*or kynureni*or kyna, and cognit*or attention or memory or executive function. only original papers were included. seventy five papers were identified using the selected terms. after a critical analysis of the abstracts, the papers excluded were either too basic, concerning chemical or biological in vitro research or animal studies with murine models with little contribution for clinical practice; or too broad, revising immunological aspects in other neuro-psychiatric disorders or concerning different symptomatological domains. papers that only inferred that immune changes could affect cognition in schizophrenia, without actual tests, were also excluded to homogenize the sample and focus on clinical and practical aspects. papers that assessed cognition and immune and/or inflammatory markers in patients with schizophrenia are displayed on table 1. clinical trials that added immuno-modulatory drugs to antipsychotic regimen are shown in table 2. four studies addressed associations between immune/inflammatory markers and cognitive functions in schizophrenia or first-episode psychosis. elevated serum c-reactive protein (crp) levels were associated with the severity of cognitive impairment, but not with positive or negative symptoms. patients with crp levels above 5 mg/l (high level group) had a lower score on the repeatable battery for the assessment of neuropsychological status (rbans), which is based on scales for immediate and delayed memory, visuo-constructional, language and attention. a recent study of the same group showed that the effects of elevated cpr levels and herpes simplex virus type 1 (hsv-1) seropositivity on cognitive impairment as assessed by rbans in patients with schizophrenia were additive and statistically independent. the largest cognitive impairment was found in the group with both factors, i.e. high levels of crp and hsv-1 antibodies, reinforcing the hypothesis that inflammation and infection may play a role in cognition. another study correlated levels of either the chemokine monocyte chemoattractant protein 1 (mcp-1/ccl-2) or oxidative stress markers (nitrites and glutathione) with performance on cognitive tasks in a group of first-episode psychosis patients. mcp-1 levels were negatively correlated to learning and memory performance, while oxidative stress markers were associated with poorer executive functioning. a recent chinese study found a positive association between interleukin 18 (il-18) levels and rbans visuospatial and constructional indexes in first episode and drug nave psychotic patients. three studies yielded data about the effect of anti-inflammatory or immunomodulatory drugs as add-on therapy to the antipsychotic regimen on cognition of patients with schizophrenia. using celecoxib (cyclooxygenase type 2 (cox-2) inhibitor) 400mg/day or placebo as add-on to risperidone, mller et al. patients using celecoxib had a greater reduction of total score in the positive and negative symptoms scale (panss) when compared to the placebo group. specific effects on cognition were evidenced after re-evaluation of data that showed that the effect on the panss cognition factor (items difficulty in abstract thinking and conceptual disorganization) was the most pronounced. the effect seems to be stronger in cases with more recent onset and shorter disease duration. another trial added aspirin (1000 mg) to antipsychotic treatment in a group of 70 patients with schizophrenia for a three month period. it was argued that aspirin has a cardioprotective profile and that its unselective inhibition of cox-1 and cox-2 enzymes would allow a wider range of action. statistically significant effects on the total score and positive subscale of panss were observed. interferon- and the effect was larger in patients with a lower type-1/type-2 cytokine balance (defined by the median interferon-/ interleukin-4 ratio), suggesting that the reduction of positive symptoms was larger in patients with an immune profile tending to the type-2 response. although aspirin seemed to improve psychiatric symptoms, cognition was not affected. a double-blind, randomized, placebo-controlled study with minocycline, an antibiotic which also exerts immuno-modulatory activity, as augmentation to antipsychotics found that it may alleviate negative symptoms and improve cognitive functioning in early-phase schizophrenia. patients using minocycline showed improvement in executive functions (working memory, cognitive shifting and planning) in comparison to the placebo group measured by the cambridge neurospychological automated battery (cantab). although the research linking cognition and inflammation in schizophrenia is still scarce, initial results are promising. mcp-1 and il-18 levels, as well as crp levels and hsv-1 seropositivity, were linked to cognition in schizophrenia. interventional studies that added immunomodulatory drugs to antipsychotic regimen also yielded interesting results, opening new possibilities to improve cognition and other psychiatric symptoms in schizophrenia. elevated high sensitivity crp may be also a marker of memory and visuospatial impairment in the elderly, being associated with dementia. crp is known to be elevated in those with risk factors common to stroke and dementia, such as diabetes, obesity and smoking. recent studies suggested that assessment of low grade inflammation by high sensitivity crp can be related to cerebral microstructural disintegration, affecting frontal lobe pathways and leading to executive dysfunction. frontal dysfunction is a common feature in schizophrenia and these findings may help explain how inflammation may undermine cognitive functions. crp assessment could also help identify groups of patients with schizophrenia that would benefit from immunomodulatory drugs, improving cognitive function or even psychiatric symptoms. as well as inflammatory markers such as crp, it appears that immune activators such as viruses could be related to cognition in schizophrenia. the concept of early-life programming of adult disease postulates that specific environmental factors acting during sensitive prenatal or early postnatal developmental periods can induce persistent changes in physiological, emotional and behavioral functions throughout life. these factors could prime not only the immune system and its responses in adult life, but cause neurodevelopmental changes and increase proneness to psychosis. prenatal maternal immune activation, inflammation, viral infections, as well as psychological stress and malnutrition appear to affect offspring development, increasing the risk of psychotic disorders later on, impairing sensorimotor gating, information processing, cognition, social function and leading to subcortical hyperdopaminergia [22, 36]. prenatal influenza infections are associated with altered neuronal migration in cortical and hippocampal neurons, which are essential for broad functioning of many cognitive domains. different kinds of viruses are known to alter the monoaminergic balance within the brain after experimental infection, particularly serotonin and noradrenaline-mediated systems. hsv-1 lifelong cycles in the brain may cause neuronal damage and dysfunction and may also be associated with longitudinal gray matter loss in the posterior cingulate gyrus and decline in executive functioning among subjects with schizophrenia. significant association between the neurocognitive summary score, verbal memory, vigilance and processing speed; and antibodies to hsv-1 was described in the catie trial (clinical antipsychotic trial of intervention effectiveness). these findings may help explain the association of hsv-1 and cognition in schizophrenia described in the results by dickerson [25, 26] and also possible underlying pathological changes. we could also hypothesize that microglial activation may occur in early neurodevelopment in these cases and be linked to first episode psychosis as well, converging previous neuro-structural abnormalities and monoaminergic imbalance to behavioral and cognitive impairment. mcp-1 is a chemokine that recruits monocytes, dendritic and t cells to the inflammation site. it was hypothesized that in psychotic patients the systemic oxidative-inflammatory status may influence cognitive performance through inflammatory mediators in cerebral vasculature or increase in blood-brain permeability. mcp-1 has already been linked with cognitive deficits associated with hiv dementia and alzheimer s disease [41, 42]. therefore, inflammatory processes initiated by mcp-1, like microglia activation and leucocyte migration, appear to be associated to cognition in schizophrenia as shown by the negative association between mcp-1 levels and learning and memory (p=0.009). this is in line with the microglia hypothesis of schizophrenia, which postulates that pro-inflammatory cytokines and free radicals produced by an activated microglia may decrease neurogenesis, result in white matter abnormalities and favor neurodegeneration, contributing to the neuropathology of the disorder. animal experiments have shown that il-6 can increase dopaminergic neurotransmission in the hippocampus, and il-2 also increases serotonin and noradrenaline-mediated neurotransmission. although no differences were found between il-18 levels in first episode patients with schizophrenia and healthy controls, there was a positive association between il-18 levels and rbans visuospatial/constructional cognitive index. these findings seem paradoxal since there are studies associating il-18 levels to worse cognitive function in alzheimer s disease and multiple sclerosis. due to the association between viral infections and schizophrenia, it is proposed that il-18 release after viral infection would lead to microglial activation with interferon gamma (ifn-) release in the brain parenchyma and viral clearance. another explanation is that the visuospatial index may be more resilient to be impaired by il-18 than other cognitive functions measured by rbans. it is still uncertain how each immune marker affects neurodevelopment, and therefore also influences cognition. damage control, for example, following viral infection in pre or perinatal period, avoiding or minimizing structural abnormalities associated to schizophrenia and cognitive impairment. most clinical trials that used anti-inflammatory or immunomodulatory drugs in addition to antipsychotics in order to verify cognitive effects yielded positive results. it is an antibiotic with anti-inflammatory, anti-oxidative and anti-apoptotic properties. its neuroprotective properties may arise from astrocytic and microglial caspase 1 inhibition, as well as nitric oxide synthase inhibition, which also affects the glutamatergic system. the dopaminergic system may also be affected, since minocycline attenuates dopamine elevation following nmda agonist administration. preclinical inflammation models also showed impact on inflammatory markers, such as tnf-, il-1, pge2 and cox-2 [32, 46, 47]. pro-inflammatory cytokines and free radicals produced by microglia can contribute to neuron degeneration. there have been reports of inhibitory effects of typical and atypical antipsychotics in inflammatory and oxidative stress mediated by the microglia; factors which have been recently associated with reduced neurogenesis and white matter abnormalities. since patients were described as having early-phase schizophrenia, authors suggest that intervention with immunomodulatory drugs may display better results in initial stages of the disease and improve clinical course. celecoxib also seems to improve cognition, although the observed improvement derived from panss cognitive factors and not neurocognitive tests per se. celecoxib is a selective anti-inflammatory drug that inhibits cox-2, but not cox-1. contrasting with its isoform cox-1, cox-2 is induced by stimulation in most tissues, but it is constitutively expressed in the central nervous system structures such as frontal cortex, amygdala and hippocampus which are critically involved in cognitive functioning [38, 48]. cox-2 activity can be stimulated by tissue damage and also by glutamate excitation, showing its high sensitivity to neuronal stress. cytokines such as il-2, il-6 and il-10 activate cox-2, and result in inflammatory response also in the central nervous system. hence, cox-2 inhibition seems to balance type 1/ type 2 immune responses by inhibition of pge2 and stimulation of type 1 immune response, and also by inhibition of kyna (kynurenic acid) production. kyna is an antagonist at the glycine site of the n-methyl-d-aspartic acid receptor (nmdar) and at the 7 nicotinic acetylcholine receptor (7nachr), both of which implicated in the cognitive impairment of schizophrenia [51, 52]. increased immune function, primarily in the blood, would raise most kynurenine metabolites and in result, elevate also kyna levels in the brain. since the kynurenine pathway of tryptophan metabolism is induced by immunological activation and stress, it can mediate the effects of environmental factors in cognition and behavior. therefore, it may be a promising metabolic pathway for developing new pharmacological interventions to treat and prevent cognitive dysfunction in schizophrenia and other neuro-psychiatric disorders. the beneficial effects of cox-2 inhibition may also be due to alteration in glutamate neurotransmission, with inhibition of the nmda receptors and activation of kainate receptors. mller et al. also proposed that cox-2 inhibitors may have a role in learning and memory by affecting long-term potentiation and long-term depression, as well as attenuating cholinergic dysfunction. therefore, many mechanisms could explain the positive effects of cox-2 inhibitors on cognition in schizophrenia. as argued by mller and schwarz when evaluating the effect of anti-inflammatory drugs on cognition in schizophrenia therapeutic studies have shown beneficial effects of anti-inflammatory drugs mostly in early stages, with little effect in late stages. mller et al. stated that patients with a shorter duration of disease improved more in the aforementioned clinical trial. these findings could reflect that an early anti-inflammatory therapy could prevent neuronal damage and structural changes caused by chronic inflammation in schizophrenic patients over many years of disease evolution. aspirin (acetylsalicylic acid) is a widely used anti-inflammatory agent that inhibits inflammatory cyclo-oxygenase pathways and suppresses prostaglandins and tromboxane.. found that the group of patients with lower th1/th2 balance benefited more from the use of aspirin than the others. this finding is in line with other studies and the hypothesis of an imbalance between pro and anti-inflammatory forces. cognition was not affected, but authors argued that it was a stable domain over time in these patients, and that a longer time could be necessary to observe changes. laan et al. also hypothesized that aspirin may act by antagonizing nmda dysfunction and that it exerts its effects by interacting with antipsychotics. as only a few studies that addressed simultaneously cognition and immunological changes in schizophrenia are available to date, the results must be interpreted cautiously. the exclusion of research or animal models may have narrowed the discussion, excluding other hypothetical mechanisms linking immune changes and cognition in schizophrenia that have not been transposed into clinical studies yet. regarding immunomodulatory drugs, many parameters are still undefined, such as duration, dose and time of initiation of a possible immunomodulatory drug. moreover, it appears that positive effects are more evident in the initial stages of schizophrenia. one of the clinical trials revised did not use standard neuropsychological assessment battery in schizophrenia (panss cognition factor). it is advised a careful selection of the neuropsychological battery used to obtain more reliable results in future trials, in order to compare drug efficacy and cognitive improvement. schizophrenia is a heterogeneous syndrome and immune changes may be another neuropathogenic mechanism among many already described. cognition is one of the main compromised domains in schizophrenia and accounts for a great part of the patient s functioning and quality of life. clinical trials have shown improvement on cognitive parameters and other symptoms with the addition of immunomodulatory drugs. it seems very likely, though, that inflammation and immune dysfunction affect neurodevelopment, increase risk of psychotic illnesses and continue to affect cognitive processes throughout life. possible underlying mechanisms include monoaminergic imbalance, microglial activation, structural abnormalities in white matter, frontal lobe, hippocampus; as well as changes in the kynurenine pathway of the tryptophan metabolism affecting glutamatergic and cholinergic neurotransmission. future clinical trials using either anti-inflammatory drugs, nicotinic or glutamatergic agents or manipulation of brain kyna levels should address cognition more directly. more basic and clinical studies are needed in order to further enlighten the mechanisms underlying cognitive deficits and pursue new cognitive-enhancing drugs to the treatment of the disorder.

objective: recent evidence has associated immune and inflammatory changes to cognitive performance in many diseases, including schizophrenia. since this is a new research field where concepts are not yet solid and new questions and hypothesis are still arising, the present study aimed at summarizing the available clinical data associating schizophrenia, cognition and inflammation/immune function. methods:a systematic review of the literature was made by searching the following terms in medline: schizophrenia or psychosis or psychotic and inflamm*or immun*or cytokine or il-*or tnf-*or kynureni*or kyna, and cognit*or attention or memory or executive function. results:seventy five papers were identified using the selected terms, and seven papers were included in the review. papers excluded focused mainly on basic research or other neuropsychiatric disorders. conclusions:recent findings link inflammatory markers to cognition in schizophrenia, suggesting that inflammation is associated with worst cognitive performance. microglial activation, monoaminergic imbalance, brain abnormalities and the kynurenine pathway are possible mechanisms underlying cognitive impairment in schizophrenia. clinical trials with addition of immunomodulatory drugs have shown promising results, opening new windows to tackle cognition in schizophrenia.

PMC4023457

pubmed-642

nowadays cardiac surgeons must face the problem of severely impaired left ventricle function almost on a daily basis. heart transplantation, due to the limited donor pool, can be offered only to a small number of patients suffering from cardiac failure [1, 2]. furthermore, the idea of long-term mechanical support as a destination therapy is still evolving. hence, efforts to optimize the results of routine cardiac surgery have to be undertaken. in a properly selected group of patients coronary surgery alone or combined with mitral surgery can improve both quality of life and long-term survival. with borderline ventricular function prior to surgery this may result in failed weaning from cardiopulmonary bypass. in isolated coronary surgery beating heart strategy, either on or off pump seems to be a safer option. its use however is hardly possible when mitral valve repair or replacement needs to be done. intra-aortic balloon pump (iabp) is a gold standard here, but some patients may require more sophisticated mechanical assistance [3, 4]. the idea of short-term, perioperative mechanical support, preferably with a low anticoagulation regime, looks very convenient in this respect. impella microaxial pumps (abiomed europe gmbh, aachen, germany) seem to meet these criteria. they use the idea of pulling the blood from the left ventricle into the aorta, which is accomplished by means of a rotating impeller positioned in the aorta with the tip of the device and inlet area positioned in the left ventricle. this results in reduction of both left ventricle end diastolic volume (lvedv) and left ventricle end diastolic pressure (lvedp). the systemic pressure and flow are increased. the less stretched left ventricle, with better subendocardial perfusion, has more time to recover. on the other hand, the impella pump can be applied either by cardiologists (impella 2.5/impella cp) in a cath lab setting or by cardiac surgeons (impella 5.0/impella ld) in an operating theatre with either peripheral vascular or central direct access. offering flow up to 5 l/min, the anticoagulation management requires only heparin infusion to keep activated clotting time (act) not shorter than 160 s. thanks to the automated controller with an intuitive, user friendly interface, operating the device can be easily mastered by doctors and nursing staff. a 64-year-old woman suffering from ischemic dilated cardiomyopathy associated with mitral insufficiency was referred for combined treatment consisting of resynchronisation therapy followed by simultaneous coronary and mitral surgery. initial echocardiography revealed impaired global contractility (ejection fraction [ef] 10-15%), dilated left ventricle (left ventricle end diastolic diameter [lvedd] 7.8 cm), significant ventricular asynchrony (50 ms) and moderate mitral regurgitation (vena contracta [vc] 6 mm) due to both restriction of the posterior leaflet and dilation of the mitral annulus (5.5 cm). there were no significant abnormalities regarding the right ventricle, pulmonary valve and tricuspid valve. clinically the patient presented with end stage cardiac failure including resting dyspnea (nyha iv). the patient underwent uneventful implantation of an icd crd (maximo ii), which resulted in improved exercise tolerance. this was consistent with post-procedure echocardiography showing no significant ventricular asynchrony (4 ms), slightly improved contractility (ef 20%) and unchanged moderate mitral regurgitation (vc 6 mm). therefore the patient was referred for combined cardiac surgery including mitral repair and coronary grafting. because of poor lv function and the complexity of the procedure, it was decided to apply short-term mechanical support during and after surgery. the impella ld microaxial pump was chosen due to the simplicity of implantation and the low anticoagulation regime. also, the impella ld does not require a hybrid theater with fluoroscopic guidance. antegrade cold blood cardioplegic solution was administered through the aortic root. the posterior descending artery (pda) and left anterior descending artery (lad) were grafted with the long saphenous vein and left internal thoracic artery respectively. the left atrium was entered in a routine manner after dissecting sondergaard's plane. on inspection the echocardiographic findings were confirmed. the only surgical issue was severe atherosclerosis of the ascending aorta affecting both anastomosis of the vein graft and implantation of the impella device. before the implantation a 10 mm dacron graft was anastomosed to the ascending aorta using a side biting clamp (fig. the impella catheter was inserted through the graft and then, under transesophageal echocardiographic (tee) guidance, was forwarded through the ascending aorta and the aortic valve into the left ventricle (figs. 2 and 3). immediately after confirming the position the impeller rotation was initialized. meticulous attention was paid to maintain proper left ventricle volume preloading in order to avoid pump malfunction. after securing hemostasis the chest was closed, letting the driveline out above the suprasternal notch and through the upper end of the sternotomy wound. the patient was transferred to intensive treatment unit (itu) in a stable condition. there she was kept sedated and ventilated electively. therapeutic activated clotting time (act) the position of the pump was checked daily and in case of any suspicion of malfunction. adequate flow was maintained most of the time, with one episode of stopping of the machine, which was easily managed with fluid infusion. the inotropic support was reduced gradually without affecting hemodynamic stability. on the 2 postoperative day the further postoperative course was mainly uneventful, although prolonged mechanical ventilation was required (8 days). the first follow-up visit two months after discharge confirmed good exercise tolerance and satisfactory echocardiographic findings (trace mitral regurgitation, lvedd 7.6 cm, ef 25%). impella device inserted through the tubing graft anastomosed to the ascending aorta impella device in the ascending aorta, approaching the aortic valve impella device finally positioned in the left ventricle doppler scan showing the flow from the left atrium to the left ventricle (blue) and within the device from the left ventricle to the aorta (red) patients with impaired left ventricle function require a particular surgical strategy. obviously, it is crucial to perform a routine procedure such as coronary grafting or valve surgery with meticulous surgical technique and good timing. therefore, these patients should be operated on by senior surgeons. however, even in experienced hands, such cases may prove quite tricky. sometimes, even perfect coronary anastomoses and the shortest ischemic time can not prevent failed weaning from cardiopulmonary bypass. the possibility of perioperative mechanical support, exceeding the intra-aortic balloon pump, is quite tempting. one option is extracorporeal membrane oxygenation (ecmo), which can support the circulation for up to a few weeks. this requires, however, both arterial and venous cannulation, an aggressive anticoagulation regime and specially trained perfusionists and nursing staff. extracorporeal membrane oxygenation is also associated with a long list of potential complications including infection, bleeding, thromboembolic events, hemolysis and finally multiorgan failure (mof). there is no doubt that ecmo is a well-established and recognized life-saving procedure; however, the question may be raised whether some patients could benefit from an easier technology used in a planned manner., there is quite a wide range of indications for their use, two in cardiology: as a rescue treatment in cardiogenic shock caused mainly by acute coronary syndrome and, electively, as a support in high-risk percutaneous interventions including pci and vt ablation [7, 8]. because of endovascular insertion and therefore limited device size (12 fr) the generated flow can not be higher than 2.5 l/min, which usually is enough in the above situations. impella pumps may be applied in post-cardiotomy shock when an intra-aortic balloon is not enough to succeed in weaning from cardiopulmonary bypass. cardiac surgeons may also use them more electively as a perioperative support in patients with significantly impaired left ventricular function. the surgical insertion can be performed either peripherally (impella 5.0) or directly through the ascending aorta (impella ld). in peripheral access the femoral or axillary artery is exposed, then the device is inserted using a tubing graft and forwarded to the left ventricle under fluoroscopic guidance. the hybrid theatre setting is required, but on the other hand it is possible to perform less invasive, thoracoscopic surgery. in direct access, after sternotomy, the implantation is quite easy under tee guidance. whatever the access, the surgical technique allows one to insert a bigger device (21 fr), which is able to generate flow up to 5 l/min (!). that means that after the procedure, which is slightly more complex than insertion of an iabp, and having a device which is almost as easy to operate as an iabp, we achieve an effect that is very close to the benefit of left ventricular assist device (lvad). the aforementioned problem of left ventricle venting is solved by definition, with impella being an intraventricular device. it should not stay in for longer than 5 days, which is more than enough to rethink and rearrange a new strategy. (impella, ecmo, lvad, recovery or transplant). when it is applied electively, as in our case, the chances are very high that only the first stage will be necessary. this being the case, the impella pump can be deservedly regarded as a short bridge to recovery.

cardiac surgeons have to face the problem of impaired left ventricle function in patients undergoing routine valve or coronary procedures. the intra-aortic balloon pump is not always effective in preventing cardiac failure. the idea of using a microaxial rotating pump as a short-term perioperative support seems to be a convenient solution. the case of a patient with dilated cardiomyopathy undergoing combined mitral and coronary surgery with elective use of the impella ld pump is presented. various options of applying the impella device are discussed, especially as a bridge to transplant or bridge to recovery.

PMC4520509

pubmed-643

hepatitis c virus (hcv) is a parentally transmitted hepatotropic rna virus that causes chronic hepatitis, which may lead to cirrhosis and hepatocellular carcinoma (hcc). evidence suggests that clearance and control of hcv infection during acute phase is dependent on vigorous and multispecific cd4 and cd8 t lymphocyte responses [2, 3]. on the contrary, the development and maintenance of chronic infection is linked to weak or absence of hcv-specific th1 response and to the presence of th2 cytokines (il-4 and il-10) [4, 5]. so, some studies have implicated il-10 in hcv pathogenesis [4, 6]. regulatory t cell 1 (tr1) was first described in 1996 [7, 8] as a further subtype of cd4 t cells, without specific markers but with cytokine profile distinct from that of th1 or th2 cells. indeed, they were shown as secreting high level of il-10, moderate levels of ifn- and il-5, quite undetectable il-4, and low amounts of transforming growth factor (tgf-). recent studies have suggested that tr1 could be induced against bacterial, viral and parasite antigens in vivo and might prevent infection-induced immunopathology or prolong pathogen persistence by suppressing protective th1 response [9, 10] but has never been directly implicated in hepatitis c viral pathogenesis. the dysfunction of the immune response against hcv could be explained by immunosuppressive mechanisms supported by tr1 via their high production of il-10. the recent identification of combined expression of cd4, cd18 (integrin 2), and cd49b (integrin 2) as specific markers for tr1 cells should facilitate their characterization in vivo. an increased frequency of tregs was recently described in the blood of patients with persistent hcv infection when compared with those who had cleared hcv [12, 13]. it has been proposed that tregs contribute to hcv persistence by suppressing hcv-specific t-cell responses [14, 15]. some studies have shown a correlation between a reduced hcv-specific t-cell response and the secretion of il-10 and tgf- by liver-infiltrating tregs and that tregs may inhibit hcv-specific t-cell activity in a cell-cell contact manner [12, 13]. it has also been shown that treg levels are significantly enhanced in recurrent hepatitis c and that treg type 1 cell (tr1) levels are specifically higher in severe recurrent hepatitis c. moreover, the opportunity of studying three histologically well-defined liver biopsies of an hcv genotype 1b infected patient followed from the chronic infection to the hcc allowed us to investigate the tr1 cells infiltration within these liver biospies. the first biopsy was performed during the chronic phase (date not specified in the file) and appeared normal without any liver injury, the second one was performed during the cirrhotic phase (march 1990), and the third one was performed during hcc (1998). thus, we examined the intrahepatic t cell response in terms of cytokines, cytokine receptors, and adhesion molecules in a patient under ifn-/ribavirin treatment and who remained persistently infected>18 years. the hcv infected patient was followed up in the hepatogastroenterology department of the necker university hospital. information concerning the patient included age alat>three times the normal and negative anti-hav, hbv, and hev igm, and positive serology for hcv (third generation microparticle eia (abbott axsym, abbott park)) confirmed by riba strip immunoblot assay (chiron corporation, emeryville, ca). genotyping of hcv was done using a multiplex pcr method with genotype-specific primers, and the concentration of hcv rna was determined by rt-pcr with the cobas amplicor hcv monitor test (roche diagnostics, branchburg, nj). the patient presented no human immunodeficiency virus (hiv) coinfection, immunosuppressive therapy, chronic liver disease due to hepatitis b virus, autoimmune hepatitis, or primary biliary cirrhosis and other causes of chronic liver disease. three liver biopsies were performed for one patient who underwent several biopsies for a longitudinal study. the three biopsies were performed as part of the routine medical followup and were obtained by the standard menghini procedure (needle diameter: 1.6 mm) with a biopsy sample length of approximately 1 cm. based on histologic fibrosis (f) and necroinflammatory (ni) scores, liver biopsies were divided into biopsies with small hepatitis and without liver lesion (f<or=1/6; n<or=1/18), biopsies with fibrosis grade and liver lesions corresponding to histologically proved cirrhosis (f=6/6), and biopsies with hcc histologically proved by anatomopathologic expertise. the study was approved by the institut de biologie de lille (cnrs) institutional review boards and informed consent was obtained in writing from the donor. extraction of total rna from frozen liver biopsy samples was performed using trizol reagent (gibco brl, invitrogen, scotland, uk) as described by the manufacturer. homogenized samples were incubated for 5 minutes at room temperature and resuspended in 60 l of chloroform (prolabo, merck eurolab, france). total rna was then immunoprecipitated with 100 l of isopropanol (acros organics, usa). the rna pellet was finally washed twice with 75% ethanol (carlo erba reagent, france) and dissolved in dnase, rnase-free distilled water (gibco brl, invitrogen, scotland, uk). before reverse transcription, we performed dnase treatment using the kit message clean (genhunter corp., france). cdna was synthetized from total rna at a concentration of 100 ng/l using random hexamers and superscript reverse transcriptase (gibco brl, invitrogen, scotland, uk). the quantification of transcripts from liver samples was performed by real-time quantitative rt-pcr using the light cycler system (roche diagnostics, mannheim, germany). the pcr mixture contained the following: taq polymerase, 1x of light-cycler dna master sybr green i (roche diagnostics, meylan, france), 3 mm of mgcl2, 0.5 mol/l of each primer, and 1 l of cdna preparation (patient cdna samples) in a total volume of 20 l. thirty-two samples were run in parallel by performing an initial denaturation at 95c for 8 minutes; the pcr reactions were cycled 35 to 40 times as follows: 15 seconds at 95c, 7 seconds at the appropriate annealing temperature (table 1), and 18 to 64 seconds at 72c according to the length of the target sequence, followed by annealing (40 s at 58c). the melting curve analysis was carried out immediately after amplification, following the manufacturer's instructions. amplification of liver cdna was successfully repeated three times with cdna from the same extraction. all primers were designed for real-time pcr use and were purchased from mwg-biotech (germany) (cf. samples are quantified using relative standard curves for each amplification reaction, and results were normalized to the internal controls -actin and g3pdh. after each light cycler run, agarose gel electrophoresis with tbe (tris-borate-edta) 1.5% agarose (sigma-aldrich, germany) gel, followed by dna staining with ethidium bromide, was performed to have an independent validation check of the presence of an amplicon. in order to control the length of the amplicon generated, a 100 bp dna ladder (gibco brl, invitrogen, scotland, uk) was used. as the lymphotropism of hepatitis c virus (hcv) has already been ascertained, and in the light of the fact that the immune defense system is an organized network composed of functionally immune cells, this study was carried out to verify the possible involvement of tr1 cells in the progression of hcv-related chronic hepatitis. in this study, we first analyzed the quantitative expression of the different intrahepatic cell populations by real-time pcr using light cycler system (figure 1), and we performed after each rt-pcr an agarose gel electrophoresis to have an independent validation check (figure 2). we observed a significant increase of cd19 expression in cirrhotic and hcc liver biopsies in comparison with the first biopsy without liver lesions. this observation confirmed data of the literature showing disturbances of b lymphocyte activation and function associated with hcv chronic infection [17, 18]. the cd8 but not cd8 gene expression was also increased in the cirrhotic and hcc biopsies. this observation was in correlation with recent data evidencing by a similar approach of real-time quantitative assays a significant increase and infiltration of cd8 during chronic hcv infection. but the major observation of our work was the significant increase of cd4 expression in the course of time and proportionally with the severity of the fibrosis. however, we did not observe a variation of ifn- or il-2 expression, suggesting that the cd4 marker detected in cirrhosis and hcc liver biopsies was probably not associated with the th1 protective phenotype. due to the absence of il-4 expression, the cd4 cells with th2 phenotype were also excluded in accordance with the work of bergamini et al.. a further subtype of cd4 t cells, with immunosuppressive function and cytokine profiles distinct from either th1 or th2 t cells, termed regulatory t cells has been described. they include tr1 cells which secrete high level of il-10 and low to moderate levels of tgf-, th3 cells which primarily secrete tgf-, and cd4cd25 cells, which inhibit immune response through cell-cell contact (9). although the level of il-10 and tgf- produced in vitro may not be significantly higher than that produced by classical th2 cells, what is important is that tr1 cells make these cytokines in the absence of significant levels of il-4. we confirmed within cirrhotic and hcc biopsies a significant increase of expression of il-10, tgf-1 and their respective receptors il-10r, il-10r, and tgf-r2 whereas we did not detect an increase of il-4. so, the production of il-10 and tgf- in the absence of il-4 argued in favor of the presence of tr1 in the cirrhotic and hcc biopsies. although liver dendritic cells also secrete high levels of il-10, in the present study we could not detect cd11c expression, confirming a tr1 cells origin for il-10 production. in this sense, functional alteration of hcv-specific cd4 t cells or failure to develop a long-lasting t helper response was recently correlated to a loss of ifn- secretion and the presence of a significant antigen-specific il-10 and tgf-, which could contribute to chronic hepatitis c persistence. moreover, tgf- plays a pivotal role in inducing fibrosis and has been proposed as its surrogate marker. indeed, a very interesting study has shown that serum tgf-1 could be used to assess therapeutic outcome and short-term prognosis of hcv-related chronic hepatitis. moreover, results obtained for tr1 specific markers cd49b and cd18 confirmed the tr1 phenotype and showed that tr1 cells increased during the progression of the pathology. despite their low proliferative capacity, cloned tr1 express normal levels of t cell activation markers such as cd25 following tcr-mediated activation. they also expressed ccr5 and t1-st2, an il-1-r-like molecule, markers previously expressed preferentially on th1 and th2 cells, respectively. we clearly confirmed here within cirrhotic and hcc biopsies the expression of cd25, ccr5, and t1-st2 in relation to the presence of tr1 cells. in accordance with this tr1 phenotype, the expression of tgf--r2 was also observed. however, we can not exclude the implication of other regulatory t cell subsets, insofar as we observed a significant increase of cd25, pselectin, and icam i expression, which were known as specific markers for cd4cd25 regulatory t cells. these observations were also conforted by other experiments we performed on liver biopsies of three well-defined cohorts of 45 hcv genotype 1b infected patients, including patients without liver lesions, patients with cirrhosis, and patients with histologically proved hcc that confirmed an increase of tr1 proportional to the aggravation of the pathology. a first implication of regulatory t cells during hcv infection was examined in peripheral blood of patients infected with hcv genotype 1b, showing a secretion of ifn- or il-10 but not il-4 by hcv core-specific cd4 t cell clones. this work demonstrated that helper type 1 and regulatory t cells were induced probably against the same epitopes on the core protein. moreover, sugimoto et al. have suggested for the first time that hcv persistence was associated with a reversible cd4-mediated suppression of hcv-specific cd8 t cells and with higher frequency of cd4cd25 regulatory t cells that could directly suppress hcv-specific type 1 cd8 t cells ex vivo. indeed, it is possible that the induction of regulatory t cells during hcv infection may suppress antiviral th1 and consequently cd8 ctl responses in vivo, and this may explain the persistence of infection and prevalence of cirrhosis. evidence of regulatory t cells installation in the liver of a chronically infected patient with cirrhosis and hepatocellular carcinoma (hcc) suggests a key role for these cells in the aggravation of the liver pathology and could potentially represent a predictive factor of liver damage aggravation. as previously described high serum il-10 levels may be related to a poor response to ifn treatment in patients with chronic hepatitis c. in this sense, it would have been interesting to investigate other markers such as spleen markers or beta2-microglobulin, although it has been described that neither spleen measurements nor serum beta2-microglobulin levels were able to predict therapeutic response to antiviral therapy. in conclusion of our study, the increase of tr1 cells, which suppressed protective th1 responses via their high production of il-10, could explain the failure of the ifn treatment observed for this patient. in a more general way, the implication of tr1 in viral persistence and associated hepatic pathologies could have significant implications for our understanding of the role of t cells in immunity to infectious diseases and for the development of new therapies for the control of immune-mediated disorders.

hepatitis c virus (hcv) is an important causative agent of liver disease, but factors that determine the resolution or progression of infection are poorly understood. in this study, we suggested that existence of immunosuppressive mechanisms, supported by regulatory t cells and especially the regulatory t cell 1 subset (tr1), may explain the impaired immune response during infection and thus the fibrosis aggravation to hepatocellular carcinoma (hcc). using quantitative real-time pcr, we investigated the intra-hepatic presence of tr1 cells in biopsies from a genotype 1b infected patient followed for an 18-year period from cirrhosis to hcc. we described a significant increase of gene expression in particular for the cytokines il-10, tgf-, and their receptors that were perfectly correlated with an increased expression of the tr1 specific markers (combined expression of cd4, cd18, and cd49b). this was strongly marked since the patient evolved in the pathology and could explain the failure of the treatment. in conclusion, evidence of regulatory t cell installation in the liver of chronically infected patient with cirrhosis and hcc suggests for the first time a key role for these cells in the course of hcv infection.

PMC4890904

pubmed-644

mung bean (phaseolus radiatus l.) is a leguminous species grown in different parts of the world, primarily especially in asia including china, india, burma, and thailand. mung bean commonly is a common source of protein in the asian diet or nutrient supplements. mung bean has been reported to possess antioxidant, antidiabetic, anti-inflammatory, antitumor and antimelanocytes, and antiangiotensin i-converting enzyme activities [28]. mung bean contains free phenolic acids, bound phenolic acids, total phenolic, and anthocyanin. correlation analyses between bioactivities and phytochemicals demonstrated that antioxidant bioactivity may be mainly contributed to phenolic compounds, whereas anthocyanins play an important role in the antidiabetic bioactivities. other reports showed that flavonoids including vitexin and isovitexin were the dominant components in mung bean [2, 9] and the content of vitexin was much higher than that of isovitexin in ethanol extracts. it has been reported that mung bean has a strong antioxidant activity and isovitexin and vitexin contribute to most of the 1,1-diphenyl-2-picrylhydrazyl, ferric-reducing antioxidant power or 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonate) radical scavenging ability. various factors including geographical location, climate change, temperature, and illumination time have important effects on the types and contents of plant chemical components, which are related to their bioactivity, functionality, and applications. light quality is one of the most important factors in the regulation of plant growth, morphogenesis, photosynthesis, metabolism, and gene expression [11, 12]. for the photobiological research, ultraviolet-visible spectrum between 200 nm and 800 nm wavelength plays an important role in changes of chemical compounds of the organisms by irradiating them, especially compounds with ultraviolet (uv) absorption property [13, 14]. compared to the ordinary fluorescent light source, the light-emitting diode (led) light sources can provide a single wavelength of light quality with high photoelectric conversion efficiency, fixed wavelength, and low heat. led light source is considered to be a new important light source in the field of plant physiology and plant cultivation. previous studies indicated that the led light sources were used in the research of photomorphogenesis, chlorophyll synthesis, and photosynthesis. recently the studies of this field attract more and more researchers focusing on the work [18, 19]. many studies indicated that there were the practical problems of insufficient light intensity and limited spectral wavelength during the process of plant cultivation in the laboratory [2022]. it is necessary to find effective ways to replace or assist the ordinary fluorescent light source, to improve research method for the plant, and to promote the plant quality. in this study, we used the red leds and ultraviolet-b (uv-b) radiation as additional light sources for the process of plant cultivation in the laboratory to determine the role of the different qualities of light source on growth and photosynthetic characteristics of mung bean. mung bean seeds were obtained from yangling breeding center of national bean engineering research center of china (shaanxi, china). the ordinary fluorescent light source (power 40 w) was purchased from philips inc. the light directly irradiated the seedling of mung bean from am 7:00 to pm 7:00 each day. the uv-b radiation was provided by filter qin brand (baoji lamp factroy, china) 30 w fluorescence sunlamps. they were filtered with 0.13 mm thick cellulose diacetate (transmission down to 290 nm) for uv-b radiation. the dose of uv-b irradiation was 0.861 kj/m per day. the lamps were suspended above the plant at the height of 40 cm perpendicular to the ground. firstly, seeds were sterilized for 10 min by 0.1% hgcl2 and were grown in petri dish (diameter 18 cm) after being washed for 50 min by flowing water. until seeds were germinated, they were transplanted in basin (diameter 25 cm) which was filled with the ratio of peat: vermiculite: perlite for 3: 1: 1. one week after seed germination, the supplementary light treatments carried out seed germination. on the 20th day and 40th day of supplementary light treatments, organisms were sampled, respectively, for various analyses. the morphology including plant height, fresh weight, dry weight, root length, and stem diameter was measured. mung bean seedlings were oven dried at 80c until constant weight and being weighed using electronic scale as biomass (g). the results of stomatal conductance, photosynthesis and water use efficiency were the mean values of the day. the method for the measure of the concentration of chl a and chl b was extracted by acetone and determined following the reported methods. intact leaf samples of seedlings (fresh weight 0.5 g), which were at 5-6 leaves stage of development, were placed in a mortar and followed by the addition of silica of 0.2 g, caco3 of 0.2 g, and 15 ml 80% acetone. after thorough grinding, the samples were filtrated with two layers of filter paper by pump air and fixed to 25 ml with 80% acetone, and then the absorbance at 663 and 645 nm was determined, respectively. chlorophyll concentration was calculated and expressed as mg/g fw. fresh samples of 0.5 g were taken from the epicotyls and extracted in 10 ml acidified methanol (methanol-water-hydrochloric acid, 79: 20: 1, v/v) for uv-b absorbing compounds, according to the procedure of mirecki and teramura. extract absorbance at 300 nm was measured with a spectrophotometer (uv-2100; shimadzu, columbia, md, usa) and the absorbance was arbitrarily used for analysis. the results were expressed as the means standard error (se) of triplicate. the data were subjected to one-way analysis of variance (anova) and the significance of difference between samples means was calculated by duncans ' multiple range test and p values less than 0.05 were considered significant. it was observed that the values of the growth parameters of mung bean seedlings were irradiated for 20th day by red leds and uv-b was not significantly different compared with that of the ordinary fluorescent light (table 2). however, the red leds treatment caused a significant increase (p<0.05) of the values of plant height, fresh weight, dry weight, and root length compared with that of the ordinary fluorescent light for 40th day. with the uv-b radiation for 40th day, an obvious decrease (p<0.05) of plant height was observed, while it induced a marked increase (p<0.05) in fresh weight, dry weight, and stem diameter. the red leds treatment induced a significant increase (p<0.05) in the values of the photosynthetic characteristics for the two durations (table 3). however, a significant decrease (p<0.05) was observed in the values of the photosynthetic characteristics of the uv-b radiation for 40th day. the concentrations of chl a and chl b may affect the values of the photosynthetic characteristics to a certain extent. it was obvious that the red leds treatment induced statistically significant increases (p<0.05) not only in the values of the photosynthetic characteristics (table 3) but also in the concentrations of chl a and chl b (figure 1) for the two durations. compared with the ordinary fluorescent light, the uv-b radiation did not cause significant differences. with the treatment of uv-b radiation, the concentrations of uv-absorbing compounds uv-b radiation-treated seedlings resulted in a notably increase in the concentrations of uv-absorbing compounds for the two durations (p<0.05). however, red leds did not cause significant difference in comparison to the ordinary fluorescent light. leds are a promising irradiation source for plant growth in space for long life, minimal mass, volume, and being a solid state device. the red leds (wavelength 650 nm) were used as a supplementary light source for the greenhouse tomato in 1982, which was reported earlier by japan's mitsubishi corporation. during the process of laboratory cultivation, it has been reported that the ordinary fluorescent light lacked the ultraviolet part of the solar spectrum background, which was essential growth factor to play an important biological role [20, 26]. however, it was difficult to use a mixed-use led light sources during the process of plant cultivation completely. therefore, we used red led and uv-b as supplementary light sources for the ordinary fluorescent light to study the role of these light sources in plant cultivation. our results showed that these light sources were obviously increased for growth and photosynthetic characteristics of mung bean. the red led light source promoted the growth of the mung bean root (table 2), which was useful to absorb the nutrients and water of the soil. chlorophyll concentrated in the chloroplast grana is the main pigment to capture the energy for photosynthesis in green plants. the results showed that the red led light source can significantly increase the concentrations of the chlorophyll (figure 1), which effectively promoted the photosynthesis and water use efficiency (table 3). on the contrary, uv-b radiation induced a notably decrease in the photosynthesis and water use efficiency. it was suggested that the uv-b treatment will reduce the stomatal opening degree of mung bean and then affect the gas exchange in photosynthesis. uv absorption compounds in leaves are an important class of pigments including flavonoid, flavonol, cinnamon, and anthocyanin, which determine the color changes of many plants and are very sensitive to light. they play an important role in protective effect as a class of secondary metabolites [28, 29], which are related to antioxidant, antidiabetic, anti-inflammatory, antitumor and antimelanocytes and antiangiotensin i-converting enzyme activities [28]. the previous studies have reported that vitexin and isovitexin were major flavonoid in the ethanol extract of mung bean and vitexin content was much higher than isovitexin in ethanol extracts from mung bean sprout [3, 9]. however, another report showed that no significant difference in levels of vitexin and isovitexin was observed in mung bean sprout of the same cultivar tested in the experiments. since uv absorption compounds have an absorption peak in uv-b radiation scope, it could be found that the uv-b treatment caused a significant increase in uv absorption compounds (figure 2). compared with the ordinary fluorescent light, red leds did not induce significant differences in uv absorption compounds. red led and uv-b as supplementary light sources have an important effect on plant growth and chemical components.

mung bean has been reported to have antioxidant, antidiabetic, anti-inflammatory, and antitumor activities. various factors have important effects on the types and contents of plant chemical components. in order to study quality of mung bean from different light sources, mung bean seedlings were exposed to red light-emitting diodes (leds) and ultraviolet-b (uv-b). changes in the growth parameters, photosynthetic characteristics, the concentrations of chlorophyll a and chlorophyll b and the content of uv-b absorbing compounds were measured. the results showed that photosynthetic characteristics and chlorophyll a and chlorophyll b concentrations were enhanced by red leds. the concentrations of uv-b absorbing compounds were enhanced by uv-b on the 20th day, while photosynthetic characteristics, plant length, and the concentrations of chlorophyll a and chlorophyll b were reduced by uv-b on the 40th day; at the same time the values of the stem diameter, plant fresh weight, dry weight, and the concentrations of uv-b absorbing compounds were enhanced. it is suggested that red leds promote the elongation of plant root growth and photosynthetic characteristics, while uv-b promotes horizontal growth of stems and the synthesis of uv-b absorbing compounds.

PMC3942199

pubmed-645

sarcoidosis is a multisystemic granulomatous disease of unknown etiology characterized by the presence of non-caseating granuloma consisting of epithelioid cells. heart, spleen, bone marrow and less often eye, skin and salivary glands are common extrapulmonary sites of disease manifestation. the liver has sarcoid nodules on occasions, however these nodules are generally multiple and diffuse. granulomatous lesions in hepatic sarcoidosis are most often located at the catchment area of glisson's capsule. in these patients, portal hypertension is the predominant symptom. the diagnosis of hepatic sarcoidosis might be easy because patients with hepatic sarcoidosis have generally pulmonary lesions or hilar lymph node swelling at the same time. we present a case of hepatic sarcoidosis with a solitary giant nodule in the liver, but with radiological findings typical of hilar cholangiocarcinoma. a 51-year-old female was referred to our institution with abnormal laboratory data of liver function and the biliary system. the laboratory data on admission showed slight elevation of alkaline phosphatase (alp) and gamma-glutamyl transpeptidase (-gtp), but a normal level of tumor marker (table 1). her physical condition upon admission was generally good; there was no anemia or jaundice, and the liver was not palpable. abdominal ultrasonography showed a low echoic lesion of 37 mm diameter with an unclear margin at segment 4. the intrahepatic bile ducts (ihbds) of the left lobe were dilated to 6 mm with obstruction by this tumor. a swelling round lymph node of 11 mm diameter was revealed in the hilar region of the liver. abdominal dynamic computed tomography (ct) revealed a 4.0 cm tumor of low density at segment 4, which partially invaded segments 1, 5, and 8., the tumor was detected as a low-density lesion with an irregular shape (fig. the contrast enhancement of the tumor was slightly intense at the margin of the tumor during the equilibrium phase (fig. 1c) and became more intense during the delayed phase, but the margin became unclear (fig. magnetic resonance cholangiopancreatography showed remarkable dilatations of the ihbds of segment 2 and 3 with an extreme stenosis of the left main hepatic duct. endoscopic retrograde cholangiopancreatography also revealed conspicuous stenosis at the junction of the left hepatic duct and the common hepatic duct as well as stenosis and rigidity of the anterior and posterior branches of the right hepatic duct. moreover, the junction of the hepatic duct draining the right caudate lobe had stenosis (fig. when deep cannulation was performed beyond the stenotic hepatic duct, dilated ihbds of segment 2, 3 and 4 were detected. 2b). extended left lobectomy with caudate lobectomy and bile duct resection was performed with a diagnosis of hilar cholangiocarcinoma located predominantly at the left main hepatic duct. some daughter nodules were found in the left lobe, which were considered to be intrahepatic metastasis. pathological findings confirmed the presence of non-caseating granuloma with multinucleated giant cells. there were multiple nodules with severe fibrosis in the resected specimen. a lot of small non-caseating granulomas consisting of epithelioid cells were found in the dissected lymph nodes. the case reported here is important because of its morphologically distinct characteristics from radiological findings for hepatic sarcoidosis. sarcoidosis is a systemic disease that primarily affects the lungs and lymphoid tissues of the body. in japan, sarcoidosis occurs mainly in the age group of 50- to 60-year-olds with a predilection for females. hepatic involvement of sarcoidosis was described in 11.5% of 736 patients in the access study. hepatic granulomas due to sarcoidosis were recognized most commonly as multiple and small nodules with less than 1.0 cm diameter. liver involvement ranges from asymptomatic incidental granulomas to portal hypertension from granulomas in the portal triad, usually with relative preserved liver function. angiotensin-converting enzyme (ace) level may be elevated in 70% of patients with sarcoidosis and is elevated in almost all patients with hepatic sarcoidosis. in general, in 2040% of patients with sarcoidosis, alp and -gtp levels are elevated, but in those with hepatic sarcoidosis the levels are usually much higher. ultrasonography findings include parenchymal echogenicity, coarsening of the liver parenchyma with or without discrete nodules, and focal calcifications as well as contour irregularity. on ct, hepatic granulomas are visualized as multiple, discrete, low-attenuating, non-contrast-enhancing nodules of variable size. as they increase in size, they tend to become confluent and have to be differentiated from various infectious and neoplasmatic conditions. magnetic resonance imaging also shows multiple diffuse, densely packed, uniform nodular foci with normal signal intensity with t1- and hypodense with t2-weighted sequence. on dynamic ct imaging, our case was compatible with hepatic sarcoidosis due to a poor enhancement effect on early arterial phase and a slight enhancement from the margin on delayed phase, even though this intrahepatic nodule was huge and solitary. in most patients with sarcoidosis, the course of hepatic involvement is asymptomatic. in a few patients, chronic intrahepatic cholestasis or portal hypertension portal hypertension could be due to obstruction of portal flow because of granulomas in the portal area, with fibrosis and hyalinization of the portal triad causing presinusoidal block. another possible mechanism was advocated in which there might be arteriovenous shunts that increase portal blood flow. asymptomatic patients with mild elevation in liver function enzymes should not be treated but should be followed with serial blood work. it has been reported that the majority of untreated patients who are clinically asymptomatic have spontaneous improvement in their liver function. indeed, perry and vuitch reported that the mortality rate of patients with sarcoidosis was 1 of 28 at autopsy and 1 of 18 with portal hypertension. for patients with refractory liver dysfunction, chronic progressive cholestatic disease and portal hypertension that develop more severe liver disease, corticosteroids should be administered. however, corticosteroids can not prevent disease progression, including development of portal hypertension in patients with early or established cirrhosis. for such cases, when medications are no longer efficacious, liver transplantation is the only option. there are other causes of hepatic granulomas, including bacterial infections such as tuberculosis, brucellosis, viral infections and parasite infections, primary liver disease such as primary biliary cirrhosis, autoimmune cholangiopathy and many other systemic illnesses. as mentioned above, there is a wide differential for granulomas in the liver. in western countries, a large portion is attributed to primary biliary cirrhosis (up to 55%) and sarcoidosis (1530%), but infective cases, like tuberculosis and brucellosis, should always be kept in mind during initial investigation [12, 13]. evaluating hepatic granulomas the majority of cases in which granulomas appear only in the liver are caused by liver disease rather than multisystemic disease. in that case, the differential diagnosis may be frequently difficult compared to other tumor-forming diseases, such as neoplasm in the liver. the diagnostic approach to hepatic sarcoidosis must be made on the histological evidence of non-caseating granulomas in a liver biopsy. in this report, we describe a rare experience with hepatic sarcoidosis, which we presented with clinical and radiological findings characteristic of hilar cholangiocarcinoma. our experience suggests that the diagnosis of patients with tumors in the liver with atypical radiological findings, who have a case history of sarcoidosis, need to be established by comprehensive and cautious consideration. there should be no hesitation to perform liver biopsy if there is difficulty in making a definitive diagnosis. despite utilizing several detailed and extensive diagnostic modalities, we may occasionally encounter a patient with a solitary tumor in the liver for whom it is extremely difficult to make a differential diagnosis from the malignant neoplasm, much like our experience. in this case, a surgical treatment including liver resection should be seriously considered, because a patient with hepatic sarcoidosis occasionally had to undergo liver transplantation in the end after a short period of observation without any treatment due to the difficulty of diagnosis like our case.

sarcoidosis is a multisystemic granulomatous disease of unknown etiology. hepatic involvement was reported in about 11% of patients with sarcoidosis. however, cases of sarcoidosis in which the granuloma is solitary and limited in the liver are very rare. a 51-year-old woman with tumors in the liver underwent extended left lobectomy with caudate lobectomy and bile duct resection. the tumor was located between segment 4 and the hilar region. some daughter nodules were found in the left lobe, which were regarded as intrahepatic metastasis. our case displayed clinical and radiologically distinct findings, which are very similar to those of hilar cholangiocarcinoma restricted to the liver. this report demonstrates that sarcoidosis can show solitary hepatic involvement in the absence of thoracic lymphadenopathy. in such a case, it is difficult to distinguish the diagnosis from other malignant neoplasms. in conclusion, the diagnosis of hepatic sarcoidosis has to be made through prudent and comprehensive investigations that include a full clinical history of sarcoidosis in other organs. despite utilizing several detailed diagnostic modalities, the definitive diagnosis of cases of solitary sarcoidosis may remain difficult. in these cases, surgical treatment including liver resection should be considered in order to avoid missing a suitable opportunity for treatment.

PMC3088740

pubmed-646

over many years, the interference of tracheostomy on swallowing has been studied; tracheostomy may increase the risk of aspiration, as it interferes directly in the pharyngeal phase of swallowing.1 the type of cervical incision, surgical technique applied, type of cannula used, and inflation of the cuff may, separately or together, contribute to a further fixing of the larynx, increasing aspiration. the loss of sensitivity of the larynx caused by lack of air traffic contributes to the reduction of coughing reflex, facilitating secretion and food aspiration.2 however, frequency of tracheostomy in traumatic brain injury (tbi) management when patients are receiving mechanical ventilation contrasts with the lack of evidence as to when tracheostomy should be removed.3 tracheostomy is indicated when there is upper airway obstruction, when the patient requires prolonged mechanical ventilation and/or has difficulty in weaning, when there are excessive tracheobronchial secretions, and when the patient needs continuous airway protection against aspiration.4 approximately 10% of critically ill patients undergo tracheostomy for respiratory support and facilitation of air passage into airways, providing better quality of life for patients and reducing hospital expenses.5 6 dysphagia presents a close relationship with tracheostomy, not only because this procedure is indicated in patients with swallowing problems and tracheal aspiration but because the tracheostomy itself may cause aspiration.1 although it is a common procedure, the presence of a tracheostomy tube is not devoid of complications. the cause of aspiration is unknown, but probably results from these multiple factors.7 five mechanisms are responsible for the aggravation of aspiration after tracheostomy: decreased laryngeal elevation, esophageal obstruction, cleaning of larynx with airflow, laryngeal desensitization, and uncoordinated laryngeal closure. this airflow deviation and the interruption of normal vocal function have great implications throughout respiratory, phonatory, and swallowing systems. in some cases, even post-tracheostomy scar, fixing the trachea to the skin, without any other disease can cause dysphagia.8 information is needed on the treatment of dysphagic and tracheostomized individuals, including information on intervention methods, which must be evaluated objectively. patients were separated into two groups: the first group received a multidisciplinary approach and the other group was retrospectively assessed through medical charts, answering to a clinical protocol. the group that underwent a multidisciplinary treatment showed a significant decrease in mean length of cannulation time to 28 days, compared with those without the approach who had an average of 33 days.9 the early speech therapy intervention in a hospital aims a fast identification of dysphagia, which is an important factor for decreasing risks involving aspiration-related pneumonia and poor nutrition and preventing complications arising from the same clinics; this may reduce hospital stay, leading the patient to an early independence and an improvement of life quality.10 speech therapy within an interdisciplinary team is responsible for therapeutic management of dysphagic patient to optimize airway protection, aiming at reintroduction of oral intake in a fast and secure way, as well as helping with the decannulation process.11 12 following specific protocols in tracheostomy prescription is common, but there is no protocol description for the decannulation process. most hospitals have an interdisciplinary team that is responsible for this process, and clinician, nurse, physiotherapist, and speech pathologist interaction reduces time of tracheostomy use, accelerates weaning, and increases safety for the patient while reducing risk of failure and complications.13 clinical speech therapy evaluation should occur prior to the actual condition before decannulation is attempted of the patient so that, under favorable conditions (high level of consciousness, adequate breathing pattern, minimum quantity of orotracheal secretions, good phonation, normal swallowing, and effective coughing), decannulation can be performed quickly and effectively, avoiding the impact of tracheostomy in the laryngotracheal region.14 recent research established some guidelines for speech pathologists prior to tracheal decannulation: the patient should be able to clean the oral tract; during cuff deflation, only minimum secretion from above the cuff should need to be suctioned; during tube occlusion, the patient must breath spontaneously and sufficiently through the upper airway with sufficient and stable oxygen saturation; the patient should have efficient spontaneous coughing with subsequent swallowing and must have improved vigilance.9 other researchers found the following criteria for tracheostomy decannulation: stable arterial gasometry, absence of respiratory distress, hemodynamic stability, absence of fever or active infection, paco2<60 mm hg, absence of delirium or psychiatric disorder, normal endoscopic evaluation or revealing lesion occupying<30% of airway, adequate swallowing, and expectoration capability.4 because little is known about how health care professionals make a decannulation decision, a survey was conducted with professionals working in this field, in large centers around the world, to determine clinical factors they believe to be important in decannulation recommendation. four determinant criteria for tracheostomy decannulation were found: level of consciousness, ability to tolerate tracheostomy tube capping, coughing effectiveness, and secretion quantity. of mild importance were the following: patient comorbidities, etiology of respiratory failure, swallowing, and oxygen rates. these factors may drive the creation of specific decannulation protocols.15 even though there are specialist recommendations to guide management decisions for decannulation, there is no objective protocol to establish guideline criteria. therefore, to aid in evaluating a tracheostomized patient's clinical state for possible decannulation and making speech therapy rehabilitation possible, the main objective of this research was to evaluate the applicability of a protocol tracheal decannulation. twenty patients were assessed in this prospective study; the patients were between 21 and 85 years of age (average 33.55), and there were 4 women (20%) and 16 men (80%). all patients had been diagnosed by a neurologist as having tbi, and anatomical region of the lesion was known (table 1). were adult patients over 18 years of age suffering from tbi, tracheostomized, alert and responsive at the time of evaluation, and with medical approval for a speech therapy evaluation. this research took place in the neurology wing of a general hospital in curitiba, brazil. subjects signed a consent form with the knowledge of the objectives, procedures, and responsibilities and received answers to any questions regarding the survey. the subjects were evaluated following tracheal decannulation criteria, through a clinical assessment protocol developed by the authors: the speech therapy tracheal decannulation protocol (appendix i). speech therapy clinical evaluation consisted of patient identification data analysis considering the variables age, gender, diagnostic, anatomical region of the lesion, and six criteria for tracheal decannulation as described: level of consciousness: measured by the glasgow coma scale, the level of consciousness was considered insufficient to protect the airway when the score was consistently less than 8 points.3 measurement was made by the medical team, and during speech therapy assessment, the patient's most recent score was noted. respiration: tube material was noted (plastic or metal), as was whether the cuff was inflated, deflated, or partially inflated and if the patient kept the cuff deflated. whether the patient maintained breathing pattern during tube tracheal secretion: secretions in the tracheostomy region were observed and amount, aspect, and color were noted. amount was quantified as little, normal, or abundant, and thickness, consistency, and color (clear or yellowish) were noted. orally responsive patients were checked for presence of wet (gurgly) voice. patients not verbalizing were evaluated for reaction to pain, moaning, coughing, and/or frequent throat clearing. swallowing: a clinical evaluation of swallowing was performed with occluded tracheostomy, offering food in various consistencies and noting the findings of swallowing phases. the functional oral intake scale (fois) graded the functional oral intake of patients at specific levels, with level 1 being nothing by mouth and level 7 being total oral diet with no restrictions.16 coughing: coughing was observed in food intake or in the event of tracheal aspiration, as was the presence of voluntary coughing and whether it was effective or ineffective, because coughing is a reflex mechanism for airway protection. statistical analysis applied the fisher exact test, which was the basis for comparative process of tabulated data. all subjects in this study were diagnosed with a severe degree of tbi, characterized by anatomical region and hemisphere of the lesion; these variables were correlated with the decannulation indicator. eleven (55%) patients had frontal lobe lesion, 1 (5%) had lesion in the temporal lobe, 3 (15%) in parietotemporal region, and 5 (25%) in the frontal-parietotemporal region. of the total, in 8 (40%) patients the lesion was located in the right hemisphere, 6 (30%) in the left, and 6 (30%) bilaterally (table 1). we did not find any positive statistical correlation between sex and decannulation, which confirms other studies.17 patients underwent a clinical evaluation to assess their clinical conditions at the time of the procedure and to establish speech therapy criteria for tracheal decannulation. for criterion level of consciousness, patients were given a glasgow coma scale score; of the 20 patients, 6 (30%) had scores less than 8, which was considered insufficient for airway protection, and at the time of the evaluation 14 (70%) had scores greater than 8. of the 14, only 2 (14%) could not start the decannulation process, because one could not maintain cuff deflation and the other could not keep the breathing pattern with closed cannula. the correlation between level of consciousness by the glasgow coma scale and decannulation was significant (p=0.0007). of the 20 individuals who participated in the survey, 3 (15%) had a metal cannula and 17 (85%) with a plastic cannula with cuff at the time of assessment. eight (47%) had inflated cuff, 1 (6%) was partially inflated, and 8 (47%) were deflated. of those with plastic cannula cuff, 11 (65%) were eligible for decannulation, and the other 6 (35%) showed no conditions were ineligible for decannulation. of the individuals with a metal cannula, 1 (33%) could be decannulated and the other 2 (67%) could not. despite being relevant information for assessment, this study revealed that these data were not significant for the rate of decannulation (p=0.3439). of the 17 subjects using a cuff, 13 (76%) were able to keep it deflated and 4 (24%) were not. two (15%) of those who were able to keep the cuff deflated were unable to be decannulated for other reasons; however, 11 (64.7%) were able to begin the decannulation process, proving this criterion to be significant (p=0.0063). patients were also observed with tracheostomy tube occluded; out of total population of the study, 12 (60%) maintained the breathing pattern after brief tube capping and 8 (40%) had alterations in the breathing pattern. consequently, 12 (60%) patients were able to close the tube and 8 could not keep it occluded, which also confirmed a significant relationship between respiration and decannulation (p=0.0000). fifteen (75%) patients had secretions in the tracheal region and 5 (25%) did not. of these 15, 8 (53%) had a small amount and could be decannulated, but those who had great amounts were not considered capable of decannulation. of the patients who had secretions in the tracheal region, 1 (7%) had thick secretions and was decannulated. four (20%) patients without secretion were able to close the cannula, and 1 (5%) patient, showing no secretion, was not able to close the cannula. still, regarding the secretion criterion, it was possible to close the tracheostomy in 7 (47%) patients who had clear secretion. of the total of 15 patients with tracheal secretions, 1 (7%) had yellowish secretion and could begin the process of decannulation. from these data, the amount, thickness, and color of the secretion in relation to decannulation indications were found to be significant (p<0.05 for every aspect). thirteen patients (65%) were capable of verbalizing and with the exception of 1 (5%), all the others were able perform the training to close the tube. in the sample surveyed, 7 (35%) were considered to have no verbal responses, and none of these could be decannulated, which also confirmed a significant relationship between phonation and decannulation (p=0.0001). of the 20 total patients, 3 (15%) had wet voice and were to occlude the tracheostomy, and 9 (45%) did not have this vocal pattern and were also able to cap the tube. it should be noted that patients with absent phonation were not evaluated for wet voice because at the time of assessment they were not verbally responsive. regarding swallowing, of 20 patients evaluated, 8 (40%) were graded as level 1 (nothing by mouth in fois) and could not have tracheostomy tube closed. only 1 (5%) was graded at the same level, and the other 11 (55%) patients were classified at levels 2, 3, 4, and 5 and could occlude the tracheostomy tube. swallowing was also assessed for signs of aspiration, considering cough reflex, dyspnea, wet voice, throat clearing, and/or discomfort at one or all offered consistencies. of the 20 patients evaluated, 11 (55%) showed no suggestive signs of aspiration during intake and of these, 9 (82%) began training to close the tube and 2 (18%) were not considered capable. of the total sample, 9 (45%) aspirated during the intake, and of them, 3 (33%) patients were able to cap the tracheostomy and 6 (67%) were not. these data showed that aspiration was significantly correlated to decannulation (p=0.0399). of the total of evaluated patients, 12 (60%) had coughing and could be decannulated, 2 (10%) had coughing but were not able to occlude the cannula, and 6 (30%) had no coughing, none of whom were able to be decannulated. the relationship between coughing and decannulation was significant (p=0.0007). however, the effectiveness of coughing was not significant (p=0.8571). this study evaluated 20 patients of both sexes, between 21 and 85 years of age, affected by tbi and tracheostomized with an open cannula. the decreased level of consciousness of the patient has been described as a related factor to oropharyngeal dysphagia and in the literature is associated with aspiration and pneumonia. this positive influence of the patient's awareness confirms the findings of other studies that a preserved level of consciousness decreases the risk of aspiration pneumonia and is considered so protective that it allows a better prognosis for improving communication and swallowing.18 19 deficient respiratory conditions have been reported as risk factors for aspiration and dysphagia, complicating the rehabilitation of patients with dysphagia. the more dependent on respiratory support, the less airway protection ability patients have, causing a greater risk to their clinical state and to speech therapy interventions with food.18 a recent study compared breathing through the tracheostomy cannula to breathing through the upper airway with capped cannula. the authors conclude that the work of breathing increases in 30% of patients due to higher ventilation requirements and therefore tracheal decannulation is extremely important.20 secretion should have acceptable volume and thickness (clear and fluid). secretion aspect (thick or fluid) in the tracheal region or aspirated indicates the level of hydration and humidification. patients with a large amount of secretion are more likely to have laryngotracheal penetration and aspiration.21 decannulation failures could be attributed to uncontrolled secretions and severe glottic stenosis.14 voice quality is the term used to denote a set of characteristics that identify the voice and aims to identify the presence or absence of wet voice after food intake by comparing the voice before and after swallowing. changes in voice quality may suggest paresis or paralysis of the vocal fold, which in addition to altering vocal quality of the patient is an indication of impaired sphincter action of the larynx when swallowing, which could cause aspiration. a wet voice confers the possibility of presence of saliva, secretions, or food in the vocal cords and into the laryngeal vestibule. it is necessary to give prominence to the wet voice with spontaneous throat clearing or hoarse/breathy voice quality, in combination with other changes observed during the assessment, as these characteristics are often associated with increased risk of aspiration.22 23 the neurologic aspects often determine a condition for vocal change.24 in 2005 the fois was validated, which grades at specific levels the amount of oral intake; the grading may be applied throughout the therapy process to monitor patients ' evolution, and so it was chosen to measure swallowing.16 tracheal aspiration occurs when the material being swallowed enters the airway below the true vocal folds. the material over the inflated cuff can drip down into trachea and lungs. when the cuff is deflated for swallowing tests, it is important to suction the area above the cuff before deflating it to remove all accumulated food and secretions.7 the identification of clinical signs of aspiration not only speeds up speech therapy but also enables airway assessment, eliminating any factor that can make weaning difficult or impossible (stenosis, granuloma, tracheomalacia, and significant dysphagia).25 26 the coughing reflex may be decreased due to both sensory (internal superior laryngeal nerve) and motor components (recurrent laryngeal nerve). the recurrent laryngeal nerve, in addition to laryngeal motricity, is responsible for cough as protection and cough in general, of a different etiology than aspiration. this distinction is very important as it allows understanding that the aspiration is likely to occur silently, without the normal response of cough and choking.18 the criteria described above were relevant to establish the beginning of decannulation process of tracheostomy. however, the data collected are based on the start of a further longitudinal research with an increase of the population assessed. this study used a protocol to establish six criteria for tracheal decannulation in patients affected by tbi: level of consciousness, respiration, tracheal secretion, phonation, swallowing, and coughing.

introduction the frequency of tracheostomy in patients with traumatic brain injury (tbi) contrasts with the lack of objective criteria for its management. the study arose from the need for a protocol in the decision to remove the tracheal tube. objective to evaluate the applicability of a protocol for tracheal decannulation. methods a prospective study with 20 patients, ranging between 21 and 85 years of age (average 33.55), 4 of whom were women (20%) and 16 were men (80%). all patients had been diagnosed by a neurologist as having tbi, and the anatomical region of the lesion was known. patients were evaluated following criteria for tracheal decannulation through a clinical evaluation protocol developed by the authors. results decannulation was performed in 12 (60%) patients. fourteen (70%) had a score greater than 8 on the glasgow coma scale and only 2 (14%) of these were not able to undergo decannulation. twelve (60%) patients maintained the breathing pattern with occlusion of the tube and were successfully decannulated. of the 20 patients evaluated, 11 (55%) showed no signs suggestive of tracheal aspiration, and of these, 9 (82%) began training on occlusion of the cannula. the protocol was relevant to establish the beginning of the decannulation process. the clinical assessment should focus on the patient's condition to achieve early tracheal decannulation. conclusion this study allowed, with the protocol, to establish six criteria for tracheal decannulation: level of consciousness, respiration, tracheal secretion, phonation, swallowing, and coughing.

PMC4435458

pubmed-647

in recent years, it has been revealed that astrocytes perform a significantly wider range of functions than previously appreciated. interest in astrocyte function has increased dramatically because of their newly discovered roles in synapse formation, maturation, efficacy, and plasticity. today, astrocytes are recognized as multifunctional cells with well-defined essential neuron supporting functions. mounting evidence suggests that these versatile cells participate in a multitude of diverse processes in the central nervous system (cns). these roles include regulating blood flow, providing much needed energy to neurons, and supplying the building blocks of neurotransmitters that fuel synapse activity. the addition of their role in synaptic function to the known repertoire of astrocyte activities over the past decade has enhanced our conception of their seminal importance in normal functioning of the adult brain. more comprehensive reviews highlighting astrocyte function include jacobs et al., wang and bordey, and kimelberg. in the developing nervous system, the assembly of synaptic circuits is a complex and dynamic process, requiring the coordinated exchange of signals between pre- and postsynaptic neurons and neighbouring glia. the formation, maintenance, and modulation of synaptic connections are required for normal cns function and ongoing plasticity. in the diseased nervous system, however, the structural and functional integrity of synaptic connections is often modified or lost, resulting in profound cognitive and behavioral deficits. yet until recently, no exact roles had been identified for astrocytes in the pathogenesis of specific cns diseases. while some aspects of the mechanisms underlying the formation, maintenance, and plasticity of cns synapses in the developing and diseased nervous system have been elucidated, many more remain enigmatic. as our knowledge about astrocytic function in normal physiology has expanded, exploration into their likely role in disease pathology has followed. in the case of fragile x syndrome (fxs), a compelling case can be made for the abnormal dysfunction of astrocytes. fxs is the most common form of inherited mental impairment, and it typically results from the transcriptional silencing of the fmr1 (fragile x mental retardation 1) gene and loss of the encoded protein, fmrp (fragile x mental retardation protein). fxs symptoms include neurodevelopmental delay, anxiety, hyperactivity, and autistic-like behavior. fmrp was once thought to be expressed solely in neurons; however, it was later shown to have specific roles in astrocytes. pacey and doering found that fmrp is expressed in early development in cells of the glial lineage both in vitro and in vivo. although few studies focus specifically on the role of astrocytes, recent work provides important examples of how a better understanding of astrocyte biology during development can enhance our knowledge about human disease. in this paper, we discuss the landmark findings and recent advances in our understanding of astrocytes and their featured roles in regulating synapse formation, maturation, and synaptic transmission. further, we assess how astrocytes contribute to the extensive plasticity that occurs during development, highlighting the dynamic morphology of astrocyte processes and their involvement in synaptic development. lastly, we explore the means by which perturbations in astrocyte function may contribute to neurological diseases, such as fxs, in the context of synaptic defects. we propose here that, by investigating the precise roles of astrocytes during neurological disease, we are likely to achieve a broader understanding of how the brain works, in addition to new insights into disease prognosis, diagnosis, and treatment. they were so named due to their stars in the night sky appearance obtained from golgi stained samples. in the late nineteenth century and the early twentieth century, camillo golgi and santiago ramn y cajal noticed that, although different astrocytes share a stellate feature, their morphology is extremely diverse, perhaps as diverse as neurons. since cajal's time, modern scientists have confirmed the morphological diversity of astrocytes in vitro and in vivo [10, 11]. astrocytes are divided into two main classes distinguished on the basis of their morphology and primary location [12, 13]. their processes, which are long, thick, and highly ramified, are closely associated with synapses as well as blood vessels. in the hippocampus, protoplasmic astrocytes ensheath more than half of the synapses, most of which are excitatory. the other subtype is composed of fibrous astrocytes found mainly in the white matter of the brain, where their processes pass between nerve fibers. in contrast to protoplasmic astrocytes, fibrous astrocyte processes are long, cylindrical, smooth, and branch infrequently. from the cell soma radiate primary branches that gradually divide into finer and finer processes to generate a dense network of delicate terminal processes, which associate very closely with synapses. a number of immunological markers have been used over the years to characterize astrocyte morphology. until recently, our understanding has been predominantly based on classical immunostaining with the widely used astrocyte marker gfap (glial fibrillary acidic protein, an intermediate filament protein), which grossly underestimates the complexity of astrocytes and their interactions with neurons and other cells. gfap only reveals the structure of primary branches, which represent a meager ~15% of the total volume of the astrocyte. other markers include aldh1ll (aldehyde dehydrogenase 1 family, member l1), glt-1 (glial glutamate transporter 1), and glast (glutamate-aspartate transporter). to date, no marker has been identified that is expressed exclusively in mature astrocytes. moreover, no pan-astrocytic marker has been identified with which to determine the fraction of astrocytes that are gfap+, although recent studies on aldh1l1 seem promising. recent physiological and gene expression profiling studies indicate that astrocytes, like neurons, are a diverse cell population with distinct properties in different brain regions and at different periods of development. they function as neural stem cells and guide axon projections; they promote synapse formation and maintain neuronal survival [20, 21]. subsets of astrocytes, or astrocyte-like cells, in the adult subventricular zone (svz) and in the subgranular zone (sgz) of the dentate gyrus of the hippocampus act as neural stem cells, whereas most astrocytes in other parts of the adult brain do not normally proliferate. heterogeneity of astrocytes, however, is not exclusive across brain regions, as it can also exist within the same areas of the brain. the number and size of astrocytes in the brain also vary between species relative to species brain size and cognitive ability. for example, the human brain contains several more populations of astrocytes than the rodent brain, and human astrocytes are threefold larger than their rodent counterparts. therefore, these classifications may not be adequate to appreciate the full extent of astrocyte diversity. astrocytes have unique cytoarchitectural and phenotypic features that ideally position them to sense their surroundings and respond in dynamic ways to changes in their microenvironment. astrocytes are, therefore, well suited to share synaptic function with neurons as they extend numerous processes, forming highly organized anatomical domains with little overlap between adjacent cells. the territory of a single astrocyte is estimated to contact between 300 to 600 dendrites and upwards of 10 synapses [16, 26]. this extensive synaptic interaction not only ensures that astrocytes are able to fulfill their metabolic support roles but also positions astrocytes to directly influence the structure and function of the synapse. while some astrocyte processes (which express a wide range of receptors and ion channels) closely ensheath synapses, others are in close contact with intraparenchymal blood vessels via specialized processes called endfeet. in line with this, the formation of synaptic contacts is paramount for the proper development and function of the cns. although most neurons are produced during embryonic stages, the major waves of synaptogenesis follow and depend on astrocyte production. given their proximity to synapses, astrocytes can directly promote and regulate these processes through both secreted and contact-mediated signals. the traditional assumption that neurons are intrinsically able to form synapses led early studies on synaptic development to focus on neuronal signals and surface molecules. remarkably, neurons cultured with media conditioned by astrocytes control the number and effectiveness of synapses [2830], indicating that soluble factors secreted from astrocytes play an important role in synapse formation. these include matricellular proteins, such as thrombospondins (tsps-14), sparc, sparc-like 1 (hevin), and tenascin c, which are all expressed by astrocytes in the cns of rodents. a possible role for glial involvement in cns synaptogenesis was first elucidated by a series of studies on rat retinal ganglion cells (rgcs). cholesterol complexed to apolipoprotein e (apoe) released by astrocytes increases the number of glutamatergic synapses in rgc cultures. when cholesterol is applied directly to cultured rgcs, the frequency of spontaneous synaptic events increases. the researchers further demonstrated that cholesterol acts to increase the quantal content of synaptic vesicles and the overall efficacy of vesicle release. this is in concert with other findings that cholesterol is an essential component of synaptic vesicle production whose presence serves as a limiting factor in vesicle formation. the rgc culture technique has also been used to identify other key synaptogenic-secreted factors including thrombospondins 1 and 2 (tsp-1 and tsp-2), members of oligomeric extracellular proteins. christopherson and colleagues identified tsps as the signals coming from astrocytes that can induce an increase in synapse number. when directly applied to rgc cultures, immunodepletion of tsps from astrocyte-conditioned medium (acm) decreased its synaptogenic effect down to control levels indicating that tsps are the key synaptogenic component of acm. the expression of tsp-1/2, which is elevated in the developing brain when the majority of synapses are formed (during postnatal week 1), ceases in the mature adult brain (by postnatal week 3). this suggests that astrocytes downregulate pathways that strongly promote synapse formation when the synaptogenic period of neurons is reduced, and other tsp genes may be functioning to stabilize synaptic structures. besides tsps 1 and 2, other tsps (tsps-35) are detected in mammals. in contrast to other tsp-s, tsp-4 expression is only detected in mature astrocytes after p17. this suggests that tsp-4 could represent the adult isoform of tsp in the cns and is important for the control of synaptogenesis and enhanced plasticity in the adult brain. recently, gabapentin receptor 2-1 has been identified as the tsp receptor responsible for mediating excitatory cns synaptogenesis. despite the critical role of tsps in promoting synaptogenesis, additional signals are likely required for synapse maturation, as tsp-induced synapses are ultrastructurally normal, but postsynaptically silent, underscoring the complexity of astrocyte contribution to synapse formation. a more recent study has identified two closely homologous glypicans, glypican-4 and glypican-6, as astrocyte-secreted proteins that are sufficient to increase ampa glutamate receptor levels on synapses, thus inducing postsynaptic function. additionally, tenascin-c (tn-c), another extracellular matrix glycoprotein, seems to play a role in synaptogenesis and synaptic function [38, 39]. tn-c is highly expressed by astrocytes during early stages of development, while its expression ceases in the adult cns, with the exception of specific cell populations, particularly those in close proximity to areas of active neurogenesis, such as the hippocampus, subventricular zone borders, and the rostral migratory stream. following stimulation of synaptic activity, tn-c was found upregulated in the hippocampus within a few hours. in tn-c knockout mice, stimulation of schaffer collaterals resulted in a reduction of long-term potentiation (ltp) at ca1 synapses, whereas ca1 long-term depression (ltd) was completely abolished [42, 43]. these expression patterns reveal important roles for tn-c in the remodeling of the cns, both during development and in adulthood. in fact, recent studies reveal astrocyte contributions to inhibitory synapse formation and function in cultured hippocampal neurons. while astrocyte-expressed extracellular matrix protein hevin has been found to induce the formation of synapses in cultured rgcs [44, 45], its homolog, sparc, which is also secreted by astrocytes, antagonizes the synaptogenic function of hevin, thereby acting as a negative regulator of synapse formation. sparc expression is typically high in early development, where it then becomes downregulated in certain parts of the brain by the time of synaptogenesis. alternatively, hevin expression increases with development in agreement with synapse formation and is also present in adulthood, most likely functioning in the maintenance of existing synapses. unlike tsp-1 and tsp-2, the expression of which is decreased during maturation, hevin and sparc mrna levels remain high even in the adult. taken together, the secretion of both positive and negative regulators of synapse formation allows astrocytes to regulate the timing and location of synapse formation with greater precision. moreover, a recent study has provided evidence that astrocytes play a role in the elimination of redundant synapses during development. in the developing postnatal brain and retina, immature astrocytes seem to be a source of a signal that triggers the expression of complement component c1q in developing neurons. c1q's best-known role in the innate immune system is to opsonize or c1q localizes to synapses that are thus tagged for elimination through the activation of the complement cascade and deposition of c3b, an opsonin derived from the proteolytic activation of the complement component c3. mice deficient in c1q or the downstream complement cascade protein c3 exhibit large sustained defects in cns synapse elimination, as shown by the failure of anatomical refinement of retinogeniculate connections and the retention of excess retinal innervation by lateral geniculate neurons. also, c1q-deficient mice show enhanced neocortical excitatory synaptic connectivity and epileptiform activity. together, these findings implicate a role for astrocytes during the critical period when neural circuits are formed. while astrocyte-secreted factors induce the formation and function of synapses, other evidence proposes further regulatory roles for astrocytes through contact-mediated mechanisms. an elegant study by hama et al. local contact with astrocytes via integrin receptors facilitated excitatory synaptogenesis through the activation of protein kinase c (pkc) in individual dissociated hippocampal neurons. the researchers observed that pkc activation, while initially focal, subsequently spread throughout the entire neuron. thus, propagation of pkc signaling could signify an underlying mechanism for global neuronal maturation following local astrocyte adhesion. astrocyte processes, which are highly mobile, contribute to the stabilization of new synapses during synaptogenesis. astrocytes may induce local structural and functional modifications of dendritic segments or individual synapses through a contact-mediated mechanism involving bidirectional ephrin/epha signaling [4951]. membrane-bound ligands on astrocytes, such as ephrin-a3, have been shown to upregulate spine morphology in the hippocampus, suggesting local activation of epha receptors on spines by astrocytic ephrin-a3. dendritic spines are small protrusions visible on dendrites of neurons that serve as postsynaptic sites for excitatory input [5254]. live imaging of organotypical hippocampal slice preparations showed that astrocytes rapidly extend and retract fine processes to engage and disengage from postsynaptic dendritic spines. studies with two-photon microscopy that tracks the dynamics of astrocyte processes and the fate of dendritic protrusions also revealed contributions of astrocyte contact. dendritic protrusions with astrocyte contacts had a longer lifetime and were morphologically more mature. thus, dendritic protrusive activity and transient contacts with astrocytes act to stabilize newborn synapses and promote subsequent spine maturation. spine dynamics are largely controlled through changes in cytoskeletal proteins. expressing a dominant negative mutant rac1, a gtpase that mediates actin motility, reduces astrocyte process motility and provides evidence that cytoskeletal rearrangements underlie motility, similar to mechanisms of spine extension and retraction [56, 57]. liu et al. showed that local contact between neurons and astrocytes significantly increased the amplitude and density of gabaa currents in developing hippocampal neurons. this contact-dependent increase in gabaergic synaptic activity relied on ca signaling in astrocytes. in addition, astrocytes were shown to regulate cl gradient in cultured spinal cord neurons and convert gabaergic synapses from excitatory to inhibitory. this finding is particularly exciting given the importance of local gabaergic inhibitory circuits in both activity-dependent wiring of developing neural circuits and the consolidation of critical period plasticity [60, 61]. overall, these studies reveal that contact-mediated signaling between astrocytes and neurons is important for the structure and maintenance of synaptic connections and suggests a model in which physical and molecular interactions between neurons and astrocytes provide instructive cues that control synapse formation, morphology, and plasticity. as our understanding of the extent of their influence at the synapse unfolds, it is much more apparent that astrocytes are well poised to modulate multiple aspects of synaptic plasticity than was previously imagined. a turning point in our understanding of astrocytes was elicited by the recognition of their active communicative properties [6264]. astrocytes, which are bidirectional, can communicate and exchange information with both pre- and postsynaptic elements. communication is primarily controlled by the change in ca concentrations, causing excitability within the astrocyte [6466]. astrocytes use their ability to respond to neurotransmitters and secrete neuromodulators to actively regulate a number of processes involving synaptic plasticity [6769]. in addition to secreting factors that influence and modulate synapse formation, astrocytes are known to release factors that can directly affect synaptic transmission. briefly, of the gliotransmitters released by astrocytes, the most well characterized and extensively reviewed are glutamate [71, 72], adenosine triphosphate (atp), and d-serine [74, 75]. glutamate serves as the principal excitatory neurotransmitter in most regions of the cns, and its release from astrocytes has been shown to modulate synaptic transmission. glutamate released from neurons activates metabotropic glutamate receptors on astrocytes, leading to an increase in astrocyte ca concentrations and a subsequent astrocytic release of glutamate. d-serine, perhaps the most interesting, is an important neurotransmitter that serves as a coagonist with glutamate, promoting nmda (n-methyl-d-aspartate) receptor activity at synapses in the hypothalamus. moreover, astrocytes release atp to communicate with each other and other glia by activating purine receptors localized on neighbouring cells. these findings have led to the establishment of a new concept in synaptic physiology, the tripartite synapse, in which astrocytes exchange information with neuronal synaptic elements [6, 67, 77]. consequently, astrocytes are an integral part of the synapse, being involved not only in passive homeostatic control of adequate conditions for synaptic function, but also actively in synaptic function. with an evident role of astrocytes in normal neural function at all cellular and molecular levels, it is not surprising that astrocytes contribute in some capacity to almost all pathological conditions of the nervous system [7984]. for most disorders, it remains unclear whether astroglial changes are causative of the disease or if they merely represent an accompanying phenomenon. accordingly, astrocyte-dysregulated function has been fundamentally linked with the progressive pathology of ischemic stroke, epilepsy, and to a number of neurodegenerative disorders including, but not limited to, amyotrophic lateral sclerosis, huntington's disease, and parkinson's disease. further involvement of astrocytes has also been implicated in the development of neurodevelopmental disorders such as rett syndrome (rtt), down syndrome (ds), fragile x (fxs), and autism. among these conditions, fxs has emerged as the prototypical disorder in which to study how altered signaling may lead to synaptic defects and dysfunctional neural circuitry underlying pathology. both dysregulated astrocyte signaling and abnormal synaptic function stand as prominent contributing factors to the learning disability phenotype expressed in fxs. fragile x syndrome (fxs) is the most common form of inherited intellectual disability. it affects approximately 1 in 4,000 males and 1 in 6,000 females and is characterized by cognitive impairments, attention deficits, and autistic-like behaviors. fxs is caused by an expanded cgg trinucleotide repeat in the 5 untranslated region of the fmr1 gene leading to gene silencing and the consequent loss of fmrp expression [87, 88]. to understand the etiology of the synaptic phenotypes that accompany fxs, it is first important to discuss the purported function of fmrp. fmrp acts as a regulator for the transport and local translation of specific synaptic mrnas in response to neural stimulation. fmrp is found in growth cones, immature axons, and mature dendrites, as well as dendritic spines. the loss of fmrp results in the aberrant expression of its mrna targets, which in turn leads to functional deficits that characterize fxs. the reason that fmrp has been implicated in synaptic plasticity is on the basis of dendritic spine abnormalities and exaggerated long-term depression (ltd) displayed by fmr1 mutant mice. this finding led to the mglur theory of fmrp, whereby synaptic signaling of metabotropic glutamate receptor 5 (mglur5) leads to the localized translation of fmr1 mrna. as such, the newly synthesized fmrp acts as a translational repressor of specific target mrnas, resulting in the downregulation of mglur5 activity through a negative feedback loop (figure 1). several exceptional reviews on the genetic and clinical features of fxs or molecular functions of fmrp include bear, huber et al. current knowledge surrounding the pathophysiology of fxs has been greatly advanced by the development of animal models. these transgenic animals do not carry the trinucleotide expansion but do have functional deletions of fmrp. the first model developed was the fmr1 knockout (ko) mouse, which recapitulates behavioural and cognitive deficits reminiscent of the human condition. drosophila and zebrafish models also exist and have contributed to our understanding of the conserved roles of fmrp in neural development [9799]. although they are not perfect models of the human disease, they have helped to reveal the cellular and molecular mechanisms underlying fxs, and they have immensely enhanced glial-neuronal research. during development, selective elimination or pruning of inappropriate synaptic connections occurs for the proper formation and establishment of neural circuitry. current models regarding the neurobiological changes that underlie fragile x have largely focused around the synapse. this is based in part on the structural synaptic changes and alterations in synaptic function, which are observed in human patients and fxs animal models. spines develop around the time of synaptogenesis and are dynamic structures that continue to undergo remodeling over time. developmental changes in the shape of dendritic protrusions reflect the progressive replacement of thin, elongated, and highly motile filopodia, characteristic of immature neurons, with more stable spines that acquire a mature morphology. spine morphogenesis is fundamental to the development of neuronal networks and the regulation of synaptic plasticity. some of the first neuroanatomical findings associated with mental impairment were alterations in dendritic spine structure. the first such evidence of altered synapse structure in fxs came from analysis of postmortem cortical tissue, which exhibited an increased number of dendritic spines relative to control individuals. this data revealed that excitatory synapse number was increased in fxs patients and further provided a potential mechanism for the increased rates of epilepsy in fxs. it was additionally noted that a large proportion of the spines of fxs patients appeared abnormally long, thin, and tortuous, a phenotype reminiscent of the immature spine precursors (filopodia), and indicative of alterations in synapse development and/or function. at this point, it was not clear if the excess filopodia-like spines in fxs represented functional synapses or immature synapse precursors. much of the evidence for a role for fmrp in synaptic and neurite pruning is derived from the drosophila melanogaster model of fxs (dfxr). during development, fmrp has been shown to control the pruning of immature dendrites in developing neurons. in support of a pruning function for dfxr, most neurons of dfxr null flies exhibit an overgrowth and elaboration of axons and dendrites into the peripheral and cns [98, 103106]. parallel to human studies, work with the fmr1 ko mouse has largely confirmed the spine phenotype observed in fxs patients. numerous studies agree that fmr1 mutant brains display an increase in long, thin, immature dendritic spines [102, 107] mirroring human neuroanatomical abnormalities. it is important to note that many of these defects in spines and in synaptic/circuit plasticity occur during critical periods of development in the first postnatal weeks, coinciding with the maximal expression of fmrp. however, the existence and/or magnitude of the spine alterations in the fmr1 ko mouse varies according to brain region, developmental age, and genetic background, indicating the complex and multifactorial regulation of spines. in a study by cruz-martin et al., spines of l2/3 layered pyramidal neurons were imaged at various developmental stages, and it was revealed that fmr1 ko mice demonstrated a delay in the stabilization of dendritic spines, due to high turnover during the second postnatal week. this happens to correspond to the time when fmrp protein expression is highest in the cortex. in the absence of fmrp, hippocampal neurons have fewer spines that colocalize with synaptic markers, which suggests a loss of functional spines this provides compelling evidence that fxs might be caused by a failure in the transition from filopodia (earliest dendritic protrusions) to mature spines, consequently resulting in an increase of immature synapses. the failure of spines to stabilize during the critical period in the barrel cortex strongly suggests that fmr1 ko mice could have problems in maintaining the proper balance between stable and dynamic connections that is necessary to establish mature synapses. since dendritic spines are the primary sites of excitatory synapses and information exchange in the cns, perturbations in their structure and function can result in synaptic and circuit alterations leading to disrupted brain function and pathology. while it has been recognized that astrocytes play multiple critical roles in the regulation of normal cns function, the possibility that astrocyte-specific dysfunction might cause diseases that manifest as pathologies of neurons is a relatively recent idea. previously, it was thought that fmrp expression in the brain was exclusively confined to neurons. our laboratory initially identified fmrp in the astrocyte lineage in the fxs mouse. when studying stem and progenitor cells from the brains of wild-type (wt) and knockout (ko) fxs mice, approximately half of the cells in culture coexpressed fmrp and gfap. parallel immunocytochemical studies in vivo also showed the coexpression of fmrp and gfap in the embryonic and adult developing hippocampus. with the identification of fmrp in astrocytes and knowledge of their role in synaptogenesis, our laboratory was prompted with further experiments to explore neuronal development and synapse formation in fxs. utilizing a coculture design adapted by jacobs and doering, hippocampal neurons (e17) and cortical astrocytes (p0-1) were independently isolated to explore four different combinations of neuronal-astrocyte cultures (wt neurons+wt astrocytes, wt neurons+fmr1 ko astrocytes, fmr1 ko neurons+wt astrocytes, fmr1 ko neurons+fmr1 ko astrocytes). the cells were grown for 7, 14, or 21 days in vitro and then processed for immunocytochemistry to analyze morphological and synaptic profiles. these experiments are novel and exciting as they are the first to establish a potential role for astrocytes in the altered neurobiology of fxs. the first group of experiments focused on neurons in each of the four combinations to elucidate the effects of fmrp on dendritic morphology and excitatory synapse expression. the neurons were studied with antibodies directed against the neuronal (dendritic) marker, map-2, the presynaptic protein synaptophysin, and excitatory postsynaptic protein, psd-95, respectively. through sholl analyses, morphological assessments were performed on neurons under parameters of dendritic branching and the area of the cell body. synaptic protein distribution was determined by the quantification of synaptic puncta (spots of intense staining). wt neurons grown on fmr1 ko astrocytes exhibited significantly altered dendritic arbor morphologies, with a shift toward a more compact and highly branched dendritic tree. specifically, wt neurons grown on fmr1 ko astrocytes resulted in a decrease in the length of the longest primary dendrite and area covered by dendritic arbor, and an overall increase in branch number and density in comparison to their wt counterparts. these neurons also displayed a significant reduction in the number of pre- and postsynaptic protein aggregates. however, when the fmr1 ko neurons were cultured with wt astrocytes, the alterations in dendritic morphology and synaptic protein expression were remarkably prevented. in fact, their morphological characteristics and synaptic protein expression approached the appearance of normal neurons grown with wt astrocytes. these experiments were the first to suggest that astrocytes contribute to the abnormal dendritic morphology and the dysregulated synapse development seen in fxs. in the next phase of this research, we wanted to determine if these altered characteristics represented a developmental delay imparted by the fmr1 ko astrocytes. focusing on wt neurons grown in the presence of wt or fmr1 ko astrocytes, we evaluated the dendritic arbor morphology and synaptic protein expression at 7, 14, and 21 days in culture. our results revealed that wt neurons grown with fmr1 ko astrocytes displayed significantly altered morphological and synaptic protein profiles at 7 days (when compared to the wt condition). strikingly, by 21 days in culture, these differences were no longer significantly different from normal. in light of these findings, it appears that astrocytes in the fxs mouse may contribute to the altered characteristics of neurons seen in fxs in a developmentally regulated manner. thus, these results suggest that timing is crucial in brain development. despite these outcomes, it is noteworthy that conclusions about synapse maturity can not be drawn. it is possible that the increase in synapses observed in the neurons grown on fmr1 ko astrocytes reflects an increased number of immature synapses. given that the dendritic spine is the site for the majority of excitatory synapses, this finding would be in agreement with numerous studies that identified neurons in fxs with an abnormally high number of immature dendritic spines. as a note, the methods used in the current study did not permit the assessment of alterations in dendritic spine morphology. understanding the role of astrocytes in human neurological diseases requires a comprehensive picture of how astrocytes develop and what roles they play in development. given these findings, it is highly plausible that fxs astrocytes lack functional fmrp, specifically at a time during development when astrocyte support of neuron growth and synapse formation is vital, and that this lack of fmrp could contribute to the abnormal neuron phenotype seen in fxs. however, it is uncertain whether the alterations in astrocytes are due to a lack of fmrp or if they are abnormal because they develop and function in a diseased microenvironment. also, if the absence of fmrp in astrocytes is the primary source of dysfunction, how are these effects translated to neurons? for instance, is astrocyte-neuron signaling disrupted due to a lack of astrocyte fmrp? how, where, and when do these signals act? is the abnormal astrocyte-neuron communication mitigated by a membrane associated or a soluble factor? could it be a combination of both direct and indirect contact? these questions, among many others, about the fxs astrocyte are now important targets for fxs research. the answers will allow us to gain a full understanding of the underlying neurobiology that contributes to the morphological phenotype seen in fxs and in the potential of a future treatment for individuals with fxs. recent evidence indicates that the interface between astrocytes and neurons is necessary for normal synapse development, including synaptic pruning. dendritic spines, which are highly dynamic during development, become more stable in the adult brain; thus, a correlation exists between age-dependent spine dynamics and the plasticity of the brain. this decrease in spine motility in the mature brain could be attributed to the close association of astrocytes with synapses, with astrocytes providing both physical constraints that inhibit spine movement as well as molecular interactions that stabilize spines. importantly, epha4r (expressed on dendritic spines) interacts downstream with members of the rho/ras pathways, suggesting that ephar/ephrin-a interactions may underlie aspects of actin-driven astrocyte motility observed during synapse formation [27, 115]. therefore, this raises the possibility that in vivo defects in dendritic spine development are at least partly related to neuron-glia interactions during development. astrocyte involvement has also been fundamentally implicated in neurodevelopmental disorders such as rtt and ds. a common finding in many of these studies is that astrocyte dysfunction has profound non-cell-autonomous effects on surrounding neurons. rtt, which is an x-linked neurodevelopmental disorder, is caused by the loss of the transcriptional repressor methyl-cpg-binding protein (mecp2). a study by ballas et al. showed that wild-type hippocampal neurons cocultured with cortical astrocytes or conditioned medium from mecp2-deficient mice had abnormally stunted dendrites, suggesting that mecp2-deficient astrocytes may dominantly affect normal neuronal development. furthermore, in ds patients, cognitive deficits have been associated with structural changes in the architecture and alterations in dendritic spine number. garcia et al. found that ds astrocytes are directly involved in the development of spine malformations and reduced synaptic density. these researchers also indicated that the astrocyte-secreted protein tsp-1 possesses a potent modulatory effect on spine number and morphology. taken together, these studies serve to identify astrocyte dysfunction as a significant factor of spine and synaptic pathology. future experiments will focus on the assessment of dendritic spines in fxs and the role of direct/indirect neuronal-astrocyte cell contact in the altered developmental sequences that we observed in our tissue culture paradigm. it is highly conceivable that the absence of astrocyte fmrp would directly affect spine morphology or dynamics via dysregulated protein synthesis, and a defect in the maturation of dendritic spines could explain deficits in the intellectual ability seen in individuals with fxs. armed with novel experimental techniques, powerful imaging tools, and a better understanding of astroglia, neuroscientists are uncovering a new view of the synapse. neuroscientists are now in a better position to explore and uncover the long-standing mysteries of astrocytes and gain new insights into the cellular and molecular underpinning of the nervous system. the recent findings discussed in this paper place astrocytes in an important position to actively exchange signals with neurons and other glial cells to coordinate synaptic networks. while studies help to distinguish the effects of astrocyte contact from secreted factors on neuronal form/function, it is unlikely that they are separate in vivo. also, given the central role of the synapse in neuronal communication and plasticity, it comes as no surprise that dysregulation of the synapse accounts for many, if not most, of pathological and developmental disorders in the brain. thus, the involvement of astrocytes and how they interface with neuronal circuitry should be taken into consideration when interpreting future studies in the pathophysiology of fxs and/or other related neurological diseases. a unifying theme from these recent findings is that astrocytes can promote the development and plasticity of synaptic circuits. much of the current literature surrounding fxs focuses on synaptic control of protein synthesis because it appears to be proximal to the biology of fmrp and the pathogenesis of the disease in multiple animal models. in addition to targeting synaptic protein synthesis, other therapeutic approaches show promise, for example, in changing the balance of excitation to inhibition by enhancing gaba signaling. whether different approaches will converge on the same pathophysiological processes or whether they will target distinct aspects of the disease remains to be determined. as we continue to expand our understanding, insights into how these mechanisms may be perturbed in fxs and other diseases states may pave the way for promising future therapeutic interventions and treatments. potential modes of pharmacological therapy should indeed concentrate on the astrocyte as a gatekeeper of neuronal health and function.

a growing body of research indicates a pivotal role for astrocytes at the developing synapse. in particular, astrocytes are dynamically involved in governing synapse structure, function, and plasticity. in the postnatal brain, their appearance at synapses coincides with periods of developmental plasticity when neural circuits are refined and established. alterations in the partnership between astrocytes and neurons have now emerged as important mechanisms that underlie neuropathology. with overall synaptic function standing as a prominent link to the expression of the disease phenotype in a number of neurodevelopmental disorders and knowing that astrocytes influence synapse development and function, this paper highlights the current knowledge of astrocyte biology with a focus on their involvement in fragile x syndrome.

PMC3403619

pubmed-648

the evaluation of vibration sensation informs the clinician about the integrity of mechanoreceptors in the skin, rapidly conducting large-diameter afferent fibers in the peripheral nerves and the dorsal column-medial lemniscus pathways in the central nervous system [1, 2]. therefore, diminished vibration sensation is an important finding in the diagnosis of disorders affecting the dorsal column-medial lemniscal system and may also be an early sign of peripheral neuropathies. it is known in everyday practice that patients, due to the physical properties of this sensory modality, can perceive vibration through layers of clothes. this recently resulted in a debate during a case presentation in our clinic in which one of the authors tried to perform the examination on a clothed subject for the sake of speed. the present study was designed under the inspiration of this dispute with the hope of providing some new insights into the characteristics of vibration sensation. fifty healthy volunteers (h group; median age was 37 years, 22 male) and 19 patients with diabetic polyneuropathy (pnp group; median age was 57 years, 5 male) were included in the study (table 1). the h group mainly consisted of volunteer relatives of patients, healthcare professionals, and medical students stratified into 4 age groups, as follows: ages 1825 (n=14), 2640 (n=13), 4155 (n=13), and 55+(n=10). their mean height was 168.5 9.1 cm (range 153 to 183) and their mean weight was 72.2 14.6 kg (range 49 to 110). a brief neurological examination including superficial sensation, muscle strength, and tendon reflexes was made and found to be normal in all the subjects in this group. those who had any neurological and systemic disease or who were using medications which could affect the peripheral or central nervous systems were excluded from the study. the pnp group consisted of patients with known type 2 diabetes mellitus and the clinical diagnosis of diabetic polyneuropathy visiting the diabetic outpatient clinic for their regular controls (median follow-up duration was 14 years; range 126). vibration sensation was measured with the common psychophysical technique, whereby the duration of vibration sensation is dependent on each subject's judgment. the subjects lay supine in a comfortable quiet room with an ambient temperature of 2025c during the measurements. a 128 hz standard tuning fork was used to assess the vibration sensation throughout the study (riester tuning fork w/clamps, aluminum, no. 5162, c 128 hz, california, usa). the socks used in this study all had the same thickness and tissue characteristics (the same model from a certain manufacturer; cotton, 70 denier linear mass density) which had been purchased in bulk and were disposed of after a single use. four sites were selected for measurements: dorsum of the interphalangeal joint of the great toe (gt) and the vertex of the medial malleolus (mm) on both sides. the tuning fork was applied vertically to these points with gentle pressure after the prongs were maximally activated (by tapping the prongs against the examiner's hypothenar eminence) for each measurement (figure 1). two investigators took the measurements; the first one applied the tuning fork while the second investigator measured with a stopwatch the duration of vibration sensation from the activation time of the tuning fork until the subject's verbal indication that the sensation was over. those who were found to have no vibration sensation at all on any of the measurement sites (usually at gt) were excluded from the study at this point. in the measurement phase, 2 measurements without and 2 measurements with socks were performed on each site in the following sequence, allowing a five-minute break inserted so as not to exhaust the subjects and to help them maintain their concentration: without, with, interval, with, without. sixteen measurements in total were taken for each subject, requiring about 20 minutes for the entire procedure. the averages of each pair of measurements with and without socks were used as the statistical input. the procedure was approved by the istanbul faculty of medicine ethics committee (2012/1244) and all participants provided informed consent. all the statistical analyses were performed using the statistical package for the social sciences (spss), version 17.0, for windows (spss, chicago, il, usa).samples were tested for their distribution with kolmogorov-smirnov test.correlations between the measurements in the same conditions (first and second measurements on the same side without socks and with socks) and between the measurements on both sides were tested with pearson's r.after showing a high correlation between the items in, the results of the two measurements for each condition and the results elicited on the right and left sides were averaged to constitute the main inputs for the statistical analyses. the difference between the mean durations of vibration sensation without and with socks in both groups was analyzed using paired samples t-test.statistically significant results elicited in were analyzed for the effect size, using cohen's d.in the h group, correlations of vibration sensation with age, height, and weight were assessed with pearson's r.independent samples t-tests were performed to evaluate the difference between the vibration sensations measured at the gt and mm.differences between the results elicited in the pnp group and those found in an age-matched subgroup of h (13 females and 9 males, aged 40 years (median 53, min 40, max 68)) were analyzed with independent samples t-tests. correlations between the measurements in the same conditions (first and second measurements on the same side without socks and with socks) and between the measurements on both sides were tested with pearson's r. after showing a high correlation between the items in, the results of the two measurements for each condition and the results elicited on the right and left sides were averaged to constitute the main inputs for the statistical analyses. the difference between the mean durations of vibration sensation without and with socks in both groups was analyzed using paired samples t-test. statistically significant results elicited in were analyzed for the effect size, using cohen's d. in the h group, correlations of vibration sensation with age, height, and weight were assessed with pearson's r. independent samples t-tests were performed to evaluate the difference between the vibration sensations measured at the gt and mm. differences between the results elicited in the pnp group and those found in an age-matched subgroup of h (13 females and 9 males, aged 40 years (median 53, min 40, max 68)) were analyzed with independent samples t-tests. the mean durations of vibration sensation and the differences between the results elicited without and with socks are given in table 1. the effect of wearing socks was found to be insignificant except that found on the mm in the h group, which was seen to have a small effect size upon further analysis with cohen's d<0.2 (d=0.17). vibration sensation was found to decline with age in the h group for two measurement points and both conditions. pearson's r in measurements without socks were 0.502 (p<0.001) and 0.432 (p=0.001), and with socks were 0.467 (p=0.002) and 0.416 (p=0.003), respectively (figure 2). the duration of vibration sensation was shorter at the mm as compared to gt in the h group (the mean difference was 4.3 s, p<0.001). a smaller difference in the same direction was also observed in the pnp group (1.5 s mean difference, p=0.012) (figure 2). the vibration sensation of only one of the 50 participants in the h group, a 68-year-old male, measured less at the gt than at the mm, whereas 3 patients in the pnp group were found to have shorter vibration durations at the gt. as compared to the age-matched subjects from the group h, the duration of vibration sensation in the pnp group had a significantly shorter vibration duration at the gt but not at the mm measurement points (p<0.01 and p=0.12, resp.). the present study reveals that the mean durations of vibration sensation measured without and with socks are nearly the same; there is practically no difference between them in normal subjects and patients with diabetic polyneuropathy. although a statistically significant reduction with socks was found in the measurements at the mm in group h, its effect size was small and the mean difference was less than one second. since clinicians generally express the duration of vibration sensation by whole seconds, without using fractions, the size of the difference found at the mm does not seem important enough to affect the results of clinical examination. as far as we know, this is the first study on humans to investigate the perception of vibration over clothing. other examples of clinical studies which investigated the effect of clothing during physical examination do exist, including the auscultation of lung sounds through thin clothing and measurement of blood pressure through sleeves using the auscultatory method. these studies all resemble each other in that the focus of interest is sounds and vibrations and the spectrums in question are also similar. kraman showed that lung sound perceived by stethoscope was somewhat deteriorated by one or two layers of indoor clothing but this effect can be reduced by force on the stethoscope head. liebl et al. did not found statistically significant difference between sleeved and nonsleeved arm during manual auscultatory sphygmomanometric and automatic oscillometric blood pressure measurement. an everyday experience was also found to be congruent with these results, that of a vibrating cell phone. it was observed that one is easily able to perceive the vibrations of a cell phone in her/his pocket through a significant amount of clothing. with this observation in mind, we measured the frequencies of incoming call vibrations of several models of cell phones and found that the range was 116 to 250 hz. it is not surprising that this range includes the optimum frequency range for pacinian corpuscles, namely, between 60 and 400 hz. all these data suggest that the interference from garments on vibration energy is little. with regard to the vibration of a cell phone in vibration sensing, the use of a pager device in its vibration mode was reported to be used for clinical vibration testing in an era when cellular phones were not so common. the tuning fork is the most readily available instrument in daily clinical practice for measuring vibration sensation and is sufficiently reliable when compared to other techniques of measurement [7, 8]. several studies report the use of the rydel-seiffer graduated 64 hz tuning fork because of its semiquantitative properties [79]. although one was available at the installation phase of the present study, it was discarded because the semiquantitative scale of this device seemed to provide insufficient time resolution for our requirements. the other reason for discarding this tuning fork was that its 64 hz was thought to be in the preferential frequency range of meissner's corpuscles rather than the pacinian afferents. the physiology of pacinian corpuscles may play a critical role in the sensation of vibration through clothing, due to their subcutaneous localization and wide field of perception [1, 10]. the shorter vibration durations at the mm than at the gt are an unexpected finding for clinicians familiar with axonal polyneuropathies, due to the distal to proximal progression of degeneration. it has been concluded that substantial tissue damping affects the tuning fork while taking measurements at the mm. this is probably caused by the solid mass of the tibia which is substantially larger than that of the toe bones beneath the gt measurement points. we think that the shorter vibration durations at the mm can be attributed to this phenomenon, which has been analyzed in detail in the article by goldberg and lindblom. mean vibration duration at the gt was higher than that at the mm even in the pnp group, although the difference was less. this may be due to the fact that this study involved only mild to moderate cases of diabetic pnp and excluded the more advanced cases in whom vibration sensation was measured as totally absent distally. it might be expected that, with more advanced disease, the gt measurement would have been able to exceed the mm in shortening of vibration sensation duration due to the distal axonopathy process. in conclusion, this study shows that no practically significant difference arises from wearing socks when measuring vibration sensation with a tuning fork; any interference thus caused is negligibly small. this is true for the healthy population over a wide age range and also for patients within the clinical context of a diabetic polyneuropathy. however, this study does not aim to recommend measuring vibration sensation over socks. in the patients with chronic polyneuropathy, careful examination of the feet is crucial for the screening of ulcers and infections and leaving the socks in place during examination is strongly discouraged. besides, the socks would still have to be removed in order to perform the other modalities of neurological examination. bearing these in mind, we hope that the results of the present study will be considered as a clinical clue indicating one of the remarkable properties of vibration sensation.

objective. to investigate the difference between the measurement of vibration sensation without and with socks. material and methods. fifty healthy volunteers (h group) and 19 patients with diabetic polyneuropathy (pnp group) were included. the sites of measurement were the great toe (gt) and medial malleolus (mm). a standard 128 hz tuning fork was used in the measurements. results. mean duration of vibration sensations without and with socks was as follows: in the h group, 19.4 4.2 and 19.5 4.2 s at gt and 15.1 3.3 and 14.6 3.3 s at mm; in the pnp group, 13.4 3.8 and 12.7 4.1 s at gt and 11.9 3.8 and 11.7 3.4 s at mm. no significant difference was found between the measurements without and with socks, except those found at the mm in group h (p=0.02). this significant difference was further analyzed in terms of effect size which was concluded to be practically insignificant (cohen's d<0.2). shorter mean vibration duration was measured at mm as compared to gt that could be explained by the damping effect. conclusions. wearing socks of moderate thickness does not have any important effect on the duration of vibration sensation. this might be considered as a reflection of the remarkable properties of vibration sensation.

PMC3824314

pubmed-649

alzheimer's disease (ad) is a prominent neurodegenerative disorder characterized by chronically progressive global cognitive decline. mild cognitive impairment (mci) has been recognized as intermediate between normal elderly cognition and dementia. a proportion of individuals with mci may progress to ad and have typical biological changes of this kind of dementia. to date, the underlying mechanisms involved in the degeneration of cerebral neurons and synapses in ad remain an enigma, but the theory about the involvement of inflammatory processes and immune dysregulation in their pathogenesis has been widely demonstrated. neuropathological and neuroradiological studies have demonstrated that inflammatory changes in ad brains are a relatively early pathogenic event that precedes the process of neuropil destruction [3, 4]. in addition, studies have demonstrated that the brains and cfs of ad and mci contained various proinflammatory substances, such as cytokines, acute phase proteins, and complement proteins [57], which have a fundamental role in inducing cognitive decline, memory loss, and dementia. it has been proposed that the autoimmune mechanism may be a trigger for ad, which was confirmed by the presence of autoantibodies and the apparently good outcome after immunotherapy as seen both in the animal model and in a few patients tested. circulating autoantibodies against a are several times higher in individuals with ad, and antibody titers correlate with cognitive dysfunction. active immunization and the use of monoclonal antibodies for passive vaccination have provided encouraging data in transgenic mouse models of ad. osteopontin (opn), also called early t cell-activation gene 1, is a negatively charged acidic hydrophilic protein that is produced by various cell types and participates in diverse physiological and pathological processes, including bone mineralization, oxidative stress, remyelination, wound healing, inflammation, and immunity [1113]. the expression of opn was elevated in the brains of rats with experimental autoimmune encephalomyelitis (eae) but not in brains of rats protected from eae, and severity of eae was significantly reduced in opn deficient mice. in concordance with those findings in animal models, opn transcripts were frequently detected and were exclusive to the multiple sclerosis mrna population, but not found in control brain mrna. in addition, the expression levels of opn in plasma and tissues are also elevated in other several inflammatory or autoimmune disorders, such as rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, and lymphoproliferation disease [1619]. it has been well studied that interactions between opn and its receptors (including v3, 51 and cd44) mediated survival, migration, and adhesion in many types of cells [20, 21]. as a proinflammatory mediator, opn plays a role in the progression of chronic inflammatory and autoimmune diseases through various mechanisms, including involving in generation of th1 and th17 cells that are pathogenic t cells for various inflammatory diseases [2224], inhibiting apoptosis of autoreactive immune cells and recruitment of leukocytes to sites of inflammation [21, 25]. recently, the important role of opn in ad has been investigated both in humans and animals model. in ad brains, there was a significant 41% increase in the expression of opn in pyramidal neurons compared with age-matched control brain, and there was a significant positive correlation between opn staining intensity and amyloid-beta load. by means of proteomic analysis of csf samples, simonsen and colleagues identified a phosphorylated c-terminal fragment of opn that was increased in patients with mci progressing to ad as compared to patients who remain stable over time and healthy controls. demonstrated that opn levels are increased in the csf of ad subjects as compared to controls and its levels are more markedly raised in the early stages of the disease and correlate with cognitive decline. in addition, upregulated opn expression has also been demonstrated in app/ps1 ki mice, an animal model of ad with severe pathological alterations. collectively, these findings strongly suggest the involvement of opn in the development of ad. to further clarify the role of opn in the progression of cognitive decline, we longitudinally assessed the opn expression changes in the plasma and csf in the same individuals. on the other hand, we transversely analyzed opn levels in patients with mci, newly diagnosed ad, and chronical ad, comparing the results with those obtained in the groups of healthy control and other noninflammatory neurologic diseases (ond). thirty-five patients affected by ad and thirty-one patients with a diagnosis of mci were selected for the study. table 1 lists the demographic data of the subjects, including gender, age, and the mini-mental state examination score (mmse), which is a general measure of cognitive performance. the clinical diagnosis of ad was performed according to nincdsadrda work group criteria and dsm iv-r [32, 33]. the mean age of ad patients (16 males and 19 females) was 78.2 years (age range 5880 years). all patients underwent complete medical and neurological evaluation, laboratory analysis, ct scan, or mri to exclude reversible causes of dementia. standard laboratory tests performed at the time of diagnosis included complete blood count, serum electrolytes, serum glucose, blood urea nitrogen, b12, folate, thyroid function tests, and serology for syphilis. neuropsychological evaluation and psychometric assessment was performed with a neuropsychological battery including mmse, digit span forward and backward, logical memory and paired associated words tests, token test, supra span corsi block tapping test, verbal fluency tasks, raven colored matrices, the rey complex figure, clinical dementia rating scale (cdr), and the hachinski ischemic scale. thirty-two patients were late and three early ad were onsets; all cases were sporadic. ad patients were divided into two subgroups according to disease duration: 17 newly diagnosed ad patients (adn, disease duration 2 years) and 18 chronical ad patients (adc, disease duration>2 years). the diagnosis of mci was based on the following unanimously adopted criteria: (1) reported cognitive decline; (2) impaired cognitive function; (3) essentially normal functional activities; and (4) exclusion of dementia [35, 36]. the mean age of mci patients (17 males and 14 females) was 74.2 years (age range 5782). all of these patients received neurological examination, laboratory test, and brain mri to exclude intracranial mass, infarcts, moderate to severe nonspecific white matter disease, and reversible causes of cognitive impairment. all patients had a follow-up visit every 6 months, and the monitoring period was 3 years. based upon subsequent diagnosis status at follow-up evaluations, mci participants can be divided into two subgroups: 13 mci patients who have converted to ad (mci converters, mcic) and 18 mci patients who have not converted to ad (mci nonconverters, mcinc). twenty patients with ond (11 females and 9 males, age 5979 years, mean age 76.4 13.1 years) were also enrolled in the study. these patients with the following conditions: 2 strokes, 5 transient ischemic attacks, 4 chronic intractable headache, 3 status epilepticus, 3 normal pressure hydrocephalus, and 3 peripheral neuropathies. plasma samples were also obtained from 24 healthy elderly subjects (hc), age and sex matched with the patient (11 males and 10 females, age 5781 years, mean age 74.7 14.5 years). these individuals were either unrelated healthy spouses of ad and mci patients or healthy volunteers, and they had no family history of dementia or evidence of acute or chronic diseases at the time of enrollment. the cognitive status of ond and hc was assessed by administration of mmse (score for inclusion as normal control subjects 28). all formal neurocognitive test scores for these participants were within 1.5 standard deviations of normative data in published studies or manuals. patients with an inflammatory or infectious disease, with a history of immunological or malignant disease, medication of immunologically relevant drugs, abnormal white blood cell count, and abnormal csf findings, were not included into the study. all study procedures were approved by the harbin medical university, china, institutional review board, and all participants or their representatives gave informed consent. all of blood and csf samples were obtained at the initial visit. in those mci converters, the second blood and csf samples were available when they received a diagnosis of ad during followup. after lumbar puncture, csf samples (2030 ml) were obtained and collected in polypropylene tubes. the samples were centrifuged at 2,000 g at 4c for 10 minutes to eliminate cells and other insoluble material and were then immediately frozen and stored at 80c pending biochemical analyses, without being thawed or refrozen. cell count was performed on the csf samples and no sample contained more than 500 erythrocytes/l. the opn protein content in serum and csf was measured using a commercial elisa according to the manufacturer's instructions (assay designs, inc. briefly, serum and csf samples were diluted, respectively, 1: 20 and 1: 50 into assay buffer provided by the manufacturer and were incubated at 37c for 1 h in microtiter plates precoated with a polyclonal n-terminal capture anti-opn antibody (assay designs). then, the plates were washed and wells were incubated at 4c for 30 min with a horseradish peroxidase labeled opn-specific monoclonal antibody (assay designs). after washing, the wells were incubated with tetramethylbenzidine-h2o2 solution for 30 min. the color reaction was stopped by adding a solution containing 1 n sulfuric acid. optical densities were measured at 450 nm with reference wavelength set at 590 nm. the opn concentrations were calculated using a standard curve of recombinant human opn provided by the manufacturer. the lower detection limit of the kit was 3.33 ng/ml. baseline and follow-up csf and plasma samples from a patient were measured on the same plate. assays were repeated when the difference between the two probes of one sample was more than 10%. normally distributed data sets were analysed by student's t-test, paired t-test, analysis of variance (anova), and linear regression and correlation analysis (using primer for biostatistics). there was no difference in sex distribution in mci, ad, and ond patients and the healthy control subjects. plasma opn concentrations in healthy controls (51.4 9.8 ng/ml) and ond (53.3 10.3 ng/ml) did not differ significantly and did not correlate with age and sex. plasma opn concentrations in mcinc (52.3 11.7 ng/ml), mcic (53.7 11.1 ng/ml), adn (74.4 13.7 ng/ml), adc (54.8 10.1 ng/ml), ond patients, and healthy controls differed significantly (figure 1(a); p<0.001). adn patients had higher plasma concentrations of opn than the healthy controls (p<0.005), whereas plasma concentrations of opn in the mcinc, mcic, and adc patients did not differ significantly from plasma opn concentrations in ond or healthy controls (figure 1(a)). we next questioned whether the plasma opn concentrations would vary within the same individual in relation to disease status. in 13 mcic individuals tested longitudinally, the plasma opn concentrations were significantly elevated when they received a diagnosis of ad during followup (figure 1(b)). the csf represents the fluid compartment that is closest to reflect the inflammatory situation in the degenerative processes of the nervous system, so we then sought to compare the concentrations of opn in csf from patients with mci, ad, and ond. csf opn concentrations differed significantly in mcinc (128.6 17.7 ng/ml), mcic (173.2 20.6 ng/ml), adn (226.5 21.2 ng/ml), adc (165.6 20.4 ng/ml), and ond patients (134.5 19.3 ng/ml) (figure 2(a)). patients with mcic, adn, and adc (p<0.001) had significantly higher opn concentrations in the csf than the neurological controls. the concentrations of opn in csf from patients with adn was significantly higher than that from patients with adc (p<0.001). we also determined whether the csf opn concentrations would vary within the same individual in relation to disease status. in 13 mcic individuals tested longitudinally, the csf opn concentrations were significantly elevated when they received a diagnosis of ad during followup (figure 2(b)). when comparing paired csf and blood samples from patients with mci, ad, and ond, the concentrations of opn in the csf were significantly higher than plasma concentrations in all patients (figure 3). first, we determined whether there was a correlation between the levels of opn in the csf and the degree of cognitive decline in ad patients. a strongly positive correlation between the csf opn concentrations and the mmse score (r=0.53, p<0.001) was observed (figure 4(a)). secondly, we explored whether there was a correlation between the levels of opn in the csf and disease duration of ad patients. our result showed that the csf opn concentration was correlated inversely with the disease duration (r=0.51, p<0.001) (figure 4(b)). no other clinical parameters, such as gender, age, a42, tau, and p tau levels, had any significant correlation with the levels of opn in the csf of ad patients. although a trend was noted toward the positive correlation between the plasma opn concentrations and the mmse score in the adn patients, it was not statistically significant. by contrast, no clear correlation was found between the csf opn concentrations of mcic and both the degree of cognitive decline and disease duration. in this study, the crosswise comparison demonstrated that csf opn concentrations were significantly increased in ad and mci converters compared to ond, and opn protein levels both in the csf and plasma of newly diagnosed ad patients were higher than that of chronical patients. furthermore, in mci converters individuals tested longitudinally, both plasma and csf opn concentrations were significantly elevated when they received a diagnosis of ad during followup. finally, opn csf levels displayed direct correlation with the mmse score and inverse correlation with disease duration. our data have shown that the plasma concentrations of opn were significantly increased in the group of newly diagnosed ad than the other groups, and there was a trend toward the positive correlation between the plasma opn concentrations and the mmse score, although it was not statistically significant. one previous study on serum opn concentrations in ad patients gave a different result from ours. found that there were no significant differences in serum opn concentrations comparing ad to controls. the results of the present study suggest that the differences between the results of the previous study may, at least partly, be explained by that we stratified patients according to their disease duration. studies demonstrated that high levels of plasma opn were well correlated with the activities of various disease conditions: plasma opn concentrations were significantly higher in systemic lupus erythematosus patients and increase in opn concentration correlated positively and significantly with sledai score in all patients; serum opn concentrations of patients with idiopathic retroperitoneal fibrosis were elevated compared to healthy controls and correlated with the transverse diameter of the periaortic cuff as determined by imaging studies. in addition, elevated plasma opn levels have been also shown to play an important role in inflammatory and degenerative processes of the central nervous system: opn plasma levels were elevated in secondary progressive ms compared to relapsing-remitting ms patients in remission and healthy controls, supporting a role for opn in the chronic disease activity; opn serum levels were elevated in parkinson's disease and higher serum levels were associated with more severe motor symptoms. prospective epidemiological studies show that elevated plasma levels of acute phase reactants can be considered as a risk factor for ad. therefore, we speculate that elevated opn plasma levels in the initial stages of ad may contribute to the progression of cognitive decline, although the exact role of opn and its underlying mechanism as a key proinflammatory cytokine in ad is not understood. th17 cells have been shown to be a pathogenic effector cell for development of various inflammatory and autoimmune diseases. shinohara and colleagues found that opn plays a critical positive role in the differentiation of th17 cells by repressing il-27 secretion in mouse dendritic cells. a recent report showed that th17 t cells were increased in ad patients, which favors the speculation that elevated opn plasma levels in ad may be associated with the differentiation of th-17 cells. accumulating evidence suggests that inflammation mainly occurs in pathologically vulnerable regions of the ad brain (such as the entorhinal, temporoparietal, and cingulate cortex), with increased expression of acute phase proteins and proinflammatory mediators. therefore, we further examined the concentrations of opn in the csf, which directly contact with brain and can accurately reflect the ongoing inflammatory process in the central nervous system. our results showed that the csf concentrations of opn were significantly increased in patients with mci progressing to ad than that in stable mci. prediction of conversion from mci to ad is of major interest in ad research, which would allow for the appropriate application of disease-modifying treatments at a point where clinical manifestations are limited. presently, there are few clinical or imaging markers for the early identification of mci which progresses to ad and mci which does not progress. recently, simonsen and colleagues found that a phosphorylated c-terminal fragment of opn was increased in the csf of patients with mci progressing to ad as compared to patients who remain stable over time and healthy controls and proposed opn as a biomarker to predict the progression of mci to overt ad. the findings of these two studies showed that not only the intact forms of opn but also the cleaved form of opn was increased in the csf of patients with mci progressing to ad. more importantly, our results showed that the csf opn concentrations of mcic were significantly elevated when they received a diagnosis of ad during followup. our findings are in agreement with another study, which showed that ad patients displayed about a two-fold increased opn levels in the csf compared to age-matched controls and it was particularly striking in the early stages of the disease. to identify protein changes during the presymptomatic phase of ad, ringman and colleagues performed proteomic analyses of csf from persons with or at risk for inheriting familial ad using high-resolution liquid chromatography-mass spectrometry. their results showed that opn was elevated in the csf of familial ad mutation carriers compared to related noncarriers, which suggest changes of opn in the csf occurring a decade before clinical dementia. in addition, increased serum and csf opn levels were also detected in the lewy dementia, and the genotypic variation of snp-66 was associated with the occurrence of the lewy body dementia. studies showed that opn seemed to act as a double-edged and might exert two opposite functions in the progression of neurodegenerative diseases. on one hand, opn functions as a neuroprotectant by upregulating myelination and remyelination. on the other hand, opn had a disease-accelerating role by triggering neuronal toxicity and death. in the current study, though we could not directly conclude that the increased levels of opn within the csf stimulate the degeneration of cerebral neurons and synapses in ad, we conjecture that opn may favor ad development because mcic patients had high levels of csf opn and the concentrations of opn in the csf and plasma were further increased when they received a diagnosis of ad. their results demonstrated that opn expression was increased in the pyramidal neurons of the ca1 region of the hippocampus of ad patients and opn staining intensity positively correlated with both amyloid-beta load and age. increased opn expression may exacerbate the abnormal immune response presented in the ad brain by enhancing the survival of activated t cells, which were detected in the brain tissues of ad patients. in conclusion, the bell-shaped curve of csf opn expression in disease progression of cognitive decline has extended the evidence for a role of opn in ad pathogenesis. it will be important to study larger cohorts of individuals with longer durations of followup, and from different centers, to further evaluate whether higher baseline level of csf opn was associated with a more marked decline of mmse over followup.

inflammatory mediators are closely associated with the pathogenesis of neurodegenerative changes in alzheimer's disease (ad) and mild cognitive impairment (mci). osteopontin (opn) is a proinflammatory cytokine that has been shown to play an important role in various neuroinflammatory diseases. however, the function of opn in ad and mci progression is not well defined. cerebrospinal fluid (csf) and plasma samples were obtained from 35 ad patients, 31 mci patients, and 20 other noninflammatory neurologic diseases (ond). concentrations of opn in the csf and plasma were determined by enzyme-linked immunosorbent assay. during a 3-year clinical followup, 13 mci patients converted to ad (mci converters), and 18 were clinically stable (mci nonconverters). csf opn concentrations were significantly increased in ad and mci converters compared to ond, and increased levels of opn in ad were associated with mmse score; opn protein levels both in the csf and plasma of newly diagnosed ad patients were higher than that of chronical patients. in mci converters individuals tested longitudinally, both plasma and csf opn concentrations were significantly elevated when they received a diagnosis of ad during followup. further wide-scale studies are necessary to confirm these results and to shed light on the etiopathogenic role of osteopontin in ad.

PMC3612435

pubmed-650

the eye is an immune privileged site where the introduction of antigens does not elicit an inflammatory immune response. the first line of protection is the blood-ocular barrier, which impedes harmful pathogens from entering the eye via the peripheral bloodstream. a regional immune system provides a second multilayered defense in case the blood-ocular barrier is breached. the anterior chamber is bathed in aqueous humor fluid, which is strongly immunosuppressive and profoundly inhibits t cell activation. in addition, low expression of major histocompatibility complex (mhc) class ii molecules limits antigen presentation to immune cells reducing the chances of an immune response. stromal cells of the iris and ciliary body have the ability to convert effector t cells to regulatory t cells and expression of death-inducing molecules results in apoptosis of immune cells keeping the immune response in check. furthermore, aqueous humor outflow regulation, which is important in glaucoma, may be impacted by the innate immune system. the glaucomas are a group of optic neuropathies with a characteristic pattern of damage to the optic nerve that leads to loss of peripheral vision (see figure 1). a leading cause of global irreversible blindness, glaucoma will impact 111.8 million individuals by 2040. the number of affected individuals is likely to be much higher than the reported number because the disease is usually asymptomatic up until major neural damage has occurred [7, 8]. it is clear that it is a heterogeneous group of disorders with multiple causative factors including gene mutations, environmental factors, and certain medications. moreover, there are several risk factors that predispose to the disease including advanced age, elevated intraocular pressure, race, and family history [912]. while lowering intraocular pressure often reduces the rate of vision loss, many patients continue to go blind despite apparently successful pressure control [10, 11, 13]. since there is no single causative factor, there are potentially many disease mechanisms behind glaucoma. some lead to higher intraocular pressures, while others affect how the optic nerve withstands either pressure fluctuations or sustained elevation of intraocular pressure. thus, glaucoma research has focused on the front of the eye, where intraocular pressure is regulated by a tissue called the trabecular meshwork, the back of the eye, where studies have focused on retinal ganglion cell physiology and optic nerve damage, and distal changes in retinal ganglion cell axons in terminal projection sites such as the superior colliculus and the lateral geniculate [1418]. diagnostic criteria for glaucoma have been revamped significantly over the past 40 years with increased emphasis on characteristic changes in the optic disc and retinal nerve fiber layer and less reliance on elevated intraocular pressure [9, 19]. normal tension glaucoma, that is, patients with statistically normal intraocular pressures, makes up 30 to 40% of glaucoma cases [13, 20]. even for these patients, the only effective treatment for glaucoma continues to be reduction of intraocular pressure levels by either pharmaceutical or surgical means. physiological intraocular pressures are established by maintaining a balance between production and drainage of aqueous humor in the anterior chamber. aqueous humor is continuously produced by the ciliary processes and it bathes tissues in the anterior chamber before exiting out to schlemm's canal via a filter-like tissue called the trabecular meshwork (see figure 2). building resistance to aqueous humor outflow in the trabecular meshwork produces a tunable system by which this filter can increase or decrease outflow when needed. dysfunction of this conventional outflow pathway leads to impaired drainage and elevated intraocular pressure, as is seen in poag patients. increased intraocular pressure places excessive mechanical stresses on the lamina cribrosa of the optic nerve in the posterior segment of the eye and loss of retinal ganglion cells ensues. since these cells are responsible for transmitting visual signals to the brain, irreversible blindness occurs. inflammatory responses may contribute to the glaucomatous process as shown by studies in humans and rodent models [2326]. in the anterior segment, certain inflammatory cytokines have altered expression levels in the aqueous humor of glaucomatous eyes compared to age-matched normal eyes. these include interleukin-6 (il-6), transforming growth factor beta-1 (tgf1), tgf2, il-6, il-8, il-10, il-12, -serum amyloid a, interferon- (ifn), and cxl9 [2733]. the source of these cytokines in the aqueous humor of glaucoma patients is not clear. however, acute elevation of intraocular pressure, such as in primary angle-closure glaucoma, damages the blood-aqueous-barrier (bab), which can lead to leakage of the cytokines into the aqueous humor. inflammation-related changes also occur in the posterior segment. in a mouse model of laser-induced ocular hypertension, there was upregulated expression of mhc-ii and glial fibrillary acidic protein (gfap) in the microglia of contralateral eyes. the authors suggest that microglial activation in the nontreated eye could be related to an immune response [35, 36]. in humans, however, a systemic autoimmune response is less convincing because the contralateral eye in patients with unilateral glaucoma does not appear to exhibit glaucomatous degenerative changes. however, proinflammatory cytokines such as tumor necrosis factor- (tnf) and its receptor are upregulated in glaucomatous human optic nerve [3840]. moreover, use of bupropion, which suppresses tnf production, significantly lowered the risk of developing poag in humans in a large retrospective study, while anti-tnf medication (etanercept) was found to be neuroprotective in a rodent model of glaucoma. thus, both in glaucoma patients and in rodent models of glaucoma, there is accumulating evidence for a potential role of the immune system in contributing to deleterious changes in anterior and posterior ocular tissues. our recent identification of a mutation in interleukin-20 receptor-b (il-20rb) supports this contention. our group mapped a gene in a large poag oregon family to chromosome 3, the glc1c locus. eighty-six family members, ranging in age from 8 to 91 years old, had extensive ophthalmic examinations. thirteen family members were diagnosed with poag and twelve of these had elevated intraocular pressure (22 to 49 mm hg). an additional nine individuals, who do not have poag at this time, had elevated intraocular pressures. after refining the region to 4 cm, we sequenced all 49 genes in the glc1c locus and identified one nonsynonymous mutation: a t104 m change in il-20rb (rs367923973). this is an extremely rare variant with a reported frequency of 0.02% in dbsnp (http://www.ncbi.nlm.nih.gov/snp). this mutation, t104 m, lies in il-20rb's active binding site for the cytokines, il-19, il-20, and il-24, which are all members of the il-20 subfamily of interleukins (see below). substitution of t104 with a methionine would replace the hydroxyl group that forms a hydrogen bond with s111 in il-20 with a sulfate group, thus disrupting the bond between the cytokine and its receptors. based on these findings, this il-20rb mutation is highly likely to impact the il-20 signaling pathway, which may contribute to the pathogenesis of glaucoma in this family. the il-20 subfamily of cytokines and receptors are members of the larger il-10 family, which are grouped together based on their utilization of common receptor subunits, similarities in their target-cell profiles, and biological functions. this subfamily consists of the cytokines, il-19, il-20, il-22, il-24, and il-26, as well as the receptors, il-20ra, il-20rb, il-10rb, and il-22ra1. il-19 exclusively signals through the il-20ra/il-20rb heterodimer, while il-20 and il-24 can use both the il-20ra/il-20rb heterodimer and the il-22ra1/il-20rb receptor. it should be noted that il-20ra and il-20rb are also known as il-20r1 and il-20r2, respectively. the il-20 subfamily participates both in amplifying inflammatory responses particularly during autoimmune and chronic inflammation and alternatively in anti-inflammatory responses, such as tissue protection and regeneration [47, 48]. thus, understanding how regulation of this subfamily is occurring is paramount in devising new treatment strategies for patients in this large poag family. both il-20ra and il-20rb are expressed in normal human trabecular meshwork cell lysates and are upregulated in response to cytokine treatment. il-20rb mrna is also expressed in moderately high levels in both the retinal ganglion cell layer and the optic nerve head in rats (dr. elaine johnson, personal communication). il-20, il-24, il-20ra, and il-20 rb are expressed in the retina and optic nerve head in the dba/2j mouse model of glaucoma (see later). however, il-19 is not expressed in the retina or optic nerve head of dba/2j mice. binding of il-20 to its receptor activates the janus kinase- (jak-) signal transducer and activator of transcription (stat) pathway (jak-stat) [51, 52]. elevation of intraocular pressure has also been shown to activate the jak-stat pathway, which is involved in retinal ganglion cell survival. activation of stat3 can be both pro- and anti-inflammatory even within the same cell type, but how the desired response is elicited remains a question. for example, il-6 and il-10 both activate stat3, but they generate different cellular responses with il-6 generating a proinflammatory response and il-10 producing an anti-inflammatory one. these differences appear to correlate with the level of stat3 over time, with il-6 producing a transient activation while il-10 generates a sustained level of stat3 activation. the stat3-induced protein, suppressor of cytokine signaling-3 (socs3), may be involved because it mediates signaling dynamics due to its ability to inhibit signals from the il-6 receptor, but not the il-10 receptor. several studies have linked il-20 signaling to mmp levels and activity. in breast cancer cells, il-20 appears to act synergistically with il-1 in these cells: higher levels of tnf, il-1, il-6, il-8, mmp-3, and monocyte chemoattractant protein-1 (mcp1) were found when disc cells were treated with both cytokines compared to exposure to il-20 or il-1 alone. induction of mmp activity decreases the resistance to aqueous humor outflow through the trabecular meshwork which, in turn, lowers intraocular pressure. to investigate whether normal downstream signaling pathways were active in mutant cells, we asked if il-20 treatment induces stat3 phosphorylation and mmp activity in normal dermal fibroblasts and in poag patient fibroblasts with the il-20rb mutation. fibroblasts from glaucoma patients with the t104 m il-20rb mutation had higher basal levels of phosphorylated stat3 compared to wild-type fibroblasts (see figure 3). stimulation of wild-type fibroblasts by il-19, il-20, or il-24 cytokines led to a significant increase in phosphorylation of stat3 after 15 minutes, but this was not found in glaucomatous fibroblasts with the t104 m il-20rb mutation. using a quenched fluorescent peptide assay that produces fluorescent signal when cleaved by a mmp, we showed that il-20 increased mmp activity in wild-type human fibroblasts, but not in fibroblasts with the t104 m il-20rb mutation (see figure 4). the differential response of mutant and wild-type cells to cytokine treatment suggested that poag cells with the t104 m mutation are unable to launch an appropriate cell signaling response upon stimulation by the il-20 family of cytokines. collectively, these observations suggest that, in normal cells, il-20 and related cytokines bind to il-20 receptors on the cell surface (figure 2(c)). this would in turn activate stat3, which would translocate to the nucleus to modify transcription of inflammatory-related genes. the anti-inflammatory response would include upregulation of il-20 receptors by trabecular meshwork cells and activation of mmps leading to remodeling of the extracellular matrix. this would lead, ultimately, to greater outflow of aqueous humor. in glaucoma patients with the il-20rb mutation (figure 2(d)), the il-20 family of cytokines would not bind efficiently to the il-20ra/rb receptor so stat3 would not be activated and translocated to the nucleus. therefore, the anti-inflammatory response would not be adequate and sustained expression of proinflammatory genes would remain. subsequently, many secondary downstream effects may contribute to the elevated intraocular pressure observed in glaucoma patients harboring the il-20rb mutation and, ultimately, to glaucomatous damage. the dba/2j mouse model of glaucoma is an inbred mouse strain that progressively develops glaucoma-like abnormalities with aging. in the anterior chamber, the mice develop a form of pigment dispersion syndrome with the primary action being an inflammatory response resulting in elevation of intraocular pressure. the dba/2j mouse has two distinct phenotypes: iris pigment dispersion, which may be involved in immune dysfunction in dba/2j eyes, and iris stromal atrophy. these phenotypes are caused by mutations in the gpnmb and tyrp1 genes, respectively, [66, 67]. gpnmb is expressed in some types of dendritic cells [68, 69], which are potent professional antigen presenting cells, whereas tyrp1 is an antigen that is involved in inflammatory eye disease. as discussed above, however, the aqueous humor of dba/2j mice lacks immunosuppressive properties and the capacity to support anterior chamber associated immune deviation. iris pigment is shed into the aqueous humor, where it enters the outflow pathways and eventually causes blockage of the drainage channels. dba/2j mice between 6 and 7 months of age begin to lose retinal ganglion cells. by 1012 months, significant retinal ganglion cell loss has occurred in the majority of dba/2j mice [72, 73]. however, if the mice are exposed to a high dose of -irradiation, the dba/2j mice are protected from developing glaucomatous damage, although they still have high intraocular pressures [73, 74]. both retinal and optic nerve morphology appear normal in the irradiated mice, whereas untreated dba/2j mice show optic nerve atrophy, as well as clear loss of retinal ganglion cell axons. a unique finding in the dba/2j mice is a high level of activated microglia in the inner central retina and optic nerve region at 3 months, which occurs well before the loss of retinal ganglion cells. il-19 is the mostly highly upregulated gene in activated microglia and, in addition, the il-20ra and il-20rb receptors are expressed. il-19 has anti-inflammatory activity in microglia via stat3 activation, which leads to an anti-inflammatory response. irradiation reduces the number of proliferating microglia in the optic nerve head along with a reduction in the levels of microglia activation in the central retina, optic nerve head, and laminar region in the dba/2j mouse [74, 77]. minocycline, a neuroprotective tetracycline derivative that suppresses chronic neuroinflammation and microglial activation, also has a protective effect on retinal ganglion cell viability in the dba/2j mouse. in dba/2j mice with moderate axon damage, il-24 expression is significantly increased in the retina, but no significant differences were seen for either il-20 or il-20rb. conversely, il-20ra levels are significantly reduced in optic nerves from eyes with severe axon damage in dba/2j mice compared to dba/2j-gpnmb./il-20rb signaling is impaired, which could ultimately lead to altered extracellular matrix remodeling by mmps via inappropriate stat3 activation as described above. changes in extracellular matrix composition and organization in the glaucomatous optic nerve head have been well documented and include deposition of extracellular matrix materials in areas formerly occupied by axons. the extracellular matrix changes likely contribute to the altered biomechanical properties of the glaucomatous optic nerve head and increase the vulnerability of the remaining axons to cell death [80, 81]. in conclusion, several lines of evidence provide compelling evidence for a role of the il-20 family of cytokines in glaucoma. first, we have identified a mutation in il-20rb at a residue that is critical for binding of the receptor to the il-20 family of cytokines in a large poag pedigree. second, il-20 stimulation of the mutant il-20rb leads to abnormal stat3 activation and mmp activity in glaucoma fibroblasts. third, il-20 family members and their receptors have altered expression levels in the retina and optic nerve head of the dba/2j mouse, which develops glaucoma-like symptoms with aging. while the il-20rb mutation most likely is not a causative factor in the majority of glaucoma cases, the identification of this mutation has revealed that defective il-20 signaling may lead to glaucoma in this large poag pedigree. future studies will focus on the role of the il-20 subfamily members in both aqueous outflow regulation in normal trabecular meshwork and their anti-inflammatory role in the posterior tissues of the eye. this may lead to the development of novel therapeutic treatments to maintain tissue homeostasis and prevent glaucomatous vision loss in this large oregon poag family.

glaucoma is a common disease that leads to loss of peripheral vision and, if left untreated, ultimately to blindness. while the exact cause(s) of glaucoma is still unknown, two leading risk factors are age and elevated intraocular pressure. several studies suggest a possible link between glaucoma and inflammation in humans and animal models. in particular, our lab recently identified a t104 m mutation in il-20 receptor-b (il-20rb) in primary open angle glaucoma patients from a large pedigree. several of the interleukin- (il-) 20 family of cytokines and receptors are expressed in ocular tissues including the trabecular meshwork, optic nerve head, and retinal ganglion cells. the dba/2j mouse develops high intraocular pressures with age and has characteristic optic nerve defects that make it a useful glaucoma model. il-24 expression is significantly upregulated in the retina of these mice, while il-20ra expression in the optic nerve is downregulated following pressure-induced damage. the identification of a mutation in the il-20rb gene in a glaucoma pedigree and changes in expression levels of il-20 family members in the dba/2j mouse suggest that disruption of normal il-20 signaling in the eye may contribute to degenerative processes associated with glaucoma.

PMC4745377

pubmed-651

primary stabbing headache is a short duration headache disorder that is characterized by brief paroxysms of sharp head pain often located in the first division of the trigeminal nerve, occurring at an irregular frequency, lacking any accompanying symptoms, and not attributable to any underlying cause. stabbing headache has also been reported to occur as a symptom of, or in association with, a variety of underlying secondary disorders, including giant cell arteritis [2, 3], pituitary tumors, meningiomas, ocular pathology, ischemic stroke [6, 7], cavernous hemangioma of the frontal bone, and varicella zoster meningoencephalitis. stabbing headache has not been reported to manifest as a direct consequence of a small well-circumscribed acute brain lesion, and its pathophysiology remains elusive. herein, a lesional cause of stabbing headache, a small acute thalamic hematoma, is presented. a 95-year-old right-handed caucasian woman presented to our emergency department with the sudden onset of a constellation of spontaneous neurological symptoms. she first noted repetitive, sharp, 12 s paroxysms of pain in strictly the right frontal and supraorbital region, occurring dozens of times per hour but at irregular and unpredictable frequencies. her pain was unaccompanied by photophobia, phonophobia, cranial autonomic symptoms, or any visual changes. she did not identify any triggers and tactile stimulation of any area of the head or face did not provoke the pain. these attacks continued until she finally fell asleep in the hospital 12 h later, and upon awakening 6 h later, did not recur. minutes after the head pain onset, she noted mildly slurred speech, which also resolved by the following morning, and upon attempting to ambulate she felt very imbalanced, which persisted for several days. her past medical history included hypertension, hyperthyroidism, gastroesophageal reflux, osteoarthritis, and a left intertrochanteric femoral fracture sustained after a fall, which was surgically repaired 4 months previously. postoperatively she developed cholangitis from choledocholithiasis, an acute pulmonary embolism, and paroxysmal atrial fibrillation, for which she was treated with intravenous antibiotics and placed on long-term anticoagulation. she had no history of any primary headache disorder, denied ever experiencing any focal neurological symptoms. her medications included warfarin, fosinopril, omeprazole, metoprolol, digoxin, and methimazole. she lived with her husband and was a retired school teacher. on review of systems, for several months she had ongoing right upper quadrant pain and occasional nausea that had been attributed to choledocholithiasis. she denied jaw claudication, episodes of visual loss, myalgias, or neck pain. her general medical examination revealed a well-appearing woman with normal vital signs, and only mild right upper quadrant tenderness. on neurological examination, deep tendon reflexes were 1+in the arms, trace at the knees, and she lacked ankle jerks. she had a white blood cell count of 12,000/l, a total bilirubin of 2.3 mg/dl, a direct bilirubin of 1.2 mg/dl, an alkaline phosphatase of 560 u/l, a sgot of 341 u/l, and a sgpt of 292 u/l. noncontrast computed tomography of the head revealed a rounded hyperdense lesion in the left lateral thalamus abutting the posterior limb of the internal capsule, consistent with an acute small hemorrhage, along with multiple subcortical lacunes and white matter ischemic changes (fig. 1). the hematoma volume was estimated to be 0.12 cm using the abc/2 method . fig. 1axial noncontrast computed tomography of the brain revealed an acute, rounded hyperdense lesion in the region of the left thalamus and likely abutting the posterior limb of the internal capsule (white arrow), corresponding to a small acute hemorrhage axial noncontrast computed tomography of the brain revealed an acute, rounded hyperdense lesion in the region of the left thalamus and likely abutting the posterior limb of the internal capsule (white arrow), corresponding to a small acute hemorrhage the patient was administered fresh frozen plasma and vitamin k to reverse her coagulopathy and ultimately had an endoscopic retrograde cholangiopancreatography, where numerous gallstones were removed. the patient was ultimately discharged to a short-term rehabilitation facility for her imbalance and deconditioning. the correspondence of the acuity of the clinical syndrome with the radiographic demonstration of an acute hematoma strongly supports its causality, although the lack of magnetic resonance imaging makes the exclusion of other acute lesions slightly less definitive. a fixed site of stabbing headache may signify an underlying symptomatic cause of the disorder, as was the case with this patient. although a stabbing pain character of headache is not uncommon in association with acute cerebrovascular disease, the diagnosis of stabbing headache has not been well described in this population. in a large prospective series of over 2,000 patients with acute ischemic stroke where headache details were captured at stroke onset, 20% of the patients described their pain character as stabbing, although the diagnosis of stabbing headache was not mentioned. pareja and colleagues mentioned the onset of stabbing headache in a patient with an ischemic stroke to the parietal lobe, but did not describe its laterality and relationship to the site of the stabbing head pain. piovesan and colleagues reported the occurrence of stabbing headache in a delayed fashion after an ischemic stroke in three patients who all likely sustained acute middle cerebral artery territory infarctions. regarding hemorrhagic stroke, in a large series of 90 intracranial hemorrhage survivors, six patients experienced stabbing headache de novo after intracranial hemorrhage, only one of whom had no premorbid headache history. acute thalamic hematomas in particular can lead to headache over half the time, but similar to other deep structures do not lead to headache nearly as often as hemorrhage in the posterior fossa or lobar structures. in this patient, stabbing headache resulted from an acute parenchymal brain lesion in the thalamus, a location vital to the processing of pain. more specifically, the location of the hematoma may have encompassed the ventral posteromedial nucleus of the thalamus, a relay center for sensory input from the trigeminal system. the association of the pain strictly occurring in the right trigeminal distribution with a lesion in the corresponding left thalamus is intriguing. some authors have proposed the disorder results from spontaneous activation of peripheral nerve branches in the trigeminal or upper cervical distribution [14, 15]. another theory is that patients with stabbing headache have segmental and fleeting disinhibition of central pain pathways [14, 15]. perhaps in cerebrovascular disease, such disinhibition occurs in an acute fashion in those patients with contralateral pain to the site of infarction or hemorrhage. in stroke patients with ipsilateral stabbing headache this report demonstrates an association of an acute thalamic lesion with stabbing headache occurring in the contralateral trigeminal distribution. particularly in an elderly patient or individual taking anticoagulants, a small thalamic hemorrhage should be considered in the differential diagnosis of new-onset stabbing headache, even when fleeting .

stabbing headache can be encountered in both primary and secondary forms, but has been infrequently reported among patients with stroke, and is not known to be associated with a small well-circumscribed brain lesion. a 95-year-old woman taking warfarin presented with the sudden onset of stabbing headache strictly in the right frontal and supraorbital regions, along with gait imbalance and dysarthria. neuroimaging revealed a small left thalamic hematoma. this association of an acute thalamic lesion with stabbing headache in the contralateral trigeminal distribution is discussed, along with a brief review of stabbing headache occurring in cerebrovascular disease.

PMC3094649

pubmed-652

alzheimer's disease (ad) is the most common cause of dementia among the aging population. early memory deficits and progressive loss of higher cognitive functions are common clinical features of ad patients. pathologically, ad is characterized by insoluble aggregates of extracellular amyloid-beta (a) peptides (senile plaques) and intracellular filaments composed of hyperphosphorylated tau (neurofibrillary tangles) in the brain. strong evidence from human genetics and transgenic mouse models has implicated a in the etiology and pathogenesis of ad. a peptides are derived from -secretase- and -secretase-mediated sequential proteolytic cleavage of the amyloid-precursor protein (app), with a140 and a142 being the most abundant species. many human mutations associated with familial ad, such as those that are found in genes encoding app and the catalytic subunit of -secretase, presenilin (ps1 and ps2), promote amyloidogenic processing of app, leading to enhanced a production. recent studies have shown that soluble oligomeric forms of a (ranging from dimers and trimers to dodecamers) exert potent and acute neurotoxic effects on the structure and function of synapses, including reduced excitatory synaptic transmission, loss of dendritic spines, and aberrant neuronal network activity [4, 5]. these deleterious effects could contribute to the cognitive deficit and memory loss associated with ad, indicating that synaptic failure is likely to be one of the earliest events that occurs in the pathogenesis of ad prior to neuronal loss [68]. the majority of fast excitatory synaptic transmission in the mammalian central nervous system is mediated by the release of glutamate from the presynaptic terminal and its binding to glutamate receptors on the postsynaptic membrane. the ionotropic glutamate receptors consist of ampa (-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), nmda (n-methyl-d-aspartate), and kainate receptors. among these, ampa receptors (ampars) are the principal receptors that mediate fast excitatory synaptic transmission in the mammalian brain. they are tetrameric assemblies of two dimers of four potential subunits (glua1glua4) encoded by distinct genes, gria1gria4. the predominant ampars expressed in the hippocampal and cortical pyramidal neurons are composed of glua1/glua2 and glua2/glua3 subunits. brief periods of high neuronal activity open nmda receptors (nmdars) and induce ca influx, leading to a long-lasting increase in synaptic efficacy, known as long-term potentiation (ltp), which is characterized by an increase in the number of ampars on the postsynaptic membrane and spine growth. in contrast, repetitive low frequency stimulation leads to the removal of synaptic ampars to produce long-term depression (ltd), that is, a decrease in synaptic strength. it has long been postulated that these forms of synaptic plasticity represent a cellular correlate of learning and memory. one of the key mechanisms underlying synaptic plasticity is the tight control of ampar number at synapses. this requires a balance between the biosynthesis (number of receptors being produced), membrane trafficking (the movement of receptors to and from the plasma membrane via exocytosis and endocytosis), and degradation of receptors (receptor turnover), all of which are dynamically regulated by ampar interacting proteins as well as by various posttranslational modifications that occur on their cytoplasmic carboxyl terminal domains [11, 12]. aberrant trafficking of ampars usually leads to impaired synaptic plasticity and deficits in learning and memory. importantly, several studies have demonstrated a role for a in promoting ampar endocytosis and hence synaptic depression [1316]. this review focuses primarily on nmdar and metabotropic glutamate receptor- (mglur-) mediated signaling. in particular, we highlight several mechanisms that underlie synaptic ltd as common signaling pathways that are hijacked by the neurotoxic effects of a. several pharmacological agents that target these pathways and are efficacious in inhibiting or reversing the neurotoxic effects of a on glutamatergic neurotransmission and synaptic plasticity are also discussed. the ability of neurons to modulate their synaptic strength is widely believed to be a cellular correlate of learning and memory. nmdar-dependent ltp and ltd are two major forms of synaptic plasticity that are best studied in the hippocampus, a region of the brain that is both critical for memory formation and highly vulnerable to a toxicity. it is well established that synthetic soluble a oligomers [17, 18] or those secreted from cell lines overexpressing app acutely and potently block hippocampal ltp at high concentration. more recent studies have further shown that soluble a dimers, but not a monomers, either prepared by chemical cross-linking or extracted directly from postmortem ad brains, are extremely potent in inhibiting hippocampal ltp both in vitro and in vivo [4, 20]. congruent with the ltp hypothesis of long-term memory, injection of these soluble a oligomers into the rat hippocampus disrupts cognitive function and learned behavior [4, 21]. most transgenic ad mouse models overexpressing different familial ad mutations, such as tg2576 (appswe; k670n/m671l), pdapp (appind; v717f), 3xtg (appswe, tau p301l, and ps1 m146v), and 5xfad (appswe, appflorida; i716v, applondon; v717i, ps1 m146l, and ps1 l286v), generally display impairments in ltp and cognition [2226]. notably, some ad transgenic mice show abnormal ltp and learning deficits well in advance of plaque formation [22, 27, 28]. collectively, these results lend support to the idea that soluble oligomeric a plays a key role in disrupting synaptic plasticity. more importantly, studies performed in human subjects have also revealed deficits in ltp-like cortical plasticity in mild-to-moderate ad patients [2931]. consistent with the fact that a induces an impairment in ltp, soluble a oligomers have been demonstrated to facilitate the expression of ltd in the hippocampus [4, 17, 32]. although the exact mechanisms underlying a-induced ltd remain equivocal, they have been shown to involve internalization of nmda- and ampa-type glutamate receptors, dendritic spine shrinkage, and eventual synaptic loss [14, 16, 33, 34]. dynamic trafficking of ampars to and from synapses is a critical mechanism underlying the induction of synaptic plasticity. defects in the endocytosis and lysosomal trafficking pathways are known to contribute significantly to ad pathogenesis. consistent with this notion, overexpression of app and a high concentration of soluble oligomeric a are able to induce the removal of surface ampars at synapses, leading to synaptic depression and inhibition of ltp [14, 19, 36, 37]. mechanistically, these neurotoxic effects of a are mediated by high levels of glutamate at synapses as a result of a disrupted glutamate reuptake process that subsequently leads to aberrant activation of nmdars, mglurs, and the cellular prion protein (prp), as well as elevated levels of ampar ubiquitination. activation of these signaling pathways in turn promotes synaptic depression, via common pathways shared with ltd as summarized in figure 1, which are discussed in detail in the following sections. nmdar-dependent ltd induced by low frequency stimulation or by direct application of nmda (chemically induced ltd) triggers ca entry into the postsynaptic compartment and activates protein phosphatase 2b (pp2b, also known as calcineurin), which in turn leads to the activation of protein phosphatase 1 (pp1) [38, 39]. pp1 and pp2b are known to mediate nmdar-induced ampar internalization by dephosphorylating the glua1 subunit of ampars at ser-845 [40, 41], a protein kinase a (pka) site that is crucial for maintaining the stability of ampars at perisynaptic sites and ltp [4244]. nmdar-dependent ltd also induces the p38 mitogen activated protein kinase (p38 mapk) signaling pathway via the activation of rap small gtpases, leading to the removal of ampars [45, 46]. emerging evidence demonstrates that toxic levels of a aberrantly enhance the activity of nmdars in favor of ltd induction, thereby preventing ltp [32, 37, 47]. in cultured neurons and acute brain slices, soluble oligomeric a induces excessive influx of ca through the glun2b-containing extrasynaptic nmdars, which subsequently activates the rap-p38 mapk signaling pathway, as well as the protein phosphatases, pp1 and calcineurin [13, 14, 16, 32, 37, 4850]. one of the consequences of a-induced activation of calcineurin is reduced phosphorylation of ser-845, which induces ampar endocytosis and impairs the synaptic incorporation of these receptors. consistent with this finding, appswe, ind transgenic mice display lower levels of ser-845 phosphorylation, a phenomenon that correlates well with the loss of ampars on the cell surface and deficits in initial learning and memory. another key substrate of pp1 the activity of which is required for the expression of nmdar-dependent ltd is glycogen synthase kinase-3 (gsk3). pp1 can activate gsk3 by a direct dephosphorylation mechanism, as well as via the modulation of the upstream caspase akt signaling pathways, which are also crucial for ampar internalization and ltd [51, 52]. interestingly, both gsk3 and caspases are enzymes that have been widely implicated in ad. indeed, it has been demonstrated that inhibition of ltp by a is mediated by the caspase 3, akt1, and gsk3 signaling pathway [32, 53]. paradoxically, however, gsk3 activity has also been reported to play a role in maintaining ampar synaptic expression under basal conditions as its inhibition leads to the loss of surface ampar expression by controlling the rate of ampar internalization. however, during nmdar-dependent ltd, gsk3 may preferentially phosphorylate other substrates including the key scaffolding protein in excitatory synapses, postsynaptic density-95 (psd-95). psd-95 stabilizes ampars at synapses through its interaction with transmembrane ampar regulatory proteins (tarps), auxiliary subunits of ampars. overexpression of psd-95 promotes synaptic maturation and enhances synaptic strength, whereas psd-95 knockdown results in the opposite effects [5660]. it appears that gsk3 phosphorylation of psd-95 at thr-19, following its dephosphorylation at ser-295 by pp1, destabilizes and mobilizes psd-95 away from the psd, resulting in increased ampar internalization [61, 62]. whether or not the phosphorylation status of psd-95 is modulated by oligomeric a via the gsk3 and pp1 signaling pathways remains to be determined. gsk3 is also a major kinase that phosphorylates the microtubule-associated protein tau [63, 64]. a causes tau hyperphosphorylation and mislocalization from axons to somatodendritic compartments, where it accumulates and mediates a-induced downregulation of surface ampars [6568]. recent studies have shown that nmdar-induced gsk3 phosphorylation of tau at ser-396 is required for hippocampal ltd by enhancing the interaction between the glua2 subunits of ampars with the protein interacting with c-kinase 1 (pick1) [69, 70], a process that is fundamental for ampar internalization and/or intracellular retention during ltd [7176]. furthermore, phosphorylation of pick1 by gsk3 at ser-416 has also been reported to augment this interaction. glua2 can be phosphorylated by protein kinase c (pkc) at ser-880 and by the protein tyrosine kinase of the sarcoma (src) family at tyr-876, both of which are required for ampar internalization and ltd [7880]. glua2 phosphorylation at these sites differentially regulates the interaction of the subunit with pick1 and glutamate receptor interacting proteins (grip) 1 and 2 [80, 81]. grip1 plays an important role in stabilizing ampars at synapses and is essential for ltd [72, 79]. given that phosphorylation of glua2 weakens the interaction of the subunit with grip1, but not pick1, it has been postulated that ltd involves destabilization and detachment of glua2 from synapses, allowing ampars to be internalized. in accord with the role of a in inducing aberrant ampar endocytosis, one study has observed that oligomeric a increases pkc-mediated phosphorylation of glua2 at ser-880 and subsequently reduces surface expression of ampars in cultured hippocampal neurons. more importantly, several molecular and pharmacological manipulations that inhibit glua2 internalization potently prevent a-induced synaptic depression and rescue memory impairment in ad mice. these include the glua2-r845a mutant, glua2-3y peptides, and a small molecule pick1 inhibitor. a new mechanism underlying the pathological action of a that involves the cyclin-dependent kinase 5- (cdk5-) activating peptide, p25, has recently been described by seo et al.. elevated levels of p25 have been implicated in many neurodegenerative diseases, including ad. in their study, seo et al. found that a induces calpain-mediated cleavage of p35 into p25 in the hippocampus, a process that requires the activity of glun2b-containing nmdars and ca/calmodulin-dependent protein kinase ii (camkii). the a-induced elevation in p25/cdk5 activity subsequently enhances the phosphorylation of dopamine- and cyclic adenosine monophosphate-regulated neuronal phosphoprotein (darpp-32) at thr-75, thereby inhibiting the activity of pka. in a synergistic manner, a also triggers dephosphorylation of darpp-32 at thr-34, presumably by calcineurin, thereby releasing its inhibition on pp1 [86, 87]. these converging mechanisms eventually lead to the loss of glua1 phosphorylation at ser-845 and induce ampar internalization and synaptic depression. remarkably, genetic inhibition of p25 generation rescues ltp and memory deficits in 5xfad transgenic mice. in addition to promoting the internalization of ampars, oligomeric a can also act through mechanisms that prevent the forward trafficking of ampars towards the plasma membrane. a has been shown to cause aberrant redistribution of camkii from the synaptic to the cytosolic fraction both in cultured neurons and in the brain of appswe transgenic mice. camkii can potentiate ampar-mediated transmission via (a) phosphorylation of glua1 at ser-831 to enhance ampar channel conductance, (b) phosphorylation of the tarp, stargazin, to facilitate synaptic recruitment of ampars, and (c) potentiation of the ras-erk (extracellular signal-regulated kinase) pathway to promote ampar insertion into the plasma membrane [45, 8991]. consistent with the role of camkii in synaptic potentiation, exposure of soluble a oligomers reduces surface glua1 clusters in cultured neurons, concomitant with decreased ampar synaptic responses in cortical pyramidal neurons recorded from acute brain slices of appswe transgenic mice. a has been shown to interact with nmdars [92, 93] and to reduce their surface expression through endocytosis. a-induced internalization of nmdars involves dephosphorylation of the glun2b subunit at tyr-1472 by step61 (striatal-enriched protein tyrosine phosphatase 61), the expression of which is upregulated in several ad mouse models, as well as in the postmortem prefrontal cortex of ad patients [33, 9496]. the fact that a enhances the internalization of nmdars seems counterintuitive given the role of nmdars in mediating ampar endocytosis, spine loss, and ultimately excitotoxicity in neurons. recent studies on the putative oligomeric a receptor, prp, have provided insights into two potential mechanisms that regulate nmdar function [97, 98]. firstly, soluble oligomeric a binding to prp activates the tyrosine kinase fyn, which initially phosphorylates glun2b and transiently enhances nmdar function, before the step61 level increases and dephosphorylates glun2b. secondly, a disrupts the ability of prp to limit excessive nmdar activity in a copper-dependent manner, potentially by chelating copper ions and preventing them from binding to prp, thereby producing large nondesensitizing steady-state nmdar currents. albeit controversial, loss of prp function has been reported to prevent a-induced ltp and memory impairment in mice [98, 101105]. despite this, the role of prp in regulating ampar trafficking has not been directly examined. recent studies by kessels and colleagues have challenged the central role of nmdar-mediated ca influx in a-induced synaptic depression. it is well established that the neurotoxic effects of oligomeric a on synapses can be blocked by the nmdar antagonist, d-apv (d-2-amino-5-phosphonopentanoic acid), which prevents glutamate binding and blocks the activation of nmdars. however, noncompetitive nmdar antagonists that block ion flow through the receptor, such as mk-801, ketamine, and 7-chlorokynurenic acid, are not able to rescue a-mediated synaptic depression [106, 107]. a similar finding consistent with the idea that a operates through shared pathways with ltd, metabotropic, but not ionotropic, nmdar function has been shown to be required for nmdar-dependent ltd in the hippocampus by activating the p38 mapk signaling pathway. in fact, ligand binding to the extracellular domain of nmdars induces conformational change and movement of their cytoplasmic tails, allowing pp1 to dephosphorylate camkii together with other signaling molecules that contribute to synaptic depression [110, 111]. although the role of metabotropic nmdars remains controversial, it does offer an explanation for the fact that the fda-approved nmdar antagonist, memantine, has poor efficacy in treating early-stage ad. further research is warranted, as delineating the metabotropic nmdar signaling pathway may shed light on new strategies for the development of future ad drugs. mglurs belong to the g-protein-coupled receptor superfamily that modulates neuronal excitability, synaptic transmission, and plasticity in the central nervous system. group i mglurs, which consist of two members, mglur1 and mglur5, predominantly localize to the postsynaptic membrane and are canonically coupled to gq/11 to activate phospholipase c (plc) that catalyzes the hydrolysis of phosphoinositides into inositol 1,4,5-triphosphate (ip3) and diacylglycerol (dag). subsequently, these second messengers trigger the release of ca from intracellular stores and activate pkc, respectively. group i mglurs, and more specifically mglur5, are the predominant receptors that mediate mglur-dependent ltd in the hippocampus and have been widely implicated in ad. it is well established that mglur-dependent ltd requires the internalization of glua2-containing ampars, leading to a long-term reduction in the number of surface ampars [115, 116]. one of the mechanisms that regulates mglur-induced ampar endocytosis involves the phosphorylation of glua2 at ser-880 by pkc, a process that is facilitated by pick1 [117119]. however, in the ca1 region of the hippocampus, internalization of ampars does not require pkc but instead relies on the dephosphorylation of glua2 at tyr-876 by step61 [120122]. dephosphorylation of glua2 stimulates the binding of brag2 (brefeldin resistant arf gef 2), which in turn activates the small gtpase arf6 through augmentation of its gef (guanine-nucleotide exchange factor) activity and promotes ampar endocytosis. in accordance with this model genetic deletion of step61 restores the number of ampars on the postsynaptic membrane, enhances ltp, and improves cognitive function in ad mice [95, 123]. a new small molecule inhibitor of step61, tc-2153, like nmdar-dependent ltd, mglur-mediated ltd also involves the rap1-p38 mapk signal transduction pathway to facilitate ampar internalization via the formation of the gdi-rab5 complex [125127]. in addition, a role for erk in mglur-dependent ltd has also been reported. one unique feature of mglur-dependent ltd is its requirement for rapid translation of preexisting mrnas (local protein translation) in dendrites. mglur-dependent de novo protein synthesis can be regulated through multiple pathways, including the pi3k-akt-mtor (mammalian target of rapamycin) and erk signaling pathways that converge on the initiation and elongation factors of protein translation. several mrna encoding proteins that regulate ampar trafficking are locally translated during mglur-dependent ltd, including the activity-regulated cytoskeleton-associated protein (arc), microtubule-associated protein 1b (map1b), and step [121, 131133]. given that grip1 stabilizes ampars at synapses, the newly synthesized map1b may sequester grip, hence loosening its interaction with glua2. on the other hand, arc interacts with the endocytic proteins, endophilin and dynamin, and is able to enhance dynamin polymerization and gtpase activity, thereby promoting ampar endocytosis [135, 136]. interestingly, soluble oligomeric a rapidly induces arc expression in neurons, which may contribute to the loss of ampars from the plasma membrane. moreover, arc also regulates the endosomal trafficking of app and bace1, as well as ps1, a mechanism that is essential for the activity-dependent production of a in the brain, and genetic deletion of arc reduces the a load in appswe;ps1e9 transgenic ad mice. this may serve as a positive feedback mechanism underlying the overproduction of a in the pathophysiology of ad. studies from several laboratories have implicated the mglur-dependent signaling pathway in the neurotoxic effects of a on synaptic function [4, 14, 139143]. notably, genetic and pharmacological inhibition of mglur5 prevents oligomeric a-induced impairment in ltp, spine loss, and cognitive deficits in ad mouse models [139, 142145]. more recently, a seminal study by strittmatter and colleagues identified an interaction between mglur5 and prp, which together act as a coreceptor for oligomeric a. they also revealed an essential role for mglur5 and prp coupling in the pathology of ad. mechanistically, mglur5 links prp to key intracellular signaling molecules, such as homer1b/c, pyk2, fyn, and camkii, all of which play major roles in synaptic plasticity [144, 146, 147]. when neurons are exposed to oligomeric a, the prp-mglur5 complex mediates the aberrant activation of pyk2, fyn, and camkii, causing altered neuronal states that lead to impaired ltp [99, 144, 146]. it is interesting to note that a also induces a biphasic alteration in camkii activity, resembling that of fyn, in a prp-mglur5-dependent manner, and that this is accompanied by the increased association of mglu5 with camkii. given that mglur5 activation enhances nmdar forward trafficking through camkii-mediated phosphorylation of glun2b at ser-1303, it is hypothesized that a-induced enhancement of the association between mglur5 and camkii may prevent synaptic potentiation. furthermore, pharmacological activation of mglur5 in the presence of prp causes a redistribution of camkii into the cytoplasm, which may have an impact on ampar trafficking. interestingly, the cross talk between mglur5 and nmdar signaling is bidirectional. not only can mglur5 potentiate nmdar currents through camkii and pkc signaling pathways [148, 149], but also activation of nmdars can potentiate mglur5 responses under physiological conditions [150, 151]. this involves the nmdar-dependent activation of calcineurin that dephosphorylates mglur5 and reduces receptor desensitization. however, a high concentration of nmda can induce pkc-dependent mglur5 phosphorylation and inhibit mglur5 responses. although the interaction of mglur5 and nmdars has been implicated in synaptic plasticity and various animal behaviors [153156], their alteration in the presence of a binding to prp and how this impacts on ampar trafficking remain unclear. posttranslational ubiquitination, a regulatory signal that controls protein trafficking and turnover, has recently emerged as an important mechanism that regulates ampar function [157, 158]. all ampar subunits undergo activity-dependent ubiquitination in cultured neurons, a process that is ca-dependent and requires the activity of l-type voltage-gated ca channels [159161]. the primary e3 ligases that catalyze the ubiquitination of glua1 and glua2 subunits are nedd4-1 and rnf167, respectively [160, 162]. while the role of protein ubiquitination on the glua1 and glua2 subunits in ligand-induced ampar endocytosis remains controversial, it is well accepted that ubiquitination of ampars regulates the intracellular sorting of receptors into late endosomes for degradation [159161, 163]. under normal conditions, the degradation of ampars is required for protein homeostasis to ensure turning over of old or used receptors in order to maintain healthy levels of ampars in neurons. however, when the ubiquitin pathway is hijacked (e.g., by elevated levels of a), there is an excessive downregulation of ampars and synaptic depression. indeed, a new finding has demonstrated a role for naturally secreted and synthetic a in promoting the ubiquitination of ampars by nedd4-1. interestingly, knocking down nedd4-1 rescued a-induced synaptic deficits, including reduced glutamatergic synaptic transmission, decreased levels of surface ampars, and the loss of dendritic spines. these findings have important implications in targeting ubiquitin e3 ligases as potential drug targets for the treatment of ad. research over the past decade has provided strong evidence that the cognitive deficit associated with ad is caused by the neurotoxic effects of soluble a oligomers on synaptic function. increasing evidence indicates that the trafficking of ampars, which is essential to multiple forms of synaptic and structural plasticity in the brain, is aberrantly dysregulated by oligomeric a and manifests as impairments in ltp, learning, and memory. it is particularly encouraging to learn that pharmacological and genetic manipulations that block endocytosis or enhance the forward trafficking of ampars can rescue ltp and reverse cognitive deficits in ad mice. given that a-induced ampar internalization requires the same adaptor proteins as the conventional trafficking pathway, it will be challenging to minimize unwanted side effects. hence, further research is needed to identify specific targets for improving the memory deficits associated with ad. rapid progress has been made in delineating the molecular mechanisms and signaling pathways underlying the loss of ampars from the plasma membrane induced by oligomeric a (figure 1). the discovery of prp as a receptor for soluble a oligomers that signals through nmda and mglur5 receptor has underscored the importance of glutamatergic signaling in the etiology of ad. it is likely that these receptors act cooperatively to mediate the synaptotoxic effects of a, highlighting the need for further investigation of the associated signaling mechanisms with a view to developing more effective therapeutic strategies for the treatment of ad.

evidence from neuropathological, genetic, animal model, and biochemical studies has indicated that the accumulation of amyloid-beta (a) is associated with, and probably induces, profound neuronal changes in brain regions critical for memory and cognition in the development of alzheimer's disease (ad). there is considerable evidence that synapses are particularly vulnerable to ad, establishing synaptic dysfunction as one of the earliest events in pathogenesis, prior to neuronal loss. it is clear that excessive a levels can disrupt excitatory synaptic transmission and plasticity, mainly due to dysregulation of the ampa and nmda glutamate receptors in the brain. importantly, ampa receptors are the principal glutamate receptors that mediate fast excitatory neurotransmission. this is essential for synaptic plasticity, a cellular correlate of learning and memory, which are the cognitive functions that are most disrupted in ad. here we review recent advances in the field and provide insights into the molecular mechanisms that underlie a-induced dysfunction of ampa receptor trafficking. this review focuses primarily on nmda receptor- and metabotropic glutamate receptor-mediated signaling. in particular, we highlight several mechanisms that underlie synaptic long-term depression as common signaling pathways that are hijacked by the neurotoxic effects of a.

PMC4814684

pubmed-653

the incidence of diabetes mellitus is increasing worldwide; this trend is particularly strong for type 2 diabetes mellitus (t2dm)1. t2 dm is a chronic disease characterized by decreased insulin sensitivity and overall poor glucose control. the systematic review by boule et al. indicates structured exercise programs have a statistically and clinically significant beneficial effect on glycemic control in patients with t2dm3. in addition, patients with t2 dm who use insulin, low-intensity exercise significantly reduces the prevalence of hyperglycemia4. despite the benefits of physical activity, many people with t2 dm for some individuals, secondary diabetes-related complications such as lower-limb amputations, neuropathies, hypertension, nephropathies, and retinopathies can either contraindicate exercise or make it more difficult. in addition, many elderly people with t2 dm do not have sufficient physical ability to perform aerobic exercise and thus have problems maintaining euglycemia5. passive static stretching occurs when sustained tension develops within a person s muscles through external forces. accordingly, blood glucose levels could decrease following a program of successive sustained muscle stretching. in addition, because passive stretching requires minimum effort by the person performing the stretch, people with t2 dm who are reluctant or unable to exercise may be willing to follow a stretching protocol6. therefore, this study determined the effect of passive static stretching on blood glucose levels in people with t2 dm. fifteen in-patients with t2 dm at a hospital in busan, korea participated in this study. patients were eligible if they were sedentary (i.e., not participating in regular aerobic or strengthening exercises 6 months before the study) and willing to commit to an 8-week supervised exercise program7. all patients were diagnosed with t2 dm confirmed by a glycated hemoglobin (hba1c) level 6.5% or higher as a criterion for the diagnosis of diabetes8. all patients meeting the inclusion criteria were given verbal and written information about this study. patients were randomized to the control group (n=7) or passive static stretching group (pss, n=8). the control group was instructed to maintain their diet and medications for diabetes and not to perform any exercise during the experiment. meanwhile, patients in the pss group followed the same instructions as the control group but received a 40-minute intervention consisting of 6 lower-body and 4 upper-body static passive stretches. for each stretch, the muscle was held in the stretched position for 30 seconds and was repeated 4 times. each repetition was separated by a 15-second relaxation period, and different stretches were separated by a minimum of 1 minute. a description of stretch is provided in table 1table 1.descriptions of the stretches used in the interventionstretchdescriptionseated knee flexor (bilateral)the patient sat on the floor with their legs extended and arms above their head. from this position, they lowered their head toward their knees while the experimenter pushed down on their back.seated knee flexor hip adductor (bilateral)the patient sat on the floor in the cross-legged position. from this position, the patient lowered their head toward the floor while the experimenter pushed down on their back.seated shoulder lateral flexor (bilateral)the patient sat in a chair with fingers interlaced behind their head. keeping their arms in this position, the experimenter stood behind the patient and pulled the elbows back toward the body s midline.supine hip flexor knee extensor (unilateral)the patient lay on their back with their leg hanging over the edge of the table with the knee flexed at approximately 90. the hip was then hyperextended by the experimenter while pushing down on the thigh.seated hip external rotators, extensors (unilateral)the patient sat on the floor with one leg extended. the opposite leg was flexed at the knee, and the foot was placed flat against the extended leg s inner thigh. the patient then lowered their head toward the extended knee while the experimenter pushed down on their back.seated shoulder extensors, adductors, retractors (unilateral)while seated in a chair, the patient extended one arm and placed it horizontally across the front of the chest. the experimenter stood behind the patient, grabbed their wrist, and pulled their arm against the chest as much as possible while keeping the arm parallel to the floor.supine knee flexor plantar flexor (unilateral)the patient lay on their back with the legs extended. the experimenter then raised one leg and simultaneously flexed the hip and dorsiflexed the ankle.prone hip flexor (unilateral)the patient lay on their stomach and flexed one knee at approximately 60. keeping the knee in the flexed position, the experimenter lifted the thigh to hyperextend the hip.seated shoulder flexors, depressors (bilateral)the patient sat on the floor with the legs extended. the experimenter then grabbed their wrists and hyperextended the shoulder by raising the arms behind the back and up toward the head while keeping the back and elbows straight.seated shoulder and elbow flexors (unilateral)the patient sat on the floor with the legs extended, with one elbow flexed and brought up near the ear. from this position, the shoulder was hyperflexed by the experimenter by pushing the upper arm down toward the floor.6. the pss group performed the stretches 3 times per week for 8 weeks. for outcome measurements, a 10-ml blood sample was collected from each patient to determine blood glucose levels using an hba1c analyzer (variant turbo, bio-rad laboratories, inc. a paired t-test was used to determine whether there were significant changes in blood glucose levels before and after the intervention. meanwhile, an independent t-test was used to analyze differences between the 2 groups. the level of significance was set at p<0.05. the baseline characteristics of the patients are shown in table 2table 2.baseline characteristics of the patientscon (n=7)pss (n=8)page (years)58.4 1.849.6 5.20.2duration of diabetes (years) 5.2 2.9 5.4 con: control group; pss: passive static stretching group; bmi: body mass index. there were no significant differences in the baseline characteristics between groups (p>0.05). the results of outcome measures are summarized in table 3table 3.outcome measurescontrol group(n=7)passive static stretching group(n= 8)pre-intervention post-interventionpre-interventionpost-interventionhba1c (%) 7.4 1.37.4 1.47.4 1.56.8 1.5*values are means sd.*p<0.05 vs. post-intervention. hba1c: glycated hemoglobin a1c .. there was no significant difference in hba1c level after the intervention in the control group (p>0.05). however, hba1c levels decreased significantly in the pss group after the intervention (p<0.05) and were significantly different between groups (p<0.05). con: control group; pss: passive static stretching group; bmi: body mass index values are means sd.*p<0.05 vs. post-intervention. as mentioned above, this study determined the effect of passive static stretching on blood glucose levels in patients with t2 dm. the results showed hba1c levels decreased significantly after an 8-week passive static stretching intervention. there are several possible mechanisms that could explain how passive stretching of skeletal muscles decreased blood glucose levels. according to a review by dohm9, glucose transport into the skeletal muscles is primarily mediated by a glucose transport protein, glut-4; accordingly, exercise can increase glut-4 levels in the skeletal muscles. furthermore, increased metabolic activity accompanying passive muscle stretching is related to the glut-4 activation pathway9, 10. therefore, passive muscle stretching could induce the incorporation of glut-4 into the stretched skeletal muscles. other studies also support the possibility of stretching-induced incorporation of glut-4 into the skeletal muscles. first, the activity of protein kinase b controls glut-4 incorporation; accordingly, protein kinase b is activated by passive stretching of isolated muscles11. report that ischemia induces the translocation of glut-4 to the plasma membrane of cardiac myocytes12; accordingly, passive stretching of the skeletal muscles can cause ischemia13. third, in an experimental study by roberts et al., exercise-induced increases in nitric oxide levels resulted in increased glucose transport14; accordingly, passive stretching can increase nitric oxide release from excised soleus muscles by 20%15. finally, mitogen-activated protein kinase activity stimulates glucose uptake in muscle cells16; martineau et al. report that the activity of mitogen-activated protein kinase directly reflects the magnitude of mechanical stress (e.g., actively or passively generated tension) applied to the muscle17. the sample size is insufficient to generalize the results to all patients with t2 dm. in addition, as hba1c reflects the average plasma glucose level over the preceding 23 months, the 8-week study period might have been too short to determine changes in blood glucose levels as a result of stretching18. therefore, further studies are required to ascertain the long-term (i.e., more than 3 months) effects of passive static stretching on blood glucose levels in a larger population of patients with t2 dm .

[ purpose] this study determined the effects of passive static stretching on blood glucose levels in patients with type 2 diabetes. [subjects] fifteen patients (8 males and 7 females) with type 2 diabetes were recruited and randomly assigned to the control group or passive static stretching group. [methods] glycated hemoglobin was measured before and after the 8-week training period. [results] glycated hemoglobin levels decreased significantly in the passive static stretching group, and there were significant differences in blood glucose levels between the 2 groups. [conclusion] passive static stretching of the skeletal muscles may be an alternative to exercise to help regulate blood glucose levels in diabetes patients.

PMC4483419

pubmed-654

cardiovascular disease (cvd) is the leading cause of mortality in both men and women in developed countries. nonetheless, sex-associated differences regarding the age of cvd onset and its progression are observed worldwide. incidence of cvd in premenopausal women is markedly lower than age-matched men in epidemiological studies (messerli et al., 1987; bairey merz et al., 2006; shaw et al after menopause, however, the incidence is comparable or even higher in women than in men (lerner and kannel, 1986; eaker et al., 1993), making cvd the primary cause of death in postmenopausal women (55 versus 43% in men), exceeding all cancer deaths (rosamond et al., 2008). the lower cvd risk among fertile women is often attributed to the protective role of estrogens at the vascular level. according to epidemiological observations and extensive basic research, estrogen and other sex steroids estrogen modulates a myriad of molecular pathways that improve vascular function, whether at the physiological level or when administered as hormone replacement therapy (hrt; grodstein et al., 2000, 2001; miller and duckles, 2008) nevertheless, some clinical studies have questioned the protective value of hrt against vascular disease. two randomized clinical trials, the women s health initiative (whi; rossouw et al., 2002) and the heart and estrogen/progestin replacement study (hers i and ii; gambacciani et al., 2002), indicate that hrt may increase cvd risk and events in postmenopausal women. the reason for this paradox could be attributable to many patient characteristics, including age. although aging occurs progressively in both men and women, the onset of menopause marks a sudden increase in the appearance of aging-associated signs in women, and more specifically in the progress of vascular aging. information about the role of age and menopause in the development of cvd in women is scarce. this review of clinical and experimental data on the effects of aging, estrogens, and hrt on vascular function of females aims to clarify how menopause and aging contribute jointly to vascular aging and how estrogen modulates vascular response at different ages. the numerous vascular effects of estrogens are triggered by complex genomic and non-genomic mechanisms. they include modulation of vascular function and inflammatory response as well as metabolic and hemodynamic effects. estradiol, the most abundant and potent estrogen in humans, mainly binds and activates estrogen receptors (ers). vascular estrogen signaling involves at least three ers identified in both vascular smooth muscle and endothelium, reinforcing the idea that estrogen has a key role in controlling vascular function. the classical subtypes er (soloff and szego, 1969) and er (kuiper et al., 1996) vary not only in their tissue distributions, but also in their agonist/antagonist profile with respect to several compounds (cano and hermenegildo, 2000). these er subtypes belong to the intracellular receptors classically defined as nuclear ligand-activated transcription factors. activation of these receptors by the corresponding hormones affects gene expression by acting on estrogen-response elements in the target genes and modulating transcriptional events (beato et al., 1995). estrogen binding to er and er regulates gene expression in a time- and tissue-dependent manner, generating controversy about the type of receptor involved in vascular protection (murphy, 2011). in the cardiovascular system, both er and er have been identified in the endothelium, smooth muscle cells, adventitia, and adrenergic nerve endings of arteries from various territories and several species, including humans (karas et al., 1994; kim-schulze et al., 1996; although it has been reported that cultured endothelial cells do not express er (toth et al., 2009), other investigators have demonstrated the presence of both er and er mrna in endothelium (wagner et al., 2001) and data from our group demonstrate the protein expression of both er and er in huvec (sobrino et al., 2009, 2010). in addition to their classic nuclear location, er can also target the plasma membrane, enabling estrogen activation of several signaling pathways, including those involved in calcium mobilization (zhang et al. prakash et al., 1999) and the phosphatidylinositol-3-kinase (pi3k) pathway (hisamoto et al., a third type of er, g-protein coupled, and mainly located in the plasma membrane, was initially named gpr30 (takada et al., 1997), then renamed gper by the international union of basic and clinical pharmacology, iuphar (alexander et al., 2008). gper is expressed in both endothelial and smooth muscle cells of human arteries and veins (haas et al., gper activates rapid signaling cascades such as extracellular signal-related kinase and pi3k (meyer et al., 2011). several rapid and non-genomic estrogen effects formerly attributed to er have now been described as gper-mediated (prossnitz and barton, 2011). the vascular protection conferred by estrogen may be mediated indirectly by its influence on the metabolism of lipoproteins or by a direct action on the modulation of molecular pathways in the vessel wall, and more specifically on endothelial cells (hermenegildo et al., 2002). vascular endothelium not only regulates vascular tone through flow-mediated mechanisms, but also confers antithrombotic and antiinflammatory properties to the blood vessel. nitric oxide (no), the primary endothelial-derived mediator, is involved in many physiological processes, including vasodilation and inhibition of thrombosis, cell migration, and proliferation (dudzinski and michel, 2007; lamas et al. estrogen is known to increase no bioavailability by mechanisms that either directly increase no generation (figure 1) or decrease superoxide anion o2 concentration, thereby attenuating o2-mediated no inactivation. mechanisms involved in estrogen-induced increases in no availability include: (1) transcriptional stimulation of endothelial no synthase (enos) gene expression (huang et al., 1997; sumi and ignarro, 2003); (2) non-genomic activation of enzyme activity via a pi3k/phosphokinase b (pkb/akt)-mediated signaling pathway (hisamoto et al., 2001); (3) increased intracellular free ca concentration ([ ca]i) in endothelial cells (rubio-gayosso et al., 2000); (4) decreased production of asymmetric dimethylarginine (adma), the enos endogenous inhibitor (monsalve et al., 2007); and (5) attenuated o2 concentrations (wassmann et al. estradiol effects on enos-mediated nitric oxide (no) production include both genomic and non-genomic effects. genomic effects include the classical intracellular estrogen receptors (er), which after binding of e2 interact with estrogen-response element (ere) in dna, resulting in an increased enos expression. moreover, e2 binding to gper leads to activation of different transcriptional factors such as camp response element (cre) which also induces enos expression. among non-genomic effects, er and gper regulate the e2-induced enos activity (modified from sobrino, 2011). estrogens such as 17-estradiol, estrone, and estriol have been described to act as reactive oxygen species (ros) scavengers by virtue of the hydrogen-donating capacity of their phenolic molecular structure (halliwell and grootveld, 1987; dubey and jackson, 2001). however, in these studies the direct effect of estrogens as scavengers can only be observed at concentrations above 1 m (arnal et al., 1996; kim et al., 1996). considering that plasma concentrations of estrogen in physiological conditions are within the nanomolar range, it is likely that direct scavenger action is not estrogen s main antioxidant mechanism. estrogen modulates ros concentration through a mechanism that involves interaction with its estrogenic nuclear receptors to decrease oxidative proteins and/or increase antioxidant enzymes expression. many studies have associated changes in estrogen levels with altered levels of antioxidant enzymes including glutathione peroxidase, catalase, and superoxide dismutase (capel et al., 1981; robb and stuart, 2011; sivritas et al., 2011). moreover, estrogen modulates nadh/nadph oxidases and at1 receptor gene expression, both of which are major sources of o2 production (wassmann et al., 2001; dantas et al., estrogen also has a modulating effect on constrictive factors and positively upregulates the production of endothelium-derived relaxing factors such as pgi2 (sobrino et al., 2009, 2010) and the endothelium-derived hyperpolarizing factors (golding and kepler, 2001), both of which are important mediators of vascular relaxation in resistance-sized arteries. the beneficial effects of estrogen on the endothelium can be partially explained by an inhibitory effect on the production or action of the cyclooxygenase (cox)-derived vasoconstrictor agents prostaglandin h2, pgh2, and thromboxane a2, txa2 (davidge and zhang, 1998; dantas et al., 1999; novella et al., 2010), and of endothelin-1 (et-1; david et al., 2001). it regulates contractile responses by a direct modulation of ca mobilization into the vascular smooth muscle cells. direct interaction of estradiol with voltage-gated maxi-k channel subunit beta, which confers higher ca sensitivity, may modulate vascular smooth muscle (valverde et al., 1999). estrogen does not inhibit ca release from the intracellular stores (crews and khalil, 1999; murphy and khalil, 1999). however, supraphysiological concentrations of estrogen impede ca influx from the extracellular space (han et al., 1995; crews and khalil, 1999; murphy and khalil, 1999) by inhibiting ca entry through voltage-gated ca channels (freay et al., 1997; kitazawa et al., 1997; crews and khalil, 1999; murphy and khalil, 1999). expression of the l-type ca channels in cardiac muscle is substantially increased in er-deficient mice (johnson et al., 1997), suggesting er-mediated regulation of ca mobilization. estrogen also exerts direct modulation on the components of the renin-angiotensin system (ras), a key regulator of blood pressure and smooth muscle cell growth. production of angiotensin ii (ang ii), the active hormone of the ras, is reduced by estrogen inhibition of angiotensin-converting enzyme (ace) expression. in animal models of menopause and in postmenopausal women, chronic estrogen replacement reduces ace activity in the circulation and in tissues including the kidney and aorta (brosnihan et al., 1999; seely et al., 2004). furthermore, estrogen attenuates expression of and tissue response to type 1 angiotensin receptor (at1) in the aorta, heart, and kidney (silva-antonialli et al. vascular aging is associated with endothelial dysfunction, arterial stiffening and remodeling, impaired angiogenesis, defective vascular repair, and an increasing prevalence of atherosclerosis (lakatta and levy, 2003; erusalimsky, 2009). aging-associated changes in structure and function of large elastic arteries are seen even in the absence of clinical cvd (moreau et al., 2003). although aging per se has detrimental effects in the vasculature, the lack of estrogen due to menopause may add an aggravating cvd risk factor in women, compared to arterial aging in men. in middle-aged females, aging-associated vascular dysfunction is potentiated by lack of estrogen due to menopause or ovariectomy and improves with estrogen replacement (harman, 2004; stice et al., 2009; novella et al., 2010 unfortunately, the onset of menopause coincides with a time when aging-associated damage may be noted, making it particularly difficult to distinguish between the contributions of aging and the lack of estrogen. vascular aging is a natural phenomenon that could be simply described as a consequence of physical stress, beginning early in life. arteries are elastic tissues, susceptible to fatigue, and fracture over time as a consequence of extension-relaxation cycles during heartbeats (avolio et al., 1983). in cross-sectional studies, postmenopausal females taking hrt have less arterial stiffness than their non-treated peers (moreau et al., 2003; radial artery distensibility fluctuates in accordance with estrogen levels during menstrual cycles (giannattasio et al., 1999). basic research using animal models of estrogen withdrawal and aging suggests a modulatory role for estrogen in the molecular mechanisms to prevent arterial stiffening (zhang et al., 2000) a recent study reported that hrt improves arterial compliance, an effect related in part to estrogen actions in the control of endothelial-dependent vasodilatory tone (moreau et al., 2012). collagen and elastin content of arterial walls is a key factor in arterial thickening and stiffening. it is mostly regulated by matrix metalloproteinases (mmp), enzymes capable of degrading components of the extracellular matrix. during estrogen replacement in ovariectomized rats increases mmp activity and restores aged arteries to structural properties similar to those of younger animals studied (zhang et al., 2000). dysfunction of both endothelial and smooth muscle molecular signaling appears during the aging process and favors vasospasm, thrombosis, inflammation, and abnormal cell migration and proliferation (lakatta and levy, 2003; briones et al. endothelial dysfunction in the elderly has been associated with malfunctioning of vascular tissue, resulting in atherosclerosis, hypertension, and coronary artery disease (lakatta and levy, 2003; herrera et al., 2010), renal dysfunction (schmidt et al., 2001; erdely et al., 2003), and alzheimer disease (price et al., 2004). in women, a slight age-related decrease in endothelium-dependent relaxation persists until middle age (around 50 years). after that, the declining response to the endothelium-dependent vasodilator hastens, even exceeding the rate experienced by men (taddei et al., the mechanisms for age-associated endothelial dysfunction are multiple, although most are associated with decreased no bioavailability (hayashi et al., 2008; santhanam et al., 2008; erusalimsky, 2009; kim et al., reduced endothelium-dependent and no-mediated vasodilation has been described in both human and animal models of aging (kim et al., 2009; virdis et al. lower no production in the elderly may be based on decreased no synthesis or increased no degradation. suggested mechanisms to explain reduced no production include: (1) decreased expression of enos (briones et al., 2005; yoon et al., 2010); (2) a lack of no precursor (l-arginine; santhanam et al., 2008) and enos cofactor tetrahydrobiopterin (bh4; yoshida et al., 2000; eskurza et al., 2005; meyer et al., 2011); and (3) increased endogenous enos inhibitor adma (xiong et al., 2001; kielstein et al., on the other hand, strong evidences support the hypothesis that age-associated increase in oxidative stress and consequent production of o2 is a potent contributor to lower no bioavailability and increased endothelial dysfunction (jacobson et al., 2007; rodriguez-manas et al., there is little information to correlate the progression of aging with the production/degradation of no in women. although several studies have described decreased expression of enos in senile female rats and mice (wynne et al., 2004; novensa et al., 2011), aging-associated effects on enos in women can be easily confounded with the effects of lack of estrogen, since most of these studies grouped women into just two time-points: premenopausal and menopausal groups. even though the decline in no bioavailability could sufficiently explain most of the changes in the functioning of vascular cells, other molecules crucial to control of vascular function are also modified by aging. in the regulation of vascular tone, cox-derived factors are particularly important as they can induce both vascular relaxation (pgi2) and contraction (txa2 and pgh2). some studies have reported a prevalence in the production of relaxing cox factors in the vasculature of young and healthy individuals (tang and vanhoutte, 2008). with aging, cox-dependent vasoconstrictors production becomes evident, leading to increased vascular contraction (taddei et al. however, the cox isoform involved in the generation of contractile prostanoids remains unclear. in functional studies developed in femoral arteries of aged rats, oxygen free radicals participate in the augmented endothelium-dependent contractions mediated by cox-derived prostanoids. both the constitutive and inducible isoforms of cox contribute to this endothelial dysfunction (shi et al. molecular studies performed in endothelial cells from aged rats showed an increase in mrna levels of cox-1, cox-2, and other enzymes involved in the synthesis of prostanoids (tang and vanhoutte, 2008), demonstrating the importance of the arachidonic acid cox cascade in the endothelial and vascular dysfunction associated with aging. moreover, functional studies have demonstrated an interaction between no and prostanoids pathways. in aorta from aged female mice, no bioavailability increases when the cox pathway is inhibited; both gene and protein expression of cox-1 are increased (novella et al., 2011). furthermore, activation of inflammatory pathways in the vascular wall plays a central role in the process of vascular aging. even in the absence of traditional risk factors for atherosclerosis, an age-associated shift to a proinflammatory gene expression profile, known as endothelial activation, induces upregulation of cellular adhesion molecules and cytokines, which increases endothelial leukocyte interactions and permeability, mechanisms considered crucial to initial steps in the development of atherosclerosis (herrera et al., 2010; seals et al., 2011). accordingly, a sex-associated difference in inflammatory responses during aging has been proposed. inflammatory atherosclerosis and associated acute coronary heart disease develop earlier in life in men than in women (roger et al., 2011) and are associated with earlier death, although men and women present the same overall plaque burden (frink, 2009). in animal models of atherosclerosis, male sex contributes to a faster and more severe progression of lipid deposition, remodeling, and aortic lesions (pereira et al. with the wide-ranging data from experimental research, estrogens might appear to promise protection against the progression of vascular aging and cvd in women. epidemiological observational studies also suggest that postmenopausal women on hrt are less likely to develop cvd than non-users at the same age (grodstein et al., 2000, 2001) nevertheless, these studies contrast with the large prospective clinical trials, hers and whi, which failed to show reduced cardiovascular events in postmenopausal women on hrt. in fact, whi suggested that hrt was associated with increased risk to the cardiovascular system (rossouw et al., 2002). possible reasons for this discrepancy have been extensively discussed and include the average age of women entering most hrt clinical trials, 65 years and older, which results in a study population with some degree of aging-associated vascular damage. in addition, participants had been estrogen-deficient for an average of 10 years before starting hrt, a relatively late start that could modify the status of ers and molecular signaling so as to attenuate the benefits of estrogen. for instance, during aging ers can undergo posttranslational modifications such as methylation, which decreases their expression and activity. we recently reported that aging contributes to increased dna methylation in female mice aorta, which could be associated with the decrease in the modulatory effects of estrogen (novensa et al., 2011). a few clinical studies also provide evidence for aging-associated dysregulation of er methylation and suggest that focal epigenetic changes in er could contribute to decreased estrogen activity and to the development of atherosclerosis in elderly women (post et al., 1999; detailed examination of whi data reveals that early initiation of estrogen replacement produces more favorable results than the later average initiation employed in the whi studies overall (grodstein et al., 2006; these findings, together with observational studies, have led scientists to create the so-called timing hypothesis that estrogen-mediated benefits to prevent cvd only occur when treatment is initiated before the detrimental effects of aging are established on vascular walls (harman, 2006). these effects include endothelial dysfunction and pathophysiological actions, such as increased vascular calcification and generalized stiffening of the arterial tree that increase the prevalence of hypertension and atherosclerosis (lakatta and levy, 2003; erusalimsky, 2009; kovacic et al., 2011). little information is available on whether and how vascular effects of estrogen are modified with aging in females. aging has been associated with significant reductions in the direct estrogen-mediated mechanisms of vascular relaxation (wynne et al., 2004; the lack of estrogen responses in these animal studies was not related to age-associated changes in the plasma levels of estrogen or activity of er, but rather to possible age-related changes in estrogen-mediated signaling pathways in the vasculature. modifications in the ratio between er and er in older female mice are associated with the lack of protective effects of estrogen on no production and with a reversal in its antioxidant effect to a pro-oxidant profile (novensa et al., 2011). moreover, clinical studies have revealed that cvd risk factors in postmenopausal women were lower among women aged 5059 years at hrt initiation (manson et al. these studies clearly establish the complexity of estrogen effects, which may be influenced by pathophysiological conditions including aging and subclinical cvd. despite convincing arguments by the followers of the timing hypothesis the potential extrapolation of the protective effects of estrogen replacement, well described in young females, to older women remains controversial. the field still lacks detailed experimental and clinical research on the long-term effects of estrogen and how it modulates cardiovascular function during aging. our society is aging progressively, and increased life expectancy enhances the risks for diseases associated with the natural fatigue of the body, including cvd. despite this undeniable reality, there is evidence that vascular aging in women does not follow the same chronology as in men. the vascular protective effects exerted by estrogens have been proposed as the major reason for reduced signs of vascular aging and cvd risk in premenopausal women, compared to men. when natural estrogen withdrawal occurs and a woman enters her climacteric stage, effects of sudden vascular aging become evident, leading to vascular dysfunction and increased risk of a cardiovascular event. the lack of crucial information from clinical trials and the discrepancies in the available data on the regulation of the female cardiovascular system can lead to inappropriate diagnosis and treatment of cvd in older women. women have been treated like men, despite the notable sex-associated differences in the elements of aging and disease processes. much research effort is still needed to understand age- and sex-related differences in cardiovascular control, establish the impact of the menstrual cycle and hrt on vascular function, and propose new therapeutic strategies to improve cvd diagnosis and treatment and the overall management of vascular senescence in women. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

aging is associated with structural and functional changes in the vasculature, including endothelial dysfunction, arterial stiffening and remodeling, impaired angiogenesis, and defective vascular repair, and with increased prevalence of atherosclerosis. cardiovascular risk is similar for older men and women, but lower in women during their fertile years. this age- and sex-related difference points to estrogen as a protective factor because menopause is marked by the loss of endogenous estrogen production. experimental and some clinical studies have attributed most of the protective effects of estrogen to its modulatory action on vascular endothelium. estrogen promotes endothelial-derived no production through increased expression and activity of endothelial nitric oxide synthase, and modulates prostacyclin and thromboxane a2 release. the thromboxane a2 pathway is key to regulating vascular tone in females. despite all the experimental evidence, some clinical trials have reported no cardiovascular benefit from estrogen replacement therapy in older postmenopausal women. the timing hypothesis, which states that estrogen-mediated vascular benefits occur only before the detrimental effects of aging are established in the vasculature, offers a possible explanation for these discrepancies. nevertheless, a gap remains in current knowledge of cardiovascular aging mechanisms in women. this review comprises clinical and experimental data on the effects of aging, estrogens, and hormone replacement therapy on vascular function of females. we aim to clarify how menopause and aging contribute jointly to vascular aging and how estrogen modulates vascular response at different ages.

PMC3368545

pubmed-655

among natural amino acids histidine is one of the strongest metal ion coordinators. hence, histidine plays an important role in metal ion coordination in proteins and peptides. histidine is a tridentate ligand, which provides ligands at the imidazole imido nitrogen, the amino nitrogen, and the carboxylate oxygen. the imidazole ring nitrogen of histidine residues often provides the primary coordination site for metal ions. copper is an essential metal ion for biological functions and is found in bound forms in metalloproteins and in low molecular weight complexes to avoid toxicity associated with free copper. copper-containing proteins often have binding sites with irregular geometries containing one or more histidine ligands. copper coordination in amyloidogenic proteins such as amyloid- (a), prion, and -synuclein is achieved through histidine residues. small changes in the coordination may have an effect on aggregation and other chemical mechanisms in the diseased state. hence, it is critical to elucidate the structural details of the different coordination modes to completely understand the biological role played by metal protein complexes. in -synuclein two cu(ii) coordination sites at the n-terminal region were identified using mass spectrometry (ms) and nuclear magnetic resonance (nmr). later circular dichroism (cd) and electron spin resonance (esr) results indicated that these sites are independent of each other. the high affinity site is anchored by the n-terminus, and the second site is anchored by the histidine at position 50. other than these two sites, recent nmr and esr results propose a low affinity cu(ii) binding site at the c-terminal region. in prion protein the presence of four binding sites at higher cu(ii) occupancy in each of the phgggwgq octarepeat regions were identified based on esr, cd, nmr, and x-ray absorption spectroscopy (xas) results. two additional cu(ii) equivalents coordinate via the histidine residues located at positions 96 and 111. additionally, the cu(ii) coordination environment in prion octarepeat domain has been extensively studied by ab initio methods. the copper coordination to a is highly heterogeneous, with the specific coordination environment depending on the ph, ionic strength, concentration of the metal ions, and the presence of other metal ions. the metal binding domain of a contains three histidine residues at positions 6, 13, and 14. the presence of all three histidine residues in cu(ii) coordination spheres was initially proposed by nmr and was later confirmed by esr. other techniques such as xas, fourier transform infrared spectroscopy (ftir), cd, ultraviolet visible (uv vis), ms, and ab initio calculations also proposed the involvement of histidine residues in cu(ii) coordination. a number of esr studies have suggested an equilibrium between two different cu(ii) coordination modes in an equimolar acu(ii) complex at ph 7.4, known as component i and component ii. component ii is dominant at higher ph (ph 8.7). these two components are believed to have a different number of histidine residues coordinated to the cu(ii) ion. component i is believed to have two histidine residues that simultaneously coordinate to cu(ii). previously we used electron spin echo envelope modulation (eseem) in conjunction with n isotopic labeling, to determine that the a component i consists of three subcomponents, at physiological ph where two of the three histidines are simultaneously coordinated to cu(ii) ion. interestingly, only two of these subcomponents, are present at lower ph values. cu(ii) coordinated to his 6his 13 and his 6his 14 are found in equal proportions, while the his 13his 14 coordination is absent at lower ph. these measurements were able to uncover the critical role of his 13his 14 in cu(ii) coordination at physiological ph and provide a possible rationale for the presence of amorphous aggregates, rather than fibrils at high cu(ii) concentrations. the number of histidine residues coordinated to cu(ii) in component ii is controversial. one hypothesis proposes that cu(ii) is coordinated to the co group of the ala 2glu 3 bond and three histidine residues. the second proposition involves the n-terminus, one n atom from one of the three histidine imidazoles, the backbone n from asp 1-ala 2 peptide bond, and the co group of the ala 2glu 3 peptide bond. the coordination of cu(ii) in component ii is believed to be highly dynamic. the elucidation of the coordination in component ii is critical as a recent research shows that, in the presence of zn(ii), cu(ii) coordination moves to a component ii-like coordination. furthermore, it has been shown that zn(ii) can only displace cu(ii) from component i coordination. one equivalent of zn(ii) ions displaces 25% of the bound cu(ii) from a(116) at physiological ph, while rearranging the rest of the bound cu(ii) in component i. in the presence of one equivalent of zn(ii) ions, components i and ii account equally for cu(ii) coordination in a. however, at excess amounts of zn(ii), component ii becomes the dominant cu(ii) coordination mode. in brain tissues affected by the alzheimer s disease, the concentration of zn(ii) is approximately three times higher than cu(ii). therefore, component ii cu(ii) coordination may be the most dominant coordination mode in vivo. shedding light into the coordination of component ii is critical to understand the behavior of cu(ii) in ad etiology. herein, we propose a method to quantify the number of n nuclei coordinated to a cu(ii) center by the use of integrated intensities of the fourier-transformed eseem. eseem spectroscopy is a pulsed esr technique that has been used to identify and quantify the number of histidine imidazoles coupled to a cu(ii) center. spectral simulations of the eseem spectra are used to determine the number of coupled n nuclei. compared the normalized integrated intensities of the frequency domain eseem spectra to calculate the intensity reduction resulting from the replacement of a n with n in a(116)cu(ii) complexes. although, the use of integration method was useful in elucidating important structural information, the validity of the method was not tested experimentally. here we obtained eseem data from simple model complexes with different numbers of imidazoles coordinated to cu(ii). the normalized integrated intensities of the model complexes increased monotonically, when the number of imidazole rings increased in model complexes. two small peptides with known cu(ii) coordination was used to test the validity of the method. the dahk finally, our method was used to distinguish between the two proposed modes of cu(ii) coordination. then, in conjunction with n isotopic labeling we quantified the subcomponent proportions in component ii. for the preparation of tetrakisimidazolecopper(ii) sulfate (see figure 1a), 40 ml of a 1 m imidazole (99% sigma-aldrich co., st. louis, mo) solution was added to 10 ml of 1 m cuso45h2o (98+% sigma-aldrich co., st. after a few days, dark blue colored crystals formed. for the preparation of bis(2-methylimidazole)copper(ii) diacetate (see figure 1b), 0.5 g of cu(ii) acetate hydrate (98% sigma-aldrich co., st. louis, mo) and 1.0 g of 2-methylimidazole (99% sigma-aldrich co., st. louis, mo) were added and dissolved in a mixture of chloroform (10 ml) and methanol (2.5 ml). then, 15 ml of diethyl ether was added to the filtrate and stirred for 10 min. then another 5 ml of diethyl ether was added and filtered under reduced pressure and washed with diethyl ether and chloroform. the solid was air-dried and recrystallized from methanol/diethyl ether. for dienimidazolecopper(ii) diperchlorate (see figure 1c), 30 ml of 2 mm cu(clo4)26h2o (98% acros organics, new jersey) in a methanol/acetonitrile (5:1) mixture was stirred with 3 ml of 2 mm triethylenediamine (97% fluka, netherlands) in water. then, 10 ml of 2 mm imidazole in methanol was added to the mixture. after being left overnight, all the solvents used in synthesis were purchased from sigma-aldrich co., st. crystals were dissolved in water to make 10 mm stock solutions. for esr experiments, samples were prepared in the presence of 25% (v/v) glycerol (99% sigma-aldrich co., st. louis, mo), with a final cu(ii) concentration of 1.25 mm. structures of the model complexes with 1, 2, and 4 imidazole rings and their crystal structures. the peptide phgggw, the cu(ii) binding domain of prion, was synthesized at the molecular medicine institute, university of pittsburgh, using standard fluorenylmethoxycarbonyl chemistry. isotopically enriched [g-n]-n-fmoc-n-trityl-l-histidine was purchased from cambridge isotope laboratory (andover, ma), in which all nitrogen atoms are n enriched. three different variants of amyloid-(116) (daefrhdsgyevhhqk) with each containing an n enriched histidine at either position 6, 13, or 14 were synthesized at the molecular medicine institute, university of pittsburgh, using standard fluorenylmethoxycarbonyl chemistry. double-labeled peptides containing two n enriched histidine residues were synthesized in the same manner. all the labeled amyloid- (116) variants were purified by high-performance liquid chromatography and characterized by mass spectroscopy. nonlabeled amyloid- (116) peptide was purchased from rpeptide (bogart, ga). the peptide fragment dahk, the n-terminus region of the human serum albumin, was purchased from fisher scientific, hanover park, il. (116) peptide was purchased from rpeptide (bograt, ga). for peptide samples, the concentration of the peptide was 1.25 mm and an equimolar amount of cu(ii) was present in both the samples. samples were prepared in n-ethylmorpholine (nem) buffer at ph 7.4 in 25% (v/v) glycerol and appropriate amounts of hydrochloric acid. x-ray diffraction data for the one-imidazole and two-imidazole model complex structures were collected using a single crystal on a bruker x8 prospector ultra ccd diffractometer with a cuk (= 1.54178 a) imus radiation source. the parameters used during the collection of diffraction data for each structure are summarized in supporting information. crystals were mounted in fluorolube oil on a mitegen mount and placed in a cold n2 stream (150(1) k) for data collection. x-ray diffraction data for the four-imidazole model complex was collected on a bruker smart apex ccd diffractometer with graphite-monochromated mok (= 0.71073) radiation at room temperature. for each structure, unit-cell dimensions were derived from the least-squares fit of the angular settings of 999 strong reflections from the data collection. each structure was solved via direct methods, which located the positions of the non-hydrogen atoms. an inspection of fo vs fc values and trends based upon sin, miller index, or parity group failed to reveal any systematic error in the data. all computer programs used in the data collection and refinements are contained in the bruker program package apex2 v.2013.100. all three crystal structures have been reported previously, and our results are in substantial agreement with those previously reported structures. a 200 l aliquot of complex samples with a concentration of 1.25 mm was transferred into a quartz tube with an inner diameter of 3 mm. all esr experiments were performed on a bruker elexsys e580 x-band or a bruker elexsys e680 x-band ft/cw spectrometer equipped with bruker er4118x-md5 and en4118x-md4 resonators, respectively. the temperature was controlled using an oxford itc503 temperature controller and an oxford cf935 dynamic continuous flow cryostat connected to an oxford llt 650 low-loss transfer tube. continuous-wave esr experiments were carried out on sample solutions at 80 k and at x-band microwave frequency. the magnetic field was swept from 2600 to 3600 g for 1024 data points. a time constant of 10.24 ms, a conversion time of 20.48 ms, modulation amplitude of 4 g, a modulation frequency of 100 khz, and a microwave power of 0.1993 mw were the other instrument parameters used for the cw experiment. the three-pulse eseem experiments were performed on the sample solutions at 20 or 80 k, by using a /2 /2 t /2-echo pulse sequence with a /2 pulse width of 16 ns. the first pulse separation,, was set at 144 ns, and the second pulse separation, t, was varied from 288 ns with a step size of 16 ns. the experiments were carried out at the magnetic field, where the esr intensity was maximum. after the baseline correction, the spectra were fast fourier-transformed. then the final spectra were obtained as the magnitude of the fourier transforms. we normalized the eseem spectra using the integrated intensity of h eseem signal (1316 mhz). possible limitations of the above method are discussed in the results section. for a single n nucleus coupled to an electron spin, the relative modulation depth is1where k is the modulation depth and subscripts 14 and 1 denote the n and h spin, respectively. superscripts and denote the and spin manifolds of the electron spin, respectively. for two equivalent n nuclei coupled to an electron spin the relative modulation depth becomes2 k14 is the modulation depth of the spin manifolds of the two equivalent n nuclei. the relative modulation depth of n increases with the addition of the n nucleus, and the factor of increase is given by3 if k14 and k14 are much smaller than 1, the factor converges to 2. the theoretical value for k14 and k14 is approximately 0.15, for a /2 pulse length of 16 ns. if two nonequivalent n nuclei are coupled to the electron spin, the relative modulation depth is given as4k14 is the modulation depth of the spin manifolds of the two nonequivalent n nuclei. the increase in the relative modulation depth with the additional n nuclei is given by5if k14, k14, k14, and k14 are much smaller than 1, eq 5 simplifies to6if two n nuclei are equivalent (i.e., k14=k14), the factor in eq 6 becomes 2. however, obtaining modulation depth information from eseem time domain data is difficult as many components are overlaid in the signal. it has been shown that integrated intensities can be used to account for the changes in the modulation depth. in the frequency domain n eseem signal is well separated from the h eseem signal, so it is possible to integrate separately. the n eseem intensity is obtained by integrating between 011 mhz, and the h eseem intensity is obtained for the region between 1316 mhz. in this work we experimentally determine the validity of the use of eseem integration intensities to quantify the number of n nuclei coupled to a cu(ii) center. to this end a series of model complexes with different numbers of imidazole ligands coordinated to copper are used. then, the eseem analysis was carried out on two small peptides with known cu(ii)histidine coordination, namely dahk and phgggw peptides. the dahk peptide is the n-terminus region of the human serum albumin (hsa) and coordinate cu(ii) in the well characterized atcun motive. the dahk cu(ii) complex contains a single n eseem active nucleus through the histidine imidazole coordination. the peptide phgggw is a truncated fragment of the octarepeat cu(ii) binding domain of the prion protein and has a known cu(ii) coordination including a crystal structure. cu(ii) complex is specifically used as there are two nonidentical n eseem active nuclei coupled to cu(ii). one n nucleus is from the histidine imidazole coordination and the other from the peptide backbone coordination. then we used eseem to distinguish between the two proposed modes of coordination for component ii of a. furthermore, the proportions of each histidine residue coordinated to cu(ii) ion as an equatorial ligand were measured with the use of systematic n-labeled histidine residues. in copper imidazole complexes, the imidazole ring has a noncoordinated distal nitrogen, which contributes toward the eseem signal at x-band frequencies (9.5 ghz), with pulse lengths used in this work (/2=16 ns). hence, the n eseem intensity will increase with the addition of imidazole rings to the coordination. in order to quantify the increase in n eseem intensity we synthesized three model complexes (shown in figure 1) containing 1, 2, and 4 imidazoles coordinated to a copper center, respectively. all three complexes were synthesized as crystals, and the structure of the complexes was verified using x-ray crystallography. all these structures contain copper centers coordinated with four equatorial ligands according to the information gained from crystal structures. in the one-imidazole and the four-imidazole complexes, structures of the counterion ligands perchlorate and the sulfate, esr measurements are less sensitive to axial ligands and are not considered in the analysis. the one-imidazole complex contains four directly coordinated nitrogens; three from the tridentate ligand, and one from the imidazole ring. directly coordinated nitrogens do not contribute to eseem at x-band, with pulse lengths used in this work. the one-imidazole model complex contains just single n eseem active nuclei, which is in the imidazole ring. as shown in figure 2, the one-imidazole complex has g and a values of (2.22 0.005) and (191 1) gauss, respectively. these esr parameters correspond to four nitrogen nuclei coordinated to the cu(ii) center in the equatorial plane, which is consistent with the structure of the one-imidazole complex, as shown in figure 1a. the two-imidazole complex has g and a values of (2.30 0.005) and (158 1) gauss, respectively, which is consistent with a two nitrogen and two oxygen nuclei equatorial coordination. for the four-imidazole complex, g and a values are (2.26 0.005) and (182 1) gauss, respectively, which again is consistent for four directly coordinated nitrogen nuclei. hence, cw-esr parameters clearly show that model complexes maintain the same cu(ii) coordination environment in solution. nuclear quadrupole interactions (nqi) of n give rise to features below 2 mhz. the broad feature around 4 mhz is due to the double quantum (dq) transition of the remote nitrogen in an imidazole ring. the intensity of the dq peak increases with the number of imidazole rings coordinated to the cu(ii) center. a peak around 9 mhz (black arrow in figure 3) is also indicative of multiple imidazole coordination. this peak is clearly observed in the two- and four-imidazole complexes. experimentally obtained three-pulse eseem spectra of the model complexes at the maximum g position. appearance of a peak around 9 mhz in two- and four-imidazole complexes is indicative of multiple imidazole coordination. the integrated intensity for n-eseem was calculated for the region between 011 mhz and then divided by the h-eseem intensity integrated between 13 and 16 mhz. details of the error calculation are provided in the supporting information (see figure s3). the normalized n-eseem intensity increases from 8.4 to 21 when going from single imidazole to two imidazoles as shown in table 1. when there are four imidazoles coordinated to the cu(ii) center, normalized n-eseem intensity is increased to 40. hence, there is a monotonic increase in the normalized n-eseem intensity with the increase of number of imidazole rings coordinated. in the text, values in the last column are referred as normalized integrated intensity. in order to test our claim in a biologically relevant system, eseem experiments were conducted on two different peptide fragments with well-known cu(ii) coordination. the four amino acid peptide dahk is the n-terminus fragment of the human serum albumin. in the dahk peptide, the imidazole ring of the histidine residue coordinates to the cu(ii) ion, and the distal nitrogen of the imidazole ring is the only eseem active nuclei as shown in figure 4. the comparison between the eseem spectra of cu(ii)dahk and one-imidazole complex is shown in figure 4. the intensity of dq peak reflects the number of imidazoles coordinated to the cu(ii) center. the normalized integrated intensity for cu(ii)dahk is 7.0 0.1 compared to 8.4 0.1 for the one-imidazole complex (table 1). only one n-eseem active nuclei is present in the reported coordination of dahk. then we used the phgggw peptide fragment, which is the cu(ii) binding domain of the prion protein. resolved the cu(ii) coordination environment of the octarepeat fragment using esr experiments and a crystal structure of the cu(ii)phgggw complex. as shown in figure 5, cu(ii) is coordinated to an imidazole nitrogen in a histidine, two backbone nitrogens from two glycine residues, and an oxygen from a carboxylic group. the structure of the cu(ii) bound hgggw pentapeptide was found to be unstable in solution due to the breaking of the axial water coordination also, eseem results have indicated that histidine imidazole coordination and backbone coordination is present in the cu(ii)phgggw complex in solution. the eseem spectra of the cu(ii)phgggw complex and the two-imidazole complex are shown in figure 5 for comparison. given the different coordination environments the two spectra do not have identical peak positions. the two-imidazole complex has a larger dq peak compared to the phgggw complex. the reported structure for the cu(ii)phgggw structure contains two n-eseem active nuclei from the distal nitrogen of the imidazole histidine and the backbone coordination as shown in figure 5. hence, the intensity of dq peak is expected to be different between the two complexes, as dq peak intensity is indicative of the number of imidazoles coordinated. however, the normalized n-eseem integrated intensity obtained for the cu(ii)phgggw complex is similar to the integrated intensity of the two-imidazole complex. this information verifies that the integration method can predict the number of n nuclei coupled to a cu(ii) center. comparison of eseem spectra of cu(ii)phgggw complex and the two-imidazole complex. the integration analysis was used to determine the number of nitrogens coupled to the cu(ii) ion in a(116) in component ii. as shown in the inset of figure 6, zn(ii) selectively displaces cu(ii) coordinated to component i. at excess amounts of zn(ii) (ten equivalents of zn(ii)), component ii accounts for 65% of the overall coordination (inset of figure 6), where in the absence of zn(ii) the percentage is only 35%. amyloid aggregates in brain tissues contain approximately three times zn(ii) than cu(ii). figure 6 shows the comparison between the two-imidazole complex and the a(116)cu(ii) complex at ph 8.7. at ph 8.7 only the component ii of cu(ii) coordination exists. the integrated intensities tabulated in table 1 suggest that two n nucleus are coupled to the cu(ii) ion. the features of the eseem spectrum clearly illustrate the imidazole histidine coordination as shown in figure 6. the backbone coordination peak is possibly due to the coupling between the cu(ii) ion with the remote backbone nitrogen nuclei of glu 3, where cu(ii) is coordinated to the carbonyl oxygen of ala 2. this suggests just a single histidine residue is coordinated to cu(ii) in component ii coordination. comparison of eseem spectra of cu(ii)a(116) complex at ph 8.7 and the two-imidazole complex. the inset shows the increase of component ii cu(ii) contribution in the presence of zn(ii). the normalized integrated intensity (n-eseem/h-eseem) of a(116) is 17 compared to 21 and 22 for the two-imidazole complex and the phgggw, respectively. the lower value for a(116) is possibly due to the smaller number of protons that interact with the cu(ii) center. the cu(ii) centers in the model complexes are solvent accessible. in a(116) the solvent cu(ii) interaction may be restricted because of the neighboring amino acids. in our analysis we have normalized the integrated area of the h-eseem peak to be the same for all spectra. therefore, we may be underestimating the n-eseem integrated intensity for a(116). nevertheless, the normalization method does suggest that cu(ii) is coupled to two n-eseem active nuclei, not one or three. hence, we can answer the crucial question of the number of histidines coordinated to component ii. three-pulse eseem spectroscopy was used in conjunction with isotopic substitution to determine the coordination of component ii. specifically the aim of these experiments was to provide more insight into the proportions of histidine residues involved in component ii coordination. two histidine residues at a time are isotopically labeled with n. upon n substitution, the modulation depths of the frequencies due to eseem active n nuclei will decrease. this decrease is because the single quantum transition of n nuclei does not substantially contribute to the eseem signal. we compare the integrated intensities of nonlabeled and n-labeled variants. as the modulation depth of h frequency is not affected by the n substitution, the h eseem peak is used to normalize the integrated intensity of n eseem. because two of the three histidine residues are labeled, the n eseem signal intensity results only from the single nonlabeled histidine. this provides a direct method to determine the extent to which each histidine residue is involved in component ii. all the samples were at ph 8.7, and the experiments were carried out at 3355 g, which corresponded to the maximum signal in the echo detected field sweep. the eseem intensities are integrated between 0 and 11 mhz in all the eseem spectra collected (figure 7). the integrated eseem intensity of the his 6,13-labeled variant was 40% of that of the nonlabeled variant. in the his 6,13 variant, his 14 is the only labeled histidine residue and the n eseem intensity results from only his 14. likewise, his 13 contributes 40% and his 6, 20%, toward the component ii cu(ii) coordination (table 2). the integrated intensities are tabulated in the supporting infomation (table s2). to further confirm the proportions of the histidine residues, we performed the experiments using single-labeled variants in which only one histidine is labeled at a time. the decrease in the signal intensity in the n eseem region with respect to the nonlabeled variant indicates the extent of the involvement of the labeled histidine in the coordination. the eseem spectra for single-labeled variants are shown in figure s4, supporting infomation. the integrated n eseem intensities (table s3, supporting infomation) for single-label variants show the similar pattern of his 14 his 13>his 6 as observed with the double-label variants. three-pulse eseem spectra of the nonlabeled and single n-labeled a(116) variants mixed with equimolar amounts of cu(ii) at ph 8.7. the decrease in intensity below 8 mhz in n-labeled a(116) variants gives the contribution of each histidine residue for component i in a(116)cu(ii). the inset shows an expanded view of the 06 mhz region for the labeled peptides. this trend can be rationalized by the pka values of the histidine side chains. his 6 has a pka of 7.1, while for his 13 and 14 the pka values are 7.7 and 7.8, respectively. as the imidazole ring in his 6 has a lower pka value, ring nitrogens will be deprotonated at lower ph values than in his 13 and 14. when the ph is raised, his 13 and 14 rings become more accessible for cu(ii) coordination. hence the proportions of his 13 and 14 are increased with the increase in ph. these results are in accordance with an x-ray examination of a(116), which suggests that his 13 and his 14 are readily accessible for metal ion coordination. the design of a curcumin scaffold was discussed in this work, which is used to compete for cu(ii) coordination with the his 13his 14 dyad. hence, we suggest only one histidine residue is involved in component ii, with a preference to his 13 and his 14 over his 6. the other residues involved in the component ii coordination are the carbonyl oxygen of ala 2, the n-terminus (asp 1), and the amide nitrogen of ala 2. the peak around 2.8 mhz in the eseem spectra is indicative of backbone coordination and further confirms the involvement of the peptide backbone in component ii coordination. his 13 and his 14 equatorially coordinate to cu(ii) more than does his 6. our eseem results performed using both the double and single n-labeled histidine residues indicate that his 13 and his 14 have a higher preference for the equatorial coordination position in cu(ii) component ii coordination. chemically, component ii cu(ii) coordination is really interesting as zn(ii) biologically, it is important to understand the component ii coordination, as component ii may be the most significant cu(ii) coordination in vivo, as zn(ii) coexists with cu(ii). the insight into the component ii coordination, more importantly the proportions of subcomponents, will shed light on understanding the role of metal ions in alzheimer s disease. we experimentally validated the use of eseem intensities to quantify the number of n nuclei coupled to a cu(ii) ion. a monotonic increase in the n eseem intensities were observed for the model complexes synthesized with different numbers of imidazole rings. then, the validity of the method was tested with two well-characterized cu(ii) binding peptides. we used our method to solve an important structural problem in acu(ii) complex. we determined that only a single histidine residue is coordinated to cu(ii) ion in component ii in a. finally, in component ii, cu(ii) uses his 13 and his 14 as an equatorial ligand over his 6. the proportions of the three histidine residues in cu(ii) coordination can be rationalized by the pka values of the histidine side chains. shedding light into the component ii coordination was critical as component ii might be the dominant cu(ii) coordination mode of a in vivo.

we validate the use of eseem to predict the number of 14n nuclei coupled to a cu(ii) ion by the use of model complexes and two small peptides with well-known cu(ii) coordination. we apply this method to gain new insight into less explored aspects of cu(ii) coordination in amyloid- (a). a has two coordination modes of cu(ii) at physiological ph. a controversy has existed regarding the number of histidine residues coordinated to the cu(ii) ion in component ii, which is dominant at high ph (8.7) values. importantly, with an excess amount of zn(ii) ions, as is the case in brain tissues affected by alzheimer s disease, component ii becomes the dominant coordination mode, as zn(ii) selectively substitutes component i bound to cu(ii). we confirm that component ii only contains single histidine coordination, using eseem and set of model complexes. the eseem experiments carried out on systematically 15n-labeled peptides reveal that, in component ii, his 13 and his 14 are more favored as equatorial ligands compared to his 6. revealing molecular level details of subcomponents in metal ion coordination is critical in understanding the role of metal ions in alzheimer s disease etiology.

PMC4120975

pubmed-656

over the past several decades, numerous studies have described pharmacologic strategies to utilize matrix metalloproteinase-inhibitors (mmp-is) to prevent connective tissue breakdown associated with various inflammatory and other diseases, for example, periodontitis, arthritis, osteoporosis, cardiovascular disease, and cancer [14]. recently, these have also included less obvious strategies such as (but not limited to) blocking mmp-mediated cleavage of insulin receptors in type-2 diabetics to improve insulin sensitivity and to reduce hba1c levels. however, to date, the only orally (systemically) administered mmp-is approved by the us-fda and other national regulatory agencies (europe and canada) are those based on the surprising nonantimicrobial properties of the tetracycline antibiotics [4, 79]. in this regard, studies on experimental animals and on human subjects have demonstrated the efficacy of nonantimicrobial tetracycline formulations, as pleiotropic mmp-is, in periodontal and other diseases [4, 7, 9, 10]. in addition to demonstrating that these medications, which include two formulations of subantimicrobial-dose doxycycline (both fda-approved), can inhibit collagenolysis, connective tissue destruction, and bone resorption in the diseased periodontal tissues, other therapeutic mechanisms have also been identified. these include suppressed expression of inflammatory mediators such as the cytokines (e.g., il-1, tnf-, and il-6), prostaglandins, reactive oxygen species (e.g., hocl), and nitric oxide, the latter reflecting the inhibition of inducible nitric oxide synthase [11, 12]. given this background, a search has been underway for new drug molecules which exhibit a similar active site for mmp-inhibition as the tetracyclines but with a different phenolic superstructure. with this strategy in mind, the therapeutic potential of the tetracycline diketonic, metal-ion binding site [8, 9] has been expanded by the recent development of a new series of compounds with a similar zinc-binding moiety, which are bicyclic rather than tetracyclic, that is, the chemically modified curcumins or cmcs. the structures of these compounds, their potency and mechanisms of action as mmp-is, and their zinc-binding (and other) characteristics have been described recently, and a lead this compound, cmc 2.24, is a phenylamino carbonyl curcumin, is triketonic (which enhances its zinc-binding characteristics) in contrast to the diketonic active site on both the tetracyclines and on traditional/natural curcumin compounds, and has shown evidence of efficacy in vitro, in cell and organ culture, and in animal models of chronic inflammatory and other diseases [1315]. as additional background, recent studies have shown that natural/unmodified curcumin administered to rats with experimentally induced periodontal disease was effective in reducing inflammatory mediators and mmps in the gingiva and periodontal ligament but was ineffective in reducing the excessive resorption and loss of alveolar bone. accordingly, the current report describes the first of a series of studies which examined the efficacy of cmc 2.24 as a pleiotropic mmp-i in several rat models of periodontitis with a particular focus on its ability to inhibit pathologic alveolar bone loss. moreover, because of the long-standing interest in the link between the oral disease, periodontitis, and systemic inflammation (the latter associated with increased risk for various diseases, notably cardiovascular disease and more severe diabetes [4, 17]), the effects of treatment with this novel compound on biomarkers in the circulation were also examined. eleven male holtzman rats (rattus norvegicus albinus) weighing 150250 g were maintained under pathogen-free conditions with controlled temperature (21 1c) and humidity (6570%) and a 12 h light-dark cycle. 30 g of lipopolysaccharide (lps) from escherichia coli (strain 055:b5; sigma chem co., st. louis, mo, usa) diluted in phosphate buffered saline (pbs) was injected into the palatal gingiva (3 l volume per injection) using a hamilton microsyringe (agilent, santa clara, ca, usa) as described by us previously. these lps injections were made into the palatal tissue between the upper 1st and 2nd molars, on the left side of the animal, three times a week for 14 days (a total of 6 injections and 180 g of lps in each site). the opposite side received injections of the same volume of pbs vehicle and served as the control site (split-mouth also at the time of sacrifice, blood samples were collected and the serum and plasma were separated by standard procedure and analyzed for mmps and cytokines as described below. the study protocol was previously approved by the institution's committees (araraquara-unesp, sp, brazil, and stony brook university, ny, usa) for experimental animal use. the effects of cmc 2.24 (a phenylamino carbonyl curcumin) were assessed in a prophylactic model (the efficacy of this compound in a therapeutic model will be assessed in future studies) in which the induction of periodontal disease by lps injections was carried out during the same period of time (14 days) as the daily oral administration of cmc 2.24 (30 mg/kg) or the vehicle-control. the test compound and the vehicle-control (a 1 ml suspension of 2% carboxymethyl cellulose) the rats and their periodontal tissues were randomly distributed into the following experimental groups as illustrated in figure 1. group 1gingiva injected with pbs in rats systemically administered vehicle alone (n=5); group 2gingiva injected with e. coli lps in the vehicle-treated rats (n=5) (note: with this split-mouth design, group 1 and group 2 tissues involve the same 5 rats); group 3gingiva injected with pbs in rats systemically administered the test medication (cmc 2.24; n=6); and group 4gingiva injected with e. coli lps in rats systemically administered cmc 2.24 (n=6) (as above, groups 3 and 4 involve the same 6 rats). however, for the -ct analysis, additional rats were added to each experimental group resulting in n=10 rats per group. the gingival tissues from the hemimaxilla of each rat were excised and pooled per experimental group (5-6 rats per group) as described by us previously [19, 20]. the pooling of gingival tissues for each group was necessary because individual rats do not yield sufficient gingiva for enzyme analyses. the gingival tissues were extracted and the mmps were partially purified as described by us previously [19, 20]. in brief, the samples were homogenized (all procedures at 4c) with a glass grinder (kontes, glass co., vineland, nj) attached to a t-line lab stirrer (model 106 taboys engineering corp., nj) in 50 mm tris-hcl buffer (ph 7.6) containing 5 m urea, 0.2 m nacl, and 5 mm cacl2 and then extracted overnight and centrifuged at 15,000 rpm for 1 h. the supernatants were collected and dialyzed exhaustively against 50 mm tris buffer (ph 7.8) containing 0.2 m nacl and 5 mm cacl2. ammonium sulfate was added to the dialysate to produce 60% saturation, allowed to stand overnight, and the precipitate containing the mmps was collected by centrifugation at 15,000 rpm for 90 min. the pellets were then dissolved in the tris buffer (ph 7.8) containing nacl, cacl2, and 0.05% brij and exhaustively dialyzed against the same buffer. the relative levels of the higher molecular weight proforms and the lower molecular weight activated forms of mmp-2 and mmp-9, in the pooled gingival extracts from each of the four experimental groups (figure 2), were determined by zymography (the gelatin zymography system was purchased from invitrogen corp., carlsbad, ca). in brief, all samples were run under nonreducing denaturing conditions on the gelatin zymography system containing polyacrylamide copolymerized with gelatin at a final concentration of 1 mg/ml. after electrophoresis, the gels were washed in 2.5% triton x-100 and incubated at 37c overnight in the assay buffer (40 mm tris, 200 mm nacl, and 10 mm cacl2; ph 7.5). clear zones of lysis against a blue background indicate gelatinolytic activity, as described by us previously [11, 21, 22]. densitometric analysis of the gelatinolytic bands was carried out using the scientific imaging system (kodak i d 3.5, rochester, ny). since this is a major outcome in the experimental periodontal disease model and since reducing alveolar bone loss is a key therapeutic goal in treating human inflammatory periodontal disease, two methods were used to assess the effects of cmc 2.24 on this inflammatory-driven bone loss model. as described previously, the soft tissues were carefully dissected to maintain the integrity of the maxillary bone specimens. these were then completely defleshed by immersion in 8% sodium hypochlorite for 4 h followed by gentle mechanical scavenging of the remaining soft tissue. after washing in running water, the specimens were immediately dried with compressed air. to distinguish the cementum-enamel junction (cej), 1% aqueous methylene blue solution (sigma-aldrich, saint louis, mo, usa) was applied to the specimens for 1 min and then washed in running water. the specimens were fixed on 3 mm thick red dental wax with their palatal surface facing up. standardized orientation was achieved by positioning the specimens with the palatal cusp tip of the first and second molars superimposed on the corresponding buccal cusp tips (i.e., occlusal plane perpendicular to the ground). to validate measurement conversions, the specimens were positioned under a stereomicroscope (leica mz6, buffalo grove, il, usa) and digital images were obtained at 25x magnification using a 6.1-megapixel color digital camera coupled to the microscope. a single examiner, who was not aware of the experimental group allocation of the specimens, carried out all morphometric measurements of alveolar bone loss by delineating the area of exposed root surface of the first and second molars using an image analysis software (leica application suite, v3.8.0, leica microsystems, buffalo grove, il, usa) and the results were converted to mm using measurement of the reference millimeter grid. the area of exposed root surface in each specimen was averaged according to the experimental groups. intraexaminer calibration was performed by evaluating repeated measurements of 10 nonstudy images presenting alveolar bone loss similar to the present study., the hemimaxillae of the rats were dissected including teeth and surrounding soft tissues, fixed for 1824 h in 10% neutral buffered formalin at 4c, washed in distilled water, and transferred to 70% ethanol. this procedure allowed us to use these same specimens for the histological assessments used in subsequent studies (guimaraes et al. these samples were scanned on a microcomputer tomograph (skyscan 1176, skyscan, aartselaar, belgium) using 18 m slices. the digital radiographic images of each sample were reconstructed into a three-dimensional model (nrecon software, skyscan, aartselaar, belgium) consisting of a matrix of 18 18 18 m and a standardized gray scale value to visualize only mineralized tissues. using the software package dataviewerctanctvol (skyscan, aartselaar, belgium), the reconstructed tridimensional matrix of each sample was initially reoriented in a standardized manner on three planes: sagittal, coronal, and transversal. subsequently, a cubic region of interest (roi) of 9.72 mm was defined using standardized dimensions and anatomical landmarks: cementum-enamel junction of the first molar as the coronal limit extending vertically 1.5 mm apically, an anteroposterior dimension of 3 mm from the distal aspect of the mesial root of the first molar, and the transversal (buccolingual thickness) dimension of 2.16 mm (120 slices of 18 m each). this roi included the first molar, half of the second molar, and also approximately 1 mm from the most palatal aspect of the first molar crown (including the palatal bone adjacent to the first and second molar teeth which was the site of lps injections). we determined the relative volume of this roi occupied by mineralized tissue in each sample. the data was averaged for each experimental group and compared by nonpaired t-tests using welch's correction for unequal variances. mmp-8 levels in plasma and gingival extracts, the latter prepared as described above, were determined by western blot analysis. in brief, samples were reduced, boiled, subjected to sds/page, and transferred to polyvinylidene difluoride (pvdf) membrane (amersham pharmacia biotech inc., piscataway, nj). blots were blocked with 5% nonfat dry milk for 2 h at room temperature. the membranes where then incubated with polyclonal antibodies specific for mmp-8 (abcam plc, cambridge, ma) overnight at 4c. blots were washed and incubated with secondary antibodies purchased from thermo scientific for 2 h at room temperature. detection of the bands was carried out on radiographic film by using supersignal west dura extended duration chemiluminescent substrate (thermo fisher scientific inc., the band densities were quantified by scanning on a laser densitometer. to assess the levels of inactive (proform) and smaller molecular weight active forms of the mmp-8 (collagenase-2), bands corresponding to both molecular weight forms were quantitated, and the data is expressed as densitometric units and as the ratios of inactive/active forms. recombinant rat mmp-8 (source: mouse myeloma cell line, nso derived) from r&d systems (minneapolis, mn) was used as a standard for western blot analysis of the rat plasma samples. this mmp-8 standard was incubated for 4 hours at room temperature, in the presence or absence of 1 mm amino phenyl mercuric acetate (apma), a known activator of higher molecular weight pro-mmps into the lower molecular weight activated forms. the level of mmp-13 was measured in the gingival tissue extracts and plasma of each rat by enzyme-linked immunosorbent assay (elisa). this assay was performed according to the manufacturer's instructions (tsz scientific llc, framingham, ma). the levels of 3 bone resorptive cytokines (il-1, il-6, and tnf-) were measured in serum and gingival tissue extracts by enzyme-linked immunosorbent assays (elisas). these assays were performed according to the manufacturer's instructions (r&d systems, minneapolis, mn), and the results were normalized to the total concentration of protein in the samples. the levels of both mmp-2 (72 kda progelatinase) and mmp-9 (92 kda progelatinase) were assessed by gelatin zymography in pooled gingival tissue from half-jaws of rats from each experimental group (figure 2). lps-induced periodontal disease dramatically increased mmp-2 and mmp-9 levels in the pooled gingival tissue, while lower levels of the pro- (higher molecular weight) and activated (lower molecular weight) forms of these gelatinases were seen in the gingival tissue from all of the other experimental groups. treatment of the rats with systemically administered cmc 2.24 appeared to normalize the pathologically excessive levels of the various molecular weight forms of these gelatinolytic mmps in the lps-injected gingiva assessed either visually (figure 2(a)) or by densitometric analysis of the zymograms (figure 2(b)). some reduction of these mmp proteinases by cmc 2.24 administration was also seen in the gingiva from the rats without lps injections (figures 2(a) and 2(b)). in a pattern reminiscent of the zymograms described above and based on morphometric analysis of alveolar bone height loss which measured the area of exposed root relative to the cementoenamel junction as a fixed anatomical landmark, lps injections into the gingiva significantly (p=0.005) increased alveolar bone loss (figure 3). moreover, when the lps-injected rats were treated by oral administration of cmc 2.24, alveolar bone loss was significantly reduced (p=0.003) back to the normal level seen in the rats not exposed to gingival lps injections. note that cmc 2.24 treatment did not affect alveolar bone loss in the control rats receiving injections of pbs vehicle rather than lps (figure 3). to confirm and expand these data on alveolar bone loss in the four experimental groups (figure 1),, these data again demonstrate that lps increased the loss of bone in the aoi and that cmc 2.24 administration reduced this bone loss to the level seen in the control rats in which the gingivae were injected with pbs instead of lps. analysis of il-1 in extracts of the pooled gingival tissues indicated that lps injections markedly increased the level of this proinflammatory cytokine since it was not detectable in the extracts of the pbs-injected gingival tissue (figure 5(a)). moreover, cmc 2.24 administration reduced the pathologically excessive levels of il-1 in the gingiva by 93% (figure 5(a)). similar concentrations of il-6 were detected in the gingival tissues from the different groups of rats; however, the lps injections did not appear to affect these levels and cmc 2.24 treatment only slightly reduced the levels of this cytokine by about 15% (data not shown). tnf- was undetectable in both gingival extracts and serum (see below). in the experimental protocol used in the current study (a split-mouth design), mmp-8 (neutrophil-type collagenase, collagenase-2) and mmp-13 (collagenase-3) were both detected in the plasma samples from the different groups of rats but neither was detected in the gingiva (see section 4). based on western blot analysis, the plasma samples from the lps-injected rats (half-jaw only) which were treated by oral administration of cmc 2.24 appeared to exhibit reduced levels of activated, lower molecular weight forms of mmp-8 compared to the plasma from the lps-treated rats administered with the vehicle alone (controls) (figure 6(a)). based on the densitometric analysis of these western blots (figure 6(a)), the plasma of the cmc 2.24-treated rats with lps-induced periodontitis exhibited a ratio of pro/active mmp-8 of 2.52 0.20 (sem) which was 89.5% higher than the ratio, 1.33 0.05, seen in the plasma from the vehicle-treated lps-periodontitis rats (figure 6(b)), and this inhibition of activation of the precursor (latent) form of mmp-8 by the cmc2.24 treatment was statistically significant (p=0.024). note that a 4-hour incubation of the standard recombinant rat mmp-8 with 1 mm apma, a known activator of pro-mmps in vitro, converted the higher molecular weight pro-mmp-8 into the smaller molecular weight activated form of this leukocyte-type collagenase (see figure 6(a)). the plasma levels of mmp-13 assessed by elisa were found to be about 1.1 g/ml. administration of cmc 2.24 to the lps-periodontitis rats appeared to slightly reduce the levels of this collagenase in the plasma; however, this effect was not statistically significant (data not shown). regarding the proinflammatory cytokines in the serum (figure 5(b)), because of the split-mouth design (see figure 1), there were no serum samples from rats without gingival lps injection. however, the levels of il-1 in the serum of these lps-exposed rats (about 30 pg/ml) were significantly (p=0.03) reduced to undetectable levels by cmc 2.24 administration, a pattern similar to that seen in the gingival tissues (figure 5(a)). il-6 showed higher concentrations in the serum (about 95 pg/ml) than il-1 in the lps-periodontitis rats, and, again, cmc 2.24 appeared to reduce the level of this cytokine. however, this lesser effect (about 18% reduction) was not statistically significant (data not shown). this paper advances a novel therapeutic strategy which uses systemically administered medications as adjunctive therapy to modulate the host response in periodontal disease (periodontal therapy has traditionally only focused on locally suppressing the pathogenic microorganisms in the oral biofilm), with applications for other chronic inflammatory diseases as well (see below). the clinical application of this strategy began with the discovery that tetracyclines (tcs), unexpectedly, can inhibit host-derived mmps, inflammatory mediators (e.g., the cytokine il-1), and collagen degradation including bone resorption; and by mechanisms not dependent on the antibacterial properties of these drugs [4, 710]. soon thereafter, doxycycline was found to be a more potent mmp-inhibitor than other tetracycline antibiotics, including minocycline and tetracycline itself, and was subsequently developed and approved as a nonantibiotic low-dose formulation for long-term administration to patients with chronic periodontitis and the dermatologic inflammatory disease, rosacea [4, 9]. based on these earlier and the current studies, the nontetracycline chemically modified curcumin (discussed below) appears to be as, or more, potent an mmp-inhibitor compound compared to doxycycline [4, 8, 9, 13]. as one example, the ic50 (the concentration of the compound required to inhibit 50% of mmp activity in vitro) of doxycycline has been reported to be approximately 15 m [8, 9]. in contrast, recent studies by our group have demonstrated ic50 levels of cmc 2.24 at even lower m levels (25 m) when tested in vitro against mmps such as mmp-8 (leukocyte-type collagenase), mmp-9 (leukocyte-type gelatinase), mmp-12 (macrophage metalloelastase), and mmp-14 (membrane-type mmp). however, a significant disadvantage of the approved subantimicrobial-dose formulations of doxycycline is that no increase in the dose of this tetracycline can be prescribed to the patient (which might be desirable in order to, possibly, enhance the efficacy of this treatment in collagen-destructive diseases, e.g., periodontitis) because the low nonantibiotic blood levels of the drug (< 1 g/ml) produced by this formulation can not be exceeded in order to prevent an important side-effect, namely, the emergence of tetracycline-resistant or pan-antibiotic-resistant bacteria. in contrast, the potential strategy of long-term administration of cmc 2.24, for inflammatory diseases, would not be undermined by this strict, low-dose, limitation because this compound is not an antibiotic like the tetracyclines. as described earlier (see section 1), natural curcumin has a similar active site (i.e., the diketone zinc-binding moiety) as the tetracyclines and can also modulate the host response including mmp-inhibition and suppression of inflammatory mediators [2531], although it is ineffective against alveolar bone loss (see below). however, the chemically modified curcumin, cmc 2.24, tested in the current in vivo study, has a modified active site which is triketonic as detailed by us in previous studies by zhang et al. recently, newer host-modulating medications have also been investigated as adjunctive treatment for periodontal disease and related medical disorders. these, in particular, have included (1) the resolvins such as the polyunsaturated fatty acids which do not suppress the acute inflammatory response required by the host to combat infection, but which do prevent the tissue-destructive prolongation of this process, and (2) the subject of the current study, the chemically modified curcumins (cmcs). of importance, the latter have shown improved solubility, zinc-binding, and biological effects in comparison with natural curcumin [13, 14]. development of these cmcs is based on maintaining a similar active site for mmp-inhibition as that of the tetracyclines but with a different phenolic superstructure, which most recently resulted in the development of a new series of compounds with a triketonic zinc-binding moiety, which are still bicyclic rather than tetracyclic, that is, the chemically modified curcumins or cmcs. a series of these triketonic cmcs have been developed including cmc 2.5 (a methoxy carbonyl curcumin) which, in turn, has been superseded by a more potent mmp-i compound, cmc 2.24, a phenylamino carbonyl curcumin; the latter has shown evidence of efficacy (and safety) in vitro, in cell and tissue culture, and in vivo models of several diseases including arthritis, diabetes, and cancer [1315, 33]. the current study is the first to demonstrate efficacy of this compound, cmc 2.24, in an animal model of experimental periodontitis. evidence of the onset and progression of this disease, induced by several injections of lps into the gingiva of the rat, included dramatic increases in several forms (both pro- and activated) of connective tissue-destructive mmp-2 (72 kda) and mmp-9 (92 kda) gelatinases, elevated levels of the inflammatory cytokine often associated with periodontitis, il-1, and, most importantly in this model, a significant increase in alveolar bone loss, assessed morphometrically and by -ct, in the same jaws as the increase in gingival inflammatory mediators and mmps (the impact of this local inflammatory disease and this experimental treatment on systemic levels of mediators is discussed below). the potent efficacy of cmc 2.24 was demonstrated by (i) the statistically significant reduction of the lps-induced, pathologically elevated alveolar bone loss down to the levels seen in the healthy controls and (ii) the essentially complete reduction of the pro- and activated, pathologically excessive levels of mmp-2, mmp-9, and il-1, in the inflamed gingival tissues back down to the un- (or barely) detectable levels seen in the control gingiva. as a result of the profound efficacy of this novel compound in this initial study, we now have a rationale to initiate studies using a modified animal model of experimental periodontal disease, which does not use split-mouth design, and in a periodontitis model in which the cmc 2.24 is administered therapeutically (after the disease has been established) rather than prophylactically as in the current study. in a more recent study in which alveolar bone loss was assessed at the cellular level histomorphometrically and histochemically, a similar pattern of change was seen, namely, that lps injection increased osteoclast-mediated bone resorption and that cmc 2.24 inhibited this mechanism of alveolar bone loss (guimaraes et al., in preparation). the potency of the biological effects of cmc 2.24 at an oral dose of 30 mg/kg is further demonstrated by the fact that, in previous experiments, we have not observed a significant decrease of inflammatory-driven bone resorption with 100 mg/kg dose of natural curcumin. interestingly, in recent experiments, we found that daily administration of 400 mg/kg of natural curcumin significantly reduced inflammatory-driven bone resorption in this model, but this dose of natural curcumin is more than 10-fold higher than the dose of cmc 2.24 administered in the current study (guimaraes et al., in preparation). regarding insights into the mechanisms, plus the impact of this local disease and its treatment on the systemic condition of the host, we also observed the following: (i) the apparent reduction of il-1 by cmc 2.24 treatment in the pooled gingival tissues was paralleled by a dramatic and significant reduction in this inflammatory mediator in the systemic circulation of the same animals, and (ii) for cmc 2.24 treatment, although it did not appear to alter the total levels of mmp-8 (neutrophil-type collagenase) in the blood samples of the lps-injected rats, it did significantly reduce the ratio of the lower molecular weight, activated, collagen-destructive forms of this collagenase relative to the higher molecular weight, inactive, proforms of this mmp (note that, in the current experiment, mmp-8 could not be detected in the pooled gingival tissue). mechanisms could include the ability of cmc 2.24 to inhibit other neutral proteinases such as plasmin, elastase, and mmp-1 which are known to cleave the amino-terminal propeptide domain of pro-mmp-8, converting it into the smaller molecular weight activated forms [9, 20]. of relevance to the mechanisms involving cmcs ability to inhibit pro-mmp activation, recent studies (s. simon et al., unpublished data) indicate that 2.24 can inhibit serine neutral proteinases (i.e., neutrophil elastase) which could explain the reduced conversion of pro- into smaller molecular weight activated mmps which was observed in the current study in the systemic circulation. still another possible mechanism involves the potential of this compound to inhibit the production of reactive oxygen metabolites (e.g., hypochlorous acid, hocl). these are known to mediate proteinase activation by dissociating the thiol group in the propeptide domain. this mechanism is significant because mmp-8 is largely derived from the degranulation of polymorphonuclear leukocytes, and, in the human periodontal pocket, mmp-8 constitutes about 8090% of the total collagenase in this lesion; mmp-13 is the second most dominant collagenase in the periodontal pocket in humans, contributing about 1020% of the total, and is thought to be derived from the junctional epithelium and bone cells [7, 34]. however, in the rat, mmp-13 is analogous to the constitutive collagenase, mmp-1, in humans and likely plays a role in physiologic turnover of collagen rather than the pathological degradation of collagen during periodontitis. in this regard, mmp-13 also could not be detected in the inflamed gingival tissues in the rats in the current study and, although it was detected in the plasma, was not reduced by cmc 2.24 treatment suggesting a preferential effect of the test compound on pathologically elevated rather than on constitutive levels of these mmps. additional mechanisms include the ability of natural curcumins to inhibit various signaling pathways and transcription factors involved in the expression of inflammatory mediators (ap-1, mapk, nf-kb, and stat3) resulting in a decrease in the expression of the inactive proforms of the mmps and of inflammatory cytokines and, ultimately, a marked change in the microenvironment [25, 35, 36]. the results of this initial study indicate that the oral administration of a novel, triketonic phenylamino carbonyl curcumin (cmc 2.24), to rats with endotoxin- (lps-) induced periodontitis, is a significant and potent inhibitor of both pathologic alveolar bone loss and its inflammatory and collagen-destructive mediators. moreover, this chemically modified curcumin appears to have additional benefits by reducing the impact of this local inflammatory disease on systemic biomarkers of the host without (apparently) negatively affecting the mediators of constitutive connective tissue turnover. studies are now underway to expand these observations in additional rat models of experimental inflammatory periodontal disease with a particular focus on cmc 2.24 effects (i) on the cellular mechanisms of alveolar bone loss; (ii) in a model in which the test medication is administered therapeutically (i.e., after the disease has been established) rather than prophylactically; and (iii) on the pharmacokinetics (such as peak blood levels; serum half-life) of this novel compound.

tetracycline-based matrix metalloproteinase- (mmp-) inhibitors are currently approved for two inflammatory diseases, periodontitis and rosacea. the current study addresses the therapeutic potential of a novel pleiotropic mmp-inhibitor not based on an antibiotic. to induce experimental periodontitis, endotoxin (lps) was repeatedly injected into the gingiva of rats on one side of the maxilla; the contralateral (control) side received saline injections. two groups of rats were treated by daily oral intubation with a chemically modified curcumin, cmc 2.24, for two weeks; the control groups received vehicle alone. after sacrifice, gingiva, blood, and maxilla were collected, the jaws were defleshed, and periodontal (alveolar) bone loss was quantified morphometrically and by -ct scan. the gingivae were pooled per experimental group, extracted, and analyzed for mmps (gelatin zymography; western blot) and for cytokines (e.g., il-1; elisa); serum and plasma samples were analyzed for cytokines and mmp-8. the lps-induced pathologically excessive bone loss was reduced to normal levels based on either morphometric (p=0.003) or -ct (p=0.008) analysis. a similar response was seen for mmps and cytokines in the gingiva and blood. this initial study, on a novel triketonic zinc-binding cmc, indicates potential efficacy on inflammatory mediators and alveolar bone loss in experimental periodontitis and warrants future therapeutic and pharmacokinetic investigations.

PMC4101223

pubmed-657

, urinary incontinence (ui) affects about one third of healthy elderly people, and up to 90% of nursing home patients. the proportion of the elderly population is increasing worldwide; it is currently around 10%, and is expected to reach 20% within 40 years. accordingly, the prevalence of conditions such as metabolic syndrome and obesity are increasing annually, and these are key risk factors for voiding dysfunction. moreover, voiding dysfunction influences patient quality of life, and can contribute to serious morbidities, such as falls and bone fractures. the average age of the south korean population is increasing more rapidly than that in other countries: the proportion of elderly people rose from 3.8% in 1980 to 9.5% in 2006. consequently, south korean society is becoming more concerned over voiding problems after middle age. for this reason, it is essential that investigators conduct epidemiologic studies into the prevalence of voiding dysfunctions in south korea. in this review, we aimed to clarify the prevalence and clinical features of voiding dysfunctions among both men and women. several extensive epidemiologic studies have been performed in western countries since the 1990s; however, in south korea, epidemiologic studies have only been conducted since the start of the 21st century. to advance epidemiologic research, longitudinal designs confer greater statistical power than cross-sectional designs, because longitudinal research uses serial data that reveal substantial realistic prevalences, as well as the potential causes of the disease. on the other hand, more epidemiologic studies involve a cross-sectional design. to ensure significant epidemiologic data, investigators must select a proper target population and adopt proper survey methods; questionnaires should be adjusted to the target population. early epidemiologic studies into voiding dysfunction were mainly performed by postal survey; this method has been commonly used since the 1960s. this approach involves lower costs and higher efficiency, and it has fewer effects on the patients mood; therefore, the postal survey served as a strong epidemiologic method. more recently, communication devices and multimedia technology have allowed telephone interviews and internet surveys to emerge as practical epidemiologic tools. telephone interviews can provide a cross section of almost the entire population, and they are cost- and time-effective. furthermore, the results of telephone interviews are less influenced by the interviewer than are those of personal interviews. nonetheless, this method is limited, because, in the past, few people in south korea had telephones. with the increased distribution of telephones,, the telephone interview can be affected by social desirability bias, which can arise in cases of delicate personal questions, especially in the field of urology. furthermore, some people feel their privacy is violated by random-dialing surveys; therefore, the nonresponse rate increases. after high-level communication and multimedia technology were developed, the internet survey was introduced to gather data. however, such an approach confers a coverage problem, because most people are still not capable of accessing surveys via the internet or electrical devices. for this reason, the internet survey must be developed further if it is to become the ideal survey design. in addition, internet surveys require comprehensive programming, which is not true of face-to-face interviews. therefore, the face-to-face interview is still frequently used in epidemiologic studies, even though more money, time, and manpower are required. the major advantage of the personal interview is that the researcher can explain the intent and exact meaning of questions; this guarantees higher response quality. it is true that every survey method has its own advantages and handicaps, and sometimes various survey methods are used concurrently. nonetheless, large-scale epidemiologic data are most frequently obtained by telephone interview or internet survey. furthermore, in both sexes, ui, lower urinary tract symptoms (luts), overactive bladder (oab), and nocturia are highly prevalent, distressing conditions. herein, we have provided an overview of the epidemiologic studies into the above voiding dysfunctions in south korea. all of the surveys included in this review were conducted after profound consideration of the various aspects. furthermore, we have reviewed in detail each specific survey method, as well as the methods for obtaining detailed clinical details of voiding dysfunction. finally, we considered the practical limitations of the survey selection processes, which are influenced by special factors. its various voiding-related symptoms, enhanced by obstruction of the bladder outlet, include nocturia, tenesmus, urgency, and hesitancy; these symptoms reduce the quality of life in affected men. the prevalence of bph increases with age, and it affects 40% to 70% of men aged between 60 and 70 years. bph is diagnosed pathologically in approximately 40% of autopsies of men in their 50s, and in 70% of autopsies of men in their 60s. hence, in this review, we have used based based definitions of bph as a voiding dysfunction. to investigate the real prevalence of bph the demarcation of what constitutes clinical bph is somewhat controversial; to date, clinicians have come to no consensus regarding the definition of bph. in addition, different characteristics of the target population, as well as the methods used to collect information, can influence the results. this diversity in the definition of bph influences the ability of investigators to analyze and compare results across studies. the first study into bph prevalence in the inland area of chungcheongbuk-do was performed in 1999, and 27.7% of 764 men were found to be affected. in this study, bph was defined when the prostate volume (pv), measured by transrectal sonography, was 20 ml, and when the maximal flow rate (qmax) was 10 ml/sec. in another study, bph was defined when the international prostate symptom score (ipss) was 8, when the pv (measured by finger) was 30 ml, and when qmax was 15 ml/sec. the rate of moderate-to-severe ipss was 49.5% (213 in 431 men). but by the definitions in this study, bph prevalence were 4.3% of men in their 50s, 13.2% of men in their 60s, and 16.3% of men in their 70s or older (overall, 11.1% of men aged over 50 years). the population-adjusted prevalence of bph in south korean men aged 50 and over was 8.7%. another community-based study defined bph in cases of an ipss 8 points and a qmax 10 ml/sec; the study found that the incidence of bph was 25.5%. on the basis of ipss, 36.3%, 49.7%, and 14.0% of the men were mildly (17), moderately (819), and severely (2035) symptomatic, respectively. in 2009, park et al. found that the prevalence of bph, defined in cases of an ipss>7 and a pv>30 ml, was 40%. this study was based on the korean longitudinal study on health and aging, a population-based, prospective study involving a randomly sampled population of 301 men aged>65 years living in seongnam, south korea. in the latest study, which involved 779 men who lived in yangpyeong county and participated in a prostate examination campaign, the wide range of bph prevalence in previous studies from 11% to 40%is mainly due to methodological differences between studies, such as differences in selection of the target population. furthermore, the proportion of the population who are elderly is higher in some districts than in others. the prevalence of clinical bph in south korea is similar to that in western and other eastern countries. specifically, the bph prevalence in scotland was estimated as 25.3% in 705 men. a cross-sectional study involving 8,466 iranian men (> 40 years old) conducted by 74 personal interviewers identified a prevalence of 23.8% when bph was defined as an ipss>7, a qmax of<15 ml/sec, and a pv>30 ml. asian data were collected from china, south korea, singapore, pakistan, the philippines, india, thailand, and taiwan. the prevalences of an ipss 8 points were 18%, 29%, 40%, and 56% among men in their 40s, 50s, 60s, and 70s or older, respectively. these rates are higher than those reported for the united states (12% 17%, 23%, and 29%, respectively). this may be due to differences in the cellular composition of prostatic tissue between asians and caucasian-americans. some studies have reported that asians have more portions that are glandular and less muscle and connective tissue, as well as a transition zone that occupies a higher volume ratio within the prostate. however, studies use various combinations of parameters to define bph, including symptom score, pv, and urodynamic parameters. therefore, it remains difficult to determine the exact in prevalence, and to make further progress in the survey method. even though ui is not life threatening, it is a severe health problem and causes psychological distress, hygiene problems, and social impairment in affected patients and their families. according to the international continence society definition, any involuntary leakage of urine constitutes ui. in general, many women regard ui as a natural consequence of childbirth and aging. even though ui can be managed in various ways by clinicians, many women tend to manage their ui personally. in addition, due to shame, many people conceal their problem rather than ask for medical help. the incidence of ui increases with aging, and with the rapid growth of the aged population, ui is gradually becoming an enormous social burden in south korea. the earliest prevalence data for ui in south korea were reported in 2004; the study comprised an internet survey involving 3,372 respondents, and the prevalence of ui in women was 21%. an additional large telephone survey reported that the overall prevalence of ui was 40.8% among 1,301 women. with regard to ui type, 22.9% of cases were stress-type, 3.1% were urgetype, and 14.9% were mixed. in 2008, the korean national survey on urinary incontinence yielded nationwide representative data; in this survey, 13,345 households were interviewed. the prevalence of ui was 24.4%, and stress ui was the most prevalent type. a further population-based cross-sectional telephone survey of south koreans included 888 men and 1,112 women. the most predominant types were other (1.3%) in men and stress (20.7%) in women. data from 9,873 women participants in the korea national health and nutrition examination survey were reported in 2014. this study reported that the overall prevalence of ui among women was 7.9%. by age, the prevalences were 10.1% and 10.3% in women over 40 and 60 years of age, respectively. the reported prevalences were 0.8%, 4.6%, 9.3%, 10.8%, 10.9%, and 9.7% among women in their 20s, 30s, 40s, 50s, 60s, and 70s or older, respectively. the 2008 actual living conditions of the elderly and welfare need survey obtained data by personal interview; this survey attempted to provide longitudinal population information using a cluster sampling method. the 8,961 women studied, 6.5% had self-reported ui and 2.3% had been medically diagnosed with ui. the most recent population-based, cross-sectional study randomly sampled 500 korean women who reside in the seoul, incheon, and gyeonggi-do areas. the survey comprised a computer-aided telephone interview, and used 16 questions to collect data; the questions sought information on demographic characteristics, information sources, disease insights, and general health-seeking behavior. among the responders, 23.8% of women experienced ui. western data report a prevalence range of 3.9% to 24.4% in women, while asian data have shown variable results: 53.7% of japanese women aged over 40 years, and 27.7% of taiwanese women aged 65 years or older. korean data on ui have also varied widely; however, the prevalence is comparable with that of worldwide data, irrespective of region or race. because korea is a conservative country, and koreans are relatively passive in coping with ui problems, ui may be more common among south korean women than is reported therefore, assessment of the real ui prevalence is the first step to creating public awareness and increasing public knowledge regarding this problem. in this way oab is a broad terminology that refers to a complex of symptoms such as urinary urgency, urge incontinence with nocturia, and frequent urination. investigation into the prevalence of oab is still in its infancy, because the definitive characters of oab are obscure in that there is no clear metabolic or histologic explanation for the symptoms. nonetheless, oab is a common voiding dysfunction, especially among older people. in addition to causing physical problems, oab may have a negative effect on the person s emotions and quality of life; furthermore, the disorder may eventually place a severe financial burden on society not only in medical terms, but also through loss of productivity. the reported incidence of oab has varied significantly among epidemiologic studies, because the diagnostic criteria with regard to each individual urological symptom are different. in south korea, epidemiologic studies into oab that had consistent diagnostic criteria began relatively recently, as did studies into the impact of the condition on the individual and society. we summarized the south korean studies into oab in table 3. in 2004, 3,757 participants completed a questionnaire via an internet survey. among the 3,372 women who responded, 429 suffered from oab (12.7%). in another study, nationwide community samples were obtained from south koreans aged 40 to 89 years. through a telephone survey that used allocation sampling, choo et al. reported that the prevalence rates in men and women were 13.3% and 16.3% for dry-type oab, and 7.5% and 15.0% for wet-type oab, respectively. in 2011, lee et al. performed a population-based survey using the international continence society (ics) definition of oab the study was cross-sectional in design and used a telephone survey method to gather data from 2,005 persons. they found that 12.2% of individuals had oab, with similar rates among men (10.0%) and women (14.3%). in a regional study in guri city and yangpyeong county, gyeonggi-do, the general oab prevalence was 14.1% (12.2% in men and 15.5% of women). the survey involved face-to-face interview of 926 residents, and defined oab when the urgency score was>2 points and the oab symptom score was>3 points. in general, south korean prevalence data range from 10% to 16%, with a higher rate in women than in men. this is similar to the prevalence found in western countries; furthermore, the prevalence of oab in south korea increases with age, just as in western countries. for instance, in a survey involving 5,204 americans, the prevalence of oab was 16.0% in men and 16.9% in women. data from six european countries revealed that the overall prevalence of oab among 16,776 people was 16.6%. referring again to asia, the prevalence among 4,570 japanese respondents was 12.4%, and it was 20.9% among 1,247 taiwanese women. oab patients are known to have a lower quality of life; however, investigators require a deeper insight into the condition s harmful influence in the community, especially in south korea. proper epidemiologic data will be a valuable resource for educating the south korean population about oab a term unfamiliar to the public at the current time. according to the 2002 ics definition, the term luts encompasses various types of storage, voiding, and postmicturition symptoms. similar to other voiding dysfunctions, luts are a common condition, and their prevalence has positive correlations with age. one problem with luts prevalence studies is that the condition is difficult to define, and most studies use criteria that overlap with those of bph, ui, or oab. until recently, when investigation into luts prevalence began, one innovative study involving south korean men was performed in yeoncheon county. this community-based epidemiologic study assessed the severity of urinary symptoms in 514 men over 50 years of age. the overall incidence was 23.2%: 17.7% in those aged 5059 years, 23.3% in those aged 6069 years, and 35.3% in those aged over 70 years. in 2001, cho et al. a total of 1,356 men between 40 and 79 years old living in the seoul area were selected using stepwise random sampling and surveyed with a questionnaire that included questions about ipss. about 16% had moderate-to-severe luts, defined as an ipss>7. the prevalence of moderate-to-severe luts increased significantly with age, 10%, 16%, 29%, and 45% in the age groups 4049, 5059, 6069, and 7079 years, respectively. one population-based telephone survey involving 2,000 individuals reported that the overall frequency of luts was 61.4% (53.7% in men, 68.9% of women). 64.4% of women, voiding luts affected 28.5% of men and 25.9% of women, and postmicturition luts affected 15.9% of men and 13.9% of women. kim et al. surveyed a random sample of men older than 40 years. responses from 1,842 men were investigated, and the overall prevalence of luts was 83.4%. storage luts (70.1%) were more common than voiding (60.4%) or postmicturition luts (38.3%). as can be seen in the above summary, the reported prevalences of luts vary widely, from 16% to 83%. nonetheless, the south korean prevalence data regarding luts are comparable to those in reports from across the world. one study in europe found that the prevalences of luts were 16.2%25.1% in men and 12.6%23.7% in women. moderate-to-severe luts were reported by 36% of men aged 5059 years, 50% of men aged 6069 years, and 60% of men aged 7079 years. however, there have been fewer epidemiologic studies into luts in asian countries than in western countries, and prevalence data focus mainly on men rather than on the general population; there are few data regarding the prevalence in women. as shown in table 4, the prevalence varies widely between studies worldwide, mainly because investigators still have no clear definition of luts. the condition is complex, and this is a severe obstacle to researchers who wish to obtain the true epidemiology of luts in south korea. nocturia is defined by the ics as the complaint that the individual has to wake at least once at night to void. getting up at night to void urine increases the risk of individual physical or mental injuries in elderly people. nocturia reduces the quality of life and is associated with sleep fragmentation, reductions in productivity, reduced general health, and falls or fractures. most urologists consider a single episode to be nocturia, but many epidemiologic studies have defined nocturia as more than two episodes of voiding per night. only a few studies into nocturia have been conducted in south korea (table 5). in 2008, choo et al. conducted a clinically validated, computer-assisted telephone survey. among the 2,005 respondents (1,005 women and 1,000 men), 33.5% complained of once-per-night voiding, and 48.2% stated that they had to void twice or more per night. interestingly, even though the prevalence of nocturia was high, the negative influence on everyday life was reported to be minimal. in 2000, data were published involving 2,000 participants (888 men and 1,112 women). according to this cross-sectional telephone survey, the prevalences of nocturia (2 voids per night) were 36.6% in men and 48.2% of women. in 2012, lee et al. investigated the prevalence of nocturia (2 voids per night) using a face-to-face interview method. the prevalence was 56.0% in this community-based study involving 439 elderly south korean men in a single city (seongnam). it is true that not all patients suffer from nocturia, and the degree of impact is influenced by the severity of the condition. to understand the exact epidemiologic nature of nocturia in south korea, it will be useful to examine to what degree cultural background has an effect on the psychological impact of nocturia. the prevalence of nocturia in south koreans appears to be higher than that in other communities. the prevalences of nocturia (2 voids per night) among japanese community-dwelling adults were 28.1% in men and 27.1% in women. a multistage, cluster sampling study with a personal interview-type design was used to obtain data from 15,988 people in the united states. the prevalences of nocturia (2 voids per night) were 9% in men and 16% in women. data collected from 1,247 women and 1,221 men in austria showed that 37.2% of men and 43.1% of women experienced 1 voids per night, and that 7.5% of men and 8.1% of women experienced 2 voids per night. these lower prevalences in other populations may have been affected by the population distributions studied, as well as by the definition of nocturia used. nonetheless, the probable causes of the high prevalence of nocturia among south koreans remain to be clarified. epidemiologic surveys are essential for understanding the potential causes and characteristics of related medical disorders. in addition, they are the necessary first steps in evaluating psychosocial impacts of a disorder. in general, most of the estimated prevalences of voiding dysfunctions in south korea were similar to those in other countries; they varied widely, primarily due to different definitions of the respective disorder. in addition to the lack of solid definitions of voiding dysfunctions, inconsistencies in integrated questionnaires and variances among target populations render population-based surveys difficult to compare. most investigations were based on self-reported symptoms, so recall bias and person-to-person social bias may have been present, especially in personal interviews. nonresponse bias could also be a problem in that nonresponders to a survey may have been significantly different from responders in terms of the respective symptoms. population-based surveys are carried out because diagnosis by strict medical assessment is not practical in such large-scale prevalence studies. nonetheless, researchers need to develop other ways to obtain more proper, valuable epidemiologic data. we think that investigators should focus on unifying all of these disease entities and definitions. more efficient investigational processes and methodologies are also warranted. from a methodological point of view, 14 of the studies (58.3%) included in the present report, involved personal interview data. despite considering the advantages of the personal interview, however, the bashful and conservative nature of koreans may prevent investigating the epidemiology of voiding dysfunction in simpler, less time-consuming ways. as a result, relatedly, the random digital dialing in telephone surveys is often perceived as violating personal privacy; for this reason, telephone surveys will not be easy to conduct in the future. due to the noteworthy progress in multimedia technology in south korea, epidemiologic studies using the internet and other applications are a promising method. using web services an internet survey can gather enormous amounts of data from large groups within a relatively short time. furthermore, videos or pictures can be used easily, at reasonable cost, and with less social desirability bias than the other study methods. mobile communication techniques may improve methothologic efficiency in various social and natural science fields, as well as in medicine. for example, bladder diary apps may exclude the disadvantages of pen-and-paper diaries in clinical application. currently, the apps available vary in quality, but there is room for medical associations to collaborate with developers in this area. even though some of the public are not familiar with surveys by internet or electrical devices, much progress will be made in epidemiologic studies due to technology, especially with regard to methodology. this review constituted a nationwide representative summary of studies on the prevalence of voiding dysfunctions in south korea. we hoped to contribute to the understanding of voiding dysfunctions by providing the epidemiologic characteristics of each condition. however, much work remains to be done in this field, because few surveys had a longitudinal design, and more investigations are needed that consider the whole range of age, sex, and location in the south korean population. given the influence of voiding dysfunction on quality of life, as well as the socioeconomic burden of the condition, more studies on the optimal approaches to epidemiologic study will be needed in the future.

this review assessed the epidemiology of voiding dysfunctions in south korea. comprehensive understanding of this epidemiology is crucial because the senior population and the social burden are increasing because of voiding dysfunctions is growing. we searched the medical records using several terms related to voiding dysfunction: benign prostatic hyperplasia, urinary incontinence, lower urinary tract symptoms, overactive bladder, and nocturia. we then estimated the prevalence of voiding dysfunctions in south korea; our data were comparable with those from other countries, with slight differences. the ranges of incidences varied widely between studies, mostly because investigators defined disorders differently. voiding dysfunction greatly affects healthcare costs and individual quality of life; therefore, more proper and valuable epidemiologic data are needed. in addition, efforts to unify the definitions of various voiding dysfunctions and progress in investigational methodologies using multimedia are warranted.

PMC4932641

pubmed-658

however, graft dysfunction remains a problem affecting up to one-third of the recipients, despite reports of good to excellent long-term outcome. the organ dysfunction is considered multifactorial, but ischemia and reperfusion (i/r) injury is probably the most important contributing factor, although the detailed steps of pathogenesis are controversially debated. i/r injury is a major cause of liver graft dysfunction resulting in adenosine triphosphate (atp) decrease, evidence of intracellular acidosis, and cell swelling of the hepatocytes. this aspect is also accompanied by or linked to bile duct loss or ductopenia, cholestasis, and biliary ductular proliferations in the posttransplant liver biopsy. however, biliary marker levels increase usually only 57 days after transplantation stimulating several fields of research in the last couple of decades. cholestasis is associated with high morbidity and mortality in patients undergoing liver transplantation, and the steps reaching this state are not completely understood. we reviewed the canine liver transplantation model as i/r injury model to delineate in detail the intermediate filaments of the cytoskeleton that are probably the determinants in changing the phenotype of hepatocytes to cholangiocytes, which seems to be a post-transplant event occurring in the liver at an earlier stage than frank cholestasis. here, we speculate that i/r liver injury through a phenotypical switch of the liver cells may contribute to the poor outcome of the liver graft. liver transplantation is widely known as the most effective therapy for both acute and chronic liver failure. in 1963, thomas starzl, an american surgeon, northwestern university medical school graduate with degrees in anatomy, neurophysiology, and medicine, was the first to perform the liver transplantation on a child suffering from biliary atresia. despite the significant success of liver transplantation, infection, poor graft function, and rejection are major problems that may still contribute to death of the transplanted patient or to deterioration of the graft. reperfusion following long ischemia of liver during liver transplantation causes severe injury, which is now universally indicated as i/r injury. i/r injury of liver is a major cause of morbidity and mortality in patients undergoing liver transplantation. several mechanisms and distinct pathways may involve i/r leading to both initial poor function and primary nonfunction of the liver allograft. three cytoskeletal proteins form a network that provide the cellular structure and fundamental integrity of the eukaryotic cells: microfilaments, microtubules, and intermediate filaments. the intermediate filament proteins have an important role in liver protection against mechanical and nonmechanical injury, which have been demonstrated in animal models. overexpression of proteins and mutations of the corresponding keratin genes have been reported to contribute to several human oncological and nononcological diseases. eukaryotic cells have a unique cytoplasmic structure labeled as cytoskeleton, which consists of three distinct kinds of cytoskeletal filaments, including microfilaments, microtubules, and intermediate filaments. besides giving the rigidity of the cell and maintaining cell shape and borders or cell scaffolding, cytoskeleton plays important and probably crucial roles in intracellular transport, cell division, gene regulation, and signal transduction of the genetic information [6, 7]. actin, which is widely accepted to be a highly conserved structure among different species, is the major protein that constitutes the microfilaments. there are three known classes of human actin gene that have been identified as -, -, and -actin. cell-cell interaction, signal transduction, cell shape maintenance, and cell motility are the central functions of actin. microtubules, which have a diameter of around 23 nm, are protofilaments that are important for the intracellular transport, movement of cilia and flagella, mitotic spindle, and cell wall synthesis. both microfilaments and microtubules have several groups of binding proteins that modulate their stability and biological functions, such as signal transduction. intermediate filaments with around 10 nm of diameter are more stable than actin filaments and function in the maintenance of cell shape by carrying tension. intermediate filaments, which include, for example, vimentin, glial fibrillary acidic protein, neurofilament proteins, keratins, and nuclear lamins, organize the internal 3d cell structure anchoring cyto-organelles and serving as structural scaffolds of the nucleus and cytoplasm. cytokeratins (ks) are the main protein family that constitutes the intermediate filaments with more than fifty members that have been identified. polypeptides of the ks are variants and have been divided into type i and type ii, or acidic and basic, respectively. studies have shown that each epithelial cell has a distinct content of cytokeratins (i.e., expression of keratins is described as tissue-specific manner) playing a major role in the tissue identification of metastasis of unknown primary carcinomas. ks usually present in cells as heteropolymers pairs composed of type i (k9k20) and type ii (k1k8). k8 (mw 52kd) and k18 (mw 45kd) are considered as the only ks that are normally expressed in human hepatocytes. moreover, both k8 and k18 have been found to be expressed in early development stage of mouse embryos. immunohistochemical studies have revealed that cholangiocytes (bile duct cells) express k7 (54 kd) and k19 (40 kd) in addition to k8 and k18, and those ks have been used as valid markers for both studies on development of the intrahepatic biliary system and for assessment of bile duct damage. it has also been well established that cholangiocytes are derived from hepatoblasts found around portal vein. these cells show a particularly intense expression of k8 and k18. as indicated above, k7 and k19 have been linked to development of the intrahepatic bile duct system in both humans and animal models. the expression of k19 is useful to identify primitive biliary cells, while the expression of k7 appears after 20 weeks of gestation in humans. both k7 and k19 are consistently expressed in the development of the intrahepatic bile duct with elevated levels until one month of postnatal age. thus, in order to phenotypically switch hepatoblasts into cholangiocytes, these cells should firstly express k19 followed by expression of k7. however, it has been shown that hepatocytes might express k7 in response to different conditions, such as ductopenia and cholestasis, and, interestingly, it has been found that hepatocellular carcinoma cells expressing k19 are significantly associated with reoccurrence of neoplasm after transplantation. a recent study has demonstrated that hepatocytes are frequently expressing k7 in case of chronic liver allograft rejection. have demonstrated that hepatocytes intensively expressed k7 and k19 early following cold ischemia in a canine isolated perfused liver transplantation model. furthermore, they demonstrated that bile duct cytokeratins are very useful markers to diagnose an early sign of cholestasis. it has become clear that cholestasis is significantly associated with high morbidity and mortality in patients undergoing liver transplantation. therefore, investigations that lead to discover the mechanisms of the cholestasis are dramatically required. i/r injury is a phenomenon that occurs when blood flow and oxygen delivery return to reperfused tissue. it is considered one of the major causes of morbidity and mortality among patients undergoing liver transplantation. i/r injury has been demonstrated in several diseases such as cerebrovascular diseases, peripheral vascular diseases, sepsis, myocardial infarction, and organ transplantation. it involves several mechanisms, which lead to organ failure, circulatory dysfunction, and, finally, death of the transplanted patient. cellular mechanisms of i/r injury include a few essential cell cascades that suggest the role of activation of endothelial cells, kupffer cells, reactive oxygen species (ros), and polymorphonuclear leukocytes (pmn) or neutrophils in the pathogenesis of i/r injury. there may also be mechanisms involving t lymphocytes that seem to have a key role in short- and long-term damage during i/r injury. t lymphocytes act as mediator in the subacute inflammatory phase of neutrophilic recruitment following i/r injury. another important constituent of tissue injury following i/r injury that needs to be emphasized is the production of oxygen free radicals (ofrs). these ofrs can arise from different sources, such as kupffer cells, pmn, and xanthine oxidase (xo) that is the most significant source. several etiological factors are involved in i/r injury, including atp reduction, activation of proteases, and alteration in the intracellular concentration of cytokines and chemokines, and cell swelling. investigations of posttransplantation surgery have indicated that reperfusion plays an essential role in primary graft nonfunction, which is one of the most serious complications of liver transplantation. i/r injury also causes early organ failure up to 10% as well as increases the acute and chronic rejection. several studies demonstrated that during i/r injury, morphological changes of liver tissues occur, and these changes can have a prognostic significance. the intermediate filament (if) cytoskeletal protein is one of the three major cytoskeletal proteins whose result is important in maintaining both cellular structure and integrity of eukaryotic cells. the other two filament proteins are microfilaments (mf) and microtubules (mt). beside their cellular functions like cell motility, division, and stress responses, they also have an essential role in human diseases due to mutations in filament proteins. if proteins consist of five types of keratins according to structure of genome and composition of amino acid, if proteins types i iv are cytoplasmic, and type v if contain nuclear lamins. the keratins are obligating noncovalently heteropolymers because they consist of one of each type i and type ii keratins as pairs. neutral to basic keratins are the largest group of if proteins classified as type i (k9k20) and type ii (k1k8). another type of if proteins is type iii if that includes vimentin, peripherin, glial fibrillary acidic protein, and desmin of mesenchymal cells, peripheral neurons, glia cells and astrocytes, and muscle cells, respectively. type iv if proteins include neurofilament proteins (nf-l, nf-m, and nf-h), synemin, nestin, syncoilin, and -internexin; lamins a as indicated above, the adult hepatocytes express k8 and k18 only compared to other epithelial cells that express 2 or more type i or type ii keratins such as bile duct, which expresses additional keratins k7 and k19. the first disease to be discovered related to keratin mutation was epidermolysis bullosa simplex (ebs) with mutations in k5 and k14. additionally, mutations in k8 and/or k18 lead to acute or chronic liver diseases. normally in hepatocytes, further studies indicated that reperfusion alone causes alteration of theses microfilaments (f-actin) leading to contraction of canaliculi, relocalization of enzymes and transporters of canaliculi, and increasing permeability of cellular tight junction. cholestatic liver disease occurs when there is a decrease in bile flow, which is an abnormal physiologic state, and retention of toxic bile acids. clear cytoplasmic hepatocytes (cholate stasis) presented in cirrhotic nodules and showed a decrease in keratin if network in the cytoplasm. studies on mice reported that keratin was overexpressed in epithelia of bile duct due to ligation of bile duct and in hepatocytes. in cholestatic liver disease, bile duct epithelial-type keratin (k7 and k19) intermediate hepatocytes express k7, while reactive bile ductules express k7 and k19. in post-transplant period, keratins of biliary type were detected in the hepatocytes in the early period of i/r injury. moreover, bile canaliculus progressively dilate after ischemia, and a change of the microvillous integrity is demonstrable. the liver is one of the organs with an incredible capacity for in vivo tissue engineering which allow restoration of the liver architecture and reestablishment of certain vital functions. the study of liver regeneration in humans arise ethical issues, and it is difficult to carry out because of a plethora of heterogeneous liver lesions. accordingly, using experimental animal models is more useful and helpful for studying liver regeneration. in vitro studies need to be followed by in vivo ones to simulate the interaction between liver cell populations. in our opinion, the use of large animals such as dog, pig, or sheep is more advantageous than small animals like rat or mouse; large animals are similar to human beings in their physiology and anatomy, and techniques of microsurgery are not necessary to carry out determinate experiments. only few studies have been carried out on i/r animal models in liver. the canine liver model following i/r injury has been used as a liver transplantation model to study excretion of bile and intrahepatic intermediate filaments expression involved in morphological changes of the biliary system. to the best of our knowledge, in a canine isolated and perfused liver model, there was the first clear-cut evidence of cholestatic changes starting early following cold ischemia despite prompt recovery of the bile flow. according to some other authors, the use of the canine liver transplantation model seems to allow a better vascular perfusion under ex vivo conditions in contrast to the rat model. 's study revealed an ischemia-dependent impairment of an experimental biliary dye excretion during early stage of reperfusion and a progressive cytokeratin expression of biliary type in the hepatocytes despite a prompt recovery of the bile flow after i/r. the recovery of the bile output after i/r represents a key event in lt. the nonstimulated bile flow rate was unaffected by cold ischemia up to 10 h in our canine isolated perfused liver model, but the decrease of the biliary dye excretion during early stage of reperfusion correlated with the peak output rate of the dye across the canalicular membrane. remarkably, the dye uptake at the sinusoidal membrane was not affected by cold ischemia, leading to a possible accumulation of toxic compounds in the hepatocytes. hepatic elimination of the dye following i/r has been analyzed mainly by plasma clearance. very importantly was the study of the hepatic elimination of the dye after a bolus injection by compartmental spectrophotometric analysis both in the perfusate by transhepatic sampling and in the bile. differential mechanisms can be suggested for the impaired biliary excretion of organic anions following i/r. previously, a positive correlation between biliary dye excretion, viability of the graft, and hepatic atp content has been found in cholestatic liver disease, and reduced biliary dye excretion has been attributed to the lack of atp. atp content does not seem to be the main limiting factor for the hepatocellular transport following i/r. an impairment of the intracellular transport or a decreased transport rate across the canalicular membrane might play a major role as contributing factor. the step across the canalicular membrane generally represents the rate-limiting one in the hepatocellular transport. in the canine liver transplantation model, the flow in the portal vein was continuous, because roller pump, oxygenator, and heat exchanger in the perfusion line were connected in series with the roller pump upstream. the oxygen content of the saline reperfusion solution was 2% in volume (10% of arterial hepatic blood and about 15% of the portal venous blood). thus, one can expect to have reactive-oxygen-species- (ros-) linked damage at the beginning of the reperfusion after 8 or 10 h of ischemia. however, the excellent reperfusion results of the control group and data of the 2 h ischemia group indicate that this is not a general problem, but may be an ischemia-time-linked problem. high o2 partial pressure in the liver stimulates ros formation after extensive long ischemia times, and this may be one of the most important limiting factors of the ischemia tolerance. electron microscopy studies in hepatocytes revealed microfilaments, which are distributed along the plasma membrane and in the region of the bile canaliculus. as indicated above, cytoskeleton filaments are also essential for the maintenance of cell shape, bile canalicular architecture, and integrity of the cellular tight junctions. the microfilaments network surrounding the canalicular pole and extending into the canalicular microvilli is damaged during cholestasis. reperfusion, but not ischemia, has been considered faulty, inducing an alteration of f-actin microfilaments, suggesting an impairment of the canalicular contraction, an increase of the tight junction permeability, and a probable relocalization of canalicular enzymes and transporters. bile canalicular size change as detected in our study may be coincident with the reorganization of the pericanalicular filaments and the colocalization of actin and myosin. in fact, contraction in this study has been associated with shortening and/or twisting of bile canaliculi. however, graft dysfunction remains a problem affecting up to one-third of the recipients, despite good to excellent long-term outcome. the organ dysfunction is considered multifactorial, but i/r injury is probably the most important contributing factor. the detailed steps of the pathogenesis of i/r injury continue to be a topic of vivid debate. i/r injury is a major cause of liver graft dysfunction resulting in atp decrease, intracellular acidosis, and cell swelling of the hepatocytes. this feature is also linked to bile duct loss or ductopenia, cholestasis, and biliary ductular proliferations in the liver biopsy. however, the plasmatic levels of biliary markers increase usually only 57 days after transplantation prompting on-going research to address new markers of early liver damage. this could also be a stimulus to support more grant approvals for liver posttransplantation studies. cholestasis is associated with high morbidity and mortality in patients undergoing liver transplantation, but the steps reaching this state are still deemed elusive. we reviewed the canine lt model as an i/r injury model and reviewed the intermediate filaments of the cytoskeleton that are probably the determinants in changing the phenotype of hepatocytes to cholangiocytes. in fact, this phenotypic switch of the liver cells, which is also sometimes called pseudo- or neoacinar transformation in diseases with cholestasis, seems to occur at a stage earlier than frank cholestasis. we emphasized that i/r injury through this peculiar phenotypic switch of the hepatocytes may be a milestone to investigate the pathways contributing to the poor outcome of the liver graft. reperfusion injury following ischemia affects graft function in liver transplantation by induction of phenotypical changes in hepatocellular keratins. keratin phenotype of hepatocytes leads to hepatocellular damage that is intimately connected to several intracellular processes, which are under intense investigation in our laboratory, and the appropriate choice of an animal model is crucial in forwarding research in very complex areas requiring not only human, but also comparative (veterinary) pathology knowledge.

liver transplantation has been a successful therapy for liver failure. however, a significant number of recipients suffer from graft dysfunction. considerably, ischemia and reperfusion (i/r) injury is the most important factor leading to organ dysfunction, although the pathogenesis has not been fully described. i/r injury have several established features that are accompanied by and/or linked to bile duct loss or ductopenia, cholestasis, and biliary ductular proliferations in the posttransplant liver biopsy. however, biliary marker levels increase usually only 57 days after transplantation. intermediate filaments are one of the three cytoskeletal proteins that have a major role in liver protection and maintaining both cellular structure and integrity of eukaryotic cells. we reviewed the canine liver transplantation model as i/r injury model to delineate the intermediate filaments of the cytoskeleton that are probably the determinants in changing the phenotype of hepatocytes to cholangiocytes. remarkably, this interesting feature seems to occur earlier than frank cholestasis. we speculate that i/r liver injury through a phenotypical switch of the hepatocytes may contribute to the poor outcome of the liver graft.

PMC3321507

pubmed-659

breast carcinoma is the most frequent cancer among women with considerable invasive and metastatic behavior. the recent increased knowledge in molecular mechanisms of this cancer and consequent targeted treatments have improved its outcome. it belongs to the family of d-type cyclins, which regulate cell cycle progression from g1 to s phase by regulating the activity of cyclin-dependent kinases (cdks). cyclin d1 and its close relatives, cyclin d2 and cyclin d3, all appear to associate with the same kinase partners. binding of cyclin d1 to cdk4 and cdk6 induces hyperphosphorylation of retinoblastoma protein (rb). this leads to the activation of e2f and transcription of several genes required for the g1 to s phase transition, thereby promoting cellular proliferation. recent findings have revealed further roles of cyclin d1 in promoting cell cycle progression through cdk-independent mechanisms such as interaction with and modulation of transcription factor activities. the well-known oncogene ccnd1, which encodes cyclin d1 is amplified in a substantial proportion of human cancers including parathyroid carcinoma, colon cancer, lymphoma, melanoma, prostate cancer and breast cancer. however, the impact of cyclin d1 overexpression on behavior of breast cancer remains controversial. although cyclin d1 has a pivotal role in promoting cell cycle progression and its overexpression in breast cancer is expected to be associated with poor prognosis, recent studies have shown both positive and negative prognostic impacts. besides inducing cell cycle progression through regulation of cdks activity, cyclin d1 promotes other regulatory molecules by cdk-independent mechanisms. cyclin d1 represses the transcriptional activity of signal transducer and activator of transcription 3 (stat3) in vitro. cyclin d1 is the intermediary molecule in other cell cycle pathways such as nuclear factor-b (nfb), rac1 and 5 adenosine monophosphate-activated protein kinase (ampk) signaling pathways. decline in rac1 levels causes inhibition of nfb signaling and induces downregulation of cyclin d1. active ampk leads to loss of cyclin d1 messenger ribonucleic acid and protein. only a minority of breast cancers with cyclin d1 overexpression show amplification of the cyclin d1 gene, indicating that pathogenic transcriptional activation of this gene by factors such as estrogen receptor (er) could be another important mechanism triggering its overexpression. using antibody against cyclin d1 protein, overexpression of the protein can be detected even in the absence of any apparent increase in copy numbers. therefore, immunohistochemical staining with the specific monoclonal antibody provides an accurate method for detection of deregulated cyclin d1 expression. er is another regulatory molecule with critical roles in proliferation of cancer cells in reproductive organs such as breast and uterus. most er positive breast cancers are low-grade and have lower metastatic potential while er negative ones demonstrate poor differentiation. er expression is in turn influenced by the expression of other genes such as mta1. silencing mta1 gene results in er expression in er negative cells. genetically modified mouse models reveal the necessity of cyclin d1 expression for postnatal mammary development in response to the sex steroids. indeed, cyclin d1 expression in mammary epithelial cells is induced by estrogen and progesterone. the present study aims to find the frequency of cyclin d1 overexpression and the relationship between cyclin d1 overexpression and some well-known clinicopathologic prognostic determinants in breast invasive ductal carcinoma. in this descriptive-analytical and cross-sectional investigation, 89 patients with breast invasive ductal carcinoma hospitalized in alzahra hospital, isfahan, iran from 2003 to 2008 enrolled in the study. patients had undergone mastectomy and axillary lymph node dissection and the status of er, progesterone receptor (pr) and human epidermal growth factor receptor 2 (her2)-neu had been determined immunohistochemically using the formalin fixed and paraffin embedded tissue samples of the primary tumor. the studied variables included age, tumor grade, tumor stage, er, pr, her2-neu and cyclin d1 status. grading was carried out based on the nottingham modification of bloom and richardson system, which considers the three parameters of tubule formation, nuclear pleomorphism and mitotic count as determining factors. staging was carried out based on the tnm classification of malignant tumours (tnm staging system) for breast carcinoma. expression of er and pr was considered as negative if lesser than 1% of nuclei were stained and as positive if 1% or higher of nuclei were stained. the antibodies used were against er (monoclonal mouse anti-human; clone: 1d5; isotype: igg1, kappa; dakocytomation, denmark) and pr (monoclonal mouse anti-human; clone: 1a6; isotype: igg1, kappa; dakocytomation, denmark). the antibody used for her2-neu study was polyclonal rabbit antihuman antibody against c-erbb-2 oncoprotein, dakocytomation, denmark. her2-neu expression was scored as 3+(positive) when strong complete staining of the membrane was present in more than 30% of the neoplastic cells, 2+(positive) if weak to moderate complete staining of the membrane was seen in more than 10% of the neoplastic cells, 1+(negative) when faint staining was detected in a portion of the circumference of the cytoplasmic membrane in more than 10% of the neoplastic cells and 0 (negative) when no membranous staining was identified or membrane staining was observed in less than 10% of the tumor cells. antibody used was monoclonal mouse anti-human antibody, clone cyclin d1 antibody (dcs-6) (isotype: igg2a, kappa), dakocytomation, denmark. four micron sections were prepared from each formalin-fixed and paraffin-embedded tissue sample to be stained with antibody against cyclin d1 using the envision method. sections were placed on poly-l-lysine slides and dried in an oven at 60 c for 60 min. subsequently, they were deparaffinized, rehydrated and rinsed in tap water before antigen retrieval. after inactivation of endogenous catalase by 3% hydrogen peroxide, the sections were incubated with primary antibody for 1 h and secondary antibody for 30 min. after each step of antibody treatment, slides were drained with phosphate buffer to eliminate excess antibody. the validity of immunohistochemistry staining was provided by using positive and negative controls in each set of staining. the intensity and distribution of cyclin d1 immunoreactivity were semiquantitatively scored using the allred scoring method. the intensity of immunohistochemical reaction by light microscopy was recorded as 0 (negative) when no staining of the nuclei was seen even at high magnification, 1 (weak) if staining was visible only at high magnification, 2 (moderate) when staining was readily visible at low magnification and 3 (strong) if staining was strikingly positive even at low power magnification. the proportion of tumor cell nuclei showing positive staining was also scored as either 0 (none), 1 (< 1/100), 2 (1/100-1/10), 3 (1/10-1/3), 4 (1/3-2/3) and 5 (> 2/3). the intensity and proportion scores were then added to obtain a total score ranging from 0 to 8 and the tumors were categorized into four groups based on their total score: negative expression: total score of 0weak expression: total score of 1 or 2intermediate expression: total score of 3-5strong expression: total score of 6-8.only nuclear staining was considered specific. negative expression: total score of 0 weak expression: total score of 1 or 2 intermediate expression: total score of 3-5 strong expression: total score of 6-8. only nuclear staining was considered specific. data was collected in prepared checklist and analyzed by version 16 of statistical package for the social sciences (spss) software (spss corp, chicago, usa) using anova and chi-square tests. the mean age of patients (sd) was 51.38 11.37 years (range: 26-83 years). 18%, 41.6% and 40.4% of the tumors were grade i, ii and iii, respectively. 7.9%, 40.4%, 18%, 30.3%, 1.1% and 2.2% of the cases fitted into stages 1, 2a, 2b, 3a, 3b and 3c, respectively. er, pr and her2-neu were positive in 60.7%, 58.4% and 36% of the cases, respectively. cyclin d1 was strong (s), intermediate (i), weak (w) and negative (n) in 19.1%, 44.9%, 14.6% and 21.3% of cases, respectively [figures 1 and 2]. negative cyclin d1 status was mostly seen in grade iii strong cyclin d1 status was more frequent in grades i&ii there was a statistically significant reverse relationship between cyclin d1 and tumor grade (p=0.009). the n status of cyclin d1 was mostly seen in grade 3 while s, i and w states were most frequent in grade 2 [table 1]. the relationship between cyclin d1 and tumor grade the relationship between cyclin d1 and er was also statistically significant (p=0.0001). the s and i states were mostly seen in patients with positive er while the n status was most frequent in patients with negative er [table 2]. the relationship between cyclin d1 and estrogen receptor a statistically significant relationship was found between cyclin d1 and pr (p=0.0001). the s, i and w states were mostly seen in patients with positive pr while the n status was most frequent in patients with negative pr [table 3]. the relationship between cyclin d1 and progesterone receptor there was no statistically significant relationship between cyclin d1 on one hand and age, stage and her2-neu on the other (p>0.05). the present study was conducted to determine the relationship between cyclin d1 overexpression and well-known clinicopathologic prognostic determinants in breast invasive ductal carcinoma. it has been shown in previous studies that cyclin d1-deficient mice are susceptible to mammary carcinomas induced by c-myc or wnt-1, but not those induced by c-neu and v-ha-ras. these findings suggest a pivotal role for cyclin d1 in a subset of breast cancers. the oncogene ccnd1 is amplified in 10-20% of breast carcinomas while the overexpression of its product cyclin d1 is more frequent and seen in about 34-81% of breast carcinomas. in our study, cyclin d1 expression was seen in 78.6% of the cases. the frequency of strong (s), intermediate (i), weak (w) and negative (n) states of cyclin d1 were found to be 19.1%, 44.9%, 14.6% and 21.3%, respectively. many researches have shown that overexpression of cyclin d1 in tumors is related to an unfavorable outcome, but others have yielded different results. in our study, there was no statistically significant relationship between cyclin d1 overexpression and tumor stage. this finding may be explained by the fact that cyclin d1 overexpression can not always be attributed to gene amplification. activation of other mitogenic pathways such as -catenin, stat 5, stat 3, nuclear factor kappa b, c-jun, e2f-1, ppar y, calveolin-1 and ras signaling may provide alternate routes to disturb regulation of cyclin d1. moreover, the promotion provided by cyclin d1 to proceed the cell through the cell cycle notwithstanding, degradation of cyclin d1 is essential for replication of deoxyribonucleic acid (dna). cyclin d1 level rises early in g1 phase of the cell cycle and continues to accumulate followed by a rapid decline by the entrance to the s phase. some studies have demonstrated that acute overexpression of cyclin d1 in fibroblasts prevents s-phase entry. cyclin d1 has been shown to repress dna replication by binding to proliferating cell nuclear antigen and cdk2. in the present study, a statistically significant reverse relationship was found between cyclin d1 and tumor grade as evidenced by the observation that negative cyclin d1 status was mostly seen in grade iii while strong, and intermediate states of cyclin d1 were more frequent in grades i and ii. this observation is the same as the finding of some other studies in this field. the grade of invasive ductal carcinoma is estimated by histological evaluation of tubular formation, mitosis and pleomorphism. low grade tumors are well-differentiated and show histological features closer to their original tissue. the reverse relationship observed between cyclin d1 overexpression and tumor grade suggests that higher expression of cyclin d1 may directly or indirectly result in maturation of tumor cells. s and i states of cyclin d1were mostly seen in patients with positive er while n status was most frequent in patients with negative er. s and i states were most frequent in patients with positive pr while n status was mostly seen in patients with negative pr. these findings further confirm the results of some previous studies in this field, which have stated a positive relationship between hormone receptor status and cyclin d1 overexpression in breast carcinoma. this is in favor of the effect of cyclin d1 on cell maturation and differentiation. however, overexpression of her2-neu has been reported to be associated with the high expression of cyclin d1. the reverse relationship between cyclin d1 overexpression and tumor grade as well as the positive relationship between the overexpression of this marker and hormone receptor status in breast carcinoma suggest a regulatory role of cyclin d1 overexpression in cell maturation and differentiation.

background: breast carcinoma is the most frequent cancer among women with considerable invasive and metastatic behavior. ccnd1, the oncogene encoding cyclin d1, is amplified in a substantial proportion of human cancers. although cyclin d1 overexpression has been reported in up to 50% of human breast cancers, its prognostic impact on breast carcinoma is still controversial. materials and methods: in this cross-sectional investigation, 89 patients with breast invasive ductal carcinoma enrolled in the study. tumor tissue samples were stained immunohistochemically for cyclin d1. the marker was semiquantitatively scored using the allred scoring method and its relationship with er, pr, and her2-neu status as well as age, tumor grade and stage was then determined. results:cyclin d1 was strong (s), intermediate (i), weak (w), and negative (n) in 19.1%, 44.9%, 14.6%, and 21.3% of the cases, respectively. estrogen receptor (er), progesterone receptor (pr), and her2- neu were positive in 60.7%, 58.4%, and 36% of the cases, respectively. there was a statistically significant reverse relationship between tumor grade and cyclin d1 (p=0.009). the relationship between cyclin d1 and both hormone receptors was also statistically significant (p=0.0001). there was no statistically significant relationship between cyclin d1 on one hand and age, stage, and her2-neu on the other (p>0.05). conclusion: the reverse relationship between cyclin d1 overexpression and tumor grade as well as its positive relationship with er and pr in invasive ductal carcinoma suggest that cyclin d1 may directly or indirectly result in maturation and differentiation of tumor cells.

PMC3908521

pubmed-660

cytokines are small proteins initially thought to be components of the immune system, but have since been found to play a much broader role in physiology. interleukin-6 (il-6) is a cytokine originally identified as a b-cell differentiation factor (bsf-2) in 1985 1, as a factor that induced the maturation of b cells into antibody-producing cells. human il-6 2 and il-6 receptor 3 were cloned soon thereafter. as with many other cytokines, it was soon realized that il-6 was not a factor only involved in the immune response. in early 1990s it was clear that besides controlling other immune cells such as t cells, il-6 was also important in the regulation of hepatocytes, hematopoietic progenitor cells, the skeleton, the cardiovascular system, the placenta and the nervous and endocrine systems 4. structural analysis has allowed the grouping of these proteins into different structural classes such as the helical cytokines, the trimeric tumor necrosis factor (tnf) family, the cysteine knot growth factors and the -trefoil growth factors 5. cytokines can also be grouped according to the type of receptor they bind, which comprise six major families: class i cytokine receptors (the largest family), class ii cytokine receptors, tnf receptors, tyrosine kinase receptors, and chemokine receptors 5. cytokine families may be named differently according to other aspects such as the sharing of a receptor subunit (i.e. the gp130 family) or its physiological roles (i.e. neuropoietic family, for its effects on hematopoietic and nervous system). il-6 is a prototypical four-helix bundle cytokine that is the founder member of the neuropoietins, a group of cytokines structurally related, that include il-6, il-11, il-27, il-31, leukemia inhibitory factor, oncostatin m, cardiotrophin-1, neuropoietin and cytokine cardiotrophin-like (also known as new neurotrophin 1 and b cell stimulatory factor-3), and two viral analogs of il-6 5-10. these cytokines bind to class i cytokine receptors, membrane proteins with a characteristic modular architecture that do not have intrinsic enzymatic activity, and that for signaling often need to recruit additional receptor proteins shared by different cytokines: gp130, c or c. this protein has also a modular architecture, part of which has features typical of the cytokine receptors (two fibronectin type iii modules containing conserved cysteine residues and a wsxws motif) 4. for il-6 specifically, a hexamer forms (two il-6, two il-6r and two gp130) that can activate intracellular tyrosin-kinases such as janus kinase (jak) and, to a lesser extent, tyk, which, in turn, activate a number of proteins including the stat (signal transducer and activator of transcription) family of transcription factors, or the ras-raf-mapk pathway, pi3 (phosphatidyl inositol-3) kinase, or irs (insulin receptor substrate) 11. the sharing of gp130 explains at least in part the redundancy of the actions of these cytokines. it was soon established that the expression of il-6r is restricted to some tissues, while that of gp130 is ubiquitous, and that il-6 may upregulate gp130 expression 12. the same study already provided evidence that extracellular, soluble il-6r (sil-6r) in the presence of il-6 could activate cells expressing gp130, and stated that naturally occurring sil-6r could be found in the murine serum. it is now widely accepted that sil-6r is formed physiologically, either by limited proteolysis of the extracellular domain of membrane il-6r (mil-6r) by metalloproteases such as adam10 and adam17, or by alternative splicing of il-6r mrna, and that sil-6r can bind both il-6 and gp130 and signal in cells with or without endogenous il-6r expression, a mechanism known as trans-signaling 13. to complicate things further, it is also known that a soluble form of gp130 (sgp130) is also formed 14, in this case only by alternative splicing of gp130 mrna; sgp130 can inhibit trans-signaling but does not affect normal signaling by mil-6r (figure 1). soon after its discovery, it was demonstrated that some astrocytoma and glioma lines expressed il-6 when stimulated with il-1, which prompted speculation that il-6 could have a role in the cns 2. the same group demonstrated that il-6 indeed was capable of inducing the neuronal differentiation of the rat pheochromocytoma pc12 cell line, to some extent similarly to the prototypical neurotrophin ngf 15, 16. it was therefore not surprising the finding that both glial and neuronal cells expressed il-6 and il-6r to various degrees throughout the brain 17-24. in vitro, microglia, astrocytes and the neuronal line n18, but not oligodendrocytes, expressed il-6r 25; in vivo, nevertheless, oligodendrocytes may express il-6 and il-6r 26. also, il-6 and il-6r were expressed in sympathetic and sensory ganglia, predominantly in neurons 27, as well as in adrenal chromaffin cells 28, of adult rats. in line with these results, in vitro studies demonstrated that dissociated sympathetic neurons and pc12 cells expressed il-6 and the two receptors, il-6r and gp130 29, 30. besides neurons and glial cells, endothelial cells produce copious amounts of il-6, which can act on surrounding cells but also in an autocrine manner regulating a number of adhesion proteins but also il-6 synthesis, particularly in the presence of sil-6r 31-33. thus, both the central and the peripheral nervous system appear to express il-6 and the corresponding receptors (figure 1). many cytokines and inflammatory factors as well as neurotranmitters and neuropeptides have been shown to affect il-6 regulation in brain cells; we will comment here some of them studied in in vitro assays. both virus-infected microglial cells and il-1 and tnf induced il-6 in cultured cortical neurons 36 and astrocytes 37, 38, in the latter involving nfb 39 and the pkc pathway 40. it is likely that membrane depolarization is one of the main mechanisms for neuronal upregulation of il-6, where ca currents (such as those elicited by the glutamate agonist nmda) and ca/calmodulin-dependent kinases are critical factors 43, 44. the major bacterial pathogen, lps, normally induces il-6 in both astrocytes and microglia 45-47, but tnf- induces il-6 in astrocytes but not microglia 45. there may be species-specific effects since in human cells in vitro lps mostly affect microglia rather than astrocytes (obtained from brains at second trimester of gestation) regarding tnf, il-1 and il-6 production, although il-1 is a potent stimulator of il-6 production in astrocytes (in microglia the 3 cytokines are upregulated) 46, 48. gm-csf stimulates microglial il-6 but not that of astrocyte 49, whereas ifn- induces il-6 (and no) in the murine microglial cell line 6-3 50; this cytokine does not induce il-6 in astrocytes unless it is coincubated with il-1 37. il-6 production by astrocytes is subjected to autocrine regulation by il-6, and the addition of sil-6r synergizes dramatically with il-1 and tnf to induce il-6 52. oncostatin m (osm) induced il-6 alone and synergized with tnf to induce il-6 52. il-17 functioned in a synergistic manner with il-6 (+ sil-6r) to induce il-6 expression in astrocytes 53. norepinephrine, vasoactive intestinal peptide (vip) and pituitary adenylate cyclase activating polypeptide (pacap38) stimulate il-6 in astrocytes, and may synergize with il-1 and tnf 54-56; in contrast any of these factors have a major effect in microglia 54. vip may induce il-6 through the pka pathway and independently of prostaglandins 57, 58. prostaglandin e1 (pge1) and pge2, but not pgd2 and pgf2 alphae2, induce il-6 in human astrocytoma cells; pge2 potentiates il-1 induction of il-6 59, 60. the synthetic ceramides c2- and c6-ceramide as well as the enzyme sphingomyelinase were able to induce il-6 in astrocytes 61. il-1, substance p and histamine induced il-6 expression in human astrocytoma cells 41, 62, through nf-b for il-1 and through nf-il-6 for sp and histamine 63. serotonin and adenosine agonists were also effective inducers in human astrocytoma cells 65 66; in mouse astrocytes, adenosine induces il-6 through activation of pka and nf-il-6 67. tgf- inhibits microglia proliferation and activation, including il-6 production 68; in contrast, it stimulates il-6 production in astrocytes 69. il-6 may affect neuronal functionality, for instance it induces the cholinergic phenotype of sympathetic neurons 30, 70, 71. sensory neurons are particularly affected by il-6 deficiency, since in normal conditions il-6 ko mice show a 60% reduction of the compound action potential of the sensory branch of the sciatic nerve and a dramatic decrease of temperature sensitivity in the frontpaw withdrawal time in the hot-plate assay 72; these neurons are also highly dependent on il-6 for functional recovery following injury 72, 73. results with il-6 ko mice imply a role for il-6 on sympathetic sprouting induced by nerve injury 74, 75. it also promotes sprouting and functional recovery of organotypical cultures of hippocampus 76, and causes a dose-dependent decrease of post-tetanic potentiation (ptp) and long-term potentiation (ltp) in the ca1 region of rat hippocampus 77. il-6 also has a notorious role in adult neurogenesis 10, 78, the process of creating new neurons and glial cells from neural stem cells (nscs) discovered almost 50 years ago 79, which is now known to be dramatically affected by a myriad of factors such as exercise, environmental enrichment, stress, or aging. not surprisingly, neurogenesis is also altered in many neuropathological situations like stroke, status epilepticus, mechanical damage, and alzheimer, parkinson and huntington diseases; in all these cases a detrimental role of inflammation has usually been suggested 80, 81, and, as stated above, il-6 will be upregulated and could have a role on neurogenesis. gfap-il6 mice, indeed, show a diminished hippocampal neurogenesis 82, and moreover, in vitro il-6 clearly decreases the differentiation of neural stem/progenitor cells into neurons 83, 84. in contrast, il-6 is involved in oligodendrogliogenesis 85, 86 and astrogliogenesis 84. yet, other studies claimed that il-6 promotes both gliogenesis (through the stat-3 pathway) and neurogenesis (mapk/creb pathway) 87, 88. besides effects on neurons, il-6 also displays a number of effects in glial and endothelial cells. early in vitro assays showed that both virus-infected microglia and astrocytes secreted il6, and that il-6 induced the secretion of the major neurotrophin ngf by the latter 34. in vitro there is some conflicting results whether or not il-6 does have proliferative effects on astrocytes 89, 90, probably it does but synergizing with other factors 91. microglia in culture consistently proliferate when stimulated with il-6 92il-6-il-6/sil-6r may alter the factors produced by astrocytes: for instance it induces specific patterns of neurotrophins 93, inhibits tnf 94, and together with sil-6r and il-17, it may shift chemokine production to that favoring t cell recruitment to the cns 95. differences in the pattern of cytokines secreted by astrocytes of il-6 ko mice are readily noticeable in vitro 96. in vivo experiments demonstrate that il-6 exerts profound effects on their differentiation, which is dependent on the brain area from which they are isolated 93. transgenic mice overexpressing il-6 show prominent astrogliosis and microgliosis 97-100, whereas the opposite is usually observed in il-6 ko mice in different models of injury 101-105. il-6 alone does not affect intercellular adhesion molecule-1 (icam-1) gene expression, but dramatically inhibits the activating effect of ifn-, il-1 and tnf in rat astrocytes, and that of ifn- in microglia, very much alike il-10 106. in human astrocytes, il-6, sil-6r or both do not affect vcam-1 or icam-1, but il-6/sil-6r complex (and h-il-6) inhibits tnf--induced vcam-1 gene expression; sil-6r upregulates endogenous il-6 production 107. this inhibitory effect of il-6 in astrocytes is in sharp contrast with the stimulatory effect it has on endothelial cells, where again sil-6r greatly affects the activation of endothelial cells, not only upregulating the adhesion proteins e-selectin, icam-1 and vcam-1 but also il-6, il-8 and monocyte chemotactic protein 1 (mcp-1); this constitutes a model where neutrophils may retrogradely signal inflammation to endothelial cells by shedding sil-6r, which can then recruit leukocytes and contribute to their extravasation 32, 108. regarding oligodendrocytes, in addition to promoting oligondendrogenesis il-6 promotes survival and myelin production of oligodendrocytes 85, 86, 109, 110. although il-6 is often related to inflammatory and pathological situations, it is a factor that contributes decisively in the normal function of the brain. thus, il-6 is involved in the control of body weight, food intake and energy expenditure 111-115, stimulates the pituitary-adrenal axis 116 117, induces fever 111, 118, 119 and is for instance very important in the control of body temperature following recovery from stroke 120. results with il-6 ko mice imply a facilitatory role for il-6 in pain 74, 75, effects on sleep-wake beavior 121, emotional reactivity 122, 123, sickness behaviour 124 and learning and memory 125-127. as stated above, soon after its discovery il-6 was shown to induce the neuronal differentiation of pc12 cells 15, 16. after these initial studies, a flurry of papers demonstrated that il-6 affected in many different ways neurons and glial cells. thus, it was shown to promote the survival of cultured basal forebrain and septal cholinergic neurons and mesencephalic catecholaminergic neurons 128, 129, retinal ganglion cells 130, sympathetic neurons and dorsal root ganglia (drg), particularly if sil-6r was added to the culture 30, 131. it has been suggested that a survival mechanism could be the inhibition of neuronal activity and release of glutamate 132. in vivo, il-6 increases with development in drg, but levels are low in adults; however, following sciatic nerve transection its expression increases dramatically, not only in the drg but also in many neurons in the corresponding motor nucleus or sympathetic ganglion 133. studies in a drop weight model of cortex lesion (closed skull) also indicate that il-6 expression increases following axonal damage, mainly in neurons 134, thus probably neuronal induction of il-6 in response to injury is a general response. in the sciatic model, il-6 expression increases potently within large and medium-sized axotomized neurons 133, whose survival is decreased by 50% in il-6 ko mice 135. il-6 probably promotes survival through inducing bdnf, and indeed in vivo il-6 ko mice do not upregulate this neurotrophin in drg following nerve injury 136. experiments after injury of the hypoglossal nerve in mice also demonstrated a nerve regenerating role of il-6 137. in contrast, in the facial nerve axotomy model no difference in neuronal survival was observed 104, whereas il-6 was shown to be detrimental in a model of optic nerve injury 138. cns il-6 is upregulated whenever neuroinflammation is expected, such as following cns infection or injury or in a number of cns diseases (figures 2 and 3). early studies demonstrated that il-6 was expressed and produced in cns during viral meningitis, in encephalitis mouse models, and in csf of patients with acute viral infections 139. il-6 was also found to be upregulated in mouse experimental cerebral malaria (ecm) 140. moreover, il-6 was highly found in csf of patients with systemic lupus erithematosus (sle) with cns involvement 141 or in those in advanced stages of patients with human immunodeficiency virus 142 infections 143. il-6 levels in csf were also significantly higher than plasma levels in patients who had suffered traumatic brain injury 144, and recently an il-6 polymorphism (-174c/g) has been associated with fatal outcome in patients with severe traumatic brain injury 145. as expected, il-6 is upregulated in several animal models of brain injury and shows a myriad of actions as suggested by studies in il-6 ko mice which show a compromised inflammatory response, increased oxidative stress, impaired neuroglial activation, decreased lymphocyte recruitment and a slower rate of recovery and healing 47, 101, 102, 104, 134, 146-148. of note, il-6 is a critical cytokine controlling the transition from innate to acquired immunity, which is imperative for dealing properly with injured (and infected) cns tissue, and where il-6 trans-signalling is dramatically important 149. in line with the results with il-6 ko mice, gfap-il-6 mice (which overexpress il-6 in the cns) showed more rapid healing and recovery after traumatic brain injury because of extensive revascularization 148, 150. the transcriptomic analysis of il-6ko mice versus wt mice 151 and that of gfap-il-6 mice 152 in a model of brain cortex cryoinjury revealed that il-6 modulates the expression of many genes involved in inflammation, apoptosis and oxidative stress among others. stroke patients also show significant elevations of csf and serum il-6 shortly after the ischemic event that correlate with brain infarct volume, and il-6 haplotype affects both infarct size and il-6 levels 153-157. ischemic brain injury involves inflammation, excitotoxicity, oxidative damage and apoptosis, and thus to some extent it is a similar scenario as that following traumatic brain injury. it is therefore not surprising that il-6 may be an important factor orchestrating the responses elicited by stroke. in animal models of cerebral ischemia it has been a consistent finding an upregulation of il-6, mostly in neurons but also in glial cells and vascular endothelium 158-161. in line with the in vitro studies detailed above, neuronal il-6 upregulation following cerebral ischemia is likely mediated by glutamate-induced neuronal depolarization 44, 162, 163. several of these studies clearly demonstrated a neuroprotective role of exogenously administered il-6, as did experiments injecting anti-mouse il-6 receptor monoclonal antibody 164 and with il-6 ko mice provided body temperature is controlled 120, 165. controlling oxidative stress 166 and angiogenesis 165 are among the attributed functions of il-6 during stroke. epileptic seizures often involve excitotoxicity, and thus it was expected to find that patients suffering of epilepsy show increased csf levels of il-6 after seizures 167, and that well-known animal models of epilepsy such as the glutamate analog kainic acid (ka) upregulate cns il-6 162. il-6 ko mice are more susceptible to various convulsant stimuli including several glutamate analogs and show clear signs of increased hippocampal damage 168-170. yet, this is a complex system, since intranasal administration of il-6 to rats 171 and transgenic il-6 expression in the brain 172 are proconvulsive. also, some authors also claim a damaging role of il-6 on neuron development in culture and on the response to nmda 173 174. the reasons for such dual effects of il-6 remain uncertain, but probably reflect that the context has a dramatic effect as often is the case with growth factors. as a prototypical cytokine with roles in the control of inflammation, il-6 it is therefore not surprising that il-6 expression is altered in the brains of alzheimer's disease (ad) patients, being increased around amyloid plaques and in cerebrospinal fluid 175-179. il-6 stimulated the synthesis of the ad beta-amyloid precursor protein 180, 181, and, conversely, il-6 is upregulated in cultured glial cells upon stimulation with the carboxy-terminal 105 amino acids of app 182. moreover, il-6 also enhances neuronal damage induced by beta-amyloid peptide in cultured rat cortical neurons 174. despite this detrimental role of il-6 seen in vitro, in vivo studies with ad transgenic mouse models rather show a beneficial role of il-6, in principle due to a massive gliosis which attenuated beta-amyloid peptide deposition and enhanced plaque clearance 183. this is not unexpected as activated microglia can efficiently phagocytize beta-amyloid peptide and delay pathology course in transgenic models 184-186. astrocytes also may be involved in the clearance of beta-amyloid peptide 187, also by regulating microglial phagocytosis 188. in humans, attempts to find important interactions between polymorphisms in specific alleles, such as the il-6-174 g/c promoter allele, and genotype frequencies, are not conclusive 189-191. the -572c/g polymorphism of il-6 gene promoter region is another polymorphism that might be associated with ad 192. multiple sclerosis (ms) is an inflammatory demyelinating autoimmune disease of cns mediated by cd4+t cells and soluble inflammatory mediators, in which il-6 has an important role. while there is significant controversy on whether or not ms is correlated with either plasma or csf il-6, sil6r and sgp130 levels 193-196, it has been demonstrated the presence of il-6 in acute and chronic active plaques of ms patients, mainly associated with astrocytes rather than macrophages or mononuclear infiltrating cells 197. yet, the association of ms with il-6 polymorphisms is again not conclusive 198, 199. one of the most common animal model of ms is experimental autoimmune encephalomyelitis (eae) 200. il-6 is upregulated in cns during eae 201, and different approaches have demonstrated a major role of this cytokine. thus, it was soon established that neutralization of il-6 with antibodies led to a reduced disease 202, which later on was seen to be due to the suppression of the mog-induced differentiation of naive t cells into th17 and th1 cells 203. a seminal series of early studies demonstrated that il-6 deficient mice were resistant to eae 204-207, highlighting the essential role of this cytokine. absence of infiltrating cells in the cns, reduction of lymphocyte proliferation, change of the cytokine profile and failure to stimulate endothelial vcam-1 were some of the reasons suggested to be responsible for resistance to eae at the time. trans-signaling is also crucial for eae induction as its blockade with gp130-fc fusion protein delayed the onset of adoptively transferred eae compared to controls due to a reduction in vcam-1 expression on spinal cord microvessels 208. since the discovery of two novel subsets of t helper cells, named treg and th17 cells, and its importance in ms, it has been shown that il-6 has a major role in th17 cell differentiation from naive cd4 t cells, particularly in the eae model 203, 209, 210. th17 cells produce il-17 (among other cytokines) which enhances il-6 production by astrocytes, which in turn induces differentiation of th17 cells in a positive feedback loop between il-17 and il-6 53, 211. although eae is considered a disease mostly induced peripherally, the fact is that the cns local milieu may have dramatic effects. thus, the gfap-il6 mouse model of chronic transgenic il-6 expression 97 presents an atypical eae due to a retargeting of the immune attack, with no signs of spinal cord damage while showing a prominent cerebellar damage 212. the mechanisms responsible for these responses to il-6 in the cerebellum are complex and likely to include an increased vascular activation, loss of integrity of the bbb and the induction of specific cytokines and chemokines. parkinson's disease (pd) is a degenerative disorder of the cns with severe motor impairment, like shaking or rigidity among many others, due to the death of dopaminergic neurons in the substantia nigra. il-6 is upregulated in pd 213, 214, and il-6 levels in the csf of pd patients are elevated 215, although an inverse correlation between severity of pd and il-6 levels is observed 216. il-6 ko mice show increased vulnerability to the mptp, a molecule that is metabolized into the complex i inhibitor mpp+ by astrocytes and is a selective dopaminergic toxin 217, whereas il-6 is capable of protecting rat dopaminergic neurons from the neurotoxicity of mpp 218. altogether, the results with these animal models of pd suggest that il-6 could be exerting a neuroprotective role during pd. huntington's disease 208 is an inherited neurodegenerative disorder with both neurological and systemic abnormalities, characterized by abnormalities in the huntingtin gene. il-6 expression is dramatically elevated in the striatum of hd patients, and in general these patients show clear signs of abnormal immune activation, il-6 being one of the cytokines affected and monocytes, macrophages and microglia (from both hd and mouse hd models) being overactive upon stimulation 219. il-6 is suggested to be neuroprotective as concluded from results with the quinolonic acid rat model of hd 220. some studies have pointed out that il-6 levels in csf but not in plasma were increased in combat veterans with posttraumatic stress disorder (ptsd) in comparison with those of controls 223, and that il-6 and/or sil-6r levels in plasma but not sgp130 were significantly higher in ptsd patients respect to controls, and higher in ptsd patients with concurrent major depression than in ptsd patients or controls 224. il-6 has also been related with schizophrenia (sz), a complex neurological disorder characterized by a breakdown of thought processes and by poor emotional responsiveness, commonly manifested with hallucinations, delusions, paranoid and mental deterioration 225, 226. thus, a single maternal injection of il-6 during pregnancy causes schizophrenia-like behavioral abnormalities in wt mice but not in il-6 ko mice 227. finally, il-6 has been found to be increased in the cerebellum of autistic brain 228 and has been suggested to mediate autism-like behaviors 229. many of its effects are caused by trans-signaling, while others are mediated by the membrane receptor; both can be essentially considered an integrated, unique system. the relevance of trans-signaling in vivo in a number of peripheral and cns diseases is widely recognized 111, 149, 230-234, and not surprisingly therapeutic approaches aiming to counteract il-6 effects not only focus in il-6 membrane receptor 235-238 but also in il-6/sil-6r complex 13, 239-241. likewise, sgp130 is also of an invaluable utility to specifically block diseases states in which trans-signaling responses exist 232, 234, 241, to the extent that the molecule has recently been structurally-optimized to better antagonize trans-signaling pathologic effects 242. still, we must keep in mind that il-6 has a role in the normal brain, and interfering with them may be a significant source of concern. finally, we need to understand in vivo what the role(s) of il-6 are to be due to specific cellular production of and response to il-6; hopefully the production of floxed mice for il-6 will help to answer these questions.

interleukin-6 (il-6) is a cytokine originally identified almost 30 years ago as a b-cell differentiation factor, capable of inducing the maturation of b cells into antibody-producing cells. as with many other cytokines, it was soon realized that il-6 was not a factor only involved in the immune response, but with many critical roles in major physiological systems including the nervous system. il-6 is now known to participate in neurogenesis (influencing both neurons and glial cells), and in the response of mature neurons and glial cells in normal conditions and following a wide arrange of injury models. in many respects, il-6 behaves in a neurotrophin-like fashion, and seemingly makes understandable why the cytokine family that it belongs to is known as neuropoietins. its expression is affected in several of the main brain diseases, and animal models strongly suggest that il-6 could have a role in the observed neuropathology and that therefore it is a clear target of strategic therapies.

PMC3491449

pubmed-661

advanced glycation end-products (ages) have been shown to accumulate in various tissues under diabetic conditions, and they participate in the development of vascular complications such as diabetic retinopathy [1, 2]. our recent results showed that even a low concentration of ages, for example, 10 g/ml, can induce neuronal apoptosis in retinal neurons and decrease the number of regenerating neurites in cultures. ages accomplish their effects by binding to specific cellular receptors [4, 5], such as receptors for ages (rages). these receptors have been found on neurons, mesangial cells, smooth muscle cells, and endothelial cells [68]. the binding of rage to ages precursors generates intracellular oxidative stress which then induces receptor-mediated production of reactive oxygen species. this then results in the activation of the free radical-sensitive transcription factor nuclear factor-b (nf-b) [9, 10]. the activated nf-b translocates into the nucleus and causes pathological changes in gene expression [9, 1113]. specificity protein 1 (sp1) is a transcription factor that either activates or represses transcription in response to physiological and pathological stimuli. it regulates the expression of a large number of genes involved in a variety of processes such as cell growth, apoptosis, differentiation, and immune responses. several genes can be regulated by a combination of nf-b and sp1, and in specific cases by direct interaction between the nf-b protein and sp1 protein. tanaka et al. found that ages can activate the rage gene through nf-b and sp1, causing enhanced age-rage interactions in human vascular endothelial cells. however, there are no reports demonstrating the relationship between nf-b/sp1 expression and regeneration in age exposed retinal neurons. growing evidence indicates that neuronal abnormalities such as neuronal cell death and vascular abnormalities are associated with the development of early diabetic retinopathy. because neuronal cell death is an irreversible change and can affect visual function of diabetic eyes, neuroprotective and regenerative therapies need to be determined. however, no reports have been simultaneously compared the neuroprotective and regenerative effects of several neurotrophic factors including neurotrophin-4 (nt-4), hepatocyte growth factor (hgf), glial cell line-derived neurotrophic factor (gdnf), and tauroursodeoxycholic acid (tudca) in age exposed retinas cultured in the same system. the purpose of this study was to examine the effect of high doses of ages on neuronal cell death and neurite regeneration in isolated rat retinas. we also determined the neuroprotective and regenerative effects of four neurotrophic factors, namely, nt-4, hgf, gdnf, and tudac in age exposed retinas. in addition, we also examined whether the expressions of nf-b and sp1 were correlated with the neuroprotective and regenerative effects of different neurotrophic factors in ages exposed rat retinas. seven-week-old male sprague-dawley (sd) rats (japan slc co., hamamatsu, japan) were used. all of the procedures were performed in accordance with the arvo statement for the use of animals in ophthalmic and vision research. the retinas were isolated under sterile conditions and cut into square pieces of 0.16 mm with sharp razor blades. then the retinal explants were cultured on three-dimensional collagen gels as described in detail [1823]. the retinal explants were incubated in 6 different types of media; (1) serum-free control culture media, (2) 100 g/ml glucose-age-bsa (cyclex co., nagano, japan) or glycolaldehyde-age-bsa (cyclex co) or glyceraldehyde-age-bsa (cyclex co) media, (3) glucose-age or glycolaldehyde-age or glyceraldehyde-age+100 ng/ml nt-4 (r&d systems, minneapolis, mn) media, (4) glucose-age, glycolaldehyde-age, or glyceraldehyde-age+100 ng/ml hgf (r&d systems) media, (5) glucose-age, or glycolaldehyde-age, or glyceraldehyde-age+100 ng/ml gdnf (r&d systems) media, and (6) glucose-age, or glycolaldehyde-age, or glyceraldehyde-age+100 m tudca (wako, osaka, japan) media. the serum-free media contained 7.5 mm glucose, 5 g/ml insulin, 16.1 g/ml putrescine, 10% bovine serum albumin, 3.7 mg/ml nahco3, 5.2 mg/l na2seo3, and 3.6 mg/ml hepes in minimum essential medium as described [21, 23, 24]. one hundred g/ml bsa was added to the control medium (n+a) as a control for the concentration of age-bsa. to determine whether apoptosis had occurred, the retinal explants were fixed in 4% paraformaldehyde after 7 days in culture and sectioned on a cryostat. then, tdt-dutp terminal nick-end labeling (tunel) staining was carried out with an apoptosis detection kit (chemicon international, temecula, ca) according to the manufacturer's instructions. sections were costained with 4,6-diamidino-2-phenyl indole (dapi, polyscience inc., warrington, pa). for quantitative analyses, the ratio of the number of tunel-positive cells to the total number of dapi-staining nuclei in the ganglion cell layer (gcl) was determined. a total of 18 sections from the 6 explants/group were studied, and the results were used for the statistical analyses. the total number of nuclei counted was 405 (n), 215 (n+a), 816 (ages), 472 (nt-4), 396 (hgf), 512 (gdnf), and 494 (tudca). the retinal explants were fixed as described and cryosections were cut. after blocking the sections in 5% goat serum and 3% bovine serum in 0.1 m phosphate buffer saline, they were incubated with antibodies against rabbit anti-phosphorylated nf-b (p-nf-b) and sp1 transcription factor (santa cruz biotechnology, santa cruz, ca) at 4c overnight. then, the sections were incubated with fluorescein isothiocyanate-conjugated anti-rabbit igg for one hour. the number of p-nf-b- and sp1-positive cells in the gcl was counted. for quantitative analyses, the number of immunopositive cells in the gcl was expressed relative to the total number of dapi-stained nuclei. the total number of nuclei counted was 301 in n, 234 in ages, 198 in nt-4, 254 in hgf, 186 in gdnf, and 276 in tudca. the number of neurites regenerated from the explants was counted under a phase-contrast microscope after 7 days in culture when the number of regenerating neurites was very high [3, 18, 21, 23, 24]. branched neurites were counted as one. the number of explants examined was 101 in the control group including that in serum-free media (n, n+a, n+nt-4, n+hgf, n+gdnf, n+tudca), 11 in the glucose-age-bsa group, 19 in the glycolaldehyde-age-bsa group, 18 in the glyceraldehyde-age-bsa group, 14 in the glucose-age-bsa+nt-4, 12 in the glycolaldehyde-age-bsa+nt-4, and 11 in the glyceraldehyde-age-bsa+nt-4 groups, 12 in the glucose-age-bsa+hgf, 14 in the glycolaldehyde-age-bsa+hgf, 13 in the glyceraldehyde-age-bsa+hgf groups, 12 in the glucose-age-bsa+gdnf, 13 in the glycolaldehyde-age-bsa+gdnf, 14 in the glyceraldehyde-age-bsa+gdnf groups, 10 in the glucose-age-bsa+tudca, 11 in the glycolaldehyde-age-bsa+tudca, and 10 in the glyceraldehyde-age-bsa+tudca groups. to determine whether ages were toxic to the retinas in culture, the number of tunel-positive cells in the gcl was counted. the majority of the tunel-positive cells were detected in the gcl because all of the retinal ganglion cells (rgcs) were axotomized to isolate the retina [3, 18, 21, 2426]. in retinas added 100 g/ml bsa more to the control medium (n+a), the number of tunel-positive cells was not significantly different from the control medium (n) (9.3 2.3% versus 9.7 6.8%, p=0.244). in retinas cultured in glucose-age-bsa, glycolaldehyde-age-bsa, and glyceraldehyde-age-bsa, the number of tunel-positive cells in the gcl was significantly higher than that in the serum-free control medium (38.1 6.2% versus 9.7 6.8%, p<0.0001; 36.1 4.9% versus 9.7 6.8%, p<0.0001; and 39.9 9.4% versus 9.7 6.8%, p<0.0001, resp.; figure 1). the addition of nt-4 decreased the number of tunel-positives cells more than in glucose-age-bsa without nt-4 (17.8 7.2% versus 38.1 6.2%; p=0.0002), in glycolaldehyde-age-bsa without nt-4 (12.8 4.2% versus 36.1 4.0%; p=0.0046), and in glyceraldehyde-age-bsa without nt-4 (13.4 3.6% versus 39.9 9.4%; p=0.0078; figure 1). addition of hgf in age-bsa did not decrease the number of tunel-positives cells compared to the number in glucose-age-bsa without hgf (34.0 5.5% versus 38.1 6.2%; p=0.7861) and glyceraldehyde-age-bsa without hgf (29.1 11.0% versus 39.9 9.4%; p=0.1722) but it significantly decreased in glycolaldehyde-age-bsa without hgf (25.4% 7.3% versus 36.1 4.0%; p=0.0039; figure 1). addition of gdnf did not decrease the number of tunel-positives cells compared to the number in glucose-age-bsa without gdnf (32.4 3.0% versus 38.1 6.2%; p=0.0935) and in glycolaldehyde-age-bsa without gdnf (35.2% 5.1% versus 36.1 4.0%; p=0.7373) and in glyceraldehyde-age-bsa without gdnf (31.2 9.78% versus 39.9 9.4%; p=0.1293; figure 1). the addition of tudca decreased the number of tunel-positives cells more than that in glucose-age-bsa without tudca (28.2 6.5% versus 38.1 6.2%; p=0.0184) and in glyceraldehyde-age-bsa without tudca (26.6 8.3% versus 39.9 9.4%; p=0.0294) but it did not decrease in glycolaldehyde-age-bsa without tudca (31.6 3.3% versus 36.1 4.0%; p=0.45; figure 1). the sections were immunostained for p-nf-b to determine whether nf-b was activated in retinas exposed to ages. the effects of the neurotrophic factors on the activation of nf-b were also examined. in retinas cultured with glucose-age-bsa, the number of p-nf-b immunopositive cells was higher than in serum-free control medium (53.2 7.2% versus 31.6 16.1%; p=0.0146; figures 2 and 3). addition of nt-4 decreased the number of immunopositive cells more than in the serum-free media (11.3 6.9% versus 31.6 16.1%; p=0.0315) and more than in glucose-age-bsa without nt-4 (31.5 5.3% versus 53.2 7.2%; p=0.0133; figures 2 and 3). addition of hgf decreased the number of immunopositive cells more than in serum-free media (26.8 5.3% versus 31.6 16.1%; p=0.049) and in glucose-age-bsa without hgf (42.6 2.7% versus 53.2 7.2%; p=0.0114; figures 2 and 3). addition of gdnf decreased the number of immunopositive cells more than in serum-free media (13.3 5.0% versus 31.6 16.1%; p=0.0495) and in glucose-age-bsa without gdnf (25.8 5.3% versus 53.2 7.2%; p=0.0011; figures 2 and 3). addition of tudca decreased the number of immunopositive cells more than in serum-free media (18.6 3.97% versus 31.6 16.1%; p=0.0024) and in glucose-age-bsa without tudca (36.2 7.2% versus 53.2 7.2%; p=0.0034; figures 2 and 3). the sections were immunostained for sp1 transcription factor to determine whether it was expressed in retinas exposed to ages. we also examined the effect of neurotrophic factors on the expression of sp1 transcription factor. in retinas cultured with glucose-age-bsa, the number of sp1 immunopositive cells was higher than in serum-free control medium (32.2 6.8% versus 6.3 1.2%; p<0.0001; figures 4 and 5). addition of nt-4 increased the number of immunopositive cells more than in serum-free media (13.0 4.3% versus 6.3 1.2%; p=0.0315) but did not decrease the number of immunopositive cells in glucose-age-bsa without nt-4 (30.2 8.4% versus 32.2 6.8%; p=0.6753; figures 4 and 5). addition of hgf increased the number of immunopositive cells more than in serum-free media (19.7 5.16% versus 6.3 1.2%; p=0.0001) but did not decrease the number of immunopositive cells in glucose-age-bsa without hgf (24.0 6.7% versus 32.2 6.8%; p=0.0926; figures 4 and 5). addition of gdnf increased the number of immunopositive cells more than in the serum-free media (12.3 1.4% versus 6.3 1.2%; p<0.0001) but it decreased compared to in glucose-age-bsa without gdnf (19.7 6.4% versus 32.2 6.8%; p=0.0007; figures 4 and 5). addition of tudca increased the number of immunopositive cells more than in serum-free media (10.2 3.9% versus 6.3 1.2%; p=0.0454) but it decreased compared to in glucose-age-bsa without tudca (19.5 5.5% versus 32.2 6.8%; p=0.0075; figures 4 and 5). in retinas added 100 g/ml bsa more to the control medium (n+a), the number of neurites was not significantly different from the control medium (n) (94.6 24.0/mm versus 97.5 34.9/mm, p=0.948). in retinas incubated with ages (glucose-age, glycolaldehyde-age, and glyceraldehyde-age), the number of regenerating neurites was less than in retinas without age (45.0 27.5/mm versus 97.5 34.9/mm, p=0.0046; 29.4 29.4/mm versus 97.5 34.9/mm, p=0.0003; 25.0 15.6/mm versus 97.5 34.9/mm, p<0.0001; figures 6 and 7). all of the retinas incubated in the neurotrophic factors (nt-4, hgf, gdnf, and tudca) had an increase in the number of regenerating neurites in serum-free media (541.9 77.5/mm versus 97.5 34.9/mm, p<0.0001; 207.5 49.1/mm versus 97.5 34.9/mm, p<0.0001; 211.3 70.6/mm versus 97.5 34.9/mm, p<0.0001; 229.4 33.8/mm versus 97.5 34.9/mm, p<0.0001; figures 6 and 7). in addition, all of the neurotrophic factors increased the number of regenerated neurites in ages exposed retinas, but the most significant regenerative effect was found in the nt-4 group: 379.4 178.0/mm versus 45.0 27.5/mm, p<0.0001, in nt-4 group; 281.3 100.6/mm versus 45.0 27.5/mm, p<0.0001, in hgf group; 148.8 35.0/mm versus 45.0 27.5/mm, p<0.0001, in the gdnf group; 247.5 56.9/mm versus 45.0 27.5/mm, p<0.0001, in tudca group supplemented with glucose-age incubated retinas; 440.0 165.0/mm versus 29.4 29.4/mm, p<0.0001, in nt-4 group; 196.3 89.4/mm versus 29.4 29.4/mm, p<0.0001, in hgf group; 161.1 53.9/mm versus 29.4 29.4/mm, p<0.0001, in gdnf group; 238.1 33.1/mm versus 29.4 29.4/mm, p<0.0001, in tudca group supplemented to glycolaldehyde-age incubated retinas, 230.6 116.9/mm versus 25.0 15.6/mm, p<0.0001, in nt-4 group; 123.8 29.4/mm versus 25.0 15.6/mm, p<0.0001, in hgf group; 137.5 48.8/mm versus 25.0 15.6/mm, p<0.0001, in gdnf group; 178.8 39.4/mm versus 25.0 15.6/mm, p<0.0001, in tudca group supplemented to glyceraldehyde-age incubated retinas (figures 6 and 7). the importance of ages in the pathogenesis of diabetic complications has been shown in animal models. two unrelated age inhibitors were found to partially block some functional and structural changes in retinas, neuronal tissues, and kidneys of diabetic animal models [2729]. our previous study showed that even a low concentration of ages, for example, 10 g/ml, induced neuronal apoptosis in the gcl and decreased the number of regenerated neurites in culture. higher doses (100 g/ml) of ages had similar effects as low concentrations and decreased the number of tunel-positive cells and significantly blocked neurite regeneration. the results of this study confirmed that the apoptosis in the gcl were caused by ages and also that neurite regeneration was significantly suppressed. all examined neurotrophic factors were able to increase the number of regenerative neurites; however the most significant regenerative effect was observed with nt-4. in addition, all examined neurotrophic factors suppressed the expression of nf-b expression in ages exposed retinas. nf-b is a primary transcription factor that plays an important role in regulating cellular responses, that is, a transcription factor that is present in cells in an inactive state and does not require new protein synthesis to become activated. other members of this family include transcription factors such as c-jun, stats, and nuclear hormone receptors. some aspects of the mechanism by which nf-b protects cells against toxins have been identified. for example, tumor necrosis factor- has been shown to protect hippocampal neurons against excitatory amino acid toxicity through nf-b activation by inducing bcl-2 and bcl-x expression. sp1 transcription factor belongs to a group of factors that are associated with gc-rich promoters that are involved in basal promoter activity. sp1 regulates the expression of different genes, including the vascular endothelial growth factor, fibrogenic cytokine, and many matrix genes. several studies reported that the interactions between age and rage cause phenotypic changes in the microvascular endothelial cells and pericytes [3238]. our results showed that ages increase the expression of the transcription factor nf-b and sp1 in retinal neurons. this suggests that ages enhanced the expression of the rages gene in retinal neurons through the increased expression of nf-b and sp1. an upregulation of nf-b and sp1 proteins in retinal neurons suggests that different protective mechanisms are important for the protection of these cells against ages-mediated cell death. tudca is a member of a group of compounds that modulate the endoplasmic reticulum (er) function, protecting the cells against er stress-induced apoptosis. earlier studies showed that tudca had protective effects on damaged retinal neurons under diabetic stress as an anti-er reagent. the neuroprotective effect of tudca was correlated with the suppression of phosphorylated jnk and phosphorylated c-jun expression [18, 26]. it is also a potent mitogen and differentiation factor for endothelial cells, and it had neurotrophic and neuroprotective activity for central nervous system neurons [41, 42].. used an optic nerve axotomy model and demonstrated that hgf prevented rgc apoptosis in vivo in a concentration-dependent manner. found that hgf promoted long-term survival and axonal regeneration of rgc after optic nerve injury. recently, gdnf was found to be a growth and survival factor for neuronal cells, and it promoted the survival of peripheral sensory and sympathetic neurons and also motor neurons. koeberle and ball studied the effects of gdnf on rgc survival and apoptosis after optic nerve transection. nt-4 is a member of the nerve growth factor family which acts on different types of nerve cells, for example, sensory, cortical, and hippocampal neurons., we investigated the neuroprotective and regenerative effects of nt-4 on retinal neurons under diabetic condition [3, 18, 21, 26]. our results showed that nt-4 promoted the survival and the regeneration of neuronal cells in the retinas incubated in high glucose media. the neuroprotective and regenerative effects of nt-4 were correlated with the reduction in the activation of caspase-9 and -3, expression of perk and chop, and c-jun and jnk expression. the results of the present study showed that all examined neurotrophic factors decreased the number of nf-b immunopositive cells in glucose-age-bsa exposed retina, but nt-4 had the highest significant effect. however, the number of sp1 immunopositive cells was increased by the addition of neurotrophic factors in serum-free media and more significantly in the hgf and nt-4 groups. thus after the addition of neurotrophic factors to glucose-age-bsa media, the level of sp1 transcription factor remained high in the nt-4 and hgf group. kanda et al. found that prostaglandin e2 promoted innervation in skin lesions by the induction of nt-4, and the induction was mediated by sp1. their results showed that the ep3, g-protein-coupled receptors, mediated by prostaglandin e2 transcription of nt-4 was dependent on the activity of sp1. thus, our findings suggest that sp1 may be related to neuronal survival and regeneration. it is possible that the increased expression of sp1 may result from an enhanced phosphorylation of sp1, because the sp1 promoter contains several sp1-binding elements and is positively regulated by its own gene product, sp1 protein. further studies are needed to identify the connection between sp1 expression and nt-4 transcription. to find a possible decrease in the effect of the ages on the retina is important. our findings that a suppression of nf-b expression in retinal neurons by several neurotrophic factors can result in neuroprotection, and reducing inflammation and oxidative stress suggests therapeutic potential of neuroprotective therapy in various ocular pathologies associated with ages accumulation. in conclusion, high-dose ages inhibit neurite regeneration which is correlated with increased expression of nf-b and sp1. nt-4 enhances neurite regeneration in ages exposed retinas more than other neurotrophic factors such as hgf, gdnf, and tudca. our results indicate the therapeutic potentials of the neurotrophic factors as axoprotectants in ages exposed retinas.

to determine the effect of advanced glycation end-products (ages) on neurite regeneration, and also to determine the regenerative effects of different neurotrophic factors (ntfs) on rat retinal explants, the retinas of sd rats were cultured in three-dimensional collagen gels and incubated in 6 types of media: (1) serum-free control culture media; (2) 100 g/ml ages-bsa media; (3) ages-bsa+100 ng/ml neurotrophin-4 (nt-4) media; (4) ages-bsa+100 ng/ml hepatocyte growth factor media; (5) ages-bsa+100 ng/ml glial cell line-derived neurotrophic factor media; or (6) ages-bsa+100 m tauroursodeoxycholic acid media. after 7 days, the number of regenerating neurites was counted. the explants were immunostained for nuclear factor-b (nf-b) and specificity protein 1 (sp1). statistical analyses were performed by one-way anova. in retinas incubated with ages, the numbers of neurites were fewer than in control. all of the ntfs increased the number of neurites, and the increase was more significant in the nt-4 group. the number of nf-b and sp1 immunopositive cells was higher in retinas exposed to ages than in control. all of the ntfs decreased the number of nf-b immunopositive cells but did not significantly affect sp1 expression. these results demonstrate the potential of the ntfs as axoprotectants in ages exposed retinal neurons.

PMC4452840

pubmed-662

a 70 year-old male presented to the emergency department of our institution after being involved in a motor vehicle accident. he had sustained a minor injury of the head (glasgow coma scale 15/15), the chest (seat belt burn) and lower extremities (bruises). his past medical history was significant for diabetes mellitus controlled with an oral hypoglycemic (metformin) for 6 years with satisfying control of blood glucose levels. the patient's medical history did not reveal any previous high-energy damage of the right ankle and clinical examination of other joints was negative for hypermobility., there was moderate edema of the right ankle region and obvious anterior-lateral dislocation of the talus with the forefoot in line. the right foot had normal skin temperature, pedal pulses were palpable and there was slight hypoesthesia of the dorsal aspect in comparison to the contralateral foot. the initial plain radiographs (anteroposterior and lateral views of the ankle joint) confirmed the diagnosis of talar dislocation, but also the absence of any fracture (fig. 1). owing that there was no fracture and major neurovascular trauma, an attempt of closed reduction was performed at the emergency department at approximately 1 hours after the injury. radiographic ankle views at the initial presentation showing the anterior-lateral dislocation of the ankle (a, b). the closed reduction was achieved by axial traction of the calcaneus with one hand while the other hand was placed at the dorsal aspect of the midfoot with the foot held in slight plantarflexion for a couple of minutes, followed by lateral compression and external rotation of the talus and, finally, dorsiflexion of the foot in a way to redirect the articular line of the ankle joint. the attempt was successful and confirmed with radiographic imaging after the closed reduction (fig. 2). maintenance of reduction was accomplished with a posterior ankle splint while the patient remained at the hospital for observation. general measures for pain and edema of the closed reduction included elevation of the right lower extremity, ice therapy, oral analgesic and non-weight bearing status to the right foot. ten days later, the edema had subsided significantly, the splint was changed to a cast and partial weight-bearing was allowed for 5 weeks followed by full weight bearing for the next 3 weeks. after 8 weeks, the lower extremity cast was removed and the patient was followed with standard and stress radiographic views. at that time, there were no signs of instability and the range of motion of the right ankle was satisfactory and painless. the patient was then allowed to full weight-bearing status without the use of additional cast applications. one year after the initial injury, the radiological findings were negative for any signs of osteonecrosis or early signs of ankle arthritis (fig. 3). the right ankle was pain-free with similar range of plantarflexion and dorsiflexion as compared to the left ankle [plantarflexion: 044 (right), 046 (left)/dorsiflexion: 015 (right), 016 (left)]. the first case of tibiotalar dislocation without an accompanying fracture of the ankle was described by peraire in 1913 (4). wilson et al. reported the first large series of 14 cases collected from the literature and added two cases of their own (4, 12). although multiple cases of ankle dislocations have been reported in the literature, there is rare mentioning of an anterior-lateral dislocation of the ankle, especially in diabetic patients. closed ankle dislocation without an associated fracture or disruption of the tibiofibular syndesmosis is rare and only a small series have been reported in the literature (1, 2, 4, 7, 11, 15, 16). the ligaments are stronger than the malleoli, so that ankle dislocation is usually accompanied by fracture (4, 9). cases of open dislocation of the ankle without fracture have been also reported and are common (1, 4, 6). ankle dislocations are caused primarily by motor vehicle accidents, with the second most common cause being sports trauma and these usually occur in young athletes (17). pertinent information about the mechanism of trauma injury is paramount in order to make an accurate and timely diagnosis (4, 18). tibiotalar dislocation is classified by fahey and murphy into five types, based on the direction of the dislocation (19). the ankle dislocation can be anterior, posterior, medial, lateral, upward or a combination of these (1). the wider anterior talus wedging results in forced widening of the joint and could be accompanied by either disruption of the tibiofibular syndesmosis or fracture of the lateral malleolus. anterior dislocation occurs when a posterior force is applied to the tibia with forced dorsiflexion. lateral and medial dislocations occur from forced eversion, inversion or rotation of the ankle (4, 12, 13). according to a study on cadavers (20), the author was able to manually dislocate the ankle medially or laterally without associated tibia or fibular fracture by placing an inversion or eversion stress on a maximally plantarflexed foot. the supporting structures that failed were the anterolateral part of the joint capsule followed by the anterior talofibular and calcaneofibular ligaments. the author assumed that once the ankle had been dislocated, the talus and the foot were pulled posteriorly by the achilles tendon. the talus has a rhomboidal shape when viewed from its superior aspect and its posterior portion is narrower than the anterior portion. plantarflexion places the narrowest portion of talus in the ankle mortise which is the most unstable position. an inversion force causes ligamentous and capsular failure which causes the potential for ankle dislocation with or without fracture. the published literature supports the non-operative treatment of closed ankle dislocation without fracture. closed injuries have been treated conservatively with prompt reduction and cast immobilization and the results were reported to be good to excellent (1, 4, 5, 7, 8, 15, 16, 21). it is absolutely necessary to check the neurovascular status of the foot before and after manipulation. after achieving a satisfactory reduction, the integrity of the ankle ligaments, especially the deltoid ligament, consideration must also be given to other anatomical anomalies, especially hypoplasia of the medial malleolus, which must be addressed as part of the final treatment plan. complications, such as the loss of ankle range of motion, stiffness or residual instability, and early arthritis, were reported following closed treatment of ankle dislocations (7). in lateral ankle dislocations, as presented in this case report, rupture of the anterior and posterior talofibular ligaments, as well as rupture of the calcaneofibular ligament have been reported as probable causes (8, 22). the association of the lateral with the anterior type of ankle dislocation could be the result of a posterior force applied to the tibia with the ankle in dorsiflexion and rupture of the talofibular and calcaneofibular ligaments. the anatomical characteristics of the talus and the positioning of the ankle mortise during dorsiflexion make this injury rare. early diagnosis and treatment of this rare type of anterior-lateral ankle dislocation is paramount in the overall patient's successful outcome. our rare case was reported in a diabetic patient that was treated conservatively with closed reduction and cast immobilization. continuous post-operative care is essential in this type of population with known diabetes related complications. the authors have not received any funding or benefits from industry to conduct this study.

anterior or anterior-lateral dislocation of the ankle is a rare condition that can be treated conservatively as well as any other similar types of ankle dislocations without associated fractures. we present a case report of an anterior-lateral ankle dislocation with a concomitant avulsion injury of the ankle's anterior capsule in a diabetic patient that was treated conservatively. at the patient's visit 12 months after the initial injury, he was asymptomatic with full range of motion of the ankle joint. to our knowledge, we could not identify this type of an injury in a diabetic patient that was treated successfully with conservative treatment in the existing literature.

PMC3369671

pubmed-663

central venous catheters (cvcs) have an essential part to play in the management of a critically ill patient. they are useful for hemodynamic monitoring, for administration of specific medications like vasoactive drugs, parenteral nutrition and for hemodialysis. these are associated with substantial risks of complications, which can be mechanical, septic and thrombotic. upper limb dvt is a well known complication of thrombosis associated with cvcs, which, in its fatal form, can sometimes lead to life-threatening pulmonary embolism. the incidence of thrombosis varies from 1.9% in subclavian cvcs to 21.5% in femoral catheters. the risk of thrombosis associated with ijv catheters is estimated to be four-times higher than that with subclavian catheters. sixty-six percent of the patients with internal jugular vein (ijv) catheters have evidence of thrombus formation either on ultrasound or on autopsy. the aim of this study was to determine the incidence of thrombosis associated with right-sided ijv catheters, the extent of thrombosis, its clinical implications and to relate it with the pharmacological prophylaxis for dvt. one hundred consecutive adult patients admitted to our critical care unit, who had right-sided ijv catheters placed for various indications, were included in the study. cvc insertion was performed by intensivists or doctors having at least 5 years of experience in cvc insertion. color doppler sonography was done on the 3 and 6 days of catheter placement. local signs of inflammation and any clinical signs of upper limb dvt or pulmonary embolism, if any, were noted. presence of pharmacological prophylaxis for dvt, presence of parenteral nutrition and other medications like mannitol and hypertonic saline were noted. patients with diagnosed or suspected malignancy, those with diagnosed prothrombotic states and those having hemodialysis catheters in ijv were excluded from the study. among the patients enrolled in our study, there were 70 males and 30 females. there was evidence of thrombus in 33 patients in color doppler duplex sonography (33.0%). in males, thrombus was found in 23 of 70 patients (32.86%) and in females thrombus was present in 10 of 30 patients (33.33%). the incidence in males was 158.62 per 1000 catheter-days and in females 185.18 per 1000 catheter-days. we categorized the thrombus arbitrarily into three groups: small, medium and large, depending on the size of the thrombus. small thrombus was just around the catheter, medium-sized thrombus was up to 4 mm in diameter and the large-sized thrombus was more than 4 mm in diameter. in 15 patients the thrombus was small, in nine figures 1a, b and c show the different sized thrombi on vascular doppler studies. the incidence of different sizes of thrombus in males and females has been shown below [table 1]. the incidence of small-sized thrombus was significantly higher in males (p=0.05) and the incidence of large-sized thrombus was significantly higher in females (p=0.05). small thrombus in the right internal jugular vein medium-sized thrombus in the right internal jugular vein large-sized thrombus in the right internal jugular vein incidence of cvc-related thrombus in males and females of 33 patients found to be having thrombus, in 21 (63.64%) it was detected on the third day after cvc insertion, whereas in 12 patients (36.36%) it was detected on the sixth day. if the thrombus was small or medium sized, then it was observed and followed-up with a color doppler sonography after 3 days. of 33 patients, 24 had low molecular weight heparin as part of the dvt prophylaxis and nine did not have the same due to some medical contraindications. we did not find any significant effect of hypertonic solutions including mannitol and 3% saline as a risk factor for thrombus formation. we did not find diabetes mellitus, hypertension or smoking as increasing the risk of thrombus formation. they provide an important invasive tool for hemodynamic monitoring. they also allow delivery of certain medications and nutrition to the patient safely. as a result, cvc insertion is one of the most frequently performed invasive procedures in the intensive care unit. unfortunately, their use is associated with many untoward complications, which increases the morbidity of the patient and can be life threatening. hirsch and coworkers detected the prevalence of dvt in critically ill patients to be 33% with the help of ultrasonography and color doppler imaging. merrer and colleagues found the incidence of cvc-associated thrombosis to be 1.9% in the subclavian route and 21.5% in the femoral route. a review showed that in such patients, the incidence of asymptomatic cvc-related thrombosis varied from 1.5 to 34.1% and that of symptomatic thrombosis from 1.2 to 13%. another review found that the prevalence of cvc-related upper torso deep venous thrombosis in asymptomatic cancer patients varies from 11.7 to 44%, whereas in symptomatic patients it varies from 6.7 to 48%. there have been some studies on catheter-related thrombosis in patients having hemodialysis catheter in situ. terrence and colleagues found the evidence of right ijv thrombus in 25.9% of the patients. apart from patients with hematological and other malignancies as well as those having hemodialysis cannula, the incidence of cvc-related thrombosis and its clinical impact have not been studied well. in this study, we found the incidence of catheter-related thrombosis in right-sided ijv catheter to be 33.0%. the etiology of thrombosis associated with cvcs can be explained on the basis of virchow's triad of endothelial damage, altered blood flow and hypercoagulability. vessel endothelium damage can be due to various factors including mechanical injury during the process of cvc insertion, number of vein punctures as well as irritation of endothelium by hypertonic solutions and drugs. within hours after insertion of the catheter there is deposition of platelets around the cvcs, reaching its peak in 34 h. this is followed by formation of a sleeve around the cvc, which is an adherent coating of fibrin and collagen. the formation of sleeve is reported to occur in up to 47% of the catheters. the fibrin sleeve in itself is benign, but promotes infection and may lead to thrombus formation. mural thrombosis can lead to subtotal stenosis or occlusion of vessel lumen leading to clinical manifestation of thrombosis and its complication. uncomplicated or noninfected cases present with pain and swelling in the neck and a cord can be palpated beneath the sternocleidomastoid muscle. described the following clinical manifestation in a large series of patients with septic ijv thrombosis: fever (83%), leucocytosis (78%), cervical pain (66%), neck swelling (72%), cord sign (39%), sepsis syndrome (39%), pleuropulmonary complications (28%), superior vena cava syndrome (11%), chylothorax (5%) and jugular foramen syndrome (6%). they are thrombogenecity of the catheter material, circumferential size of the catheter, catheter tip position, side of insertion, puncture site of insertion, multiple venipuncture attempts, composition of infusate, thrombophilic abnormalities, cvc-related infection and duration of catheter placement. borow and crowley studied the adhesion of chromium-51-labeled platelets to the different cvc materials and found that the least thrombogenic catheters were hydromer-coated polyurethane catheters. the incidence of cvc-related thrombosis is found to be higher in patients in whom the catheter tip is placed in the innominate vein or proximal superior vena cava as compared with the distal superior vena cava/right atrial junction. showed a 2.6-fold higher risk when the catheter was located in the superior vena cava compared with the right atrium. other important risk factors are side of insertion of the catheter and puncture site of cvc. tesselaar et al. also showed that the placement of cvc on the left side was associated with a 3.5-times higher risk of thrombosis as compared with the right side. in children, the incidence of catheter-related thrombosis was 44% in subclavian vein cvcs compared with 20% in jugular vein cvcs. this difference in the relative risk can be possibly explained by the anatomy of the venous system in the upper torso. as compared with the right side, the left brachiocephalic vein is longer and has a more horizontal course, leading to a sharper angle to the superior vena cava. further, compared with the jugular cvcs, the subclavian cvcs follow a sharper curve into the superior vena cava, facilitating wall adherence. the subclavian cvcs enter where the vein passes between the clavicle and the first rib, which may cause vein compression and kinking of the cvc. cvc-related infection is also an important risk factor that can lead to increased propensity for thrombus formation. van rooden et al. showed an increased incidence of cvc-associated thrombosis in patients with cvc-related infection as compared with those without infection in a population of patients with hematological malignancy. the major contributing factor for both of them is the formation of a fibrin sheath around the catheter. they also produce a coagulase enzyme that enhances the thrombogenic process. on the other hand other modalities used to diagnose upper limb dvt are computed tomography scan with contrast, magnetic resonance imaging and nuclear medicine scan. color doppler duplex sonography is an noninvasive, safe and convenient means of diagnosing upper limb dvt and cvc-related thrombus. a systematic review of studies reported a sensitivity of compression ultrasound ranging from 56 to 100% and a specificity of 94100% for the diagnosis of upper limb dvt. it still remains a matter of debate whether antithrombotic prophylaxis is effective in preventing cvc-related thrombosis. there have been studies using unfractionated heparin, minidose warfarin and low molecular weight heparin for antithrombotic prophylaxis but, due to lack of well-designed prospective studies, it still remains a matter of debate. treatment strategies consist of thrombolytic therapy, initiation of systemic anticoagulation, removal of the catheter or both. for cvc-related thrombosis, the preferred treatment is a combination of low molecular weight heparin followed by oral anticoagulant for 36 months, but no prospective randomized studies have been published on this subject. it appears to be more common in patients with hematological and other malignancies and in patients having hemodialysis cannula in situ. many risk factors have been identified in different studies that have a strong association with thrombus formation. in this study, those patients who had cvc in the right ijv were only included. males had a significantly higher incidence of small-sized thrombus. in 63.64% patients, the low molecular weight heparin used for dvt prophylaxis was not effective in preventing the cvc-related thrombus. the risk of thrombosis was not increased with the use of hypertonic solutions or with the presence of factors like diabetes mellitus, hypertension and history of smoking. there is no consensus regarding management of asymptomatic cvc-related thrombus, and more prospective randomized studies are required. till then, knowledge of the different risk factors for the thrombus, its prevention and effective treatment of cvc-related infection remains the mainstay to avoid cvc-related thrombosis and its dreaded complications.

background and aims: central venous catheters (cvc) are essential in a critical care setting. thrombosis is one of the very important associated complications that can lead to increased morbidity and mortality. the aim of this study was to find out the incidence of thrombosis in right-sided internal jugular vein (ijv) cvc with the help of color doppler duplex sonography, its extent, risk factors and clinical impact. materials and methods: one hundred consecutive patients having right-sided ijv cvc were included in the study. color doppler sonography was performed on the 3rd and 6th days after cvc insertion. the size of the thrombus was noted. presence of diabetes mellitus, hypertension or smoking was noted. presence of any hypertonic solution and thromboprophylaxis for deep vein thrombosis (dvt) were also noted. results:thrombus was detected in 33 of 100 (33.0%) patients. the incidence in males was 32.86% and in females was 33.33%. males had a significantly higher incidence of small thrombus (p=0.05), whereas females had a significantly higher incidence of large thrombus (p=0.05). dvt thromboprophylaxis was not effective for cvc-related thrombosis. hypertonic solution, presence of diabetes, hypertension or history of smoking did not increase the risk of thrombosis. conclusion:cvc-related thrombosis is common and has the potential for serious complications. females appear to be at a higher risk for larger thrombus formation. dvt thromboprophylaxis does not confer protection for cvc-related thrombosis. color doppler duplex sonography provides with an easily available, noninvasive means of detecting a thrombus. more studies are needed to establish a consensus for prophylaxis and treatment of asymptomatic cvc-related thrombosis.

PMC3338233

pubmed-664

in united states, about 595,800 establishments make up the healthcare industry. according to the american hospital association, in 2009 about 21 percent of hospital jobs are in service occupations, such as nursing, psychiatric, and home health aides, or cleaners. the international labour organization's (ilo) key indicators of the labor market suggest that wages for work such as performed by cleaners is low in comparison to most other occupations. more specifically, employment statistics on jobs in the us show that the mean annual wage estimates of housekeepers are approximately one-half of the mean annual wage estimates for all occupations. in 2008, the total number of nonfatal occupational injuries in hospitals (private industry, state government, and local government) was 323,200. the rate of occupational injuries and illnesses among cleaners was 3.9 per 100 full-time workers. such injuries and illnesses affect the job performance of the cleaners thus affecting their efficiency. due to the reduced efficiency and absenteeism, the employers have to incur losses and the treatment and rehabilitation of these employees are costly to society. many of the cleaners develop conditions like arthritis, tendonitis, carpel tunnel syndrome, and other musculoskeletal conditions. the tasks performed by cleaners are labor intensive and most of the cleaners have to work under time constraints increasing the physical and mental stress. a study carried out in four countries of the european union showed that the prevalence of health problems like musculoskeletal problems, skin problems, and psychosomatic disorders among cleaners is high compared to the average wage earners in general. occupational physical risk factors responsible for musculoskeletal morbidity include repetitive work, working with hands above shoulder height or below knee height, carrying heavy loads, and operating vibrating tools [8, 9]. all of the above-mentioned risk factors are prevalent in cleaners and may therefore contribute toward musculoskeletal morbidity in the cleaning occupation. the bureau of labor statistics provides the number of nonfatal occupational injuries and illnesses for cleaners; however, there is no record of these among cleaners specific to hospitals. it is apparent from the review of literature that little work has been done on the cleaning workers specific to hospitals. a large number of studies have investigated the association of musculoskeletal disorders and physical workload [1012]. most studies have suggested the evidence of physical work factors as determinants of musculoskeletal disorders of the upper extremity and the low back [10, 13, 14]. association between physically demanding work tasks and disorders of the lower extremity is evident in some studies [1517]. therefore, the specific objectives of this study were to (a) determine the rates of nonfatal occupational injury and illness in hospital cleaners in one hospital in texas and (b) investigate the prevalence of musculoskeletal symptoms in this population in one texas hospital. the focus of this study is on injuries related to slip/trip/fall, material handling, and work-related musculoskeletal aches, pains and discomfort. this study explores information about incidence rates among hospital cleaners categorized by various factors such as type of injury and source of injury. the study sample comprised of 106 of 108 housekeeping employees of a hospital located in texas, united states giving a response rate of 98.14%. the mean age of the sample was 46.36 years (sd 13.89 years), and median age was 48.5 years. the cleaners worked for an average of 8 h per day in one of three shifts. 51% worked during the first shift (7 am to 3 pm), 35% worked during the second shift (3 pm to 11 pm), and 14% worked during the third shift (11 pm to 7 am). the number of years they worked in cleaning jobs ranged from 1 year to 40 years with a mean of 14.2 years (sd 9.84 years). the cleaners perform repetitive cleaning of assigned patient rooms, dismissals, units, x-ray rooms, surgical unit, recovery rooms, all offices, and other areas using standard cleaning supplies and disinfectants. as part of their job profile, they spend 40% of their time cleaning patient rooms, clinics, and offices, 25% of their time emptying trash and linen, and 10% of their time vacuuming and shampooing carpets. they spend 5% of their time on mopping floors, cleaning furniture, refinishing floors, moving furniture, and responding to hospital emergency drills, respectively. the cleaners carry supplies of linens, towels, toilet items, and cleaning materials, using carts and keep storage areas clean and tidy and carts well stocked. cleaning patient rooms, clinics, and offices involves pushing/pulling beds, turning the mattress, moving furniture, dusting furniture, sweeping and mopping the floor. moving trash involves lifting trash bags and dumping them into a cart, walking long distances pushing the cart to the dump station, and emptying the trash into the electric dumpster. cleaning bathrooms involves bending over and stooping to clean toilets and tubs, washing sinks, mopping floors, refilling soap, and loading toilet paper. water from the mop is squeezed by wringing the washcloths using a wringer on the mop bucket. washing windows, walls, ceilings, woodwork, and waxing and polishing as necessary is also done. cleaning rugs, carpets, upholstered furniture, and/or draperies, using vacuum cleaners and/or shampooers are common housekeeping tasks. the cleaners are also required to prepare rooms for meetings, arrange media equipment, and furniture for social or business functions in auditoria of the hospital. the linen room has three employees who sort, count, fold, and store linen in closets; they also move linen carts to various units of the hospital., employee injury and illness reports were obtained from the occupational health services of the hospital. the report consisted of information about 117 incidents among cleaners from 2004 to 2008. the second part consisted of a cross-sectional survey using an interview-based modified nordic musculoskeletal questionnaire to study the extent of musculoskeletal symptoms of different body parts among the housekeeping employees. responses to the questionnaire were noted. the questionnaire comprised of four sections: demographic information, information on pain and discomfort, types of work tasks, and work organization. the demographic information included age, gender, height, body weight, number of hours worked each day, areas of the hospital assigned to clean, number of years working in the hospital, and total number of years in the cleaning work. subjective complaints of pain and discomfort from neck, shoulders, elbows, wrist/hands, upper back, lower back, hips/thighs/buttocks, knees, and ankles/feet during the last 12 months were obtained. the employees who reported pain or discomfort were asked to identify the painful body parts on a body map showing the body regions and the level of pain on a scale of 0 to 10 ranging from no pain at all to worst imaginable pain. the level of pain was categorized as mild (score: 1 to 3), moderate (score: 4 to 7), and severe (score: 8 to 10). the third section included questions on buffing, vacuuming, lifting equipment, mopping floors, carrying garbage and linen, cleaning bathrooms, making beds, moving furniture, carrying heavy loads, and working with the back, neck, or arms in awkward postures. in the study, the employees were asked about the frequency of their work tasks. the answers were categorized into three classes (1=frequently, 2=sometimes, 3=never). the work organization section asked participants if they had to work fast, work intensively and if they had enough time to do their work. participants were grouped as per gender, age, number of years in cleaning occupation, race, body mass index, and shift work. the prevalence of musculoskeletal aches, pains and discomfort among cleaners in the past 12 months in association to the above-mentioned demographic variables and selected body parts was calculated. incident rates among the housekeeping staff and the rest of the hospital staff were extracted. the incidence rates in this sample represent the number of injuries per 100 full-time employees (fte). the incidence rates were calculated as (n/h) 200,000 where n=number of injuries and illness, h=total hours worked by all employees during period covered, and 200,000=represents the equivalent of 100 employees working 40 h/week, 50 weeks/year. the incidence rates for all injuries, for injuries due to slip/trip/fall, and injuries due to material handling among cleaners and rest of the hospital staff were compared. the 117 incidents among cleaners were categorized as strains, contusion/abrasion, laceration/scratch, puncture wound, chemical exposure, others and the number of cases in each category was calculated. the frequency of execution of different tasks performed by housekeepers was extracted thus calculating the percentage of housekeepers performing tasks frequently, sometimes, and never. background demographic characteristics and the prevalence of musculoskeletal pain for the study population are shown in table 1. female cleaners had a higher prevalence of musculoskeletal symptoms in the last 12 months than male cleaners. cleaners in age group from 39 to 58 years, housekeepers working in housekeeping jobs for over 31 years, housekeepers with body mass index (bmi) less than 18.5, and those working in the first shift had the highest prevalence of musculoskeletal pain in the last 12 months. this being a hospital setting, the workload for cleaners during the first shift is a lot more as compared to other shifts. this could be attributed to the influx of patient admissions during the first shift and the cleaning of surgical units and other units where the workload is highest in the morning hours. the incidence rate for total occupational injuries in cleaners for each year from 2004 to 2008 was significantly higher than that for all other employees of the hospital with the highest incidence rate during 2005 (75.18 per 100 fte) (table 2). the drop in the incidence rates in the subsequent years can be attributed to preventive measures that were taken by the occupational health services of the hospital to address the high incidence rate. mandatory slip resistant footwear, lowering of threshold plates for those areas where carpet to tile transitions (to reduce pushing/pulling injuries), ergonomic mops, soiled laundry receptacles that emptied from the bottom, department-specific education about ergonomics, and enforcement of use of appropriate personal protective equipment were some of the measures taken to reduce the injuries and illnesses among cleaners. nursing services were also included in training so that laundry hampers were not overfilled, which left an msd hazard for the cleaners. the five-year incidence rate of total injuries for the housekeeping employees was found to be 28.32 per 100 fte and that for rest of the employees of the hospital was 13.54 per 100 fte (p<.05). the incidence rate of slips/trips/falls among the housekeeping employees and rest of the employees of the hospital was 4.39 per 100 fte and 2.37 per 100 fte, respectively (p<.05). the incidence rate of injury due to material handling in the housekeeping employees and rest of the employees of the hospital was 5.45 per 100 fte and 1.08 per 100 fte, respectively (p<.05) (table 2). the most common type of injury among housekeepers was due to strains followed by contusion/abrasion (table 3). the most common cause of injury was being struck by an object (table 3). symptoms in the lower back were most prevalent (49%) followed by the right wrist (43%), ankle, left knee, left wrist (35% each), right knee (34%), right shoulder (25%), and other selected body parts (table 4). similarly, current pain and discomfort was reported by 42% of the cleaners. in this sample, 82% of those who complained of pain in the last 12 months reported that the pain was related to work. 4% of the participants complained of mild pain in the wrists, whereas 16% had moderate pain and 11% had severe pain in the wrists. the frequency of execution of different tasks by housekeepers was expressed as percent (table 5). the prevalence of musculoskeletal pain was highest in participants who worked with the back in the awkward posture (65%), followed by employees who worked with their arms and neck in the awkward posture, respectively (64%). the employees who cleaned bathrooms had a prevalence of 64%, followed by those who mopped floor and carried/emptied garbage (63%). the prevalence of musculoskeletal pain in the participants who carried heavy loads, made beds, moved furniture, and used the vacuum ranged between 61% and 63% (table 6). in order to determine the number and incidence rate of nonfatal occupational injuries and illnesses among hospital housekeepers, the authors used the injury data from the housekeeping employees of a hospital to extrapolate possible number of injuries in this occupation in healthcare industry. as per the 2008 statistics, in the united states, cleaners held about 1.5 million jobs. hospitals employed about 17 percent of these workers, thus making it approximately 255 thousand cleaners in hospitals. in 2008, there were 22 injuries among 108 housekeeping employees studied. considering approximately 255,000 cleaners in the hospitals, in 2008, cleaners would have over 52,000 injuries. since musculoskeletal disorders (msds) affect a significant proportion of the workforce, they are a major problem in several economic activity sectors in industrialized countries this includes an estimated $155 billion for occupational injuries. in this study, for 108 housekeeping employees the cost incurred by the hospital in 2008 for occupational injuries and illnesses was $127,955, thus extrapolation of this rate makes it approximately over $302 million for claim costs for all the hospital cleaners in us in 2008. the true economic burden of work-related musculoskeletal disorders is likely to be even greater because many cases are not reported to the workers ' compensation system. a study of individuals diagnosed with work-related musculoskeletal disorders of the upper extremity, neck, or low back reported that only 25% filed a workers ' compensation claim. a population survey in connecticut, estimated that one-tenth of working-age adults have a work-related musculoskeletal condition, but only 10% of these workers file compensation claims. the cleaners in this study had higher rates of injury due to slip/trip/fall and material handling. slips and falls were reported as the leading cause of death in the workplace and source of more than 20% of all disabling injuries. occupational injuries associated with slip and fall accidents pose a significant problem to industry both in terms of human suffering and economic losses. in terms of the labor costs, over one-quarter of overall fall-related injuries resulted in 31 days or more workdays being lost, costing us economies nearly $10 billion/year. in the self-reported work-related illness in 1995 survey, musculoskeletal disorders were by far the most commonly reported class of work-related illness, and it was estimated that 52% of the 1.2 million reported a work-related musculoskeletal disorder due to manual handling associated with their job. manual handling is not only one of the frequent workplace activities, but is also one of the most common causes of workplace injury. the number of sprains and strains for all private industries put together (416,620 in 2008) was nearly 4 times the number of bruises and contusions (93,650 in 2008), the second highest type of injury of interest to ergonomists. injuries to the back were the highest category of injuries in the service occupations (about 31%). these findings are similar to those noted in this study. in terms of their contribution to total workers ' compensation costs, at the workplace, back problems are the single most costly injury. in 1992, back cases represented 24% of us workers ' compensation claims and 31% of costs. after low back pain, the next common body part affected in this sample was the wrist. in a similar study it was found that wrist/hand, elbow and knee pain were more prevalent among cleaners; neck, shoulder, low back, hip, and ankle pain/discomfort was similar to other referent groups, while upper back pain was reported less by cleaners. an occupational injury profile of the american industry states that upper extremity injuries, including injuries to the wrists, the hands, and the fingers, were the next highest category of injuries after back injuries. this correlates to the presence of pain in the back and wrist in descending order in the participants of this study. the prevalence of musculoskeletal pain and the trend in incidence rates in this sample were similar to the prevalence in other studies [24, 30, 31]. this can be explained by the high prevalence of pain among participants who worked with their back in an awkward posture. as it is seen in other studies, it is evident from this study too that the cleaning occupation has a high prevalence of musculoskeletal pain which can predispose the cleaners to musculoskeletal disorders and injuries. several studies have indicated the various risk factors for musculoskeletal ill-health, high workload, repetitive motion, speed and intensity of work, and weak organizational strategies to name a few. there is evidence that exposure to each of these ergonomic factors causes msds in one or more body regions: repetitive upper extremity motion patterns; forceful exertions, nonneutral body postures; and vibration. the risk is especially pronounced when a job includes exposure to a combination of two or more of these risk factors. all authors found that the most significant risk factors associated with the physical work of cleaners are static muscle loads (much of which involves bending and twisting of the back) and repetitive movements of the arms and hands using a high output of force. these types of prolonged static and repetitive muscle activities cause muscle fatigue and may lead to musculoskeletal disorders. a sizable proportion of msds among exposed workers are preventable, and protective action is both warranted and necessary. concentrating only on physical ergonomic factors like equipment design, reduction in forces, and improvement in postures may not achieve as much benefits in terms of reduction in sickness rates as a more holistic approach would, which also takes account of work organizational risk factors. a holistic approach is essential to address these issues and reduce the incidence rates of occupational injuries and illnesses. health and safety researchers and practitioners are constantly trying to control workplace injuries by trying to understand the causal mechanisms underlying workplace injuries and by designing safer working conditions through job, equipment and workplace design, educating employees, and matching employees to jobs. a review by bigos et al. on preventing episodes of low back pain suggests a strong evidence that exercise programs are effective, whereas other interventions including education alone (ergonomic, back school, and stress management), back supports (back belts), and shoe inserts were not effective daily sessions of exercise and relaxation techniques before the employee starts work will help reduce the tension in the muscles thus preparing the body for various work related postures. a study by toivanen et al. showed that regular relaxation training at the workplace to provide stress management diminished tension in the neck-shoulder region efficiently and decreased absenteeism. inter correlations were found between the neck-shoulder tension, psychosocial factors, depression, and the absentee rate. thus a continued application of a combination of regular exercise, relaxation training, participatory ergonomic training programs, team work, work organization changes, and effective communication between the cleaners and the supervisors may help in curbing musculoskeletal ill-health among the housekeepers. first, the findings may not be generalizable as they are not based on representative random sample drawn from hospital cleaners around the country. however, a response rate of 98.1% clearly indicates the validity of findings at that institution. another limitation is that our study assessed work-relatedness of pain or injury (and the severity of these conditions) by self-report. data were not available to validate doctor visits for work-related pain or workers ' compensation claim reporting and acceptance rates. however, the self-report data was supported by medical or administrative records making it more reliable, and also self-reports can be a more reliable source for determining the frequency of work-related pain. the questions were answered based on the memory of the participant thus the possibility of recall bias exists. recall may have been influenced by such factors as presence of pain or negative affect. further research is required to study the risk factors associated with musculoskeletal pain and discomfort in each body part to develop better intervention strategies .

objectives. to determine the prevalence of musculoskeletal disorders in hospital cleaners. methods. injury data on all hospital employees were extracted from occupational health records and compared. additionally an interview-based modified nordic questionnaire (response rate 98.14%) was conducted. results. the mean total injury rate for cleaners was 35.9 per 100 full-time equivalent (fte), while that for other employees was 13.64 per 100 fte. slips/trips/falls and mmh contributed 4.39 and 2.37 per 100 fte among cleaners and rest of the hospital employees, respectively. the most common type of injury was strain while the most common cause of injury was a striking object. conclusion. the cleaners have higher injury rates and morbidity as compared to other employees of the hospital. the lower back was most commonly affected.

PMC3136138

pubmed-665

lead poisoning is one of the most serious environmental health hazards, with a particularly acute effect on young children. the u.s. centers for disease control and prevention estimated that approximately 2.5% of children aged 15 years in the u.s. have elevated blood lead levels. the percentage of affected children in other countries is expected to be even higher. lead poisoning causes learning disabilities, behavioral problems, and, at very high levels, seizures, coma, and even death. the mechanism of pb toxification mainly involves binding of pb to proteins and the subsequent inhibition of the proteins physiological functions in blood and tissues. the documented proteins targeted by pb include several zinc enzymes or proteins (such as -aminolevulinic acid dehydratase (alad), acetylcoline esterase, cys2his2 zinc-finger proteins, and acid phophatases) and calcium-binding proteins (calmodulin, calbindin, and troponin c). pb can replace zinc and calcium at the oxygen/nitrogen/sulfur-rich active sites of these proteins, thereby inhibiting the protein functions by altering their coordination chemistry and native structures. for example, the function of alad, an enzyme involved in the second step of heme biosynthesis, is altered by pb binding at the active site via a trigonal pyramidal geometry. consequently, the hemoglobin synthesis is blocked, leading to anemia. to reduce the pb-induced toxicity, organisms have developed various defensive mechanisms with species such as metallothioneins (mts), glutathione, phytochelatins, and lead-binding proteins (pbbps, which are non-mt acidic proteins that have not been fully characterized). mts, a class of thiol-rich (up to 30% of its amino acid residues), low-molecular-weight proteins whose abundance is particularly high in the liver and kidneys of mammals, are perhaps the most important species for lead detoxification. mts is crucial to a better understanding of the chemical stabilities, biological functions, and detoxification mechanism of pb mts. however, due to the absence of single crystals of pb mts, information about the pb the coordination and binding stoichiometry between mts and metals are dependent on the type of metal ions. usually, divalent metal ions, such as cd and zn, are tetrahedrally coordinated by four cysteine sulfurs and bind to mts with a stoichiometry of 7:1. it can be complexed by a combination of s, o, n, and p-donor ligands with a coordination number ranging from 2 to 9. when pb binds to sulfur-rich proteins, three sulfurs in a trigonal pyramidal geometry and the pb 6s lone-pair electrons occupying the axial position (hemidirected) constitute the coordination sphere. a number of complexes containing the pb s3 coordination have been observed using spectroscopic and mass spectrometry (ms) methods. in the presence of biomolecules possessing several distinct donor ligands (n, o, and s), the pb coordination chemistry is diverse and includes formation of pbsxoy and pbsxny. owing to the presence of o- and s-donor ligands in mts and the unique electronic configuration of pb, coordination of pb with mt is of higher complexity than that of zn or cd. visible (uv vis) spectra and microcalorimetry have suggested that different pb palacios et al. demonstrated with electrospray ionization mass spectrometry that the metal content in pb mt2 complexes is dependent on the solution ph (neutral or 4.5). using extended x-ray absorption fine structure (exafs), vasak et al. however, crucial questions such as the difference of pb coordination and the chemical stability and physiological function of the two pb theoretical calculations have been widely used for the prediction of structures and spectra of metalloproteins. the combination of quantum mechanics and molecular mechanics-based hybrid (qm/mm) method allows two or more computational methods to be performed in a single calculation, making it possible to investigate the chemistry of complex systems with high precision. in this context, the oniom (our own n-layered integrated molecular orbital+molecular mechanics) scheme is a general approach because it can combine any number of molecular orbital and molecular mechanics methods. the oniom method has been successfully utilized in the elucidations of structural and functional properties of many metalloproteins such as cyt c and azurin. in our study, optical methods (uv vis absorption and circular dichroism (cd) spectrometry) and nmr were used in tandem with the two-layer oniom method to investigate the effect of ph on the structures of two different pb the differences in the coordination chemistry of the metal centers and the protein structures at various ph were deciphered. we also investigated the chemical stabilities and structural flexibility of these two complexes in proteolytic processing to gain insight into the lead detoxification process involving mts. zn-containing mt2, isolated from rabbit liver, was purchased from hunan lugu biotechnology co. (changsha, china). individual metal-free domains (apo-mt2 and apo-mt2) of both rabbit liver and human mt2s, and the corresponding mutants (d25n-apo-mt2 and d2n-apo-mt2) were synthesized by shanghai apeptide co. (shanghai, china). all the domains and their mutations were confirmed by mass spectrometry performed by the vendor, and the corresponding purity values (> 95%) were determined with hplc-ms. pb (99.1%) was acquired from isoflex usa (san francisco, ca). lead nitrate, 5,5-dithio-bis-(2-nitrobenzoate) (dtnb), and cathepsin b were purchased from sigma-aldrich (st. deionized water with resistivity of 18.2 m cm was collected from a millipore simplicity 185 system (millipore co., billerica, ma). all solutions were prepared with deionized water and degassed with nitrogen for at least 30 min. a 3 kda cutoff millipore (ym-3) membrane (millipore, billerica, ma), equilibrated with 0.01 mol l hcl, was used to separate zn from zn7mt2. after spinning at 13 000 rpm for 30 min at room temperature in an eppendorf 5417r centrifuge (eppendorf, hamburg, germany) complete removal of zn from zn7mt2 was confirmed by the disappearance of the characteristic absorption of zn7mt2 at 220 nm. the apo-mt2 concentration was determined by assaying thiol groups with ellman s reagent, dtnb. mt2 solution was stored in a nitrogen-saturated flask to avoid thiol oxidation in apo vis absorption experiments were carried out on a uv-2450 spectrophotometer (shimadzu, japan) in quartz cuvettes (1 cm path lengths). cd data were obtained with a jasco-810 spectrophotometer (jasco corporation, japan). zeta potentials of individual apo-mt2 domains or their mutants were measured at room temperature in a folded capillary cell with a zetasizer nano zs instrument (malvern instruments, southborough, uk). pb was dissolved with 0.15 m trace metal grade nitric acid (fisher scientific) at 250 c, and the pb(no3)2 precipitate was collected, dried, and weighed. appropriate amounts of mt2 (250 m), kcl (10 mm), and d2o were mixed under n2 atmosphere. both pb7mt2 complexes were prepared by adding pb(no3)2 to obtain an mt2/pb stoichiometry of 1:7 at corresponding ph (7.0 or 4.0). the resultant solutions were allowed to incubate for 1 h, and the solution ph was brought up to ph 7.0 with koh. d2o was added to a final volume of 1 ml, and the solution was transferred to an nmr tube. all pb nmr spectra were recorded on a bruker advance drx-400 mhz spectrometer at 25 c using 60 pulses, a 2 s relaxation delay, and a 0.12 s acquisition time (spectral width of 555.6 khz). a linear prediction was performed to remove the noise, and the real free induction decay (fid) was determined before data processing. after zero-filling, the data (128 000 data points) were processed with an exponential line broadening of 5 hz using the software topspin nmr. the pb nmr chemical shifts are reported downfield from tetramethyllead (= 0 ppm; toluene) using 1.0 m pb(no3)2 salt (fisher) as an external standard (= 2990 ppm, d2o, 25 c; relative to tetramethyllead). both pb7mt2(i) and pb7mt2(ii) complexes were freshly prepared and diluted in 10 mm kcl solution (ph 7.0) at 37 c to desired concentrations. cathepsin b (1.8 ng), with a specific activity of 3000 pmol min g, was mixed with 504 pmol of pb7mt2(i) or pb7mt2(ii) (the final concentration of pb was 57 m) in 10 mm kcl solution (ph 5.0). oniom calculations were performed to predict the optimal geometries and electronic absorption spectra of pb7mt2 complexes. the initial atomic coordinates of pb4mt2 and pb3mt2 were taken from the corresponding pb substituted cd4mt2 and cd3mt2, respectively. the structures of cd4mt2 and cd3mt2 were retrieved from the rcsb protein databank (pdb i d: 1 mrb for the -domain and 2 mrb for the -domain), in which the absent hydrogen atoms were added using gaussview 4.0. the protonation states of titratable residues (e.g., aspartic acid and lysine) of pb4mt2 and pb3mt2 at ph 4.0 and 7.0 were determined using pka values estimated with propka 2.0. the electronic structures of pb4mt2 and pb3mt2 were modeled with the inclusion of the protein environment, using the two-layer oniom (qm/mm) model. the qm region comprises the active sites of pb4mt2 and pb3mt2, and the mm region contains the protein environment (cf. spin unrestricted density functional theory (dft) with the becke s three-parameter hybrid exchange functional and the lee yang parr correlation functional (b3lyp) or pure functional bp86 were used for the qm system, and the dunning basis set aug-cc-pvtz-pp was used for pb and 6-31+g*basis set for other atoms for the qm calculations. for the mm region, the protein molecule was treated using the universal force field (uff). an electronic embedding scheme was adopted to deal with the electrostatic interactions between the qm and mm regions in the qm/mm calculations. on the basis of the optimized geometries, vertical excitation energies were computed within the oniom scheme by employing the time-dependent density functional theory (tddft). tddft calculations were carried out with the density functional b3lyp and bp86. the trizeta basis set aug-cc-pvtz-pp was used for pb, and 6-31+g** was used for others. the results were transformed via the swizard program (version 4.6) into each uv spectrum using gaussian functions with half-widths of 3000 cm. uv vis absorption and cd spectra were recorded during the titration of rabbit liver apo-mt2 with pb at ph 7.0 (figure 1). for simplicity, we termed the pb mt2(i) exhibits a characteristic peak centered at 330 nm (figure 1a). the time-resolved absorbance changes (inset) indicates that the complexation reaction is fast (completed in less than 10 min). during the titration of apo mt2 with pb at neutral ph, some pb(oh)2 (ksp=1.4 10) precipitate was formed and affected the absorbance value of pb7mt(i) at 330 nm, as evidenced by the small fluctuation even after 30 min. the precipitation can be avoided by lowering the solution ph below 6.0. as shown in the inset of figure s1 in the supporting information, the absorbance value of pb7mt(i) remains stable after the complexation reaction is completed. as indicated by the mt2/pb stoichiometry (figure 1b), the maximum binding stoichiometry is 1:7, consistent with results reported from the substitution experiment of zn7mt2 with pb. the absorption wavelength (330 nm) and extinction coefficients (3500 m cm) of pb7mt2(i) are analogous to values reported for several pbs3 complexes. the secondary structural variation from apo-mt2 to pb7mt2(i) is shown in figure 1c. this is in contrast with the intense cd peaks of cd7mt2 and zn7mt2 between 210 and 290 nm. in zn7mt2, the characteristic cd band centering at 244 nm is attributed to the zn(sr)4 chromophore. similarly, the cd bands of cd7mt2 at 242 and 262 nm can be attributed to the excitation coupling between adjacent pairs of the cd(sr)4 chromophore. since the m(sr)4 chromophore leads to the appearance of these cd peaks, the absence of any obvious cd peaks in figure 1c suggests that pb7mt2(i) adopts a different metal coordination geometry from those of cd7mt2 and zn7mt2, as alluded to in the introduction. the individual-and -domains display uv vis and cd spectral features (data not shown) similar to those of apo-mt2 during the titration with pb, indicating that the metal centers in the two different domains have similar coordination spheres. quantitative analysis confirms that the pb/mt2 stoichiometric ratios are 4:1 and 3:1 in the-and -domain, respectively. time-dependent (a) uv vis absorption and (c) cd spectra in 10 mm kcl solution (ph 7.0) containing 7.20 m rabbit liver apo-mt2 and 20 mol equiv of pb. (b) dependence of uv absorption peak at 330 nm upon addition of pb to apo-mt2. the pb7mt2(ii) complex displays dramatically different uv vis absorption and cd spectra (figure 2) from those of pb7mt2(i). in the uv vis absorption spectra, two intense peaks at 325 and 375 nm appear, with the latter having a shoulder peak at 400 nm. in the cd spectra (figure 2c), a strong envelope with maxima at 240 (+), 265 (+), 320 (), 350 (+), 370 (+), and 395 () is produced isodichroically (280, 340, and 375 nm). positions of all cd peaks are invariant with the pb/apo mt2 ratio (increased stepwise from 1:1 to 7:1), but the peak intensity increases with the ratio. the cd spectra of cd7mt2 have two bands at 240 and 260 nm split from the 250 nm band, which corresponds to the conversion of isolated cd(sr)4 to the (sr)3cd sr thus, the unsplittable pb7mt2(ii) cd peak is indicative of the absence of excitation coupling between adjacent chromophores. the multiple peaks in the uv vis absorption and cd spectra suggest that pb in the pb7mt2(ii) complex has binding modes that are distinctively different from cd in cd7mt2. similar to pb7mt2(i), the pb/mt2 stoichiometric ratios in the pb7mt2(ii) complex are 4:1 and 3:1 in the-and -domains, respectively. the uv vis absorption peaks of pb4mt2(ii) are slightly shifted, with higher intensity than those of pb3mt2(ii) (figure 3d). these differences suggest that the metal centers in the two domains have different coordination geometries. to pinpoint the ligands responsible for coordination of pb in the two different domains at neutral and acidic ph time-dependent (a) uv vis absorption and (c) cd spectra in 10 mm kcl solution (ph 4.5) containing apo-mt2 (7.20 m) and 20 mol equiv of pb. (inset) the time-resolved absorbance changes at 325 and 375 nm, respectively. (b) the dependence of uv absorption peaks at 325 and 375 nm on the addition of pb to apo-mt2. structures and electronic absorption spectra of the-and -domains in (a, c) pb7mt2(i) and (b, d) pb7mt2(ii) obtained by experimental and oniom methods. in panels a and b, the dark gray spheres are pb, yellow spheres are s, red spheres are o, gray spheres are c, and white spheres are h atoms. the oniom method has been successfully used to predict metalloprotein structures. to validate the method for the studies of mt2 structures, structural optimization of cd4mt2 and cd3mt2 models compared to the nmr results, the computed structures of the metal clusters display little deviation (e.g., the bond lengths have a root-mean-square deviation of only 0.040.05 when compared to the experimental data) (figure s2 and table s1 in supporting information). therefore, we conclude that the oniom method is viable for the studies of the two pb7-mt2 complexes. the theoretical calculations were performed separately on the-and -domains on the basis that the two domains are structurally independent. the initial atomic coordinates of each domain in pb7mt2 were respectively adopted from the pb-substituted cd4mt2 and cd3mt2 because a well-established model of apo-mt2 is not available. the protonation states of titratable residues (e.g., asp and lys) at different ph were determined from the pka values estimated with propka 2.0. in the two-layer oniom model of pb7mt2(i), the qm region consists of four pb atoms, and the side chains consist of eleven cysteine residues in the -domain. as for the -domain, the three pb atoms and the side chains of nine cysteine residues constitute the qm region. figure 3a displays the optimized molecular geometries of the-and -domains in pb7mt2(i), which are quite different from the well-characterized cd7mt2 and zn7mt2 structures. in cd7mt2 and zn7mt2, each metal adopts the tetrahedral coordination with terminal and bridging thiolates to form a metal ligand six-membered ring in the -domain and two fused six-membered rings in the -domain.(cf. figure s2 in supporting information) however, all seven pb ions in pb7mt2(i) are trigonally coordinated by three cysteine sulfurs (pb s3) without any metal these significant differences can be attributed to the pb 6s lone-pair electrons, which disrupt the tetrahedral coordination by occupying the axial position with a significant stereochemical activity. this point is in line with the report by godwin and co-workers, who stated that pb s4 is not a preferred coordination and that trigonal pyramidal geometry in all-sulfur coordination is predominant. moreover, due to the pb s3 coordination, only one bridging cysteine (pb6s18pb7 in the -domain) remains, preventing a pb according to the corresponding structural parameters (table 1), the average length of the pb s bonds is 2.68, close to the reported exafs value (2.65). the electronic absorption spectra (red line curves), simulated by tddft using the b3lyp functional, are overlaid with the experimental results (black line curves) in figure 3c. the agreement between the simulation and experimental data in the oscillator strengths indicates that the optimized molecular geometries of pb4mt2(i) and pb3mt2(i) are reasonable. detailed analysis of vertical excitation energies, oscillator strengths, and molecular orbital contributions (table s3 in supporting information) indicated that four transitions contribute to the absorption peak at 330 nm. these transitions are homolumo, homo-1lumo, homo-2lumo, and homo-1lumo+1. from the frontier molecular orbitals (figure s3 in supporting information), lumo and lumo+1 (the final state of the transitions) s bonds, while homo and homo-1 (the initial states of the transitions) are largely localized at the sulfur atoms. therefore, the electronic absorption band at 330 nm can be attributed to the spb ligand-to-metal charge transfer (lmct). in the two-layer oniom model of pb7mt2(ii), we initially assigned the qm region of each domain to be the same as that of pb7mt2(i). however, the optimized molecular geometries revealed two unexpected short pb/o distances (5.52 for pb1 with the carbonyl oxygen of asp2 in the -domain and 4.31 for pb6 with the carbonyl oxygen of asp56 in the -domain). these short distances should result from the electrostatic interaction between the protein surface (the mm region) and the negatively charged pb acidic ph conditions, the protein surface is neutral or positively charged, causing the mm and qm regions to move closer, thereby shortening the distance between the pb moreover, based on the pka values of asp (4.00), both asp residues are neutral in weakly acidic solution, which are more favorable than the negatively charged (deprontonated) form to the positioning of the negatively charged pb these pb/o distances are close to the sum of the van der waals radius of pb and o (3.54), indicating the pbo interaction must be taken into account. we therefore modified the initial qm region in each domain by including the respective asp residue. as shown in figure 3b, the optimized molecular geometry of pb7mt2(ii) shows an entirely different coordination sphere from that of pb7mt2(i). the overall coordination sphere includes the trigonal pyramidal pb s3 mode, the distorted trigonal pyramidal pb s2o1 mode in the -domain, and the distorted quadrilateral pyramidal pb s3o1 in the -domain. the pb 6s lone pair electrons occupy the axial position with a significant stereochemical activity, resulting in hemidirectionality in the pb ligand coordination. moreover, due to protonation of the peptide side chain, several cysteine sulfurs (especially in the -domain) are closer to the protein exterior and increase the effective radius of the metal center and the pb s bond length (cf. the loosened structure of the metal center renders a higher flexibility to pb7mt2(ii). the simulated oscillator strengths are in good agreement with the experimental data (figure 3d), indicating that the optimized molecular geometries of pb4mt2(ii) and pb3mt2(ii) are reasonable. the small deviation between the electronic absorption spectra of the two domains (cf. detailed vertical excitation energies and molecular orbital contributions (table s5 and figure s4 in supporting information) indicate that all three bands are primarily associated with the spb lmct. note that all of the above calculations are based on b3lyp, a hybrid functional to fit data primarily for main-group elements. because of the large exact exchange component (20%), b3lyp is known to favor loose electron densities and low transition energies. to confirm the reliability of b3lyp, the pure functional bp86, a generalized-gradient approximation (gga) class with zero exact exchange, is used as a control study on the geometry optimization and calculations of the excited states for both pb7mt2 complexes. the simulated structures of both pb7mt2 complexes from bp86 pure functional (table s69 in supporting information) are similar to those from b3lyp hybrid functional. however, the spectra from bp86 pure functional display a significant deviation from the experimental data (figure s5 in supporting information), indicating that the b3lyp hybrid functional is a better choice. the b3lyp hybrid functional produces better spectral accuracy, which can be attributed to its increased amount of hartree local minima in the qm optimization. in the qm/mm calculation of biomolecules, the vast size of the available configuration space may cause the qm optimization to stop at local minima. one way to circumvent this problem is to use a reliable structure (e.g., a structure deduced from nmr or x-ray crystallography) at the beginning of the qm optimization. have used the x-ray structure of azurin as the initial structure in the qm/mm calculation of metal-substituted azurins and obtained noticeable structural changes on the active sites when cu in azurin was substituted by metal ions such as co, ni, or zn. we adopted this approach by using the pb-substituted cd4mt2 and cd3mt2 structures deduced from the nmr experiments as the initial structures. the theoretical spectra agree well with the experimental results, suggesting a high level of reliability of the calculation and in the predicted structures. to provide more experimental evidence to our computational results about the two different pb7mt2 complexes, we conducted pb nmr in solutions of the two complexes (figure 4). pb7mt2(i) displays one pb peak at 5679 ppm, while pb7mt2(ii) exhibit two peaks at 5820 and 4348 ppm. thus it is clear that the pb coordination in these two complexes are different. furthermore, the pb signal of pb7mt2(i) at 5679 ppm is well within the chemical shift region (from 5600 to 5800 ppm) where pbs3 species with the trigonal pyramidal coordination are observed. thus we conclude that pbs3 is the binding mode in pb7mt2(ii). for pb7mt2(ii), the peak at 5820 ppm is also assigned to the pbs3 coordination, given its close vicinity to the 56005800 ppm region. compared to the pb7mt2(i) peak, the shift by 141 ppm can be attributed to the changes of the aforementioned pb s bond length and the s pb s bond angle in the pbs3 coordination. the lower intensity is indicative of the decreased number of pbs3 clusters in pb7mt2(ii). the conversion of metal centers in the pbs3 coordination to a different binding mode contributes to the appearance of the peak at 4348 ppm. it is well-known that the nmr signal of pb bound to o-containing ligands is shifted upfield with respect to that bound to s-containing ligands. pb nmr spectra of (red) pb7mt2(i), (blue) pb7mt2(ii), and (black) pb(no3)2 at ph 7.0. the carbonyl group of asp is the only o-donor ligand in mt2. to further verify the formation of the pb o bond in pb7mt2(ii), a titration was performed by adding pb into a peptide solution whose asp residue had been mutated with asn (i.e., d25n-apo-mt2 and d2n-apo-mt2). zeta potential and cd measurements did not show discernible changes in the surface charge and structure of both mutants (figure s6 and s7 in supporting information), confirming the viability of these mutants for coordination studies. at neutral ph, both uv vis absorption and cd spectra of the two mutants show features similar to those in the same spectra of pb4mt2(i) and pb3mt2(i), verifying that asp does not participate in the pb coordination in pb7mt2(i). at acidic ph (ph<5), the uv vis absorption spectra of both mutants display only a single peak at 330 nm, and the cd spectra did not reveal any changes. while these features are rather different from the corresponding spectra of pb4mt2(ii) and pb3mt2(ii), they are analogous to those of pb4mt2(i) and pb3mt2(i). we therefore conclude that asp is involved in the coordination of pb in pb7mt2(ii). the unique uv vis absorption spectra of pb7mt2(ii), with multiple peaks and the presence of a new pb nmr peak at 4348 ppm, are well-correlated with the mutational study. another piece of evidence for the formation of pb o bond is the transition from a single peak to three peaks in the time-dependent spectra of a mixture of pb/apo-mt2 at ph 4.55.0 as well as the appearance of multiple peaks in the cd spectra (cf. however, because of the negative impact of acidity on the formation of the pb s bond (cf. the proton-releasing process shown in reaction 1) and the rearrangement of the peptide chain, the pb therefore, under more acidic conditions (ph<4.5), the time-dependent peak transition disappears, and the spectra show features typical of pb7mt2(ii).1 time dependence of (a) uv vis absorbance and (b) cd spectra in a kcl solution after the addition of 7 mol equiv of pb to an apo-mt (7.20 m) solution at ph 5.0. reaction times from bottom to top: 0, 1, 3, 5, 7, 10, 15, 20, 25, 30, 45, 60, 75, 90, 105, 120, 135, 150, 175, and 180 min. to study the acid tolerance of both pb7mt2 complexes, a series of spectrophotometric titrations at different ph was performed. the corresponding complex was confirmed by the appearance of characteristic uv vis absorption peaks of pb7mt2(i) or pb7mt2(ii). the results show that pb7mt2(i) is formed above ph 5.0, whereas pb7mt2(ii) is produced at more acidic ph. pb7mt2(i) can be transformed to pb7mt2(ii) by adjusting the solution ph to 5.0 or lower. however, once pb7mt2(ii) is formed, it remains stable at neutral ph. our demetalation experiments (figure s8 in supporting information) indicate that ph 2.5 is sufficiently low for the complete removal of pb from pb7mt2(i), but stripping pb completely of pb7mt2(ii) requires a ph as low as 2.0. these results (summarized schematically in figure 6) indicate that pb7mt2(ii) has a higher tolerance toward an acidic environment than pb7mt2(i) has. moreover, pb7mt2(ii) remains stable even in the presence of apo-mt at neutral ph (data not shown). we believe that the greater acid tolerance and higher structural stability of pb7mt2(ii) results from the pb o bond. such a finding has a significant implication to the lead detoxification process in physiological milieu. the pb-inflicted toxicity stems from its tight binding to a variety of sulfur-rich proteins, such as gata proteins and the steroid receptor dna-binding domains. mt2 reduces the pb-inflicted toxicity by seizing free pb or sequestering pb from these proteins to recover the native protein function. higher acid tolerance and greater structural stability render mt2 a greater power in effectively scavenging pb in different environments. as shown in reaction 1, mt2 accompanies the release of h, which in turn increases the acidity in a highly localized region (e.g., in cytosol). moreover, it has been reported that elevated pb concentrations induce the stress level of various organisms, and acidity is also correlated with the stress level. we posit that pb7mt2(ii) is more effective than pb7mt2(i) in lead detoxification, given its greater structural stability and acid tolerance. elucidation of the metabolism of the pb-mt complexes is vital for understanding the lead detoxification by mts. some reports have suggested that exogenous mt is processed mainly by the lysosomal protease, and the rate of mt degradation is dependent on the types of metals bound by mts. four different cathepsins have been identified in lysosomes, and the cysteine protease (cathepsin b, l, and h) is the principal protease for mt degradation. variations in the concentrations of the two pb7mt2 complexes in the presence of cathepsin b were measured by uv vis absorption spectrometry. in figure 7, the pb7mt2 concentrations were normalized with respect to their initial concentrations. within 120 min, pb7mt2(ii) such a higher degradation rate can be attributed to the more flexible structure of the pb7mt2(ii) complex. cathepsin b is an endopeptidase that cleaves internal peptide bonds and favors a large hydrophobic side chain in the substrate protein. side chains on the amino acids dock into the cathepsin s subsites, whose interaction with the protein substrate is dependent on the flexibility of substrate protein. in mts, the existence of bridging cysteine sulfurs compacts the metal center, which dominates the protein folding. in pb7mt2(ii), due to the lack of bridging cysteine sulfurs, the metal center is loosened, which improves the flexibility of the protein. moreover, when the pb s3o1 become distorted. consequently, pb ions are positioned farther from the cysteine sulfurs, and the pb s bonds are weakened. both processes facilitate the conformational adjustment of pb7mt2(ii). the significantly improved flexibility of pb7mt2(ii) facilitates the protein in the induced-fit model with cathepsin b. as a result, the proteolytic processing of pb7mt2(ii) is greatly accelerated. in living organisms, the acidity in lysosome is about 5, which is sufficiently low to cause the structural conversion from pb7mt2(i) to pb7mt2(ii) and to accelerate the degradation of pb mt2 complexes. time-dependent proteolytic processing of (black) pb7mt2(i) and (red) pb7mt2(ii) by cathepsin b. the coordination of pb with individual human apo mt2 or apo mt3 domains at different ph was also studied by uv vis absorption and cd spectrometry. mutational studies revealed that the asp residue is also essential for the ph-dependent structural variation. in line with the data observed for the rabbit liver mt2, the structure-dependent chemical and biological activities of pb7hmt2(ii) formed between human mt2 and pb have higher acid tolerance, more coordination stability, and faster proteolytic processing than pb7hmt2(i) (figure s10 in supporting information). our results suggest that there exists a commonality in the pb coordination chemistry among mammalian mt2s. in this work, the ph-dependent coordination chemistry between pb and mt2 was systematically studied. the combination of spectroscopic studies and oniom calculations provided a detailed description of the two different pb7mt structures. the results and structures were further verified by pb nmr and mutational experiments. the similar structural, chemical, and biological properties between rabbit liver pb7mt2(ii) and human pb7mt2(ii) suggest a commonality in the pb coordination chemistry among mammalian mt2s. the higher acid tolerance, greater coordination stability, and faster degradation rate of pb7mt2(ii) have significant implications for the pb detoxification process. specifically, mt2 reduces the pb-inflicted toxicity by seizing free pb in the cellular milieu or by sequestering pb from pb-inflicted proteins. the unique properties of pb7mt2(ii) render mt2 a greater power to effectively scavenge pb in different environments (e.g., in a localized acidic cytosol region). moreover, the greater flexibility of pb7mt2(ii), resulting from the absence of bridging cysteine sulfurs, helps to accelerate its processing by lysosomal protease. the structural conversion from pb7mt2(i) to pb7mt2(ii) is likely to occur in the acidic environment of lysosome, facilitating the effective detoxification and metabolism of pb.

lead is a toxic heavy metal whose detoxification in organisms is mainly carried out by its coordination with some metalloproteins such as metallothioneins (mts). two pb mt complexes, named as pb7mt2(i) and pb7mt2(ii), form under neutral and weakly acidic conditions, respectively. however, the structures of the two complexes, which are crucial for a better understanding of the detoxification mechanism of pb mts, have not been clearly elucidated. in this work, coordination of pb2+with rabbit liver apo mt2, as well as with the two individual domains (apomt2 and apomt2) at different ph, were studied by combined spectroscopic (uv visible, circular dichroism, and nmr) and computational methods. the results showed that in pb7mt2(i) the pb2+coordination is in the trigonal pyramidal pb s3 mode, whereas the pb7mt2(ii) complex contains mixed trigonal pyramidal pb s3, distorted trigonal pyramidal pb s2o1, and distorted quadrilateral pyramidal pb s3o1 modes. the o-donor ligand in pb7mt2(ii) was identified as the carboxyl groups of the aspartic acid residues at positions 2 and 56. our studies also revealed that pb7mt2(ii) has a greater acid tolerance and coordination stability than pb7mt2(i), thereby retaining the pb2+coordination at acidic ph. the higher flexibility of pb7mt2(ii) renders it more accessible to lysosomal proteolysis than pb7mt2(i). similar spectral features were observed in the coordination of pb2+by human apo-mt2, suggesting a commonality among mammalian mt2s in the pb2+coordination chemistry.

PMC3993925

pubmed-666

we applied the blastp program (5) to identify, for each predicted protein sequence in the h37rv genome (genbank accession no. ae000516). following genbank annotations, we compared 3,972 predicted proteins in h37rv with 4,187 predicted proteins in cdc1551. we used a strict search criterion (e=10-50) to identify truly orthologous gene pairs. we aligned (6) the putative orthologous pairs of amino acid sequences (n=3,428), then imposed this alignment on the dna sequences. visual inspection of amino acid alignments showed that certain alignments, usually near the n-terminus or c-terminus, had regions of very low sequence identity. examination of the dna sequences of the corresponding genes showed that these regions of low identity were typically caused by a frameshift in one of the two genomes relative to the other. whether these frameshifts are biologically real or result from sequencing error was uncertain; therefore, we eliminated 119 such gene pairs from our data set. for the remaining gene pairs (n=3,309), we computed the proportion of synonymous substitutions per synonymous site (ps) and the proportion of nonsynonymous substitutions per site (pn) by using nei and gojobori s method (7). because values of ps and pn were very low in most cases, we did not correct for multiple hits. because ps values appeared to fall into two groups (see results), we used a simple probabilistic model to separate these two sets of gene pairs. we assumed that the probability of synonymous substitution followed two separate binomial distributions, designated models a and b, with probabilities of success (i.e., of a synonymous difference) designated pa and pb, respectively. using the bayes equation, for each gene pair with a given ps value, we computed the probability that model a applies, given the observed ps: p(a|ps)=(psa) fa /[(psa) fa+(psb) fb], where fa is the frequency of cases to which model a applies, fb the frequency of cases to which model b applies, psa is the binomial probability of obtaining the observed ps, given the number of synonymous sites in the gene and a probability of a synonymous difference equal to pa; and psb is the binomial probability of obtaining the observed ps, given the number of synonymous sites in the gene and a probability of a synonymous difference equal to pb. of 3,309 pairs of putatively homologous protein-coding genes in the h37rv and cdc1551 genomes of m. tuberculosis, 2,662 (80.5%) showed no synonymous or nonsynonymous nucleotide differences between the two genomes, and 3,010 (91.0%) showed no synonymous differences between the two genomes. however, in a small number of gene pairs, the proportion of synonymous differences per synonymous site (ps) was surprisingly high. in 13 (0.4%) gene pairs, ps was>0.01, and in 3 gene pairs ps exceeded 4%. these extreme ps values seen in a small number of gene pairs are much higher than generally observed between alleles at neutrally evolving loci in eukaryotes (8). thus, the comparison of protein-coding genes between the two m. tuberculosis genomes suggested the existence of two distinct groups of gene pairs: a large group having few or no synonymous differences and a much smaller group with a substantial degree of synonymous divergence. we used a simple probabilistic model (see methods) to separate these two sets of gene pairs, designated group a and group b, respectively (figure). the application of this method showed 11 loci with unusually high ps values and probabilities of assignment to group a of<50% (figure). a plot of p(a|ps), the probability of assignment of a locus to group a given the observed ps value, as a function of ps at 3,309 loci compared between the h37rv and cdc1551 genotypes of mycobacterium tuberculosis. the plot shows the bimodal nature of the distribution of ps values, with overall higher values of ps at the 11 loci having p(a|ps)>50%. we assumed that group a members are truly orthologous gene pairs that diverged at the time of the common ancestor of the h37rv and cdc1551 genomes. group a included 3,298 pairs, with mean ps for all genes of 0.000328 0.000022 standard error. when ps was estimated for the 3,298 genes concatenated together (a total of 934,413 synonymous sites), an estimate of ps=0.000348 0.00019 was obtained. the range of ps values in group a was between zero and 0.012; a total of 288 loci in group a had ps values other than zero. these results show a substantial level of nucleotide diversity, approximately half the level of nucleotide diversity in humans (9). rates of nucleotide substitution per unit time are difficult to estimate in bacteria given the lack of calibration from the fossil record (10). to obtain an estimate of the rate of synonymous nucleotide substitution, we used published data on comparisons of escherichia coli and salmonella typhimurium (11,12), which are believed to have diverged approximately 100 million years ago (13,14) (table 1). this procedure yielded estimates for the last common ancestor of h37rv and cdc1551 in the range of 34,00038,000 years (table 1). these estimates are approximately twice previous estimates of the age of the common ancestor of worldwide m. tuberculosis (2,3). to obtain the observed mean ps value between h37rv and cdc1551 within 15,00020,000 years would require a rate of synonymous substitution approximately twice that observed in enterobacteria. based on synonymous substitutions between escherichia coli and salmonella typhimurium, assumed to have diverged 100 million years ago (13,14). estimates are shown standard error, based on standard error of mean ps. group b consisted of 11 gene pairs with mean ps of 0.0286 0.0050 (table 2). paired sample t-test of the hypothesis that ps=pn, p<0.01. the quantities ps and pn are the proportion of nucleotide difference per synonymous site and per nonsynonymous site, respectively. in enterobacteria, a negative correlation exists between observed proportions of synonymous difference and codon bias (11). in the case of mycobacterium, codon bias results mainly from the very high third position g+c content of most genes (15). in our data, however, we observed no correlation between ps and proportion g+c at third codon positions (r=-0.010; not significant). a number of additional possibilities may explain the occurrence of gene pairs with higher than expected ps values: 1) balanced polymorphism. selectively maintained polymorphisms are expected to be much older than neutral polymorphisms and may even predate speciation events (16). in the case of haploid organisms such as bacteria, balancing selection would take the form of frequency-dependent selection rather than overdominant selection. 2) differential deletion. in a multi-gene family, if one member of an orthologous pair of genes were deleted in one genotype, the gene pairs would involve paralogous, not orthologous comparisons. 3) horizontal gene transfer. a gene obtained by one of the two genotypes from another bacterial species one indication of a balanced polymorphism is a higher rate of nonsynonymous than synonymous substitution (8). there was no strong evidence of such selection in the present case; ps was greater than pn at 10 of the 11 loci, and pn exceeded ps only slightly at one locus (table 2). in addition, we compared ps and pn in sliding windows of 30 codons along the length of these genes. no regions were observed in which pn was greater than ps (data not shown). thus, there was no evidence of positive selection acting on specific regions of these genes. on the other hand, differential deletion can probably explain some cases, most notably members of the pe multi-gene family (11) (table 2). the remaining gene pairs are possibly cases of horizontal gene transfer (table 2), for which there is some recent evidence in m. tuberculosis (17). our results did not support the hypothesis that the common ancestor of m. tuberculosis was relatively recent (2). rather, the pattern of nucleotide substitution at synonymous sites suggested a divergence time for the two available genotypes of this species approximately 35,000 years ago. since h37rv and cdc1551 represent two genotypes sampled from within the species, they are probably not the most divergent genotypes possible. thus, the last common ancestor of m. tuberculosis likely occurred considerably earlier than 35,000 years ago. while the difference between an estimate of 15,00020,000 years and one of 35,000 years is not large on an evolutionary time scale, such a difference is substantial on the scale of human history. for example, the existence of two genotypes in the current population of m. tuberculosis with a common ancestor at 35,000 years is evidence against the hypothesis that m. tuberculosis arose, presumably from m. bovis, at the time of human domestication of cattle (18). our result is thus consistent with phylogenetic analyses based on insertion-deletion events, which suggest that the m. tuberculosis lineage was a human pathogen well before the origin of m. bovis (19). thus, along with recent evidence of an ancient origin and extensive polymorphism in the malaria parasite plasmodium falciparum (20,21), our study provides evidence against the long-held view that virulent pathogens are invariably evolutionarily recent (22). our estimate is conservative because the rate of synonymous substitution may actually be lower in mycobacterium than in enterobacteriaceae, given the highly skewed g+c content in the former. furthermore, our estimate of the mean proportion of synonymous difference was conservative because we excluded 119 loci with potential frameshifts between the two genotypes as well as a set of 12 loci with unusually high ps values. in addition to the 12 loci assigned to our group b, certain other loci might also have originated from horizontal gene transfer. however, even if horizontal gene transfer has occurred at other loci besides those in group b, eliminating further loci with relatively high ps values from group a will not affect the results greatly. for example, if we eliminate the 10 loci with highest ps values from group a, mean ps will be reduced only to 0.000299, and the estimated age of the common ancestor will be barely affected. the degree of polymorphism observed in this study is unlikely to have been substantially influenced by sequencing errors. the error rate for finished sequences from the institute for genomic research (where cdc1551 was sequenced) has been independently estimated at<1 in 88,000 bases (23). assuming a similar error rate for both 4.4 mega-bp m. tuberculosis genomes, we would expect to see approximately 100 differences between them due to sequencing errors. approximately 21 such differences would be expected in the 938,778 synonymous sites in group a and group b genes. in fact, 411 synonymous differences were observed at these sites; thus, even if present, sequencing errors are likely to have made up only a small fraction (approximately 5%) of the total synonymous polymorphism. at such a rate, sequencing errors would have little effect on our estimates of nucleotide diversity at synonymous sites or the age of the common ancestor of the two genomes. in addition, the hypothesis that the single nucleotide polymorphisms (snps) observed between these genotypes are real received strong support from a recent study that observed a number of the same snps in clinical isolates (24). moreover, since sequencing errors are expected to occur at random with respect to the reading frame of coding sequences, the fact that mean ps exceeded mean pn in both group a and group b was strong evidence against the hypothesis that a substantial proportion of the observed polymorphism was due to sequencing error. simple considerations of probability can explain why earlier studies produced relatively low estimates of this species age. if we assume that the per-site probability of a synonymous difference between two m. tuberculosis genomes is equal to the mean ps observed between h37rv and cdc1551 (0.000328), then the probability is approximately 95% that no synonymous differences will be seen in a gene with 150 synonymous sites. the probability that no synonymous differences will be seen in 10 such loci chosen at random is approximately 60%, and the probability that no synonymous differences will be observed at 20 such loci is approximately 37%. on the other hand, the probability that no synonymous differences will be seen at 100 these calculations emphasize the need to examine a very large number of nucleotide sites to obtain a reliable estimate of nucleotide diversity and thus of the age of the most recent common ancestor in cases where the frequency of substitution is less than one in a thousand. even when the frequency of substitution is between one in a thousand and one in a hundred, substantial stochastic error is possible if the number of loci examined is small. thus, any study that estimates population parameters from nucleotide sequence data needs to survey a substantial number of loci. these considerations are particularly important in the case of pathogenic microorganisms, where a number of factors (including both natural selection and horizontal gene transfer) may lead to substantial differences among loci with respect to the level of nucleotide diversity. comparison of two complete genomes of m. tuberculosis showed a greater extent of sequence polymorphism than would be expected on the basis of previous studies, in turn suggesting that analysis of additional genomes will likely show further polymorphism. polymorphism in any species of pathogen may complicate therapeutic strategies because it implies the existence of variation on which selection can act, including selection imposed by human vaccines and pharmacologic agents (20). on the other hand, known polymorphisms may prove useful to investigators in reconstructing the evolutionary relationships among clinical isolates and in providing markers for understanding the genetic basis of complex phenotypic traits.

comparison of the pattern of synonymous nucleotide substitution between two complete genomes of mycobacterium tuberculosis at 3,298 putatively orthologous loci showed a mean percent difference per synonymous site of 0.000328 0.000022. although 80.5% of loci showed no synonymous or nonsynonymous nucleotide differences, the level of polymorphism observed at other loci was greater than suggested by previous studies of a small number of loci. this level of nucleotide difference leads to the conservative estimate that the common ancestor of these two genotypes occurred approximately 35,000 ago, which is twice as high as some recent estimates of the time of origin of this species. our results suggest that a large number of loci should be examined for an accurate assessment of the level of nucleotide diversity in natural populations of pathogenic microorganisms.

PMC2738538

pubmed-667

cytomegalovirus (cmv) is a significant cause of morbidity among solid organ transplant recipients. active infection can result in the well-defined direct effects of either cmv syndrome or tissue-invasive disease, while the indirect effects include potential immune-mediated injury of the allograft as well as an increased propensity for coinfections. ganciclovir and its prodrug, valganciclovir, each have been effective in both prevention and treatment of cmv disease [24]. however, the emergence of ganciclovir-resistant (gcv-r) cmv has posed a more significant threat due to an aggressive disease course and a greater mortality risk. treatment options for gcv-r cmv are limited, with foscarnet being recommended as the initial treatment option followed by cidofovir. although these agents are known to have activity against cmv, both are associated with substantial side effects, the most notable of which is nephrotoxicity. significant renal injury with these agents has been reported in 3060% of patients and may even occur after only 1-2 doses [7, 8]. furthermore, cidofovir is contraindicated in patients with an estimated creatinine clearance of<55 ml/min or a serum creatinine of>1.5 mg/dl, which are common findings among the solid organ transplant population. herein, we report a successful strategy of reduced-dose cidofovir in combination with cmv-hyperimmune globulin (cmv-igg) in four consecutive kidney transplant recipients with varying degrees of renal impairment and mild cases of genotypically confirmed gcv-r cmv. we chose to use cidofovir instead of foscarnet in these cases based on our own disappointing experience with the latter agent, having observed a high rate of acute renal failure requiring dialysis, in patients with severe gcv-r cmv disease. the rationale for reduced-dose cidofovir was the impaired baseline renal function observed in our patients as well as the ability to use probenecid and hydration as nephroprotective measures. cmv-igg was provided as an adjunct to antiviral therapy, as well as for its potential immunomodulatory properties. use of this regimen resulted in viral clearance, preservation of graft function, and avoidance of rejection in all four cases. four consecutive kidney transplant recipients with documented ganciclovir-resistant cmv were included in this review. immunosuppression was administered per center protocol: antithymocyte globulin (atg) induction was administered to three patients based on high immunologic-risk status (panel reactive antibody greater than 20% and/or african american race) while basiliximab induction was given to one patient with low-immune risk status. all four patients were high serologic risk for cmv (donor igg positive/recipient igg negative) and were to receive valganciclovir 450 mg daily for 6 months with routine cmv polymerase chain reaction (pcr) screening at prespecified time points (1, 3, 6, 9, and 12 months) after transplant and when clinically warranted. treatment of cmv disease in clinically stable patients consisted of a valganciclovir 900 mg twice daily (adjusted for renal function) induction period for 2128 days, followed by a 900 mg/day maintenance phase for an additional 28 days or until two consecutive negative pcrs (defined as<300 copies/ml) were achieved two weeks apart. interestingly, all four patients included in this report developed breakthrough viremia in conjunction with symptoms during the period of prophylaxis. therefore, valganciclovir dosages were increased to 900 mg twice daily (adjusted for renal impairment). genotypic analysis for ul97 or ul54 mutations was performed upon failure to eradicate detectable viremia on treatment dosages of valganciclovir. all patients demonstrated confirmation of ul97 mutations and absence of ul54 mutations with documented resistance only to ganciclovir. each patient was then initiated on combined therapy with intravenous (iv) cidofovir 1-2 mg per kg in 5001000 ml of normal saline and cmv-igg (cytogam, csl behring ag, king of prussia, pa) 100 mg per kg. oral probenecid was administered at a dosage of 2 grams, 1 hour prior to and 4 hours after the completion of each cidofovir infusion. in addition, maintenance immunosuppressants were reduced to attain tacrolimus levels of 48 ng/ml and mycophenolate mofetil dosages of 500 mg twice daily. combination therapy and pcrs were repeated every 2 weeks until two consecutive negative pcrs were achieved, at which point treatment was discontinued and pcr monitoring became less frequent. the patients were young, with a mean age of 33 years, and all were male. three of four patients were african american and the same three received deceased donor kidneys. one patient (patient 1) had an episode of acute rejection in the first posttransplant month requiring atg treatment prior to cmv detection. the mean time from transplant to the first detectable cmv pcr was 114 days. because low-dose valganciclovir in some patients may actually be appropriate dosing based on the presence of renal dysfunction, we evaluated renal function using both estimated 4-variable glomerular filtration rate (gfr) and cockcroft-gault equations. at the time of detectable cmv replication, only one patient (patient 4) had a gfr greater than 60 ml/min/1.73 m. however, 3 of 4 patients had a creatinine clearance greater than 60 ml/min using the cockcroft-gault method. therefore, according to approved labeling of valganciclovir, three of the four patients were indeed receiving low-dose genotypic assessment and diagnosis of gcv-r cmv were made at a mean of 103 days from the initial cmv pcr detection date, after persistence of detectable pcrs despite treatment dosages of valganciclovir. the average duration of valganciclovir exposure at the time of resistance diagnosis, including posttransplant prophylaxis, was therefore 217 days. all patients experienced cmv syndrome, with a predominance of fever, leukopenia, or malaise. in addition, patient 1 had gastrointestinal symptoms, although a biopsy was not performed to evaluate for tissue invasion. two consecutive negative pcrs were attained in all patients, occurring after an average of 5 treatments and a median of 42 (range 14 to 105) days from the date of the first infusion (figure 1). following discontinuation of therapy, patients 1 and 4 each developed recurrent viremia. in each patient, the recurrence was not associated with symptoms; however, treatment was reinitiated. viremia subsequently cleared again after 2 additional courses of cidofovir/cmv-igg in patient 1 and after 10 doses of cidofovir alone in patient 4. in the latter patient, a repeat genotype was obtained, demonstrating the same ul97 mutation site and conferring resistance only to ganciclovir. the mean change in gfr from the time of gcv-r diagnosis to the completion of therapy was 1 ml/min. at the time of this writing and after a mean follow-up of 21 months since the final dose of cidofovir, all patients are currently alive with functioning grafts. none of the patients developed recurrent disease and none experienced acute rejection throughout the follow-up period. available data on the use of cidofovir for treatment of cmv in solid organ transplant recipients is scarce. while cidofovir is contraindicated in patients with reduced renal function, several reports describe its safety at lower dosages for the treatment of polyomavirus-induced nephropathy. furthermore, based on limited pharmacokinetic data in renally impaired patients, reduced dosages of cidofovir may provide adequate antiviral concentrations. this led us to investigate the ability to use reduced-dose cidofovir to eliminate mild cases of gcv-r cmv disease. in addition, each patient received cmv-igg for two reasons: first, to augment antiviral treatment by providing cmv antibodies in previously seronegative patients and, second, to take advantage of the potential immunomodulatory effects in an effort to prevent rejection. in all four patients, this regimen led to resolution of gcv-r disease without deterioration of renal function or rejection. antiviral-resistant cmv has been reported in up to 14% of transplant recipients, with the highest rates occurring in high-risk serostatus recipients and lung transplant recipients [5, 1214]. ganciclovir, a guanosine analogue, requires three stages of phosphorylation in order to exert its inhibitory effect on cmv dna polymerase. the initial phosphorylation occurs via viral ul97 phosphotransferase, which is followed by two subsequent phosphorylations by host cellular kinases. resistance can be conferred by mutations at the ul97 kinase and ul54 polymerase genes [15, 16]. mutations in ul97 alone can result in resistance to ganciclovir and other nucleoside analogues by inhibition of the initial phosphorylation step. mutations in ul54, albeit much less common, can confer resistance to cidofovir and foscarnet. inadequate drug exposure and prolonged duration of antivirals have been attributed to the development of resistant cmv strains. in addition to its significant morbidity and mortality, treatment options for gcv-r cmv are also associated with severe adverse outcomes. recommended options such as cidofovir and foscarnet are associated with a high incidence of nephrotoxicity, often resulting in renal failure in both kidney and non-kidney transplant recipients [12, 13, 18, 19]. additionally, rejection may also occur as a result of reduced immunosuppression and/or indirect effects associated with cmv infection. while foscarnet is considered the preferred treatment for gcv-r, our own experience with the agent has been disappointing due to a very high incidence of acute renal failure requiring hemodialysis in patients with more severe disease. given our familiarity in using cidofovir as a treatment option for polyomavirus nephropathy and the ability to use probenecid and hydration as nephroprotective measures, we chose this agent in these four cases of milder disease. cidofovir is approved in the united states for cmv retinitis in patients with acquired immunodeficiency syndrome (aids). the drug requires phosphorylations to monophosphate and diphosphate (active) forms by pyrimidine nucleoside diphosphate kinase and nucleoside diphosphate kinase, respectively. the final metabolite, cidofovir diphosphate, inhibits cmv dna polymerase, a product of gene ul54. while each of our patients demonstrated mutations only in ul97, development of resistance to cidofovir was an obvious concern. however, due to the relatively mild nature of disease and the presence of renal dysfunction in each patient, we felt that a reduced dose would be appropriate. a genotype was repeated in one patient with a slight rise in pcr after resuming cidofovir and was confirmed negative for the ul54 mutation. an interesting finding in our cohort was that each patient developed their initial infection while on valganciclovir prophylaxis. this suggests that breakthrough cmv viremia during prophylaxis should warrant testing for antiviral resistance. treatment of gcv-r cmv in solid organ transplant recipients is challenged by dose-limiting toxicities of effective agents and potential graft injury due to indirect viral effects or rejection associated with lowering of immunosuppressive agents. in our experience, 4 consecutive patients with documented ul97 mutations conferring resistance to gcv were successfully treated with a regimen of reduced-dose cidofovir and cmv-igg. it is important to highlight that all patients were diagnosed relatively early in the course of their infections, making this conservative approach more plausible. undoubtedly, a more aggressive treatment would be required in patients with greater disease severity. we also can not rule out the impact and importance of reduction of immunosuppression in the management of patients with gcv-r cmv. nevertheless, all patients experienced viral eradication, and, importantly, none developed nephrotoxicity or rejection. we conclude that this regimen in addition to reduction of immunosuppression may serve as a treatment option in patients with mild gcv-r cmv disease.

ganciclovir-resistant cytomegalovirus (cmv) is associated with significant morbidity in solid organ transplant recipients. management of ganciclovir-resistant cmv may be complicated by nephrotoxicity which is commonly observed with recommended therapies and/or rejection induced by indirect viral effects or reduction of immunosuppression. herein, we report a series of four high serologic risk (donor cmv positive/recipient cmv negative) kidney transplant patients diagnosed with ganciclovir-resistant cmv disease. all patients initially developed breakthrough viremia while still receiving valganciclovir prophylaxis after transplant and were later confirmed to exhibit ul97 mutations after failing to eradicate virus on adequate dosages of valganciclovir. the patients were subsequently and successfully treated with reduced-dose (1-2 mg/kg) cidofovir and cmv-hyperimmune globulin, given in 2-week intervals. in addition, all patients exhibited stable renal function after completion of therapy, and none experienced acute rejection. the combination of reduced-dose cidofovir and cmv-hyperimmune globulin appeared to be a safe and effective regimen in patients with mild disease due to ganciclovir-resistant cmv.

PMC4058803

pubmed-668

a pure ground-glass nodule (ggn) in the lung may represent an early lung malignancy, such as adenocarcinoma in situ (ais) or minimally invasive adenocarcinoma (mia) (1). approximately 20-30% of patients with adenocarcinoma and a predominantly pure ggn who undergo surgery have additional ggn lesions, either in the same or different lung lobes (2). in such cases, multiple limited resection may be an appropriate treatment if a few pure ggn lesions are scattered in multiple lobes or peripherally in one lobe, and the patient has sufficient pulmonary functional reserve (3). however, alternative options should be considered if several ggn lesions are scattered in both lungs with multiple lobes involved, and there is a risk for insufficient pulmonary functional reserve after resection. chemotherapy has been suggested as an additional therapeutic option, if surgery is not possible due to a lack of pulmonary functional reserve (4). another strategy is to rely on serial computed tomography (ct) scans until the nodule develops a definite solid portion. if curative therapy is not performed, there remains a significant chance for progression (1). cryoablation has been explored as an option for treating metastatic lesions in patients with non-small cell lung cancer who can not undergo surgery or chemotherapy (5). however, no clinical reports of cryoablation techniques applied to multiple ggns in the lung have been published. here, we describe a patient with lung cancer and a pure ggn in the left lower lung lobe that persisted after several limited resections and was successfully treated with cryoablation. a 59-year-old woman visited our hospital due to abnormal chest radiography findings on a health maintenance exam at another hospital. all pre- and post-procedural ct scans were performed using a multi-detector ct scanner (somatom definition flash, enlargen siemens, forchheim, germany). the scanning parameters were: 120 kvp, 200 mas, 0.75-mm collimation, 2-mm reconstruction intervals, smooth algorithm, and pitch of 1. several ggns were detected by ct scan: a 12-mm-sized, partially solid nodule in the right middle lobe (fig. 1a), a 7-mm-sized pure ggn in the right upper lobe, and two 5-mm-sized pure ggns in each of the lower lobes (fig. the patient underwent a right middle lobectomy and wedge resection of the right upper lobe using video-assisted thoracoscopic surgery. the partially solid nodule in the right middle lobe was diagnosed as adenocarcinoma, and the pure ggn in the right upper lobe was diagnosed as a mia. the two pure ggns in both lower lobes remained without significant change on a 10-month follow-up ct scan. an additional wedge resection was performed for the pure ggn in the right lower lobe, and this nodule was diagnosed as ais. after these surgeries, the patient did not have sufficient pulmonary functional reserve to tolerate any additional surgery. thus, we decided to perform cryoablation on the pure ggn in the left lower lobe. the patient fasted for 6 hours before the procedure, and her blood coagulation parameters were checked. we used an argon and helium gas-based system (seednet, galil medical, yokneam, israel), a 1.5-mm 17-g cryoprobe, and a thermal sensor. the procedure was performed in a fluoroscopy room by an interventional radiologist. after placing the patient in the prone position on the table, a cross-shaped radio-opaque skin marker was applied to determine the needle insertion site under fluoroscopy. subsequently, a c-arm cone-beam ct (cbct) scan (dynact, enlargen siemens) was taken (fig. 1d-f) to obtain 1.5-mm thick axial, coronal, and sagittal images. based on these images, we measured the distance from the center of the skin marker to the target ggn. the area of the skin marker was draped, and a 1% lidocaine solution was injected into the skin down to the pleural surface. after localizing the needle tip, a cbct scan was performed to confirm the probe insertion site. a 17-g cryoprobe (seednet, galil medical) was carefully introduced into the pure ggn under fluoroscopic guidance. we used a fluoroscope and cbct to visualize the size of the ice ball during the procedure. we took a ct scan to confirm that the ice ball had reached sufficient size, as indicated by a low attenuation region with an envelope of ground-glass opacity and a margin of 1 cm beyond the ggn. a small pneumothorax was noted after removing the cryoprobe; however, the patient was asymptomatic with stable vital signs. we performed a follow-up ct scan 1 day after cryoablation to assess any initial changes in the ablated lesion and procedural-related side effects (fig. the scan showed a 3-cm sized ablated zone in the left lower lobe with no major procedure-related complications, such as hemothorax. the size of the ablated zone had decreased markedly on a follow-up ct scan 2 months later. the pure ggn in the left lower lobe was judged to have been successfully ablated without recurrence on a 6-month follow-up ct scan (fig. our results show the potential of cryoablation as a new treatment method for ggn, particularly in patients in whom surgery is contraindicated. some controversy exists as to whether some nodules should be resected or followed by serial ct scans. kim et al. (6) noted that several pure ggn lesions detected in patients undergoing surgery for bronchioloalveolar carcinoma did not change in size or features during follow-up. they suggested that surgical resection should be considered in selected cases in which nodules exhibit significant changes in size or appearance during follow-up. (7) found that all ggn lesions>10 mm were carcinomas and insisted that these lesions should be resected rather than followed. partially solid ggn lesions should be resected regardless of size because they represent more invasive lesions than pure ggns. thus, a non-surgical alternative is needed for patients with insufficient pulmonary functional reserve or in patients in whom surgery is technically not feasible. minimally invasive ablation techniques, such as radiofrequency ablation (rfa) and cryoablation, may be useful adjunctive treatment options for lung cancer or metastatic lesions and preserve pulmonary function (5, 9, 10). minimally invasive ablation techniques for lung cancer have several advantages, including selective damage, minimal morbidity and mortality, minimal loss of lung function, repeatability, low cost, excellent monitoring during treatment, less pain, and shorter hospital stay (11). ablating lung tumors is currently used for curative treatment of primary lung cancer and metastatic lung malignancies or cytoreduction (12). ablation is also an option in patients with lung metastases from colorectal and renal cell carcinoma, melanoma, hepatocellular carcinoma, or primary sarcoma (13). although rfa is a contemporary tumor ablation technique, it may cause serious complications, such as air embolism or uncontrolled pain (9, 14). cryoablation is a thermoablational technique that consists of alternating cycles of decreasing (freezing) and increasing (thawing) temperatures, leading to direct cellular and vascular injury. cryoablation works by forming an ice ball, which causes increased extracellular osmolarity and, as a consequence, water diffuses from the intracellular space into the extracellular space. thus, cryoablation may be a better option for patients with extensive emphysema (10). however, percutaneous or surgical biopsy was difficult due to the small size and multiplicity of the ggn lesions. nevertheless, localized ggns can include invasive disease, such as ais or mia (1). in our case, two similar nodules in the right upper and right lower lobes were identified during surgery as mia and ais, respectively. cryoablation may reduce the likelihood that a remnant ggn will progress to advanced cancer and does not require additional surgery. thus, long-term follow-up may be necessary to determine the outcome of cryoablation for treating pure ggns. in conclusion

treatments for pure ground-glass nodules (ggns) include limited resection; however, surgery is not always possible in patients with limited pulmonary functional reserve. in such patients, cryoablation may be a suitable alternative to treat a pure ggn. here, we report our initial experience with cryoablation of a pure ggn that remained after repeated surgical resection in a patient with multiple ggns. a 5-mm-sized pure ggn in the left lower lobe was cryoablated successfully without recurrence at the 6-month follow-up.

PMC4435997

pubmed-669

the inflammatory bowel diseases (ibds) are chronic illnesses affecting the gastrointestinal tract: they predominantly comprise crohn's disease (cd) and ulcerative colitis (uc). although cd and uc share some features, they have distinctive endoscopic and histological characteristics and disease patterns. furthermore, the clinical manifestations and outcomes of these two conditions vary. at present, there are no specific medical cures for cd and uc: current therapies aim to control the disease and prevent adverse outcomes. although ibd is uncommon in the first decade of life, presentation in infancy is well recognized. ibd is more common in the second decade, particularly in adolescence [2, 3]. there are clear demonstrations of increasing incidence of ibd around the world over the last decades, with increased rates also noted in areas with previous very low rates [38]. the changing incidence of ibd has also been noted in paediatric populations, with particular increases in cd [9, 10]. a history of weight loss or poor weight gain is commonly recorded at the time of diagnosis with ibd in children and adolescents. poor linear growth may also be evident at the initial assessment, leading to growth failure in some. impaired nutrition may lead onto short- and long-term consequences that include delayed puberty, micronutrient deficiencies, impaired adult height, and bone disease. regular monitoring and detailed attention to nutritional support remains a central facet of the management of children and adolescents with ibd. this paper will focus on the impact of ibd upon the nutritional state of children and adolescents and will highlight recent advances in this area. numerous cohort studies have demonstrated weight loss or poor weight gains at the time of initial diagnosis of ibd. reports indicate that most children with cd have a history of weight loss or absent weight gains prior to diagnosis. in a cohort of 386 canadian children diagnosed with cd over a ten-year period, 80% had a history of weight loss. a retrospective chart review of a cohort of 61 children diagnosed with cd at sydney children's hospital, sydney, australia showed that 51% had a history of weight loss at the time of diagnosis. in a prospective uk study of 379 paediatric cd patients, 58% had weight loss at diagnosis and 27% had weight weight loss tends to be seen less commonly in children with uc up to 31% of 172 children in the same uk cohort had a history of weight loss at diagnosis. maintaining appropriate weight gain also continues to be an ongoing issue after diagnosis. in a cohort of 41 children with longstanding cd recruited to assess dietary intakes and nutrition, the 18 children with active cd had lower mean z scores for weight, height, and bmi than the 23 children currently in remission. the reasons for altered weight gains are likely multifactorial, with decreased caloric intake being the most common [1419]. poor intake may be the consequence of the anorexic effects of proinflammatory mediators, such as interleukin-1 or tumour necrosis factor (tnf)- [20, 21] or result from early satiety, pain, or nausea. pain associated with eating, leading to poor tolerance of foods, and the fear of diarrhoea following meals are additional reported reasons for reduced intake. small bowel involvement may lead to disaccharide intolerance resulting in shorter gut transit times, pain, and exacerbation of diarrhoea. malabsorption of food components and the diversion of calories to sites of gut inflammation may be further contributing factors leading to impaired growth. in addition to presentation with loss of body weight or poor weight gains, children also commonly have altered patterns of linear growth. decreased height velocity was noted in up to 88% of a group of 50 children at the time of diagnosis with cd a number of these children had impaired linear growth prior to the onset of gut symptoms. impaired linear growth tends to be more pronounced in boys than girls this is principally related to the male pubertal growth spurt occurring later and lasting longer. furthermore, the limited duration of puberty limits the time before impaired linear growth becomes irreversible [23, 24]. cd is commonly associated with impaired linear growth either at diagnosis or as a long-term outcome [2229]. some earlier studies reported that despite growth retardation occurring during teenage years, many of these patients eventually achieved heights within the normal range for the general population [30, 31]; however, the genetic component of linear growth was not considered in these studies. markowitz et al. retrospectively assessed the growth patterns of 48 adults with ibd (38 with cd and 10 with uc: 73% male) who had previously been diagnosed during childhood. these investigators used two height prediction methods and employed the american national centre for health statistics growth charts: 31% of this group were found to have permanently impaired linear growth. in their study of 132 children and young adults aged between 525 years, sentongo and colleagues argued that height adjusted for genetic potential was a more powerful way of assessing height status in children with cd. in these children, the z scores based on adjusted heights were significantly lower than z scores based on measured heights. again, males in this group were most severely affected, with the average adjusted height for age z score one standard deviation less than expected. lee et al. recently undertook a prospective assessment of height in 295 children with cd and uc who had been diagnosed between 2002 and 2008 in boston, usa. twenty-two percent of these children (90% of whom had cd) had linear growth impairment (height for age z-score of 1.64 or less) documented on one or more measurements from the time of diagnosis. the final mean adult height z score in this cohort was 0.39.the main predictors of final adult height in this cohort were found to be lower parental height and the patient's lowest height z score. a recent british study also assessed the final adult height of a group of 23 individuals who had been diagnosed with cd prior to their 16th birthday. this group had a final adult height z score of 0.29: very similar to that observed in the north american study. the mean final height of these patients was 2.4 cm less than the parental or target height (range of 20.0 to+9.0 cm). twenty percent of the group had a final adult height greater than 8.0 cm below their target height. jejunal disease and the length of symptoms prior to diagnosis (diagnostic delay) were predictive factors of final adult height in this cohort. in this cohort, exposure to corticosteroids, surgical intervention, and parental height jejunal disease location has previously been suggested to be associated with linear growth impairment [32, 33]. a danish population-based assessment of growth in children with ibd highlights the differential effects of cd and uc upon growth. forty-four children with cd were included in this study, along with 50 with uc and four with ibd unclassified (ibdu). the children with uc had similar height and bmi scores for age to control data. in contrast, the children with cd had height for age z score of 0.77: also they were shorter than the children with uc (p=.005). fifty percent of the children with cd had a height less than the 25th centile for age. elegant studies in rodent models show that approximately 40% of growth impairment is consequent to the effects of inflammatory proteins, especially il-6 [38, 39]. further, tnf- and il-6 both independently suppress levels of insulin-like growth factor (igf)-1, a critical mediator of the local actions of growth hormone. male gender, age at diagnosis, disease location, disease severity, and treatment modality may also all affect final height acquisition. severity of disease (defined by various measures such as steroid requirements, use of immunosuppressives, and cumulative period of hospitalisation) was correlated with impaired weight and height gains in a cohort of israeli children. interestingly, the presence of the nod2 genotype was not associated with impaired weight or linear growth, despite associations between ileal location and mutations in this gene. a recent report highlighted the importance of genetic influences upon linear growth in children with ibd. in a cross-sectional study, 951 subjects, comprising 317 cd patient-parent trios, linear growth impairment in the children with cd was associated with a polymorphism in the dymeclin gene dym (odds ratio 3.2). delayed puberty is also frequently observed in children with ibd, especially those with cd [43, 44]. in one cohort of children diagnosed with cd prior to the onset of puberty, menarche was noted to occur at or later than 16 years of age in almost three-quarters of the girls. delayed pubertal development is more common in children who have never achieved remission or who have had repeated disease relapses, emphasizing the interactions between disease control and growth in children. although the most concern in regard to the nutritional impact of ibd in children is towards under nutrition, some children with ibd may be overweight (bmi>85%) and/or obese (bmi>95%). in a cohort of 166 children with newly diagnosed ibd from wisconsin, usa, 12.1% of those with cd and 17.6% of those with uc were overweight/obese. in a group of 1598 american children with known ibd overweight/obesity in this multicentre cohort was associated with african-american ethnicity and medicaid insurance status. in addition, a prior surgical intervention was linked with overweight/obese status in the children with cd, suggesting that the presence of over nutrition may be associated with a more severe disease course. corticosteroids, for example, can lead to numerous side effects, including nutritional consequences. increased appetite and fluid retention are commonly seen after commencement of steroids. consequently, apparent improvements in weight during a course of corticosteroids may reflect fluid retention and not an improvement in the underlying nutritional status. steroids also lead to enhanced bone resorption and decreased new bone formation, adversely affecting bone health [48, 49]. further, daily corticosteroid therapy can suppress igf-1 activity, contributing to inhibition of linear growth and impaired final height. sulphasalazine may lead to folate malabsorption; however, daily folate supplementation does not appear necessary. folate supplementation is required, however, when methotrexate is used to avoid folate deficiency due to the inhibition of the conversion of folate to the active moiety tetrahydrofolate. cholestyramine, when required for bile-salt diarrhea, may impede absorption of vitamin b12. in contrast, some interventions, such as exclusive enteral nutrition (een) and biological therapies, have numerous positive nutritional effects. een is a specific nutritional therapy that involves the administration of a liquid formula as sole nutritional intake (with exclusion of normal diet) for up to 8 weeks. this therapy is efficacious in active cd, with remission rates similar to those seen with corticosteroids. een leads to positive improvements in weight and linear growth, normalization of igf-1 levels, and stabilization of bone turnover. een is also associated with improved vitamin d status compared to children treated with corticosteroids. biological therapies in children with cd examined the growth patterns of 32 children treated with infliximab (28 responders) for severe cd. these children had improved height velocity, and height centile increases after infliximab, with timing prior to early puberty being important.. found that improvements in linear growth after infliximab can be independent of pubertal status and reduction in corticosteroids. these effects of infliximab in children with cd may be due to improved inflammation with mucosal healing or due to direct effects upon growth hormone and igf-1 signaling. in a recent report, cromb et al. demonstrated that the height z scores of a group of 41 french children responding to infliximab improved from 0.57 1.18 to 0.25 0.99 (p=.04) over the period of followup. in contrast, those children not responding to this drug had no catch up growth. given the frequency and patterns of disturbed growth in children with ibd (and especially those with cd), the management of such children must include a clear and constant focus upon growth and nutrition. resumption of normal growth patterns should be seen as an element of the success of therapy. baseline assessment of nutritional status at the time of diagnosis should include detailed standard anthropometry, along with clear documentation of preceding growth patterns and familial growth patterns (especially parental heights). furthermore, close monitoring of growth during followup must be considered a mandatory part of the multidisciplinary care of children and adolescents with ibd. there is now plentiful evidence demonstrating the significant impact of ibd upon growth and nutrition in children. recent studies have helped to demonstrate the importance of nutritional, inflammatory, and genetic factors in growth outcomes. consequently, a key aspect of the ongoing management of children and adolescents with ibd should be the close and constant attention to growth .

the inflammatory bowel diseases (ibds) are chronic inflammatory processes affecting the gastrointestinal tract. when diagnosed in childhood and adolescence, ibd almost always impacts adversely upon the nutritional state of the patient. weight loss and impaired linear growth may be present at diagnosis or subsequently. further potential nutritional consequences in childhood ibd include malnutrition, anaemia, osteopaenia, and delayed puberty. understanding the nutritional aspects of ibd is paramount in growing children, especially those entering and advancing through puberty. this paper focuses upon the nutritional impacts of ibd in children and adolescents.

PMC3263571

pubmed-670

bleomycin a2 (blm, 1, figure 1) is the major component (70%) of the clinical anticancer drug blenoxane, which is currently used for the treatment of hodgkin s lymphoma, melanomas, head and neck carcinomas, and testicular cancers. blm exerts its biological effects through a metal ion and oxygen dependent dna cleavage that occurs selectively at 5-gc or 5-gt sites. although capable of producing single-strand and double-strand breaks, the latter is believed to be the most biologically relevant event. despite being an effective therapy for numerous malignancies, blm treatment is often limited by dose-dependent pneumonitis, lung fibrosis, and skin toxicities, some of which have been attributed to the c-terminal bithiazole appendage. thus, there remains an interest in identifying synthetic blm-based analogs that lack off-target toxicity and maintain efficacy, which can be used as single component oncology drugs. each structural unit of blm contributes an important role in the dna binding and cleavage cascade. systematic evaluation of its structure through single site deep-seated modifications prepared by total synthesis has delineated the function of each subunit and the role of their individual substituents. these studies, in conjunction with nmr-derived structural models of dna bound blm, continue to define the remarkable combination of functional, structural, and conformational properties integrated into the natural product. the pyrimidoblamic acid subunit not only participates in the metal chelation and o2 activation but also is responsible for the dna cleavage selectivity via triplex-like hydrogen bonding to guanine at the 5-gc and 5-gt cleavage sites. in such studies, we have additionally shown that removal of the pyrimidine c5 methyl group, enlisting the functionalized pyrimidine found in p-3a, has no impact in the functional activity of the resulting blm analog. from these studies, ()-pyrimidoblamic acid (2, figure 2) and the related peptide-derived natural product, p-3a (3, figure 2), have been identified as key subunits for the continued preparation of improved or simplified bleomycin analogs. total syntheses of ()-pyrimidoblamic acid were originally described by umezawa, hecht, and later by us. although elegant and pioneering in their own regards, these previous efforts have suffered from the inability to fully control the stereochemistry at the benzylic pyrimidine c2 tertiary center (scheme 1). similarly, our initial total synthesis of p-3a, whose pyrimidine core differs from that of pyrimidoblamic acid by only the lack of the c5 methyl group, relied on a late stage diastereoselective introduction of the benzylic pyrimidine c2 tertiary center that proceeded with modest diastereoselectivity (87:13). recently, we defined the cycloaddition scope and productive reactivity of substituted electron-deficient 1,2,3-triazines with various dienophiles, including the first report of their ability to participate in previously unexplored [4+2] cycloaddition reactions with amidine or imidate heterodienophiles to provide highly substituted pyrimidines. these latter studies provided the basis for the potential development of second generation asymmetric total syntheses of ()-pyrimidoblamic acid and p-3a, in which all necessary stereochemistry is installed prior to a late stage [4+2] cycloaddition introduction of the pyrimidine, addressing the common limitation of previously reported routes, permitting the late stage divergent modification of their structures, and further expanding the scope of the 1,2,3-triazine inverse electron demand diels we envisioned that the pyrimidine core of ()-pyrimidoblamic acid would arise from an inverse electron demand diels alder reaction between the highly functionalized amidine 4 and 1,2,3-triazine 5a (scheme 2). additionally, the same amidine 4 and its cycloaddition with 1,2,3-triazine 5b would produce the pyrimidine core found in p-3a.this late stage convergent assemblage of the pyrimidine cores not only allows the early stage synthesis of the stereochemically rich c2 side chain common to both pyrimidoblamic acid and p-3a but also permits the late stage divergent synthesis of the natural products utilizing two different 1,2,3-triazines. amidine 4 was expected to arise from the reductive alkylation product of n-(triphenylmethyl)-l-asparagine (6) with aldehyde 7. key to the construction of 4 is the manipulation of intermediates, especially the desired free-based amidine, without sacrificing the stereochemical integrity of the -stereocenter, as similar amidines have demonstrated a propensity to epimerize. this concern dictated the late stage amidine introduction in order to minimize the potential impact of amidine epimerization. 1,2,3-triazines 5a and 5b, the electron-deficient heterocyclic azadienes for use in the key cycloaddition reaction, were anticipated to arise from an oxidative ring expansion of n-aminopyrazoles 9a and 9b. a key caveat to the approach is that although the two electron-withdrawing substitutents on the 1,2,3-triazines would be expected to enhance their cycloaddition reactivity, it was unknown whether their placement at the c4 and c6 positions would sterically slow the reaction and electronically redirect the mode of cycloaddition (c4/n1 vs c5/n2) with amidines. the requisite n-aminopyrazole 9a for oxidative ring expansion to the 1,2,3-triazine 5a was prepared from 8a that was accessed by a palladium-mediated tandem cross-coupling/electrocyclization reaction of commercially available ethyl diazoacetate and the vinyl triflate derived from t-butyl acetoacetate (scheme 3).n-amination of 8a was accomplished upon treatment with o-4-nitrobenzoylhydroxylamine in the presence of potassium t-butoxide (t-buok) in n-methylpyrrolidone (nmp), which yielded n-aminopyrazole 9a in 73% yield in a near 1:1 mixture of inconsequential regioisomers. in an analogous fashion and with similar yield (72%), the requisite n-aminopyrazole 9b used to access 1,2,3-triazine 5b was prepared from pyrazole 8b, which was generated from a [3+2] dipolar cycloaddition between ethyl diazoacetate and t-butyl propiolate. although a range of oxidants have been reported for transforming n-aminopyrazoles into 1,2,3-triazines, we have found that the optimal reagent for this oxidative ring expansion reaction is substrate dependent. the ring expansion is often suggested to occur through a nitrene intermediate, which initiates n n bond migration and inserts the central nitrogen of the 1,2,3-triazine. adaptation of conditions developed by ohsawa and colleagues, in which an n-aminopyrazole is treated with iodine (i2) in the presence of aqueous potassium bicarbonate (khco3), provided the 1,2,3-triazine 5a in excellent yield (75%). these conditions also proved effective for the production of 5b, yielding the 1,2,3-triazine in 68% yield. notably, 1,2,3-triazines 5a and 5b proved stable to extended storage at room temperature and purification by flash chromatography using silica gel. as summarized in figure 3, the most commonly used oxidants for this transformation (i.e., pb(oac)4, ni2o3, and mno2) proved unsuccessful in converting 9a to the desired 1,2,3-triazine 5a, instead resulting in n silver(i) nitrate, a commonly utilized nitrene transfer reagent in aziridination, alone failed to provide 5a but produced 5a in modest yield (34%) when used in the presence of phenyliodine diacetate (pida) as a co-oxidant. similarly, sodium periodate (naio4), which promotes the oxidative ring expansion efficiently for 4-substituted n-aminopyrazoles, afforded the 1,2,3-triazine 5a in moderate yield (28%) with a majority (70%) of the material isolated being a mixture of unreacted starting material (9a) and free pyrazole (8a). addition of a phase transfer catalyst improved the conversions to as high as 70%, but was less consistent, and the use of tetrabutylammonium periodate failed to generate any desired compound. amidine 4 was prepared from commercially available n-(triphenylmethyl)-l-asparagine (6) and aldehyde 7 (scheme 4). reductive alkylation of 6 with 7 in the presence of excess nabh(oac)3 provided 10 in 84% yield. treatment of 10 with 1-hydroxybenzotriazole (hobt) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (edci) followed by nh3/thf, provided the primary amide 11. dehydration of 11 to provide the nitrile 12 was accomplished by treatment with n-propanephosphonic acid anhydride (t3p) in the presence of hunig s base (i-pr2net). all intermediates up to this stage, including 13, exhibited a robust stability and no epimerization was observed throughout the reaction sequence. treatment of 13 with raney nickel (ra ni) in the presence of acetic acid (acoh) in meoh generated the acetate salt of the desired amidine, which could be filtered through celite, redissolved in ch2cl2, and treated with 20% aqueous naoh to yield the free-based amidine 4. chromatographic purification of the amidine 4, as either the acetate salt or the free base, resulted in partial epimerization. as a result, the crude amidine was used without purification and produced immediately prior to its use. notably, the free-based amidine epimerized much more readily than the protonated amidine in solution, and if the adjacent secondary amine was protected as the t-butylcarbamate (boc), even more significant slow epimerization of the -stereocenter occurred upon amidine formation. therefore, the free secondary amine is essential to the observed stereochemical stability of the adjacent amidine chiral center. effective access to the requisite intermediates 4 and 5a set the stage for examination of the key inverse electron demand diels alder reaction. consistent with the electron-deficient character of the 1,2,3-triazine, the cycloaddition reaction was found to proceed at room temperature or lower. significantly, the intrinsic regioselectivity of the amidine cycloaddition and 1,2,3-triazine cycloaddition mode were unaltered by the c4/c6 substitution with electron-withdrawing groups, and the amidine was found to add exclusively across c4/n1 (c6/n3) with no evidence of a redirected c5/n2 mode of cycloaddition. by design and because of the pseudo-c2-symmetric nature of the product pyrimidine, the cycloaddition across c4/n1 versus c6/n3 are indistinguishable, permitting the differential ester protection. although not all conditions surveyed are included, figure 4 summarizes representative examples in the optimization of the reaction between 4 and 5a to provide the desired pyrimidine 14. in accordance with our previous studies, which utilized 2.0 equiv of amidine, as summarized in figure 4, an increase in reaction time and temperature significantly increases the overall yield (entries 14). however, increasing the reaction temperature diminished the diastereomeric purity of the product, resulting from competitive amidine epimerization. notably, we found that the reaction produces a single diastereomer of 14 when conducted at 5 c in acetonitrile (entry 5). because the 1,2,3-triazine 5a is the simpler of the two reaction components, we focused on identifying conditions that employed amidine 4 as the limiting reagent (entries 8 and 9 vs 6 and 7). when the reaction was conducted with 1.0 equiv of amidine 4 and 2.0 equiv of the 1,2,3-triazine 5a, the yield increased to a respectable 46%. finally, if the reaction was allowed to stir at 5 c for 14 h and then warmed to 25 c for 6 h, 14 was obtained as a single diastereomer in 54% yield. optimization of the [4+2] cycloaddition reaction. with an effective route to 14 in hand, the secondary amine of 14 was protected as the t-butylcarbamate to provide 15 to eliminate oxidation risks later in the synthesis. chemoselective hydrolysis of 15 with 1 m aqueous naoh in thf: meoh (3:1) was followed with a curtius rearrangement to provide 16 (78% yield over two steps). removal of the acetonide protecting group with p-toluenesulfonic acid monohydrate (p-tsohh2o) in meoh yielded the boc-protected amino alcohol 17, which was subsequently treated with jones reagent to give the carboxylic acid 18. conversion of 18 to amide 19 was accomplished upon treatment with hobt and edci followed by nh3/thf, which provided the fully functionalized and protected ()-pyrimidoblamic acid 19 in 96% yield. although attempts to promote a global deprotection with 4 m hcl in etoac proved unsuccessful, as the tritylated carboxamide exhibited an unusual stability to these traditional reaction conditions, treatment of 19 with a 3:1 mixture of trifluoroacetic acid (tfa) and ch2cl2 followed by deliberate counterion exchange with a 1 m aqueous hcl workup provided ()-pyrimidoblamic acid (2) in quantitative yield and identical in all respects with authentic material. although the similarity in the pyrimidine cores of ()-pyrimidoblamic acid and p-3a is striking and may suggest that extrapolation of the approach to p-3a is straightforward, it was not clear what the impact of the c5 substituent might be. with electron-withdrawing groups at the c4- and c6-positions of 5a and 5b, it was still unknown whether the c5 methyl group affects the efficiency of the cycloaddition reaction of 5a and if its absence in 5b would alter the mode of cycloaddition. thus, before embarking on the synthesis of p-3a, we first examined the reactions of the two 1,2,3-triazines 5a and 5b in parallel. remarkably, the less substituted 1,2,3-triazine 5b was even more reactive than 5a, providing the product pyrimidine at a faster rate and in a higher yield in its reaction at room temperature with the aliphatic amidine substrate, where good conversion to the product was observed even within 5 min (figure 5). slower and a more comparable reactivity between 5a and 5b were observed with the aryl amidines. most significant in these studies is the fact that 5b showed no evidence of a potentially competitive cycloaddition across c5/n2 (vs c4/n1) despite the lack of a c5 substituent. with this knowledge in hand, comparison of the cycloaddition reactions between 5a or 5b and amidines. in accordance with the model substrates, the cycloaddition reaction between 4 and 5b (25 c, ch3cn, 12 h) yielded the desired pyrimidine 26 in 76% yield as a single diastereomer (scheme 6). in contrast to the reaction between 4 and 5a, which requires 5 c for 12 h prior to warming to 25 c to prevent competitive epimerization and preserve the amidine stereochemical integrity, the faster reaction between 4 and 5b can be conducted at 25 c without compromising the diastereomeric purity of the product and provided the stereochemically pure pyrimidine 26 in even higher yield (76%). following a synthetic route modeled on that used to complete the synthesis of ()-pyrimidoblamic acid, compound 26 was advanced (scheme 6). boc-protection of the secondary amine provided compound 27, which was subjected to saponification of the ethyl ester. however, attempts to selectively hydrolyze the ethyl ester by treatment with 1 m aqueous naoh in thf/meoh provided a mixture of compounds, which included those derived from transesterification or hydrolysis of the t-butyl ester. by substituting t-butanol (t-buoh) for meoh as the reaction cosolvent the competitive reactions were avoided and the reaction cleanly provided the requisite carboxylic acid, which was subjected to curtius rearrangement conditions to provide 28 in good yield (82% over two steps). removal of the acetonide protecting group with p-tsohh2o in meoh afforded the amino alcohol 29, which could be transformed to the primary carboxamide 31 in two steps. global deprotection of 31 was effected by treatment with tfa: ch2cl2 (3:1) to provide 32, a key analog of ()-pyrimidoblamic acid lacking only the c5 methyl group, in quantitative yield. additionally, treatment of 31 with 1 m aqueous naoh in thf: meoh (3:1) results in t-butyl ester hydrolysis and provided carboxylic acid (33), which was coupled with n-boc-l-his-l-ala-o(t-bu) to provide the fully assembled but protected p-3a (34). notably, this coupling reaction enlisted a more highly protected derivative of 32 than our prior efforts and proceeded more smoothly, providing higher yields of the product 34. global deprotection of 34 with tfa: ch2cl2 (3:1) followed by counterion exchange with a 1 m aqueous hcl workup provided p-3a (3), in quantitative yield and spectroscopically identical in all comparable respects with authentic material. convergent total syntheses of ()-pyrimidoblamic acid and p-3a were detailed based on the early stage preparation of the chiral highly functionalized c2 side chain and subsequent late stage, divergent construction of the pyrimidine cores through use of a powerful inverse electron demand amidine/1,2,3-triazine [4+2] cycloaddition reaction conducted at 25 c. in addition to permitting full control of the natural product stereochemistry, the approach provides the opportunity for the late stage divergent synthesis of modified analogs bearing deep-seated changes in either the pyrimidine core (c4 and c5) or the highly functionalized c2 side chain. such investigations are in progress and will be reported in due course. the examination of the key 1,2,3-triazine cycloaddition reaction with amidines defined nonobvious 1,2,3-triazine substituent effects that maintain or enhance the heterocyclic azadiene reactivity without altering the intrinsic regioselectivity or mode of cycloaddition. these observations with 1,2,3-triazines extend the utility of the inverse electron demand cycloaddition reactions of electron-deficient heterocyclic azadienes that includes the complementary 1,2,4- and 1,3,5-triazines, 1,2,4,5-tetrazines, and 1,2-diazines in the synthesis of highly substituted and functionalized heterocycles found in complex natural products.

total syntheses of ()-pyrimidoblamic acid and p-3a are disclosed. central to the convergent approach is a powerful inverse electron demand diels alder reaction between substituted electron-deficient 1,2,3-triazines and a highly functionalized and chiral primary amidine, which forms the pyrimidine cores and introduces all necessary stereochemistry in a single step. intrinsic in the convergent approach is the potential it provides for the late stage divergent synthesis of modified analogs bearing deep-seated changes in either the pyrimidine cores or the highly functionalized c2 side chain common to both natural products. the examination of the key cycloaddition reaction revealed that the inherent 1,2,3-triazine mode of cycloaddition (c4/n1 vs c5/n2) as well as the amidine regioselectivity were unaffected by introduction of two electron-withdrawing groups (co2r) at c4 and c6 of the 1,2,3-triazine even if c5 is unsubstituted (me or h), highlighting the synthetic potential of the powerful pyrimidine synthesis.

PMC3940392

pubmed-671

the process of hydrated excess proton solvation and transport in aqueous systems displays many unique characteristics due the unique nature of the net positive charge defect that an excess proton creates. by altering the covalent bonds and hydrogen bonds of surrounding solvent molecules, the hydrated excess proton charge defect is strongly delocalized and creates a series of dynamically interchanging structures (i.e., the zundel h5o2 and eigen h9o4 cations). proton (or more accurately stated, the charge defect) is also able to hop between neighboring water molecules by structural diffusion via successive hopping events involving the rearrangement of the local bonding topologies., is crucial to a number of fundamental processes in chemistry, physics, biology, and materials science. in biology, protons are widely used for the transduction of signals and energy (e.g, in channels, transporters, and enzymes). they are transported through both protonatable residues and buried water molecules via grotthuss shuttling. hence, studies on proton transport (pt) in biological systems have almost always started with the assumption that pt follows aqueous (already hydrated) pathways, which can be obtained from experimental data or by computational predictions. however, hydrophobic regions are commonly found in proteins, which complicates the interpretation of pt. carbon nanotubes (cnts) have provided insight into the solvation and ion transport properties of homogeneous hydrophobic spaces. experiments and computer simulations have shown that water molecules can stably occupy the interior of cnts. theoretical studies and recent experiments have further shown that proton diffusion through nanoconfined spaces, such as hydrophobic channels and nanotubes, can be facile (and possibly even faster than in bulk water). these results provide support for the supposition that the hydrophobic spaces in biomolecules may also transiently contain water molecules capable of proton conduction. however, a common assumption is that hydrophobic spaces must be solvated prior to pt. this logic has led to numerous mechanistic predictions based on the existence of a quasi-stable water wires (e.g., see refs (3133)). however, it has also been clearly demonstrated that the presence of an excess proton greatly influences the local water solvation structure (see ref (4) for a discussion). in the present work, it is in fact found that as soon as a charge defect associated with a hydrated excess proton charge nears a hydrophobic space, the associated solvating water can experience a strong driving force to fill that space. moreover, this finding naturally leads to the possibility that when an excess proton deprotonates from a peripheral amino acid residue or is solvated outside a hydrophilic (or amphiphilic) region, it can initiate additional solvation of such a region, which will in turn be coupled to the pt process through it. herein, multiscale reactive molecular dynamics (ms-rmd) is used to study pt through a cnt penetrating a graphene sheet. as described in the previous paragraph, a surprising phenomenon is revealed in which an excess proton charge defect creates its own aqueous transport pathway by shuttling water molecules through it into the hydrophobic nanoconfined space. this process can be described as a variant of grotthuss shuttling, wherein water molecules travel through a hydrated excess proton charge defect. the induced wetting is shown to be excess proton specific; it does not happen when the excess proton (h) is replaced by k or even a the two-dimensional free energy surface reveals a three-step mechanism by which the protonic charge defect is transiently stabilized at the nanotube entrance, facilitates nanotube wetting via grotthuss-facilitated water migration through the charge defect, and then traverses a lower free energy barrier for grotthuss shuttling proton permeation via activated (infrequent event) dynamics. this finding has widespread implications for pt through hydrophobic (and likely other) regions in molecular systems such as proteins, by demonstrating that protons can dynamically create their own solvation pathways that would not be detected by experimental or computational means in absence of an explicit protonic charge defect. the system studied in this work consists of a 29.4 (z-dimension) armchair-type (6,6) single walled carbon nanotube (cnt) with a single layer of graphene and bulk water on either side (figure 1a). the space between the graphene layers was left empty (it is merely intended to provide a low dielectric environment). the graphene layers extend 41.82 and 42.5 in the x and y dimensions and are replicated under periodic boundary conditions. the two slabs of water molecules on either side of the graphene sheets contain seven pairs of k and cl ions collectively. this type of (6,6) cnt has been the focus of previous computational work, partially because its 8 diameter accommodates a single-file chain of water molecules, which is similar to the solvation structure found in some biological channels. as previously reported, the use of standard force field parameters enables spontaneous wetting of a cnt of this diameter. thus, to mimic hydrophobic environments, the lj lj parameter for the cnt carbon atoms was reduced to provide a mostly dry (hydrophobic) cnt in unbiased molecular dynamics simulations, with only two to four water molecules transiently entering the mouth regions (figure 1a). (a) construct of the simulation system. the armchair-type (6,6) cnt structure is assembled between two graphene single layers that separate the bulk water. (b) overview of the proton induced wetting process along with the motion of the excess proton from bulk tube interface into about 4 of the nanotube. (c) real-time densities traces of the channel water molecules starting from a partially dry nanotube with the existence of the excess proton. each bright line can represent the trace of the oxygen atom in a water molecule. (d) simulation with the k inside the nanotube, which remains mostly dry. the hydrated excess proton was treated explicitly with the multiscale reactive molecular dynamics (ms-rmd) method developed by the voth group. the multistate empirical valence bond version 3 (ms-evb3) model and the spc/fw water model were used. all other parameters (for ions and carbon atoms) were taken from the standard charmm22 force field. the depth of the lennard-jones potential well for cnt carbon atoms is scaled to 80% of the standard value to make the cnt more hydrophobic as noted above and hence dry in the absence of ions restrained to be in the cnt. the simulations were run with raptor, an in-house extension of the lammps software. the particle particle, particle mesh method was used to treat long-range electrostatics. the nos hoover thermostat at 300 k and a time step of 1.0 fs were used. to construct the free energy surface for the wetting process inside the cnt, we defined a water occupancy collective variable:1where nh2o is total number of the water molecules and ni is the occupancy of the ith water molecule in a defined rectangular box:2the dimensionless coordinate ri, is defined as3where r0 and ri are the positions of the center of the cnt and the oxygen atom of the ith water molecule, respectively. a box similar to the shape of the cnt was defined by bx=by=4.0 and bx=14, while d was chosen to be 5.0 to allow a smooth transition of the water occupancy from zero (when the molecule is at the graphene water interface) to 1 (when the molecule is inside the cnt). the umbrella-sampling technique and the wham method were used with the bias potential defined by4applied to two independent reaction coordinates: the z-axis position of the positive charge (h, k, or h3o) and the water occupancy number in the cnt. the resulting 2d free energy surface was constructed from 325 sampling windows covering the range of charge locations from 9.0 to 21.0 in 1.0 intervals and for water occupancies ranging from 2 to 14 with an interval of 0.5. the bias force constants were chosen to be 10.0 kcal mol and 5.0 kcal mol, respectively, to ensure sufficient window overlap. as described in the section methods, the cnt studied herein was modified to be more hydrophobic and hence mostly dry. in unbiased md simulations only one to two water molecules transiently enter the mouth regions on either side of the cnt (figure 1a). these waters are partially stabilized by interactions with the graphene atoms, which were simulated with the standard lj parameters. the hydrated excess proton (or more accurate the net positive charge defect associated with an excess proton) was described with the ms-rmd method, which as noted earlier has been shown to successfully model pt in numerous aqueous and biomolecular systems. since there is a large free energy penalty for any ion to shed its solvation shell and enter a nanoconfined hydrophobic region, umbrella sampling was used to calculate the free energy profiles for ion transport through the cnt as described in methods. figure 1 shows how the charge defect associated with the hydrated excess proton strongly influences the cnt hydration. we note that hereafter this net positive charge defect will be referred to as just the hydrated excess proton or the proton even though it is in fact a positively charged defect having more than one proton and water molecule involved in its definition (see ref (5) for more information). when the proton is far from the mouth of the nanotube, no significant change in the hydration can be observed. as the proton approaches the entrance (within a few), the number of pore water molecules increases from 2 to 4. as the proton enters the channel, it drags a few waters with it. surprisingly though, once the proton has entered the mouth of the nanotube, the number of water molecules in the cnt continues to increase by having the waters shuttle through the excess proton charge defect. in other words, the proton shoots waters into the nanotube, thus creating its own water wire for subsequent transport. by the time the excess proton is 23 into the cnt, the tube transitions to the fully hydrated or wet state (figure 1b). to analyze the origin of water molecules that wet the cnt relative to the position of the excess proton, we traced the positions of the water oxygen atoms over the course of a simulation (figure 1c). this demonstrates that water molecules originate on the same side of the graphene sheet as the excess proton defect and hence have to pass through it to enter the cnt. the length of the water wire ahead of the proton fluctuates until it connects with waters from the other side to fully wet the nanotube. therefore, the transport of water molecules into the nanotube is enabled by an unusual manifestation of the grotthuss shuttling mechanism involving the rearrangement of covalent bonds as water molecules pass through the relatively stationary protonic charge defect near the mouth of the channel. to check whether other monatomic ions can also induce a similar wetting process, the same simulations were carried out with k in place of an excess proton. although k pulls four water molecules into the nanotube, two on each flanking side, to form its hydration structure, it does not induce a full wetting transition (figure 1d). the free energy profiles for the ion permeation (figure 2a) are also different for h and k. the energy barrier for k to penetrate 5 into the cnt is over 30 kcal/mol, while that for the excess proton is less than 15 kcal/mol (see figure 2a). the large difference in these two free energy profiles is consistent with the principal that the free energy barrier for an ion to enter a nanoconfined region is strongly influenced by the cost of dehydration. since the excess proton can effectively keep part of its solvation shell as dynamical h5o2 and h7o3 structures, it has a lower barrier for cnt penetration. free energy profiles of the ion permeation and the water occupancy in different simulation systems. the free energy of h (the hydrated excess proton) has much lower energy barrier than the others, displaying the unique features of the excess h. (b) free energy profiles of the water occupancy with the h charge defect (green), k (red), or classical h3o (yellow) fixed at z= 12.0 (23 inside the mouth of the nanotube), compared to the result without the ions present (blue). the induced wetting process is only seen in the system with h. the average errors are (a) 0.45 kcal/mol and (b) 0.30 kcal/mol calculated from 500 ps block averages. moreover, the excess proton charge defect can transiently delocalize the net positive charge over even more water molecules and therefore reduce the dehydration penalty in this fashion. similar to the situation with k, replacing the excess proton with a classical approximation of a hydronium cation (a simple ion with no possibility of grotthuss shuttling and charge defect delocalization) also results in a large free energy cost for cnt penetration (> 25 kcal/mol; see figure 2a). thus, the excess proton s ability to distribute the charge defect to surrounding water molecules within the cnt clearly also contributes to its decreased translocation barrier. to further study the underlying mechanism of this proton induced cnt wetting process, a water occupancy reaction coordinate (see methods, eqs 13) was defined to calculate the free energy profiles for nanotube wetting as well as those for ion transport at different hydration levels. first, we obtained the free energy profile of the wetting process in the absence of an ion (figure 2b, blue curve). similar to previous work, the free energy profile of water occupancy has dry and wet minima (at nw=4 and nw=13, respectively) separated by a barrier (near nw=12). consistent with a dry cnt, the fully wet (water occupied) state is 1 kcal/mol less stable than the dry state. however, when an excess proton is located in the region around z=12 (just 23 inside the cnt), the wetting free energy profile changes dramatically, as seen in figure 2b, green curve. the wet minimum becomes 6 kcal/mol more stable such that wetting becomes spontaneous, and the transition along this wetting coordinate is nearly barrierless. when a k ion is located in the same location (z=12), the barrier for wetting is increased to 5 kcal mol and the wet minima is destabilized by 2 kcal/mol (figure 2b, red curve). the case for the classical h3o ion is even more dramatic, replacing the wet minimum with a 6 kcal/mol barrier (figure 2b, yellow curve). the collected results described above highlight the excess proton s unique ability to self-solvate and project outward a water wire into a hydrophobic space, thus wetting a dry region and thereby decreasing the barrier it has to overcome to transport through it. such a phenomenon may have significant implications for understanding pt through hydrophobic regions of biomolecules and materials. the free energy profiles of figure 2 indicate that the excess proton can induce the wetting of the hydrophobic nanotube and also that the proton has higher conductance (i.e., lower transport barrier) compared to k. are the two processes related? to answer this, two independent collective variables, the water occupancy number and the position of the excess proton charge defect, were sampled from 325 simulation windows to construct a two-dimensional (2d) free energy surface. from the 2d surface (figure 3), a fascinating coupled, stepwise mechanism is clearly seen for the wetting and proton permeation. first, the excess proton is trapped close to the mouth of the nanotube in a free energy minimum 1 kcal/mol more stable than bulk. hummer and collaborators have reported similar results with a proton minimum at the entry of the cnt. this result is also consistent with the amphipathic nature of the hydrated proton, as reported in several theoretical studies. the vacuum and nanotube wall are hydrophobic while bulk water and the graphene surface are more hydrophilic creating an amphipathic interface where the excess proton is stabilized. second, after being trapped at the entry of the nanotube, the proton initiates the wetting transition described earlier by greatly lowering the free energy barrier for water penetration and stabilizing waters in the previously dry space (cf. when the proton is 23 into the cnt (zcec 1213 in figure 3), the relative free energy for wetting becomes very favorable with a negligible barrier (figure 4a, blue, green, and red curves), transitioning the hydrophobic nanotube to the fully wet state. as the cnt hydration level increases, the barrier for the proton transport into the nanotube also greatly decreases (figure 4b) and the proton can progress further into the cnt and eventually through via activated (barrier surmounting) dynamics. indeed, the free energy penalty for the proton moving into the nanotube is greatly reduced from when the nanotube is dry. in fact, the free energy for proton permeation goes from>36 kcal/mol for the dry cnt, which is close to the energy barrier for k, to 18 kcal/mol for the fully wet cnt (figure 4b). it is interesting to note that the lowest barrier for a constrained solvation state is 18 kcal/mol for nc=13 (i.e., the fully wet state), while the free barrier with no solvation constraints is only 15 kcal/mol (figure 2a). thus, the proton induced wetting and proton permeation are dynamic and inherently coupled. the horizontal axis represents the z-position of the hydrated excess proton charge defect, while the vertical axis represents the level of cnt water occupancy. the white dotted line (z=14.7) indicates the position of the graphene layer and the mouth of the nanotube. a stepwise but coupled mechanism can be observed from the 2d free energy surface, in which three steps, trapping wetting permeation, are highlighted by yellow dashed arrows. 1d free energy profiles extracted from the 2d free energy surface in figure 3. (a) free energy profiles of the wetting process when the excess proton charge defect is located at different positions along the nanotube axis. (b) free energy profiles of proton permeation while the nanotube is in different hydration states where nc is defined as the number of confined waters in the nanotube. the 2d free energy surface in figure 3 reveals the sensitivity of the cnt s solvation structure and stability to the position of the excess proton charge defect. the excess proton changes the water occupancy in the cnt even before it enters. although the excess proton is weakly attracted to the entrance to the nanotube, it does not block water penetration as other ions will do. instead the excess proton defect shuttles waters through it into the nanoconfined space via an unique manifestation of the grotthuss shuttling mechanism, thereby lowering its own barrier for subsequent permeation. the 2d free energy surface for k permeation versus nanotube hydration (figure 5a) highlights two important differences between h and k. first, there is no energy minimum that indicates trapping at the mouth of the cnt. this is consistent with the common finding that k prefers to be fully solvated in the bulk rather than at an interface. in fact, the wetting free energy shift (and barrier) remains positive no matter where the k ion resides and is highest (least favorable) when k is at the mouth of the cnt. consistent with previous studies, the water molecules in the cnt are highly mobile in the absence of ions, entering and leaving the tips of the nanotube frequently. however, when k is near the entry of the nanotube, it blocks the water flux, which is entropically unfavorable. in contrast, water molecules in the presence of the excess proton charge defect at the entrance of the nanotube retain their ability to move in and out of the channel through the grotthuss mechanism. thus, delocalization of the excess proton charge defect is essential to decreasing the cost of wetting and ion permeation. 2d free energy surfaces for (a) a k cation and (b) a classical h3o (non-grotthuss shuttling) cation showing the free energy for ion permeation relative to nanotube water occupancy and prepared in the same way as for figure 3. to confirm this hypothesis, we conducted another set of free energy surface calculations with a classical hydronium model (simple h3o cation), in which the charge defect delocalization and grotthuss shuttling are disabled. as discussed above, the free energy profiles for h3o permeation and wetting (shown in figure 2) are more similar to the results for k than to those for h. the 2d free energy surface (figure 5b) reveals that h3o also has a sort of trapping state near the mouth of the cnt as seen for h. however, the classical hydronium does not exhibit the favorable wetting transition (compare figure 3 to figure 5b). in fact, just as with k, water molecules are blocked from entering and leaving the cnt. in order to quantify the degree of charge defect delocalization of the excess proton in the cnt, we analyzed the magnitude of excess positive charge owned by each water molecule in the simulations with h restricted at z=12.5. this is the case when the proton defect center of excess charge coincides with the second cnt water molecule. when the nanotube is dry (nw=4), about 99% of the positive charge is distributed on three water molecules with the percentages 67%, 20%, and 12%, respectively. when the nanotube is fully wet (nw=14), the distribution shifts to 55%, 33%, and 10%. in the bulk, eigen cation, h9o4, with 62% of the charge on the central water molecule and the three surrounding waters that possess 19%, 10%, and 5% of the excess charge. thus, although the charge defect delocalization in the cnt is weaker than it is in the bulk system, because of the strong spatial confinement, it is still significant. moreover, it shifts to a more zundel-like delocalized species in the fully wet nanotube, which will contribute to the stabilization of the fully wet state. thus, charge defect delocalization is again confirmed to be essential to the wetting mechanism and decreases the free energy barrier for ion permeation. for many years, studies of pt in biological systems have largely focused on the identification and the analysis of aqueous pathways interlaced with protonatable residues through which excess protons might migrate (especially via grotthuss shuttling). great effort has been devoted to characterizing internal hydration structures that connect protonatable residues to form such pathways. when hydrophobic cavities are encountered, i.e., those lacking crystallographically resolved water molecules, simulations have often been used to try to identify states of the system (e.g., via oxidation state or conformational changes) that induce wetting. in this manner, mechanisms of pt have been proposed based on the existence and stability of hydrogen-bonded water wires. however, the simulations presented herein suggest that the excess proton itself is strongly coupled to the solvation structure and stability in nanoconfined spaces and hence must be explicitly included in the analysis of internal solvation. by simulating pt through a nanotube penetrating a graphene sheet with ms-rmd, we have discovered that a hydrated excess proton charge defect can induce wetting into a previously dry hydrophobic space. this is a novel wetting process in that water molecules actually pass through the protonic charge defect via grotthuss-like shuttling. other ions have the opposite effect, blocking the diffusion of water into the nanotube when they are close to the nanotube s entrance. thus, this wetting process, which relies on charge defect delocalization and grotthuss shuttling, is unique to a hydrated excess proton (though something similar may be possible for the hydroxide anion). although the present simulations have focused on pt through a cnt, our findings have broad implications for pt in biomolecular and materials systems. just as our simulations have demonstrated in a cnt, an excess proton may actually transiently induce wetting in biomolecular hydrophobic cavities when it is located near a peripheral residue or water cluster. in this manner, the excess proton can create its own aqueous pathway for subsequent charge transport. as shown in this work, water rearrangement around a confined excess proton can be fast (from hundreds of picoseconds to several nanoseconds) relative to the rare events of biomolecular pt (typically microseconds or longer). thus, with the strong correlation between solvation rearrangement and the position of a hydrated excess proton, the two processes of pt and wetting are likely to be strongly coupled (as demonstrated in the cnt). indeed, the concept that pt requires an existing water wire, e.g., one seen in an x-ray crystal structure or md simulations without an explicit excess proton, should be questioned. what is more, computer simulations probing pt mechanisms should include an explicit treatment of the hydrated excess proton (along with its full physics of grotthuss shuttling and charge defect delocalization) to properly capture the coupling between water dynamics, hydration, and pt. using solvation structures to interpret pt mechanisms in the absence of an explicit excess proton can quite possibly lead to incorrect conclusions.

grotthuss shuttling of an excess proton charge defect through hydrogen bonded water networks has long been the focus of theoretical and experimental studies. in this work we show that there is a related process in which water molecules move (shuttle) through a hydrated excess proton charge defect in order to wet the path ahead for subsequent proton charge migration. this process is illustrated through reactive molecular dynamics simulations of proton transport through a hydrophobic nanotube, which penetrates through a hydrophobic region. surprisingly, before the proton enters the nanotube, it starts shooting water molecules into the otherwise dry space via grotthuss shuttling, effectively creating its own water wire where none existed before. as the proton enters the nanotube (by 23), it completes the solvation process, transitioning the nanotube to the fully wet state. by contrast, other monatomic cations (e.g., k+) have just the opposite effect, by blocking the wetting process and making the nanotube even drier. as the dry nanotube gradually becomes wet when the proton charge defect enters it, the free energy barrier of proton permeation through the tube via grotthuss shuttling drops significantly. this finding suggests that an important wetting mechanism may influence proton translocation in biological systems, i.e., one in which protons create their own water structures (water wires) in hydrophobic spaces (e.g., protein pores) before migrating through them. an existing water wire, e.g., one seen in an x-ray crystal structure or md simulations without an explicit excess proton, is therefore not a requirement for protons to transport through hydrophobic spaces.

PMC4515783

pubmed-672

hepatitis c virus (hcv) is a major public health problem all over the world; it is currently estimated that about 85% of those infected with hepatitis c virus will become a chronic carrier and may develop severe end stage liver diseases including cirrhosis and hepatocellular carcinoma. currently, the most important risk factor for infecting by hepatitis c is intravenous drug use (idu) which is the most associated with the sharing of drug injection equipments especially needles, syringes and other paraphernalia. infection with hcv among prison inmates is usually higher than that among the general population mainly because of past history of intravenous drug use and possibly high risk addiction-related behaviors in prison. during the last few years, much attention has been given to the prevalence of blood born diseases including hepatitis c among prisoners. according to limited research studies in iran, prevalence of hcv infection among prisoners with a history of drug injecting varies between 31.5% to 47% in different parts of the country.[68] however, a study in a local prison in fars province revealed 78% prevalence rates of hcv infection among incarcerated drug users. to address the often hidden phenomenon of hcv infection in prisons, considering the limited data on the epidemiology of hcv infection and related risk behaviors in our region, in this study we have focused on this infection and transmission risk factors among prisoners with history of idu in isfahan that could potentially be incorporated into current and future harm reduction initiatives us in iran. in a cross-sectional study, according to self-report and confirm by head of prison's healthcare, the prison inmates who had intravenous drug history or current idus entered into study at march 2009. since the most of our enrollees had sclerosis in peripheral vessels, blood sampling was mostly taken from the femoral vein by the health personnel of the prison setting. blood samples were then sent to the laboratory of infectious diseases research center during 3 hours in cold box to be tested by eilsa (diapro-italy) for the presence of hcv antibody. incarcerated with idu histories were also asked about their demographic characteristics and hcv-related risk behaviors with an interviewer assisted questionnaire. face and content validity of the questionnaire were evaluated by specialists and its reliability was confirmed by chronbach alpha=0.74. the research protocol was approved by the ethical committee of isfahan university of medical sciences in iran. all available means were used to guarantee privacy during interviews and confidentiality. instead of a block for the patient's name, each questionnaire and test tube had an anonymous identification code, which was used for reporting laboratory results, too. statistical analysis was performed using spss for windows (version 16.0, 2007, spss inc, chicago, il, usa). univariate analysis was used to assess association between being hcv positive and related risk factors. the variables that were significant in the univariate analysis (p-value<0.05) were included in the multiple logistic regression to estimate adjusted odds ratio (aor) and 95% confidence intervals (ci). a total of 943 incarcerated idus (938 male and 5 female) participated in the study. the median age of participants was 32.6 years (range: 18-67). the majority of participants (98.6%) were iranian and 523 (55.5%) had ever been married. among the married persons 451 (92%) had been married once, 35 (7%) had been married twice and 5 (1%) had been married more. 143 (15.1%) cases mentioned a history of traveling to another countries. the socio-demographic characteristics of participants overall, the prevalence of hcv-ab seropositivity was 41.6% [table 1]. the median frequency of iv injections was 30 times per month (range 1500), the median duration of addiction was 12 years (range 0.5-57), the median frequency of incarceration was 3 times (range 1-37), and the median of total duration of incarceration was 3.66 years (range 0.08-35). frequency of hcv transmission related factors within study population 71.76% of the participants had illegal sex (contact with other than spouse). among whom, 43% of men had history of having sex with another man (msm), 64.2% had an intercourse with commercial sex workers and 30.9% of them had idu sexual partner. according to self reports, only 31% of them had ever used a condom during sex. within all studied samples with idu history, 13.3% had a single sexual partner and the others had 2 or more, in their lifetime. table 3 shows the odds ratio for some risk characteristics of idus using the logistic regression model. the results of this study indicated that overall prevalence of hcv ab seropositivity among idu inmates in isfahan province is 41.6%. in similar studies in iran, similar results were observed. in zakizad study, the seroprevalence of hepatitis c infection in sari addict prisoners has been reported as 30.8%. in another study by khani et al. the prevalence of hcv was 47.7% among drug addict prisoners in zanjan. in a study which was done in 3 provinces in iran (isfahan, chahamahal bakhtiary and lorestan) in 2003, 34.7% of male prisoners who had been imprisoned for various drug-related offenses including purchase or sale and consumption, were hcv ab positive. in mashhad, the seroprevalence of hcv in incarcerated idus had been reported as 59.4%. in mohammad alizadeh study, the hcv antibody positivity among drug abusers in the central prison of hamedan was 30%. in a study on drug abusers admitted to prison in guilan province, of 460 inmates, 45.4% were hcv antibody positive which in intravenous drug abusers the prevalence reached to 88.9%. the prominent aspect of our study in comparison with other similar studies in iran, is the high number of participants whom all of them had history of intravenous addiction. as a matter of fact, this is one of the first large prevalence studies of blood- born infections among incarcerated idus in iran. in this study, the history of shared drug injection inside prison was one of the main incarceration-related risk factors. this association, has also been reported in other studies in the world.[1315] it seems that, the lack of access new needle/syringe in prisons and consequently needle sharing practice is the main reason of the hepatitis c epidemic in prisoners. our data, highlight for comprehensive and integrated interventions for incarcerated idus to prevent hcv transmission among idu population and community. effectiveness of needle programs in reducing needle sharing among idus has been shown in many countries. in our country, an outreach program for blood born infections prevention was supported by the united nation and the ministry of health of iran in 2003. multiple incarcerations was the another independent risk factor in our subjects which show that the high rates of arrests and incarcerations lead to drug-related offences or other confounding factors inside prison, such as violence. our findings suggest that preventive interventions are compelling now for idus to ensure their safe passage throughout incarceration. in our study, other important modes of transmission were frequency and length of drug injection. according to these results, harm reduction strategies need to be expanded to prevent new hcv infections, particularly among young injectors.[1920] in our study, history of marriage was a protective factor for hcv-infection in the subjects after controlling for all other variables in the logistic-regression model. sexual transmission could play a role in sporadic or community-acquired hcv infections. however, there is contradictory finding regarding the association between marriage and hcv infection in the world. vandelli and colleagues indicated that the risk of sexual transmission of hcv within heterosexual monogamous couples is extremely low or even null. it is shown that, those who have multiple sexual partners, including female sex workers, men having sex with men, and attendees of sexually-transmitted diseases (stds) clinics are the main risk groups for hepatitis c. in the acute hepatitis surveillance study in usa, 18% of newly infected individuals reported sexual contact with an hcv-infected person or multiple sexual partners as their only risk factor for hcv acquisition. for the individual with chronic hcv infection, the estimated risk of sexual transmission of virus was 0% to 0.6% per year for those in monogamous relationships, and 1% per year for those with multiple sexual partners. so, it will be useful to encourage young people to get married to prevent sexually transmitted diseases in our society. in conclusion, according to progressive reported prevalence rates of hcv in the world and the importance of the current and potential burden of hcv-related complications, it is important to primary prevention of hcv infection that will be addressed through implementation of safe injection practices. the mortality associated with hepatitis c is expected to double in the next 10-20 years. it is conservatively estimated that the direct costs related to hepatitis c will be $10.3 billion during the years 2010-2020. the indirect costs of hepatitis c (eg, the loss of productivity during that era) are estimated to total another $54.3 billion due to premature death and $20.6 billion due to disability. in addition, the total cost of therapy for hepatitis c is estimated to be $10,000-$12,000. so, primary prevention will be more cost-effective than secondary prevention of morbidity and mortality from hcv infection through provision of interferon-based therapy. it is good opportunities in prison to have large number of idus over longer period to do specific preventive activities such as needle/syringe exchange and counsel imprisoned idus.

objectives: hepatitis c virus (hcv) infection is a major public health problem worldwide with serious complications. according to the importance of intravenous drug use (idu) as the main risk factor for hcv infection and transmission and prison as the main source of risky behaviors, this study conducted to define hcv infection and related risk factors in prison inmates with history of idu in isfahan province, iran. methods:this is a cross sectional study which the prison inmates with idu history in voluntary basis were enrolled. a validated questionnaire was asked and blood sample was obtained from each subject for the presence of hcv antibody. odds ratio and logistic regression were used for data analysis and p-value<0.05 considered significant. results:i943 inmates with history of idu participated in the study. the overall prevalence of hcv antibody was 41.6%. the main independent risk factors were number of injection in the month [or: 1.006 (1.002- 1.011)], length of drug addiction [or: 1.05 (1.004-1.098)], multiple incarceration [or: 1.15 (1.05-1.23)] and use of needle/syringe share inside prison [or: 4.19 (2.22-7.9)]. in our study, marriage was a protective factor for hcv infection [or: 0.34 (0.18-0.64)] as well. conclusions:according to relatively high prevalence of hcv infection and associated risk factors which observed in this study it is important to primary prevention in prisons through syringe/needle exchange and counsel with imprisoned idus.

PMC3399300

pubmed-673

stainless steel alloys have remained the material of choice despite the emergence of the more recent titanium, composite and polycarbonate orthodontic brackets. stainless steel alloy contains 8%-12% nickel, 17%-22% chromium and other elements such as copper, iron molybdenum, manganese, silicon and sulfur[3-5] in the oral environment, orthodontic brackets are subjected to mechanical and chemical damaging which results in susceptibility to corrosion. corrosion leads to loss of substance from the material, change in its structural characteristics, or loss of structural integrity. due to the electrolytic capabilities of saliva various types of brackets are commercially available and each demonstrates a unique pattern of corrosion. in soldered brackets, this corrosion is due to the presence of dissimilar metals (i.e. the silver solder and the stainless steel), a phenomenon termed galvanic corrosion. metal injection molding (mim) brackets are manufactured as a single unit and therefore do not demonstrate galvanic corrosion. corrosion can have detrimental effects on the surface of stainless steel brackets due to the continuous loss of metal ions. corrosion can increase the surface roughness of the bracket which leads to elevated friction forces between the bracket and the archwire. this increase in friction results in unfavorable distribution of forces and reduces the effectiveness of archwire guided orthodontic tooth movement.[7-8] moreover, by means of increased stress, the friction would further accelerate the corrosion process. the release of metal ions following the corrosion of brackets has concerned clinicians and has instigated research in this field. among these metal ions, furthermore, direct and prolonged contact of orthodontic appliances and the resulting corrosion products have been shown to cause local pain and swelling in the adjacent tissues. edema, gingivitis, gingival hyperplasia, perioral stomatitis, dna instability and altered cellular metabolism are among other reported side effects. corrosion also has detrimental effects on the teeth, and permanent staining around the bracket base has been reported. due to the wide diversity of options regarding bracket selection, orthodontists look for brackets which possess satisfactory biomechanical properties while presenting with a reasonable price. it was mentioned that corrosion has a detrimental effect on the physical properties of brackets and causes unwanted biological side effects; therefore, brackets with minimal corrosion tendencies are more desirable. the present study aimed to investigate five different brands of stainless steel brackets and compare their tendency towards corrosion by measuring ion release in an in-vitro setting. five different brackets (dentaurum, 3 m, ortho organizer, cobas and o.r.g) were selected based on their popularity among faculty members of the orthodontic department. (table 1) ten central incisor brackets were selected from each brand. in order to simulate conventional orthodontic treatment, 8 mm of 0.016 stainless steel archwire (dentaurum, germany) was tied in each bracket using 10 mm of 0.25 mm ligature wire (dentaurum, germany). once the brackets were prepared, they were placed in poly-ethylene capped vials containing 10 ml of artificial saliva at a ph of 7.2. the vials were incubated at 37c for 6 weeks and then they were subjected to thermocycling with 500 temperature cycles from 5c to 55c to simulate the effect of temperature changes in the oral cavity. the brackets were immersed in each bath for 30 seconds with 2 seconds at air temperature in-between the immersions. the details of the brackets selected for the study after thermal-cycling the solutions from the vials were analyzed to determine the amount of nickel, chromium, manganese, and iron using an inductively coupled plasma (icp) spectrometer (icp-oes; varian, vista-pro model, mulgrave, victoria, australia; 1400w applied power). for the purpose of analysis, the icp device had to be calibrated for each of the metal ions. calibration was performed using standard stock solutions (100 mg/ml) prepared by dissolving nitrate salts of the aforementioned ions in distilled deionized water. the standard stock solutions were then diluted to render the concentrations necessary (0.1- 10 mg/ml). once the icp device was calibrated and a standard calibration curve obtained, the samples were analyzed and the readings were recorded. before each reading a drop of 65% nitric acid the addition of nitric acid facilitates the stabilization of the released ions by producing nitrate salts. a vial containing only the artificial saliva was used as the device blank so that the ions present in the saliva itself do not compromise the readings. statistical analyses were performed using spss software for windows (version 11; spss inc, chicago, iii). the kruskal-wallis test was used to analyze the differences among mean ion concentrations in the 5 groups. the median levels of ion release were measured for chromium, iron, manganese and nickel using an icp device (figure 1). the overall amount of ion release was greatest in the cobas bracket, while ortho organizer and org brackets demonstrated minimal corrosion. ion release from brackets made by 5 different manufacturers measured in milligram per liters (mg/l) the level of chromium release was highest in the cobas bracket followed by 3 m, dentaurum, org and ortho organizer. it should be noted that the difference between cobas and 3 m was statistically insignificant (p>0.05) as was the difference between org and ortho organizer (p>0.05). iron release presented the same order that was observed for chromium; however, the levels were generally higher for iron especially in the cobas bracket. considering manganese, it can be observed that the levels are higher in 3 m followed by cobas, dentaurum, org and ortho organizer. the differences between all brackets except dentaurum and org were statistically significant (p<0.05). nickel release however was more pronounced in cobas followed by 3 m, org, ortho organizer and dentaurum. only the cobas bracket demonstrated statistically significant differences with the other brackets (p>0.05). from the ions that were analyzed, iron was released the most, followed by nickel, manganese and chromium (figure 2). a: total iron release, rendered by adding the median values for the analyzed brackets. b: percentage of total ion release attributed to each bracket if we consider looking at the brackets separately, iron release was found to be greater than the other ions in every bracket. due to the importance of the biocompatibility of orthodontic armamentaria, in the present study we chose five popular brackets among orthodontists and determined their susceptibility to corrosion by measuring ion release in anin-vitro environment. the brackets and wires in this study were all manufactured from 18-8 stainless steel (18% chromium, 8% nickel). based on the results of this study, we found that except for manganese, the cobas bracket had the highest level of ion release. furthermore, we observed that among the studied brackets, iron was released more than the other ions. and in contrast with the findings of huang et al. in which nickel release was more profound. moreover the amount of ion release in our samples was higher compared to similar studies.[3,16-17] this difference in ion release may be due to the fact that we chose to tie a segment of archwire to the bracket to better simulate conventional orthodontic treatment. first, by increasing the amount of alloy available to be subjected to corrosion and second, the potential for galvanic corrosion increases since the alloy for the archwire is different from the alloy used in the bracket. another reason for greater levels of ion release may be due to the technique used for detection of ions. the values obtained in the present study closely match the values presented by kuhta et al. who elected to use an icp device for ion detection. the results of the present study indicated that soldered brackets do not necessarily demonstrate less resistance to corrosion when compared to brackets made by metal injection molding (mim). when the org (soldered) and the dentaurum (mim) bracket are compared we observed that, except for iron which was released more by the dentaurum bracket, the other ions were not significantly different. while it has been argued that soldered brackets present a tendency towards galvanic corrosion, mim brackets have increased porosity rendering them susceptible to another kind of chemical breakdown, termed pitting corrosion. this may be the reason why similar studies also found no measurable advantage regarding the utilization of mim brackets. the presence of an archwire may be a confounding factor which may induce galvanic corrosion to mim brackets that cancels out any possible differences. the results of the present study indicated that iron was released more than the other ions, followed by nickel. while this was the overall pattern, in the 3 m bracket, it was observed that manganese was released more than nickel. this finding is in contrast to the findings of karnam et al. who reported that nickel was the major ion released from orthodontic brackets. the findings of this study also suggest that there were no statistically significant differences between the 3 m and dentaurum brackets in any of the ions studied. these differences could be due to study design, addition of an archwire element, testing conditions and measurement methods. it is noteworthy that in the present study, an icp-ms was used to measure the released ions. icp-ms has shown lower detection limit compared to atomic absorption spectrometry used in similar studies. another advantage of the icp device over atomic absorption is that it can measure several ions simultaneously without interference from other ions. this study in agreement with previous findings declares that the values of ions released from orthodontic brackets falls short of the permissible daily doses determined by the world health organization. the maximum tolerable dose of nickel, iron, manganese and chromium for a 60-kg individual is 1.2, 15, 10 and 50-200 mg/day, respectively. however the chronic nature of metallic exposure resulting from orthodontic treatment is a cause for concern. it has been reported that chronic exposure to metal ions can cause alterations in cell morphology and metabolism leading to inflammation and dna instability.[21-26] iron ions in particular have been demonstrated to cause mitochondrial dysfunction in cells with active mitochondrial activity. they have also been linked with lysosome instability which could result in cell death and apoptosis. regarding irreversible damage to oral tissues resulting from orthodontic treatment however, hafez et al. concluded that 6 months after the removal of the orthodontic appliance, no difference was observed between the orthodontic patients and the controls. the findings of this study are based on an in-vitro experimental design, and therefore suffer from the inadequacies related to the simulation of the in-vivo environment. a bracket in the oral environment is subjected to many kinds of chemico-thermal insults including rapid changes in ph, the intake of hot and cold beverages and mechanical challenges. while in many senses the in-vitro design is a drawback, in measuring ion release such a setup is rather useful. not all of the ions released from orthodontic brackets are found in the oral fluids. some of the ions are absorbed into the oral and gingival tissues, while others are ingested and could disperse into distant organs. the advantage of the in-vitro design is that it presents the total ions released from the bracket, which could better show the true effect of this phenomenon. another drawback to this study is that we did not evaluate the effects of different wire-bracket combinations and only used a single wire for all of the brackets. based on the findings of the present study, regarding ion release levels, the org and ortho organizer brackets were superior while dentaurum and 3 m did not demonstrate any significant differences.

statement of the problem: stainless steel brackets release metallic ions following the process of corrosion in the oral environment. these released ions have potential adverse effects on health, friction between wire and bracket, staining, strength of brackets. choosing a bracket with favorable corrosive properties; therefore, should be a goal of every practitioner. purpose: the goal of this study is to compare the amount of corrosion among five different brands of brackets using inductively coupled plasma (icp) mass spectrometry. materials and method: five different brands of brackets (dentaurum, 3 m, ortho organizer, cobas and o.r.g) were chosen and ten brackets were selected from each brand. a piece of stainless steel wire was ligated to each bracket. the bracket-archwire complex was then immersed in artificial saliva. subsequently, the samples were analyzed using an icp device and the levels of iron, chromium, nickel, and manganese ions were measured. results: the findings of this study demonstrated that iron was released the most from the tested brackets, followed by nickel. we also found that the cobas bracket had the most ion release among the tested brackets (p<0.05), while ortho organizer and org performed favorably. there was no significant difference between dentaurum and 3 m (p>0.05). conclusion: based on the results, ortho organizer and org brackets are suggested in terms of resistance to corrosion.

PMC5103473

pubmed-674

it is well known that the stress transfer between an implant device and a bone is not homogeneous when young's moduli of the implant device and the bone are different; this is defined as stress shielding. in such conditions, bone atrophy occurs and leads to the loosening of the implant and refracturing of the bone. therefore, it is desirable if the stiffness (young's modulus) is not too high compared to that of bone. implant devices are mainly made from metallic biomaterials such as stainless steels, co-cr alloys, and titanium (ti) and its alloys. young's moduli of these metallic biomaterials are generally much greater than that of the bone. young's moduli of the most widely used stainless steel for implant devices, sus316l stainless steel and co-cr alloys, are around 180 gpa and 210 gpa, respectively. young's moduli of ti (pure titanium) and its alloys are generally smaller than those of stainless steels and co-cr alloys. for example, ti and its alloy, ti-6al-4v eli, which are widely used for constructing implant devices, have a young's modulus of around 110 gpa. however, this value is still higher than that of the bone, that is, 1030 gpa. ti alloys are grouped into -, (+ )-, and -type alloys. young's moduli of-and (+ )-type titanium alloys such as ti and ti-6al-4v eli are higher than those of -type titanium alloys. therefore, -type titanium alloys are advantageous for the development of titanium alloys with low young's modulus for biomedical applications. researchers have been focusing on reducing young's moduli of -type titanium alloys for use in biomedical applications because they are composed of toxicity- and allergy-free elements. a number of -type titanium alloys mainly composed of toxicity- and allergy-free elements and with low young's moduli have been developed or are still being developed. their young's moduli are approximately below 80 gpa in solution-treated conditions. young's modulus of a material can be different depending on the type of measurement methods used, such as tensile tests, three-point bending tests, and free resonance methods. the lowest value of young's modulus reported for the polycrystal -type titanium alloy, ti-35nb-4sn, or ti-24nb-4zr-7.9sn, subjected to severe cold working, is around 40 gpa. the authors have also developed a -type titanium alloy, ti-29nb-13ta-4.6zr, referred to as tntz, that is composed of toxicity- and allergy-free elements and that has a low young's modulus. young's modulus of tntz subjected to solution treatment and measured by a resonance method has been found to be around 60 gpa. this value is lowered to around 55 gpa by severe working such as severe cold rolling and cold swaging. the strength as well as young's modulus of titanium alloys is a very important factor for their long-term use in implants for biomedical applications. in particular, dynamic strength such as fatigue strength is highly important. developing the fatigue strength and simultaneously lowering young's modulus is somewhat difficult because they are opposite natures when the fatigue strength of -type titanium alloys used in biomedical applications with maintaining the young's modulus as low as possible is currently being developed. concerning young's modulus, the level of the value of young's modulus, which is effective to prevent the stress shielding between the implant made of the low-young's modulus -type titanium alloy and bone should be proved. the effect of young's modulus on bone atrophy has previously been investigated using implants made of titanium alloys with different young's moduli. this paper chiefly describes the simultaneous improvement of the dynamic strength and lowering young's modulus of the -type titanium alloy, tntz. the effect of young's modulus on bone atrophy and bone remodeling has also been discussed in this paper. improving static strength such as tensile strength can be achieved by employing strengthening mechanisms such as work hardening, grain refinement strengthening, precipitation strengthening, and dispersion strengthening. one of the best ways to increase tensile strength while maintaining a young's modulus low is to generally introduce many dislocations by ordinal severe cold working such as severe cold rolling and swaging and special severe cold working such as high pressure torsion (hpt), accumulative roll-bonding (arb), and equal channel angular pressing (ecap). figure 1 shows the relationships between the tensile properties and working ratio of tntz subjected to cold working by general cold rolling or swaging. the relationships between young's modulus and working ratio of tntz subjected to severe cold working by general severe cold rolling or swaging are shown in figure 2. the tensile strength and 0.2% proof stress increase with an increase in the working ratio and become almost equal to those of ti-6al-4v eli (having tensile strength of around 800 mpa) with good elongation of tntz subjected to both cold rolling and cold swaging. young's modulus of tntz subjected to cold rolling or cold swaging is almost constant with increasing working ratio. young's modulus of tntz subjected to cold rolling tends to decrease when the working ratio is high because the trend of the formation of the texture becomes significant. figure 3 shows the tensile properties of tntz subjected to hpt as a function of the number of rotations, n. it also shows the tensile properties of tntz subjected to solution treatment and severe cold rolling. the tensile strength under hpt increases significantly with an increase in the number of rotations, in contrast, the elongation decreases with an increase in the number of rotations. young's modulus of tntz subjected to hpt is almost constant with increasing the number of rotations although it decreases a little with increasing the rotation number. as shown in figure 4, the dynamic strength, that is, fatigue strength of severe cold worked tntz is not high as compared to that of tntz subjected to solution treatment. the fatigue strength is significantly improved by conducting aging treatment after solution treatment or thermomechanical processing including severe cold working and aging treatment. the phase or the phase precipitates in the matrix phase by aging treatment. therefore, the fatigue strength can be significantly improved by precipitation strengthening caused by the precipitation of the or phase. however, as seen in figure 5, young's modulus increases by the precipitation of the or phase because young's moduli of these phases are much higher than that of the matrix phase. the phase precipitation significantly increases the strength and young's modulus of the alloy as compared to the phase precipitation although the phase enhances its brittleness. therefore, a small amount of phase precipitation is expected to improve the fatigue strength of tntz while maintaining its young's modulus fairly low. for this purpose, short-time aging at a fairly low temperature, which enhances phase precipitation by a small amount, is effective. figure 6 shows young's moduli of tntz subjected to solution treatment (st), severe cold rolling (cr), and aging after solution treatment at a temperature of 573 k(at) as a function of the aging time. up to an aging time of around 10.8 ks, young's modulus is below 80 gpa, which is a tentative target value for low young's modulus. figure 7 shows fatigue properties (s-n curves) of tntz subjected to solution treatment (st), severe cold rolling (cr), and aging for 3.6 ks (at3.6) and 10.8 ks (at10.8) at a temperature of 573 k. the fatigue strength of tntz is improved by aging treatment for 10.8 ks (at10.8), whereas young's modulus is lower than 80 gpa. thus, employing the proper precipitation method for the phase, that is, short-time aging at a relatively low temperature improves the fatigue strength of tntz while keeping young's modulus low. the addition of a small amount of ceramic particles in the matrix is also expected to improve the fatigue strength of -type titanium alloys while maintaining low young's modulus. figure 8 shows young's modulus of tntz with y2o3 additions subjected to severe cold rolling as a function of y concentration. young's modulus is nearly constant at around 60 gpa with increasing y concentration. figure 9 shows the s-n curves of tntz with 0.2 mass% and 0.5 mass% (tntz-0.2ycr and 0.5ycr, resp.) subjected to severe cold rolling after solution treatment and the s-n curves of tntz subjected to solution treatment or cold rolling after solution treatment. relationships between tensile strength and elongation of tntz added with different amounts of y2o3 subjected to severe cold rolling (0.05ycr: tntz-0.05ycr, 0.1ycr: tntz-0.1ycr, 0.2ycr: tntz-0.2ycr, 0.5ycr: tntz-0.5ycr, and 1.0ycr: tntz-1.0ycr) and tntz subjected to severe cold rolling after solution treatment (tntcr) are shown in figure 10. the balance of the tensile strength and elongation of tntz with y2o3 additions is excellent. while using low-modulus titanium alloys, some surgeons specializing in spinal diseases, such as scoliosis, spondylolisthesis, and spine fracture, pointed out that the amount of spring back in the implant rods should be small so that the implant offers better handling ability during surgeries. the implant rods undergo bending when they are manually handled by surgeons within the small space inside the patient's body for in situ spine contouring. it is considered that the amount of spring back depends on both the strength and young's modulus of the implant rod. if two implant rods having the same strength but with different young's moduli are used, the implant rod having lower young's modulus shows greater spring back. implant rods made of low modulus titanium alloys exhibit lower young's modulus, resulting in greater spring back. thus, low young's modulus, which is one of the key features of -type titanium alloys such as tntz as a metallic biomaterial, is obviously a desirable property for patients but becomes an undesirable property for surgeons. titanium alloys, which satisfy the requirements of both surgeons and patients with regard to young's modulus of the implant rod, are currently being developed. the amount of spring back is considered to be small for the alloy having higher young's modulus as compared to that of one having low-young's modulus as schematically shown in figure 11. therefore, the low-young's modulus -type titanium alloy, whose young's modulus varies and becomes high only at the deformed part, is considered to reduce spring back and satisfy the low-young's modulus condition as well. this concept is called self-adjustment of young's modulus. in general, the young's modulus of metals and alloys does not drastically change by deformation. however, in the case of certain metastable -type titanium alloys, nonequilibrium phases such as,, and phases appear in the matrix during deformation. if young's modulus of the deformation-induced phase is higher than that of the original phase, young's modulus of only the deformed part of the implant rod increases, whereas that of the nondeformed part remains low. in orthopedic operations performed for the treatment of spinal diseases, the implant rod is bent by the surgeons so that it corresponds to the curvature of the spine. therefore, if a suitable titanium alloy is employed as the implant rod material, spring back can be suppressed by the deformation-induced phase transformation that occurs during bending in the course of operation, while low young's modulus can be retained for patients. in general, young's modulus of phase is much greater than those of the, ,, and phase. among these phases, the,, and phase one of the candidate alloys with self-adjustable young's modulus for biomedical applications has been reported to be ti-12cr. figure 12 shows young's moduli of ti-12cr subjected to solution treatment (ti-12cr-st) and severe cold rolling (ti-12cr-cr) along with those of tntz subjected to solution treatment (tntz-st) and severe cold rolling (tntz-cr). ti-12cr-st exhibits low young's modulus of<70 gpa; this value is comparable to that of tntz-st, which has been developed as a biomedical -type titanium alloy having low young's modulus. tntz-cr also shows low young's modulus almost equal to that of tntz-st. however, in the case of ti-12cr, young's modulus increases by cold rolling and that of ti-12cr-cr is>80 gpa. the deformation-induced phase was detected in ti-12cr, but no induced phase was detected in tntz. therefore, the increase in young's modulus of ti-12cr is probably the deformation-induced phase transformation. figure 13 shows the tensile properties of ti-12cr-st, ti-12cr-cr, tntz-st, and tntz-cr. the tensile strengths of both ti-12cr-st and tntz-st show an increase, but the elongation due to cold rolling tends to decrease. further, the tensile strengths of ti-12cr-st and ti-12cr-cr can be higher than those of tntz-st and tntz-cr, respectively. moreover, the elongations of ti-12cr-st and ti-12cr-cr are>10% and ~10%, respectively. high strength is an essential requirement from the viewpoint of practical application, although such high strength could lead to undesirable spring back. therefore, the fundamental composition of ti-12cr makes it one of the preferred candidates for use in spinal fixation devices as a biomedical titanium alloy with the ability to self-adjust its young's modulus. in the case of some types of internal fixation devices implanted into the bone marrow such as femoral, tibia, and humeral marrow, in the case of screws used for bone plate fixation, and in the case of implants used for children, which otherwise would grow into the bone, it is essential to remove the internal fixation device after surgery owing to certain specific indications; these indications include significant local symptoms such as palpable hardware, wound dehiscence/exposure of hardware, or athletes returning to contact sport [16, 17]. the assimilation of removable internal fixation device with the bone due to precipitation of calcium phosphate might cause refracture of the bone during the removal of the fixation device. therefore, in these cases, it is essential to prevent the adhesion of the alloys with the bone tissues. hence, considering this requirement, it is essential to inhibit the precipitation of calcium phosphate. it is reported that zr, which is nontoxic and allergy-free element, has the ability to prevent precipitation of calcium phosphate and ti alloys with zr contents exceeding 25 mass% prevent the formation of calcium phosphate, which is the main component of human bones. thus, ti-30zr-mo has been proposed as low young's modulus titanium base biomaterials for use in removable implants. figure 14 shows young's moduli of ti-30zr-xmo alloys subjected to solution treatment and those of the alloys considered for comparison. young's modulus of ti-30zr-xmo is lower than that of the alloys considered for comparison except tntz. young's modulus of 6mo shows a minimum value of around 60 gpa, and tntz also shows low young's modulus. in orthopedic applications, ideal biomedical implant materials are required to have high strength and low young's modulus. the elastic admissible strain, defined as the strength-to-modulus ratio, is a useful parameter considered in orthopedic applications. the higher the elastic admissible strain is, the more suitable are the materials for such applications. figure 15 shows the distribution of the as-solutionized ti-30zr-xmo alloys and the alloys considered for comparison in the plot of elastic admissible strain against elongation. 6mo and 7mo exhibit larger elongation and higher elastic admissible strain than those of the other ti-30zr-xmo and sus316l, cp ti, ti64 eli, and tntz. figure 16 shows the density of the cell cultured in 7mo and the alloys considered for comparison, for 24 h. 7mo has the highest value of cell density. therefore, 6mo and 7mo show promising potential to be new candidates for use in biomedical applications. it is very important to prove that the implant has young's modulus similar to that of the bone, which will inhibit bone atrophy and good bone remodeling [1, 4]. of course, the geometry of implant is another factor to control young's modulus. studies have been conducted on the implantation of intramedullary rods and bone plates made of low-young's modulus -type titanium alloy (tntz), conventional practical (+ )-type titanium alloy (ti-6al-4v eli), and conventional stainless steel (sus316l) into fracture models made into the tibiae of rabbits [8, 22]. young's moduli of tntz, ti-6al-4v eli, and sus316l stainless steel used for intramedullary rods, which were measured by three-point bending tests, were 58, 108, and 161 gpa, respectively. in both cases, the implantation of intramedullary rod and the implantation of bone plate, bone atrophy, and bone remodeling have been reported to be the least and the best, respectively, for tntz. figure 17 [8, 22] shows the x-ray photographs of the fracture models implanted with intramedullary rods made of tntz and sus316l stainless steel at 24 weeks after implantation. bone atrophy can be observed at the upper rear portion of the tibia for the intramedullary rod made of sus316l stainless steel, but no bone atrophy is seen for the intramedullary rod made of tntz. large bone formation can be observed at the front part of the tibia for the intramedullary rod made of sus316l stainless steel, but very small bone formation can be observed at the front part of the tibia for the intramedullary rod made of tntz. therefore, low young's modulus is effective in inhibiting bone atrophy and leads to excellent bone remodeling. further study on the effect of young's modulus on the bone remodeling has been done by implanting bone plates made of tntz, ti-6al-4v eli, and sus316l stainless steel into the fracture models made in tibiae of rabbits. only for the case of the bone plate made of tntz, the increase in the diameter of the tibia and the double-wall structure in the intramedullary bone tissue has been reported to be observed as shown in figure 18. for figure 18, the inner wall bone structure is the original cortical bone, namely, the remained old cortical bone and the outer wall bone structure is newly formed one. this is the possible result of the bone remodeling with the low-young's modulus bone plate. furthermore, it is necessary to understand the level of young's modulus, which is effective for inhibiting bone atrophy and enhancing bone remodeling. figure 19 shows the profiles of the extracted bone plates made of tntz subjected to solution treatment (tntz-st), tntz subjected to aging after solution treatment (tntz-at), and sus316 l stainless steel (sus316l) attached to the tibiae of rabbits at 52 weeks after implantation. young's moduli of tntz-st, tntz-at, and sus316l, which were measured by three-point bending tests, were 58, 78, and 161 gpa, respectively. the upper and side surfaces of each bone plate made are covered by newly formed bone, but a fairly large amount of newly formed bone can also be observed on the heads of the screws made of tntz-st and tntz-at, which are surrounded by circle marks. figure 20 shows the optical micrographs of the bone state beneath the bone plates made of tntz-st, tntz-at, and sus316l. bone atrophy can be observed in all cases but becomes more distinct with increasing young's modulus; it is the highest for sus316l, followed by tntz-at and tntz-st. it is expected that titanium alloy with even lower young's modulus will be advantageous in inhibiting bone atrophy and lead to much better bone remodeling. titanium base alloys with low young's modulus have been proved to be effective for inhibiting bone atrophy and enhancing bone remodeling by conducting animal tests in rabbits. therefore, low young's modulus titanium alloys are expected to be useful in practical applications such as implant devices used for replacing failed hard tissue. however, considering the ease of the operation, other properties such as small spring back, which is effective in maintaining the bending shape of the implant in the body, and low yielding stress and high ultimate strength, which lead to ease in achieving permanent deformation of the implant in the narrow space in the body, are also important. metallic biomaterials, which satisfy the demands of both patients and surgeons, are highly required.

-type titanium alloys with low young's modulus are required to inhibit bone atrophy and enhance bone remodeling for implants used to substitute failed hard tissue. at the same time, these titanium alloys are required to have high static and dynamic strength. on the other hand, metallic biomaterials with variable young's modulus are required to satisfy the needs of both patients and surgeons, namely, low and high young's moduli, respectively. in this paper, we have discussed effective methods to improve the static and dynamic strength while maintaining low young's modulus for -type titanium alloys used in biomedical applications. then, the advantage of low young's modulus of -type titanium alloys in biomedical applications has been discussed from the perspective of inhibiting bone atrophy and enhancing bone remodeling. further, we have discussed the development of -type titanium alloys with a self-adjusting young's modulus for use in removable implants.

PMC3132537

pubmed-675

morphine and codeine are naturally occurring alkaloids in opioid plants, have long been used as a drug, and are also abused. while the presence of illicit drugs or their metabolites in urine is an evidence of intake, their concentrations in blood are expected to correlate with their effects on the central nervous system. codeine is a potent -opioid receptor agonist which is used for the treatment of adult cough. simultaneously, there have been athletes in sports competitions who use a larger dose in order to improve performance. this practice is contrary to the principle of fair competition and also harmful to the health of the athletes ' body. heroin as one of the most widely abused drug, rapidly metabolized to 6-monoacetylmorphine (6-mam) once inside the human body. this specific heroin metabolite 6-mam is detected at a higher concentration usually within 2 to 4 hours, and after six hours, has not been detected in the urine. the absence of 6-mam in urine, however, morphine is both a well-known pharmaceutical agent and an important metabolite of codeine and heroin which have relatively long a detection time. morphine and codeine analysis of urine is used in forensic toxicology to study drug addiction. there are numerous papers published about the simultaneous determination of morphine and codeine in human fluids, including the micellar electrokinetic chromatography (mekc) method, disposable pipette extraction (dpx) method, high performance liquid chromatography method, liquid chromatography-mass spectrometry, and liquid chromatography/triple quadrupole tandem mass spectrometry (lc/ms/ms) method [68]. several gas chromatography-mass spectrometry (gc-ms) methods have been developed for the analysis of codeine, morphine, or other opiates. much attention has been directed to the confirmation of morphine and codeine in urine by gc-ms. a few methods have been developed specifically for the analysis of 6-acetylmorphine (6-am) with morphine and codeine because all three drugs are often present after heroin use. assays of morphine and codeine by gc-ms are capable of high sensitivity, specificity, and selectivity. gc-ms is superior to other analytical methods which provide important diagnostic value to study the drug abuse. the aim of this study was to establish methods and seek out more reliable identification and quantitation of morphine and codeine for detection addicts sample. although through in saliva is another approach; the reliability of saliva analysis is limited by the fact that analyte levels, and even the availability of required sample volume, are again dependent on several physiological factors, nutrition and fluid intake, while the biological effects of the consumed illicit substance may also be a significant factor. the identification of chronic consumers or the late verification of a single intake is feasible using hair as a matrix, but it is not suitable for the early verification of consumption. urine is a preferable matrix for analytical purposes in comparison with saliva because of the minimal discomfort caused to sampled individuals, so it is widely available. the simple and effective ethyl acetate extraction was employed in our work, and ethyl acetate was adopted because of its high extraction efficiency. propionic anhydride was chosen as the derivatization reagent because it exhibited better effect than acetic anhydride or trifluoroacetic acid anhydride, which could provide preferable stability, and the disadvantage of acetyl derivatives indistinguishable from morphine and the 6-am can be avoided.. evaluated propionic anhydride, mbtfa, hfaa, and bstfa for gc-ms analysis of 6-am. they concluded that propionic anhydride gave accurate, precise, and sensitive results while providing compatibility with other methods on the same gc-ms instrument. residual derivatization reagent in the injector will react with drugs in other methods not intended for derivatization. the derivatization procedure accommodates the analysis of opioids commonly requiring gc-ms confirmation in urine. concentrations of the analytes in the samples were expected to be smaller than the low end of the therapeutic range (25 ng/ml), which highlighted the importance of efforts aimed at increasing the sensitivity of detection. the relative standard deviation of the retention parameters of the target compound was required not to exceed 5% relative standard deviation. morphine [10 g/ml in methanol] and codeine [10 g/ml in methanol] solutions were obtained from the institute of forensic science under the ministry of justice (shanghai, china). sodium hydroxide (purity>98.0%) was purchased from sigma-aldrich trading co (shanghai, p.r., china), and ethyl acetate (purity>98.0%) was purchased from siyou chemical reagent co., ltd (tianjin, china), and propionic acid anhydride (purity>98.0%) was purchased from sinopharm chemical reagent co., ltd (beijing, china). pyridine was from shenbo chemical co., ltd (shanghai, china). while methanol was obtained from siyou chemical reagent co., ltd (tianjin, china). ultrapure water was prepared by a milli-q purification system from millipore (bedford, usa). analysis was performed on an agilent 6890n gas chromatograph (gc) coupled with an agilent 5975b mass spectrometer (ms, agilent technologies, wilmington, de, usa). the capillary column used was a hp-1ms [30 m 0.25 mm, 0.25 m]. helium was the carrier gas at a flow rate of 1.0 ml/min. the temperature program was: initial temperature, 100c for 1.5 min; ramp at 25c/min to 280c and held for 15 min; injection temperature, 250c; and transfer line, 280c. electron impact ionization was performed at 70 ev energy and at a 230c ion source temperature. sim mode was applied to quantify analyzes using target ions at m/z 341, 397, and 268 for morphine propionyl compound and m/z 229, 355, and 282 for codeine propionyl compound (figure 1). the primary standard stock solutions of morphine (100 g/ml) and codeine (100 g/ml) were separately prepared in 10 ml volumetric flasks with urine; 10% naoh was added dropwise until ph 9.09.2 was reached, and 1.0 ml of borax buffer solution was added. to this, 3 ml of extraction solvent (ethyl acetate) was added and vortex-mixed on a vortexer for 2.0 min, followed by centrifugation at 3000 r/min for 5 min. the supernatant organic layer was transferred into a 5 ml glass test tube and dried under air stream at 60c. the dried residue was reconstituted in 50 l of propionic anhydride and 20 l of pyridine. all reagents were vortex-mixed, then heating for 3 min at 80c and dried under air stream at 60c. the dried residue was reconstituted in 50 l of methanol, and 1 l of this solution was injected into gc-ms. specificity was determined by analysis of blank urine, without addition of morphine and codeine to determine possible interference with these compounds. to evaluate the linearity, the calibration curves were generated using the analyte peak area by linear regression on three consecutive days. the lloq was estimated in the process of calibration curve construction and was defined as the lowest concentration for which precision (rsd) was better than 20%. qc samples at three concentration levels (50, 200, and 1600 ng/ml for morphine and codeine) were analyzed to assess the accuracy and precision of the method. again, the assays were performed on three separate days, and on each day six replicates of the qc samples at each concentration level were analyzed. the assay precision for each qc level was determined as the relative standard deviation (rsd) of the measured concentrations. the intra- and interday precisions were required to be below 15%, and the accuracy was required to be within 15%. stability in urine was assessed in the autosampler at room temperature for 12 h. the effect of three freeze-thaw cycles was also investigated. figure 2 shows the typical chromatograms of a blank urine sample spiked with morphine and codeine. no interfering endogenous substances were observed at the retention times of the morphine and codeine. calibration curves for morphine and codeine were generated by linear regression of peak area ratios against concentrations, respectively. the regression equation for the calibration plot were y=2270.9c+202.3 with r=0.9974 for morphine, and y=3099.0c+31625.7 with r=0.9958 for codeine (y is the peak area of analyte, and c is the concentration of analyte in human urine), and concentrations are in the range 252000 ng/ml for morphine and codeine, respectively. the lloq for morphine in human urine was 25 ng/ml and the precision and accuracy at lloq were 10.5% and 87.6%, respectively. the lloq for codeine in human urine was 25 ng/ml and the precision and accuracy at lloq were 13.8% and 88.9%, respectively. the precision of the method was determined by calculating rsd for qcs at three concentration levels over three validation days. intraday precision was 12% or less and the interday precision was 13% or less at each qc level. the aforementioned results demonstrate that the values are within the acceptable range and the method is accurate and precise. the recovery of morphine and codeine was evaluated by comparing peak area ratios of extracted qc samples with those of reference qc solutions reconstituted in blank urine extracts. all the stability studies of morphine and codeine in human urine were conducted at three concentration levels (50, 200, and 1600 ng/ml for morphine and codeine) with three replicates for each concentration. the stability results showed that morphine and codeine in human urine were stable during three freeze-thaw cycles. stability of morphine and codeine extracts in the sample solvent on autosampler was also observed over a 12 h period. a stable, selective, and sensitive gc-ms method has been developed for the simultaneous determination of codeine and its metabolite morphine in human urine. this developed method with derivatization for sample preparation was successfully applied for the determination of morphine and codeine in human urine for methodological study.

a sensitive and selective gas chromatography-mass spectrometry (gc-ms) method was developed and validated for the determination of morphine and codeine in human urine. the gc-ms conditions were developed. the analysis was carried out on a hp-1ms column (30 m 0.25 mm, 0.25 m) with temperature programming, and helium was used as the carrier gas with a flow rate of 1.0 ml/min. selected ion monitoring (sim) mode was used to quantify morphine and codeine. the derivation solvent, temperature, and time were optimized. a mixed solvent of propionic anhydride and pyridine (5: 2) was finally used for the derivation at 80c for 3 min. linear calibration curves were obtained in the concentration range of 252000.0 ng/ml, with a lower limit of quantification of 25 ng/ml. the intra- and interday precision (rsd) values were below 13%, and the accuracy was in the range 87.2108.5%. this developed method was successfully used for the determination of morphine and codeine in human urine for forensic identification study.

PMC3810378

pubmed-676

disporopsis pernyi (hua) diels, which belongs to disporopsis genus of liliaceae family, mainly grows in south asia such as vietnam, laos, thailand, and yangtze river basin of china. it was a well-known traditional chinese medicine which has been widely used for the treatment of abnormal sweating, chronic cough, women postpartum weakness and irregular menstrual cycle, and so forth. the medical value of disporopsis genus plants has not got much attention until the beginning of 21st century. the roots of the plant are rich in bioactive substances which are possible to have considerable medicinal value and can be used for hypertension cough, inflammation, and tumor. study suggests that a total of 4 polyphenolic compounds are found in extract including rutin, luteolin, quercetin, and betulinic acid, and the phenolic compounds contribute significantly to the antioxidant and antimicrobial activities [2, 3]. rutin has both antihypertensive effect and antidiabetes effect [4, 5]. luteolin which has anti-inflammatory, antibiosis, and anticancer properties has been used for relieving cough and eliminating phlegm. in addition, it has potential anti-hiv effect [6, 7]. quercetin can be used for relieving cough and eliminating phlegm and for hypertension and hyperlipemia; also, it has neuroprotective and antiproliferative activities [8, 9]. acid can kill human melanoma cell without hurting healthy cell and inhibit the hiv-1 infection. additionally, recent study shows that it also has the inhibition effects of cerebroma and leukocythemia [10, 11]. the four compounds are major bioactive constitutes in the extract of disporopsis pernyi (hua) diels roots. so far, there is no report on the content of the 4 polyphenolic compounds in disporopsis pernyi (hua) diels. uplc is a simple and quick tool for the quantitative determination of the bioactive constituents in pharmaceutical industry [1214]. as rutin, luteolin, quercetin, and betulinic acid the hplc-grade methanol and acetonitrile used were purchased from caledon (canada) and formic acid was obtained from dima company (beijing, china). the rutin, luteolin, quercetin, and betulinic acid were purchased from the national institute for the control of pharmaceutical and biological products in china. the purity of the standard compounds was 98%; their chemical structures are shown in figure 1. disporopsis pernyi (hua) diels were collected from songtao which is in guizhou province of china. standard stock solutions of rutin, luteolin, quercetin, and betulinic acid were dissolved in methanol, at concentration of 1.0 mg/ml. all standard solutions were filtered through 0.22 m syringe filter. the extraction was carried out using 5.0 g of powdered roots. it was dissolved in 50 ml of 70% ethanol solution and back-flow for 60 min. after filter and rotary evaporation to no ethanol smell, 50 ml acetic ether was added and extraction was done three times. the extract and washing liquid were combined and filtered and then evaporated to dryness under reduced pressure in a rotary evaporator. the dried extract was dissolved in methanol and diluted to a 5 ml volumetric flask. the hplc system used was a shimadzu nexera x2 uplc (kyoto, japan) chromatograph equipped with a solvent delivery unit (lc-30ad), an autosampler (sil-30ac), a column oven (cto-20a), a degasser (dgu-20a5r), and a photodiode array detector (spd-m20a). separation was conducted on a shim-pack xr-ods column (2.0 75 mm, 1.6 m; shimadzu cooperation, japan). the column temperature was set at 30c. the mobile phase consisted of water containing 0.1% formic acid (a) and acetonitrile (b). the composition of the mobile phase was 5% (b) for 02 min, 5%10% (b) for 24.5 min, 10%40% (b) for 4.511 min, 40%60% (b) for 1113 min, 60%70% (b) for 13-14 min, 70%80% (b) for 1416 min, 80%90% (b) for 16-17 min, 90% (b) for 1720 min, and it was held for 3 min and then reequilibrated to 5% (b) until the end of the analysis. the flow rate was 0.2 ml/min and the injection volume was 5 l. the detection wavelengths of all standards and samples were in the uv at 210, 254, and 280 nm. the 4 standard compounds were accurately weighed and dissolved in methanol to prepare stock solutions at a concentration of 1.0 mg/ml. stock solutions of the compounds were serially diluted to construct calibration curves. the diluted concentrations of compounds were plotted against the peak area on the calibration curves and the linearity was measured from the correlation coefficient. blank samples were analyzed in triplicate and the area of the noise peak was calculated as the response. the lod and loq were calculated as lod=3.3 sd/s and loq=10 sd/s, where sd is the standard deviation of the response and s is the slope of the calibration curve. the precision was calculated by analyzing sample extracts containing low and high concentrations of the compounds. the precision was represented by the relative standard deviation (rsd), which was calculated using the equation rsd=(standard deviation/mean) 100. the precision was measured three times in a single day (intraday precision) and over three consecutive days (interday precision). the recovery was calculated as follows: recovery (%)=(( detected concentration initial concentration)/spiked concentration) 100. we used pda detector in this experiment, which could select each wavelength of chromatogram. in our study, we took into account the question that most of the components we studied also have good absorption at wavelength of 350 nm and 370 nm [15, 17]. by comparing the resolution and response at different wavelength, the results showed that the resolution and response of the four components at 350 nm and 370 nm are not as good as the wavelengths 210, 254, and 280 nm we chose. also, we chose 210 nm to detect betulinic acid for its good response. combined with the literature reports, methanol-water, acetonitrile-water, and methanol-acetonitrile-water were examined as mobile phase as well as the type (formic acid and glacial acetic acid) and concentration (0.01%, 0.05%, and 0.1%) of the acid. a shim-pack xr-ods column was employed for the simultaneous determination of the 4 compounds, as it has been the most frequently used technique in the chemical analysis of herbal medicines by uplc. peak resolution and shape of the compounds were considered better indicators when 0.1% formic acid was used as a modifier. taking peak shape, degree of separation, the symmetrical factor, and other factors into consideration, acetonitrile-0.1% formic acid water solution was determined as gradient elution process. the linearity of the calibration curve was measured by the correlation coefficient (r), which ranged in value from 0.9992 to 0.9997 for each compound. the lod and loq were 0.1370.264 g/ml and 0.4560.881 g/ml, respectively (table 1). the intra- and interday precision, which were represented by the rsd values, were rsd<2.0%. the recoveries of the 4 compounds were in the range 96.2%102.6%, with rsd<2.0% (table 2). the results indicate that the developed analytical method was accurate and precise for the analysis of the 4 compounds in disporopsis pernyi (hua) diels. the method we developed was applied to determine the 4 compounds in disporopsis pernyi (hua) diels successfully. the calculated contents of the four compounds were 5.63 g/g for rutin, 2.51 g/g for luteolin, 3.87 g/g for quercetin, and 2.41 g/g for betulinic acid. the uplc method mentioned here represented an excellent technique for simultaneous determination of rutin, luteolin, quercetin, and betulinic acid in the extract of disporopsis pernyi (hua) diels roots, with good sensitivity, precision, and reproducibility. the method gives a good resolution among the four components with the analysis time (25 min). furthermore, the method can be used as quality control of polyphenolic compounds in disporopsis pernyi (hua) diels roots and will play a reference role on the determination of polyphenolic compounds in other medicinal plants or pharmaceutical preparations.

disporopsis pernyi (hua) diels, which belongs to genus disporopsis, has been widely used for the treatment of abnormal sweating, chronic cough, and so forth. an ultra-performance liquid chromatography (uplc) analysis was developed for the determination of rutin, luteolin, quercetin, and betulinic acid in disporopsis pernyi (hua) diels roots. uplc analysis was conducted by using a shim-pack xr-ods column with gradient elution with the mobile phase of acetonitrile and water containing 0.1% formic acid and with a flow rate of 0.2 ml/min, detected at 210, 254, and 280 nm. the method was precise, with relative standard deviation<2.0%. the recoveries for the four components in disporopsis pernyi (hua) diels were between 98.5 and 100.9%. the average contents of rutin, luteolin, quercetin, and betulinic acid in roots were 5.63, 2.51, 3.87, and 2.41 g/g, respectively. the method was accurate and reproducible and it can provide a quantitative basis for quality control of disporopsis pernyi (hua) diels.

PMC4700192

pubmed-677

osteoarthritis is the most common joint disorder in the united states and many other industrialized countries (1). in adult individuals aged 60 years and above, the prevalence of symptomatic knee osteoarthritis is estimated at about 10% in men and 13% in women (2). however, the prevalence of osteoarthritis varies according to the definition used, the specific joints under investigation, and the characteristics of the study population (2). the age-standardized prevalence of radiographic knee osteoarthritis in adults aged 45 years or above was 19.2% among the participants in the framingham study and 27.8% in the johnston county osteoarthritis project (3). in addition, in the third national health and nutrition examination survey (nhanes iii), about 37% of participants aged 60 years and above exhibited radiographic knee osteoarthritis (2, 3). furthermore, the age- and sex-standardized incidence rates of symptomatic hip, knee, and hand osteoarthritis were estimated at 88, 240 and 100 (per 100,000 person-years), respectively, according to a study conducted in massachusetts, usa (4). the etiology of osteoarthritis is multi-factorial and is generally considered as a product of interaction between systemic and local factors (1, 2). from this point of view, certain individuals may have a genetic predisposition to develop osteoarthritis, but this condition (osteoarthritis) will be established only if an injury to the joint takes place. essentially, the systemic risk factors for osteoarthritis include age (1,3,5), sex (6, 7), race and ethnicity (8, 9), genetics (10, 11), congenital and other developmental factors (12, 13) and diet (14, 15). on the other hand, local factors consist of obesity (1, 2), injuries (2, 16), occupation (2, 17), physical exercise (2, 18), mechanical factors, alignment, or laxity (2, 19, 20). yet, the relative importance of each risk factor may vary for different joints affected, for different stages of osteoarthritis, for the development (establishment) versus the progression of disease, and for radiographic compared to symptomatic disease (2). it should be noted that the number of individuals who develop osteoarthritis is expected to increase due to the aging of the population and the obesity epidemic (2). a similar aging and obesity situation is evident also in albania, a post communist country in south eastern europe which is experiencing a significant socioeconomic transition in the past twenty five years. due to the demographic transition in the past couple of decades, in albania, the proportion of individuals aged 65 years and above has increased up to 11% (21). this proportion is going to increase further given the decline of fertility rates (21), a gradual increase in life expectancy (21, 22) and the ongoing emigration of young people (22). in addition, overweight and obesity pose a serious public health concern in view of the rapid changes in lifestyle/behavioral patterns, where processed foods are increasingly replacing traditional foods in albania (22). according to the world health statistics 2014 report, the prevalence of obesity in albania is 21.7% in males and 20.5% in females (23). the burden of musculoskeletal disorders has also increased in albania in the past two decades. overall, the share of these disorders accounted for 8.5% of the total burden of disease in 1990, whereas in 2010 it increased up to 11.0% (22, 24). in relative terms, there was evidence of a stronger increase in females (with 3.7% increase in the proportional burden of disease) compared to males (only 2.0%) (22, 24). however, specific information on osteoarthritis including its prevalence and associated risk factors is scarce for albania. in this context, we aimed to describe the distribution of the main risk factors among primary health care users diagnosed with osteoarthritis in albania, which was the most isolated communist country in europe until 1990. our study involved all individuals who were diagnosed with osteoarthritis over a two-year period (from january 2013 to december 2014) in several primary health care centers in tirana, the albanian capital. selection of the primary health care centers included in this study was based on the probability proportional to size principle. on the whole, during this two-year period, 1179 adult individuals were diagnosed with osteoarthritis (521 men aged 60.110.6 years and 658 women aged 58.19.6 years). according to the recommendations of the american college of rheumatology for the clinical diagnosis of osteoarthritis, the diagnosis of osteoarthritis was based on the following criteria (25, 26): (i) history of the disease: self-reported presence of pain, aching, stiffness, or other symptoms in the joints affected by osteoarthritis; (ii) physical examination: difficulties in flexion/extension and rotation of the joints (range of motions), tenderness, crepitations, or enlargement of the joints; (iii) laboratory findings: erythrocyte sedimentation rate, rheumatoid factor, c-reactive protein and uricemia, and; (iv) radiological findings: joint space narrowing and presence of osteophytes. fisher s exact test was used to compare the distribution of the major risk factors (smoking, alcohol intake, body mass index, genetic factors, major trauma, weight lifting, heavy physical exercise and preexisting inflammatory diseases) among primary health care men and women diagnosed with osteoarthritis. conversely, binary logistic regression was used to assess the sex-differences regarding the main risk factors among primary health care users diagnosed with osteoarthritis in tirana. initially, crude (unadjusted) odds ratios (ors) and their respective 95% confidence intervals (cis) were calculated. next, multivariable-adjusted ors and their respective 95% cis were calculated in logistic regression models controlling (adjusting) simultaneously for all the main risk factors for osteoarthritis (smoking, alcohol intake, body mass index, genetic factors, major trauma, weight lifting, heavy physical exercise and preexisting inflammatory diseases). statistical package for social sciences (spss, version 17.0) was used for all the statistical analyses. table 1 presents the distribution of risk factors among primary health care users diagnosed with osteoarthritis in tirana during the period 2013-2014. it was substantially higher in men than in women (37.2% vs. 13.1%, respectively; p<0.001). similarly, the prevalence of alcohol intake was remarkably higher in men compared to women (78.1% vs. 19.6%, respectively; p<0.001). there was no significant difference in the prevalence of overweight or obesity among male and female patients with osteoarthritis (p=0.111). on the other hand, the prevalence of the predisposing genetic factors was significantly higher in women compared to men (35.1% vs. 23.6%, respectively; p<0.001). major trauma experienced in life (different types of accidents) were somehow equally distributed between men and women (p=0.195). conversely, the proportion of men reporting weight lifting was significantly higher compared to women (17.5% vs. 7.0%, respectively; p<0.001). likewise, the prevalence of self-reported heavy physical exercise (at work, at home, or at leisure time) was considerably higher in male patients compared to their female counterparts (36.3% vs. 18.5%, respectively; p<0.001). on the other hand, the prevalence of preexisting inflammatory conditions (including rheumatoid arthritis, ankylosing spondylitis, metabolic arthropathy, or other inflammatory diseases) was significantly higher in women than in men (11.9% vs. 8.1%, respectively; p=0.033) (table 1). distribution of risk factors among primary health care users diagnosed with osteoarthritis in tirana during 2013-2014. table 2 presents the sex-differences with regard to selected risk factors between primary health care men and women diagnosed with osteoarthritis in tirana during the period 2013-2014. in crude (unadjusted) logistic regression models, there was an inverse association of female gender with smoking (or=0.25, 95%ci=0.19-0.34) and especially with alcohol intake (or=0.07, 95%ci=0.05-0.09). on the other hand, there was evidence of a positive association between female gender and constitutional (genetic) factors (or=1.75, 95%ci=1.35-2.27). there was no significant association with past major trauma experiences (p=0.341), but an inverse association of female gender with weight lifting (or=0.36, 95%ci=0.24-0.52) and heavy physical exercise (or=0.40, 95%ci=0.31-0.52). sex-differences regarding risk factors among primary health care users diagnosed with osteoarthritis in tirana during 2014-2015.*odds ratios (or: women vs. men), 95% confidence intervals (95%cis) and p-values from binary logistic regression. overall p-value and degrees of freedom (in parentheses). upon simultaneous adjustment for all the risk factors presented in table 2, female gender was inversely associated with smoking (or=0.39, 95%ci=0.27-0.56), alcohol intake (or=0.08, 95%ci=0.06-0.10), overweight but not obesity (or=0.65, 95%ci=0.46-0.91 and or=0.74, 95%ci=0.46-1.18, respectively), weight lifting (or=0.38, 95%ci=0.22-0.66) and heavy physical exercise (or=0.69, 95%ci=0.46-1.03). on the other hand, female gender was positively related to genetic factors (or=2.17, 95%ci=1.55-3.04) and preexisting inflammatory diseases (or=1.53, 95%ci=0.93-2.53) (table 2). our main findings relate to a negative (inverse) association of female gender with lifestyle/behavioral factors including smoking and alcohol intake, overweight (not obesity though), weight lifting and heavy physical exercise. on the other hand, female gender in this study conducted in tirana similar to our findings, women have been largely shown to be at a higher risk for osteoarthritis compared to men including also development of more severe forms of this condition (2, 6, 7). regarding the genetic predisposition, several studies have demonstrated that osteoarthritis is inherited and this tendency may vary by joint site (2). in addition, some studies have reported an inverse (negative) association between general joint hypermobility, a lone benign trait, with hand and knee osteoarthritis and serum cartilage oligometric matrix protein levels (2). the association with obesity in our study is compatible with prior international reports (1, 2). obesity and overweight have long been recognized as important risk factors for osteoarthritis, especially of the knee (1, 2). according to the arthritis, diet, and activity promotion trial, weight loss combined with exercise, but neither weight loss nor exercise alone, were effective in decreasing pain and improving function in obese elderly individuals with symptomatic knee osteoarthritis (27). as for the role of physical activity, there are conflicting findings in the international literature. interestingly, the overall level of physical exercise may in itself increase the risk of osteoarthritis (2). findings from the framingham study indicate that older individuals who engaged in high levels of physical exercise (consisting of leisure time walking and gardening) had a threefold greater risk of developing radiographic knee osteoarthritis than their sedentary counterparts (2, 18). similar results were obtained in another study in which women who had a high lifetime level of physical exercise had also a high prevalence of hip osteoarthritis (28). conversely, some other studies indicate that, in the absence of acute injury, recreational (but moderate) long distance running and jogging does not increase the risk of osteoarthritis (29). our study may have some limitations, mainly related to the recruitment of participants and the information obtained through the interview. in our study, we included several primary health care centers (on a probability proportional to size basis) which are assumed to be representative of the overall primary health care facilities in tirana. our data collection period lasted for two years and included all male and female adults diagnosed with osteoarthritis in the primary health care centers under investigation. hence, the sample of patients with osteoarthritis included in our study is assumed to be representative of the overall primary health care users in tirana. nonetheless, representativeness of our study sample can not be assumed for the overall adult population of albania given the fact that we did not include in our study health centers from other districts of albania. from this point of view, our findings should be interpreted with caution and should be limited to adult primary health care users of the albanian capital namely tirana. in our study, the diagnostic criteria for osteoarthritis were based on the instruments and criteria recommended by the american college of rheumatology (25,26), which is a clear strength pointing to employment of standardized and well-validated tools for diagnosis of osteoarthritis in the albanian population. on the other hand, in our study, data on behavioral/lifestyle determinants was based on interview, which may have been subject to different information biases. thus, male and female participants may have tended to report differently about their lifestyle characteristics. if so, we can not entirely exclude the possibility of reporting bias for the self-reported information about lifestyle/behavioral factors including smoking, alcohol intake and physical exercise. potential limitations aside, this study offers useful evidence about the distribution of the main risk factors for osteoarthritis in adult individuals diagnosed with osteoarthritis in albania. this information may support health professionals and decision-makers in albania for evidence-based health planning and policy formulation in order to control the toll of osteoarthritis in this transitional society.

aim: we aimed to describe the distribution of the main risk factors among primary health care users diagnosed with osteoarthritis in albania, a post-communist country in south eastern europe. methods:our study involved all individuals who were diagnosed with osteoarthritis over a two-year period (january 2013 december 2014) in several primary health care centers in tirana, the albanian capital. on the whole, during this two-year period, 1179 adult individuals were diagnosed with osteoarthritis (521 men aged 60.110.6 years and 658 women aged 58.19.6 years). according to the criteria of the american college of rheumatology, the diagnosis of osteoarthritis was based on the history of the disease, physical examination, laboratory findings and radiological findings. binary logistic regression was used to assess the sex-differences regarding the major risk factors among individuals diagnosed with osteoarthritis. results:in multivariable-adjusted logistic regression models, female gender was inversely associated with smoking (or=0.39, 95%ci=0.27-0.56), alcohol intake (or=0.08, 95%ci=0.06-0.10), overweight but not obesity (or=0.65, 95%ci=0.46-0.91 and or=0.74, 95%ci=0.46-1.18, respectively), weight lifting (or=0.38, 95%ci=0.22-0.66) and heavy physical exercise (or=0.69, 95%ci=0.46-1.03). conversely, female gender was positively related to genetic factors (or=2.17, 95%ci=1.55-3.04) and preexisting inflammatory diseases (or=1.53, 95%ci=0.93-2.53). conclusion: this study offers useful evidence about the distribution of the main risk factors for osteoarthritis in adult individuals diagnosed with osteoarthritis in albania. this information may support health professionals and decision-makers in albania for evidence-based health planning and policy formulation in order to control the toll of osteoarthritis in this transitional society.

PMC4500384

pubmed-678

the genus flavivirus currently includes 86 viruses, of which 73 are grouped into 53 species (1). more than 40 of these flaviviruses are known to be pathogenic for humans and other vertebrates, in which they cause a variety of clinical diseases from mild febrile illness to severe encephalitis and/or hemorrhagic fever. flaviviruses are small enveloped viruses with positive-sense single-stranded rna genomes consisting of an open reading frame (orf) flanked by 5 and 3 noncoding regions (ncr). the polyprotein encodes three structural proteins (the capsid [c], membrane [m], and envelope [e] proteins) and seven nonstructural (ns) proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5) (2, 3). flaviviruses have extensive geographic distributions and diverse arthropod vectors, and many of them infect vertebrate hosts (4). among the arthropod-borne flaviviruses there is a correlation between phylogenetic relationships and virus-vector-host interactions (58). on the basis of virus neutralization studies and, separately, the association of arthropod vectors with vertebrates, 4 major groups of flaviviruses are recognized: the tick-borne flaviviruses (tbfvs), the mosquito-borne flaviviruses (mbfvs), no-known-vector flaviviruses (nkvs), and no-known-vertebrate-host flaviviruses (5, 6, 9, 10). the mosquito- and tick-borne borne flaviviruses contain important animal and human pathogens, including yellow fever virus (yfv), dengue virus (denv), west nile virus (wnv), st. louis encephalitis virus (slev), japanese encephalitis virus (jev), and tick-borne encephalitis virus (tbev), which, in total, annually cause millions of human infections worldwide. subsequently, on the basis of phylogenetic analysis of a relatively limited number of viral envelope gene sequences, the mosquito-borne flaviviruses were subdivided into the aedes- or culex-associated viruses. some of the aedes-transmitted viruses induce hemorrhagic fevers in humans and primates, whereas many of the culex-transmitted viruses are associated with encephalitic infection in avian species (5). in addition to nkvs, sokoluk virus (sokv), entebbe bat virus (entv), and yokose virus (yokv), which share ancestral roots with the aedes-associated mbfvs, were isolated from vertebrates but not from arthropods. they form a clade normally referred to as the entebbe bat virus group, which is closely related to the yfv and edge hill virus (ehv) clades. it was previously suggested that these viruses may have lost their association with arthropods during their evolution and divergence (11). in addition to these three groups of flaviviruses which infect vertebrates, additional groups have been isolated only from mosquitoes or sand flies and under experimental conditions appear to infect and replicate only in insect cell lines. accordingly, they have tentatively been defined as insect-specific flaviviruses, until appropriate taxonomic criteria are devised and approved. the cell-fusing-agent virus (cfav) was the first of many insect-specific flaviviruses (classical insect-specific flaviviruses [clsfs ]) to be identified. cfav was first isolated from an aedes aegypti cell line in 1975 (12), and its genomic sequence was characterized in 1992 (13). cfav and a subsequently identified heterogeneous group of related clsfs form a distinct lineage in flavivirus phylogenies. these viruses have subsequently been isolated from a wide range of mosquito species in many countries throughout the world (1422). an additional separate group of flaviviruses that do not appear to infect vertebrate cells currently consists of nine viruses: lammi virus (lamv) (23), ilomantsi virus (ilov) (24), marisma mosquito virus (mmv) (19), donggang virus (donv) (unpublished data; genbank accession number nc_016997), chaoyang virus (chaov) (25, 26), nounane virus (nouv) (27), barkedji virus (bjv) (28), nhumirim virus (nhuv) (29), and nanay virus (nanv) (30). these nine viruses form a distinct clade that sits within the mbfv group of viruses. moreover, flavivirus-like genomic sequences integrated within the genomes of aedes mosquitoes (21, 31) have also been identified., tamana bat virus (tabv), ngoye virus (ngov), and mogiana tick virus (mgtv), are also considered to be flaviviruses because they share similar genome organizations with the recognized flaviviruses (1, 32). the only representative of a sand fly-borne flavivirus, saboya virus (sabv), sits in the group of mosquito-borne flaviviruses primarily associated with aedes mosquitoes in africa (5, 33). flavivirus rna has also been discovered in phlebotomine sand flies from algeria (34) and portugal (unpublished data; genbank accession number hm563684). we report here on the detection, isolation, complete genome sequence, and phylogenetic assignment of a novel sand fly-borne flavivirus in psathyromyia abonnenci (pa. abonnenci) sand flies. we propose the name ecuador paraiso escondido virus (epev), based on the village where the sand flies were collected. we also discuss the possible significance of the discovery of a new world (nw) sand fly-associated virus that shares a common ancestral lineage with nonvectored old world (ow) flaviviruses. sand flies were trapped during march 2011 in the locality of paraiso escondido (008503n, 791749w), pichincha province, ecuador. the identified sand flies were pooled on the basis of species and sex with up to 50 individuals per pool and placed in 1.5-ml tubes for storage at 80c. pools of sand flies were ground in 600 l of eagle minimal essential medium (supplemented with 7% fetal bovine serum, 1% penicillin-streptomycin, and 1% l-glutamine [200 mm ]) in the presence of 3-mm tungsten beads using an mm300 mixer mill (qiagen, courtaboeuf, france) as described previously (37). a 200-l aliquot was used for viral nucleic acid (na) extraction with a biorobot ez1-xl advanced virus extraction minikit (qiagen) and eluted in a 90-l volume. five microliters of this solution was used for sybr green reverse transcription (rt)-pcr and seminested pcr assays with the primers and by the protocol described previously (34, 38). bands of the expected size were purified (amicon ultra centrifugal filters; millipore) and directly sequenced. a 100-l volume of the pcr-positive sand fly pool was inoculated onto c6/36 cell monolayers in 25-cm tissue culture flasks after being mixed with 900 l of l15 medium enriched with 1% penicillin-streptomycin, 1% l-glutamine (200 mm), 5% kanamycin, 3% amphotericin b (fungizone), and 5% tryptose phosphate broth solution. after incubation at room temperature for 1 h, 5 ml fresh medium containing 5% fetal bovine serum (fbs) was added. the flasks were incubated at 28c and examined daily for the presence of a cytopathic effect (cpe). cell lines of different vertebrate species, including human (sw13), hamster (bhk), monkey (vero), and amphibian (xtc), were inoculated with the supernatant medium of epev-infected c6/36 cells obtained at passage 6. two flasks were inoculated for each cell line and incubated at either 32c or 37c. a 100-l volume of the pcr-positive sand fly homogenate was also inoculated onto vero cells. in the absence of a cpe, regardless of the absence of a cpe, 5 serial passages were performed, and each was tested by real-time rt-pcr (38) for the presence of epev rna. a total of 15 l of undiluted epev-containing supernatant medium (passage 4) or 15 l of epev-containing supernatant medium (passage 4) diluted 1:10 with minimal essential medium was injected intracerebrally into 2-day-old newborn of1 mice. nucleic acids were purified from the brain tissues and used for the detection of epev rna by a specific real-time rt-pcr assay (38). additional mice were injected with supernatant medium containing a pool of infected brain tissue from the previously infected mice. they were observed for 14 days and then euthanized, and nucleic acids were purified from the brain tissues and used for detection of epev rna by a specific real-time rt-pcr assay (38). veterinary services of the ministry of agriculture has approved animal experimentation under the number a1301309. the epev strain (passage 6 in c6/36 cells) and the original homogenate of the epev-positive sand fly pool were used independently for complete genome characterization through next-generation sequencing (ngs). briefly, 140 l of each sample was incubated at 37c for 7 h in 30 u of benzonase endonuclease (catalog number 70664-3; novagen) to eliminate cellular dna and rna and preserve encapsidated viral particles. the encapsidated viral particles were then processed for rna extraction using a biorobot ez1-xl advanced viral rna minikit (qiagen) without an rna carrier. random amplification was performed using a tagged random primer for rt and using tag-specific and random primers for pcr amplification (applied biosystems). the pcr products were purified (amicon ultra centrifugal filters; millipore), and quantification was done using a qubit fluorometer. two hundred nanograms of the sample was processed for sequencing using an ion pgm sequencer (life technologies sas, saint aubin, france) (39). analyses of the sequencing data were performed using the clc genomics workbench program (v6.5; qiagen). the clarified supernatant medium of epev-infected c6/36 cells was incubated at 4c overnight in a polyethylene glycol 6000 (sigma-aldrich) solution, to concentrate the virions. the concentrate was centrifuged at 3,000 rpm for 30 min, and the pelleted sediments were resuspended and ultracentrifuged at 30,000 rpm for 3 h using a fiberlite f14-6 250y fixed-angle rotor with a thermo scientific heraeus multifuge x3r ultracentrifuge. the phase 4 fraction of the centrifuge tube was collected to provide the sample that was then used for viral rna extraction. a 400-l volume was used for viral rna extraction as mentioned above and treated with 10 u/l tobacco acid pyrophosphatase (epicentre); circularization was performed with 5 u/l t4 rna ligase (ambion) at 10c overnight. specific primers were designed to perform rt-pcr and to amplify the extremities using an access rt-pcr one-step kit (promega). the positive samples were gel purified using a qiaquick gel extraction kit (qiagen), and both strands were sequenced. flavivirus sequences available in the genbank database were collected to obtain a data set including a representative of at least one sequence for each species of the genus flavivirus available as an amino acid sequence of the complete orf. alignments of the amino acid sequences of the complete orf were generated using both the clustal w2 (40, 41) and muscle (42) programs, available at the embl server (http://www.ebi.ac.uk/tools/), and refined manually for comparison. the effect of mass removal of regions of ambiguous alignment by use of the gblocks algorithm (43) was also investigated. phylogenetic trees were reconstructed using markov chain monte carlo (mcmc) analysis implemented in the mrbayes software program (v3.1.2) (44). the analysis was performed using the wag substitution model with a gamma-distributed rate variation among sites and default priors. five independent markov chains were run for 10 million generations, with the first 10% of samples being discarded as burn-in. stationarity was confirmed on the basis of effective sample sizes of>400 using the tracer program (v1.4.1) (45). a maximum clade credibility tree was summarized using the treeannotator program, which annotates all nodes with posterior probability support values. the amino acid distances among the representatives of the flaviviruses were calculated for the complete orf and ns5 protein by use of the p-distance method in mega software (v5) (46). nucleotide distances were also calculated for the 1-kb region in the ns5 protein reported by kuno et al. putative cleavage sites from amino acid sequence alignments that included epev and other flaviviruses were deduced and compared according to the proteolytic processing cascade pattern previously described for the flavivirus orfs (47). predicted glycosylation and conserved cysteine residue sites were determined for epev and compared with those of known flaviviruses. conserved enzymatic patterns of epev were also analyzed and compared with those of known flaviviruses. possible ribosomal frameshifting sites on the epev genome were investigated using the methods described previously (4850). the complete genomic sequence of epev has been submitted to the genbank database and may be found under accession number kj152564. one thousand three hundred sand flies (1,150 females and 150 males) were collected in paraiso escondido village, and the following species were identified morphologically: nyssomyia trapidoi, psychodopygus panamensis, psathyromyia aragoi, pa. abonnenci, lutzomyia hartmanni, trichophoromyia reburra, and a trichophoromyia sp. twenty-six pools were tested by pcr for the presence of flavivirus rna. abonnenci sand flies contained flavivirus rna when the pan-flavivirus rt-pcr (38) was used, and on the basis of quantitative real-time pcr, the pool contained>10 genome copies. importantly, this epev-positive pool consisted exclusively of females that were neither gravid nor engorged, thus precluding the possibility that a blood meal from a vertebrate host might be the source of epev in the tested sample. on the basis of the fact that all the other sand fly pools were negative and the positive pool contained nonengorged sand flies, it seems reasonable to assume that a single sand fly might have been positive for epev rna. thus, the evidence strongly supports the concept that epev must have infected and reproduced efficiently in the positive sand fly. however, we can not rule out the possibility that epev might have been detected in mosquitoes if they had been collected from the same area where the sand flies were trapped. c6/36 cells infected with the sand fly homogenate from the pcr-positive pool showed a cytopathic effect within 3 days and 1 day at the 4th passage and 6th passage, respectively (fig. amplification of the virus using the pan-flavivirus rt-pcr assay (38) for the first three passages was also confirmed, with threshold cycle values being 18.32, 16.21, and 19.01, respectively. in order to test whether or not epev could infect human and/or other vertebrate cells in culture, vero, bhk, sw13, and xtc cell cultures moreover, no cpe was detected following five serial passages of vero cells inoculated with the original epev-positive sand fly homogenate. viral rna detection tests using the pan-flavivirus rt-pcr assay also produced negative results, indicating that epev did not replicate in any of the vertebrate cell lines tested. finally, newborn mice inoculated with the original epev-positive sand fly homogenate showed no signs of disease, and epev rna could not be detected in the brains of these newborn mice. thus, under the conditions employed in these experiments, epev did not infect or replicate in vertebrate cells or newborn mice. initially, an almost complete sequence of c6/36 cell-derived virus was obtained by ngs. unfortunately, the sand fly homogenate-derived virus gave very poor sequence readouts after ngs. reads with minimum lengths of 30 nucleotides and with a minimum quality of 99% per base were trimmed using the clc genomic workbench program (v6.5) and mapped to reference sequences previously obtained by sanger sequencing. parameters were set to ensure that each accepted read mapped to the reference sequence for at least 50% of its length and had a minimum of 80% identity to the reference sequence. thus, the reported sequence was derived from ngs of the c6/36 cell-derived virus and sanger sequencing of the gaps. missing regions, including segments of the capsid protein, the pr protein, and the 5 and 3 noncoding regions, were successfully amplified after cyclization of the genome and sequencing of the concatenated 3 and 5 termini. this resulted in determination of the complete genomic sequence of epev (genbank accession number kj152564), which was 10,761 nucleotides (nt) in length and which had an orf of 10,323 nt (positions 120 to 10,442). the single complete orf was predicted to encode a polyprotein of 3,441 amino acids (aa). the lengths and positions of the genes or genomic regions are shown in table 1. genome organization of epev at each terminus of the genome, 2 nucleotides which are conserved among members of the entire flavivirus genus (5153), i.e., 5-ag and ct-3, were detected. mutagenesis studies have previously shown that ct-3 is probably a recognition site for the replication complex to initiate rna synthesis (54, 55). the 5 and 3 ncrs are thought to function as recognition sites for virus translation, replication, and possibly, assembly (56). the lengths of the epev 5 and 3 ncrs were 119 and 316 nt, respectively. on the basis of comparative alignment of the nucleotide sequences of all flaviviruses, the epev 316-nt 3 ncr is significantly shorter than that of any of the previously studied flaviviruses and lacks a number of conserved sequences found in the mosquito-borne flaviviruses. the only conserved sequence detected, conserved sequence 1 (cs1), was 5-catattgacaccagggaaaagac-3, which is located near the 3 terminus of the 3 ncr. cs1 is complementary to a conserved sequence within the n-terminal coding region of the capsid protein. this complementarity results in a long-range rna interaction that promotes cyclization of the flavivirus genome, forming a panhandle-like rna structure required for viral rna synthesis (57). in the epev genome, the first 12 nucleotides of cs1 are complementary to a region in the capsid protein. conserved sequence 2 (cs2) is also found in all mosquito-borne flaviviruses. the nucleotides in a short stretch prior to cs1 in epev showed very poor homology with the nucleotides at the equivalent positions of other flaviviruses, and the degenerate reverse primer 3utr-mos (5-ggtctccwmtaacctctag-3), which anneals to cs2 (58), was unable to anneal to this related region in the epev 3 untranslated region. the highly conserved pentanucleotide motif 5-cacag-3 at the terminus of the 3 ncr was identified. this pentanucleotide is conserved among almost all mosquito- and tick-borne flaviviruses (the exception is 5-caccg-3 in murray valley encephalitis virus [mvev]), and it has been reported to be an element essential for viral rna synthesis, whereas its functional role is not known (5961). the predicted pentapeptide cleavage sites and conserved domains of epev were compared with the corresponding sites of all available flavivirus polyprotein sequences generated using amino acid sequence alignments. a comparison of these cleavage sites with those of other flaviviruses is shown in table s1 in the supplemental material. the similarities detected are consistent with the phylogenetic position of epev, which is explained below in phylogenetic analysis and genetic distances. putative polyprotein cleavage sites of epev polyprotein the mature virion capsid protein (virc) is cleaved from the nascent capsid protein (anchc) after a dibasic amino acid sequence at positions 101 and 102 and before a c-terminal hydrophobic domain (cthd) by the viral serine protease (vsp) in the cytoplasm of the host cell. for epev the proposed residues arg-arg were also identified in other flaviviruses. however, asn at position 1 has not been reported for virc/anchc but it was reported for other cleavage sites. the cthd protein is cleaved from the prm polyprotein after cys (at position 119 for epev) by a host signalase (hs) in the lumen of the endoplasmic reticulum (er). cys at position 1 was not reported for flaviviruses, but it is consistent with the (3, 1) rule of von heijne (62). pr is cleaved from the m protein by the host cell furin or a similar enzyme (63, 64) in the lumen of the er. a specific pattern of arg-x-(arg/lys)-arg was reported for this cleavage site (65). for epev, we propose the signature his-arg-(arg/arg)-ser after the amino acid at position 230 or ser-pro-arg/ser-ile after the amino acid at position 234. the m protein is cleaved from the envelope protein after gly at position 308 by an hs in the lumen of the er, which is consistent with the rule of von heijne (62). the envelope protein is cleaved from the nonstructural ns1 protein after ala at position 799 by a signalase (hs) in the lumen of the er, which is consistent with the rule of von heijne (62). the ns1/ns2a cleavage site occurs after ala at position 1153, which is consistent with the rule of von heijne (62). the ns2a/ns2b cleavage site occurs after arg at position 1384, which is cleaved by the viral serine protease, but thr at position 2 is not consistent with the dibasic requirement at positions 1 and 2. experimental evidence is required to determine whether the proposed cleavage site is functional or not. the ns2b/ns3 cleavage site occurs after two arg residues at positions 1514 and 1515, which are cleaved by the viral serine protease. the ns3/ns4a cleavage site occurs after a double arg at positions 2138 and 2139, which are cleaved by the viral serine protease. the ns4a/2k cleavage site occurs after gln and arg at positions 2264 and 2265, respectively, which are cleaved by the viral serine protease. the 2k/ns4b cleavage site occurs after ala at position 2288, which is cleaved by a host signalase. the ns4b/ns5 cleavage site occurs after a double arg at positions 2537 and 2538, which are cleaved by the viral serine protease. the pr gene region contains 1 possible n-linked glycosylation site and 6 cys residues which are conserved among other flaviviruses except the clsfs, the e-gene region contains 2 possible n-linked glycosylation sites and 12 cys residues which are conserved in all other flaviviruses except the clsfs, and the ns1 protein contains 2 possible n-linked glycosylation sites and 12 cys residues which are highly conserved. cysteine residues are required for stabilizing disulfide bridges in all mosquito-borne viruses (3). a sequence homologous to the fusion peptide, a 14-aa motif thought to be involved in virus fusion with cellular membranes (66), is present at positions 406 to 419 in the ns1 protein. it conforms with the asp-arg-gly-trp-x-x-(gly/his)-cys-x-x-phe-gly-lys-gly motif observed for all mosquito-borne flaviviruses (67, 68). the n-terminal sequence of the flavivirus ns3 protein contains conserved regions (boxes 1 to 4) that have significant similarity to serine proteases and belong to the trypsin superfamily (69, 70). this protease activity was reported to be required for the polyprotein processing of flaviviruses. on the other hand, the c-terminal sequence contains conserved regions (motifs i, ia, and ii to vi) which are similar to rna helicases of the dead family (71). compared with the sequences of other flaviviruses, these patterns are highly conserved in the epev genome. the epev ns5 protein contains the conserved sequences (motifs a to d) associated with rna-dependent rna polymerase activity (72) and also the gly-asp-asp sequence of motif c, which is the active site of the polymerase. in the n-terminal domain, two conserved motifs, viz. the numbers 1 to 4 indicate the conserved enzymatic motifs in the proteins encoded by the ns3 and ns5 genes. slashes, gaps; residues in yellow, nonconserved positions. with the ultimate objective of generating a phylogenetic tree, the nucleotide and amino acid sequences of the entire genome of epev were aligned with the corresponding sequences of all available flaviviruses. despite the fact that epev was genetically significantly different from the other flaviviruses, the results of the alignment clearly identified epev to be a flavivirus. nevertheless, the epev genome was distinct from the genomes of all other recognized flaviviruses in that the capsid protein contained a unique sequence of 17 amino acids from positions 141 to 157. use of the blast search engine revealed no equivalent sequence in any known virus or cellular sequences. however, fragments from this sequence of up to 10 amino acids matched fragments of sequences found in a variety of bacterial enzymes. we then prepared a truncated flavivirus alignment containing the amino acid sequences of only the capsid and prm, and we attempted to align manually a 26-aa fragment that contained the 17-aa fragment of the unique epev sequence. this truncated alignment was manually adjusted as described previously when the untranslated regions of the flaviviruses were analyzed (7376). using this method, residual amino acids within the 26-aa fragment aligned with amino acids in the upstream region of the prm protein, particularly with those of yfv, sepik virus (sepv), and wesselsbron virus (wslv), i.e., the most closely related viruses (fig. strain names and genbank accession numbers are given after the names of the viruses, which are abbreviated as follows: kunv, kunjin virus; kouv, koutango virus; yaov, yaounde virus; usuv, usutu virus; alfv, alfuy virus; cpcv, cacipacore virus; itv, israel turkey meningoencephalitis virus; bagv, bagaza virus; ntav, ntaya virus; tmuv, tembusu virus; stwv, sitiawan virus; dedsv, duck egg drop syndrome virus; rocv, rocio virus; ilhv, ilheus virus; iguv, iguape virus; aroav, aroa virus; bsqv, bussuquara virus; njlv, naranjal virus; kokv, kokobera virus; strv, stratford virus; nmv, new mapoon virus; denv_1 to denv_4, dengue virus serotypes 1 to 4, respectively; zikv, zika virus; spov, spondweni virus; kedv, kedougou virus; dgv, donggang virus; ugsv, uganda s virus; liv, louping ill virus; ssev, spanish sheep encephalitis virus; tsev, turkish sheep encephalitis virus; ggev, greek goat encephalitis virus; ohfv, omsk hemorrhagic fever virus; lgtv, langat virus; ahfv, alkhurma virus; kfdv, kyasanur forest disease virus; rfv, royal farm virus; ksiv, karshi virus; ggyv, gadgets gully virus; kadv, kadam virus; meav, meaban virus; srev, saumarez reef virus; tyuv, tyuleniy virus; ppbv, phnom penh bat virus; bcv, batu cave virus; mmlv, montana myotis leukoencephalitis virus; rbv, rio bravo virus; jutv, jutiapa virus; modv, modoc virus; mosfv, mosquito flavivirus virus; qbv, quang ninh virus; cv_theileri, culex theileri flavivirus; nakv, nakiwogo virus; pcv, palm creek virus; krv, kamiti river virus; hankv, hanko virus. the definitions of the other abbreviations for viruses are provided in the text. the topology of the phylogenetic tree based on the genomic sequence is in agreement with that in previously published analyses (11, 23, 33, 58, 77, 78) and clearly demonstrates the segregation of the major clusters consisting of the mosquito-borne, tick-borne, and no-known-vector flaviviruses with 100% support. the mbfvs showed a divergence of the two major clades, i.e., the viruses primarily associated with either aedes or culex mosquitoes (as delineated in fig. interestingly, the most recent common ancestor for epev was shared with the nonvectored entv clade and the larger mbfv clade that includes sepv, yfv, and several other viruses. it is also important to note that, apart from epev, in these three clades that share an ancestor, only yfv is found in the nw, where it is believed to have been introduced during the slave trade. nevertheless, epev diverged from these other lineages and was clearly distinct from these other flavivirus species, the closest of which was wslv, with which it had a genome sequence homology of 52.8%. in view of the discovery of a unique sequence fragment in the epev capsid region and the knowledge that some flavivirus genes possess regions with ambiguous, highly variable sequences, it was decided to conduct two phylogenetic analyses. in the first, a tree was constructed in which all ambiguous/highly variable sequence regions, including the unique epev capsid sequence, were retained in the alignment. in the second tree, these ambiguous regions were removed using gblocks trimming. despite this cautious approach, the overall topology, branching patterns, and support for the trees were closely similar (data not shown). moreover, the relative position of epev remained the same in both trees with 100% support. independent phylogenetic analyses based on the complete orf and, separately, the e-gene sequence also resulted in a similar position of epev, in which it appeared outside the entv and yfv clusters (data not shown). however, in trees based on the ns3 and ns5 gene sequences, epev was positioned between the entv and yfv clusters (data not shown). the pairwise distances of the amino acid sequences between epev and other flaviviruses are shown in table s2 in the supplemental material. more than 15 years ago, in a pioneer study, it was proposed that virus species and clades within the genus flavivirus should be based on nucleotide distances using a 1-kb region of the ns5 gene (4), and cutoff values were set at 69% and 84% for discriminating clades and species, respectively. using these values, epev, which is at best 68% similar to the 13 most closely related viruses (sokv, entv, yokv, sepv, wslv, yfv, ehv, bouboui virus [bouv], banzi virus [banv], uganda s virus, jugra virus [jugv], potiskum virus [potv], and sabv), would represent a new species. the maximum 68.0% pairwise nucleotide sequence identity of epev with these viruses was<69.0%, which was the cutoff value that grouped viruses in the same clade, and<84%, which was the cutoff to consider two viruses to be the same species. moreover, the maximum pairwise amino acid sequence identities of the complete orf and ns5 gene (52.8% and 65.4%, respectively) between epev and the yfv-related clade were lower than the minimum pairwise amino acid identities of the complete orf and the ns5 gene (61% and 69.4%, respectively) between the clades defined at that time (4). in a separate analysis, the cutoff value based on complete orf sequences was recorded to be<55% amino acid sequence identity for a number of arthropod-borne flaviviruses (77), which is also higher than the maximum identity of 52.8% with epev. many rna virus genomes contain specific sequences that direct a proportion of ribosomes into an alternative reading frame. where functionally utilized, this is known as programmed ribosomal frameshifting (prf). the most common type of prf involves 1 tandem slippage of the p- and a-site trnas on a slippery heptanucleotide sequence with the consensus motif x_xxy_yyz, where x_xx represents any three identical nucleotides (with some exceptions, such as u_cc and g_ga); y_yy represents a_aa or u_uu; z represents a, c, or u; and underscores separate zero-frame codons. for efficient 1 prf to occur, an extra stimulatory element is required, and this normally takes the form of a downstream rna stem-loop or pseudoknot structure separated from the heptanucleotide shift site by a spacer sequence of 5 to 9 nt (79). ribosomal 1 frameshifting in the gene expression of jev serogroup viruses (33, 48, 80) and the clsfs (i.e., cfav, culex flavivirus [cxfv], and relatives) has been described previously. on the basis of comparative genomic analysis, 1 prf has also been predicted to be utilized by viruses in the chaov-lamv-donv-ilov and wslv sepv flavivirus clades (33, 49). these frameshift sites occur within the region of the genome encoding ns2a-ns2b, with the site being conserved within a clade but not obviously conserved between clades, suggesting that 1 prf in the flaviviruses may have evolved independently on several occasions. on the one hand, the genome contains several slippery heptanucleotide motifs, a few of which have potential downstream rna structures. however, predictions based on a single sequence are inadequate, as such motifs have a high probability of occurring in random sequences. none of the potential 1 prf sites identified aligned with the previously predicted 1 prf site in the related wslv sepv clade. moreover, the high degree of divergence of epev from all other sequenced flavivirus species precluded robust comparative genomic analyses to identify (or tentatively rule out) the occurrence of purifying selection (i.e., evidence for functionality) at other sites with a potential 1 prf. during the past 10 years, thousands of sand flies (phlebotomus and sergentomyia spp.) have been collected in europe, africa, and the middle east and analyzed for the presence of flavivirus rna. none of these analyses produced evidence of infection by epev or any other flavivirus, suggesting that the well-known flaviviruses are rarely (if at all) vectored by sand fly species, at least in these regions of the ow. in the current study, we analyzed pools of sand flies collected in ecuador for the presence of flavivirus-related rna. abonnenci sand flies yielded a pcr product, the sequence of which suggested a genetic relationship with viruses in the genus flavivirus. this pcr-positive pool of sand flies was also inoculated onto c6/36 cells, and a cytopathic virus was subsequently isolated. however, attempts to isolate the virus using vero cells failed, implying that epev is another example of an insect-specific flavivirus. thus, epev appears to be unique, in that it was isolated in ecuador, i.e., the nw, from pa. detailed comparative analysis of the entire viral rna sequence of epev revealed a genomic organization very similar to that of viruses in the genus flavivirus (2, 3). however, alignment of the corresponding regions of epev and other flaviviruses revealed in epev a unique insert of 51 nucleotides (17 amino acids) between amino acid positions 141 and 157 (as determined from the 5-terminal starting codon), i.e., near the boundary between the viral capsid and prm genes. the lack of hydrophobic amino acids in this region indicates that this unique sequence is more representative of the newly formed n-terminal segment of the prm protein than the hydrophobic transmembrane (tm) domain of immature c protein. the tm domain is proteolytically cleaved to generate mature c and prm proteins following coordinated signalase- and ns2b/3-mediated cleavage (81, 82). by manually adjusting the alignment as described previously (7376), residual amino acids within the region unique to epev aligned with amino acids in the upstream region of the prm protein, particularly but not exclusively with those of yfv, sepv, and wslv. importantly, no similar nucleotide sequences were identified among other virus and cellular sequences when the blast search engine was used. this unique epev sequence therefore appears to contain ancestral elements of the flaviviral prm sequence which have presumably survived during the insertion and deletion process of rna evolution, as described previously (7376). the unique single polyprotein with conserved cleavage sites, the 5-to-3 orientation of structural and nonstructural proteins, and the highly conserved amino acid sequence domains in the virus genome-encoded enzymes justify the inclusion of epev in the genus flavivirus. the cysteine residues, 6 in the prm, 12 in the envelope, and 12 in the ns1, proteins which play an important role in protein folding through disulfide bridges, are all conserved among other flaviviruses. there are also n-glycosylation motifs which were speculated to be very important for viral replication, virulence, and maturation of viral particles (8386) in the prm, envelope, and ns1 proteins, some of which are conserved in epev. it contains only conserved sequence 1, which is found in all mbfvs, whereas conserved sequence 2 was highly variable. this conserved sequence 2 was reported to have a minor effect on viral rna synthesis but seems to decrease viral pathogenicity (8790). the presence of a given virus in an arthropod does not confirm that the corresponding vector species plays a role in the natural cycle of the virus. however, in the case of the epev-positive sand fly pool, the pa. abonnenci females were neither gravid nor engorged, and an extremely high viral load was detected. thus, pa. abonnenci and possibly other nw sand flies may play a direct role in the natural infectious life cycle of epev. epev is not the first flavivirus isolated from sand flies, although few viruses have been found in phlebotomine flies until now. saboya virus was isolated from sand flies in senegal (91), although it was originally isolated from the gerbil species tatem itempi in saboya village in senegal (92) and was later recovered from rodents of the mastomys, aivicantis nilaticus, and mus musculus species (93, 94) and chiropters in the republic of guinea (95). in addition, two flavivirus sequences were detected in phlebotomus perniciosus sand flies in algeria. these formed a monophyletic group more closely related to culex-associated insect-only flaviviruses (34). flavivirus rna has also been discovered in phlebotomine sand flies from portugal (unpublished data; genbank accession number hm563684). the phylogenetic analysis showed that epev diverges from the common ancestral lineage of aedes-associated mosquito-borne flaviviruses that include yfv and also the nkv-like flaviviruses (yokv, entv, sokv). epev is more distantly related to the other group of aedes-borne flaviviruses that include dengue viruses and the culex-associated group that includes wnv, slev, and zika virus. interestingly, epev is also only distantly related to the clsfs (cfav, kamiti river virus, hanko virus, and cxfv) and dual-host-affiliated isfs (disfs) (nouv, lamv, chaov, donggang virus (dgv) and ilov) and the nkvs that include bat- and rodent-associated flaviviruses, such as modoc virus and montana myotis leukoencephalitis virus. epev appeared alone as a lineage which was separated from a major phylogenetic clade that included the yfv, ehv, and entv groups. the phylogenetic analysis showed that epev appears at the root of these groups with 100% bootstrap support. however, the phylogenetic position among the mosquito-borne viruses of epev conflicts with its lack of an ability to infect and replicate in vertebrate cell cultures or newborn mice. the insect-specific flaviviruses nouv and lamv also appear to infect and replicate only in insect cell lines (23, 27), although they are phylogenetically grouped with mosquito-borne flaviviruses. these data therefore support the identification of the first representative of a novel group of nw sand fly-borne flaviviruses. when the origins of epev and the other flaviviruses are considered, most of the earliest phylogenetic lineages of the mosquito-borne viruses, tick-borne viruses, and nkvs are found in europe, asia, australia, and, in particular, africa. on the basis of the close relationships of the nw and ow mbfvs, the current opinion is that these viruses were introduced to the nw at various times before, during, or after the period of slave trading (33). the only representative of the tick-borne flaviviruses in the nw is powassan virus (powv) and its relatively recent descendant variant, deer tick virus (dtv). on the basis of historical, ecological, anthropological, and molecular epidemiological evidence, it was proposed that powv was introduced into the nw between 15,000 and 11,000 years ago, possibly during the tick and mammalian migrations, when the bering land bridge connected asia and north america (78, 96). among the nkvs, the ow virus apoi virus (apoiv), which roots all other nkvs, is the only rodent-associated virus related to the nkv group, whereas the bat-associated nkvs are found in both the ow and the nw. this supports the idea of a single dispersion into the nw, most likely through bats (11, 33). it has recently been suggested that the introduction of viruses into the nw occurred several times during two evolutionary time periods (33). it seems highly probable that the most recent introductions occurred from the period of increasing slave and commercial trading across the atlantic ocean from the 16th to the 19th centuries. the most convincing evidence of such dispersions includes the mosquito-borne viruses yfv, denv, and, in 1999, wnv. for each of these viruses there are historical records and robust phylogenetic data (97102). in the case of the potentially earlier introductions to the nw, which include aroa virus, ilheus virus, slev, cacipacore virus, and the nkvs, their dispersion appears to have occurred thousands of years before slave and commercial trading (33). whether or not these introductions to the nw occurred during the period when the bering land bridge connected asia and north america can not be determined. however, it seems unlikely, since there are no recognized ow sand flies in the nw. on the other hand, it is recognized that estimation of the time of occurrence of such assumed introductions is highly dependent on the choice of dates used for calibration (33, 78). as analytical methods improve and more viruses are identified and characterized, it should become possible to provide more robust estimates for these virus dispersion patterns. the genus flavivirus comprises both vectored and nonvectored viruses, raising the possibility that arthropod-mediated transmission is either a derived trait or a secondary loss. the former traditional hypothesis is supported extensively (4, 5, 10, 77, 103, 104), but the alternative hypothesis should not be discarded at this stage of our comprehension. the insect-specific flavivirus group consisting of nouv, bjv, donv, ilov, chaov, lamv, and the newly discovered nanv from peru clusters phylogenetically with the mbfvs, but none of these insect-specific viruses have been found to replicate in mammalian cells (23, 24, 104). in contrast, the other nonvectored viruses, entv and yokv, replicate in mosquito cells. this reflects the need for more in-depth field investigations to identify the possible arthropod vectors of these viruses. for example, the isolation of sokv, a close relative of yokv, from argasidae ticks, birds, and bats and serological evidence of human infections by yokv were recently reported (105). our phylogenetic analysis places epev at the root of the yokv, sokv, and entv clade, which itself roots the aedes-associated mosquito-borne flaviviruses (ehv, bouv, uganda s virus, banv, jugv, potv, sabv, wslv, sepv, and yfv). in contrast to epev, all of these viruses have been found only in the ow. as previously elaborated (106), sampling issues should be taken into consideration in generating these apparently conflicting results. the situation with epev is further complicated when one considers that it was isolated from a pool of nw sand flies. however, it replicates in mosquito cells but does not appear to replicate in mammalian cells, and it is ancestral to the entv and yfv clades. since epev roots clades of ow viruses, is closely related to yfv, and has also been shown to replicate to high titers in mosquito cells, we can speculate that the vectors for the transmission cycle of epev in the ow may include mosquitoes. in this case, the introduction of epev might then be attributable to the slave trade, in common with several other flaviviruses (33). this argument is further supported by the compelling evidence that the alphavirus chikungunya virus (chikv) caused many disease outbreaks in humans about 200 years ago in the americas (107). it therefore seems highly likely that chikv was also introduced into the nw during the period of slave trading across the atlantic ocean. in this case, however, the introduced chikv does not appear to have become permanently established in nw reservoir hosts. the alternative possibility, that epev was introduced into the nw via infected sand flies from the ow, seems unlikely, since ow sand fly species have never been found in the nw. clearly, mosquito trapping and screening are required in the region where the epev-positive sand fly pool was collected to discover whether or not epev circulates among the indigenous mosquito species. if this proves to be the case, it would support the hypothesis of an early divergence of the vector-borne viruses during the evolution of the flaviviruses. moreover, the insect-specific flaviviruses (lsfs) also replicate only in mosquito cells, and the number of viruses in this group has been rapidly increasing. most flaviviruses are associated with mosquitoes and vertebrates, but the isf group is associated only with mosquitoes, appearing at the root of the mbfv, nkv, and tbfv branches. the highly divergent tabv, isolated in 1973 from an insectivorous bat in trinidad (108), is now included as a tentative flavivirus (1). recently, it appears to be highly divergent but associated with the genus flavivirus on the basis of a partial ns3 and ns5 analysis. tabv and mgtv are highly divergent from each other, but they were both isolated in the nw. nevertheless, they are related, albeit distantly, to the other flaviviruses. thus, together with epev, tabv, and mgtv in the nw, viruses in the genus flavivirus appear to have evolved following their introduction into the nw from the ow. however, the wide variety of different flaviviruses and their attributes are potentially biased by the small number of representatives of different lineages that have currently been identified (24). at this stage, we can not rule out the possibility that epev may be vectored by mosquitoes. however, the results in this study suggest that epev represents a new flavivirus species and could be the first recognized representative of a novel clade including other yet-to-be-discovered sand fly-borne flaviviruses. there is support for this proposal: (i) epev has been isolated from pa. abonnenci, a species of nw sand flies that is known to be present in bolivia, brazil, colombia, french guiana, panama, peru, suriname, venezuela, and ecuador (cipa: computer-aided identification of phlebotomine sand flies of the americas, http://cipa.snv.jussieu.fr), (ii) pa. abonnenci is not listed among the species of medical interest (110), and (iii) the absence of viral replication in various vertebrate cells and in the brains of newborn mice suggests that epev could be an insect-only flavivirus rather than an arbovirus. further studies are required to understand if there are other sand fly species infected with epev in the region where the pa. alternatively, if mosquitoes are the natural vectors, it would seem sensible to look first in the region of ecuador where epev was discovered.

abstracta new flavivirus, ecuador paraiso escondido virus (epev), named after the village where it was discovered, was isolated from sand flies (psathyromyia abonnenci, formerly lutzomyia abonnenci) that are unique to the new world. this represents the first sand fly-borne flavivirus identified in the new world. epev exhibited a typical flavivirus genome organization. nevertheless, the maximum pairwise amino acid sequence identity with currently recognized flaviviruses was 52.8%. phylogenetic analysis of the complete coding sequence showed that epev represents a distinct clade which diverged from a lineage that was ancestral to the nonvectored flaviviruses entebbe bat virus, yokose virus, and sokoluk virus and also the aedes-associated mosquito-borne flaviviruses, which include yellow fever virus, sepik virus, saboya virus, and others. epev replicated in c6/36 mosquito cells, yielding high infectious titers, but failed to reproduce either in vertebrate cell lines (vero, bhk, sw13, and xtc cells) or in suckling mouse brains. this surprising result, which appears to eliminate an association with vertebrate hosts in the life cycle of epev, is discussed in the context of the evolutionary origins of epev in the new world.importance the flaviviruses are rarely (if ever) vectored by sand fly species, at least in the old world. we have identified the first representative of a sand fly-associated flavivirus, ecuador paraiso escondido virus (epev), in the new world. epev constitutes a novel clade according to current knowledge of the flaviviruses. phylogenetic analysis of the virus genome showed that epev roots the aedes-associated mosquito-borne flaviviruses, including yellow fever virus. in light of this new discovery, the new world origin of epev is discussed together with that of the other flaviviruses.

PMC4645344

pubmed-679

odontogenic myxoma (om) is considered to be a relatively rare benign tumor of mesenchymal origin. it is found in the skin and subcutaneous tissue, heart and also in various sites of the head and neck region. myxomas of the jaw bones have been traditionally considered to have an odontogenic origin due to the close relation to teeth. according to the literature histologically, it is composed of spindle- or stellate-shaped cells in an abundant mucous intercellular substance, with little collagen. those cases with higher amounts of collagen om exhibits aggressive infiltration of the adjacent tissue as it is not encapsulated and complete surgical removal is difficult. it has a high tendency to recur and can transform into malignant lesion; hence, radiographic and histopathological interpretation are important to establish appropriate surgical management. the treatment options can include curettage with peripheral ostectomy, segmental resection and radical resections for the more aggressive lesions. a 17-year-old male patient visited us with a complaint of swelling in the left maxillary molar region, which enlarged to the present size within a span of 3 months. extraoral swelling was evident in the left side of the maxilla [figure 1]. multilocular radiolucency extending from the distal aspect of the canine to the maxillary tuberosity region was observed on panoramic [figure 2a] and occlusal radiographs [figure 2b]. the computed tomographic (ct) scan showed swelling with bony expansion and thinning of the cortical plates with strong enhancement of the mass lesion in the anterior maxilla [figure 3]. the microscopic examination of hematoxylin and eosin (h and e)-stained section showed fine fibrillar mucoid stroma with evenly spaced spindle- and stellate-shaped cells, and a mild to moderate amount of collagen was observed [figure 4]. the mucoid nature was confirmed with a positive reaction with alcian blue stain [figure 5], and periodic acid-schiff stain was negative. subsequently, the lesion was diagnosed as om and surgical resection followed by prosthetic reconstruction was proposed. swelling present in the left maxilla (a) orthopantomogram showing multilocular appearance in the left maxilla, (b) occlusal radiograph showing multilocular radiolucency extending from the distal aspect of the canine to the third molar region computed tomography scan showing the extent of the lesion hematoxylin and eosin section showing stellate-shaped cells in the fine fibrillar stroma (100) alcian blue-positive reaction (40) the histogenesis of om is related to the odontogenic ectomesenchyme of a developing tooth or undifferentiated mesenchymal cells in the periodontal ligament. the odontogenic origin has been supported by the following reasons: exclusive occurrence in the tooth-bearing areas of the jawsassociation with an unerupted tooth or a developmentally absent toothfrequent occurrence in young individualshistological similarity between om and pulpal ectomesenchymeoccasional presence of sparse amounts of odontogenic epitheliumits uncommon occurrence in other parts of the skeleton. exclusive occurrence in the tooth-bearing areas of the jaws association with an unerupted tooth or a developmentally absent tooth frequent occurrence in young individuals histological similarity between om and pulpal ectomesenchyme occasional presence of sparse amounts of odontogenic epithelium its uncommon occurrence in other parts of the skeleton. the majority of cases are reported in the second and third decades of life. according to kaffe om is usually a central lesion, and the radiographic appearance is important to establish the diagnosis. multilocular radiolucency with either distinct or poorly defined margins is observed in adults and in the posterior part of the jaw. zang et al. examined the radiographic appearances of 41 om that were divided into six types. multilocular (including honeycomb, soap bubble and tennis racquet patterns); type iii involvement of local alveolar bone; type iv involvement of the maxillary sinus; type v osteolytic destruction; and type vi a mix of osteolytic destruction and osteogenesis. kaffe et al. in his radiographic study revealed an interesting correlation between size and locularity; unilocular lesions were smaller than 4 cm and multilocular lesions were larger than 4 cm. in the present case, it is difficult to differentiate solid ameloblastoma, odontogenic keratocyst and om using radiographs as all these lesions exhibit multiloculation. dental radiographs are a bidimensional projection of a tridimensional structure, and therefore superimposition of anatomic landmarks can masquerade important findings. asuami et al. examined the dynamic magnetic resonance imaging (mri) features to differentiate these lesions; solid areas of ameloblastoma showed an earlier enhancement than the whole areas of the om. these results indicated that the dynamic mri features of the tumor substance of ameloblastoma differs from om. because of the scarcity of studies using mri, the characteristics of the om have not been established satisfactorily. they found that the tumor borders were generally well defined with a smooth margin both for bony and soft tissue structures. cortical plate continuity was lost in numerous patients and intralesional trabeculations were observed. in the present case, thinning and erosion of the cortical plates was present in the anterior and posterior regions of the maxilla and intralesional trabeculations were seen. macdonald-jankowski suggested that both ct and radiographs should be used in the investigation of an om. ct assesses perforation and pattern of septa while radiographs allow a better assessment of the degree of definition of the lesion's margins with the adjacent normal bone. on gross examination microscopically, it is made up of loosely arranged spindle- and stellate-shaped cells, many of which have long fibrillar processes that tend to intermesh. in cases of myxofibroma, the amount of collagen in the mucoid stroma is more prominent. the mucoid nature was confirmed with a positive reaction with alcian blue staining and negative periodic acid-schiff staining. epithelial islands are not commonly observed in the myxomas of the jaws that do not play a significant role in om. akihiro kimura et al. reported a case of om, in which the interesting feature was the presence of active-looking and irregularly proliferating epithelial islands with a microcystic appearance. immunohistochemical positivity with ck 19 supports the odontogenic origin and high labelling index for ki-67 indicates active epithelium, which has never been reported. oms are extensively described as case reports; however, the invasive behavior of these lesions has not been explained. surgical procedures vary from currettage, enucleation, local excision and partial and total jaw resection. the lack of a capsule and infiltrative growth pattern is responsible for a high rate of recurrence when conservative enucleation and curettage are performed. boffano et al. proposed the protocol to perform conservative surgery by enucleation and curettage when lesions were smaller than 3 cm, whereas a segmental resection with immediate reconstruction is preferred in patients affected by a bigger tumor. resection of the jaw was planned for this patient as the lesion in the maxilla is in close relation to vital structures; resection procedures minimize the risk of involvement of these structures and also reduce the recurrence rate. om and other odontogenic tumors share common features on conventional radiographs that lead to a diagnostic dilemma. in order to establish a treatment protocol ,

odontogenic myxoma (om) is a relatively rare benign odontogenic tumor of mesenchymal origin. om is more common in the mandible than in the maxilla. it is an asymptomatic lesion that shows an infiltrative growth pattern. when the maxillary sinus is involved, it often fills the entire antrum. odontogenic tumors are uncommon in the maxillary molar area, which often leads to diagnostic dilemma as this region of the maxilla is in the vicinity of vital structures, and radiographic overlapping of structures is always present. we present a similar case of a 17-year-old male patient who reported with a swelling in the left maxilla that infiltrated the maxillary sinus in a short duration of time.

PMC4260388

pubmed-680

intravascular lymphomatosis (ivl) is an uncommon systemic disease in which malignant proliferation of lymphocytes occurs exclusively in the vessels. the clinical symptoms are nonspecific in most cases, making an early diagnosis difficult [1, 2]. a unique characteristic of this disorder is that the proliferation of malignant lymphocytes occurs only within the vessels, in contrast to primary central nervous system lymphoma (pcnsl). we herein report a patient with ivl who presented with fever of unknown origin (fuo) and in whom a brain mass developed, mimicking pcnsl. a 75-year-old right-handed female patient was admitted to our hospital for the evaluation of recurrent fevers. she had been well until 3 weeks before admission, when fever and fatigue developed. she had a history of cholecystectomy, spinal compression fracture, and an old cerebral infarction of the left middle cerebral artery. after the brain infarction, she developed aphasia and right hemiplegia and became dependent on a wheelchair. she showed paroxysmal atrial fibrillation, and warfarin was prescribed to prevent a second stroke. on examination, her blood pressure was 150/70 mm hg, her pulse 90 beats per minute, and her body temperature was 37.5c. there was a small decubitus ulcer on the sacrum and a nonpitting edema in both legs. her blood levels of lactate dehydrogenase (ldh), c-reactive protein, and soluble interleukin-2 receptor (sil-2r) were high at 458 iu/l (normal<229 iu/l), 16.8 mg/dl (normal<0.8 mg/dl), and 3,458 u/ml (normal<515 u/ml), respectively. her blood levels of electrolytes, glucose, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, amylase, brain natriuretic protein, and vitamin b1 were normal, as were her renal and thyroid function. lumbar puncture was performed; there was no pleocytosis in the cerebrospinal fluid, but the protein and igg levels were increased to 85 mg/dl (normal<50 mg/dl) and 17 mg/dl (normal<4 mg/dl), respectively. a contrast-enhanced ct of the chest, abdomen, and pelvis failed to reveal any abnormal lesions that could have caused the fever. a brain mri obtained 3 weeks after admission showed an old infarction but was otherwise normal. tests for antibodies to hepatitis b, hepatitis c, human immunodeficiency virus, human t-lymphotropic virus, and the treponema pallidum were negative. antibiotic treatments including ciprofloxacin at 600 mg/day, panipenem/betamipron at 1 g/day, and ceftazidime at 2 g/day combined with arbekacin at 200 mg/day were undertaken, but all were ineffective. the fever continued for more than 3 weeks and fulfilled the criteria for fuo. because the serum levels of ldh and sil-2r remained high, we suspected a hematogenous tumor such as myeloma, and bone marrow puncture was performed, but the result was normal. although there were no skin lesions, we strongly suspected ivl and thus performed a random skin biopsy. the biopsy was obtained from 1 lesion of the purpura on the abdomen and 2 regions of the skin with a normal appearance on the abdomen and left thigh. histological examination revealed atypical cells lodged in the intravascular lumens in the subcutaneous tissue of the normally appearing left thigh region (fig. higher magnification revealed vessels filled with large malignant lymphoid cells and eosinophilic nucleoli (fig. 1b); the cells were stained with cd20 antibodies but not with cd3 (fig. the patient was thus diagnosed with ivl (intravascular large b-cell lymphoma). an mri obtained 7 weeks after admission showed a mass lesion involving the right basal ganglia, thalamus, corona radiata, uncus, and crus cerebri (fig. 2). the mass was isointense on a t1-weighted image, hyperintense on a t2-weighted image, and exhibited mild restricted diffusion on a diffusion-weighted image (dwi). an mri obtained 9 weeks after admission showed an enlargement of the mass (fig. ivl is an uncommon systemic disease in which malignant proliferation of lymphocytes occurs exclusively in vessels. studied 10 cases of ivl in a single institution and reported that fuo and a mental status change were the most common signs, being present in 60% of cases. they reported that the duration of fuo ranged from 2 to 6 months. in our patient, fuo had been present for 7 weeks until the diagnosis of ivl was made. in patients with long-standing fuo histological confirmation of the malignant proliferation of lymphocytes in vessels is required for the diagnosis of ivl. although the number of reported cases of ivl in which antemortem diagnosis was achieved by biopsy is small, such biopsies reportedly involved the brain, adrenal gland, kidney, liver, spleen, lung, bone marrow, muscles, and the skin. because dermatological manifestations including nodules, plaques, and macules are present in up to one third of patients with ivl, skin biopsies should be conducted when any skin lesions are found in patients in whom ivl is suspected. the involvement of clinically unaffected skin has been reported in autopsy findings, and this involvement serves as the basis for a random skin biopsy. in our case, we performed a random skin biopsy because of the strong suspicion of ivl based on the high serum levels of ldh and sil-2r. the mri findings of ivl have not been well characterized because the number of reported cases is relatively small. however, the most commonly reported mri findings are multifocal lesions that are hyperintense on t2-weighted images, mimicking small infarctions. baehring et al. recently evaluated 5 patients with ivl in whom serial diffusion-weighted mris were obtained. they reported that multifocal dwi lesions are the most common observations, suggesting the presence of small-vessel ischemia that is probably caused by the occlusion of vessels by malignant lymphomatous cells. a brain mass developed in the present patient. the mass was isointense on t1-weighted images and hyperintense on t2-weighted images and it showed a restricted diffusion on dwi and homogeneous gd enhancement. the differential diagnoses for a brain lesion appearing hyperintense on t2-weighted images with gd enhancement may include pcnsl, toxoplasmosis, glioblastoma multiforme, abscess, metastasis, tumefactive multiple sclerosis, and primary angiitis of the central nervous system (pacns). of these etiologies, homogeneous gd enhancement and restricted diffusion on dwi make pcnsl most likely, and the serological findings suggesting a lymphomatous disease could have allowed for a diagnosis of pcnsl in the present case if the brain biopsy had not been performed. moreover, when the mass appeared, there were no definitive ischemic lesions, which are common radiological findings of ivl. antibiotic treatment was not effective and blood culture was negative in this case; however, there was still a probability of developing a brain abscess after admission. despite this, the lack of a characteristic thick rim of contrast enhancement around the lesion was considered to make a brain abscess unlikely. pacns is a rare vasculitis of unknown etiology that exclusively involves the central nervous system. commonly reported mri findings of pacns are multifocal deep gray and subcortical hyperintensities on t2-weighted images. recently, some cases of pacns have been reported to present as brain masses, mimicking brain tumors. the low sensitivity of the characteristic angiographic signs and the lack of specific findings in general medical laboratory investigations make a brain biopsy necessary for the diagnosis of pacns. the findings in our case suggest that if pacns is suspected in a patient with a brain mass, a random skin biopsy prior to brain biopsy should be considered to allow for the avoidance of a more invasive brain biopsy. however, some authors have reported that extravasation of malignant lymphocytes can occur to some degree in the late stage of ivl [1, 7]. to the best of our knowledge, there have been 4 cases of ivl in which postmortem examination revealed a brain mass and 5 cases of tissue-proven ivl in which mri showed a brain mass (table 1) [8, 9, 10, 11, 12, 13, 14]. our case is notable because it is the first description of dwi of a brain mass in a patient with ivl. dwi is a series of t2-weighted sequences that detect the movement of protons in water. the causes of restricted diffusion on dwi include acute ischemia, cerebral abscesses, diffuse axonal injury, active demyelination, and highly cellular tumors such as lymphoma. dwi change in acute ischemia is commonly known to occur, and marked restricted diffusion in acute ischemia generally remains for 35 days. in our case, the restricted diffusion enlarged over 2 weeks, supporting the high cellularity of the mass as the cause of restricted diffusion. although there are no pathognomonic presenting symptoms or signs in pcnsl, focal neurological deficits such as personal changes or cerebellar signs are reportedly rather common. patients with pcnsl may infrequently present with fuo as the initial clinical manifestation. the majority of patients with pcnsl reportedly develop a solitary supratentorial tumor, and the most common sites are the frontal lobe, temporal lobe, parietal lobe, deep nuclei, occipital lobe, cerebellum, or the brain stem. considering the fact that there are no specific symptoms or signs of pcnsl and that the brain mass noted in our case was a solitary mass located in the basal ganglia, which is a common site of pcnsl, the present case could have been misdiagnosed as pcnsl instead of ivl if the random biopsy had not been performed. a combination of cyclophosphamide, doxorubicin, vincristine, and prednisone with the recombinant anti-cd20 antibody rituximab (r-chop) is the most commonly employed treatment in patients with ivl. on the contrary, chop is either ineffective or toxic in the treatment of pcnsl, and methotrexate with brain radiation therapy is usually employed. if the skin biopsy had not been performed, this patient could have been treated by methotrexate, which is not effective against ivl. mri showed a brain mass mimicking pcnsl; however, a random skin biopsy confirmed the diagnosis of ivl. although postmortem examination was not performed, the brain mass noted on mri was considered to be a collection of malignant lymphomatous cells invading from the vessels.

we herein report a 75-year-old female patient with intravascular lymphomatosis (ivl) who presented with fever of unknown origin. examination, including contrast-enhanced ct and 67ga scintigraphy, failed to show any lesions. her blood levels of lactate dehydrogenase and soluble interleukin-2 receptors were high, suggesting a lymphomatous tumor. a bone marrow puncture was negative, and a random skin biopsy revealed a monoclonal proliferation of naked, large lymphocytes in the vascular space of the subcutaneous tissue, confirming the diagnosis of ivl. mri, performed 7 weeks after admission, showed a brain mass mimicking primary central nervous system lymphoma. the mass was considered to be a collection of malignant lymphocyte cells invading from the vessels. without the random skin biopsy, this case may have been misdiagnosed as primary central nervous system lymphoma.

PMC3995396

pubmed-681

for more than 40 years, metal ceramic restorations (mcrs) have been widely used in the fabrication of fixed partial dentures (fpds) and still represent the gold standard nowadays.1 the success of these mcrs depends on the effective bonding between the veneering ceramic and metal substructure, which is believed to develop from chemical bond of the metal oxides.2 however, although the conventional mcrs have demonstrated superior fracture resistance, metal-free restorations have gained importance and their development has been accelerated by increasing interest in aesthetic dentistry.3 the introduction of zirconium dioxide as a framework structure or coping for ceramic restorations has initiated lengthy discussions on the design and limitations of these restorations. furthermore, the bonding mechanism between zirconia framework and veneering ceramic is not yet well understood since no documented evidence of the bonding mechanism between these materials is available.4 however, a few studies have looked into methods to increase the surface roughness of the zirconia.56 as the surface roughness increases, the bonding between zirconia coping and veneering ceramic increases, thus leading to success of the restorations. various surface treatments (e.g. sandblasting, acid etching, glazing, heat treatment, and application of liner onto coping materials) have been recommended to enhance the bonding efficiency between veneering ceramic and coping material. however, none of these treatments have been determined to produce the highest bond strength. airborne-particle abrasion or sandblasting, an important treatment procedure for achieving strong adhesion of veneering ceramics, works by increasing surface roughness and providing undercuts.7 for ceramic surface treatment, acid reacts with the glassy matrix that contains silica and forms hexafluorosilicates.8 as a result, the surface of the ceramic becomes rough, which is advantageous for micromechanical retention on the ceramic surface. combination of surface treatments, such as sandblasting with alumina oxide-particle and acid etching may substantially increase the surface area for micromechanical retention.9 this will subsequently increase the bond strength. therefore, the purpose of this study is to investigate shear bond strength (sbs) of veneering ceramic to different types of coping material (metal alloy, zirconia oxide, and lithium disilicate) after various pre-surface treatments such as sandblast and acid etch. the null hypothesis is that the shear bond strength of these veneering ceramic will not be different after various pre-surface treatments. ninety-six disc specimens measured 14 4 0.2 mm were prepared from three different type of coping materials (n=32): i) metal alloy (ma), ii) zirconia oxide (zo), and iii) lithium disilicate (ld) pre-cast wax patterns were prepared using silicone mold (metrosil, metrodent, huddersfield, england) and were invested with investment materials., ramat-gan, israel) pre-heated to 850 with alumina plunger for 90 minutes. for ma group, the mold was filled with molten ni-cr alloy (4all, ivoclar vivadent ag, schaan, liechtenstein) using a casting machine (fornax, bego gmbh, bremen, germany). for ld group, ips e.max press (ivoclar vivadent ag, liechtenstein) ingots were softened at 920 and were automatically pressed into the mold in a furnace (ep 600, ivoclar-vivadent, liechtenstein). after pressing and cooling to room temperature finally, the specimens were cleaned in an ultrasonic bath with distilled water for 15 minutes and air-dried for 10 seconds. for zo group, the specimens were machined out of a block of pre-sintered zirconia (cercon, degudent gmbh, hanau-wolfgang, germany). the specimens were milled using cercon brain expert (degudent gmbh, germany) machine in an enlarged proportion according to the data installed from manufacturer's software. the milled specimens were then sintered in the cercon heat plus furnace (degudent gmbh, germany) at 1,350 for six hours, producing final specimens sized 14 4 mm. the specimens from each coping material group were randomly divided into 4 subgroups with 8 specimens in each subgroup (n=8). four pre-surface treatments were prepared for each subgroup; a) no treatment or control (c), b) sandblasted (sb), c) acid etched (ae), and d) combination of sandblasted and acid etched (sbae). the specimens in subgroup c were grounded with diamond disc with no further treatment applied to the surface. the specimens in subgroup sb were treated with 50 m alumina (al2o3) particles at 0.2 mpa for 10 seconds and at the 10 mm distance from the nozzle to the specimen. the specimens in subgroup ae were etched with 5% hydrofluoric acid (hf) (ips ceramic etching gel, ivoclar vivadent ag, liechtenstein) for 20 seconds. the specimens in subgroup sbae were sandblasted with 50 m alumina (al2o3) particles at 0.2 mpa for 10 seconds and at the 10 mm distance from the nozzle to the specimen. all specimens were cleaned in an ultrasonic bath containing acetone, alcohol, and distilled water for 10 minutes. two thin layers of opaque veneering ceramic in paste-liquid form were applied to the prepared surfaces of the specimens from group ma (ips inline, ivoclar vivadent ag, liechtenstein) and zo (ceram kiss, degudent gmbh, germany). the specimens were fired in the furnace (programart p500, ivoclar vivadent ag, liechtenstein) according to the recommendations of the manufacturers. a silicone mold (duplicone, shofu inc., kyoto, japan) was fabricated with a slit measured 4 4 mm for the placement of veneering ceramic. the silicone mold was placed on top of the specimen disc. veneering ceramic for specimens ma (ips inline, ivoclar vivadent ag, liechtenstein), zo (ceram kiss, degudent gmbh, germany) and ld (ips emax ceram, ivoclar vivadent ag, liechtenstein) in the study were manipulated as recommended by the manufacturer. liquid was added to veneering ceramic powder until paste consistency was obtained and condensed in the mold. after condensation was completed and excess moisture was removed with absorbent paper, the silicone was carefully removed. after the first firing cycle, another layer of dentin ceramic was applied and excess water was removed using absorbent paper. the specimens were fired for the second time to compensate for the shrinkage generated during the first cycle. at the end of these firing cycles, the veneering ceramic surface was ground flat and parallel to the coping surface using diagen turbo grinder (bredent gmbh, senden, germany). a universal testing machine (utm) (autograph ag-x, shimadzu, kyoto, japan) was used for the sbs test at a crosshead speed 0.5 mm/min. a shear load was applied until failure occurred and failures fractures were determined from the chart recorder. the data were compiled and analyzed using the statistical package for social science (spss) software version 20.0 (ibm inc., armonk statistical analyses using two-way anova and tukey's multiple comparisons tests were used. the mean sbs values for all groups of coping materials ranged from 11.61 4.84 mpa to 27.89 5.64 mpa (table 1). shapiro-wilk normality test showed that the data had a normal distribution (p>.05). two-way anova revealed that there were statistically significant interactions for the types of coping material groups, pre-surface treatment groups, and among the groups (p<.05) (table 2). furthermore, there was a statistically significant interaction between the effect of coping materials and pre-surface treatments on shear bond strength, as shown by non-parallel lines within the graph (p<.05) (fig. tukey's multiple comparisons showed a statistically significant difference in sbs of veneering ceramic within the coping material groups and the pre-surface treatment groups as shown in table 3 and table 4 (p<.05). among various types of coping materials tested, group zo produced significantly higher sbs than group ma, while group ma produced significantly higher sbs than group ld. on the other hand, the specimens treated with sbae showed significantly higher sbs than the specimens treated with sb alone or with no treatment. the specimens treated with ae were also showed significantly higher shear bond strength than the specimens treated with sb. for relationship between coping material and pre-surface treatment, tukey's comparisons test showed a statistical significant interaction between the groups with p<.05 (table 5). in group ma, the specimens treated with ae showed significantly higher shear bond strength than other pre-surface treatments, while, in group zo and ld, the specimens treated with sbae showed significantly higher shear bond strength than other pre-surface treatments. international standard of organization (iso) has standardized the bond strength measurement of metal ceramic system through the schiwickerath crack initiation test; the mean debonding strength should be greater than 25 mpa. however, due to the brittleness of all-ceramic coping materials, this test can not be applied to all-ceramic multilayered system.10 to date, there is no standardized test or minimum bond strength requirement for all-ceramic system.11 according to some authors, sbs values of 10 mpa is the minimum value for clinical flaw to happen between metal and ceramic.1213 therefore, sbs values more than 10 mpa indicate excellent bonding clinically. assuming that shear stresses are generally responsible for the clinical failure of the coping-ceramic interface, sbs test was adopted in this present study. in addition, this test required simple preparation of specimens and the testing could be performed easily. important aspects should be considered, including storage condition, type of substrate, specimen preparations, rate of load application, cross-sectional surface area, and experience of the researcher.11 in the present study, when the ceramic was veneered to ma, sbs values ranged from 14 to 24 mpa with mean value of 19.00 6.39 mpa. this finding was lower than sbs of base metal group recorded by al-dohan et al .. 11 the specimen's size could be accounted for the differences. they used smaller diameter of veneering ceramic, which could have given higher bond strength value as the formula was calculated by dividing the maximum applied force by the bonded cross-sectional area. in other studies, sbs between nickel-chromium or cobalt-chromium metal alloy were reported higher than the sbs finding in the present study.141516 however, it is difficult to compare the results of the present study with those obtained in other studies because different methods were used to evaluate the sbs. furthermore, some authors suggested that the failure of the bonded interface occurred when a crack propagated from a flaw of a considerable size found in an area subjected to high tensile stresses.10 the sbs values reported for zo group in this study ranged from 22 to 27 mpa with mean value of 24.45 5.14 mpa. this finding was slightly lower than the sbs values obtained from other similar studies.1718 the other studies used the circular interface test, which was different from the sbs test in the present study. guess et al.19 found the mean sbss of veneering ceramic to zr to be ranged from 9.4 to 12.5 mpa, which were lower than the values in the present study. the different findings were attributed to different testing method, particularly related to the size and form of the specimens tested. the mean sbs value for ld group was 13.62 5.12 mpa, which the lowest sbs value obtained among all coping materials. the present study is in agreement with study done by umer et al.20 the finding of the current study showed that application of veneering ceramic onto the coping material lowered the strength of the bilayer specimens. however, several authors have reported that the mean sbs value for ld group was higher than zirconia and base metal groups in their studies, which contradicted the current finding.1121 there are several methods used for surface grinding, which include grinding using an abrasive paper or wheels (sic or al2o3), particle air-abrasion using al2o3 or other abrasive particles ranging in size from 50 to 250 m, and grinding using a diamond bur.22 the highest mean sbs value for sandblasting treatment was obtained from group zo, followed by ma and ld groups. this finding is in agreement with other studies.2324 the other studies reported that sbs value for sandblasting group was higher than the other groups and concluded that sbs of veneering ceramic on zirconia treated with sandblasting was significantly higher than that subjected to other pre-surface treatments. contrary to the current finding above, some researches have shown that surface grinding techniques have no significant effect on increasing the bond strength of zirconia to veneering ceramic.25 another possible problem with sandblasting is that it can create surface microcracks that can initiates bigger crack. these cracks later can decrease strength and cause fracture toughness of the material.26 since acid etching was first suggested as a ceramic presurface treatment for resin bonding, many different etching periods have been advocated and used. the most profound ceramic surface roughness and the highest bond strength data at the ceramic resin interface have been obtained by 2-minute acid etching, as reported by chen and suh in 1998.27 the present study found that the mean of sbs value for acid etching treatment was highest in ma group, followed by zo and ld group. the application of hf acid to metal was capable of roughening the surfaces, therefore increasing mechanical retention. the hf acid can also cause diminishing of oxide layer to the degree that would not affect the bonding.28 the statement might explain why the sbs value of ma group was still the highest after acid etching treatment was done. on the contrary, smielak and klimek found that etching zirconia copings with 5% hf showed no significant difference to the surface roughness, as the nature of the material was not glass-like.5 the present study found that the mean of sbs value for the combined treatment of sandblasting and acid etching produced the highest sbs value in ma and ld group compared with other pre-surface treatment. sandblasting with 50 m alumina particles changed the surface by increasing the number of pits per unit area. application of hf acid following acid etch was able to remove the glass matrix and the second crystalline phase, thus creating irregularities within the ld crystals.29 zirconia had the highest shear bond strength value, while lithium disilicate had the lowest shear bond strength value among the coping materials tested. combination of sandblasting and acid etching produced the highest sbs value in zirconia and lithium disilicate .

purposepre-surface treatments of coping materials have been recommended to enhance the bonding to the veneering ceramic. little is known on the effect on shear bond strength, particularly with new coping material. the aim of this study was to investigate the shear bond strength of veneering ceramic to three coping materials: i) metal alloy (ma), ii) zirconia oxide (zo), and iii) lithium disilicate (ld) after various pre-surface treatments. materials and methodsthirty-two (n=32) discs were prepared for each coping material. four pre-surface treatments were prepared for each sub-group (n=8); a) no treatment or control (c), b) sandblast (sb), c) acid etch (ae), and d) sandblast and acid etch (sbae). veneering ceramics were applied to all discs. shear bond strength was measured with a universal testing machine. data were analyzed with two-way anova and tukey's multiple comparisons tests. resultsmean shear bond strengths were obtained for ma (19.00 6.39 mpa), zo (24.45 5.14 mpa) and ld (13.62 5.12 mpa). there were statistically significant differences in types of coping material and various pre-surface treatments (p<.05). there was a significant correlation between coping materials and pre-surface treatment to the shear bond strength (p<.05). conclusionshear bond strength of veneering ceramic to zirconia oxide was higher than metal alloy and lithium disilicate. the highest shear bond strengths were obtained in sandblast and acid etch treatment for zirconia oxide and lithium disilicate groups, and in acid etch treatment for metal alloy group.

PMC5099125

pubmed-682

radiofrequency ablation of the slow pathway is considered to be the gold standard treatment for patients with atrioventricular nodal reentrant tachycardia (avnrt). pulmonary artery agenesis is a rare anomaly that may occur during embryological development of the heart.1 this agenesis may be accompanied by a complete or partial absence of the lung. in the great majority of the cases, the diagnosis is usually made at or soon after birth and it can be associated with multiple anomalies. an otherwise normal heart may be displaced into the right hemithorax by unilateral pulmonary agenesis.2 catheter ablation of avnrt in the setting of dextrocardia is potentially challenging. there have been a few reports of successful slow pathway ablation in patients with dextrocardia, but usually at the expense of prolonged procedure and fluoroscopy times.37 to our knowledge, slow pathway ablation of a patient with dextrocardia due to pulmonary agenesis has not been described to date. a 35-year-old woman was admitted to our cardiology clinic with complaints of palpitation. she had a 7-year history of paroxysmal palpitations. the tachycardia, at a rate of 170200 bpm, was associated with dyspnea and chest tightness and lasted for up to 60 minutes at a time. her physical examination revealed no breathing sound on the right and a normal breathing sound on the left hemithorax, while the heart s sound was heard from the right hemithorax. the chest radiogram incidentally showed a rudimentary, opacified right hemithorax with mediastinal shift and herniation of the contralateral lung. except for dextroposition of the heart, a 12-lead resting electrocardiogram (ecg) showed sinus rhythm at a rate of 64 bpm, a positive qrs complex in avr, a positive p wave in avr and a biphasic p wave in avl, and a prominent r wave in v1 with undetermined horizontal axis (figure 1). the clinical tachycardia was a regular, narrow qrs tachycardia at a rate of 170 bpm, during which the p wave was indiscernible (figure 2). computed tomography showed a complete absence of the right lung with dextroposition of the heart (figure 3). electrophysiological study was performed after obtaining written informed consent and discontinuation of all drugs for five half-lives. the right atrium and the right ventricle were imaged on left lateral and anteroposterior views by contrast injection through inferior vena cava to reveal the cardiac anatomy (figure 4). then, a 4 mm-tipped ablation catheter (cordis webster, baldwin park, ca, usa) was advanced through the 6f sheath into the right atrium and a four-pole fixed-curve diagnostic catheter was placed on the bundle of his. during the electrophysiological study, a narrow qrs tachycardia with a cycle length of 270 ms was reproducibly induced by atrial premature stimulation with an extrastimulus (figure 5a). the ventriculoatrial interval, measured from the onset of ventricular activation on the surface ecg to the earliest deflection of the atrial activation in the his bundle electrogram, was<60 ms, and the diagnosis of typical slow fast avnrt was confirmed. his bundle records were taken with an electroanatomi-cal approach. then, slow pathway potentials were recorded (figure 5b). fluoroscopic views of the catheters during the ablation and mapping of the coronary sinus ostium are shown in figures 6 and 7. during ablation, the radiofrequency energy was adjusted to obtain a catheter tip temperature between 50c and 60c. the current was applied for 60 seconds after junctional tachycardia was observed during ablation (figure 8). after the ablation, there was no residual slow pathway conduction and the clinical arrhythmia was not inducible without or with infusion of isoproterenol. the total procedure time was 30 minutes with a total radiofrequency delivery time of 8 minutes and fluoroscopy time of only 6 minutes. no complication occurred after the procedure and the patient was free of symptoms at 1-year follow-up. dextrocardia or complex cardiac anatomy of the heart may be challenging to electrophysiologists during catheter ablation procedures. there have been only a few case reports of catheter ablation of supraventricular tachyarrhythmias in patients with dextrocardia.37 pulmonary agenesis can be localized to a single lobe and it can affect an entire lung or, in rare cases, both lungs.1 although a majority of patients with unilateral agenesis die soon after birth or in early childhood, in some extreme cases, patients can survive up to adulthood, like our patient. the most common and familiar form is the mirror image of normal or mirror-image dextrocardia, in which the migration of the apex of the l-bulboventricular loop is into the right hemithorax, as expected. the anterior posterior relationship of the various parts of the heart is normal, but their right-to-left orientation is reversed. the second most common type is dextroversion, in which the heart appears to be rotated into the right hemithorax relative to its normal position. 3) the third type of dextrocardia is dextroposition, in which an otherwise normal heart is displaced or shifted into the right hemithorax by other extracardiac causes such as agenesis or fibrosis of the right lung. our case was a type 3 dextrocardia. to recognize the ecg findings of dextrocardia necessitates a clear understanding of the electrical axis. global negativity in lead i, a positively deflected qrs complex in avr, and right axis deviation and reverse r wave progression in precordial leads are the mean ecg findings in dextrocardia. the finding of a positively deflected qrs complex in avr and a negatively deflected ors complex in lead i may lead to misdiagnosis. the most common cause for this finding is reversed electrode placement of arm leads (left and right). during a standard catheter ablation procedure, the catheter is targeted to the ablation region based on typical local electrogram characteristics (slow-pathway potentials) and anatomical landmarks. in contrast to patients with normal anatomy, localization of the slow pathway and stabilization of the catheter are more difficult in patients with dextrocardia. therefore, the time spent in the catheterization laboratory and the fluoroscopy time are markedly longer than in cases with normal hearts. our case illustrates the role of imaging by contrast injection in a patient with dextrocardia. the identification of the accurate anatomy using imaging modalities such as computerized tomography and magnetic resonance imaging and three-dimensional image reconstruction using mapping systems may be useful not only to help the electrophysiologist, enabling a safe and successful catheter ablation procedure, but also to understand the complex anatomical structures and to guide optimal catheter access. however, these modalities are quite expensive and are not easily accessible in every unit. before the ablation procedure, we easily localized the coronary sinus ostium and his bundle position. as such, we did not need an additional catheter in this ablation procedure, but an additional catheter might further facilitate the procedure. we are presenting this pulmonary agenesis case in view of its atypical presentation in adulthood, which is exceptionally rare. as demonstrated in this case, revealing cardiac anatomy by contrast injection or other imaging modalities may shorten the procedure time and decrease the complication rates.

radiofrequency catheter ablation of the slow pathway is considered to be the treatment of choice for patients with atrioventricular nodal reentrant tachycardia. we report a 34-year-old female with mirror image dextrocardia due to unilateral pulmonary agenesis who underwent successful slow pathway ablation for typical atrioventricular nodal reentrant tachycardia. using contrast injection, cardiac anatomy was identified in a short time and successfully ablated.

PMC4321640

pubmed-683

t cells and b cells derived from the lymphoid lineage belong to the adaptive immune system. function via production of effector cytokines after differentiation and activation. in comparison to the cytotoxic feature of both adaptive cd8 t cells and innate cnk cells, the helper feature of cd4 th cells was considered to be a unique characteristic of the adaptive system acquired during evolution. however, over the past few years several groundbreaking works on a novel member of the innate immune system, the innate lymphoid cell (ilc), have dramatically changed our knowledge about the composition of the innate lymphoid lineage and led us to reconsider the relationship between innate and adaptive lymphoid lineages in the context of evolution. like other lymphocytes, ilcs also develop from the common lymphoid progenitors (clps) found in fetal liver and adult bone marrow. they were not discovered and classified as a new lymphocyte family until recently, partly due to their distinct enrichment in nonlymphoid tissues such as mucosal tissues, skin, and adipose tissues, with scarce distribution in lymphoid tissues. their lack of any known lineage surface markers may also contribute to their belated discovery. in actuality, scientists noticed certain subsets of ilcs such as lymphoid tissue inducers (ltis) as early as the 1990s [3, 4], but it was not until three independent reports on type 2 cytokine producing innate lymphoid cells (ilc2s) in 2010 [57] that people began to recognize the possible existence of an innate population with a helper feature mirroring adaptive th cells. the nomenclature of innate lymphoid cells (ilcs) was then formed based on the existence of helper lymphocyte in the innate arm of the immune system. similar to the classification of th cells, mature helper-like ilcs can be categorized into three groups based on their master regulator expression and signature effector cytokine production. ilc2s, the innate counterpart of th2 cells, express high levels of gata-3 and are capable of producing type 2 cytokines such as il-5 and il-13 [57]. ilc3s express rort and are capable of producing il-22 and il-17, similar to th17/th22 cells [810]. the ccr6 lineage includes lymphoid tissue inducer (lti) cells, while ccr6ilc3 can give rise to a special population of rort ilc3s that express the natural cytotoxicity receptor (ncr) nkp46 (encoded by ncr1) in mice and nkp44 (encoded by ncr2) in humans. ccr6ilc3s can also express t-bet which drives further development of this lineage into the ncr stage [1113]. finally, ilc1s are the innate counterpart of th1 cells. they are t-bet positive and better ifn- producers than cnk cells [14, 15]. absence of rort and eomes expression in ilc1s distinguishes them from rort-expressing ncr ilc3 and eomes-expressing cnk cells. initially, the nomenclature of ilcs included cnk cells, a notion many scientists still hold. but in this review, we limit ilcs to helper-like ilcs to distinguish these special innate lymphocytes from cnk cells. a defining function of ilcs is their production of a similar set of effector cytokines as those produced by cd4 th cells in the adaptive immune system. this feature of ilcs enables them to mount robust immune responses at the innate stage via acting on other immune or structure cells. through production of il-5 and il-13, ilc2s may induce the first wave of eosinophil recruitment and stimulate epithelial and smooth muscle cells, during type 2 responses to helminth infection or allergen inoculation [17, 18]. steady state production of il-22 by ilc3s is crucial for the homeostasis between host and commensals within the mucosal tissues. upon infection, ilc3s are also the major innate source of il-22 after receiving il-23 stimulation [8, 10]. ilc1 cells are relatively scarce in the gut but enriched in the liver in steady state [14, 20]. during early type 1 responses, ilc1s are better ifn- producers than cnk cells and they provide the initial protection in mice infected with t. gondii. ilc2s and th2 cells may collaborate to mount robust type 2 immune responses during the effector phase. some ilc2s express mhc class ii and thus are able to stimulate th2 cells to produce il-2, which in turn promotes ilc2 proliferation and cytokine production [21, 22]. however, possibly due to the lack or low level of costimulatory molecules cd80/cd86 on ilc3s, this type of antigen presentation functions through a suppressive mechanism to maintain the homeostasis of commensal specific th cell in the colon. ilcs have additional functions, which may or may not be shared by th cells. for example, ilc2 can produce amphiregulin, which facilitates the repair and reorganization of damaged tissues after viral infection. for example, ilc2s are enriched in adipose tissues and contribute to the beiging of white adipose tissue through production of cytokines and/or methionine-enkephalin [26, 27]. through il-22 and lymphotoxin production, ilc3s can induce intestinal epithelial cell expression of fucosyltransferase 2 (fut2) and thus regulate the epithelial fucosylation, which provides the metabolic substrates for commensals [28, 29]. the development, maturation, and maintenance of distinct ilc subsets are regulated by a set of specific transcriptional regulators, including t-bet, gata-3, and rort, similar to the regulation of effector th cell differentiation. a master regulator that determines th cell differentiation towards a particular subset also seems to direct the development of the related ilc subset. in addition, other factors such as ror, bcl11b, and ahr are involved in regulating the development and functions of ilc subsets. mirroring their critical functions during th1, th2, and th17 differentiation, the master regulators t-bet, gata-3, and rort are also implicated in fate determination of ilc subsets. furthermore, gata-3 is required for the maintenance and function of fully developed ilc2s [30, 31]. deletion of gata3 in mature ilc2s results in dramatic diminution of il-5 and il-13 production followed by the rapid disappearance of these cells. gata-3 is also expressed in ilc1 and ilc3 cells as well as in ilc progenitors, in which gata-3 has a critical function; we will discuss gata-3 function during early ilc development in detail in the progenitor section below. t-bet induction in ilc3s may be driven by notch signals, il-23 stimulation, and the microbiota. the gradient increment of t-bet levels directs further development of ccr6ilc3s into ncr ilc3s. some cells may even turn off rort expression to become t-betncr ex-ilc3s. in addition, t-bet regulates ifn- production by the ncr ilc3s. besides ccr6ilc3s accordingly, t-bet deficient mice lack ilc1s and ncr ilc3s but have normal ilc2s and ccr6 ilc3s. ilc1s were previously confused with cnk cells as both express t-bet and are capable of producing ifn-. however, cnk cells express eomes while ilc1s do not. in addition, ilc1s may also express cell surface markers such as cd49a, cd160, and/or cd127, which are usually absent on cnk cells. ror is highly expressed by ilc2s and is necessary for their development [32, 33]. ror deficiency results in dramatic reduction of ilc2s but does not affect the development of other ilc subsets. thus, mice reconstituted with ror deficient bone marrow are useful tools for studying ilc2 functions during immune responses. bcl11b is a critical factor during early stages of t cell development [34, 35]. but during ilc development, bcl11b deficiency only blocks the development of ilc2s as shown by two independent studies published this year [3638]. bcl11b is expressed as early as the common ilc progenitor stage and it may suppress the development of ilc3s. the mechanism of how bcl11b specifies ilc2 fate is yet to be determined. unlike gata-3 function in mature ilc2s, bcl11b deletion does not affect the maintenance or function of mature ilc2s despite bcl11b being highly expressed in mature ilc2s. the aryl hydrocarbon receptor (ahr) is well known to be involved in th17 cell differentiation and function. ahr is also expressed by both ccr6 and ccr6 ilc3s [40, 41]. in adult ahr deficient mice, ilc3 cell number in the gut is dramatically reduced, probably due to the defective accumulation and/or enhanced apoptosis of ilc3s. ahr is also critical for the function of ilc3s by regulating il-22 production and the ahr effects on ilcs seem to be ilc3 specific. ilcs, cnk cells, and t cells all develop from clps found in fetal liver and adult bone marrow. at late stages of t cell development, nave cd4 and cd8 t cells develop from the cd4cd8 double positive cells in the thymus whereas cd4 t effector th cells are differentiated from nave cd4 t cells in the periphery. considering the functional similarity between mature ilcs and cd4 th cells thus, a hypothesis concerning ilc development is that, like t cells, there may be a common progenitor for all innate lymphocytes, including ilcs and cnk cells, after the clp stage. in a subsequent stage, a common ilc progenitor would be capable of giving rise to all ilcs, in parallel with the potential of nave cd4 t cells to become different th effector cells. this hypothesis is supported by the fact that certain gene deletions affect the development of all innate lymphocytes and/or all ilcs. indeed, during the last year, we have witnessed a few breakthrough studies in identifying the common progenitors for ilcs [14, 42]. id2 is required for the development of all ilc populations since its deficiency results in the loss of all ilcs. using an id2 reporter mouse strain, a lineagecd127flt-3integrin47 population has been reported in a stage after clp and has lost the potential to become t, b, and cnk cells. id2 expression within this population has been shown by expression of an id2 fluorescent reporter. previously reported immature ilc2s in bone marrow are also id2; however, they can be excluded by cd25 staining. the multipotential capacity of these id2cd25 progenitors for all ilc subsets was also confirmed by in vitro single cell development assay. these lineagecd127flt-3integrin47id2cd25 progenitors are thus termed as common helper-like innate lymphoid progenitors (chilps). in addition to the critical role in maintenance and function of ilc2s, gata-3 is also crucial in the general development of all ilcs. it has been recently reported that gata3 deficiency prior to the clp stage affects the development of all ilcs in a cell intrinsic manner, indicating that gata-3 is a critical regulator for ilc development. in mice carrying a cre construct driven by the vav1 promoter to conditionally delete gata3 at the hematopoietic stem cell stage, dramatic defects in all il-7r-expressing ilcs in the periphery are noted. moreover, these gata3 conditional-deficient mice do not generate lymph nodes or peyer's patches, consistent with the finding that the development of functional lti cells is defective at the fetal stage. they also succumb to citrobacter rodentium infection, consistent with another report showing that gata-3 mediates the development of ilc3s by using chimera mice with hematopoietic precursor cells from e12.513.5 gata3 embryos. the essential role of gata-3 during ilc development is consistent with its indispensable function during cd4 t cell development after the cd4cd8 stage in the thymus. consistent with its critical role during ilc development, gata-3 expression levels in the common ilc progenitors, such as chilps, are comparable to that in ilc2s. thus, gata-3 is likely a master regulator for the development of common ilc progenitors. given that both id2 and gata-3 are highly expressed in ilc progenitors, it is reasonable to speculate that while id2 may direct the acquisition of the innate feature of ilcs similar to cnk cells gata-3 may play an indispensable role in directing the helper feature of ilcs similar to cd4 t cells. because of this, gata-3 may distinguish the helper lineage from cytotoxic cnk lineage during innate cell development. altogether, id2 and gata-3 coexpression during the common ilc progenitor stage may establish a special regulatory network that determines the innate and helper features of the ilcs. by analyzing a plzf fate mapping mouse strain, researchers found that the majority of mature ilcs have expressed plzf with the exception of the ccr6 ilc3 lineage. plzf is hardly detectable in mature ilcs but is transiently expressed by a subset of the chilp population in fetal liver and adult bone marrow. these plzf progenitors are multipotential cells and are able to give rise to all ilcs except ccr6 lti cells. furthermore, these plzf progenitors do not develop into t cells, b cells, or cnk cells. plzf progenitors may develop after the chilp stage and have lost the potential to develop into lti lineage. the function of plzf during ilc development remains elusive particularly because plzf is only transiently expressed during the early development stage. furthermore, although zbtb16 mice with plzf deficiency have altered ilc development especially for ilc2s, all the ilcs can still be detected in these mice. an obvious issue for these defined common ilc progenitors is that they are actually heterogeneous populations. within the chilp cells, there should be some ccr6 ilc3 progenitors within the plzf population, which will never turn on plzf expression. even within the plzf population, only a minority of the cells have the potential to become multiple ilcs during in vitro single cell development assay. it is likely that both id2 chilps and plzf common ilc progenitors contain a large fraction of partially committed ilc immediate progenitors. common progenitor multipotent for all ilcs is still required. meanwhile, in addition to fetal liver and adult bone marrow, the common ilc progenitors may also exist in other tissues, such as fetal intestine, as was shown in a recent study using arginase-1 reporter mice. this report suggests that we should not limit ourselves to study fetal liver and adult bone marrow cells to gain further knowledge on ilc progenitors. as mentioned above, many ilc progenitors found in fetal liver and adult bone marrow may have already committed partially to a specific ilc lineage. indeed, bcl11b-expressing progenitors appear within the chilp population; yet these cells have already committed to the ilc2 fate. there are also abundant immature ilc2s, presumably the most immediate ilc2 precursors, present in the bone marrow; these cells express mature ilc2 markers sca-1 and cd25. plzf fate mapping analysis indicates that most immature ilc1s have expressed plzf and thus are derived from the plzf ilc progenitors rather than the cnk progenitors. a couple of other transcription factors, nfil3 [4851] and tox, have also been found to be involved in the development of both ilcs and cnk cells. since their expression is detected earlier than id2 expression, which occurs at the chilp stage, the expression of these transcription factors may mark even earlier common progenitors for both ilcs and cnk cells, similar to cd4cd8 thymocytes during t cell development. however, the phenotypes of ilc development in mice deficient in either of these regulators are not as dramatic as those resulting from id2 or gata3 deficiency. the mechanisms through which nfil3 and tox function during ilc and cnk cell development require further investigation. given the functional similarities between the innate ilcs and cnk cells and the adaptive cd4 and cd8 t cells, it is reasonable to propose that ilcs and cnk cells follow a similar developmental pathway to that of t cells. a clp may give rise to a common progenitor for all innate lymphocytes including ilcs and cnk cells, which subsequently gives rise to chilps. distinct ilc subsets are further developed from the plzf chilp stage as the differentiation of th effectors from nave cd4 t cells. thus, the development of innate lymphocyte subsets, including ilcs and cnk cells, to a certain extent, mirrors that of t cells (figure 1). as discussed above, more and more regulators are being found to be involved in the development of ilcs. interestingly, most of these regulators are also involved with t cell development. however, distinct from t cell development, the regulatory functions of the various transcription factors during ilc development are not related to tcr-mediated thymic selection, which, to some extent, may explain why the deficiency of certain regulators has distinct effects on ilc and t cell development. although deletion of either one of the newly identified genes, including nfil3, tox, or tcf7 [53, 54], results in defective ilc development to a various extent, none of these defects are as severe as that resulting from id2 or gata3 deficiency. thus, it is possible that in the early progenitors of the ilc lineage, these regulators may function through a network designed for fine-tuning ilc development. id2 and gata-3 might form the core complex, while other regulators are involved in tuning the optimal function of the core components. id2 or gata3 deficiency dramatically affects ilc development, whereas deletion of other regulatory components results in various incomplete defects of ilc development. nuclear factor interleukin-3 (nfil3; also known as e4-binding protein 4, e4bp4), a basic region leucine zipper transcription factor, is critical for cnk cell development. based on this, it is possible that nfil3 is expressed and functions at a progenitor stage common to both ilcs and cnk cells. nfil3 deficient mice showed dramatic defects during the transition from clp to chilp stage [48, 51]. however, ilc development is not completely abolished in nfil3 deficient mice; in the periphery, there are still substantial numbers of ilcs that have escaped the developmental block, although the population is too small to control infectious challenges. nfil3 expression is induced by il-7 signaling, which is also critical for the development of ilcs. after development, nfil3 maintains a very high level of expression in mature ilcs, a level higher than that in cnk cells. nfil3 is dispensable for ilc3 maintenance; however, its function in other mature ilcs remains unclear. thymocyte selection-associated high-mobility group box protein (tox) is a member of the high-mobility group box superfamily. it contains a dna-binding domain and is required for the development of t [56, 57], nk, nkt, and lti cells. a new study this year showed that tox also has broad effects in ilc development. in tox deficient mice, the chilps as well as mature ilcs were severely reduced compared with these cells in wild type mice. there is an intrinsic defect in the notch signaling pathway in tox deficient chilps, which may explain the defect in ilc development in tox deficient mice. t cell factor 1 (tcf-1) is a critical transcription factor for t cell fate specification at the early development stage. tcf7 (gene encode tcf-1) deficiency affects both ilcs [53, 54] and cnk cells. similar as during t cell development, tcf-1 is regulated by notch signaling and may be involved in the induction of il-7r and gata-3 expression during ilc development [53, 54]. based on the sequential expression of nfil3, tox, tcf-1, id2, and gata-3 and the knowledge we have concerning their relationships in different systems, it is likely that nfil3 expression initially increases after the clp stage, which in turn regulates the expression of tox and id2. after id2 is turned on, the core assembly involved in directing the innate features of ilc development begins to function. although gata-3 is expressed at low levels at the clp stage, its function during ilc development requires high expression levels. tox is involved in the regulation of notch signaling pathway, which in turn regulates tcf-1 and gata-3. tcf-1 is then required for optimal expression of gata-3. upon increased expression of gata-3, the helper transcription factor assembly nfil3, tox, and tcf-1, in connection with the notch pathways, form a network to prepare for the upregulation of id2 and gata-3, which together form the executive regulatory network to direct lineage fate determination of ilcs, possibly with continued assistance from the initiation transcription factors such as tcf7, tox, and nfil3 (figure 2). additional experiments are required to confirm this proposed regulatory network model and it is likely that additional regulators for ilc development may soon be discovered. studies on the newly identified ilcs in recent years have enriched our knowledge on the innate lymphoid lineage development and have provided us with more evidence supporting the close relationship between the innate and adaptive immune systems during evolution. the classification of the ilc population was initially based on their capacity to produce a similar set of effector cytokines compared to th cells. subsequent studies have revealed that the development of ilcs is also regulated by a similar set of key transcription factors required for t cell development. after the clp stage, innate lymphoid lineages and t cells lineages start to develop separately but the regulatory mechanisms may still be shared. nfil3, tox, and tcf-1 expression at an early stage immediately after clp in the innate lymphoid lineage may mark the common progenitor for both ilcs and cnk cells. ilc and cnk lineages are further separated by the induced expression of id2 and gata-3 in the common ilc progenitors. at later stages, additional regulators are upregulated to drive the distinct fates of different ilc subsets. in conclusion, the ilc cell fate is precisely regulated during development by a network of multiple serially expressed regulators, which may form a regulatory network during development and render the developed ilcs with both innate and helper features.

recent studies on innate lymphoid cells (ilcs) have expanded our knowledge about the innate arm of the immune system. helper-like ilcs share both the innate feature of conventional natural killer (cnk) cells and the helper feature of cd4+t helper (th) cells. with this combination, helper-like ilcs are capable of initiating early immune responses similar to cnk cells, but via secretion of a set of effector cytokines similar to those produced by th cells. although many studies have revealed the functional similarity between helper-like ilcs and th cells, some aspects of ilcs including the development of this lineage remain elusive. it is intriguing that the majority of transcription factors involved in multiple stages of t cell development, differentiation, and function also play critical roles during ilc development. regulators such as id2, gata-3, nfil3, tox, and tcf-1 are expressed and function at various stages of ilc development. in this review, we will summarize the expression and functions of these transcription factors shared by ilcs and th cells. we will also propose a complex transcriptional regulatory network for the lineage commitment of ilcs.

PMC4563091

pubmed-684

thymic hyperplasia is defined as the enlargement of the thymus with a histologically normal cortical and medullary component. the hyperplastic gland can become very large; however, besides its larger size, the imaging appearance of the enlarged thymus is normal (1). no child cases of pericardial lipomatosis have been reported in the english literature. to the best of our knowledge, this is the first case report of a child with thymic hyperplasia simulating a fat containing mass accompanied by pericardial lipomatosis and right facial hemihypertrophy. an 8-year-old boy visited the outpatient clinic for evaluation of facial asymmetry and narrowing of the right external auditory canal. upon physical examination, the patient showed right side facial enlargement, cranial protrusion, gingival hypertrophy, and soft tissue thickening of the external auditory canal. there was no evidence of asymmetry of the body trunk and limbs or any subcutaneous/cutaneous lesions noted. the patient denied any prior history of medical, surgical problems, or any family history of hemihyperplasia. a chest posteroanterior film identified a large mediastinal mass draping over the right cardiac shadow (fig. chest ct revealed a large mass occupying the anterior mediastinum showing no mass effect or displacement of adjacent structures. it had a bilobular configuration with asymmetric mass-like enlargement of the right lobe which contained curvilinear or nodular areas of fatty component. concomitant diffuse fat attenuation filling the pericardial space suggestive of pericardial lipomatosis was also noted. 1c-f). a fat containing benign mediastinal mass including thymolipoma or lipoblastoma was suspected. a malignant mass such as liposarcoma or malignant germ cell tumor was also included in the differential diagnoses. as it was difficult to determine whether the more predominantly enlarged right lobe was the only portion involved or both the right and left lobes were pathologic, mediastinal mri was performed to further determine the precise extent of the lesion. the mri results revealed a large anterior mediastinal mass that showed heterogeneous iso-signal intensity to the chest wall muscles on t1- and t2-weighted images and heterogeneous enhancement on fat saturated t1-weighted images. there were whorls or nodular areas of high signal intensity within the thymus on both t1- and t2-weighted images which showed suppression on fat saturated contrast enhanced t1-weighted sequence. the pericardial space was also filled with high signal intensity on both t1- and t2-weighted images and showed fat suppression on fat saturated contrast enhanced t1-wieghted images (fig. 1 g, h). for evaluation of facial asymmetry and narrowing of the right external auditory canal, contrast-enhanced ct of the paranasal sinuses was also performed. paranasal sinus ct showed hypertrophy of the right side parotid gland, adenoid tissue, temporalis muscle, and sternocleidomastoid muscle. moreover, mild narrowing of the right external auditory canal was noted due to soft tissue thickening along the posterior wall of the canal (fig. the operative findings revealed a large anterior mediastinal mass showing no invasion into the mediastinal structures and the parietal pleura. on gross specimen, the mass was yellowish with a lobulating contour confined within a thin fibrous capsule. the mass was about 17 13 3 cm in size and weighed 280 g (fig. the cut surface showed multifocal areas of light-yellow spots of fatty tissue scattered within the thymic mass. there was no evidence of hemorrhage or areas of cystic, necrotic changes (fig. microscopically, the tumor showed normal distribution of the cortex, medulla and hassall's corpuscles. it had fibrous and myxoid stroma with focal areas of adipose tissue admixed in various proportions (fig. the postoperative course was uneventful and the patient was discharged to follow-up on an outpatient basis for further management of right facial hemihypertrophy. the patient was followed up for about a year and a half and has not shown any evidence of progressive course or cutaneous/subcutaneous lesions thus far. thymic hyperplasia is defined as an enlarged thymus beyond the normal upper limit for any given patient age. hence, the imaging appearance is similar to a normal thymus except for its larger size (1). moreover, thymic hyperplasia shows homogeneous attenuation similar or slightly lower than the muscles on noncontrast ct and homogeneous enhancement of 20-30 hounsfield units after contrast enhancement. on mri, thymic hyperplasia show slightly brighter si than that of the muscles on both t1-weighted and t2-weighted images (1-3). opposed-phase images may show diffuse decreased signal intensity (4). a normal thymus has a homogeneous appearance in childhood and begins to appear heterogeneous on imaging studies at about the age of thirty when the adipocytes become dominant. hence, any condition in which the thymus appears heterogeneous in a child should raise suspicion of a pathologic condition (3). in our case, the enlarged thymus had a heterogeneous appearance with areas of fat admixed within the thymic tissue. it was an unusual finding for a child and a fat containing anterior mediastinal mass such as thymolipoma, lipoblastoma was to be considered. malignant lesions such as malignant germ cell tumor or liposarcoma was also considered as a possible differential diagnosis. the mass was confirmed to be a hyperplastic thymus composed of normal thymic components and cellular organization suggestive of a true thymic hyperplasia on histologic examination. although it is well known that the thymus can undergo hyperplastic changes after suppression from recent stressful conditions such as chemotherapy for neoplasm, grave's disease, corticosteroid therapy for cushing's disease, irradiation or thermal burns and some autoimmune diseases (systemic lupus erythematosus, hashimoto thyroiditis, addison's disease, acromegaly), our patient did not have any of these well known systemic stresses or conditions causing thymic hyperplasia (5). however, the patient did have facial hemi-hypertrophy on the right side. as it has been reported that the left lobe of the thymus is usually larger than the right, the predominant enlargement of the right lobe with more prominent fatty component on the right side shown in our case may be related to the underlying right side hypertrophic condition of this patient (2, 3). complimentary to this finding, the patient also had diffuse pericardial lipomatosis, which is also an extremely rare finding with only a few cases reported in adult patients (6, 7). pericardial lipomatosis, whether isolated or accompanied by other diseases, has not been reported in the pediatric population. the combination of thymic hyperplasia, pericardial lipomatosis, and facial hemihypertrophy seen in our case is also the first to be reported, both in children and adults. although the patient did not undergo any genetic studies, considering that the patient has facial hemihypertrophy, thymic hyperplasia, and deep soft tissue lipomatosis, there is a possibility that the clinical and imaging features of this patient could be a spectrum of hemihyperplasia syndromes. hyperplasia syndromes include a heterogeneous group of disorders with overgrowth of one or more limbs or body parts as a predominant finding and includes disease entities such as beckwith-wiedemann syndrome, proteus syndrome, hemihyperplasia multiple lipomatosis syndrome (hhml), cowden/bannayan-riley-ruvalcaba syndrome (brrs) among others. beckwith-wiedemann syndrome is the most well known hemihyperplasia syndrome and is associated with anomalies such as macroglossia, abdominal wall defects, hypoglycemia, and increased risk of embryonal tumors. it is also well known because of its genotypic abnormalities of the distal region of chromosome arm 11p. proteus syndrome is a rare, highly variable hamartomatous syndrome which is clinically diagnosed if the patient satisfies the necessary diagnostic criteria of disproportionate overgrowth, connective tissue nevi, dysregulated adipose tissue, and vascular malformation. cutaneous lesions such as connective tissue nevi, tends to appear over time and may delay the diagnosis. some report patient with proteus syndrome showing mutation of pten (phosphatase and tensin homologue deleted on chromosome 10) and glypican 3 (gpc3)gene, while others cast doubts over its involvement. hhml syndrome is thought to be a distinct form of proteus syndrome which does not satisfy the diagnostic criteria and shows less severe hyperplasia of the limbs or body parts. brrs is characterized by macrocephaly, mild mental retardation, cutaneous lipomas, or hemangiomas, while pigmented macules of the glans penis and the onset is noted at birth or shortly thereafter. cowden syndrome is an adult-onset condition with mucocutaneous signs and increased risk of cancer (9, 10). among the disease entities of the hemihyperplasia syndromes listed above, the latter three are those which are known to show both hyperplasia and lipomas as in our case. however, as the asymmetric overgrowth in our patient was not from birth and the patient did not show any sign of mental retardation, brrs may be excluded and the possible diagnosis could be narrowed down to proteus syndrome or hhml syndrome. until now, our patient was followed up for about a year and a half and the most fitting diagnosis appears to be hhml. however, although the patient did not show cutaneous, epidermal nevi or capillary, venous, and lymphatic malformation, they may develop over time and show progressive enlargement. hence, further clinical observation and imaging studies including a serial skeletal survey will be necessary for the diagnosis or exclusion of proteus syndrome. unfortunately, genetic studies that may support the diagnosis of hemihyperplasia syndrome was not performed. although the diagnosis and exclusion of several hyperplastic syndromes may be available with genetic studies, it may not be of any help in differentiating proteus syndrome from hhml since there are no molecular studies available at this moment. until molecular mechanisms of the hemihyperplasia syndromes are further elucidated, thorough observation of any change in the clinical findings will remain essential for the diagnosis of patients. in conclusion, we have reviewed and discussed the imaging findings of a rare combination of thymic hyperplasia, pericardial lipomatosis and facial hemihypertrophy in a pediatric patient.

we report a case of thymic hyperplasia accompanied by pericardial lipomatosis and right facial hemihypertrophy in an 8-year-old boy. on imaging studies, the hyperplastic thymus had prominent curvilinear and nodular fatty areas simulating a fat-containing anterior mediastinal mass, which is an unusual finding in children. to our knowledge, this is the first report on a child with a combination of thymic hyperplasia, pericardial lipomatosis, and right facial hemihypertrophy. the radiologic findings are presented with a brief discussion.

PMC3088855

pubmed-685

mastitis represents a major economic cost to dairy farmers with losses of up to 190 per case depending on severity and 60 per cow for the average milk supplier. the losses associated with mastitis include discarded milk, increased number of culled cows, cost of antibiotic treatment and reduced milk quality and price. even though, it has been shown that factors such as genetic characteristics, impaired immune-function, feeding regimes and machine milking are related to mastitis, poor milking hygiene has been associated with increased somatic cell count (scc), reduced milk production and inferior milk quality. machine milking may also be considered as a major cause of bacterial cross contamination from cow to cow. however, a good premilking hygiene routine can decrease the cow infection ratio by not only reducing udder bacterial contamination from the environment, but also reducing bacterial contamination from other infected animals. newbould. showed a positive relationship between bacteria presence on teats and new intramammary infection. staphylococcus aureus is one of the major and more virulent pathogens that can cause mastitis infection and lactating cows can be considered one of the main reservoirs of this species. moreover, staphylococcus aureus colonisation of teat skin increases the risk of staphylococcus aureus intrammmary infection. the aim of any teat cleaning routine is not only to reduce mastitis infection risk, but also to enhance milk quality.. also showed that spore concentration in milk was highly correlated with spore concentration on teats; hence milk bacterial spores most likely originated from dirt and faeces attached to the teats at the time of milking. many methods of pre-milking udder preparation are practiced by producers and overall, one of the most important aspects of pre-milking udder hygiene is udder dryness at the time of machine attachment (visser et al. bacteria contaminated water can also increase milk bacterial counts (visser et al. 2007). a commonly used pre-milking teat preparation method involves washing teats by hand with water and drying teats with a paper towel just before the machine is attached. there is strong evidence that among all pre-milking procedures, wet cleaning treatment, followed by paper towel manual drying will result in the lowest bacterial counts: this practice is particularly effective in reducing milk bacterial contamination during the winter housing period. similar seasonal bacterial infection trends linked to the pasturing/housing routine have been observed by hutchison et al.. as an alternative to washing and drying teats, many producers now dip teats pre-milking with various disinfectant products such as iodophor solution, iodine based gel, sodium hypoclorate, dodecyl benzene sulfonic acid (ddbsa), chlorine, chlorhexidine, phenolics and alcohol. showed that both iodine based gel and 0.5% iodophor solution significantly reduced milk bacterial count and clinical mastitis occurrence compared to teat washing and drying with paper towels. however, showed that pre-milking disinfection with 0.25% iodine dip or phenolics solution did not reduce the incidence of clinical mastitis when compared to post-milking disinfection only. however, pankey et al. reported that predipping reduced the rate of intrammamary infection with major mastitis pathogens such as staphylococcus aureus, streptococcus agalactiae and coliforms. in addition to iodine products, chorhexidine when used as a pre-dip, significantly decreased scc values in herds infected with mastitis. furthermore, gibson et al. concluded that a chlorine based dip followed by a dry wipe was an effective pre-milking treatment for controlling cow mastitis. the benefit of using some disinfectant products pre-milking in reducing new mastitis infection has been demonstrated. a study by roberson et al. demonstrated that teat orifices colonised with staphylococcus aureus were 3.3 times more likely to have intramammary infection. therefore, reducing the microbial count on teats prior to milking is an important step in the prevention of mastitis. the type of disinfectant product used as a pre-dip may have varying degrees of success in reducing the microbial count on teats. however, there is no knowledge on the effect of pre-milking disinfection using a range of newly-formulated teat disinfectants in reducing the microbial counts on teats. thus, the objective of this study was to investigate the effectiveness of six different pre-milking teat preparation procedures on lowering the staphylococcal, streptococcal and coliform bacterial count on teat skin. six pre-milking teat preparations were applied to spring calving holstein friesian cows during two herd management periods, while cows were housed (indoors) and while cows were grazed on pastures (outdoors). during the indoor period and for the previous three months, cows were housed in an easy-feed slatted house with matted cubicle beds and dressed with lime daily to maintain a dry bed. during the outdoor period and for the previous three months, cows were grazed under a rotational grazing system and moved every 24 hours to new pasture. ten cows which were free from clinical mastitis infection were randomly chosen for each pre-milking preparation for a period of five days per treatment. the six pre-milking applications applied are identified as ' washing and drying ', ' iodine ', ' chlorhexidine ', ' chlorine ', ' wipes ' and ' no preparation '. description of pre-milking teat preparation treatments an iodine (2.0% w/v) teat disinfectant (1-4 mix) containing emollients was applied as a post-milking teat disinfectant to the following treatments, ' iodine ', ' washing and drying ', ' no preparation ' and disinfectant ' wipes ' using an in-parlour sprayer. ' chlorhexidine ' teat foam and ' chlorine ' teat foam were used for post-milking disinfection, for the respective pre-milking treatments. the same foam products were used for post-milking disinfection, as it was considered more likely that where the foam products are used on farms as a pre-milking disinfectant, they would also be used for post-milking disinfection. the possibility of a teat skin reaction if different pre- and post-teat disinfectant products were used was also considered. analysis of the bacterial counts on teats prior to teat preparation showed no effects of using different post milking disinfectant products. the ' chlorine ' teat foam product was prepared daily by mixing a foam active base and activator (50/50). the entire circumference of the teat was covered in teat foam by immersing each individual teat in the teat cup. ' iodine ' was applied as a pre-milking disinfectant by spray, as this was considered the most common application method on irish farms. approximately, 15 mls of iodine was applied to the teats of each cow when used as a pre and post milkinf disinfectant. teats disinfected pre-milking were dried with individual disposable paper towels approximately 30 seconds after the disinfectant was applied and prior to milking. cows were milked in a 20-unit, 80-degree side-by-side milking parlour and were milked in the morning at 07:30 h and in the afternoon at 15:30 h. cows were exposed to each pre-milking preparation for a period of five days, for each management period at both the morning and afternoon milking. on day four and five, all teats from each cow were swabbed using one sterile swab (cultiplast, lp italian spa, via carlo reale, 15/4, 20157, milano, italy) per cow before teat preparation and repeated after teat preparation at the morning milking (table 2). the sterile swab was rubbed across the teat orifice and down the side of each teat avoiding contact with the udder hair or cows flank at all times. a small number of teats (< 7%) that were considered excessively soiled with faeces, and where swabbing of the teat skin was not possible, in addition, by omitting these soiled teats, the potential contamination of the agar plates was avoided. in those instances, there were no differences in the number of excluded teats between treatments or management periods. swabbing procedure carried out before and after teat preparation for six pre-milking teat preparation procedures *=one swab per cow,**=all repeated after teat preparation immediately after teat swabbing was completed, the swabs were placed in individual sterile bottles containing 5 mls of tryptic soy broth (bd. bbl trypticase soy broth (soybean-casein digest broth). the broth was manufactured by becton, dickinson and company, sparks, md 21152 usa.38800 le pont-de-claix, france. the broth was prepared in 500 ml amounts and autoclaved at 121c for 15 minutes, and then distributed into 5 ml aliquots in a laminar flow cabinet. the sterile bottles containing the swabs were frozen (-20) awaiting laboratory analysis for the presence of staphylococcal, streptococcal, and coliforms. the swabs were subsequently plated on three separate selective agars: baird parker (staphylococcal), edwards (streptococcal), and macconkey (coliforms). specific bacteria types within each category were not defined. following incubation at 37c for 24 hours, microbial counts (cfu/ml) for each pathogen type were manually counted and assigned to one of six categories depending on the bacterial counts measured. (1=no pathogen present, 2=1 to 10, 3=11 to 20, 4=> 20, 5=numerous, 6=infinite). the results were analysed by logistic regression using sas. preliminary analysis of the results for before and after treatment was by fitting generalised logits for the multinomial response because, while the response was ordinal in nature, the data did not meet the assumptions of a proportional odds model. 1=lower category count after treatment and 0=same or greater category count after treatment. standard maximum likelihood estimation of the regression could not proceed because of the technical condition of quasicomplete separation for the effects. an alternative strategy due to heinze and schemper was implemented using their sas macro code there were no differences in the levels of staphylococcal, streptococcal and coliform bacterial counts measured on teats (assigned to different teat preparations) ' before ' teat preparation. however, there was a significant reduction (p<0.001) in the levels of staphylococcal pathogens on teats after teat preparation with all treatments except the ' no preparation ' pre-milking treatment. the probability of a reduction in the staphylococcal counts tended to be higher (p<0.06) for cows managed outdoors compared too indoors. treatment with ' chlorine ' teat foam resulted in a 30% reduction in staphylococcal counts when used on cows at pasture compared to its use on cows indoors, likewise ' iodine ' and ' wipes ' resulted in an increased staphylococcal count reduction of 18% and 20%, respectively, when used on cows at pasture (table 3). on the other hand ' chlorhexidine ' teat foam had a 20% greater reduction in bacterial counts when used on housed cows, compared to cows at pasture. however, the median reduction in the staphylococcal count for all treatments was 21% better for cows on pasture compared to its use on housed cows. the probability of a reduction in streptococcal bacterial counts in response to overall teat preparation was not significantly different between indoor and outdoor management periods. however, some pre-milking treatments resulted in greater reductions in counts when used on cows housed compared to when used on cows at pasture and vice versa. the treatment containing ' iodine ' resulted in a 26% greater reduction in streptococcal counts when used on cows indoors compared to when used on cows at pasture (table 3). similarly, the ' chlorhexidine ' teat foam treatment had a 20% greater reduction when used indoors, compared to when used on cows outdoors. however, both the ' chlorine ' and disinfectant ' wipes ' teat preparation treatments resulted in a 30% greater reduction when used outdoors compared to when used on cows indoors. the reduction observed for ' coliform ' counts was low for both indoor and outdoor management periods (table 3). however, the probability of a greater response to teat preparation for ' coliform ' bacteria is more likely outdoors (or=0.27; p<0.05) compared to indoors (table 4). effect of pre-milking teat preparation treatment in reducing staphylococcal, streptococcal and coliform bacteria counts on teats at two time periods (indoor and outdoor) (% reduction) estimated odds ratios (or) and their 95% confidence intervals (ci) for the effect of pre-milking teat preparation and management period on staphylococcus spp. (str) and coliform (col) counts table 4 shows the association between pre-milking teat preparation procedure and management period on the probability of a reduction in the microbial levels of staphylococcal, streptococcal and coliform pathogens. ' washing and drying ' had a higher probability (p<0.001) of reducing both the staphylococcal and streptococcal counts on teats compared to ' no preparation ', as would be expected. both ' chlorhexidine ' (or=4.46) and ' wipe ' treatments (or=4.46) had an increased probability (p<0.01) of reducing the staphylococcal count on teats compared to ' washing and drying '. treatments with ' chlorine ' (or=3.45) and ' wipes ' (3.45) had the highest probability (p<0.01) of reducing the streptococcal count compared to ' washing and drying '. both ' iodine ' (or=1.24) and ' chlorhexidine ' (or=1.65) also tended to have greater probability of reducing streptococcal counts compared to ' washing and drying '. ' washing and drying ' had a higher probability (p<0.05) of reducing the coliform count on teats compared to ' no preparation '. any of the remaining treatments did not enhance reduction in coliform numbers (p>0.05) compared to ' washing and drying '. no differences in the microbial counts were observed on teats, regardless of the post-milking disinfectant products used at the previous milking. therefore, using different products as a post disinfectant in this study did not influence the outcome of the study. in this study, the use of some disinfectant products for pre-milking teat preparation had beneficial effects on reducing the levels of staphylococcal and streptococcal pathogens on teat skin compared to ' washing and drying ' and ' no preparation '. where teat preparation is omitted, increased teat colonisation could be expected and this may result in new intramammary infection. this would concur with the findings of pankey et al. who reported that pre-dipping can reduce the rate of intrammamary infection with major mastitis pathogens. furthermore, gibson et al. concluded that most pre-milking teat cleaning treatments reduce the teat total bacterial count, but that cleaning effectiveness was influenced by the type of disinfectant and the application methods. while commercially available disinfectant products may appear to use similar ingredients, the levels and strength of ingredients with additional emollients may influence the success of a product in reducing somatic cell count and improving teat condition over a longer period. when ' iodine ' was used as a pre-milking disinfectant, while it did not significantly reduce bacterial numbers, it was 2.3 times more likely to reduce staphylococcal and 1.24 times more likely to reduce streptococcal counts on teats compared to ' washing and drying ' or no preparation treatments. this result tends to agree with the findings of ingawa et al. who demonstrated that iodine reduced the bacterial count on teats compared to washing and drying. the use of a 0.25% iodine solution pre-milking has also been shown by oliver et al. to reduce major pathogen intramammary infections resulting from streptooccus uberis and dysgalactiae by as much as 49%. however, including ' iodine ' as a pre-milking teat preparation treatment may have implications for milk residues as pre or post dipping with an iodine product can increase iodine levels in milk. therefore, correct disinfectant concentration and drying after application with paper towels must be advised to reduce milk residues. ' chlorhexidine ' teat foam which is a new product sold on the irish market was 4.46 times more likely to reduce the staphylococcal count on teats prior to milking when compared to washing and drying. this is of particular signifance to irish dairy farmers as staphylococcus aureus pathogens are to be found in 51% of irish bulk milk samples. staphylococcus aureus colonisation on teat ends has been shown to increase the risk of intrammmary infection. therefore, a reduction in staphylococcal numbers on teat ends may reduce the new infection rate on irish farms. additionally, it has previously been demonstrated that a disinfectant product containing chlorhexidine, when used as a post disinfectant and when used as a pre-milking disinfectant over a long time period, reduced the somatic cell count. the ' chlorine ' teat foam product used in this study was 3.45 times likely to reduce the streptococcal count on teats prior to milking when compared to the ' washing and drying ' treatment. this is in agreement with gibson et al. who showed that a similar chlorine based dip followed by a dry wipe was a most effective treatment for controlling cow mastitis and reducing milk contaminants. the positive effect observed with ' wipes ' in reducing both staphylococcal and streptococcal counts on teats may be due to the physical manipulation of teats combined with the disinfectant. dry wiping without disinfectant has been shown to reduce the total bacterial count in bulk milk. however, the use of dry or wet towels by themselves did not have any significant effect on reducing coliform counts in milk. in this study, the reduction in the coliform count on teats with any of the teat preparation treatments used was low. this may be influenced by a low initial level of coliform pathogens present on teats prior to treatment. teat washing combined with drying with individual paper towels reduced staphylococcal, streptococcal and coliform pathogens compared to ' no preparation ' and was particularly effective in reducing streptococcal counts when used on cows indoors compared to outdoors. this is in agreement with the findings of mckinnon et al. who concluded that washing and drying teats prior to milking significantly reduced milk bacterial counts during the winter housing period, compared to the pasture summer period. however, the results of this study would indicate that the probability of a reduction in staphylococcal and streptococcal counts could be expected to be greater where a disinfectant is used pre-milking compared to ' washing and drying ' or ' no preparation ' treatments. this study shows that the use of some disinfectant products for pre-milking teat preparation can have beneficial effects on reducing the levels of staphylococcal and streptococcal pathogens on teat skin. therefore, the possibility of bacterial transfer from cow to cow during milking could be expected to be reduced compared to many farm situations where ' no preparation ' is normally practiced. the study time period was not sufficient to come to any conclusions on the effect of premilking teat disinfection with regard to teat condition or somatic cell count. in conclusion, bacterial numbers, specifically staphylococcal and streptococcal numbers on cow teat surfaces, were significantly reduced when disinfection products were applied to teats. the use of wipes was particularly effective due to the physical wiping action in conjunction with the disinfectant application. while the practice of washing and drying did reduce bacterial numbers compared to not cleaning teats at all, it could not be considered to be as effective as cleaning with disinfectant products. given the level of bacterial numbers on non-prepared teats and the reduction observed with chemical disinfectant, it may be advisable to include this process as part of the milking routine. however, pre-milking teat disinfection must be followed by teat drying using individual paper towels to minimise the possibility of chemical residues in milk.

a study was carried out to investigate the effect of six pre-milking teat preparation procedures on lowering the staphylococal, streptococcal and coliform microbial count on teat skin prior to cluster application. the teat preparations included ' iodine ', ' chlorhexidine ' teat foam, ' washing and drying ' with paper, ' no preparation ', ' chlorine ' teat foam, and disinfectant ' wipes '. teat preparations were applied for five days to 10 cows for each treatment during two herd management periods (indoors and outdoors). teats were swabbed on day four and five before teat preparation and repeated after teat preparation. the swabs were plated on three selective agars: baird parker (staphylococcus spp.), edwards (streptococcus spp.), and macconkey (coliform). following incubation, microbial counts for each pathogen type were manually counted and assigned to one of six categories depending on the microbial counts measured. the results were analysed by logistic regression using sas [28]. the main analysis was conducted on binary improvement scores for the swabbing outcomes. there were no differences for staphylococcal, streptococcal and coliform bacterial counts between treatments, measured ' before ' teat preparation. treatments containing ' chlorhexidine ' teat foam (or=4.46) and ' wipes ' (or=4.46) resulted in a significant reduction (p<0.01) in the staphylococcal count on teats compared to ' washing and drying ' or ' no preparation '. ' chlorine ' teat foam (or=3.45) and ' wipes ' (3.45) had the highest probability (p<0.01) of reducing streptococcal counts compared to ' washing and drying ' or ' no preparation '. there was no statistical difference between any of the disinfectant treatments applied in reducing coliforms. thus, the use of some disinfectant products for pre-milking teat preparation can have beneficial effects on reducing the levels of staphylococcal and streptococcal pathogens on teat skin.

PMC3113755

pubmed-686

an increased man-made radiation exposure-risk from the use of high-dose imaging modalities such as computed tomography and angiographic suites is now being observed in many health-care centers with over 3.6 million diagnostic examinations performed annually worldwide. interventional procedures are performed by cardiologists, radiologists, endovascular surgeons, operation theater staff, etc., due to the well-known benefits in medicine. however, it is crucial for the referring clinician and the interventionalist (radiologist/cardiologist/clinician in the operation theater) to assess the potential benefit-risk ratio for various interventional procedures as some of the procedures involve a high radiation exposure due to prolonged fluoroscopic screening. all interventional cardiological procedures are invariably performed using dedicated fluoroscopy and angiography suites equipped with either image intensifier (ii)-based or flat panel detector (fpd)-based systems. the ii-based systems have been used for fluoroscopy for more than two decades. on the other hand, the fpd-based systems for medical imaging emerged in the 1990s initially for two-dimensional (2d) projection x-ray image, and subsequently for a real-time interventional angiography suites equipped with ii or fpd have the potential to impart high radiation doses to patients if optimization strategies are not well-implemented. stringent optimization involves orientation of staff, consistent restriction of frame rates during image acquisition, using low dose settings, judicious use of magnification, etc. it is also necessary to understand the potential risks due to radiation from different interventional procedures. for this reason, it is necessary that one should be knowledgeable in the magnitude of radiation dose associated with each intervention. this can be achieved by measuring real-time doses using devices such as a dose area product (dap) meter. most of the newer angiography machines are equipped with a dap meter fitted on the collimator assembly of the machine. dap is particularly a useful method for assessing and comparing the radiation dose from screening procedures and acts as a surrogate for radiation risk. entrance surface dose (esd) is also used for measuring radiation doses. from the dose descriptors, it is possible to estimate organ doses as well as effective doses for each procedure. radiation doses from interventional procedures have been widely reported in literature, with more emphasis on doses from ii-based systems. however, there are only a few reports on radiation doses from fpd systems as it may be a transition period from ii to fpd for most of the interventional users. some patient and phantom-based studies reported in literature state that doses from fpd are higher than ii systems. few other studies report that radiation doses from fpd are lower than ii systems. in comparing conventional and digital systems, hence, it is not clear whether fpd imparts lower radiation dose than ii and whether there would still be a need to further optimize doses in the newer fpd systems. the purpose of this article is to review and compare radiation doses from ii and fpd-based systems in interventional cardiology in a tertiary referral center that has introduced fpd system recently. it is anticipated that this information will be useful for those performing cardiological interventions and for those who are on a transition from ii to fpd. cardiovascular interventions were performed using two dedicated catheterization labs, each equipped with philips allura fd10 flat panel system (netherlands). the dose area product (dap) values for coronary angiography (cag, n=222) and percutaneous transluminal coronary angioplasty (ptca, n=75) procedures performed during a one year period 20122013 were prospectively recorded using a built-in calibrated dap meter (transmission ionization chamber). the ptca procedure was invariably performed by a senior interventionalist assisted by two junior cardiologists, while the cag was performed either by the senior interventionalist or by junior cardiologists. for a similar comparison of clinical protocols adopted in the institution, dap values from cag and ptca performed using philips integris h3000 and h5000 ii-based systems (netherlands) reported earlier all the x-ray systems were on periodic qa programs and conformed to the manufacturer's specifications. the ii and fpd systems had low-, normal-, and high-dose settings, respectively, for fluoroscopy. these machines incorporated a total filtration of 2.5 mmal with possibility of selecting spectral filters such as 0.1 mm, 0.2 mm, 0.4 mm cu for dose reduction. during the course of the study, low dose setting with 0.4 mm cu filter was invariably selected during fluoroscopy. in the earlier work using ii-based system, a 23-cm image intensifier format (iif) was used during fluoroscopic screening in cag and ptca procedures for tracing the path of the catheter from the region of arterial puncture and to the screening of the cardiac valve region. a 17-cm iif was used for the oblique, caudal, cranial, and lateral projections delineating the coronary anatomy. in the fpd system, 25 cm fluoro format was used during screening and 20 cm was used for other projections to delineate coronary anatomy. a transition from ii to fpd system for a catheterization lab would require adequate justification in terms of radiation dose, image quality, maintenance, and investment. it has been reported that the fpds designed specifically for fluoroscopic purposes provide superior image quality and dose efficiency compared to the ii systems, except at the lowest fluoroscopic dose levels. prieto et al., reports that even after upgrading to the fpd from ii, significant increase in patient doses were observed though the fluoroscopic time and number of images remaining the same in both cases during the initial transition period. as only a few studies on radiation doses are available for fpd systems; more work is required on optimization strategies in the fpd systems. table 1 shows the dap values and fluoroscopic time duration for ii and fpd systems from the referral center where the study was conducted. the dap values for ii-based systems represented in table 1 is from the use of optimized protocol as reported in the previous published article from the same referral center where the study was conducted. prior to optimization in ii systems, the doses were above 50%; however, it was possible to optimize dose by halving the entrance dose ratesby selecting 0.4 mm cu filtration (generally recruited in pediatric protocols) during fluoroscopy. selection of 0.4 mm cu filtration did not suffer significant loss of image quality; however, tube potentials were increased from 80 kvp to 103 kvp during fluoroscopy. in the fpd system, tube potentials ranged from 90 kvp110 kvp when 0.4 mm cu filtration was selected with low dose settings during fluoroscopy. having adhered to the same optimization strategies in both systems, doses were similar owing to the fact that further optimization is warranted in fpd system. reported dap values of 31 and 33 gycm from cag with the use of ii and fpd system, respectively. they have also reported that the total dap from fluoroscopy and cine for ii and fpd are not significantly different and the image quality from fpd is better than ii in cine mode with no difference in the imaging performance in fluoroscopy mode. fluoroscopic time and dose area product values from ii and flat panel detector (fpd)-based systems from cardiological interventions tables 2 and 3 shows dap values for cag and ptca procedures from various studies in literature. the arithmetic mean dap and fluoroscopic time duration using ii system as reported in literature was 39 gycm for 6.6 min and 61.2 gycm for 17 min for cag and ptca respectively [table 2]. with the use of fpd, mean dap and fluoroscopic time duration were 28.4 gycm for 7 min and 61 gycm for 18.05 min for cag and ptca, respectively [table 3]. from tables 2 and 3, radiation doses from fpd were significantly low for cag but were similar for ptca when compared to ii systems. it should be noted that time duration for cag and ptca was not available for some reported studies in tables 2 and 3. wide variation of doses is observed from these studies which may be attributed to the angiographic system used, time duration of the procedure and work environment. doses of the order of 492 gycm were recorded in ptca procedure from fpd system which was higher than the ii systems. chida et al., conducted studies from various ii and fpd system and the average entrance doses of cine angiography and fluoroscopy in fpd systems were not significantly different. though fpds possess good detective quantum efficiency, they did not inherently reduce the radiation dose. jensen et al., observed that patient doses from fpd were lower than ii systems; however, the eye lens doses for radiologists were higher in fpd than in ii due to the use of high filtration and recruitment of high tube potentials. in our study, high tube potentials were recruited when low dose settings involving high filtration were selected, which may have the potential to increase staff doses. radiation doses from cardiological interventions performed using image intensifier-based systems radiation doses from cardiological interventions performed using flat panel detector-based systems it is prudent to adopt stringent optimization measures in fpd as the dose may be higher than the ii systems as reported by prieto et al. and dose reduction is possible in either ii or fpd systems. during the initial stages of our study, the doses from fpd were similar to the ii system though high filtration was used. reports that it is possible to achieve mean dap of 6.2 and 10.4 gycm for cag and ptca procedures performed using ii system. they further report that the reduction of doses was by influencing the quality of fellowship training, consistent restriction to mean values of 171 frames per cag, 165 frames per ptca, low-level fluoroscopy, training in the use of fluoroscopy-free blind positioning to the region of interest, restrictions to achieve lower ii entrance dose for adequate image quality. from tables 1 and 3, the mean dap for cag from fpd were higher than those reported by kuon et al. tsapaki et al., reported the doses from fpd were increased by 35% compared to ii when fluoroscopy levels were changed from low to high mode; they also recorded a minimum dap value of 6.1 and 14.3 gycm for cag and ptca, respectively, with the use of low dose fluoroscopy settings in fpd. dekker et al., reported that the new generation fpds incorporated with good image processing and noise reduction techniques resulted in reducing patient doses by 43% without compromising image quality and staff doses by 50% during electrophysiological interventions. though fpd has reduced entrance dose rates, it does not automatically reduce radiation doses in clinical practice. further work is necessary to study the possibilities of dose reduction in fpd so as to be implemented in the clinical set up. the patient dose and image quality in any newer modality needs to be permanently monitored and transition from ii to fpd requires careful attention. this is a preliminary study as the institution where the study was conducted recently moved from ii to fpd-based systems. though radiation doses for cardiological interventions from fpd were similar to the ii-based system achieved after optimization, the advantages of fpd in terms of good image uniformity, improved patient imaging accessibility due to smaller size, absence of geometric distortion/veiling glare or vignetting make the fpd superior to the ii systems. to achieve improved patient dose reduction, it is advisable to strictly adhere to low dose protocols with high filtration in fpd systems. in addition, more attention for staff doses is warranted especially for interventionalists when this stringent patient dose reduction is employed. it is recommended to follow stringent dose reduction strategies right from the period of initial installation when there is a transition from ii to fpd systems. further studies are required to develop dose optimization in fpd, though use of high filtration is already in place.

flat panel detector (fpd) technology in interventional cardiology is on the increase due to its varied advantages compared to the conventional image intensifier (ii) systems. it is not clear whether fpd imparts lower radiation doses compared to ii systems though a few studies support this finding. this study intends to compare radiation doses from ii and fpd systems for coronaryangiography (cag) and percutaneous transluminal coronary angioplasty (ptca) performed in a tertiary referral center. radiation doses were measured using dose area product (dap) meter from patients who underwent cag (n=222) and ptca (n=75) performed using fpd angiography system. the dap values from fpd were compared with earlier reported data using ii systems from the same referral center where the study was conducted. the mean dap values from fpd system for cag and ptca were 24.35 and 63.64 gycm2 and those from ii system were 27.71 and 65.44 gycm2. transition from ii to fpd system requires stringent dose optimization strategies right from the initial period of installation.

PMC4471641

pubmed-687

molecular dynamics simulations have been used as a powerful tool to study lipid membranes but are limited by the length and time scales of the probing systems. in particular, the lateral diffusion rate of a lipid in a membrane, although varying among different experimental techniques, is in the range 1010 cm s. this means that it will take a lipid about hundreds of nanoseconds to microseconds time scale to cover a 1 nm area. furthermore, lipids are observed to move collectively with their neighbors, and therefore, the rate at which a lipid swaps position with its neighbor is even slower. this poses a serious problem when we simulate lipid bilayers with multiple components, since the lateral organization of different components requires extensive simulation to attain equilibrium and common accessible simulation time scales may provide configurations ensemble biased toward the initial conditions. temperature replica exchange molecular dynamics (t-remd) is one of the methods that has been widely used to accelerate equilibration in simulations and achieved numerous success in protein folding. however, its application to lipid bilayers is rare since the number of replicas scales with the degrees of freedom (dofs) of the whole system, and lipid membranes usually have many more dofs than the protein systems, making it computationally prohibitive to use t-remd to study lipid bilayers. one promising method to get around the poor scalability of t-remd with system size is replica exchange with solute tempering (rest), which was initially proposed by berne and co-workers. rest is a specific variation of a generalized hamiltonian replica exchange method. by changing the solute solute and solute solvent interactions in the system although rest has been demonstrated to sample the conformational ensemble of the alanine dipeptide successfully, its efficiency in folding larger proteins remains unclear, which impedes a wide adoption of the method. in this work, we show that rest can be used as an efficient way to accelerate lateral equilibration in a mixed lipid bilayer. we applied constant pressure rest to a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (dppc) bilayer with 50 mol% cholesterol (chol). constant pressure in rest simulation is used because volume expansion at high temperature increases lipids lateral diffusion. by carefully choosing the tempering solute, we managed to simulate the system with only 12 rest replicas, which compares favorably to the requirement for 100 replicas with t-remd at the same replica exchange rate. the relative diffusion rate between molecules in rest is, on average, an order of magnitude faster than the rate in standard md simulation. we also show that the lateral radial distribution function between all molecular pair types (chol chol, chol-dppc, dppc finally, we use rest to obtain the gibbs free energy profiles between different molecular pair types from the corresponding lateral radial distribution functions. let us start with a brief review of replica exchange with solute tempering (rest). rest can be derived from a more general form of replica exchange called hamiltonian replica exchange. in hamiltonian replica exchange, replica m is simulated with potential energy em, at temperature tm (the corresponding inverse temperature will be referred as m, where m=1/kbtm and kb is the boltzmann constant) and constant pressure pm. in an isothermal isobaric ensemble, the probability of configuration xm with volume vm in replica m is1where zm is the corresponding partition function. the exchange between replica m and n can be treated as the change from state i to state f in the generalized ensemble,2and we use t(i f) to denote the transition probability for i f. applying the detailed balance condition3gives the ratio of the transition probabilities4where5 if the replicas are simulated at the same temperature t0 and pressure p0 but a different potential energy, eq 5 can be further reduced to6 the detailed balance condition guarantees the boltzmann sampling of all replicas when sufficiently long simulations are performed. in rest, each replica is simulated at the same temperature (t0) and pressure (p0) but a different potential energy function. we can divide the system potential energy into three terms:7where each term, in order, represents solute solute, solute solvent, solvent solvent interactions. for replica m, we scale its potential energy according to8 following from eq 1, the boltzmann distribution for replica m becomes9equation 9 shows that, thermodynamically, we can interpret the solute of the system as if it is simulated at the scaled potential (m/0)ess at temperature t0, or at the original potential ess at an effective temperature tm. in each rest setup, we should always ensure that there is one replica simulated with tm=t0. we will refer t0 also as the target temperature because it is the temperature of interested for the studied system. replicas simulated with tm t0 are used for the sole purpose of enhancing sampling at t0 and configurations obtained from these additional replicas do not represent any thermodynamic ensembles that have experimental counterparts. first, the definition of solute and solvent in a system is not absolute. the solute can generally present a part of the system whose sampling we want to accelerate, while the solvent is defined as the rest of the system. second, the potential function of replica m can be of a form different from eq 8. rest is just a specific form of hamiltonian replica exchange, and there is no restriction in the form of the potential energy function used in each replica in hamiltonian replica exchange. the advantage of using eq 8 is that there is a physical interpretation associated with it, but this is not required. therefore, the choice of the prefactor in front of esw is not unique. we choose (m/0) for the ease of implementation, as suggested by terakawa et al. following eq 6, the exchange ratio between replica m and n is determined by10it is clear from the above equation that the exchange ratio is independent of solvent solvent interactions (eww). therefore, one can enhance the solute dynamics and simultaneously reduce the number of tempered degrees of freedom, which results in a reduction of the number of required replicas. we want to point out that in our implementation of rest we use eq 6 to calculate exchange rate rather than eq 10 for practical reasons. specifically, the total potential energy is easily available from the simulation code. in the following text, we describe how we scale the potential energy function according to eq 8, using separate force field parameters for each replica. in common molecular dynamics force fields, the potential function consists of11where the first two terms are coulomb and lennard-jones interactions and the third and the following terms are bonded interactions which define the bond length potential, the bond angle potential, the torsion potential, and so forth. the scaling of the potential in replica m descried in eq 8 can be done as follows: (1) for the bonded interactions in the solute, scale the spring constants by (m/0), (2) scale the charges in solute by (m/0), and (3) scale ij by m/0 if both i and j are in the solute, and by (m/0) if only i or j is in the solute. we scale ij directly because it can be applied to any combination rule. by these the system we studied consisted of 144 dppcs, 144 cholesterols (chol), and 14k water molecules. each monolayer in the bilayer was built independently by randomly placing 72 dppcs and 72 chols on 12 12 planar grids. we define the z axis as the bilayer normal and refer the values of z>0 nm and z< 0 nm as the upper and lower monolayers. then, we equilibrated the system for 20 ns at 323 k and 1 atm. the resulting configuration was used as the initial structure for all replicas in rest and the standard md simulations. we note here that every replica in rest has the same starting configuration as in the md simulation. in rest, we chose dppc molecules as the solute and treated cholesterols and waters together as solvent. the explanation for this choice is provided in the results section. in total, 12 replicas were used and a 25% exchange rate was achieved between the neighboring replicas. each replica was simulated at 323 k (t0), while the effective temperatures of dppc were set at 323, 341, 360, 380, 400, 421, 445, 471, 500, 531, 564, and 600 k (tm). each replica in rest was simulated for 60 ns, while the md simulation was conducted for 400 ns. we implemented hamiltonian replica exchange in gromacs 4.5.7 software package to conduct rest. the default hamiltonian replica exchange in gromacs is done through thermodynamic integration, and it suffers from a great performance loss if the potential function involving a large portion of the system is altered. systems were simulated under periodic boundary conditions, at constant temperature and pressure. for temperature coupling, dppc and chol molecules we note here that in rest, each replica is simulated at 323 k and the heating of solute is done by reducing the solute rahman barostat at 1 atm was used and the pressure in the plane of the bilayer was coupled separately from the pressure normal to the bilayer. the temperature and pressure time constants of coupling were 0.1 and 2.0 ps, respectively. the spc/e model was used for water and the 43a1-s3 force field was used for dppc and chol. settle was used to constrain water molecules and lincs was used to constrain all other bond lengths in the system. the sixth-order particle mesh ewald (pme) method was used for electrostatic interactions with a fourier spacing of 0.15 nm. the real space coulomb interactions and pair-list calculations were set to 1.0 nm. a 1.0/1.6 nm twin-range cutoff scheme was used for vdw interactions and the pair-list was updated every 10 steps. to study how molecules diffuse relative to one another, we calculated the relative diffusion coefficient dij between each pair of molecules i, j. the relative diffusion removes the contribution from collective motions of molecules to the diffusion and should give a better estimate of how fast the system samples various lateral configurations. we define the mean squared relative displacement between molecule i, j in a time interval t as1213where ri(t) is the position of molecule i at time t and the bracket means an average over different starting times t. then we linearly fit rij2(t) as a function of t for larger t. dij was assigned as /4 (two-dimensions) the slope of the curve. we note here that in rest, the relative diffusion rate is an average over temperatures (due to the exchange among replicas, replicas jump in the temperature ladder); however, it should still provide a meaningful description of how fast the simulations sample bilayer lateral configurations in general. as mentioned in the methods session, the choice of solute for rest simulations is not absolute. the solute can generally be the part of the system whose dynamics we want to accelerate, while the solvent is the rest of the system. since in this study we want to accelerate the dynamics of lipid bilayers, it is natural to choose both the dppc and chol as the solute. before we conduct rest, it is always a good practice to test the system at the highest temperature that we want to simulate in rest. we found that when we simulated the dppc and chol at 600 k (tm), the chol moved out of the monolayers and formed a third layer sandwiched by the dppc bilayer. a snapshot of the system is shown in figure 1. a possible explanation of this can be obtained by carefully examining chol molecular structures. chol has a small hydrophilic alcohol headgroup and a bulky hydrophobic body. at low temperature, the hydrophilic interactions between the chol alcohol group and water favors aligning chol along the bilayer normal. when the effective temperature of chol increases, the entropic effect becomes dominant and chol will gain entropically by placing itself in the middle of the bilayer. on the other hand, dppc has a larger hydrophilic headgroup than chol; thus, even at 600 k, it still can anchor itself upright to form the bilayer. snapshot of the system where both dopc and chol molecules are tempered at 600 k. water is colored blue. the oxygen atom in chol and the phosphate atom in dppc one way is to lower the highest temperature in rest to keep chol aligned with the bilayer normal. another way is to use only dppc as solute and treat both chol and water as solvent. we want to note that we should be able to just conduct rest with both dppc and chol set to 600 k, even though chol moved out of the monolayers at this temperature. however, this would be very inefficient. configurations similar to figure 1 obtained from high solute temperature replicas will have vanishingly small probabilities in the target temperature replica ensemble (t0=323 k) due to the detailed balance condition (eq 6). as a result neale et al. has observed such phenomena in another hamiltonian replica exchange system. in this case, high temperature replicas will not enhance the sampling in the target temperature replica but consume computing time. in this work, we take the second approach in which only dppc is chosen as the tempered solute. since this reduces the dofs in the solute, it further decreases the number of replicas required to span our temperature range. by choosing dppc as solute, we managed to heat dppc from 323 to 600 k with 12 replicas. the exchange rates between neighboring replicas are 25%, 22%, 24%, 26%, 27%, 22%, 23%, 22%, 22%, 24%, and 22%. in order to compare the efficiency between rest and t-remd, we estimate how many replicas we need in our system to conduct t-remd to maintain a 25% exchange rate between neighboring replicas. figure 2 shows that if 12 replicas are used in t-remd, we can only cover the temperature ranging from 323 to 340 k. this would hardly accelerate the simulation as t-remd usually gains simulation efficiency by increasing the enthalpy barrier crossing rate in high temperature replicas. comparatively, rest has a significant advantage over t-remd as we can heat the dppc to a much higher temperature with the same number of replicas. number of replicas required to obtain a 25% exchange rate between neighboring replicas in rest and t-remd (temperature replica exchange). below, we compare the sampling efficiency between rest and a single long md simulation. in our work, we ran rest for 60 ns and standard md for 400 ns. the equilibrium sampling of lipid bilayers, especially of bilayers with different components, depends on the ability to sample different lateral organizations. the faster the system can explore various lateral configurations, the quicker equilibrium will be reached. therefore, the lateral diffusion coefficient plays a key role in determining the equilibrium rate. however, lipids usually move collectively in bilayers. this collective motion generally does not facilitate the sampling of various lateral configurations but contributes significantly to the lateral diffusion coefficient of each individual molecule. therefore, we calculated the relative diffusion coefficient between every pairs of molecule as it removes the contribution from collective motion among lipids. dppc) of relative diffusion coefficients are calculated from the md and rest simulations. it clearly indicates that in all molecular pair types, molecules in rest diffuse an order of magnitude faster than in standard md. also, we observe that the diffusion between chol and chol is the fastest while the diffusion between dppc and dppc is the slowest. dppc, on the other hand, has two acyl tails that are usually entangled with other dppc, which reduces the diffusion. it is also worth noting that even though we only increase the effective temperature of dppc, the diffusion of chol increases as well. by omitting the chol from the tempered solute, we further reduced dofs in the solute and therefore reduced the number of replicas required for the system. this also suggests that the efficiency of rest may be further optimized by carefully choosing the tempering solute, which is also pointed out in the original paper. we note that the total simulation time in rest (60 12=720 ns) is almost twice as much as the time in md (400 ns). however, considering an order of magnitude increase in the lipid diffusion, rest is quite efficient. probability density of all pairwise relative diffusion coefficients between chol chol (c c), dppc chol (d c), and dppc dppc (d d) in md and rest simulations. the radial distribution function (rdf) quantitatively describes the lateral organization of molecules in a bilayer. we define the lateral distance between two molecules as the center of mass (com) distance between the molecules projected in the x y plane. as the coupling between separate monolayers is weak, the rdf calculated from the upper and lower monolayers should converge to the same distribution. therefore, we can judge the convergence of a simulation by the rdf difference calculated from separate monolayers. figure 4 and supporting information figures s1 and s2 show the rdfs among chol chol, chol the rdfs are calculated from separate monolayers (blue, upper monolayer; red, lower monolayer) from the rest and md simulations using different block sizes. in all cases chol rdfs (figure 4) for example, with a 10 ns block size, the rdfs in rest show reasonable convergence between separate monolayers (figure 4a c). however, this is not the case for the md simulation (figure 4d f). the rdf difference in md simulation in the last 10 ns block (figure 4f) is even larger than the difference in the first 10 ns block (figure 4d). we reason that as molecules diffuse slowly in the md simulation, the sampled lateral configurations are highly correlated in time; thus, a larger block size is required to obtain independent configurations. therefore, we increased the time block size to 120 ns to calculate the rdf from the md simulation. figure 4g i show that rdf converges better when the block size is increased. the converged rdf in md (figure 4i) has a very similar shape with the rdf obtained from rest (figure 4c). the blue/red line in each subplot represents the rdf calculated from the upper/lower monolayer, respectively. supporting information figures s1 and s2 show the rdfs between chol-dppc and dppc dppc pairs, respectively. we observe that the rdf of dppc dppc does not converge as well as the rdf of chol chol or chol-dppc. dppc pairs diffuse the slowest in both md and rest; therefore, we can expect that the rdf of dppc dppc takes the longest time to converge. to quantitative analyze the convergence rate, we define the rdf difference between separate monolayers (rdfdiff) as14where rdfu(i) and rdfl(i) are the value of the ith bin of rdfs from the upper and lower layer, respectively. figure 5 shows the convergence of rdf as a function of time block size in rest and md simulations. figure 5 shows that the rdf in rest converges an order of magnitude faster than in md. difference between rdfs calculated from the upper and lower monolayers as a function of block size. error bars are estimated from consecutive blocks with the same block size. in this session, we compare several bilayer structural properties calculated from the rest and md simulations. for rest, only the replica simulated with solute temperature at tm=323 k is used for analysis, as it represents the system with the original hamiltonian. normally, the area per lipid serves a good indicator on bilayer structural properties. however, since the bilayer we studied has both chol and dppc, we calculated the average area per molecule (aapm) instead. aapm is defined as the projected area of the bilayer in the x y plane divided by the number of molecules in a monolayer. the aapm are 43.1 0.6 in rest and 42.8 0.4 in md. another important bilayer property is the deuterium order parameter (scd) of the lipid acyl tails. the order parameter of a methylene at position i is defined as15where i is the angle between a c d vector of the ith methylene in an acyl chain and the normal of the bilayer (z axis). our calculation suggests that rest and md simulations have similar bilayer properties, which is a good validation of the rest method. deuterium order parameters |scd| of dppc palmitoyl chains at 323 k calculated from the rest and md simulations. chol is well-known for its condensing effect on lipid bilayers composed of lipids with saturated acyl tails. it smooths the lipid liquid/gel phase transition to the lipid disordered/ordered phase transition. it is reported that the average value of |scd| for dppc at liquid ordered phase and disordered phase are 0.36 and 0.21, respectively.supporting information figure s3 shows the |scd| of dppc at different solute temperatures in rest. at high solute temperatures, the dppc acyl tails are more flexible and the |scd| indicates that the bilayer is in the liquid disordered phase. the range of sampled |scd| in rest suggests that rest works well even when the system experiences the liquid disordered/ordered phase transition. based on the high resolution rdf obtained from rest, we calculate the total gibbs free energy g(r) and excess gibbs free energy g(r) profiles between chol chol, chol dppc, and dppc dppc. g(r) and g(r) are defined as16and17g(r) is the lateral radial distribution function (rdf), and r0 is the reference distance where we set g(r0)=0. g(r) is the contribution to the g(r) due to the jacobian or area effect in the two-dimensional space. this means that neither chol or dppc tends to aggregate at the 50% chol concentration. chol gibbs free energy profile in dilute conditions and found that g(r) drops below zero in the range 1.0<r< 1.5 nm. dppc g(r) is almost flat when r>1.0 nm, suggesting a random distribution of dppc at large distance. chol g(r), indicating preferential interacting locations for chol chol pairs. this supports some phenomenological models, such as the supperlattice model and umbrella model, which suggest long-range ordering for chol. however, the barriers between the free energy minimums are of the order of kt scale, suggesting that the ordering of cholesterols is sensitive to the temperature. as the derivative of g(r) is the mean force, the g(r) we obtained can be used as a reference for various coarse grained models for this system. total free energy g(r) (solid line) and the excess free energy g(r) (dashed line) profiles between different molecular types as a function of the lateral molecular center of mass distance. a general guideline is to choose the part of the system for which we want to accelerate the dynamics as the solute. for example, if we study the lipid protein interactions and are interested in the affinity of different lipid components to the protein, we can temper the lipids to accelerate their diffusion. on the other hand, if we are interested in how the protein adapt its conformation to the bilayer environment, we can temper the protein instead. with a good choice of solute, rest can accelerate the dynamics of the system with fewer replicas (compare to t-remd). the sole purpose of the replicas with the scaled potentials (tm t0) is to sample configurations that are likely to occur in the target temperature ensemble (tm=t0). therefore, if we scale the potential in such a way that the system samples configurations with low probability to populate at the target temperature ensemble (configurations such as figure 1, in our case), rest will be less efficient. however, no matter what the choice of the solute is, we always have one replica in rest that is simulated at the original unscaled potential. choosing the solute that makes rest efficient may require an intuitive trial and error approach. in the past usually, coarse grained systems have fewer degrees of freedom than their atomic counterparts, which results in smoother free energy landscape and faster lipid diffusion. another method, developed by tajkhorshid and co-workers, is a membrane mimetic model, which separates the lipid headgroup from its hydrophobic tails. this method facilitates headgroup diffusion while maintaining a hydrophobic core and has been used to study the insertion of peripheral proteins. wang et al. also developed a method based on the accelerated molecular dynamics (amd) method, which accelerates lipid diffusion by adding a boost potential to the original system. rest provides an efficient way of accelerating the equilibrium of lipid bilayer systems while simulating at least one copy of an unperturbed potential and maintaining atomistic details. in this work, we applied replica exchange with solute tempering (rest) to a cholesterol dppc bilayer system. in rest, part of the system is chosen as solute and the solute solute and solute solvent interactions are scaled such that thermodynamically, the solute is effectively sampling at a different temperature. we found that choosing both cholesterols and dppcs as solute is not efficient because most of the cholesterols moved out of the monolayers to form a third layer at high temperature. therefore, we chose to temper the dppc molecules only. since the number of replicas for rest only scales with the degrees of freedom in the solute, we managed to use 12 replicas to sample dppc at temperature ranging from 323 to 600 k, which, otherwise, would require 100 replicas in the traditional temperature replica exchange molecular dynamics (t-remd). the relative diffusion coefficients between all molecular pair types (chol chol, chol dppc) in rest are, on average, an order of magnitude larger than in standard md simulation, indicating a better sampling of lateral structures in rest. dppc, and dppc dppc radial distribution function (rdf) between separate monolayers in the rest and md simulations. since the coupling between monolayers is weak, the rdf should converge to the same distribution from different monolayers. our results show that the rdf converges much faster in the rest than in the md simulation. bilayer structural properties such as average area per molecules and deuterium order parameters are similar between the rest and md simulations. finally, we obtained the lateral free energy profile between different molecular types from rest, which could be used as a reference to coarse-grained models of the system. chol lateral free energy has several local minima and shows a long-range ordering, but the free energy barriers between minima are on the kt scale, indicating the ordering may be sensitive to the temperature. while we see a significant advantage of using rest to accelerate lateral equilibrium in mixed lipid bilayers, we believe that rest, or more generally, hamiltonian replica exchange will have broader applications. for example, the preferential interactions between different lipids and membrane proteins can be studied by tempering the lipids while leaving the protein and solvent at the target temperature. also, combined with umbrella sampling, rest can be used to accelerate relaxation on degrees of freedom orthogonal to the reaction coordinate, which is reported as a hurdle in free energy calculations for lipid membranes.

the lateral heterogeneity of cellular membranes plays an important role in many biological functions such as signaling and regulating membrane proteins. this heterogeneity can result from preferential interactions between membrane components or interactions with membrane proteins. one major difficulty in molecular dynamics simulations aimed at studying the membrane heterogeneity is that lipids diffuse slowly and collectively in bilayers, and therefore, it is difficult to reach equilibrium in lateral organization in bilayer mixtures. here, we propose the use of the replica exchange with solute tempering (rest) approach to accelerate lateral relaxation in heterogeneous bilayers. rest is based on the replica exchange method but tempers only the solute, leaving the temperature of the solvent fixed. since the number of replicas in rest scales approximately only with the degrees of freedom in the solute, rest enables us to enhance the configuration sampling of lipid bilayers with fewer replicas, in comparison with the temperature replica exchange molecular dynamics simulation (t-remd) where the number of replicas scales with the degrees of freedom of the entire system. we apply the rest method to a cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (dppc) bilayer mixture and find that the lateral distribution functions of all molecular pair types converge much faster than in the standard md simulation. the relative diffusion rate between molecules in rest is, on average, an order of magnitude faster than in the standard md simulation. although rest was initially proposed to study protein folding and its efficiency in protein folding is still under debate, we find a unique application of rest to accelerate lateral equilibration in mixed lipid membranes and suggest a promising way to probe membrane lateral heterogeneity through molecular dynamics simulation.

PMC4196747

pubmed-688

bartter syndrome (bs) is a rare inherited renal tubular disorder characterized by renal salt wasting, hypokalemic metabolic alkalosis and normotensive hyperreninemic hyperaldosteronism1, 2). although bs is a typical tubular disorder, there have been several case reports of patients who developed focal segmental glomerulosclerosis (fsgs), a glomerular lesion, during the course of bs3-7). such cases support a possible link between the two diseases, and indicate that stimulation of the renin-angiotensin system (ras) and increased angiotensin ii in response to chronic glomerular hyperfiltration due to salt-losing tubulopathy may lead to secondary fsgs3, 4, 8). in addition to fsgs, several other factors have been reported to influence the renal survival of patients with bs, including recurrent episodes of dehydration during early infancy, chronic hypokalemia, long-term use of non-steroidal anti-inf lammatory drugs, nephrocalcinosis, and mutations in the bsnd gene9-11). however, there have been only a few case reports of patients with bs who underwent renal transplantation6, 12-15). here, we present a case of a patient with bs who developed fsgs and subsequently underwent renal transplantation due to aggravation of his renal function. we also review previously reported cases of both diseases and cases of individuals with bs who underwent renal transplantation. there were no perinatal problems including polyhydramnios, and the family history was unremarkable. at age 3 months, the patient visited a hospital due to recurrent febrile episodes and poor weight gain. the patient was diagnosed as bs, and potassium supplement and indomethacin medication were started. however, his compliance to the drug was very poor. detailed clinical and laboratory data from that time are unavailable. at age 7, proteinuria was incidentally detected upon routine follow-up urinalysis. a renal ultrasonography revealed diffusely increased parenchymal echogenicity without definite evidence of nephrocalcinosis or nephrolithiasis. at age 8, a renal biopsy was conducted at the hospital due to persistent proteinuria (24-hour urine protein excretion 3,840 mg/day, serum albumin 3.4 g/dl, and serum creatinine 1.2 mg/dl). his proteinuria did not respond to 4 weeks of oral prednisolone (2 mg/kg/day) treatment. at age 10, the patient's height was 123 cm (< 3rd percentile), weight was 36 kg (90-97th percentile), and blood pressure was 117/69 mmhg at the time of admission. the laboratory examination revealed a serum creatinine level of 3.1 mg/dl, a sodium level of 138 mmol/l, a potassium level of 3.7 mmol/l, a chloride level of 103 mmol/l, a bicarbonate level of 30 meq/l, a magnesium level of 2.0/ml/hr (normal range 1-2.5), an aldosterone level of 558 pg/ml (normal range 50-194), and a spot urine calcium (mg) to creatinine (mg) ratio of 0.02. additionally, the patient's bone age was 6 years 5 months upon admission. genetic analysis revealed a homozygous p.trp(tgg)610stop(tga) mutation in exon 16 on the clcnkb gene. his renal function deteriorated progressively for one year thereafter, at which point hemodialysis was started. subsequently, the patient underwent renal transplantation successfully from a deceased donor at age 16. the post-transplantation course has been uneventful for more than 3 years, with complete disappearance of bs without recurrence of fsgs. clinically, bs can be classified into two variants, antenatal/neonatal bs and classic bs according to the onset age. genetically, bs can be classified into at least 5 subtypes according to underlying mutant genes, all of which are expressed in the tubular epithelial cells of the thick ascending limb of the loop of henle. bs type i is caused by loss-of-function mutations of slc12a1 encoding the apical sodium-potassium-chloride cotransporter (nkcc2), while bs type ii is caused by loss-of-function mutations of kcnj1 encoding the apical inwardly-rectifying potassium channel (romk), bs type iii is caused by loss-of-function mutations of clcnkb encoding the basolateral chloride channel (clc-kb), bs type iv is caused by loss-of-function mutations of bsnd encoding barttin, and bs type v is caused by gain-of-function mutations of casr encoding the basolateral calcium sensing receptor (casr)2). bs is an inherited tubular disorder; however, our patient developed fsgs, an acquired glomerular lesion, later in the disease course. there have been five similar case reports of bs complicated by fsgs3-7) (table 1). in addition, there is an additional report of a patient with classic bs who developed end-stage renal disease due to glomerulopathy12). however, this patient was excluded from the review because the renal pathology was described as ' diffuse ' glomerulosclerosis. as shown in table 1, patients with bs and fsgs show development of fsgs independently of gender, clinical variants of bs or genetic subtypes of bs. one patient had nephrocalcinosis, and three patients had a history of long-term non-steroidal anti-inflammatory drug treatment prior to the onset of proteinuria. three patients had progressed to chronic renal failure, but the rate of progression was variable. one other patient revealed mildly decreased renal function one year after diagnosis with fsgs5). normal renal function was maintained in the remaining two patients, and, interestingly, proteinuria resolved completely in one of these patients after one year treatment with potassium supplements, spironolactone, and indomethacin3). the renal pathologic findings were described in detail in three cases, two of which had perihilar type fsgs. the perihilar variant is common in patients with secondary forms of fsgs mediated by glomerular hyperfiltration or other adaptation after loss of renal mass8, 16). these findings suggest that fsgs complicated by bs may be a secondary lesion due to adaptive response to chronic hyperfiltration by chronic salt-loss and resultant chronic stimulation of the renin-angiotensin system. several case reports of gitelman syndrome complicated by fsgs support this explanation17, 18). case reports of patients with bs who underwent renal transplantation are also rare, and the causes of esrd include progression of fsgs or other causes including complication of long-term non-steroidal anti-inflammatory drug treatment6, 12-15). in one of the patients, pre-emptive bilateral native nephrectomies and renal transplantation were conducted prior to the onset of esrd due to severe, clinically brittle, neonatal bs15). in all of the cases, transplantation was successful and bs disappeared completely after transplantation as shown in our patient. in summary, bs may be complicated by fsgs due to adaptive response to chronic salt-losing nephropathy, and fsgs may cause esrd in some patients.

bartter syndrome (bs) is a clinically and genetically heterogeneous inherited renal tube disorder characterized by renal salt wasting, hypokalemic metabolic alkalosis and normotensive hyperreninemic hyperaldosteronism. there have been several case reports of bs complicated by focal segmental glomerulosclerosis (fsgs). here, we have reported the case of a bs patient who developed fsgs and subsequent end-stage renal disease (esrd) and provided a brief literature review. the patient presented with classic bs at 3 months of age and developed proteinuria at 7 years. renal biopsy performed at 11 years of age revealed a fsgs perihilar variant. hemodialysis was initiated at 11 years of age, and kidney transplantation was performed at 16 years of age. the post-transplantation course has been uneventful for more than 3 years with complete disappearance of bs without the recurrence of fsgs. genetic study revealed a homozygous p.trp(tgg)610stop(tga) mutation in the clcnkb gene. in summary, bs may be complicated by secondary fsgs due to the adaptive response to chronic salt-losing nephropathy, and fsgs may progress to esrd in some patients. renal transplantation in patients with bs and esrd results in complete remission of bs.

PMC3040364

pubmed-689

exploiting the incretin effect [1, 2], glucagon-like peptide-1 (glp-1) analogues are primarily utilised for their glucose lowering abilities through stimulation of insulin in a glucose-dependent manner [3, 4]. nevertheless, glp-1 analogues provide additional benefits which include the control of glucose excursions by attenuating the rate of gastric emptying, inhibition of glucagon secretion, and promotion of weight loss by augmenting the sensation of satiety [5, 6]. most promisingly, glp-1 analogues promote the proliferation, survival, and neogenesis of pancreatic -cells, a facet not shared by existing antihyperglycaemic agents [79]. with such beneficial properties, glp-1 analogues are commonplace in the glycaemic control of patients with type 2 diabetes mellitus (t2 dm) and presently four drugs within this class are licensed for use in t2 dm: exenatide (byetta), liraglutide (victoza), lixisenatide (lyxumia), and prolonged-release exenatide (bydureon). as established by the national institute of health and care excellence (nice), glp-1 analogues are utilised as a third-line therapy when both first-line metformin and second-line sulfonylureas fail to provide adequate glycaemic control despite appropriate diet and exercise interventions. to date, several clinical trials have demonstrated their efficacious ability to induce weight loss and improve glycaemic control in type 2 diabetic patients [1424]. nonetheless, within such studies, glp-1 analogues also fail in a cohort of individuals due to adverse effects associated with these agents (primary failure) or by not achieving the defined end-goal (secondary failure). although the definition of primary failure is standardised between healthcare institutes, different healthcare systems adhere to differing end-goal definitions. within the uk, nice define an individual who is said to have responded to therapy if their baseline weight and glycated haemoglobin (hba1c) have reduced by 3% and an 11 mmol/mol (1%), respectively, after six months of glp-1 analogue administration. if such criteria have been satisfied glp-1 analogue therapy is continued whereas if not the next appropriate therapy, typically insulin, is considered. despite the required weight loss ensuring that glp-1 analogues are used cost-effectively, an 11 mmol/mol (1%) drop in hba1c is known to reduce the development of microvascular complications, such as diabetic retinopathy, nephropathy, and neuropathy, by 25%. however, limited information is currently available which delineates factors that predict whether individuals will respond or not to glp-1 analogues. at present, the few studies in this area of research have identified that baseline hba1c appears to be the strongest predictor of response to glp-1 analogues [2631]. nevertheless, such studies are hindered by both small sample sizes and a lack of comparisons at multiple time points. furthermore, identification of individuals unlikely to respond to glp-1 analogues early on would nullify the theoretical risk of exposing patients to a 6-month period of side-effects, ranging from nausea and vomiting to acute pancreatitis [1417], and the significant expense associated with glp-1 analogues without any clinical benefit. therefore, the possibility of differentiating between responders and nonresponders to glp-1 analogues based upon patient characteristics and/or changes in anthropometric and metabolic parameters remains a topical subject amongst clinicians. subsequently, the primary aim of our study was to determine whether we could identify, in a group of patients with t2 dm initiated on the glp-1 analogue exenatide, any predictors of response by identifying any differences between parameters in those that achieved the nice required reduction in hba1c (responders) and those that did not (nonresponders). additionally, our secondary aim was to translate any identified predictors of glycaemic response into a quantifiable figure, whereby this figure could be used to distinguish between individuals who would then achieve the targeted hba1c reduction from those that would not. this retrospective observational study was registered and conducted at the countess of chester nhs foundation trust (coch), cheshire, united kingdom. participants were identified following review of patients with diabetes mellitus who were initiated on a glp-1 analogue from 2008 to 2014 at the endocrinology department at the coch. medical records for participants fulfilling the inclusion criteria were then accessed using the programme meditech (medical information technology, massachusetts). data collected included the patient's age, gender, and the number of years diagnosed with t2 dm, concurrent diabetic medications, and the patient's weight, body mass index (bmi), and hba1c at baseline (date initiated on exenatide), 3 months, and 6 months after exenatide initiation. all variables explored in this study were included in the audit protocol, thereby conforming to the ethical standards and framework set by the coch. patients were included if they met the following criteria: diagnosis of t2 dm made using the clinical criteria established by the nice, 18 years of age, initiated on exenatide, and required data available at baseline, 3 months, and 6 months after exenatide therapy. exenatide was chosen as the sole glp-1 analogue under analysis due to it being the most prescribed glp-1 analogue at the coch and our experience with this agent in clinical practice. additionally, prolonged-release exenatide was not considered due to the increased time period required for this drug to be efficacious; thus our defined time points would not necessarily reflect any glycaemic benefit. patients were excluded if diagnosed with other subsets of diabetes mellitus (dm), such as type 1 diabetes mellitus (t1 dm) and gestational diabetes, exposure to previous glp-1 analogue treatment, prescribed glp-1 analogues that did not include exenatide, prescribed weight reducing medications alongside exenatide, and if complete data was not available at the aforementioned time points. participants who satisfied the inclusion criteria were divided into two groups: responders and nonresponders. responders refer to individuals whose baseline hba1c fell by the nice instigated 11 mmol/mol (1%) reduction after 6 months of exenatide use, whereas nonresponders categorise patients who failed to achieve this decrease. the additional 3% weight loss requirement also stated by nice was not included in the response definition, as the 25% reduction in microvascular risk associated with a 11 mmol/mol reduction in hba1c is, in our opinion, of greater clinical importance. furthermore, we also categorised another group of participants as those with primary failure to exenatide treatment. this group included individuals that experienced side effects within the first two weeks of initiating exenatide resulting in the participant discontinuing further treatment. results obtained from the study are presented as mean standard deviation (sd) or as percentages (%) for continuous and categorical variables, respectively. the significance of any differences between mean values obtained from both responders and nonresponders was determined using the independent student's t-test for continuous data and chi-square test or fisher's exact for categorical data; fisher's exact test was used if sample size within categories was less than 5. to determine if any changes observed in weight, bmi, or hba1c over the three time points for the entire cohort, responder group, and nonresponder group were of statistical significance, repeated measures analysis of variance (rm-anova) was used. if statistical significance was observed, tukey's test was used to correct for multiple comparisons. in order to develop a model that could predict and define the relationship between statistically varying variables and attainment of the defined hba1c reduction, both linear regression and binary logistic regression analysis were used. results were deemed statistically significant if the p value was<0.05 and all statistical analyses were conducted using the statistical software package graphpad prism 6. 253 individuals were excluded on the basis of incomplete data (n=206), prescribed a glp-1 analogue other than exenatide (n=46; liraglutide=31; lixisenatide=7; prolonged-release exenatide=8), or diagnosed with t1 dm (n=1). this left 112 participants available for statistical analyses with 63 responding and 49 not responding to exenatide, as defined by a reduction 11 mmol/mol (1%) in hba1c from baseline to six months. none of the 112 participants reported any adverse effects and none reported any symptomatic hypoglycaemia. initial comparisons of patient demographics between responders and nonresponders identified no significant differences in gender (percentage male), age of participants within the cohorts, and duration of t2 dm (table 1). additionally, differences in medications prescribe alongside exenatide were similar between the two groups with the exception of the number prescribed exenatide with insulin. nonresponders were shown to have a significantly greater proportion of participants on exenatide with insulin compared to responders (20.4 versus 3.17%, p=0.0047). weight for the entire cohort reduced from 113 17.9 kg at baseline to 109 18.1 kg at 3 months and 106 18.9 kg at 6 months (p<0.0001). similarly, the bmi for the entire cohort reduced from 39.2 5.85 kg/m at baseline to 37.7 5.91 kg/m at 3 months and 36.8 6.31 kg/m at 6 months (p<0.0001). hba1c across the study period reduced from a baseline value of 80.9 14.6 mmol/mol to 70.4 13.4 mmol/mol by 3 months and 67.8 14.9 mmol/mol at 6 months (p<0.0001). analysis of these changes between time points showed it to be statistically significant (p<0.0001). for both responders and nonresponders, there was a significant reduction in both weight and bmi from baseline to six months (p<0.001) (table 2). although responders had a higher baseline weight compared to nonresponders (114 18.6 versus 111 16.3 kg) (39.4 5.63 versus 38.4 5.7 kg/m), both groups achieved the same mean weight by 3 months (106 20.6 versus 106 16.8 kg) and responders had a lower bmi by 6 months compared to nonresponders (36.5 6.57 versus 37.1 6 kg/m) (table 2). comparisons in the hba1c change within responders identified a reduction from 85.1 15.4 mmol/mol at baseline to 67.8 13.4 mmol/mol by 3 months and 60.7 12.2 mmol/mol by 6 months (p<0.001). however, nonresponders demonstrated an initial reduction in hba1c by 3 months (73.7 12.9 mmol/mol) from baseline (75.5 11.4 mmol/mol), yet by 6 months the hba1c had risen to a level greater than baseline (77 13.2 mmol/mol). although the overall change in the nonresponders was shown to be significant (p=0.028), following correction for multiple comparisons, it was observed that the only statistically significant difference was the elevation in hba1c from 3 months to 6 months (p=0.032) (table 2). analysis of weight between responders and nonresponders identified no significant differences between the two cohorts at baseline (114 18.6 versus 111 16.3 kg, p=0.54), 3 months (109 19.1 versus 108 16.9 kg, p=0.86), and 6 months (106 20.6 versus 106 16.8 kg, p=0.83) (figure 1(a)). similarly, any differences in the bmi between responders and nonresponders at baseline (39.4 5.63 versus 38.4 5.7 kg/m), 3 months (37.6 5.91 versus 37.7 5.97 kg/m), and 6 months (36.5 6.57 versus 37.1 6 kg/m) were also not significant with p values of 0.75, 0.92, and 0.61, respectively (figure 1(b)). however, the hba1c recorded for responders and nonresponders was shown to significantly differ at baseline (85.1 15.4 versus 75.5 11.4 mmol/mol, p<0.001), 3 months (67.8 13.4 versus 73.7 12.9 mmol/mol, p=0.021), and 6 months (60.7 12.2 versus 77 13.2 mmol/mol, p<0.001) (figure 1(c)). linear regression analysis identified a significant relationship between the baseline hba1c and changes in hba1c from baseline to 6 months (p<0.0001), with the x-intercept at 60.3 14 mmol/mol (figure 2). therefore, individuals with a baseline hba1c<60.3 mmol/mol were not likely to respond, yet those with a baseline hba1c 60.3 mmol/mol were more likely to respond to exenatide therapy over the 6-month period. additionally, those with a higher baseline hba1c experienced a greater relative and absolute reduction in their hba1c over the study period. no significant relationship was observed for any other variable (p>0.05). of the several variables included within the binary logistic regression, only baseline hba1c predicted response as defined by a reduction in 11 mmol/mol after 6 months of exenatide therapy (table 3). our model demonstrates that individuals with a higher baseline hba1c have a 5% increased odds of being classed as responders to exenatide by 6 months (p=0.004). although our model identified exenatide with insulin therapy as increasing the odds of responding compared to exenatide alone, the wide confidence interval range makes this variable unlikely to predict response to exenatide therapy. in order to determine the change in hba1c required for response to exenatide therapy, we quantified the percentage difference in hba1c from baseline to 3 months against the proportion of participants responding and not responding to exenatide by 6 months (table 4). comparisons within categories revealed that a reduction of 1520% in hba1c by 3 months compared to baseline was the first statistically significant difference that resulted in a greater proportion of responders compared to nonresponders (p=0.033). therefore, this would indicate that if the patient's baseline hba1c decreases within a range of 1520% (or more) by 3 months, they are more likely to achieve the 11 mmol/mol reduction in hba1c by 6 months. furthermore, a reduction in the hba1c from baseline between 0 and 5% resulted in the first significant difference that resulted in a greater proportion of nonresponders compared to responders. subsequently, those who have a reduction in hba1c between 0 and 5% (or less) by 3 months are more likely to not reach the 11 mmol/mol reduction in hba1c by 6 months. the clinical benefits of exenatide as a monotherapy or in combination with either existing oral antihyperglycaemic agents (ohas) and/or insulin have been extensively documented elsewhere [1417]. our study reflects similar improvements as exemplified by the significant reduction observed overall in the mean hba1c, weight, and bmi across the entire cohort of participants. therefore, our study reinforces the utilisation of exenatide as a glp-1 analogue in the management of t2 dm and additionally emphasises the efficacious properties of this glp-1 analogue with regard to improvements in both weight and glycaemic control. however, within the aforementioned studies there is a significant proportion of the initial cohort that either experience side-effects preventing them from continuing exenatide (primary failure) or fail on the basis of not achieving the defined end-point (secondary failure). our study reflects this phenomenon as demonstrated by 18.8% of the initial cohort discontinuing therapy following primary failure due to intolerable nausea and vomiting. additionally, of the participants that did complete 6 months of exenatide therapy, 43.8% failed to achieve the required 11 mmol/mol reduction in hba1c stipulated by nice. such observations reflect those reported by the limited literature presently available with studies reporting primary failure rates within the range of 11.433.6%, in addition to secondary failure rates ranging from 39 to 61% [26, 27, 2931]. although the rates of both primary and secondary failure rates observed within our study lie within such ranges, it reiterates the clinical significance and need of identifying measures which can delineate whether an individual will respond or not respond to exenatide therapy. by doing so, those distinguished as unlikely to benefit from exenatide therapy can be initiated on the next most suitable therapeutic adjunct appropriate for managing their type 2 diabetes, thus reducing the cost and exposure to side effects associated with exenatide with no clinical benefit. within our study, levels of hba1c were the only parameter to significantly differ between responders and nonresponders with our regression analysis identifying that higher levels of hba1c at baseline were associated with greater reductions in hba1c over the study period. such findings have been reported in the majority of the literature within this field [26, 27, 30, 31]; however, song et al. also noted significant correlations between changes in hba1c and the patient's age, levels of serum fasting glucose, and parameters that measure pancreatic- cell function such as homeostatic model assessment- (homa-), levels of insulin at baseline, and both basal and stimulated c-peptide levels. as we were unable to measure variables of insulin secretion, we can not ascertain whether such a relationship in the changes in hba1c and these parameters would have existed in our study. however, with regard to age, no other study has identified such a relationship and despite the study by shin et al. also including cohorts of south korean type 2 diabetics only therefore, this association may simply be exclusive to the study sample used by song et al.. additionally, our predictive model noted that baseline hba1c was the sole indicator of response to exenatide therapy with higher baseline values of hba1c corresponding to 5% greater odds of exenatide response by 6 months. although several studies have reported additional parameters as predictors of response, the most reproducible variable has been baseline hba1c. however, within the existing literature the predictive role of baseline hba1c determining response to exenatide report that a higher baseline hba1c reduces the odds of response [27, 29], yet studies by anderson et al. and observed a greater likelihood of response with higher levels of baseline hba1c [26, 31]. although results from our study correspond to the latter studies, we believe the differing predictive role of baseline hba1c observed by preumont et al. and anichini et al. is a result of the definition used to determine response to exenatide. both preumont et al. and defined response to exenatide as participants achieving an absolute hba1c value 58 mmol/mol (7.5%) and hba1c 53 mmol/mol (7%), respectively. however, in both our study and those conducted by anderson et al. and shin et al., response to exenatide was defined by a relative loss in hba1c [26, 31]. subsequently, the variations in the predictive value of baseline hba1c may be in part attributed to whether an absolute or a relative value was used as the response definition. one explanation for this is that despite individuals with a higher baseline hba1c reporting a greater relative reduction in their hba1c, it is more difficult for these individuals to reach the absolute hba1c response cut-off due to the large reduction required to reach that point. in contrast, those with a baseline hba1c that marginally differs from the hba1c response value are, despite having only a relatively small reduction in their hba1c, more likely to reach this end-point. we believe this policy of defining response based upon attainment of an absolute value should not be used to define response to exenatide, but rather a relative reduction. studies such as the uk prospective diabetes study (ukpds) have shown that an 11 mmol/mol (1%) reduction in hba1c reduces the risk of microvascular complications by 25%; thus it would be clinically unwise to omit individuals with a high baseline hba1c from receiving exenatide despite knowing these are more likely to show a significant relative reduction overall. in our study we also identified patients with a baseline hba1c<60.3 mmol/mol (7.7%) as unlikely to respond to exenatide, whereas those with an hba1c at baseline 60.3 mmol/mol (7.7%) were likely to respond. such findings have been reported by anderson et al. which noted that individuals with a baseline hba1c<56.3 mmol/mol (7.3%) were less likely to respond to exenatide compared to those with a baseline hba1c 56.3 mmol/mol (7.3%). the significance of these observations correlates to the requirements for use of exenatide as defined by nice. nice state that exenatide should be utilised when both first-line metformin and second-line sulphonylureas have failed, in addition to the patients ' hba1c being 58.5 mmol/mol (7.5%). subsequently, our results coincide with such criteria for exenatide use as patients with a baseline hba1c<60.3 mmol/mol had no response to exenatide; thus clinicians should avoid utilising exenatide in such scenarios as it only exposes patients to the possible side effects and the financial implications associated with exenatide with no improvement in glycaemic control. although identifying individuals with a baseline hba1c value<60.3 mmol/mol (7.7%) served as one means of delineating nonresponse to exenatide therapy, it only accounted for 12.2% of the cohort that eventually failed to respond following 6 months of exenatide administration. unlike the previous studies within this area, we are the only study to utilise three time-points, that is, baseline, 3 months, and 6 months. therefore, analyses in the percentage change in hba1c at 3 months compared to baseline identified another measure of predicting response to exenatide therapy. in our study, which we believe is the first to report such an observation, a reduction in the baseline hba1c by 1520% (or greater) at the 3-month period resulted in a greater proportion of the cohort following on to be classed as responders at 6 months. furthermore, individuals who have had an hba1c reduction between 0 and 5% (or less) at 3 months resulted in a significantly greater proportion being deemed as nonresponders by 6 months. at present, it is only at this point that clinicians are deemed appropriate to make a judgement regarding whether an individual is suitable to continue exenatide therapy and if not, an alternative therapy will then be commenced which will also be monitored to observe for any glycaemic benefit. this attitude within healthcare to periodically observe and in some situations fail to alter or adequately titrate upwards treatment strategies in the face of an uncontrolled disease is referred to as therapeutic inertia. in our study we identified that a significant proportion of the initial cohort could be identified as responders by the 3-month period simply by quantifying the percentage difference in hba1c from baseline. therefore, in the clinical setting clinicians would only have to wait for 3 months to determine whether an individual was likely to respond, thus allowing for the rapid intensification or alteration of treatment if shown to be not working whilst also ameliorating a further three-month period of exposure to the theoretical side effects and cost attributed to exenatide. during the course of this study we encountered limitations which may have influenced our results. firstly, we did not envisage such a large proportion of the identified sample to be excluded on the basis of incomplete data. this in turn led to a reduced sample size available for statistical analyses and thus may have resulted in fewer variables being identified as predictors of response. secondly, all our participants were derived from one institute and so this puts into question whether our predictive model is applicable to other institutes. furthermore, the patients we included within our study were identified from a cohort under the care of the endocrine department at the coch, thereby exposing our study to selection bias. as such patients in our clinics reflect individuals with difficult cases of t2 dm, and not the typical cases seen in primary care, inclusion of such cases m ay have skewed our results to reflect a predictive model only applicable for such treatment resistant cases. another limitation of our study is that we did not measure adherence to exenatide during the study period. as exenatide is associated with both side effects and the possibility of stigma arising due to use of injections, patients may have not adhered to the agreed upon treatment plan. this limitation may be responsible for the significant rise in hba1c identified between 3 months and 6 months in the nonresponder group. additionally, in our study we can not ascertain whether the weight loss observed is a direct consequence of exenatide suppressing appetite or as a result of concurrent dietary and lifestyle interventions undertaken by the patient. finally, despite identifying other medications prescribe alongside exenatide, we did record the initial dosage and whether medication regimens changed over the course of the study. therefore, this may have meant that any observed changes may have been, in part, attributed to the additional medications given alongside exenatide and not exenatide alone. although our study provides another dimension, applicable to the real world setting that clinicians should consider when determining if an individual will respond to exenatide, we believe additional research examining the other glp-1 analogues is warranted; there is currently only one study on predictors of response to liraglutide. furthermore, it would be of clinical relevance to determine if the predictors of response to exenatide differ depending upon inclusion of the 3% weight to our existing definition of response to exenatide. patients with t2 dm represent a diverse heterogeneous population with varying demographic, anthropometric, and metabolic characteristics. despite exenatide being shown to be efficacious, there remains a subset of individuals who fail to respond. our results reinforces what has been documented previously with regard to the negative linear relationship between baseline hba1c and changes in hba1c but additionally sheds further insight into the conflicting nature of baseline hba1c as a predictor of response. furthermore, our study provides a novel insight into the possibility of using the percentage change in hba1c observed at 3 months of treatment to predict response by 6 months. we hope that our findings reinvigorate this field of research as with supplementary research we can ultimately develop a predictive model which both outlines and quantifies a series of predictor variables and changes allowing clinicians to identify, with a significant degree of confidence, individuals likely to respond to exenatide therapy.

exenatide is a glp-1 analogue used in the management of t2 dm yet within a subset of patients fails due to adverse side effects or from failure to attain the end goal. this retrospective observational study aimed to determine whether we could predict response to exenatide in patients with t2 dm. 112 patients on exenatide were included with patient age, gender, duration of t2 dm, medications alongside exenatide and weight, bmi, and hba1c at baseline and 3 and 6 months of exenatide use being recorded. 63 responded with 11 mmol/mol reduction from baseline hba1c after six months and 49 did not respond to exenatide. hba1c solely differed significantly between cohorts at baseline, 3 months, and 6 months (p<0.05). regression analyses identified a negative linear relationship with higher baseline hba1c correlating to greater reductions in hba1c by 6 months (p<0.0001). hba1c was the sole predictor of exenatide response with higher baseline hba1c increasing the odds of response by 5% (p=0.004). patients with hba1c reductions 1520% by 3 months were more likely to be responders by 6 months (p=0.033). our study identified that baseline hba1c acted as the sole predictor of exenatide response and that response may be determined after 3 months of exenatide administration.

PMC4320857

pubmed-690

to report a case of recurrent idiopathic frosted branch angiitis (fba) successfully treated with adalimumab. a 14-year-old otherwise healthy boy was referred to the uveitis clinic for bilateral panuveitis with diffuse retinal vascular sheathing and severe macular edema. two years ago, a similar episode had been treated with oral prednisolone, however it was complicated by adverse psychiatric effects. the progressive course of the condition mandated considering other therapeutic measures; adalimumab was chosen based on its purported efficacy for treatment of childhood uveitis and a favorable safety profile. the patient responded dramatically to a single subcutaneous injection of adalimumab without any side effect during and after injection. to the best of our knowledge, this is the first case of idiopathic fba treated successfully with adalimumab without adjunctive steroid therapy. frosted branch angiitis (fba) is a rare disease characterised by visual disturbance associated with anterior chamber and vitreous inflammation and severe sheathing of retinal vessels resembling the appearance of frosted branches of a tree. it was originally described in a six-year-old child by ito in 1976.1 fba can be associated with ocular and systemic diseases. cytomegalovirus retinitis, acquired immunodeficiency syndrome, and toxoplasma chorioretinitis are frequent ocular associations, while systemic lupus erythematosus, crohn's disease, behcet s disease, large cell lymphoma, and acute lymphoblastic leukemia have been described as systemic associations.2 the idiopathic type typically occurs in young and healthy persons and is associated with good recovery of vision. a 14-year-old boy, who was otherwise healthy, was referred to the uveitis clinic for progressive blurring of vision in both eyes since 1 month before. on examination, anterior segment examination revealed mild ciliary injection and anterior chamber reaction (2+cells) but no keratic precipitates or posterior synechiae. both eyes had inflammatory reaction in the vitreous (2+cells) but there were no snow balls or snow bank formation. fundus examination disclosed extensive retinal vascular sheathing and scattered intraretinal hemorrhages with severe macular edema in both eyes (fig. 1). systemic work-up and investigations, including a complete blood count (cbc), erythrocyte sedimentation rate (esr), c-reactive protein (crp) titer, angiotensin converting enzyme (ace) titer, tuberculin skin test (tst), urine analysis and chest x-ray were unremarkable. rheumatologic tests including antinuclear antibodies (ana), anti double strand dna antibody (anti ds-dna), rheumatic factor (rf), antiphospholipid antibody, anti-neutrophil cytoplasmic antibodies (ancas), and cryoglobulin assay were all negative. blood cd4 and cd8 counts were within normal range and serology for the human immunodeficiency virus was also negative. blood serology for possible infectious causes including herpes family viruses, i.e. herpes simplex virus (hsv) type 1 and 2, varicella zoster virus (vzv), cytomegalovirus (cmv), and epstein-barr virus (ebv), hepatitis b and c viruses, treponema, borrelia, and toxoplasma were checked. immunoglobulin g (igg) levels against hsv type 2 and cmv (but not igm) were elevated. we tested the aqueous specimen after an anterior chamber paracentesis to investigate the presence of deoxyribonucleic acid (dna) of hsv type 1 and cmv viruses. the result of polymerase chain reaction (pcr) on aqueous samples was negative for both viruses. until the results of blood tests and aqueous pcr became ready, vision continued to deteriorate down to 20/1000 and 20/800 in the right and left eyes, respectively. based on the inconclusive work-up a diagnosis of idiopathic fba the patient s parents mentioned that two years ago, for treatment of a similar condition, oral prednisolone 1 mg/kg had been commenced but soon after initiation, the patient developed severe mood changes which persisted long after discontinuation of treatment. the progressive nature of the condition mandated considering other therapeutic measures to prevent macular scarring. due to the safety profile of anti-tumor necrosis factor (tnf) agents and their rapid onset of effect, adalimumab was considered as an alternative to conventional treatment for which the parents provided written informed consent. a single dose of 40 mg pre-filled adalimumab (humira, abbott laboratories, north chicago, il, usa) was injected subcutaneously in the patient s right thigh. no local or systemic adverse effect was noted at the time of injection or later on. three weeks after injection, visual acuity improved to 20/200 and 20/120 in the right and left eyes, respectively. anterior chamber and vitreous reaction decreased to 1+cell, and vascular sheathing and macular edema resolved dramatically. repeat fluorescein angiography and optical coherence tomography (oct) confirmed the improvement (figs. 2 and 3). 10 weeks after injection, visual acuity improved to 20/40 and 20/30 in the right and left eyes, respectively. in both eyes, clinical improvement continued up to the last visit (6 months after the injection) when visual acuity reached 20/25 in both eyes with no sign of active inflammation. fba is basically divided into three different subgroups.3 the first comprises patients affected by lymphoma and leukemia presenting with a frosted branch-like appearance in the fundus. the second group includes patients with associated autoimmune or viral disease which can present with fba in association with the underlying disease. the characteristic feature of this latter type is its occurrence in otherwise healthy young individuals with good recovery of vision. clinically, there is severe sheathing of retinal vessels appearing like the frosted branches of a tree, and acute visual disturbance associated with anterior chamber and vitreous inflammation; fluorescein angiography shows leakage from sheathed vessels without signs of occlusion or stasis.4,5 systemic steroids are the main and only suggested treatment for this condition. in our patient however, prior adverse psychiatric effects of steroid treatment encouraged us to employ another therapeutic option: adalimumab, a fully humanized monoclonal anti-tnf antibody, was chosen based on its suggested effect for treatment of refractory childhood uveitis including juvenile idiopathic arthritis.6-8 our patient responded dramatically to a single subcutaneous injection of adalimumab. the improvement was rapid and long lasting and the patient did not report any side effect during follow up. review of the literature revealed no other therapeutic option for treatment of idiopathic fba except systemic steroids. to our best knowledge, this is the first case of idiopathic fba treated successfully with an anti-tnf agent without adjunctive steroid therapy.

purposeto report a case of recurrent idiopathic frosted branch angiitis (fba) successfully treated with adalimumab. case reporta 14-year-old otherwise healthy boy was referred to the uveitis clinic for bilateral panuveitis with diffuse retinal vascular sheathing and severe macular edema. extensive work-up including aqueous sampling for detection of viral causes was inconclusive. two years ago, a similar episode had been treated with oral prednisolone, however it was complicated by adverse psychiatric effects. the progressive course of the condition mandated considering other therapeutic measures; adalimumab was chosen based on its purported efficacy for treatment of childhood uveitis and a favorable safety profile. the patient responded dramatically to a single subcutaneous injection of adalimumab without any side effect during and after injection. the therapeutic effect was rapid and relatively long-lasting. conclusionto the best of our knowledge, this is the first case of idiopathic fba treated successfully with adalimumab without adjunctive steroid therapy.

PMC3957044

pubmed-691

dentures are worn by around 20% of the population, yet if displaced they may enter the gastrointestinal or respiratory system, sometimes with grave consequences. further understanding of the epidemiology, and possible consequences of such events could lead to more informed prescription of appropriate denture design, and hence reduce these risks. tooth replacement is carried out to improve masticatory efficiency and aesthetics, reduce over-eruption and drifting of unopposed teeth, and to restore phonetics. prosthodontics may be of the upper or lower jaw, may be removable or fixed, and may be made out of plastic, metal, ceramic or a combination. an appropriate prescription for tooth replacement takes into account many factors including patient s aesthetic requirements, amount of remaining alveolar bone, dental health of remaining teeth, strength of the gag reflex, and financial cost. assessment of a patient s risk of aspirating or swallowing a particular denture is also an important part of this process. once a denture passes posteriorly into the oropharynx, or down into the hypopharynx, it may either continue into the aerodigestive system or be expelled back to the oral cavity by the usual gag reflex. any factor which inhibits the gag reflex could be reasonably considered to increase the risks of aspiration or swallowing of a denture. such factors may include neuromuscular disorders such as stroke, multiple sclerosis or parkinson s disease. from the hypopharynx, the denture may pass into the larynx and then into the respiratory system, or it may enter the oesophagus. in the larynx, the vocal cords may prevent further ingress, or the denture may pass inferiorly to the carina. from there, the more direct path is into the right bronchus, and then, depending on size, into the bronchioles. in the oesophagus there are four well documented sites where foreign bodies may lodge: a) at the level of the cricoid cartilage, b) where the oesophagus is compressed by the aortic arch, c) compressed by the left main bronchus, d) at the lower oesophageal sphincter. the stomach presents few obstructions to the foreign body, though the small bowel progressively decreases in diameter up until the ileocecal valve. different designs of dental prostheses may lodge in different areas, or may pass through the gastrointestinal tract altogether. hooked on the mucosa of the aerodigestive tract, whilst small fixed dentures may pass out in stool. different designs may also require varying surgical strategies for removal whilst short fixed dentures may be safely monitored as they traverse the intestines, larger dentures may require endoscopic removal, and those which have become attached to the mucosa may require open surgery. dentures made entirely of acrylic present a particular challenge as they can not be easily identified on plain x-rays, and therefore a ct or mri scan may be necessary to determine their position. there is clear potential for morbidity and even mortality from a swallowed or aspirated denture. therefore it is important to minimise the risk of aspirating or swallowing dentures, and mitigate the potential damage. we review recent published literature in order to identify the epidemiology of patients and characteristics of swallowed and aspirated dentures, and propose strategies to minimise these risks. protocol and registration a systematic review was carried out using the prisma 2009 guidelines (http://www.prisma-statement.org/prismastatement/). the following focus question was developed according to the population, intervention, comparison and outcome (pico) study design: what are the common design features, morbidities, therapeutic strategies and epidemiology of swallowed and aspirated dentures reported in case studies? information sources and search the authors searched embase and medline, with date limits 1 january 2000 to 31 december 2015 inclusive, using the mesh terms this date range was chosen as it represented a pragmatic and contemporary sample since the widespread introduction of internet based journals. the remaining articles were screened by title and abstract by the primary author (sk). for papers where the full text could not be retrieved, types of publications the review included case studies and series published in the english language. types of participants/population the patients in the case studies had all experienced an event of swallowing or aspirating a dental prosthesis. the case series all described at least one event of either swallowing or aspirating a dental prosthesis. inclusion and exclusion criteria the full text of all case series of relevance was obtained for assessment against the following inclusion criteria: described at least one case of either swallowing or aspirating a dental prosthesis. the following exclusion criteria were applied to case series: case series in which data were not presented as a case by case series but were given as overall observations and averages. data extraction and data items data were collected using a specially designed data extraction form on: 1) the circumstances of the event, 2) any patient factors identified in the case study which may have contributed to the event, 3) the presenting symptoms of the patient, 4) the design of the denture (material, clasps, maxillary or mandibular), 5) the anatomical position at which the denture got lodged, 6) whether the denture caused perforation of the viscus, 7) the procedure required to remove the impacted denture. any available photos of the dental prostheses were also collected, and analysed for type of denture, presence of clasps, size of the denture, and material. risk of bias assessment assessment of risk of bias was not undertaken, as the studies were descriptive case studies and were not subject to bias within themselves. data were collected on a microsoft excel spreadsheet (microsoft, version 14.3), and analysed using statistical package (spss, ibm, version 9.2). associations tested included whether the procedure used to retrieve the denture varied by presence of clasps, whether presence of clasps affected likelihood of spontaneously passing the denture, and thirdly whether dentures with clasps were more likely to cause perforation. 86 case reports and case series were reviewed [6-91]. in twelve of these the full text could not be retrieved but sufficient information was included in 11 of the abstracts to include in the final analysis. two of the case series were large, including 47 and 15 patients, and these were not included in the final analysis because of the way the results were presented and the absence of information on denture design. therefore 83 case reports and case series which included sufficient information on 91 separate swallowing and aspirating events were identified from 28 countries. from these all of the papers were case series and reports, and therefore the quality of evidence was rated as no risk of bias was identified in any of the studies (table 1). the mean age of patients was 55 (sd 16.9) years (range 15-93 years), with 74% male. there was no identifiable causative patient factor in 51 of the patients (56%), with no recording of this information in 5 (6%). of the remaining 35 patients, eight had a stroke, seven had dementia, four had epilepsy, and the rest were split between alcohol or opiate intoxication, learning difficulties, schizophrenia, parkinson s disease and multiple sclerosis. the circumstance of swallowing or aspirating the denture was known in 53 (58%) situations. of those that were recorded or known, 28 (31%) were whilst eating, 6 occurred during a general anaesthetic, 5 occurred during trauma (which ranged from a simple fall to a shotgun wound to the face), 4 were whilst sleeping and 4 were during seizures. the remainder occurred whilst intoxicated with alcohol or drugs, whilst fitting the denture or during a stroke. one letter (which was not included in the final analysis as it did not report on denture characteristics, demographics of the wearer, or retrieval method) reported a swallowed denture whilst diving into a swimming pool. the most common anatomical site for the denture to be lodged was the oesophagus, with 33 in the upper gastrointestinal tract (proximal oesophagus to stomach). no differentiation was made between the hypopharynx and larynx as most case reports did not differentiate. nineteen lodged in the airways (trachea to lungs), seventeen in the middle and lower gastrointestinal tract (ligament of treitz to rectum) and seven passed through the gastrointestinal tract. in thirteen cases the patient had suffered from a perforation of the gastrointestinal tract, and in two cases a trachea-oesophageal fistula had formed. endoscopic (n=38 procedures, 42%) included flexible and rigid bronchoscopies, flexible and rigid oesophagoscopies, gastroscopy and colonoscopy. open procedures (n=33, 36%) included bowelresection and enterotomy (16), cervical oesophagectomy (8), transthoracic oesophagectomy and bronchotomy (4), tracheostomy (4), and hartmann s procedure. in three cases the denture was not retrieved due to death and due to the dentures were divided broadly into fixed (n=18, 20%) or removable (n=60, 67%). thirty one (34%) were maxillary and 21 (23%) mandibular, and it was unclear in 39 (43%) cases. the fixed prostheses were mainly tooth supported bridges, between two and eleven units, with an average of four units. the most common material was acrylic (n=24, 27%), followed by acrylic and metal (n=17, 19%). there was no statistically significant relationship between denture design (clasped vs unclasped) and procedure used for retrieval (p=0.13), or between denture design and perforation (p=0.37), or between denture design and spontaneously passing a denture in stool (p=0.55) (table 2). swallowed and aspirated dentures are responsible for significant morbidity and occasional mortality. with an ageing population and a growing global middle class who can afford and desire tooth replacement, our systematic review examines the epidemiology of publications relating to this issue and identifies common design characteristics. limitations of this study include the methodology- by only examining case reports we have missed all of the incidents that were not published. straightforward situations have been missed in our systematic review, and what we have identified is in fact the tip of the iceberg. therefore we are likely to have a selection bias for cases which fall out with the norm, be that in terms of the magnitude of intervention required, the time a denture was left undiscovered, or even the circumstances of the event. in the author s own experience, removing a denture endoscopically from the hypopharynx of a clinically stable patient who has some dysphagia and reports swallowing their denture is a relatively unremarkable presentation to the emergency department, and this is supported by bandopathy s case series, in which 43 dentures were removed endoscopically with only one requiring cervical oesophagectomy. however, by examining the reported cases over the past 15 years we are able to draw some limited conclusions on the seriousness of the problem, and design characteristics which these cases had in common. firstly, a common theme running through many of the case reports was the diagnostic uncertainty involved in managing patients with swallowed acrylic dentures. when the patient had no recollection of the event, or was unable to express this to the clinicians, the diagnosis was often not made for days or even, in five patients [7-11], years. this could be easily remedied by making the acrylic used in denture production radio-opaque, or adding a metallic foil strip into the denture. this has been suggested by a number of authors, but has never been widely adopted. we conclude that by including radiopaque material in the acrylic, many of the cases of swallowed or aspirated dentures would have been resolved more quickly and with less morbidity. a second theme identified in the review was that there was no statistically significant difference in perforation rates or requirement for open surgery between clasped and unclasped dentures. it seems logical that metal clasps might act as a fish hook on the epithelium of the aerodigestive tract, and may therefore require surgical removal, and perforate the gastrointestinal tract. whilst clasped dentures are radiopaque due to the metal components, they are difficult to remove endoscopically, and after initial attempts to do so patients often required a second visit to the operating theatre for an open procedure. however, in the literature we reviewed [6-91] there was no difference in rates of perforation and open surgery, suggesting that where clasps are required to provide better retention their benefits outweigh perceived risks of morbidity. a third conclusion is that a number of the dentures identified were damaged at the time of the event. whilst some of these were broken during the incident (e. g. the acrylic fragments cause by the shotgun blast), most were damaged previously but still worn by the patients [14-18]. this highlights the importance of warning patients not to continue to wear dentures which have been damaged or are loose, the importance of adequate, understandable post insertion instructions and regular recall for denture maintenance. a fourth conclusion of interest was the number (56%) of people who did not have any identifiable predisposing factors to swallowing or aspirating a denture. one paper suggested that the method of eating and drinking could have an effect, with a high number of indian cases attributed to a particular method drinking (pouring liquids into the mouth from a height, to avoid lip contact with the container, seemed likely to dislodge the denture). whilst dentists may be aware of the importance of providing a suitable dental prosthesis to patients with oropharyngeal incompetence secondary to epilepsy or stroke, the fact that so many of the cases identified involved patients with no risk factors should highlight that this is a problem which could affect anybody wearing a dental prosthesis. the diagnosis of swallowed or aspirated dentures is often difficult to make, and may be simplified by addition of radiopaque material to acrylic components. secondly, damaged dentures should not be worn as they appear to be aspirated and swallowed more frequently. thirdly, all patients, regardless of comorbidity, are at risk of aspirating a swallowing a denture, and fourthly that inclusion of clasps does not appear to statistically increase the morbidity caused by the denture. the development of a reporting system would assist in identification of cases, and identification of higher risk denture designs. further research involving prospective study of large groups of patients after denture fitting, and guidelines in denture design which aim to minimise the risks and mitigate the damage caused by swallowed and aspirated dentures are required. the authors would like to thank the ear nose and throat department at aberdeen royal infirmary for suggesting the review, and miss frances parkinson for her help in organising the data.

abstractobjectivesdentures are worn by around 20% of the population, yet if they become displaced they may enter the gastrointestinal or respiratory system, sometimes with grave consequences. the aim of this study was to review recent published literature in order to identify the epidemiology of patients and characteristics of swallowed and aspirated dental prostheses, and propose strategies to minimise these risks. material and methodsa fifteen year retrospective of published case series and case reports was carried out. photographs, radiographs and descriptions of the dental prostheses were gathered, as well as the patient s presenting complaint, the anatomical site where the denture was caught and the procedure required to remove the denture. resultsninety one separate events of swallowed or aspirated dentures were identified from 83 case reports and series from 28 countries. average age was 55 years, and these were 74% male. photographs were retrieved for 49 of these dentures. clasps were present in 25 of the dentures. there was no significant difference between clasped and unclasped dentures for perforation rates, need for open surgery and spontaneously passed dentures. conclusionswe discuss the implications of this study regarding denture designs, specifically the importance of using a radiopaque acrylic, using clasps when required even if there is a risk of aspiration, advising patients to return if a denture is loose or damaged, and finally that all patients who wear a denture are at risk of aspiration and swallowing events, and associated morbidity and mortality.

PMC4970503

pubmed-692

regulation of protein expression is orchestrated through the interaction of trans-acting factors with cis-regulatory elements. different factors are targeted to specific sequence regions or secondary structures in the transcript to promote different outcomes, including initiation of translation and degradation. key regulatory steps in generating differential protein expression include the co-transcriptional events of pre-messenger rna (mrna) splicing, capping of the mrna 5 end, and polyadenylation. post-transcriptional control can be exerted through de-capping, deadenylation, and cytoplasmic polyadenylation. additionally, mrnas in the cytoplasm will often undergo transport, storage, and quality control, which influence the location and level of protein expression. all of these processes can be modulated by interactions with rna-binding proteins and non-coding rnas. for over half a century, research has focused on how these processes function individually and synchronously to control when and where proteins are made. biochemical experiments using oocytes, embryos, yeast, and cultured cells first identified the factors involved and established the role of proteins in post-transcriptional regulation. diligent exploration of individual rnas then enabled detailed models to be built for the different mechanisms of regulation. a burst in genome-wide and big-data approaches is now challenging previous assumptions and is uncovering levels of complexity in post-transcriptional control of protein expression. based on recent breakthroughs, this article will discuss how new types of data are reshaping our understanding of post-transcriptional regulation in embryogenesis, with a focus on poly(a) tails and the 3 untranslated region (utr). i will consider outstanding questions such as the following: how do length, inclusion, and exclusion of the 3 utr influence mrna fate? does poly(a) tail length directly influence translational outcomes? how is the balance between polyadenylation and deadenylation managed in the cytoplasm? when and at what level do micrornas (mirnas) function through mrna decay, repression, and deadenylation? the priority of resolving these and other queries is bolstered by increasing data linking human disease progression and severity to transcript regulation [24]. in the future, unraveling the nuances and intricacies of post-transcriptional regulation will once again require mechanistic analyses of individual transcripts. the scope of this article is limited, and the aim is to provide a glimpse of the emerging questions. in the early 1970s, fractionation experiments on mammalian cells isolated polysome-associated mrnas. an intriguing observation was made when these mrnas were cut with rnases specific to c and u as well as g; there was a long, resistant fraction. this fraction was proposed to consist of a poly(a) sequence not encoded by the dna template. this was confirmed upon the discovery of poly(a) polymerase (reviewed in). during the same period, researchers showed that poly(a) elongation commonly takes place in oogenesis, providing an important biological model. by the end of the 1980s, experiments in frog and mouse oocytes showed that regions of the 3 utr of mrna had the capacity to confer polyadenylation and initiate translation. this was somewhat surprising as the convention at the time was that the 5 utr regulated translation. it was quickly established, in both vertebrates and invertebrates, that specific mrnas were dependent on lengthened poly(a) tails for translation and that deadenylation, in some cases, directly resulted in translational repression (figure 1). moreover, the binding of trans-acting factors to the regulatory element in the 3 utr can result in recruitment of different downstream proteins and enzymes. changes in the poly(a) tail length are important for translation and mrna stability. abbreviations: mrna, messenger rna; utr, untranslated region. in many organisms, there is a delay after fertilization before the zygotic genome is transcribed, and this is why maternal mrnas and post-transcriptional regulation are so important. this remains the case until inhibitors are removed or outcompeted by other factors for elements in mrna utr. interaction between the 5 and 3 utr-associated proteins often circularizes the mrna, thus preventing the small ribosomal subunit from positioning on the transcript and initiating translation. the oocyte and early embryo of drosophila have been the focus of decades of research on mrna translational control. with a broad-scale approach taken by many researchers in the field, gene expression was recently measured by microarray and demonstrated that a specific poly(a) polymerase is required for the elongation of thousands of mrnas at the transition from oocyte to embryo. a post-fertilization wave of gene expression due to maternal mrna polyadenylation is also observed in frogs by high-resolution profiling of gene expression. these data are consistent with other work and suggest that cytoplasmic polyadenylation is a general regulator of translation at this developmental transition. once maternal mrnas have been translated, they are degraded and the onset of zygotic transcription, known as the maternal-to-zygotic transition, follows. how these mrnas are cleared was tested by transcriptome-wide analysis in oocytes, one-cell embryos, and two-cell embryos of c. elegans and revealed that clearance of mrna following fertilization is mediated by poly(c) motifs in the 3 utr of the mrna. it will be important to test what, if any, other components are also required at this stage and whether this mechanism of clearance is unique to worms. both the identification and authenticity of the c. elegans 3 utr have been areas of debate. differing methods resulted in contrasting size, site specificity, and the number of 3 utrs reported. the evidence seems clear now that worm 3 utrs are much shorter than those of mammals (approximately one sixth) but are twice as dense in conserved mirna sites. as work in worms moves forward, it has important links to the evolution of the genome architecture and is generating new techniques for studying post-transcriptional gene regulation. the same method used to accurately map poly(a) regions in c. elegans was used in a vertebrate model, zebrafish, to show that mrnas experience cleavage and polyadenylation through development. a large wave of cytoplasmic polyadenylation is detected 2 hours post-fertilization, but why and exactly how this event takes place remain unclear. one possible model is that transcripts could be sites of competitive de-adenylation and re-adenylation, resulting in differential expression or decay. in mouse development, mrnas with short 3 utrs are preferentially targeted for degradation, demonstrating again the importance of regulating the length in the 3 utr. it is clear that translational regulation of mrnas is essential in early animal development and is a highly conserved mechanism for temporal control of gene expression. although the presence of a non-templated poly(a) tail on mrna is presumed to lead to translation, a recent advancement in globally and accurately measuring the length of poly(a) tails has led to new models for the outcome of polyadenylation. in a survey of the individual length of millions of rnas from different plants, animals, and developmental stages therein, the results show that on large-scale poly(a) tail length and translation are not always coupled. as predicted, frog and zebrafish embryos displayed a strong correlation between poly(a) tail length and translation prior to gastrulation but, importantly, not after. this work challenges the assumption that poly(a) elongation is synonymous with translation. as the understanding and importance of post-transcriptional regulation, especially of the poly(a) tail and utr, have expanded, research has increased in depth as well as breadth. examples of ongoing broad research in the field include the following: work on the maternal 3 utr of atlantic cod seeking to increase egg viability, an ongoing debate in plants on how factors that control poly(a) site choice are regulated, and testing of therapeutics that alter mrna half-life through manipulation of utrs, which could potentially block the progression of breast cancer, bipolar affective disorder, hereditary thrombocythemia, fragile x syndrome, and alzheimer's disease. an emerging mechanism adding a level of complexity to the transcriptome is alternative polyadenylation (apa) (figure 2). this is a general term inclusive of multiple classes of events that through cleavage and polyadenylation generate mrna isoforms with different 3 utrs. in addition to changes in the 3 utr, less common classes of apa involve alternation of introns and exons. the full implication of apa is unclear; however, more targeted mechanistic analysis in the future will be key to understanding this process and its outcomes. experimental observations that could lead to further breakthroughs include work showing that ubiquitously transcribed genes in human cells gain tissue specificity through apa and that the rna localization of brain-derived neurotrophic factor and camkii both involve apa. it is exciting to hypothesize that apa could be involved in mrna localization in oocytes and embryos as well. moreover, during cell proliferation, apa has been shown to play a role in generating mrnas with shorter 3 utrs and in turn less mirna target sites, likely conferring unique regulation. alternative polyadenylation can change the length of the mrna 3 utr resulting in the inclusion or exclusion of regulatory elements. this results in the binding of different trans-acting factors, including proteins and mirnas, which confer specific post-transcriptional regulation. abbreviations: apa, alternative polyadenylation; mirna, microrna; mrna, messenger rna; utr, untranslated region. a direct link to embryogenesis has been shown in the mouse model. as a result of apa, during the progression of development, mrnas with longer 3 utrs are detected. with longer 3 utrs and more au-rich sequences, it is suggested that mrnas later in development are likely to undergo more extensive post-transcriptional regulation. there is also evidence from zebrafish that high levels of apa occur throughout development and in drosophila where additional regulation is conferred in neural tissue by the inclusion of more repressor-binding sites in the 3 utr of mrna. deep sequencing of the transcriptome has been paramount in identifying and annotating apa regulation in varied conditions, but the core biological mechanism remains elusive. despite these examples, few transcripts show stability changes in association with apa. therefore, questions remain as to what the role of apa is with respect to localization and translational efficiency. however, over half of human genes are predicted to generate variable 3 utrs this way, raising the possibility of major medical implications through manipulation of apa function. therefore, the ability to shorten or lengthen the 3 utr by apa could lead to the inclusion or exclusion of different types of regulatory sequences in the mrna. in this way for example, a neuronal isoform with an extended 3 utr might contain mrna stability elements, whereas in a different cell type, polyadenylation at an upstream site would generate an isoform that has a short half-life. given the many different regulatory sequences present in 3 utrs, including cytoplasmic polyadenylation elements, (a+u)-rich elements, and rna localization elements in addition to proteins binding the 3 utr, non-coding rnas also recognize and bind target sequences. individual mirnas have the capacity to downregulate many different target mrnas and in some examples promote clearance of maternal mrnas in embryogenesis [2730] (figure 1). to test for the global impact of mirnas in protein production, quantitative mass spectrometry on cultured cells was used to assay changes in protein levels before and after the addition of mirnas. the results show that destabilization was the major cause of repression for the highly repressed mrna. this work also led to the proposal that mirnas act to make fine-tuning adjustments on protein levels. correlation of ribosomal profiling data with measured mrna levels tested this question in mammalian cells. this work showed that lowered mrna levels account for a majority of the change in protein abundance. ribosomal profiling was also used to decipher how mirnas regulate gene expression in zebrafish embryos. it was shown, by testing the ribosome occupancy on transcripts in different conditions, that the specific mirna tested functioned first to regulate initiation of translation and then in decay of the target mrna. these examples highlight a level of complexity to the system where one factor can influence multiple aspects of post-transcriptional regulation. moreover, mirnas have been shown to function by different post-transcriptional mechanisms of regulation depending on the stage of development. in pre-gastrulation zebrafish embryos, mirnas function to reduce poly(a) tail length, resulting in translational inefficiency, and after this stage mirnas are thought to regulate mrna though destabilization. although a full discussion is outside the scope of this article [3437], unraveling precisely how, and to what extent, non-coding rnas regulate mrna is crucial when considering their prevalence. given all of the possible layers of regulation in a 3 utr and the mechanisms of generating the utr, post-transcriptional regulation is a complex and compelling area of research. the genome era introduced new approaches to longstanding questions in post-transcriptional control, many of which began in yeast. for example, microarray-based measurements correlated the length of mrna poly(a) tail lengths and the binding of a key protein with general translation in budding yeast. also, ribosomal profiling enabled genome-wide analysis of translation and showed substantial translational control in response to environmental stress and total protein levels. owing to conserved mechanisms, research in yeast is important for building the foundation for further understanding in mrna regulation in embryogenesis. one intriguing mechanism of regulating gene expression from yeast shows a connection between synthesis and decay of mrna through shuttling of mrna decay factors back into the nucleus. whether similar factors are found in other systems to have a dual role is not clear, but this leads to the exciting hypothesis that feedback from the cytoplasm is regulating events in the nucleus. a second possible research area is comparative dynamic transcriptome analysis, which can monitor mrna levels without perturbing metabolism. in specific yeast strains tested, it was shown that when mrna degradation rates are altered, the cell's synthesis rate was reduced as well, resulting in buffering of the system. this work again suggests a level of interconnected surveillance for registering mrna levels in the cell to take subsequent action and could to be applied outside of yeast. the application of approaches from different systems, as well as establishing new protocols for testing changes in 3 utr composition, is important to decipher the molecular mechanism of these regulatory processes. however, establishing how different mechanisms are coordinated and how they relate to one another will be key to fully understanding post-transcriptional regulation not only in the early embryo but in all cells. questions about post-transcriptional regulation are being asked more rapidly than answered. however, as demonstrated by a global comparison of mrna and protein abundance in different nematode species, separated by 30 million years of evolution, these mechanisms are important and widespread. genomic techniques are uncovering exciting levels of control previously unknown and are important to the progression of the field. alongside this work, classic biochemical and genetic approaches are essential to fully understanding mechanisms of mrna post-transcriptional regulation.

gene expression is controlled by diverse mechanisms before, during, and after transcription. chromatin modification factors as well as transcriptional repressors, silencers, and enhancers all feed into how eukaryotes transcribe rna in the nucleus. however, there is increasing evidence that post-transcriptional regulation of gene expression is as widespread as transcriptional control if not more so. studies of specific transcripts in oocytes and embryos are at the core of our mechanistic understanding of many post-transcriptional events. coupled with genome-wide and large-scale experimental approaches, research is bringing to light how these regulatory events function independently and in concert to regulate protein expression.

PMC4371236

pubmed-693

the current research on human diseases primarily focuses on the molecular, microbiological, immunological, and pathological influences. the mechanical basis of disease is now often being explored to decipher any direct contributions toward the physiological response [1, 2]. in functionally loaded tissues such as cartilage and bone, cells (chondrocytes and osteocytes) experience multiaxial forces (hydrostatic, compressive, tensile, and shear), which play a significant role in modulating the biological function through maintenance of the phenotype and production of a neotissue. conversely, abnormal mechanical forces (either static or dynamic) can lead to altered cell behavior resulting in pathological matrix synthesis, increased catabolic activity (degradation), and ultimately osteoarthritis or osteoporosis (apoptosis). our previous investigations have indicated that chondrocytes and likely other cell types respond to their stress-strain environments in a temporal and spatial manner. it has also just been shown that individual cellular mechanical properties may indicate the regenerative potential of mesenchymal stem cells. investigations of the biomechanics at the cellular level have also identified the biomarkers of disease. cytoskeletal stiffness of metastatic cancer cells was reported as more than 70% softer than the benign cells that line the body cavity in patients with suspected lung, breast, and pancreatic cancer. these approaches highlight the utility of single-cell biomechanics as a critical component of advancing microscale therapeutics. advancements in laser technology and microfluidics now allow the use of optical tweezers or traps and fluid mechanics to manipulate isolated single cells [8, 9]. isolated loads can be applied experimentally to single cells in culture to quantify cellular and cytoskeletal biomechanics. one can then apply forces and displacements as small as pico-newtons and nanometers, respectively [1012]. the local microenvironment can therefore be precisely manipulated to facilitate biomechanical test sequences on individual biological cells and molecular structures. in order to explore the connection between external mechanical stimulation and cellular regeneration or degeneration, we developed a three-dimensional, multiphysics computational model to fully characterize a unique micromechanical environment. the applied stress state within our custom-fabricated optical and hydrodynamic (optohydrodynamic) trap have been mathematically developed from the fundamental equations describing microfluidic creeping flows past a suspended sphere. the objective described in the following paper is to explore the full-field cellular strain response to a range of applied stresses and cellular moduli. the described computational framework will now allow us to develop more realistic cellular models, whose intracellular structures are distinctly identified. living non-adhered, suspended osteoblasts, chondrocytes, fibroblasts, and myoblasts have recently been isolated and mechanically manipulated [12, 16]. primary cultures of chondrogenic and osteogenic tissues were generated directly from rat long bones, while muscle cells were acquired from the mouse-derived myoblast c2c12 cell line (atcc, crl-1772, manassas, va, usa). all cells were tested at room temperature experiments (~20.5c) in a flow media consisting of a physiological buffer resulting in a media viscosity of ~1 mpa s. this single cell biomechanical manipulation was made available by combining optical trapping with microfluidics to create the optohydrodynamic trap. this work was facilitated by an instrument, which integrates two laser-based techniques for the mechanical characterization of cellular and biomolecular structures [8, 12]. the optical tweezers or the trap component of the device utilizes an infrared laser (= 1,064 nm) to suspend micron-sized objects with nanometer position control and pico-newton constraining forces. in the mie regime, where objects are larger in dimension than the wavelength of the trapping laser (here biological cells), a ray optics description indicates the transfer of refracted light and the associated momentum into trapping forces (figure 1). micron-particle image velocimetry can be engaged by incorporating two frequency-doubled lasers (= 532 nm) aligned through the same optical path as the ot for full-field flow velocity characterization. however, the nanoparticles associated with velocity imaging have proven deleterious to cellular health, but provide useful experimental validation of flows around synthetic micron-sized particles. the hydrodynamic component of this approach is facilitated through a microfluidic chip design configured in the form of a cross-junction channel (figure 2). this geometry creates an extensional flow environment and a stagnation point at the channel's geometric center. cells are positioned at the centroid with the optical trap and manipulated with microfluidics, thus creating the optohydrodynamic trap. the cell experiences a total drag force equal to zero, confirmed by integrating the stress tensor as defined by the normal (form drag) and shear (friction drag) stresses (figure 3). this reflects the mechanical stability or the saddle-point nature of the optohydrodynamic trap. previous studies describe chip fabrication in detail including the control of the gravity-driven flow initially associated with microfluidic manipulation. the two- and three-dimensional stress states were previously developed as applied to the surface of a nonrotating spheroid cell of radius a, within the optohydrodynamic trap [2, 12, 14, 15]. briefly, the full-field fluid velocity vector u was constructed from the constitutive equations describing a non-rotating sphere suspended in a general linear flow with viscosity and pressure distribution p. in the polar-spherical components (r-- magnitudes and er-e-e vectors), the generalized flow field produces the individual velocity components: (1)u=u(ra52(ar)2+32(ar)4)sin2cos2er+u(ra(ar)4)sincoscos2e+u(ra(ar)4)sincos2e including the pressure distribution (2)p=p5ua(ar)3sin2cos2. the velocity gradients can be converted into the applied fluid stresses by applying the constitutive equation for an incompressible, newtonian fluid: (3)t=pi+2e, where t is the stress tensor and i is the identity matrix associated with the local isotropic (hydrostatic) pressure distribution p. the strain rate tensor e can be characterized by the flow velocity gradient tensor and its transpose: (4)e=(12)[u+ut]. by incorporating the velocity and pressure fields into the gradient analysis and then in turn into the constitutive equation, the fluidic stress tensor can be fully defined as (5)trr=p+ua(2+15(ar)312(ar)5)sin2cos2er, tr=ua(25(ar)3+8(ar)5)sincoscos2e,tr=ua(25(ar)3+8(ar)5)sinsin2e. defining the stress tensor at the cellular surface, r=a, produces the volumetric fluidic stress state applied to the cell: (6)tr=ter=rrer+re+re, where the full-field stress state can then be defined in terms of distinct normal, rr, and shear, r and r, stress components, respectively, in polar spherical coordinates: (7)rr=p+5uasin2cos2,r=5uasincoscos2,r=5uasinsin2. a three-dimensional presentation of the stresses was developed (matlab, mathworks, inc., natick, ma) for demonstration of the site-specific nature of the stress distributions (figure 4). the six deviatoric stresses were combined as an effective stress value, eff, as a means to model the three-dimensional stress state: (8)eff=12[(xy)2+(yz)2+(zx)2 +6(xy2+yz2+zx2)]1/2. here, the maximum polar coordinate derived stresses were converted to the cartesian coordinate stresses such that the maximum normal stresses are located along the x-y-z axes and the maximum shear stresses are defined along the 45 orientation off-axis locations. volumetric strain, e, based on the strain invariants, ii, was defined to encompass the full-field deformation response of the cell within the multiaxial fluidic loading environment and does not apply the small-displacement theory assumption. the cartesian axes associated with the maximum normal strains were determined from a mohr's circle analysis [12, 20]: (9)e=i1+i2+i3=x+y+z+xy+yz+zx+xyz. the experimental approach described earlier is a planar-wise measurement technique; thus the optical depth strain value, z, can be determined through a transposition of the planar loading scenario, again through a mohr's circle analysis: (10)z=1(x+y). however, in this modeling presentation here, the full-field stress state and the corresponding strains are fully characterized. the optohydrodynamic deviatoric stress state was applied to an isotropic, homogenous biological cell (20 m in diameter) within a multiphysics computational environment (comsol v4.0, palo alto, ca, usa) in order to determine the individual principal strains and in turn the volumetric strains. in this work, we continue to examine the cellular biomechanics induced within a controlled micromechanical environment. three-dimensional cellular strains were computationally modeled in silico with multiphysics software applying the analytically defined stress state (figure 5). volumetric stress and strain relationships indicated both the nonlinear response of the spherical cellular structure as well as the logarithmic increase in strain with subsequently softer cellular moduli (figure 6). this relationship is further explored when examining the direct relationship of strain with elastic moduli (figure 7). the extreme strain response induced in diseased cells indicates their further vulnerability when passively or actively resisting applied stresses. we explored the full-field cellular strain response to a range of applied hydrodynamic stresses and cellular moduli, representing various degrees of functional loading and health/disease, respectively. the computational framework now allows us to develop more realistic cellular models with intracellular and membranous structures, distinct in spatial, elastic, and active transport characteristics. the mechanical properties of a single cell are often formulated using either macroscopic or microscopic approaches. macroscopic approaches, as partially described in this work, homogenize every cellular component to produce an isotropic or anisotropic yet homogeneous continuum model so that the mechanical properties of cells can be formulated using temporal and spatially continuous partial differential equations. future efforts will incorporate a more microscopic approach, which regards the cell as a biocomposite material consisting of randomly or uniformly spaced anisotropic reinforcement cytoskeletal materials within an isotropic medium. the microscopic approach generally obtains the biomechanical response of a single cell by applying the mechanical boundary conditions at the individual fiber and matrix level, scaling up to the cellular level. microscopic approaches often provide much more detail into the subtle interaction between the cytoskeletal fibers and matrix, which potentially leads to a more accurate model of the cellular behavior, such as characterizing the irreversible deformation of the cellular skeleton. unfortunately, refined microscopic models suffer from inhibitory computational and storage costs [26, 27]. when interpreting the potential geometric changes in cellular shape associated with mechanical loading, the cell may experience some interesting membrane transitions triggering unique biologic cues. under suspension and hydrostatic loading, the cell's volume (v) can be defined as a sphere, v=3/4abc, with radii a=b=c. as seen here during the hydrodynamic extensional loading state, the isotropic, homogenous cell model deformed into a scalene ellipsoid (a b c) with equal and opposite tensile and compressive strains in the horizontal plane. however, with the future inclusion of intracellular structures and organelles as well as nonlinear elastic properties assigned to the membrane and cytoskeleton, cellular models may also deform into either an oblate spheroid (formed when an ellipse is rotated about its minor axis, a=b>c) or a prolate spheroid (formed when an ellipse is rotated about its major axis, a=b<c). the resulting shape here replicated a scalene ellipsoid likely due to different maximum stresses applied in the three orthogonal axes. ongoing experiments and modeling will continue to explore multiaxial single-cell biomechanics as well as the biologic triggers associated with geometric shape-shifting. the described multiphysics computational framework will facilitate more realistic cytoskeletal model interpretations, whose intracellular structures can be distinctly defined, including the cellular membrane substructures, nucleus, and organelles. future results will provide mathematical outcomes supporting the ongoing investigations in tissue and cellular engineering.

controlled external chemomechanical stimuli have been shown to influence cellular and tissue regeneration/degeneration, especially with regards to distinct disease sequelae or health maintenance. recently, a unique three-dimensional stress state was mathematically derived to describe the experimental stresses applied to isolated living cells suspended in an optohydrodynamic trap (optical tweezers combined with microfluidics). these formulae were previously developed in two and three dimensions from the fundamental equations describing creeping flows past a suspended sphere. the objective of the current study is to determine the full-field cellular strain response due to the applied three-dimensional stress environment through a multiphysics computational simulation. in this investigation, the multiscale cytoskeletal structures are modeled as homogeneous, isotropic, and linearly elastic. the resulting computational biophysics can be directly compared with experimental strain measurements, other modeling interpretations of cellular mechanics including the liquid drop theory, and biokinetic models of biomolecule dynamics. the described multiphysics computational framework will facilitate more realistic cytoskeletal model interpretations, whose intracellular structures can be distinctly defined, including the cellular membrane substructures, nucleus, and organelles.

PMC3523158

pubmed-694

the sample consisted of sixty-three female wistar rats (190 1 g), kept in individual cages, receiving water and food (ad libitum) (purina-brazil), in an artificially illuminated ambience, with a 12 hour day-night cycle and mean temperature of 24c. initially, all animals were submitted to an adaptation period (5 days) of swimming for 5 minutes in water at thermo-neutral temperature in a pool (50 cm 45 cm). all the animals at the start of experimental procedure weighed between 180 g and 220 g and were in their reproductive maturity period. the determination of maximal work load was performed in a swimming pool with water at thermo-neutral temperature. the work load was increased every 3 minutes by weights attached to the animal's tail and corresponding to 1%, 2%, 3%, etc. of the total body weight, until the maximal work load was reached by the animal's exhaustion (unable to surface for 10 s). all rats were kept in cages (3-4 females for each male) during 12 hours (night period). the copulation was confirmed the following morning by the presence of sperm in vaginal smears. the pregnant rats (n=63) were then separated into three groups: sedentary rats (ps, n=21; 188 2 g) submitted to water immersion but did not perform exercise during pregnancy, exercised rats (pe, n=21; 193 2 g) submitted to swimming sessions at 80% of maximal work load supported for 30 minutes during 19 days of pregnancy, trained rats (pt, n=21; 188 2 g) submitted to swimming sessions during 45 days before and during pregnancy for 30 minutes, for 19 days of pregnancy, at 80% of maximal work load supported into water, which was calculated for each rat before pregnancy. each group was divided into three subgroups (n=7), regarding water temperature: 28c, 35c, and 39c. all swimming sessions were carried out between 8:00 a.m. and 12:00 p.m. in order to determine weight gain during pregnancy, the rats were weighed on the first and 20th day of the gestational period (ohaus balance). the rats were decapitated on the 20th day of pregnancy in order to collect a blood sample (5ml) for determining plasma levels of triglycerides and glucose by the enzymatic method.8 just after decapitation, a caesarean section was carried out and the offspring were counted and weighed. mean values standard error for rectal temperatures are shown in table 1. comparing rectal temperatures, prior and post conditions, showed that, there was a decrease in rectal temperature for all experimental groups at 35c and 28c, while an increase was found at 39c, and temperature (post-prior) was significantly higher at 28c than at 35c and 39c. mean values standard error of rectal temperature (c) determined during 19 days of pregnancy for sedentary rats (ps), rats exercised during pregnancy (pe) and trained rats exercised before and during pregnancy (pt), prior to and post-immersion or swimming (80% of maximal work load supported) into water at temperatures of 28c; 35c or 39c. (n=7 for each group) mean values standard error of weight gain (g) for sedentary pregnant rats submitted to immersion (ps), rats exercised during pregnancy (pe) and trained rats, exercised before and during pregnancy (pt) at 80% of maximal work load supported into water at 28c, 35c or 39c. (n=7 for each group) at 28c, there was no difference between ps, pe and pt groups, but at 35c, pt and pe presented smaller weight gain than the ps group, and at 39c, only pt presented a smaller gain. plasma glucose was higher and plasma triglycerides were lower at 28c and 39c than at 35c for sedentary and exercised rats. trained rats did not present differences in these variables at any of the water temperatures and had different values than sedentary rats at 35c and 39c (table 3). the litter size was not different among the groups at any of the water temperatures (table 4). mean values standard error of plasma glucose (mg/dl) and triglycerides (mg/dl) on the twentieth day of pregnancy, determined in blood samples collected 24 hours after the last immersion or swimming session (80% of maximal work load supported) of pregnant sedentary rats (ps), rats exercised during pregnancy (pe) and trained rats exercised before and during pregnancy (pt) in water at temperatures of 28c, 35c or 39c. (n=7 for each group) mean values standard error of offspring weight (g) and size of litter in pregnant sedentary rats (ps) submitted to daily immersion, rats exercised during pregnancy (pe) and trained rats exercised before and during pregnancy (pt) at 80% of maximal work load supported into water at 28c, 35c or 39c. the literature demonstrates that pregnant rats have lower glucose levels than non-pregnant ones submitted to the same exercise conditions, which means they are not influenced by water temperature.8 our findings indicate that, under thermal stress (28c or 39c), sedentary and exercised pregnant rats present higher plasma glucose values than at 35c. these results suggest that they have either a smaller uptake or an increase of glyconeogenesis from lactate, alanine and piruvate.1415 an increase in homeostasis alterations (exercises in high or low temperatures) induces higher increases in catecholamine and cortisol release.1619 a raise in adrenalin induces pancreas beta cells to reduce the insulin release, which induce alpha cells to increase glucagon.20 catecholamines, cortisol and glucagon are gluconeogenese and hepatic glycogenolysis inducers, contributing to glicemic levels rise.16192122 yet, for trained animals, plasma glucose was kept steady, suggesting that daily thermal stress did not modify the regulatory mechanism of plasma glucose in these animals. the results obtained from pregnant sedentary rats and exercised rats showed alterations of plasma glucose according to the temperatures to which they were submitted daily. it suggests that the duration of immersion or swimming was more important than the temperature concerning the determination of plasma glucose levels, since in the previous study, which used more extreme temperatures with a shorter duration of exposition, pregnant rats showed steady levels of glucose.8 by determining the glucose level during exercise performed by pregnant women, greater hypoglycemia was recorded compared to that observed in resting condition, but this hypoglycemia is smaller during water exercises than land exercises.22324 glucose returned to resting level within 24 hours of the last session of exercise, indicating the efficiency of the glucose level adjustments.25 this quick regulation is made mainly by the hyperglycemiant hormones action (catecholamine, cortisol and glucagon) that have their serum concentrations increased by exercise.1622 in the present study, trained rats had lower values of plasma triglycerides at 35c than sedentary rats, indicating an increase of lipid utilization in this condition. plasma triglyceride levels were also lower at extreme temperatures rather than at 35c for exercised and sedentary rats, but trained rats did not present differences in plasma triglycerides at any of the water temperatures. when the current results were compared to our previous study,8 in which pregnant rats were submitted to more extreme temperatures but for a smaller duration, it was observed that both glucose and triglycerides were more influenced by the duration of exposure (30 minutes) rather than by the temperature, which was warmer than in the previous research. increased serum concentrations of triglycerides during the exercise are also induced by the hormones cited above. catecholamines, cortisol and glucagon act on adiposity cells rising the lipolysis and lipids release to blood. concomitantly the decrease in insulin concentration increases the hormone-sensitive lipase, augmenting the fat acids availability too.26 increased triglyceride levels were observed in pregnant rats, when compared with non-pregnant animals, especially at the end of pregnancy.225 this increase was produced by the endogenous triglycerides from the blood stream.27 therefore, a lipid represents an alternative substrate for the mother, safeguarding glucose for the fetus. on the other hand, aerobic training modifies the substrate utilization during exercise, increasing lipid mobilization and safeguarding glucose.1 in a previous study carried out in our laboratory,8 we did not observe any alteration in weight gain of rats submitted to daily swimming sessions of 10-15 minutes, at different water temperatures (22c, 35c and 40c). in the present study, using water at more agreeable temperatures for a longer exercise period, it was observed that exercised rats (pe) at 35c and trained rats (at 35c and 39c) had smaller weight gain than sedentary pregnant rats, indicating that substrate mobilization for exercise interferes with total weight gain. a smaller weight gain was found by some authors.252833 nonetheless, other authors did not observe any alterations.3436 in the present study, as in our previous paper,8 after immersion or swimming, pregnant rats modified their rectal temperature with relation to water temperature: decreasing at 28c and 35c and increasing at 39c. in spite of these alterations, a reduction in the litter size was not observed at extreme temperatures, although the duration of immersion or exercise was greater than that employed in our previous study. nevertheless, if the length of exposure is much longer, where the rats exercise daily for one hour at temperatures of 34.6c and 37.6c, fetal abnormalities and reabsorption can be found, probably due to an increase of maternal core temperature.37 regarding exposition to the cold and heat during pregnancy, some studies showed that hypothermia as well as hyperthermia could produce deleterious effects in fetus.673842 there is a temperature gradient between mother and fetus (the fetus presents a temperature 0.4 to 0.6c greater than the mother), allowing heat transference from the fetus to the mother.43 during high-intensity exercise, this gradient could be reversed, modifying the heat exchange between mother and fetus.67 this response could lead to fetal hyperthermia and consequent deleterious effects on offspring.44 when the weight of offspring was investigated, it was verified that sedentary pregnant rats submitted daily to water immersion at 28c presented litters with smaller weight in relation to offspring from mothers submitted to immersion at 35c and 39c. this result suggests that maternal hypothermia, leading to reduction in utero-placentary flow, could induce an inadequate delivery of substrate to the fetus and, in consequence, decrease its weight. besides, such an effect was not found in exercised and trained rats, suggesting that compensatory mechanisms to safeguard an adequate delivery of nutrients to the fetus were developed by training. artal et al45 published a guideline for exercise during pregnancy of the american college of obstetricians and gynecologists and advocated the benefits of an exercise program to the pregnant and to the fetus. in a recent study barakat et al46 concluded that the regular practice of exercise during the pregnancy did not influence the gestational age, but this study included only sedentary participants. our results demonstrated that an exercise program before and during the pregnancy produced better results than an exercise program only during pregnancy. further studies are necessary to find the effect of pre pregnancy training on other variables (hormonal responses, cardiovascular and respiratory responses, gestational age, etc.) in animal and human models, considering the great similar physiology between rat and human pregnancy. we concluded that physical training before pregnancy induces a greater stability of plasma triglycerides and glucose, regardless of daily swimming sessions at different water temperatures. these exercise conditions before pregnancy interfere with total weight gain during pregnancy, without deleterious effects on offspring. dalo and idcp carried out the design and coordinated the study, participated in most of the experiments and prepared the manuscript. jsc, akr and wr carried out all the experiments and participated in manuscript preparation. mm and rp provided assistance for all experiments and participated in manuscript preparation specially the interpretation and description of the results and discussion. all authors have read and approved the content of the manuscript.

background: the aim of this study is to assess the effect of pre-pregnancy physical training on metabolic responses and its effects on offspring. methods:three groups of rats (n=7 in each group): sedentary pregnant rats (ps), exercised during pregnancy (pe) and pregnant rats trained before and during pregnancy (pt) were compared. they were separated into three subgroups regarding water temperature: 28c, 35c or 39c. plasma triglycerides and glucose levels, weight gain during pregnancy and rectal temperature pre and post exercise (swim), as well as the offspring size and weight were analysed. results:rectal temperature post exercise was lower than pre exercise at 28c and 35c, and higher at 39c. weight gain was lower at 39c for the pt group and at 35c for the pt and pe groups compared to the ps group. plasma glucose, at 28c and 39c for ps and pe groups, was higher than those obtained at 35c, while triglycerides were lower. for trained rats, plasma glucose and triglycerides were similar at all water temperatures. trained rats presented lower triglyceride values at 35c, and higher triglyceride values at 39c compared to ps group. glucose presented inverse results. none of the groups presented fetal reabsorption. however, in the ps group, the offspring presented lower weight gain at 28c than at 35c and 39c. conclusions:these results suggest that pre-pregnancy physical training induces steady values of triglycerides and glucose during exercise at all water temperatures.

PMC3129109

pubmed-695

the simple gas ethylene has been recognized as a plant growth regulator for a century (crocker and knight, 1908; knight et al., 1910; neljubov, 1901). ethylene influences many aspects of plant growth and developmental processes, including germination, fruit ripening, flower senescence, leaf abscission, nodulation, lateral root initiation, and the response to a variety of abiotic and biotic stresses (abeles et al., 1992; mattoo and suttle, 1991). the russian scientist neljubov first demonstrated that ethylene is the responsible component caused the early defoliation of the tree nearby a leaking illuminating gas main in a small german town in the late1800 s (neljubov, 1901). neljubov used pea seedlings to determine that ethylene is the active component of the illuminating gas by exposing the filtered illuminating gas to pea seedlings. the pea seedlings exhibited distinctive morphology changes which include shortening of hypocotyl and root, swelling and thickening of hypocotyl, and formation of exaggerated hook. this seedling phenotype was later defined as the triple response, which is a hallmark of the ethylene response of dark-grown seedlings (knight et al., 1910). 30 years later, gane showed that ethylene is naturally produced by plants (gane, 1934). through the efforts of yang and co-workers, 1) (kende, 1993; yang and hoffman, 1984; zarembinski and theologis, 1994). ethylene is synthesized from the amino acid methionine via two intermediates, s-adenosyl methionine (sam) and 1-aminocyclopropane-1-carboxylic acid (acc) (adams and yang, 1977; lieberman and mapson, 1964). in the first step conversion of sam to acc, which is catalyzed by a family of acc synthase (acs) enzymes, is the first committed and rate-limiting step in ethylene biosynthesis (boller et al., 1979; yang and hoffman, 1984). during this conversion, 5-methylthioadenosine (mta) is generated as a by-product which is readily recycled back to the yang cycle to conserve the methlythio group of mta into the methionine (murr and yang, 1975). this salvage step enables plants to maintain a constant level of cellular methionine, which can be utilized during rapid ethylene production (sauter et al., 2013). acc is then converted to ethylene by acc oxidase (aco), a member of the oxygenase/oxidase superfamily of enzymes (dong et al., 1992). the levels of free acc are regulated by formation of acc derivatives, malonyl-acc, -glutamyl-acc, and jasmonyl-acc, which likely affect the pools of free acc for ethylene biosynthesis (van de poel and van der straeten, 2014). as gaseous ethylene is diffusible and not degraded in plant cells, strict regulation of ethylene biosynthesis is necessary to its diverse function in plant growth and development. due to its role in the rate-limiting step of the ethylene biosynthesis pathway, acs has been considered as a major point of the regulation in the pathway. regulation of the transcript levels of acs genes appears to be a key mechanism to control changes in ethylene production in plants (argueso et al., 2007; harpaz-saad et al., 2012. however, recent studies suggest that post-translational modifications, such as phosphorylation and ubiquitination, serve as an important mechanism to regulate the stability of the acs proteins, thus controlling the levels of ethylene in plants (argueso et al., 2007; chae and kieber, 2005; chae et al., 2003). the family of acs proteins can be grouped into 3 types, type-1, type-2, and type-3, based on the presence of distinct consensus sequences including phosphorylation target sites near the non-catalytic c-termini (fig. 2) (chae and kieber, 2005; mcclellan and chang, 2008; yoshida et al., 2005). type-1 acs proteins contain a relatively long c-terminal domain that shares highly conserved sequences and the target sites for a mitogen-activated protein kinase (mapk) and calcium-dependent protein kinase (cdpk) (hernndez sebasti et al., 2004; kim et al., 2003; liu and zhang, 2004). however, type-2 acs proteins contain a unique regulatory motif called a target of ethylene overproducer 1 (eto1) (toe) which overlaps with the cdpk target site. toe motif mediates interaction with eto1 e3 ligase and its two paralogs, eto1-like (eol1 and eol2) and is required for degradation of type-2 acs (chae et al., 2003; christians et al., 2009; wang et al., 2004; yoshida et al., 2005; 2006); type-3 acs contains no target sites for a cdpk and mapk, with only a short stretch of amino acids in the c-terminal extension (chae and kieber, 2005). here, i focus on recent advances and current knowledge on the protein turnover regulation of acs in ethylene biosynthesis. new insights into the role of phosphorylation, ubiquitination and recently identified novel components governing the stability of acs proteins are discussed. finally, i conclude with the prospects regarding the cross-talk between ethylene and other cellular biosynthetic/signaling pathways. transcriptional regulation in ethylene biosynthesis has been well documented, therefore not discussed in this review. treatment of arabidopsis etiolated seedlings with ethylene results in the triple response (guzman and ecker, 1990). the triple response has been extensively used to identify arabidopsis mutants with defects in ethylene perception and signaling, as well as mutants affecting ethylene biosynthesis. the ethylene biosynthesis mutants can be further categorized into two groups: (1) cytokinin-insensitive (cin) mutants which are impaired in response to cytokinin, resulting in reduced levels of ethylene production in response to cytokinin; (2) ethylene-overproducer (eto) mutants which show a constitutive ethylene response due to increased ethylene biosynthesis (chae et al., 2003; the recessive cin5 mutant was identified as the first example of the cin mutants, and further characterization of the mutant revealed that the corresponding cin5 mutation is a loss of function allele of the acs5 gene. the cin5 mutant is severely insensitive to exogenous cytokinin, and as a result, it fails to display the triple response. however, it shows normal triple response to ethylene, suggesting acs5 is the primary target of cytokinin-mediated ethylene induction in etiolated seedlings (vogel et al., 1998). other phytohormones, such as auxin, brassinosteroids and aba are also known hormonal triggers that increase ethylene production (arteca and arteca, 2008; woeste et al., 1999a; yi et al., 1999 auxin promotes ethylene production mainly through the increase of mrna levels of specific acs genes in various plant species. in arabidopsis, the most of acs genes are transcriptionally induced in response to auxin, and auxin treatment also alters the spatial expression pattern of the acs genes (tsuchisaka and theologis, 2004). aba has been shown to regulate ethylene production in apples, tomato and various plant tissues (lara and vendrell, 2000; tari and nagy, 1996; zhang et al., 2009). in tomato, ethylene levels increase remarkably after aba treatment and this coincides with the increase in the expression of leacs2, whereas application of ndga, an inhibitor of a key enzyme in aba biosynthesis, suppresses the expression of leacs2 (zhang et al., 2009). brassinosteroid is another phytohormone that enhances ethylene production by increasing the transcript abundance of acs genes, but brassinosteroid in part promotes ethylene production by stabilizing acs protein (hansen et al., 2009; yi et al., 1999 cytokinin, however, stimulates ethylene production by acting on the stability of acs proteins, thereby increasing the ethylene production in plants (chae et al., 2003; hansen et al., 2009 analysis of eto mutants has provided further evidence that the stability of acs proteins is regulated. three eto mutants have been identified via genetic screens based on the constitutive triple response phenotype due to ethylene overproduction: eto1, eto2, and eto3 (chae et al., 2003). etiolated eto mutants exhibit the constitutive triple response and this phenotypes is rescued by treatment of mutant seedlings with ethylene biosynthesis inhibitor aminoethoxyvinylglycine (avg). the dominant eto2 and eto3 mutations alter the c-terminal domain of acs5 and acs9, both type-2 acs proteins, as the result of a single base insertion and a missense mutation, respectively. the eto2 and eto3 mutants significantly produce more ethylene than wild-type seedlings, but this increase in ethylene production is not correlated to the acs5 or acs9 gene expression, thus suggesting that the mutants control the acs function at the post-translational level similar to the action of cytokinin. these results reveal that the c-terminal domain of both acs proteins is a target for post-translational modification for degradation (chae and kieber, 2005). characterization of the eto1 revealed that ubiquitination via the 26s proteasome pathway is involved in regulating ethylene biosynthesis by modulating the protein stability of type-2 acs proteins. recessive eto1 produces a nearly 10-fold excess of ethylene compared to wild-type etiolated arabidopsis seedlings and exhibits the constitutive triple response (woeste et al., 1999b). epistasis analysis demonstrates that acs5 acts downstream of eto1; the eto1cin5 double mutant produces significantly reduced amount of ethylene compared to eto1 itself (chae et al., 2003), indicating eto1 plays a role as a negative regulator by acting through acs5 in ethylene biosynthesis. eto1 encodes a novel plant-specific protein that contains a broad-complex, tramtrack, bric--brac (btb) and tetratrico-peptide repeat (tpr) with a coiled-coil domain (wang et al., 2004). proteins containing btb motifs have been shown to participate in substrate recognition via their protein-protein interaction motifs and bridge substrates to cullin 3 (cul3)-based ubiquitin ligase complexes for degradation (albagli et al., 1995; pintard et al., 2004). the tpr motifs are involved in diverse protein-protein interactions, and also serve as a scaffold for the assembly of high-order protein complexes (blatch and lassle, 1999). eto1 interacts with acs5 and cul3, and in the case of acs5, this interaction is dependent on the c-terminus of acs5, as eto1 fails to interact with acs5 (pintard et al., 2004; wang et al., 2004). consequently, these studies suggest that eto1 serves as a substrate-specific adaptor to bridge acs5 to the cul3 to regulate acs5 protein degradation. analysis of eto1 function in plants shows that eto1 suppresses cytokinin-induced acs protein stabilization via the c-terminus of acs5. overexpression of eto1 inhibits cytokinin-induced ethylene biosynthesis, and overexpression of acs5 results in a partial constitutive triple response which is suppressed by co-expression of eto1 (wang et al., 2004). however, additional data indicate that cytokinin-mediated stabilization and eto1-mediated destabilization are at least partially independent effects; exogenous cytokinin treatment still increases acs5 protein stability in etiolated eto2 and eto3 seedlings, suggesting cytokinin partially acts via an alternative mechanism that is independent of eto1 and acs5 c-terminus (hansen et al., 2009). other factors that modify e3 ligase function involved in ethylene biosynthesis are the related to the ubiquitin (rub) and rub1 conjugating enzyme (rce1) (bostick et al., 2004; larsen and cancel, 2004). like ubiquitin, rub functions through a covalent attachment to target proteins. in arabidopsis, rub attaches to the cullins, thereby promoting the activity of the scf (for skp, cdc53p/cul1, and f-box protein) ubiquitin ligase complex for polyubiquitination of target proteins. interestingly, rna interference lines of rub exhibit the partial triple response in etiolated seedlings due to the increase in ethylene biosynthesis, implying that conjugation of rub to cul3 is required for the activation of eto1 containing cul3 e3 ligase complex (bostick et al., 2004). analysis of the rce1 mutant also demonstrated that the modification of rub is required for regulating ethylene biosynthesis (larsen and cancel, 2004). rce1 encodes a rub conjugating enzyme, and has been shown to conjugate rub to the scf complex to modify the activity of the complex. interestingly, the basis of ethylene overproduction phenotype of rce1 is not due to enhanced acs activity, but due to an increase of aco activity. aco is generally not a rate-limiting step in ethylene biosynthesis during vegetative arabidopsis tissues; however, ethylene biosynthesis in r. palustris is limited by aco activity during submergence. elevated ethylene levels have been shown to increase aco activity, which could explain the increased aco activity in rce1 (vriezen et al., 1999). xbat32, a ring domain-containing ankyrin repeat subfamily of e3 ligases, also plays a role in ethylene biosynthesis by controlling the turnover of acs4 (type-2) and acs7 (type-3) (lyzenga et al., 2012). xbat32 was previously identified as a positive regulator of lateral root development and the xbat32 mutant produces significantly less ethylene than wild type (nodzon et al., 2004; prasad et al., 2010). xbat32 interacts with the acs proteins and it catalyzes the attachment of ubiquitin to the acs proteins (prasad et al., 2010), suggesting xbat32 negatively regulates the ethylene biosynthesis by regulating the stability of acs proteins. phosphorylation is one of the most abundant post-translational modifications which affect many important aspects of protein function, including activity, stability, subcellular localization and protein-protein interaction (holt et al., 2009). mounting evidence suggests that ethylene biosynthesis is regulated by phosphorylation events which likely influence acs protein turnover. studies from the application of kinase and phosphatase inhibitors to tomato suspension cell cultures and tissues indicated that phosphorylation regulates the activity and/or turnover of acs (kamiyoshihara et al., 2010). a cdpk present in the extracts of wounded tomato fruits phosphorylates leacs2 (mayfield et al., 2007). the extract containing cdpk activity phosphorylates the c-terminal domain of leacs2 in vitro, but the activity of the leacs2 does not show a significant increase, suggesting phosphorylation regulates the turnover of acs rather than affecting the activity. the c-terminus of leacs2 contains a consensus phosphorylation target site for a cdpk, and this cdpk recognition site is present in a subset of acs isoforms (fig. 2) (kamiyoshihara et al., 2010). unlike the target sites of mapk in the c-terminal domain of the type-1 acs proteins, phosphorylation of the cdpk target site, which lies immediately upstream from the target site for mapk has not been shown to be phosphorylated in vivo and in vitro. among three types of acs, protein stability of the type-1 and genetic and biochemical studies of a mapk pathway have revealed that pathogen-activated arabidopsis mpk6 phosphorylates the type-1 acs2 and acs6, which leads to increased accumulation of these acs proteins and, hence, increases ethylene production (joo et al., 2008; liu and zhang, 2004). mpk6 phosphorylates 3 serine residues residing within the consensus mapk target site in the c-terminus in vitro, suggesting mpk6-mediated phosphorylation of acs2 and acs6 prevents their degradation, resulting in an increase in ethylene biosynthesis in response to pathogen attack. 2014) report a similar result that rice mpk3 and 6 are involved in ethylene production via the salt-intolerance 1 receptor-like kinase (sit1), but acs stability was not discussed in their work. until recently, the effect of phosphorylation on type-2 acs protein stability was not clear; neither direct phosphorylation nor responsible kinase has been identified. however, a recent study demonstrated that a casein kinase isoform 1.8 (ck 1.8) phosphorylates the type-2 acs5 protein, which in turn promotes the interaction between eto1 and acs5, resulting in the degradation of acs5 protein (tan and xue, 2014). the ck1.8 mutant displays the constitutive triple response due to overproduction of ethylene similar to the eto mutant. interestingly, the triple response of ck1.8 seedlings is only observed in the hypocotyl and hook, not in the roots, implying that ck1.8 affects specific aspects of ethylene-mediated seedling growth responses. several recent studies have identified novel regulatory factors which target multiple acs isoforms belonging in different types of acs (fig. 3). this regulatory feature is distinct from that of previous known regulatory proteins with a type-specific targeting (e.g. eto1/eol e3 ligase for type-2 and mapk3/6 for type-1 acs). 14-3-3 proteins, novel regulator of ethylene biosynthesis, target all three types of acs (fig. 14-3-3 proteins are a family of evolutionarily well-conserved dimer proteins that specifically interact with phosphoproteins and are involved in a diverse array of physiological processes (darling et al., 2005; dougherty and morrison, 2004; freeman and morrison, 2011). upon interaction with target proteins, 14-3-3 proteins change their localization, stability, and activity, resulting in changes in physiological responses (freeman and morrison, 2011). there are 13 functional 14-3-3 genes in arabidopsis and their encoded proteins possess a highly conserved target binding domain, which can recognize a short stretch of peptide containing phosphoserine or phosphothreonin on target proteins (aitken et al., 1992; de boer et al., 2013; denison et al., 2011 this could allow different 14-3-3 isoforms to function redundantly by recognizing similar sets of target proteins. however, increasing results suggest that a defined subset of 14-3-3 isoforms display specific functions, such as the regulation of stomatal opening, flowering time, and phytochrome signaling in arabidopsis (mayfield et al., 2007; paul et al. it is unclear whether this is a result of biochemical specificity or simply differences in expression patterns of 14-3-3 isoforms in plants. 14-3-3 interacts with all three types of acs proteins via a non-c-terminal domain of the proteins and there is no specificity in the interaction between 14-3-3 isoforms and acs proteins in bimolecular fluorescence complementation assay (yoon and kieber, 2013a). 14-3-3 stabilizes acs protein by direct interaction and by negatively regulating the stability of the e3 ligases, eto1/eols, which specifically target the type-2 acs proteins for degradation (yoon and kieber, 2013a). studies from mammalian and yeast systems have suggested that the stability of f-box proteins which promote ubiquitination in the ubiquitinproteasome pathway, is regulated based on the availability of substrates through an autocatalytic process (ho et al. it is possible that 14-3-3 proteins preferentially interact with type-2 acs proteins, which in turn leads to the depletion of the acs proteins for the eto1/eols, thus regulating the turnover of both sets of proteins. alternatively, the interaction with dimeric 14-3-3 proteins may cause the eto1/eols to dimerize, thereby promoting self-ubiquitination and subsequent degradation. finally, the interaction with 14-3-3 proteins could enhance the interaction with distinct e3 ligases, such as xbat32, leading to the ubiquitination and subsequent degradation of the eto1/eols. intriguingly, in mammalian cells, 14-3-3 interacts with and regulates the protein stability of a short-lived p53 tumor suppressor protein and its cognate e3 ligases cop1 and mdm2 (su et al., 2011; yang et al., 2007). 14-3-3 stabilizes p53 by down-regulation of mdm2 and cop1 protein stability. this 14-3-3-mediated inverse stability regulation on p53 and mdm2 and cop shows a similar regulatory mechanism by which arabidopsis 14-3-3 control the protein stability of acs5 and eto1/eols, suggesting that the function of a subset of 14-3-3 isoforms in protein stability regulation is evolutionarily conserved between mammalian and plants. several findings imply that 14-3-3 also regulates acs stability independently of eto1/eols (yoon and kieber, 2013a; 2013b). first, 14-3-3 interacts with acs5, a type-2 acs with a lack of toe motif for eto1/eol interaction. secondly, 14-3-3 directly interacts and stabilizes the sole type-3 acs7 and type-1 acs2, whose protein stability is not regulated by the eto1/eols. thus, there is at least one other system acting to degrade type-2 acs proteins in addition to the eto1/eols, but in the manner dependent on 14-3-3 function. this is consistent with the observation that cytokinin and brassinosteroid increase type-2 acs function partly through a toe-independent mechanism (hansen et al., 2009). while 14-3-3 positively regulates acs protein stability (yoon and kieber, 2013a; 2013b), studies from characterization of the rare cold inducible 1a (rci1a), which encodes a 14-3-3 isoform suggest that 14-3-3 negatively regulate the protein stability of acs in response to cold stress (catala et al., 2014). rci1a mutant displays increased levels of acs6 protein in response to cold treatment, and this change is not due to the changes of the acs6 mrna levels. however, the direct effect of 14-3-3 on acs protein stability was not demonstrated in the study. suggest that the discrepancy of two studies might be contributed to the functional specificities of 14-3-3 isoforms; yoon and kieber (2013) used 14-3-3, while catala et al. (2014) used 14-3-3 isoform (bornke, 2005; fu et al., 2000; a recent study also reported that single 14-3-3 isoform could have distinct functions depending on the binding sites on a given target protein (ganguly et al., 2005). identification and characterization of the role of ck1.8 have also brought new insights into the post-translational regulation in ethylene biosynthesis (tan and xue, 2014). ck1.8 is a conserved serine/threonine protein kinase that plays role in various physiological processes, including blue light signaling, flowering, microtubule organization and brassinosteroid signaling in rice (ben-nissan et al., 2008; dai and xue, 2010; liu et al., 2003; tan et al., 2013). as briefly discussed in the previous section, the ck1.8 mutant overproduces ethylene resulting from accumulation of acs proteins, suggesting ck1.8 is a negative regulator of acs protein stability. ck1.8 phosphorylates acs5 at threonine residue 463 (t463) which is located within the toe motif, and phosphorylation on this site promotes the interaction with eto1, indicating the phosphorylation of t463 on acs5 plays a negative role in acs5 protein stability. it can be found in only in a subset of arabidopsis type-2 acs (acs5 and acs9), type-1 (acs6), and tomato type-1 (leacs2), suggesting that ck1.8 could target different acs types rather than committing to regulate a specific type of acs (fig. the role of ck1.8 is somewhat in contrast to what has been observed from other studies indicating that phosphorylation promotes acs protein stability. the roots curl in 1-n-naphthylphthalamic acid 1 (rcn1) gene encodes one of three regulatory/scaffolding a subunits of arabidopsis pp2a, and targets the type-1 protein acs2 and acs6 for regulating their stability (skottke et al., 2011). genetic studies revealed that the function of rcn1 requires acs2 and acs6 and the rcn1 mutant exhibits increased accumulation of the acs6, suggesting phosphorylation promotes the protein stability of type-1 acs. strikingly, rcn1 shows different effects on type-2 acs5 protein stability. in etiolated rcn1 seedlings, the accumulation of myc-tagged acs5 is significantly reduced, whereas accumulation and turnover of the myc-acs5 is not affected, indicating that rcn1 plays a positive role in acs5 stability through the toe motif of acs5. however, rcn1-directed dephosphorylation on acs5 has not been evidenced, suggesting the possibility that rcn1 may dephosphorylate the eto1 complex. both rcn1 and ck1.8 regulate the stability of acs5 through the toe domain, but it is not clear whether the effects of rcn1 on acs5 are dependent on ck1.8 or eto1/ eols. due to the lack of regulatory motifs in the c-terminal domain, including phosphorylation sites, it was considered that acs7, the sole type-3 acs, may not be subjected to proteasome-mediated degradation pathway, and that it may be more stable than other acs proteins (chae and kieber, 2005). however, lyzenga et al., recently showed that the protein stability of acs7 is also governed by the ubiquitin-mediated proteasomal degradation, and that degradation requires the ring-type e3 ligase xbat32 (lyzenga et al. interestingly, xbat32 also confers protein instability to the type-2 acs4, suggesting xbat32-mediated degradation mechanism is not specific for the type-3 acs. a cell-free degradation assay shows that changes in 4 lysine residues in the c-terminal domain of acs4 results in accelerating degradation of acs4 protein. this result is similar to the observation that k435r in the c-terminus of flag-acs7 promotes the turnover rate of the acs7, suggesting the c-terminal lysine residues are not for ubiquitination, but for stabilization of acs4 and acs7. shortly after, xiong et al. they showed that destabilization sequences of the acs7 are located in the n-terminus of acs7. the n-terminal 54 residues of the acs7 confer significant instability to acs7-gus and first 14 amino acids are responsible for negative regulation of the acs7 protein stability (xiong et al., 2014). one possible explanation for this may be due to the nature of the c-terminal fusion of the acs7-gus used in the study. traditionally, the n-terminal fusion of acs has been routinely utilized for studying the turnover of acs to avoid masking the c-terminal regulatory domain and this may be blamed for concealing the destabilization signals located at the n-termini and making only the c-terminal signals available to degradation machinery. it is interesting to further study the role of the n-terminal domain of other types of acs whether they also contain putative degradation sequences in their n-termini. several studies indicate that there are molecular components acing on the non-c-terminal domain of acs proteins to regulate their stability. cytokinin and brassinosteroid stabilize acs5 and acs9 and the effects of these two hormones on the protein stability are additive, suggesting cytokinin and brassinosteroid act through distinct toe-independent mechanisms on these acs proteins (hansen et al., 2009). genetic studies showed that cytokinin-mediated acs stabilization requires a functional cytokinin signaling pathway (hansen et al., 2009). mutation in the signaling components, cytokinin receptors, ahps, and type-a and type-b transcription factors, in the cytokinin signaling pathway produce reduced amounts of ethylene in response to exogenous cytokinin. the effect of brassinosteroid in ethylene biosynthesis is somewhat distinguished from the typical triple response that has been observed with cytokinin treatment; br treatment results in a shortened and thickened hypocotyl formation; but it does not induce an exaggerated hook formation; and shortening of the root and hypocotyl is less severe than for cytokinin-treated seedlings. interestingly, the ethylene-insensitive mutant ein2-5 still shows cytokinin and br induced hypocotyl phenotypes, although the extent is not as severe as in wild type, indicating this process is independent of the ethylene signaling pathway. although it has not been demonstrated whether gibberellin regulates the turnover of acs proteins, studies from the characterization of a gai;eto2 (gibberellin insensitive;ethylene overproducing) double mutant showed that ga signaling is required for acs stabilization via the toe-independent manner, as the overproduction phenotype of eto2 is abolished in the gai;eto2 double mutant (de grauwe et al. furthermore, 14-3-3-mediated eto1/eol-independent stabilization of acs proteins also indicates the existence of an alternative mechanism to stabilize acs proteins (yoon and kieber, 2013a). it is not clear whether the eto1/eol-independent mechanism acts in the same pathway that is utilized by the other factors, but is it of great interest to further study to identify molecular elements involving in these regulatory pathways. together, these studies indicate that there are molecular components that act as the points of cross-talk between ethylene biosynthesis and other hormonal signaling pathways. identification of these elements will bring new insights into understanding the mechanism by which protein turnover of acs is regulated to coordinate and merge different hormonal inputs to regulate ethylene production which effects on many diverse ranges of plant growth and development.

biosynthesis of the phytohormone ethylene is under tight regulation to satisfy the need for appropriate levels of ethylene in plants in response to exogenous and endogenous stimuli. the enzyme 1-aminocyclopropane-1-carboxylic acid synthase (acs), which catalyzes the rate-limiting step of ethylene biosynthesis, plays a central role to regulate ethylene production through changes in acs gene expression levels and the activity of the enzyme. together with molecular genetic studies suggesting the roles of post-translational modification of the acs, newly emerging evidence strongly suggests that the regulation of acs protein stability is an alternative mechanism that controls ethylene production, in addition to the transcriptional regulation of acs genes. in this review, recent new insight into the regulation of acs protein turnover is highlighted, with a special focus on the roles of phosphorylation, ubiquitination, and novel components that regulate the turnover of acs proteins. the prospect of cross-talk between ethylene biosynthesis and other signaling pathways to control turnover of the acs protein is also considered.

PMC4507024

pubmed-696

the lung is an organ that performs a multitude of vital functions every second of our lives. this fact leads to considering lung abnormalities, life-sustained diseases that have high priority in detection, diagnosis, and treatment if possible. our focus in this paper will be on two popular abnormalities within the lung, which are pulmonary edema and lung tumor. pulmonary edema (water in the lungs) is caused by fluid building up in the air sacs of the lungs [1, 2]. on the other hand, lung cancer/tumor is a disease where uncontrolled cell growth in tissues of the lung occurred. computer-aided diagnosis (cad) schemes for thoracic computed tomography (ct) are widely used to characterize, quantify, and detect numerous lung abnormalities, such as pulmonary edema and lung cancer [4, 5]. an accurate lung segmentation method is always a critical first step in these cad schemes and can significantly improve the performance level of these schemes. although manual or semiautomatic lung segmentation methods for ct images were used in some early cad schemes [610], they are impractical for current cad schemes because multidetector ct (mdct) scanners can generate hundreds of ct slices for a patient. an automated method for lung segmentation in addition, the eye identification/detection of the abnormality type (pulmonary edema or tumors) in computed tomography (ct) images is very difficult even for the experienced clinicians because of its variable shape along with low contrast and high noise associated with it. as the final stage of treating the lung cancer is surgical removal of the diseased lung, hence it is necessary to identify the cancer location, which can be useful before they plan for the surgery. the aim of our work is to develop an automated novel texture analysis based method for the segmentation of the lungs and the detection of the abnormalities, whether pulmonary edema or lung tumor. haralick's features based on the gray level cooccurrence matrix (glcm) are applied to capture textural patterns in lung images. the objective of this work is the selection of the most discriminating and finding out the significant texture features that can differentiate between these two types of abnormalities, in comparison to normal. these measurements are utilized to describe the overall texture of the image using measures such as entropy and sum of variance. propose an approach, based on haralick's features, to detect and classify colon cancer cells. a study to investigate the feasibility of using haralick features to discriminate between cancer and normal subimages within a patient is illustrated in. in this paper, ct images are first preprocessed for noise reduction and image enhancement, followed by segmentation techniques, as the tools to segment the lungs, and finally haralick texture features [1315] are calculated. statistical analysis is done to detect the most significant haralick features that will characterize the type of the abnormality within the lungs. despite the low contrast and high noise existence in the images, the proposed algorithms introduce promising results in detecting the abnormality of lungs in most of the patients in comparison with the normal. this paper presents a new automatic lung cancer detection system based on haralick texture features extracted from the slice of dicom lung ct images. the proposed system is accomplished in four stages: image preprocessing, lung image segmentation, feature extraction, and classification. statistical analysis is used to obtain the best features for classification to differentiate between lung cancer patients, ordered edema patients, and control subjects. patients with either a lung cancer tumor or pulmonary edema were encompassed in the study. this study included two datasets, the first dataset referred to the radiology department at new elkasr elainy teaching hospital, university of cairo. the other dataset was obtained from the cancer imaging archive (tcia) sponsored by the spie, nci/nih, aapm, and the university of chicago. ct image, we separate the left lung from the right lung automatically, and each separated lung is labeled as normal or edema/cancer based on the dataset information. the main goal of preprocessing is to improve the quality of an image as well as make it in a form suited for further processing by human or machine. this is accomplished by enhancing the visual appearance of an image besides removing the irrelevant noise and unwanted parts in the background. the proposed enhancement process, which is based on combining filters and noise reduction techniques for pre- and postprocessing as well, is carried out applying histogram equalization (he) [1820] followed by wiener filtering [21, 22]. figure 1 presents the enhancement in the lung image contrast attained by applying the histogram equalization. however, the obtained gray scale image contains noises such as white noise and salt and pepper noise. thus, wiener filter is utilized to remove these noises from the enhanced lung image. figure 1(c) shows the effect of applying weiner filter on the contrast enhanced lung image. lung segmentation step aims to basically extract the voxels corresponding to the lung cavity in the axial ct scan slices from the surrounding lung anatomy. this technique is based on the fact that there is a large density difference between air-filled lung tissues and surrounding tissues. furthermore, both lungs are almost looking like mirror images of themselves in a human body. the segmentation of lung regions is achieved through the following steps. in the first step, the preprocessed ct image is converted into a binary image; a threshold of 128 was selected. values greater than the threshold are mapped to white, while others less than that are marked as black. consequently, the two lungs are marked and the area around them is cropped out. second, an erosion morphological operation is employed in order to eliminate any white pixels within the two lungs. afterward, black pixels for each region in the eroded image are counted; the region with the largest black area will be deemed as a lung mask. the attained lung mask is reflected in the opposite direction. as a result these masks are multiplied with the corresponding original image regions; this will project the lung masks on the original two lungs images. finally, update each black pixel in the obtained images by its original value; other pixels are set to 255. figure 2 illustrates the lung extraction process. feature extraction is the process of obtaining higher-level information of an image such as color, shape, and texture. statistical texture methods analyze the spatial distribution of gray values, by computing local features at each point in the image and inferring a set of statistics from the distributions of the local features. this technique has been widely used in image analysis applications, especially in the biomedical field. the glcm is computed in the first step, while the texture features based on the glcm are calculated in the second step. glcm shows how often each gray level occurs at a pixel located at a fixed geometric position relative to each other pixel, as a function of the gray level. the horizontal direction 00 with a range of 1 (nearest neighbor) was used in this work. the 9 texture descriptions used are presented in (4) to (13), where ng is the number of gray levels, pd is the normalized symmetric glcm of dimension ng ng, and pd(i, j) is the (i, j)th element of the normalized glcm. contrast (moment 2 or standard deviation) is a measure of intensity or gray level variations between the reference pixel and its neighbor. large contrast reflects large intensity differences in glcm:(1)contrast=ijij2pdi, j.homogeneity measures how close the distribution of elements in the glcm is to the diagonal of glcm. as homogeneity increases, the contrast, typically, decreases:(2)homogeneity=ij11+ij2pdi the value of entropy is the largest when all elements of the cooccurrence matrix are the same and small when elements are unequal:(3)entropy=ijpdi, jlnpdi, j.energy is derived from the angular second moment (asm). the asm measures the local uniformity of the gray levels. when pixels are very similar, the asm value will be large. consider(4)energy=asmasm=ijpd2i, j.correlation feature shows the linear dependency of gray level values in the cooccurrence matrix: (5)correlation=ijpdi, jixjyxy, where x; y and x; y are the means and standard deviations and are expressed as(6)x=ijipdi, jy=ijjpdi, jx= ijix2pdi, jy=ijjy2pdi, j.the moments are the statistical expectation of certain power functions of a random variable and are characterized as follows. moment 1 (m1) is the mean which is the average of pixel values in an image and it is represented as(7)m1=ijijpdi, j.moment 2 (m2) is the standard deviation that can be denoted as (8)m2=ijij2pdi, j.moment 3 (m3) measures the degree of asymmetry in the distribution and it is defined as (9)m3=ijij3pdi, j.and finally moment 4 (m4) measures the relative peak or flatness of a distribution and is also known as kurtosis:(10)m4=ijij4pdi, j.furthermore, difference statistics that are a subset of the cooccurrence matrix are also used. these features are based on the distribution of probability pxy(k) which is defined as follows:(11)pxyk=cdi, j, k=0,1, ,ng1,where cd(i, j) is the (i, j)th element of the glcm. the most basic difference statistic texture descriptions are the asm, mean, and entropy:(12)asm=kpxyk2.when the pxy(k) values are very similar or close, asm is small. asm is large when certain values are high and others are low:(13)mean=kkpxyk.when pxy(k) values are concentrated near the origin, mean is small and mean is large when they are far from the origin:(14)entropy=kpxyklogpxyk.entropy is smallest when pxy(k) values are unequal and largest when pxy(k) values are equal. the calculation of the haralick texture features using the previous equations for the ct images volume sequences for every segmented lung (right and left) separately was performed. for each participant the gray level cooccurrence texture features: contrast, homogeneity, entropy, energy, correlation, and m1, m2, m3, and m4 accompanied by the difference statistical features: asm, contrast, mean, and entropy were obtained for each segmented lung (right and left). for the purpose of random lung assignment in healthy volunteers, the left lung represented the diseased lung in the same percentage of cases as the patient population. for the acute data, two single factor analyses of variance (anova) tests were conducted for each haralick texture feature measurement between affected (either left or right) and fellow lung (either left or right) for both categories cancer and edema patients. a single factor analysis of variance (anova) other between-subject single factor analyses were conducted to find out the significant haralick features that could differentiate cancer from edema., we separate the left lung from the right lung automatically as discussed before in section 2.3, and each separated lung is labeled as normal or edema/cancer based on the dataset information. the haralick texture features measurements for each lung separately are calculated (the gray level cooccurrence texture features: contrast, homogeneity, entropy, energy, correlation, and moments along with the difference statistical features: asm, mean, and entropy). the mean and the standard deviation of the haralick texture features measurements calculated as well as the anova results are given for tumor patients affected lung versus fellow lung in table 1 and for pulmonary edema patients in table 2. the anova summary of statistics for either pulmonary edema or tumor patients versus normal is given in table 3. the significant haralick texture features that can differentiate between pulmonary edema and tumor are found in table 4. from table 1, we can conclude that haralick texture features measurements (homogeneity, energy, correlation, and entropy) of the affected cancer lung were significantly different than that of the fellow lung. the homogeneity, energy, and correlation were significantly less than those of the normal fellow lung. while entropy of the cancerous lung is approaching being significantly more than that of the fellow lung, moment 3 and the difference statistical feature asm (diff_asm) texture feature measurement of the cancerous lung is approaching being significantly less than that of the normal lung. table 2 showed that haralick texture features measurements (homogeneity, entropy, and moments calculated from the cooccurrence matrix as well as mean and asm computed from the difference statistics) of the pulmonary edema affected lung were also significantly different than those of the control subject lung; moreover contrast and entropy computed from the difference statistics were significantly more than those of the fellow lung. considering tables 1 and 2, we can conclude from table 3 that the homogeneity, energy, entropy, m3, m4, diff_asm, diff_mean, and diff_entropy are good biomarkers to significantly differentiate between diseased and normal lungs without any disease specification. on the other hand, the results illustrated in tables 1, 2, and 4 show that entropy and the entropy calculated from the difference statistics would be a good candidate to significantly differentiate between pulmonary edema and cancer. the texture features analyses are well known approaches to quantify and express the heterogeneity that may not be appreciated by clinical naked eyes, and it was presented before as good imaging biomarkers to differentiate between diseases. in this paper an evaluation of the haralick texture features is done in order to identify the most significant features that can be used in order to detect and differentiate abnormalities within the lungs for cancer and edema versus normal. our results indicate that entropy determined by gray level cooccurrence matrix and asm is significantly different in edema patients versus normal while it is not in cancer patients versus normal. since the entropy is the degree of randomness or the degree of disorder in the image, and the angular second moment represents the uniformity in the image, this may be interpreted as the cancer disease causing a localized heterogeneity in the diseased specified area in the lung while the edema causes heterogeneous disorder in the whole lung image. high entropy values calculated implies that the elevated level of disorder and disorganization occurred due to the edema diseased lung versus the cancer diseased lung. the energy feature that is derived from the angular second moment measures and representing the local uniformity of the gray levels is a good biomarker to differentiate between cancer and edema diseases. from table 2, contrast is a good biomarker for the pulmonary edema disease and this agrees with the texture feature meaning which means high contrast values for heavy texture changes. gray level cooccurrence matrix textural properties such as homogeneity, correlation, mean, and moments are good significant biomarkers for diseased lung versus normal ones in general without any specification for the disease type. our results agree with other articles indicating that textural analysis has the potential to develop into a valuable clinical tool that improves the diagnosis, tumor staging, and therapy assessment. while our results are promising, there is still further work that can be done in the detecting of the abnormality within the lungs to detect the type of that abnormality whether it will be a lung cancer or edema. a preliminary investigation has been done using statistical analysis to identify the most useful texture features that can be fed to any classification technique later. this statistical analysis is done using anova. after selecting these features we can feed them for better localization and classification as further work.

the haralick texture features are a well-known mathematical method to detect the lung abnormalities and give the opportunity to the physician to localize the abnormality tissue type, either lung tumor or pulmonary edema. in this paper, statistical evaluation of the different features will represent the reported performance of the proposed method. thirty-seven patients ct datasets with either lung tumor or pulmonary edema were included in this study. the ct images are first preprocessed for noise reduction and image enhancement, followed by segmentation techniques to segment the lungs, and finally haralick texture features to detect the type of the abnormality within the lungs. in spite of the presence of low contrast and high noise in images, the proposed algorithms introduce promising results in detecting the abnormality of lungs in most of the patients in comparison with the normal and suggest that some of the features are significantly recommended than others.

PMC4617884

pubmed-697

precise control of the adsorption of proteins on solid surfaces is a key to a wide variety of biological and technological applications. proteins are commonly immobilized on surfaces both in microarrays and other studies of protein function and in the creation of biosensors and biocatalysts. the success of these applications depends on proteins maintaining their native state and function when adsorbed to the surface and on the prevention of nonspecific protein binding. protein adsorption also plays an important role in the outcome of biomaterials (e.g., biomedical implants, artificial tissue scaffolds, and nanoparticles for drug delivery) in vivo, as proper protein adsorption contributes to cell adhesion and the integration of the biomaterial with the circulatory system, while the adsorption of undesired proteins can contribute to failure due to immune responses or fouling. surface interactions are also relevant to understanding many diseases, as they are the first step in many biological processes, including blood clotting and the formation of protein aggregates, such as the amyloid plaques found in alzheimer s disease. because of the importance of protein adsorption in these many applications, investigation of the interplay between the folding and adsorption processes and how adsorption impacts protein conformations is highly valuable. experiments, including a variety of spectroscopic methods, have been able to show that surface adsorption can result in changes in the conformation and thermodynamic stability of a protein and that these changes are dependent on a variety of factors, such as temperature, ph, protein concentration, and the hydrophobicities of the protein and surface. however, experiments have not been able to fully address many aspects of the relationship between protein adsorption and conformational changes and are complicated by the complex heterogeneity of interactions between real protein chains and surfaces. therefore, theoretical and computational efforts that often utilize simplified, coarse-grained protein models have been used to supplement experiments and provide a basic understanding of protein adsorption. one minimalist model used to investigate protein folding and adsorption is the hydrophobic-polar (hp) model, in which protein monomers are modeled on a lattice as either hydrophobic (h) or polar (p) beads. within the hp model, the many thermodynamic factors underlying complex processes, such as protein folding and adsorption, are reduced to a few basic terms (i.e., enthalpic interactions between chain segments or between the chain and surface and entropically excluded volume interactions). one route through which the hp model can be used to understand protein adsorption and/or folding is the study of two transitions, the coil globule and the critical adsorption transitions, that are the result of a balance between these thermodynamic terms. the coil globule or collapse transition is one of the first steps in the protein folding process and occurs when attractive interactions between hydrophobic protein monomers become strong enough to balance the conformational entropy lost by the protein adopting compact globule conformations. a recent experiment using synthetic polymers containing hydrophobic and polar monomers, mimicking hp model chains, confirmed that attractions between hydrophobic monomers are sufficient to be the driving force of the collapse transition. the critical adsorption point (cap) on the other hand marks the transition of a protein which prefers being in solution to being adsorbed on the surface and also involves a balance between entropic and enthalpic effects. the cap is the point at which a polymer just becomes adsorbed to a surface and occurs when the conformational entropy lost by a polymer chain near a surface is offset by attractive interactions with the surface. thus, the thermodynamics and potential conformational changes of the process through which a folded protein adsorbs on a surface, for example, can be understood in terms of these two transitions, as some hydrophobic interactions underlying the collapsed conformation of the protein can unravel to allow for additional chain surface attractive interactions. over the last quarter century, the hp and other simple coarse-grained models have been successfully used to provide insight into the conformational changes of proteins and other macromolecules during adsorption both in terms of these transitions and in a variety of other ways. first, the adsorption of hp-like chains with various sequence types on surfaces with various patterns has been studied to understand pattern recognition. these studies have revealed that the adsorption of copolymers on heterogeneous surfaces can proceed via an initial nonspecific adsorption similar to the critical adsorption transition, followed by a reorganization in which the surface pattern is recognized by the copolymer, and that such a two-stage adsorption process depends on the chain sequence, surface pattern, and interaction parameters. additionally, moghaddam and chan investigated the adsorption of block copolymers on patterned surfaces and showed that the sharpness of the adsorption transition was enhanced through the introduction of additional either attractive or repulsive chain surface interactions. however, these studies did not include intrachain interactions and, therefore, could not consider the balance between intrachain and chain surface interactions that underlies protein adsorption. second, the adsorption of a homopolymer with intrachain interactions (i.e., basically, a chain consisting of only the h beads of an hp chain) has been considered, and it was shown that the presence of the surface promoted chain collapse and increased the internal structural organization (e.g., helices and antiparallel sheets) in the chain. also, chains with strong intrachain interactions were shown to undergo two types of adsorption transitions: a docking transition, in which a collapsed chain does not deform upon adsorption, for weak chain surface attractions and a flattening transition, in which the chain adopts two-dimensional conformations after adsorption for strong chain surface attractions. finally, several studies have examined the adsorption of hp chains directly. rybicka and sikorski compared the adsorption of several hp sequences with that of a homopolymer containing only h-type beads on a homogeneous surface. they showed that the collapse of chains weakly adsorbed on a surface was roughly independent of the chain sequence; however, under strong adsorption, chains underwent a sequence-dependent rearrangement similar to the previously discussed flattening transition. studies of hp chains interacting with surfaces have also confirmed the experimental observation that the presence of the surface can significantly alter the lowest energy conformation of a folded protein and have been used to determine conformational pseudophase diagrams of hp chains near a surface that shows how temperature and the strength of attractive interactions impact adsorption and folding. in this work, we seek to increase understanding of the relationship between protein adsorption and conformation change through a systematic examination of the interplay between the collapse and critical adsorption transitions. specifically, we study the protein folding and adsorption processes by determining the temperatures of collapse, tc, and critical adsorption, tcap, transitions for hp chains end-grafted to a solid surface that equally attracts h and p monomers. in contrast with most previous simulations of the adsorption of hp chains that have used a specific short sequence with a well-defined ground state, we use a simple alternating hp sequence and vary the chain length from 10 to 400, allowing us to investigate the potential influence of chain length on collapse and adsorption. the current study also investigates the behavior of the chain at more intrachain attraction strengths and over a wider temperature range than was considered in previous studies of the relationship between chain collapse and surface adsorption. we show that, while the critical adsorption point can always be observed and is roughly independent of chain length, tcap is affected by the presence of intrachain attractions in some cases. specifically, if tc>tcap (i.e., the chain is already collapsed when the adsorption transition is attempted), the critical adsorption transition occurs at a higher temperature than a corresponding homopolymer without intrachain attractions, indicating that collapsed chains are more easily adsorbed. in contrast, if tc<tcap (i.e., the chain is already adsorbed when the collapse transition is attempted), tc is suppressed and it is more difficult to collapse the adsorbed chain than a corresponding chain that is free in solution. finally, we examine how the strength of the hydrophobic intrachain and chain surface attractions impact chain conformations. our simulation system is embedded in the three-dimensional (3d) simple cubic (sc) lattice. a self-avoiding walk (saw) other sequences representing different protein types could have been used; however, since the current study focuses on the interplay between the two transitions, we focus on a generic alternating hp chain that can be easily extended to long chains. additionally, we note that both the collapse and adsorption of an alternating hp sequence have been shown to differ from a homopolymer with intrachain attractive interactions. the protein of length n is composed of n/2 h monomers and n/2 p monomers. bond lengths between monomers fluctuate among 1, 2, and 3 lattice constants. the bond can be taken from 26 allowed bond vectors obtained from the set {(1,0,0), (1,1,0), (1,1,1)} by symmetry operations of the sc lattice. however, bond crossing is not allowed. in this coarse-grained model, the monomers do not correspond to specific atoms in a polymer but rather to small groups of atoms, and the bonds do not represent specific covalent bonds between two atoms but, instead, the linkages between monomers. the simulation box is a cuboid with sizes lx, ly, and lz in the x, y, and z directions, respectively periodic boundary conditions are employed in the x and y directions, while the z direction is confined by an infinitely large flat surface located at z=0. the surface is impenetrable to the polymer, so polymer monomers are restricted to lie in the upper half space (z>0). polymer chain lengths studied are in the range of n=10 to 400. the simulation box is always large enough to ensure no finite size effects on the simulation results. to this end, the dimensions of the simulation box in all three directions are always larger than the chain length n. the first monomer, which is always an h, is considered to be adsorbed to the impenetrable surface and is grafted at the center of the z=1 layer. then the chain is subjected to brownian motion achieved by the dynamic monte carlo (mc) technique. polymer monomers that are located on the z=1 layer are considered to be adsorbed to the surface. surface interaction is assigned for all monomers on the z=1 layer next to the surface. a two-dimensional (2d) sketch of our 3d simulation system is presented in figure 1. 2d sketch of our 3d simulation model for an end-grafted hp protein model chain. monomers are numbered from 1 to n for a polymer with length n. the first monomer h is grafted to the surface. the nearest neighbor interactions are ehh, ehp, epp, ehs, and eps, as shown. the energy of a conformation is a summation of all nearest-neighbor (nn) contact interactions among the chain and all nearest-neighbor contact interactions between the chain and surface. rij is the spatial distance between two nonbonded monomers i and j and zi is the distance of monomer i away from the surface. it is known that the adsorption of a copolymer chain is influenced by the properties of the surface. here we set ehp=epp=0, while the value of ehh is negative and varied. therefore, the energy of polymer can be expressed as2where nhh, nhs, and nps represent the nn contact numbers of h h, h surface, and p surface pairs, respectively. |ehs| is used as the unit of energy while |ehs|/kb is the unit of temperature, where kb is the boltzmann constant. h interaction ehh and temperature t. variation in ehh from zero to negative numbers will allow us to investigate the adsorption of the hp chain on the surface in the absence (ehh=0) or presence of the intrachain hydrophobic interaction. the brownian motion of the polymer chain is attributed to local moves of chain monomers. polymer dynamics is achieved by bond fluctuation, similar to that used for one-site and eight-site polymer models on the sc lattice. for each trial move, a monomer is chosen randomly to move to one of its six nn sites. if the chosen site is already occupied by another monomer, or such a move will violate bond crossing and bond length restriction, the trial move is abandoned. otherwise, the trial move will be accepted with a probability p=min[1, exp(e/kbt)], where e is the energy difference between new and old configurations. it has been pointed out that the metropolis method may have problems in describing the behavior of hp chains at low temperatures (kbt<0.3|ehh|) in comparison with the wang landau method. at these low temperatures, the sampling efficiency using the metropolis algorithm can be poor, as the polymer can become trapped in low-energy states. as we use the metropolis method in this work, we focus on polymer behavior near tcap and tc, which are higher than 0.3|ehh|. the results of the metropolis and wang landau methods are very similar for kbt> 0.3|ehh|, and ergodicity can be satisfied by a long simulation run using the metropolis algorithm. the time unit is one monte carlo step (mcs) during which n 1 trial moves are attempted since the first monomer is always adsorbed. to avoid correlation between two configurations, we measure the chain s statistical properties only after a regular time interval =n mcs. typically, each simulation is run as long as 1000, and 5000 independent runs are simulated. we also adopted an annealing process in simulations of chain configuration at different temperatures. simulations begin at a high temperature with the chain in a desorbed state and in a random coil configuration. the temperature decrement step is not a constant but is specially chosen in advance for clearly presenting the collapse and adsorption transitions and for saving calculation time simultaneously. to this end, we first roughly estimate the two transition temperatures using a simulation with a large temperature decrement step and then adopt a small temperature decrement step around the transition temperatures in a second simulation. at each temperature, the final configuration at the previous temperature was used as the initial configuration for the subsequent temperature. every independent simulation run ends at a low temperature far below the cap, where the chain is in a deeply adsorbed state. we at first determine the collapse transition of the alternating hp model chain in a dilute solution. the dependence of the mean square end-to-end distance r on the temperature t is presented in figure 2 for the case where ehh=1. the inset shows that the scaling r n at high temperature changes to r n at low temperature, indicating a collapse transition from a random coil to a compact sphere. r/n has the steepest decrease at t=0.75, that is, the temperature at which dr/dt is at a maximum. therefore, we identify a coil globule transition at tc=0.75 when ehh=1 for the hp chain in dilute solution. since the temperature t and ehh are interrelated through the boltzmann factor, a variation in ehh would shift tc according to tc=0.75|ehh| for the hp chain in dilute solution. here globule transition temperature of the hp chain in the dilute solution in the absence of any surface. dependence of mean square end-to-end distance r on temperature t for the free hp chain with ehh=1 in dilute solution. the inset presents the log log plot of r as a function of the chain length n at t=2, 1, and 0.5 (from top to bottom). the straight lines are the best fits with slopes 1.19, 1.12, and 0.65 for t=2, 1, and 0.5, respectively. next, we simulate the adsorption of the end-grafted hp chain with ehh=1 by annealing the chain with the head h monomer grafted on a flat surface and estimate the collapse transition and the critical adsorption transition temperatures. the critical adsorption transition temperature is estimated from the temperature dependence of the mean surface contact number m of the chain. m as a function of chain length at different temperatures is plotted in log log scales in figure 3. on the basis of the eisenriegler, kremer, and binder (ekb) scaling theory, the scaling relation m n is satisfied at the critical adsorption point tcap. meirovitch and livne have estimated that tcap=3.44 0.01 and the crossover exponent =0.530 0.007 for a saw chain with fixed bond length (b=1) on the sc lattice with mc simulations. the plot of m versus chain length n has a concave upward curve at temperatures below tcap and a convex downward curve at temperatures above tcap. for the present bond-fluctuation saw hp model, we estimate tcap=1.65 0.02 and =0.54 0.01. the results are close to that estimated for the adsorption of a bond-fluctuation saw homopolymer without intrachain attractive interactions, where tcap= 1.625 and =0.52 have been estimated by using a finite-size scaling formula m c)n)], indicating that hydrophobic interactions have little effect on tcap as long as the attractive interactions with the surface are the same for both h and p monomers. log log plot of the surface contact number m versus chain length n at temperatures t=1.55, 1.6, 1.65, 1.7, and 1.75 for an hp polymer with ehh=1. the statistical error of each monte carlo datum is smaller than the symbol size. dependence of mean square end-to-end distance r on temperature t for the end-grafted hp chain with ehh=1. chain lengths are n=50, 100, 200, and 400 from bottom to top. the vertical straight lines show the locations of tcap=1.65 and tc=0.5, respectively. the inset presents the heat capacity per monomer for the end-grafted hp chain with n=200 and n=400. globule transition temperature for an end-grafted chain is estimated from the temperature dependence of the mean square end-to-end distance r. figure 4 shows the dependence of r on t for the end-grafted hp polymer with ehh=1. at the critical adsorption temperature tcap= 1.65, we find that r tends to be a local minimum, which is in agreement with the results of adsorption of a homopolymer chain. however, unlike the adsorption of a homogeneous saw polymer on a surface where the r increases monotonically as the temperature is lowered below tcap due to the flattening of the chain on the surface, r for the end-grafted hp chain increases when t is lowered from tcap to about t=1 and then decreases afterward. the sharp decrease of r below t=1 is a result of the coil in contrast to the coil globule transition of free chains (figure 2), the plots of r/n versus t for different chain lengths of end-grafted chains do not cross, and we, therefore, can not use the crossing point to define the coil we can however still find the steepest decrease of r, which takes place at about t=0.5 and is roughly independent of the chain length. we also find a peak in the heat capacity at t=0.5 for the hp chain as shown in the inset of figure 4. we therefore identified this temperature as the collapse transition temperature of a surface-absorbed chain tc=0.5. this tc=0.5 of the end-grafted hp chain is lower than tc=0.75 for the free hp chain. we therefore conclude that the collapse transition of the hp polymer is suppressed by being adsorbed to the surface. when tc<tcap, the chain undergoes the adsorption transition before the collapse transition can occur. surface adsorption makes it more difficult for the chain to adopt conformations that provide a sufficient number of h h contacts for collapse to occur, reducing tc from its value in a bulk solution. figure 5 presents the number of h h contacts for the hp chains in dilute solution and end-grafted on the surface. above tcap of the end-grafted chain, nhh is small and the same for both chains. at temperature tc<t<tcap, nhh of the end-grafted hp chain is slightly larger than that of the free chain, indicating that the adsorption of the chain promotes the formation of h h pairs. below tc, however, we find that nhh of the end-grafted hp chain is significantly smaller, clearly indicating that surface adsorption prevents the collapse of chain at low temperatures. this reduces tc for the end-grafted hp chain. on the other hand, the critical adsorption of the polymer is not influenced by the collapse transition of the polymer if tc<tcap. from the heat capacity the temperature at the shoulder is consistent with the cap, determined from the location of the minimum of r, for the finite chain. dependence of the number of h h contacts, nhh, on the temperature t for hp chains in dilute solution and end-grafted on a surface. the length of the hp chain is n=400, and the h comparing data in figures 2 and 4 for t<tc, one can notice that r/n in figure 4 increases with n and is much bigger than that of a free hp chain in solution, as shown in figure 2. the reason is that the adsorbed chain adopts a roughly 2d conformation at t tcap, and the subsequent coil globule transition driven by the intrachain hydrophobic attraction now occurs within this 2d conformation. as has been previously shown for a homopolymer with intrachain attractions, the collapse of a chain in 2d takes place at a lower temperature than collapse in 3d, since a 2d chain conformation will have less pairwise attraction. as will be shown, we find that the adsorbed chain is anisotropic, since its asphericity parameter a is even bigger than that of an adsorbed hp chain without h h attraction. since tc tcap, the chain at tc is already trapped in the random coil state achieved at tcap; this would probably result in a more anisotropic conformation because the collapse would likely occur at higher density of h monomers. h attractions (ehh=2, 3, and 4), while the monomer surface attractions are fixed as ehs=eps=1. globule transition of the hp chain when free in solution to higher temperatures, following tc=0.75|ehh|. therefore, tc occurs at 1.5, 2.25, and 3 for ehh=2, 3, and 4, respectively. these conditions allow us to examine the interplay between the transitions when the collapse transition occurs at a higher temperature than the critical adsorption transition (ehh= 3 and 4) and when the transitions occur at approximately the same temperature (ehh=2). first, we determine the collapse transition of end-grafted chains when intrachain attractions are stronger than chain surface attractions (ehh<ehs=eps) and find that tc is not influenced by the presence of the surface. as shown in figure 6, a plot of r/n versus temperature for ehh=2 and 4 shows the same crossover point as expected for the transition temperature of free chains based on tc=0.75|ehh|. this behavior is different from the case where ehh=ehs=eps=1 shown in figure 4, where the chains with different lengths do not cross over with each other. although tc is not influenced by the surface, the conformational size of the chain r is influenced by the attractive surface, as can be observed through comparison of figures 2 and 6. we next examine the impact of increasing the strength of intrachain attractions on the critical adsorption transition. figure 7 presents the surface contact number as a function of chain length at different temperatures for ehh=4. a scaling relation m n is observed at tcap=2.55 with an exponent =0.34. we find that both tcap and are different from those of ehh=1. the results of tc, tcap, and for the hp chains with different intrachain interactions ehh are listed in table 1. the tcap for ehh=2, 3, and 4 is obviously affected by the collapse of chain when tc is close to or larger than tcap of the hp chain with weak h h interactions. the simulation results show that the presence of intrachain interaction shifts the tcap to a higher temperature. dependence of mean square end-to-end distance r on temperature t for the end-grafted hp chain with ehh=2 (black) and 4 (red). the vertical straight lines show the locations of tc=1.5 for ehh=2 and tc=3.0 for ehh=4, respectively. log plot of the surface contact number m versus chain length n at temperatures t=2.3, 2.4, 2.5, 2.55, 2.6, and 2.7 for an hp polymer with ehh=4. the statistical error of each monte carlo datum is smaller than the symbol size. surface interactions ehs=eps=1. at the end of this subsection figure 8 shows the coil globule transition line and the adsorption/desorption transition line for the end-grafted hp chain with polymer surface interactions ehs=eps=1. there is a specific interaction, named e*hh, at which the two lines intersect. for intrachain interactions stronger than e*hh, as temperature decreases, the hp chain changes from a desorbed 3d coil at high temperature to a desorbed collapsed structure at tc and, finally, to an adsorbed collapse structure at tcap. for interaction strengths below e*hh, the hp chain changes from a desorbed 3d coil at high temperature to an adsorbed 2d coil at tcap and at last to an adsorbed collapse structure below tc. here e*hh is estimated to be about 2.6 times the polymer surface attraction, and the corresponding temperature is about 2.0. symbols are estimated from simulation, while lines are guides for the eyes. to further investigate the interplay between the two transition temperatures, figure 9a presents the mean surface contact number m at different temperatures t for different intrachain interactions ehh. at high temperature t>tcap, the chain is in a desorbed state with m=0. at low t, one of the ways to observe the tcap is the substantial increase in m as t is lowered. at t=0, we have m=n for the case ehh=0, indicating that all monomers are adsorbed on the surface, whereas for ehh<0, m is less than n due to the collapse of the hp chain, indicating that the conformation of the adsorbed polymer is of a multilayer structure because of the intrachain attraction. the number of h h contact pairs, nhh, always increases with the decrease of temperature as shown in figure 9b. moreover, we find that nhh increases with |ehh|, whereas m decreases with |ehh|. similar to behavior that has been observed for homopolymers with intrachain attractions, there are less surface contacts but more intrachain contacts as the intrachain attraction increases. this reflects the fact that the adsorbed chain adopts more compact spherical shapes for stronger intrachain interactions. dependence of (a) mean surface contact number m and (b) the number of h h contacts nhh on temperature t for different internal interactions ehh. the length of the hp chain is n=400. from table 1, we find that the crossover exponent in the scaling relation m n is about 0.5 for tc<tcap while it decreases for tc>tcap at strong h h attraction. for the former case (tc<tcap), the conformation of the chain is a random coil near tcap, and m behaves similarly near tcap as shown in figure 9a, resulting in the same value of the crossover exponent for tc<tcap. for the latter case (tc>tcap), the contact number of the compact chain at tcap is reduced, since the contact monomers are located on the globule surface, as can be observed by comparing data plotted in figures 3 and 5. for the same reason, the crossover exponent is reduced for the case tc>tcap. the reason is that the difference between tc and tcap increases with ehh as shown in table 1, and the chain becomes more compact at lower temperature below tc. in order to learn more about the conformation of the chain, we have monitored the mean square end-to-end distance r and its two components parallel to the surface rxy and normal to the surface rz at different internal interactions ehh, as shown in figure 10. different behaviors are exhibited for three different cases: (1) a chain with no collapse transition when ehh=0, (2) tc<tcap with ehh=1, and (3) tc tcap with ehh=2 and 4. for the first case in the absence of intrachain interaction, a slight minimum in r is found at tcap that is a result of two changes, as a sharp decrease in rz is partially offset by a sharp increase in rxy. as the temperature is further reduced, the increase in rxy outcompetes the decrease in rz, resulting in an overall increase in r. the behavior of r is similar to an earlier finding by exact enumeration of all configurations for a short homogeneous saw chain. for the second case where tc<tcap, r first increases as the temperature is lowered just as in the previous case but then r decreases because of the collapse of the chain. in this second scenario, r exhibits a maximum at a temperature close to tc, a distinct feature absent in the other cases. the maximum is also presented in the plot of rxy as a function of temperature. for the third case where tc tcap, we find that r, rxy, and rz all decrease monotonically with the decrease of t. the chain is already in a compact state at tcap; therefore, it deforms little when it adsorbs on a surface, similar to the docking transition for a compact chain adsorbed on a weak attractive surface. dependence of (a) the mean square end-to-end distance r and (b) its components parallel and normal to the surface, rxy and rz, respectively, as a function of temperature t for hp polymers with different h h interactions. l1, l2, and l3 are three eigenvalues of the radius of gyration tensor4where si=col(xi, yi, zi) is the position of monomer i of polymer in a frame of reference with its origin at the center of mass. the asphericity parameter a ranges from zero for 3d spherically symmetric chain conformations, 0.25 for 2d circular shapes, and one for rod-shaped. it was found that a 0.391 for a linear rw chain and a 0.431 for a linear saw chain. values a of the hp chain in dilute solution (i.e., free hp chain) and the end-grafted hp chain are calculated. the dependence of a on temperature t is presented in figure 11 for the end-grafted hp chains with different intrachain h h interactions. plot of the asphericity parameter a vs temperature t for free hp chains with ehh=1 and end-grafted hp chains with different h the arrows indicate the location of tc and tcap, and the value in parentheses is ehh. for the free hp chain with ehh=1, a is about 0.44 at t tc and decreases steeply at tc=0.75. a is about 0.12 at low temperatures (t<tc), clearly showing that the chain is roughly a sphere at temperatures below tc. for the end-grafted hp chain, the behavior of a, like that of size r, is dependent on the intrachain attraction ehh. moreover, the behavior of a is quite complicated due to the competition between tc and tcap in the hp chain. for ehh=0, a increases at tcap due to the transition from a 3d random coil to a 2d random coil. for ehh=1, a first increases at tcap=1.65 and has a second increment at tc=0.5. the collapse at tc tcap happens locally and makes the chain configuration more aspherical. for the case with ehh=2 where tc is close to tcap, we find that a begins to increase when the temperature drops below tcap and continues to increase at tc. but, as temperature continues to decrease further below tc, we find that a begins to decrease due to the strong collapse of chain as |ehh|>|ehs|. for ehh=4, a decreases at tc because of collapse and then increases at tcap because of adsorption; finally, a plateaus as the chain becomes frozen at low temperatures. from these four behaviors, we conclude that adsorption of the chain increases a, whereas the effect of collapse is dependent on the strength of intrachain h if the intrachain h h attraction is weak where we have tc tcap, the adsorption of the chain increases a and the adsorbed configuration is a random coil. for this case, the collapse at low temperature will induce extra anisotropy and increase a. if the intrachain h h attraction is moderate where we have tc tcap, a is increased due to the adsorption as well as collapse of the chain but will decrease at low temperatures below tc. finally, if the intrachain h h attraction is strong where we have tc>tcap, a first decreases due to the collapse and then increases due to the adsorption of the chain. moreover, for the last two cases, a at low temperature reaches a plateau with a value close to 0.25, indicating that the adsorbed configuration is roughly a 2d circle. we have studied the interplay between the critical adsorption of a lattice hp protein with alternating h and p monomers and the coil globule transition with the dynamical monte carlo method. simulations are carried out in the simple cubic lattice where bond length can be fluctuated among 1, 2, and 3 lattice units. we find that the critical adsorption temperature tcap is influenced by the presence of intrachain attractions responsible for the collapse transition of the polymer. if the coil globule transition tc is lower than tcap then tcap for the hp polymer is roughly the same as that of a homopolymer without monomer monomer attractions, but the coil it is therefore more difficult for a surface absorbed hp polymer chain to go through the coil. on the other hand, if the intrinsic coil globule transition temperature tc is higher than tcap, the tcap for the hp polymer occurs at a higher temperature than a homopolymer without monomer monomer attraction; that is, a collapsed chain can be more easily adsorbed. the conformational properties of the end-grafted hp chain are strongly influenced by the pairwise h first, the hp model itself is limited in that it does not consider several factors, such as desolvation effects, that have been shown to be relevant to the behavior of real proteins, such as the cooperativity observed during the folding of many proteins. second, the sequence we have studied is the hp polymer with a fully alternating sequence in which the surface interactions of h and p monomers are treated as the same. this is a significant simplification and probably does not represent the real experimental situation very well. in most applications, a further extension of our study is to treat the surface interactions of h and p monomers differently. however, the overall conclusion about the mutual impact on the coil globule transition and the critical adsorption transition would probably still be valid.

an end-grafted hydrophobic-polar (hp) model protein chain with alternating h and p monomers is studied to examine interactions between the critical adsorption transition due to surface attraction and the collapse transition due to pairwise attractive h h interactions. we find that the critical adsorption phenomenon can always be observed; however, the critical adsorption temperature tcap is influenced by the attractive h h interactions in some cases. when the collapse temperature tc is lower than tcap, the critical adsorption of the hp chain is similar to that of a homopolymer without intrachain attractions and tcap remains unchanged, whereas the collapse transition is suppressed by the adsorption. in contrast, for cases where tc is close to or higher than tcap, tcap of the hp chain is increased, indicating that a collapsed chain is more easily adsorbed on the surface. the strength of the h h attraction also influences the statistical size and shape of the polymer, with strong h h attractions resulting in adsorbed and collapsed chains adopting two-dimensional, circular conformations.

PMC4280116

pubmed-698

grey mould, caused by botrytis cinerea (sclerotiniaceae family), is an important plant disease that affects a large number of plant species and is particularly important in greenhouse production of tomatoes in the mediterranean basin. in greenhouse tomato, the fungus infects flowers, fruits, and leaves and can grow through the petiole into the stem [2, 3]. this problem is one of the major stresses especially in arid and semiarid regions and can severely limit plant growth and productivity [5, 6]. in algeria, a wide range of environmental stresses (such as high and low temperature, drought, alkalinity, salinity, and pathogen infection) soil salinity and irrigation water are two of the main serious problems hindering the development of most plant species. thus, the effect of these factors may result from structural and physiological changes in the plant, an increased incidence, and severity of diseases caused by various species pathogen. reference showed that relatively low levels of salinity (2550 meq) could increase the severity of phytophthora root rot of tomato with high na: ca ratios (10: 1), phytophthora [912], f. oxysporum f. sp. the ability of ca to form intermolecular linkages gives it an important role in maintaining the integrity and structure of membranes and cell walls. ca is also used as a second messenger in many signal transduction pathways within the cell. several studies have reported that ca treatment of plant tissue induces an increase in tissue ca content, resulting in reduced fungal diseases. the mechanisms by which calcium salts inhibit the development and severity of diseases are not known. one hypothesis is that high external ca concentrations may increase the concentration of ca in the cytosol, which can be toxic to the fungus. the ability of calcium to reduce the development of postharvest diseases of fruit has been attributed mainly to the formation of calcium cross-linkages in the cell wall, resulting in decreased effectiveness of cell wall-macerating enzymes secreted by the pathogen. reference also demonstrated a relationship between increasing levels of calcium in the cell walls of potato tubers and a reduction in the macerating activity of erwinia carotovora. all the studies on the effect of ca on botrytis showed that it has an inhibitory effect on growth of this fungus at high concentrations [2325]. this effect is thought to be mainly due to the role of calcium in ameliorating physiological disorders and thus indirectly reducing pathogen activity [26, 27]. reference has indicated that calcium chloride reduced germination and germ tube elongation of b. cinerea and penicillium expansum in vitro. reference also has reported a similar effect of calcium in reducing the susceptibility of rose flowers to gray mold caused by botrytis cinerea. for the most effective control of disease, it seems necessary to examine the impact of salinity on the development of pathogen. the objective of this study was to determine the in vitro effect of sodium and calcium salts on spore production, conidia germination, and mycelial growth of b. cinerea. b. cinerea isolates were obtained from decayed tomato (lycopersicon esculentum) in northwestern algeria. the leaf fragments were placed on filter paper moistened with sterile water in a petri dish. conidia were harvested from 14-day-old cultures by agitating small pieces of agar, bearing mycelia and conidia, in a glass tube. conidia of b. cinerea were obtained from 2 week old pda cultures incubated at 25c in 12/12 hours light/dark. culture plates were vortexed in a tube containing 10 ml sterile distilled water and 0.05 ml tween 80. a sterile magnetic stir bar was placed on the agar and set stirring for 5 minutes to loosen the spores. the suspension enriched in spores finally the conidial concentration was determined using a malassez cell and adjusted to 10 spores per ml. to determine the influence of nacl and cacl2 on spore germination of b. cinerea, a drop containing 100 conidia was transferred onto water agar plates enriched with nacl and cacl2: 0, 50, 100, 150, and 300 meq. a conidium was considered as germinated if the germ tube length was at least twice the length of the conidium. the influence of nacl and cacl2 on the diameter growth was determined by growing the isolates in a pda medium at four nacl and cacl2 levels (50, 100, 150, and 300 meq); control medium was not amended with salts. mycelium growth inhibition was evaluated by placing a plug (4 mm diameter) from an actively growing culture in the centre of a pda agar plate of 9 cm plastic petri dishes. cultures were incubated for 714 days at 25c in the dark, and each treatment had four replications. all statistical analyses were analyzed by the software of statistics (statbox 6.0.4, grimmersoft). statistical significance was assessed at the level of p=0.05 or p=0.01. all the isolates exhibited variation in their colony characteristics such as color, shape, and texture (figure 1). b. cinerea colonies from tomato on pda at 25c were visually classified into three morphological groups, gi, gii, and giii, based on colony color and pycnidial distribution. gi (f27) isolate produced white to light grey colonies, where colony texture was generally cottony, and was present at the center of the petri dish. gii (fa13, s27, b27, and tr13) isolates had off-white to pale gray color. based on the morphological parameter of length, pycnidia can be classified into two groups: length greater in isolates fa13, s27, b27, and f27 and length less in isolates tr13 and r13. they scattered all over the medium in petri dish, covering the entire surface of the agar (b27 and tr13 and f27). in some isolates sclerotia were produced on concentric rings, formed along the edges of the petri dish (fa13, s27, and r13). this study evaluated the activity of 2 salts against b. cinerea in vitro at 4 concentrations. the effect of salts at different concentrations on mycelial growth of colonies of six isolates of b. cinerea in pda was observed after 3 days. all concentrations, except 300 ppm, of nacl significantly stimulated growth of b. cinerea., reduced growth has been correlated with the increasing in the nacl of the medium. the calcium chloride at 50 and 100 ppm stimulated mycelial growth of b. cinerea relative to the control. however, higher concentrations of calcium chloride (150 and 300 ppm) caused f27, b27, r13, tr46, s27, and fa13 to reduce growth by 26.72, 24.26, 23.24, 34.78, 15.07, and 17.04%, respectively. there was a significant reduction in growth of isolates (p<0.001) with increasing calcium salt. at 300 ppm, calcium chloride was the most inhibitory, reducing growth on pda by 34.78% for the isolate. the interaction between salt and concentration was significant for r13, tr45, s27, and fa13 (p<0.001), but not significant for f27 and b27 isolates. all concentrations were significantly different from the control (p<0.001); 150 ppm cacl2 and 300 ppm nacl or cacl2 were similar to each other and different from other concentrations in reducing mycelial growth. in the absence of salt (table 2), isolates of b. cinerea do not present the same profile of conidial production. the optimum density for spore production of this fungus was from 1.97 10 to 2.2 10 spores/ml, of r13 and b27, respectively. data in table 2 indicate that application of chloride salts and sodium salt caused a significant increase in the production of conidiogenesis at various concentrations tested compared with control (p<0.001). however, these observations indicate that the conidial production of the six isolates might increase, even at high salinity. there was a significant increase in these characters between the control and 50 ppm concentration of salinity. under these salt conditions, sodium chloride is most favorable to the sporulation of all isolates of b. cinerea, especially at high concentrations of the culture medium. by adding 300 ppm of nacl to the culture medium, an increase in spore production by 1.21 10 spores calcium chloride stimulates little sporulation of the isolates, with only 9.2 10/ml to isolate r13. the amount of spore production is in direct proportion to the concentration of salinity in the culture medium. the interaction between salt and concentration was significant for all salts isolates (p<0.05) except fa13 (table 2). the results for the percentage germination of different isolates studied in terms of germination capacity under the effect of different salt concentrations are shown in table 3. the six isolates presented dissimilar percentages of germination in the presence of sodium and calcium chloride. the analysis shows that salinity affects the percentage of germination for each value of salt. the highest germination percentage, 78.33% and 63.67%, was obtained in the absence of salt after 24 h incubation at 25c, from isolates fa13 and tr46, respectively. concerning the salts, the 50 and 100 ppm also increased the conidial germination except tr13 and fa13 isolates, which was significantly different from the stimulation caused by nacl. conversely, spore germination was decreased for sodium concentrations of 150 and 300 ppm relative to the control. the application of lower concentrations of cacl2 (50 ppm) to wounds did not inhibit their percentage germination. the toxicity of calcium chloride (ec50=100 ppm) to spores was higher than that of sodium chloride (ec50=150 ppm). the interaction between salt and concentration was significant for all isolates (p<0.001) except f27 and tr46. the purpose of this study was to compare the effect of sodium and calcium salts against b. cinerea. our in vitro tests showed that sodium chloride stimulates the development of the fungal up to 150 ppm. in contrast, only calcium salts were effective at low concentrations as compared to sodium chloride. however, higher concentrations of calcium chloride reduced mycelial growth of b. cinerea pda medium. our data indicate that salinity stimulated growth of all six isolates at high concentration (nacl at up to 150 ppm). on the contrary, high salinity (more than 300 ppm) of several reference showed that increasing the salinity of the medium promotes the in vitro mycelial growth of phytophthora citrophthora and p. parasitica, agents of root rot of citrus, with an optimum between 1.44 and 3.11 bars. similarly, showed that all calcium salts tested (except formate, calcium pantothenate, and dibasic calcium phosphate) reduced the growth of monilia fructicola responsible for brown rot of peach, on amended potato dextrose agar (pda). in the comparison of the inhibitory effect of the various salts, higher concentrations with 300 ppm to nacl or cacl2 reduced mycelial growth to 16 and 23%, respectively. regragui and lahlou showed that the stimulator effect of salinity was observed on the mycelial growth, conidia production, and conidia germination of the tested stain of v. dahliae, respectively, in concentrations 170, 120, and 256 mm of nacl. oppositely, pelizza et al. showed that the presence of nacl in the medium culture reduces the growth of an isolate of leptolegnia chapmanii. however, reid et al. reported that sodium chloride was more effective than other chloride salts (calcium chloride, ammonium chloride, and manganese chloride) in controlling fusarium crown and root rot caused by f. oxysporum f. sp. van bruggen and semenov reported that on a long-term basis there is a decrease in the genetic diversity of fungi as a result of stress. on the other hand zahran mentioned that the hydric stress has to deal with the increase in osmotic pressure and may therefore change their physiology and morphology in response to this. two strategies used by microorganisms to adapt to osmotic stress were described by killham, both of which result in an accumulation of solutes in the cell to counteract the increase in osmotic pressure. one is the selective exclusion of the solute incorporated (e.g., na, cl), thus accumulating the ions necessary for metabolism (e.g., nh4). the results of the present study demonstrate that calcium salts also have been shown to reduce mycelial growth in vitro; the percentage of reduction varied between 15 and 34% as compared to the control. several studies have reported that calcium applications can suppress diseases caused by several pathogens [23, 27, 28, 36]. our result further supports the results of maouni et al., who found that calcium chloride significantly reduced pear fruit decay caused by a. alternata and penicillium expansum when used at 4 and 6%. tian et al. recorded that calcium chloride at 2% inhibited the growth and spore germination of r. stolonifer, although cacl2 was tolerated by alternaria alternata and p. expansum in vitro. it was reported that 1,000 mg of calcium (calcium chloride) enhanced the growth of botryosphaeria dothidea. calcium salts also have been shown to reduce mycelial growth in vitro and reduce incidence and severity of infection of peach fruits and shoots by monilinia fructicola and leucostoma persoonii, respectively [23, 40]. reported that mycelia cultured with 100 to 200 mm ca exhibited a lower viability compared with mycelia grown with 10 mm ca for some isolates of botrytis spp. while little information is available on the role of ca in fungi, results of experiments with yeasts have shown that mutants that have defective intracellular ca transport systems or defective vacuolar h-atpase that produces the proton motive force necessary for the activity of the vacuolar ca/h exchanger could not grow in high ca concentrations [4245]. maintenance of low basal concentrations of free cytosolic ca, in the submicromolar range, is essential for normal cell functions [46, 47]. in the case of the evaluation the effect of nacl and cacl2 on the spore production by the fungus, all isolates of b. cinerea are able to sporulate in salinity tested, but to varying degrees. in fact, sporangium formation of phytophthora parasitica in vitro appeared to be stimulated by salinity, as the numbers of sporangia were generally higher (120% to 225%) in the salt-amended treatments than the distilled water controls. with regard to conidiogenesis, reference showed that stimulation of sporulation under the effect of salinity is due to a specific effect of ions. according to this author, na and cl stimulate the production of sporangia of p. citrophthora and p. parasitica while the osmotic effect inhibits biological activity. in verticillium, increased sporulation under the effect of the salt appears to be not only solely due to the effect of na and cl ions, but also due to the osmotic effect. however calcium salts did not reduce spore production of b. cinerea spores in this study. similarly, a minimum concentration of calcium is necessary for the production of zoosporangia or zoospore release by phytophthora spp., it was reported that, at low concentration (50 ppm), the germination capacity for most isolates increased compared with the control in both types of salt. beyond this concentration, similarly, a low reduction of conidial germination was observed for two salt types at the maximum concentration used. reference showed that increasing the concentration of calcium chloride (25175 mm) causes a decrease in germination and germ tube growth in vitro of b. cinerea and penicillium expansum, respectively, causing the gray and blue mold in apples stored. incubating b. cinerea spores in increasing concentrations of cacl2 (426 calcium was effective in inhibiting spore germination of c. gloeosporioides, rhizopus stolonifer, and alternaria alternata and penicillium expansum. physiologically, the maintenance of low basal concentrations of internal ca is essential for normal cell functions of organisms, and the inability to regulate ca may affect the organisms ' normal growth. calcium ions may reduce the incidence of fungal infection by directly inhibiting fungal growth and by inhibiting cell wall degrading enzymes produced by the pathogens [45, 54, 55]. the effects of calcium in reducing spore germination were probably due to toxicity, with high concentrations likely affecting the osmotic balance in fungal cells.

six isolates of botrytis cinerea were isolated from leaves and stems of different tomato varieties taken from four areas in the northwest of algeria where tomato is mostly grown in greenhouses and high tunnels. the purpose of this research was to determine the effect of two salts, nacl and cacl2, on three stages of botrytis cinerea's life cycle. all isolates tested were stimulated in 50 to 150 ppm; nacl was the most effective treatment to increase mycelial growth at two tested concentrations. however, at 300 ppm concentration, cacl2 completely inhibited the growth of mycelium; they reach 34.78% for the isolate tr46 and 26.72% for isolate f27. the sodium and calcium salts stimulated conidia production in liquid culture. we noticed that the effect of calcium chloride on sporulation was average while sodium chloride. in the medium containing 50 ppm, calcium chloride and sodium chloride increased the germination capacity of most isolates compared with the control. other calcium salts, at 100 or 300 ppm, decreased the germination percentage of the conidia. with the exception of sodium salts, the inhibitions of germination reduce at 150 or 300 compared with the control. conidial germination was slightly inhibited by sodium chloride only when the concentration was over 300 ppm.

PMC4391690

pubmed-699

vitamin d deficiency is a global health problem even in sunny regions as the middle eastern countries (1).vitamin d is a steroid hormone and plays a key role in minerals metabolism, specially calcium and phosphorus, as well as in bone strength (2). the active type of this vitamin is synthesized in the skin, liver and kidneys. in addition to its multi-functions in the body, vitamin d reinforces the immune system. receptors for vitamin d exist in most organs including pancreas, stomach, genital system, skin, brain etc (3-8). 25-hydroxy vitamin d (25(oh) d) level is the best index to determine vitamin d status in the body, with a half-life of 2-3 weeks (9). adequate exposure to sunlight and dietary intake of foods rich in vitamin d are necessary to satisfy the daily need of the body and prevent hypovitaminosis d (10,11). many studies have indicated the association of serum 25(oh) concentrations with serum levels of other vital elements. from the several elements found in the body, only a few number as zinc, copper, magnesium, iron and calcium play important roles in the body's chemical and physiological functions. zinc has several important roles in the human body; for instance, it is used in the process of synthesizing insulin and some enzymes as superoxide dismutase. zinc is a trace element, which after iron, has the highest amount in the body. it is mainly accumulated in the muscles but can also be found in the blood cells, retina, bones, skin, kidneys, liver and the pancreas. zinc is the second most vital element after iron and its deficiency during pregnancy may cause serious feto-maternal complications (12) including impaired cognitive development in the infant during the first six months of life, immunological complication in fetus, low birth weight, prematurity, miscarriage, fetal or infant death, postdate pregnancy, premature rupture of membranes, cleft palate and neural tube defects in the fetus (13). a study on 150 iranian pregnant women showed that 37% of them were vitamin d deficient and 23% were zinc deficient, with a statistically significant association between serum zinc and 25 (oh) d levels (14). such experience is scarce in the pediatric age group. given the high prevalence of hypovitaminosis d and zinc deficiency, as well as their possible interactions, this study aimed to assess the relationship of serum zinc and 25 (oh)d levels in adolescents. this study was conducted as a sub-study of the national survey of school students high risk behaviors, which was performed as the third survey of the school-based surveillance system entitled: childhood and adolescence surveillance and prevention of adult non-communicable disease study (caspian-iii). caspian-iii is a school-based nationwide health survey that includes 5,528 students aged 10-18 years from 27 provinces in iran. in this case-control study, as a sub-study of the caspian iii study, 165 students with hypovitaminosis d as the case group and 165 normal students without hypovitaminosis d as the control group were randomly selected from the sample of frozen sera (70c) of the participants in which 25-hydroxy vitamin d (25(oh) d) was checked (15). serum concentration of 25(oh) d was analyzed quantitatively by direct competitive immunoassay chemiluminescene method applying liason 25 oh vitamin d assay total (diasorin, inc.), with a coefficient of variation of 9.8%.25 (oh) d levels of less than 10ng/ml as vitamin d deficiency and levels between 10 and 30ng/ml as vitamin d insufficiency (16). sera of the two groups of participants with or without vitamin d deficiency were randomly selected. zinc level of the samples was determined by atomic absorption spectrophotometer using hollow cathode lamps of zn. sera were analyzed for glucose and lipid profile including total cholesterol, high-density lipoprotein cholesterol (hdl-c), ldl cholesterol (ldl-c) and triglycerides (tg), as well as liver function tests including serum glutamate oxaloacetate transaminase (sgot) and serum glutamate pyruvate transaminase (sgpt) using pars azmoon reagent kits (tehran, iran). the research and ethics committee of isfahan university of medical sciences, isfahan, iran, approved this study. statistical analysis: the relationship between serum vitamin d and zn levels was measured through linear regression analysis considering zn as an independent variable and 25(oh) d as a dependent variable. chicago, il, usa) by applying t test, one-way analysis of variance (anova), and pearson s correlation tests. to assess the relationship between serum zn concentration and vitamin d level, logistic regression was used to calculate odds ratios (or) for vitd level between various quartiles of serum zn concentration; the mean age of the participants was not significantly different among groups with and without hypovitaminosis d (14.74 2.52 vs.14.74 2.66 years, respectively, p>0.05). no significant difference was observed in the baseline characteristics of the participants of the two groups (table 1). bmi: body mass index, sbp: systolic blood pressure, dbp: diastolic blood pressure, hdl-c: high density lipoprotein-cholesterol, ldl-c: low density lipoprotein-cholesterol, tg: triglyceride, fbg: fasting blood glucose, sgot: serum glutamate oxaloacetate transaminase, sgpt: serum glutamate pyruvate transaminase, sd: standard deviation the serum meansd level of 25(oh) d was 6.341.47ng/ml in the group with hypovitaminosis d and 39.276.42ng/ml in the control group (p<0.001). the meanzn level in hypovitaminosis d group was significantly lower than in controls (1.15 0.26 vs. 1.430.32g/ml, respectively, p<0.0001). the pearson correlation of serum zn concentration and vit d with metabolic factors and existence of metabolic disease showed that zn levels had a significant positive correlation (p<0.001) with vit d level. as demonstrated in table 2, the or of higher levels of vit d levels was higher in the second (q2) and fourth (q4) quartiles significantly as compared to the reference quartile (q1). the main aim of this study was to determine the relationship between serum vitamin d and serum zn levels in children and adolescents. we found a positive correlation between serum zn and serum vitamin d levels, and this finding was consistent with a previous study conducted on children (14). this might have been caused by the prominent effects of lifestyle factors as inadequate exposure to sunlight, air pollution and poor nutrition. aside from hazardous effects on the respiratory system and pollution caused diseases, living in populated cities means less sunlight for children, especially during cold seasons when there is higher air pollution and weaker sunlight intensity (16,17). a nationwide study in iranian adolescents demonstrated a high prevalence of hypovitaminosis d and an association of serum 25(oh) d levels with cardiometabolic risk factors. it showed an inverse association of 25(oh)d with systolic blood pressure, diastolic blood pressure, total cholesterol and low-density lipoprotein cholesterol. in contrast, this association was significantly positive with high-density lipoprotein cholesterol, but not with fasting plasma glucose and mets (19). the first evidence of zn deficiency in humans was reported from central part of iran (21). zn deficiency has several etiologies; one of them might be the consumption of white flour and white rice as the main dishes in iran. the zn intake from these diets is high at 15 mg a day, but it is not available because of the high phytate content of this kind of diet. phytate, the phosphorus storage compound of plant seeds, bind zn and other bivalent ions in insoluble complexes, making them unavailable to human and other monogastric species (22). iron, when present in large amounts in the diet, also inhibits zn absorption. epidemiological studies in iran showed a considerably high prevalence of zn deficiency, as high as 31.1% among school students and 28.1% in children (23). the most important causes of zn deficiency are soil content of zn, dietary deficiency (phytate containing whole grains), malabsorption due to impaired transport across intestinal absorptive surface, damaged or absent intestinal absorptive surface, increased loss, increased utilization, and chronic disease (24). the vitamin d receptor (vdr) binds zinc, and the activity of vitamin d dependent genes in cells is influenced by intracellular zinc concentrations. it is also important to ensure that the calcium from foods or supplements is used in your bones. hence, low level of both zn and vitd can affect many important body functions (25). however, studies in animals have shown positive effects of vitd on zinc increases (26) the effects of zinc and vitd on absorption-desorption and eventually the amount of each other, but their basic metabolic pathways in humans are not clear. our results revealed that hypovitaminosis d was accompanied with low serum zn level. comparing our results with those of other studies, it could be concluded that hypovitaminosis d is probably due to inadequate exposure to sunlight. in addition, it seems inappropriate diet and lack of absorbable zn in our foods lead to zn deficiency in our schoolchildren. finally, conducting future studies to identify the factors affecting vitamin d and zinc deficiency in schoolchildren, their relationship and the real causes of simultaneous lack of vitd and zinc study limitations and strengths: the main limitation of this study was its cross-sectional nature. moreover, we did not obtain the detailed dietary intake of the participants. the strengths of the study were its novelty in the pediatric age group and recruiting a nationally representative sample.

background: vitamin d (vitd) deficiency is a common worldwide problem. some previous studies have shown that both zinc (zn) and vitd deficiency are prevalent in iran. this study aimed to assess the relationship of serum zn and vitamin d levels in a nationally representative sample of iranian children and adolescents. methods: this case-control study was conducted as a sub-study of a school-based surveillance program entitled " the caspian-iii study ". an equal number of individuals with and without hypovitaminosis d including 330 participants aged 10 to 18 years were selected. the correlation of serum 25 hydroxy vitamin d (25(oh) d), cardiometabolic factors and zn concentrations was determined. statistical analysis was done using one-way analysis of variance (anova), pearson's correlation, linear regression, and logistic regression. results: the mean age was not significantly different in participants with and without hypovitaminosis d (14.742.52 vs. 14.742.66 years, respectively, p>0.05). the mean 25(oh) d level was 6.341.47ng/ml in the group with hypovitaminosis d and 39.276.42ng/ml in controls. the mean zn level was significantly lower in the hypovitaminosis d group than in controls (1.150.26 vs. 1.430.32g/ml, respectively, p<0.001). the pearson's analysis showed a positive and significant correlation between zn and 25(oh) d serum levels (p<0.0001). odds ratios analysis for vitd level between various quartiles of serum zinc concentration for all participants showed that the odds of higher levels of vitd increased by higher levels of zn. conclusion: we found significant associations between low serum concentrations of zinc and 25(oh) d. food fortification or mineral supplementation should be considered in future health programs.

PMC5307609

pubmed-700

kienbck s disease, defined as avascular necrosis of the lunate, is a relatively rare condition with a poorly understood etiology. the disease is a relatively rare condition in which the etiology and natural history of this condition are poorly understood and even more poorly documented in the literature. a wide array of treatment recommendations is available, and reported results vary, which may hinder consistent treatment recommendations. conservative and invasive treatments for kienbck s disease include the following: wrist immobilization with a splint during early disease stages, surgical joint-leveling procedures to attempt to reduce loading on the joint and to promote revascularization and increased blood flow, vascularized bone grafting, proximal row carpectomy, and total wrist arthrodesis. proximal row carpectomy and total wrist arthrodesis attempt to alleviate arthritis pain due to carpal collapse; however, these 2 interventions greatly limit range of motion. none of these treatments effectively address the chronic pain, loss of range of motion, and decreased grip strength associated with kienbck s disease. the staging distinguishes only the state of collapse in an ordinal classification scheme and does not allow localization or indicate partial involvement of the lunate, which the image contrast from mri may provide. osteochondral transfer for treatment of kienbck s disease may be possible for individuals with segmental or incomplete avascular necrosis of the lunate. previous investigators have acknowledged the importance of donor site cartilage thickness to aid in minimizing postoperative abnormal stresses and resulting poor function. selection of donor cartilage with an appropriate thickness may be difficult due to source availability and donor site location relative to the load-bearing region of a joint, which may lead to donor site morbidity. an alternative to matching cartilage thickness is the matching of curvature of the articular surface. matching the curvature of donor and recipient sites has been shown to maintain preoperative contact stresses for a given loading regimen. in this short communication, we report the treatment of a patient s kienbck s disease by combining mri with mathematical modeling to optimize the congruency between the curvature of donor and recipient sites of an autologous osteoarticular plug transfer. the purpose of this study was to describe the feasibility of a novel surgical technique. the results indicate that donor site selection for autologous osteoarticular transfer using a quantitative evaluation of articular surface curvature may be beneficial for optimizing the likelihood for restoring the radius of curvature and thus joint articulation following cartilage repair. a 48-year-old, right hand dominant woman had left wrist pain associated with decreased wrist flexion since september 2009. in november 2009, the patient experienced severe wrist pain after falling on her hand. after acquiring standard radiographs, the woman was diagnosed with kienbck s disease, and a proximal row carpectomy was recommended by an initial orthopedic surgeon. the patient sought a second opinion after understanding that the carpectomy would not only reduce pain during activities of daily living but would also limit range of motion. upon presentation to our institution, physical examination of the left wrist revealed no focal swelling, dystrophic changes, or evidence of muscular atrophy. the left wrist range of motion was 20 of flexion and 30 of extension; the patient had full, painless range of motion in her right wrist. grip strength in her left hand was 111.2 n as compared with 333.6 n in her right hand. mri of the wrist was performed utilizing a clinical superconducting 3-t imaging unit (sigma hdx, general electric healthcare, waukesha, wi) and a dedicated wrist coil. pulse sequencing included a 3-dimensional gradient-recalled sequence with tr/te of 39/14 milliseconds, field of view of 9 cm, slice thickness of 1 mm with no gap, flip angle of 10, and band width of 41.7 khz. coronal, sagittal, and axial cartilage sensitive fast spin echo sequences were obtained with a long tr (4,000-5,500 milliseconds), moderate te (31-38 milliseconds), field of view of 8 to 9 cm, slice thickness of 2 to 3 mm, acquisition matrix of 512 512 (320-352), 2 excitations, a band width of 14.7 to 41.7 khz, and echo train length of 10 to 12. coronal fast inversion recovery sequence was performed with a tr of 5200 milliseconds, te of 23 milliseconds, inversion time of 170 milliseconds, field of view of 9 cm, slice thickness of 2.5 mm, matrix of 288 192, 2 excitations, band width of 31.2 khz, and echo train length of 12. the examination demonstrated collapse of the proximal pole of the lunate, with subchondral sclerosis and a mild bone marrow edema pattern (fig. 1). foci of completely devitalized marrow were noted in the proximal pole with an associated intense synovitis. moderate partial wear of cartilage was seen over the collapsed lunate with preservation over the radial side of the lunate fossa. a best-fit circle was manually placed on the lunate with its articulation with the distal radius to determine the joint curvature. preoperative coronal (a) and fat-suppressed (b) fast spin echo images of the wrist demonstrate sclerosis and partial collapse of the lunate (arrows) with hyperintensity in the adjacent cartilage. one-year postoperative sagittal (d) and fat-suppressed (e) fast spin echo images and computerized tomographic evaluation (f) demonstrate partial bony incorporation expected for the time interval. computerized tomographic examination was performed utilizing axial acquisition with a slice resolution of 0.8 mm, subsequently reformatted into sagittal and coronal reformations. concurrent computerized tomography was performed to quantify the extent of subchondral collapse, which measured up to 1 mm. mri of the left knee was subsequently acquired to provide a digital template for the planned osteochondral transplantation. a 3-dimensional t1-weighted spoiled gradient echo data set with frequency-selective fat suppression was acquired for cartilage segmentation: tr of 13.6 milliseconds, te of 3.0 milliseconds, field of view of 16 cm, flip angle of 20, slice thickness of 1.5 mm, matrix of 512 512, receiver bandwidth of 41.7 khz, and 1.2 excitations. the cartilage was segmented from the image data using custom-written semiautomated software (general eelectric healthcare), with minimal manual editing performed as needed. a 3-dimensional mesh representation of the segmented lateral condyle image data was constructed and smoothed for curvature calculations. curvature calculations were performed on the 3-dimensional surface mesh using a custom-written matlab (mathworks, natick, ma) program. 2a), and a best-fit paraboloid (z(x, y)=ax+bxy+cy) was fit to each vertex and the surrounding 2-ring neighborhood (fig. 2b). the analytical solutions for maximum curvature (max) and minimum curvature (min) were determined from the best-fit paraboloid (fig. this process is repeated for each vertex on the 3-dimensional surface mesh to generate max and min maps. regions with large values of min display concavities on a surface, and regions with large values of max display convexities of a surface. the model of a paraboloid was chosen due to the computational efficiency as well as the verified accuracy of the curvature calculation as compared to other numerical methods. (a) each mesh vertex (red point) had a 2-ring neighborhood (blue points) calculated. (b) the coefficients of a best-fit paraboloid through the vertex and 2-ring neighborhood were used to calculate the maximum and minimum curvatures. the directions of maximum curvature (red arrow) and minimum curvature (green arrow) were also calculated. (c) the direction and magnitude of the maximum curvature and minimum curvature at the vertex are shown by the orientation and radius of the pink and green circles, respectively. the radius of each circle () is the inverse of the calculated curvature (= 1/). at the indicated vertex (red dot), the anterolateral femoral condyle had the maximum curvature in the mediolateral direction and minimum curvature in the anteroposterior direction. the patient underwent osteochondral transfer from her left femoral condyle to her left lunate. using a tourniquet to control bleeding, the necrotic area of the lunate, which measured approximately 8 mm 1 mm 1 cm, was debrided. after thorough debridement of the avascular necrotic bone, intraoperative fluoroscopy was used to identify and better assess the defect in the lunate. the donor site was filled with a synthetic acellular biphasic copolymer plug (trufit, smith and nephew, andover, ma). using the digital template acquired from the algorithm, an osteochondral plug from the left femoral condyle that closely matched the radius of curvature of the lunate, 12.7 mm from the manual measurement, was harvested during knee arthroscopy (fig. the location of the plug was assessed from the previously constructed 3-dimensional model of the articular surface, relative to the local anatomy. a 10 blade and rongeur were used to fit the dimensions of the osteochondral plug with those of the lunate. 2 fiberwire (arthrex, naples, fl) stitch was placed through the scapholunate ligament. internal fixation was performed with a 1-mm drill, drilled antegrade through the lunate to fix the osteochondral plug; a 1.5-mm 8-mm screw was then compressed across the plug with fixation in the distal aspect of the lunate. there was no block in flexion, extension, or radial deviation, and the osteochondral fragment remained stable through a full range of motion. the patient was placed in a sling and discharged with instructions to return for regular follow-up visits. maximum (left) and minimum (right) principal curvature maps of the anterior portion of the lateral femoral condyle. postoperative radiographs demonstrated that the wrist was in good alignment and the hardware was intact. follow-up radiographs and physical examinations were performed at 2, 4, 6, 8, and 10 weeks; 3, 4, 7, and 10 months; and 1 year postoperatively. we treated kienbck s disease with the aid of a 3-dimensional model of femoral articular surface and calculated the principal curvatures of the surface. this was performed to determine the optimal congruency of the chondral donor and recipient sites. surgical challenges for autologous osteoarticular transfer include restoration of the articular surface as well as the tidemark. koh et al. have shown in a preclinical model that plugs left elevated or angled result in increased contact pressures, thus presenting potential risk to the cartilage on the opposite side of the joint. the mesh model created in the knee allowed for objective assessment of the articular surface to ensure more optimal plug alignment, provide reconstitution of joint architecture, and promote biologic incorporation of the plug into the recipient site. we did not seek to evaluate the functional outcome of the surgical procedure in this short report but to introduce the feasibility of this novel surgical planning technique for osteochondral repair. this case report presents the unique application of mri and mathematical modeling when applying the autologous osteoarticular transfer procedure to the lunate. these techniques require only an additional limited 3-dimensional model of the knee with minimal scan time beyond the routine examination of the wrist for assessment of marrow viability and the integrity of the articular surfaces. additional applications would include osteochondral lesions of the distal femur, capitellum, talus, and potentially, over the femoral head. future studies will refine the image acquisition and 3-dimensional modeling processes for optimal selection of the osteochondral plug based on local tissue morphology.

kienbck s disease, defined as avascular necrosis of the lunate, is a relatively rare condition with a poorly understood etiology. conservative and invasive treatments for kienbck s disease exist, including wrist immobilization, surgical joint-leveling procedures, vascularized bone grafting, proximal row carpectomy, and total wrist arthrodesis. staging kienbck s disease using radiography assumes near complete avascularity of the lunate. the staging distinguishes only the state of collapse in an ordinal classification scheme and does not allow localization or indicate partial involvement of the lunate, which the image contrast from mri may provide. in this short communication, we report the treatment of a patient s kienbck s disease by combining mri with mathematical modeling to optimize the congruency between the curvature of donor and recipient sites of an autologous osteoarticular plug transfer. follow-up mri and radiographs at 1 year postoperatively demonstrated gradual graft incorporation and bone healing. the purpose of this study was to describe the feasibility of a novel surgical technique. the results indicate that donor site selection for autologous osteoarticular transfer using a quantitative evaluation of articular surface curvature may be beneficial for optimizing the likelihood for restoring the radius of curvature and thus joint articulation following cartilage repair.

PMC4297129