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work - related musculoskeletal disorders (wrmds) are the most common cause of chronic pain and physical disability that affect contemporary workforces.13 in this context, musculoskeletal injuries are considered one of the largest health problems among physiotherapists, because the nature of the work that therapists expose themselves to has a high risk of pain.4,5 although physiotherapists have expert knowledge of musculoskeletal injuries and injury prevention strategies because of their training and continuous professional development, physiotherapists still report a high incidence of work - related injuries during their professional practice. the nature of the work in a physiotherapy practice is physically demanding, and it involves repetitive tasks, high - force manual techniques for treating patients, techniques that exert direct pressure on certain joints during treatment, awkward positioning of joints during certain maneuvers and prolonged constrained postures.13 these physical factors expose physiotherapists to various work - related musculoskeletal injuries.6 a paucity of information is available on the extent of this problem among physiotherapists because an enormous amount of research on wrmd among nurses are available. past studies have used lifetime prevalence, 12-month prevalence and one - week prevalence in measuring the magnitude of wrmds among physiotherapists.79 regardless of the time frame for recall, the prevalence of wrmds has been found to be high, with the lifetime prevalence reported to be 40% to 91%10,11 and the 12-month prevalence to be in the range of 58% to 91%.2,9 lower back (48%), neck (33%), upper back (23%) and thumb injuries (23%) are the various injuries that have been reported as work - related injuries in physiotherapists.1 apart from the nature of the job of therapists, working in certain specific clinical specialties in physiotherapy is also reported to contribute to injuries during work. it has been suggested that musculoskeletal outpatients (31%), neurological rehabilitation (14%) and elderly care (12%) are the three major clinical areas producing serious work - related injuries among therapists.2 the therapists who work in general medicine, pediatrics, elderly care, psychiatry and outpatient burns had a 46% greater likelihood of getting work - related injuries during the course of their work.11,12 therefore, the area of practice for the physiotherapists is an important factor for understanding the occurrence of work - related injuries among therapists. the occurrence of injuries at work differs between junior- and senior - level therapists. previous studies9,13 have cited that the incidence of work injuries is the highest within the first 5 years of practice, and it is common in junior physiotherapists and newly qualified graduates. the occurrence of injuries in junior physiotherapists also correlates with the need for intervention services that are aimed at reducing injury rates among this particular group.11 some research results have indicated that new physiotherapists are involved in the rotation of clinical postings in various specialties that may also expose them to a higher risk of injuries during their work. the incidence of work - related injuries is also associated with the gender of the therapists because more female therapists report spinal symptoms than male therapists.9 most of the available information on physiotherapy - based work injuries are reported from western and european countries where manual therapy is widely practiced. little information exists regarding wrmds among the physiotherapists in asian work places, and this study reports the occurrence of such injuries among physiotherapists in the asian work culture. a difference in the clinical practice among therapists exists in the asian work place because manual therapy is less commonly practiced among therapists in this region, where electrotherapy and exercises are the most commonly used treatment modalities. however, manual therapy use is increasing among asian nations, especially in malaysia, and this may expose the therapists to a high level of risk for work - related injuries. because the physiotherapists are treating an average of 15 to 20 patients per day for 8 working hours, the risk of work - related injuries may be more significant because of this high workload. therefore, the main aim of the current study is to establish information on wrmds experienced by physiotherapists in malaysia and to explore the influence of factors such as gender, body mass index (bmi), years of work experience and clinical placement areas on the occurrence of wrmds. the national professional association and the professional governing body can use this information to take adequate measures to minimize the exposure risk to injuries and to develop new injury management strategies. the current work was a cross - sectional study completed among physiotherapists during 20082009. subjects were recruited using the convenience sampling method from the physiotherapy departments of three main public hospitals in selangor and kuala lumpur, malaysia, because of the relatively high number of physiotherapists working there and their wide range of clinical specialties. tuanku ampuan rahimah hospital, which is located in the selangor state, has 850 beds, 20 clinical disciplines and 20 physiotherapists. kuala lumpur hospital, which is the largest medical facility in malaysia, has 27 clinical disciplines, 83 wards, 2302 beds and 53 physiotherapists of different grades working in various wards and in outpatient physiotherapy units. the university of kebangsaan, malaysia medical centre, is a 1050-bed hospital with 32 physiotherapists, and it is an important public hospital in kuala lumpur. all physiotherapists who worked at a full - time clinical practice, regardless of age, gender and work experience, were included in this study. physiotherapists who had musculoskeletal problems prior to the start of work, those who acquired musculoskeletal problems during their career because of other causes, such as motor vehicle injuries, sports injuries and trauma, and those who were in administrative positions without having significant involvement in daily clinical practice were excluded from the study. a self - administered questionnaire that was adapted from the nordic musculoskeletal questionnaire (nmq) was used to assess wrmds. a pilot study using the questionnaire was carried out among six physiotherapists, who responded well to the questionnaire, indicating the clarity of the questions asked. subjects were given two weeks to complete the questionnaire, and the completed questionnaires were returned to the respective managers for collection by the researchers. all subjects received an information sheet and signed an informed consent form prior to completing the questionnaire. ethical approval was granted by the research and ethics committee of the national university of malaysia and by the clinical research centre of ministry of health, malaysia. the data obtained were analyzed using the statistical package for social science (spss) version 14 software. the analysis was completed using descriptive statistics, and differences in responses between the subgroups of interest were identified using the chi - square test. out of a total of 105 questionnaires distributed, 81 questionnaires (77%) were returned. among the respondents who participated in the study, 22.2% (n = 18) were males, and the remaining 77.8% (n = 63) were females. furthermore, 48.1% (n = 39) were from klh, 34.6% (n = 28) were from ukmmc and 17.3% (n = 14) were from tarh. table 2 displays the occurrence of wrmds in general and the occurrence according to the subgroups of gender, bmi, work experience and clinical area of practice. the overall prevalence of wrmds reported among the therapists during the past 12 months was 71.6% (n = 58). the prevalence of wrmds was 73% (n = 46) among female therapists and 66.7% (n = 12) among male therapists. the chi - square analysis found significant differences between the proportion of female physiotherapists who reported wrmds and those who did not report wrmds (= 13.3, p 25 (= 9.0, p = 0.003) and the group with a bmi of 1825 (= 7.8, p = 0.006). analysis of wrmds in terms of the subgroups of clinical placements showed that the incidence of wrmds was highest among therapists working in pediatric areas (87.5%). the other clinical areas that reported higher rates of wrmds were cardiopulmonary practice (71.4%), neurology (70%) and musculoskeletal physiotherapy (71.4%). the body areas that were affected by wrmds and the physiotherapy techniques identified by the respondents that contributed to work - related injuries are shown in figures 1 and 2. the lower back was the site of the highest percentage (51.7%) of work - related injuries, followed by the neck (46.5%) and the thoracic spine (44.8%). the elbow (8.6%) and the hand - wrist (12.0%) were rarely affected by injuries. perceived manual techniques (58.6%) and the lifting or transferring of patients (41.3%) were listed as the two techniques with the largest contribution to work - related injuries in this study. this study found that the prevalence of wrmds among malaysian physiotherapists during the past 12 months was 71.6%, which is higher than the previously reported data from studies in other regions of the world.2,14 the higher 12-month prevalence observed in this study can be explained by the conditions under which physiotherapists practice in malaysia, particularly in public hospitals. because of the limited number of physiotherapists, most physiotherapy units are understaffed, such that a high clinical workload is unavoidable. although the ministry of health (moh) of malaysia has attempted to ensure that all basic hospital equipment is ergonomically appropriate, other factors, such as increased patient - to therapist ratios, limited therapist - patient contact time and therapists working in all specialties, might contribute to the higher incidence of wrmds. furthermore, because therapists were working with more than one patient at a time in some specialty areas, the high clinical work - load challenges the physiotherapists physically and makes them more susceptible to injuries.15 the prevalence of work - related injuries was significantly higher among female physiotherapists (73%, = 13.3, p = 0.000). this finding is in agreement with findings from several studies.5,7,16 these studies recorded wrmd prevalence among female physiotherapists to be in the range of 73% to 100%. in general, females are physically weaker than males, and this may place them at a disadvantage during patient care tasks, particularly when lifting and transferring patients.8 females are also exposed to pregnancy - related stress, which commonly affects the lower back region. even though the current study did not include any pregnant therapists, a previous study stated that changes in spinal posture and a weakening of joint structure related to a history of pregnancy increases the risk for wrmds.8 these reasons may explain why the female therapists reported a higher incidence of injuries than their male counterparts. physiotherapists with a bmi over 25 reported the highest prevalence of work - related injuries (80%). a significant difference was found between the proportion of physiotherapists who had wrmds and those who did not have wrmds in the group of patients with a bmi that was greater than 25 (= 9.0, p = 0.003). this finding is in contrast with that of a past study7 that reported a higher prevalence of wrmds among physiotherapists with a low bmi. although a review from a previous study13 found a weak association between being overweight and wrmds, with the exception of carpal tunnel syndrome, a recent work14 on wrmds noted that a high bmi was one of the important risk factors for the development of wrmds. the therapists who are overweight may not be physically active, such that they may be more susceptible to wrmds. furthermore, wrmds were more prevalent among pediatric physiotherapists and among those who practiced in musculoskeletal areas. in the pediatric specialty, all except one physiotherapist reported work - related injuries during the past 12 months, and 66.7% of the musculoskeletal physiotherapists reported wrmds during the same period. although the prevalence of injuries among pediatric physiotherapists is underreported in the literature, evidence of a high prevalence of wrmds among physiotherapists working in the musculoskeletal specialty is well documented.8 this result may be related to a higher use of manual therapy techniques. manual therapy has been implicated as a risk factor for wrmd, and physiotherapists who routinely performed manual therapies were 3.5 times more likely to have had musculoskeletal injuries than physical therapists who did not routinely perform manual therapies.8 the lower back region was the most common site for wrmds among physiotherapists (51.7%) in this study, followed by the neck (46.5%) and the thoracic region (44.8%). this result is consistent with findings from previous studies, which found that the prevalence of lower back wrmds during the past 12 months was between 45% and 69.8%.1,8 injuries to the lower back have been identified as the most prevalent type of wrmd among therapists, followed by injuries of the upper back and neck.7 the cause of the high incidence rate of lower back injuries among physiotherapists is directly related with patient - care activities, such as lifting and transferring patients, prolonged standing, frequent twisting and bending.16 the above claim is supported by our study because the physiotherapists who reported wrmds were involved in manual therapy techniques (58.6%), such as mobilizations, manipulations and massage, and lifting or transferring activities (41.3%) were the two most likely contributing factors to wrmds. the findings of this study support those of previous studies,7,10,17 which identified lifting patients, transferring patients and performing manual techniques as the top three problematic tasks that put physiotherapists at risk for injuries. it is also interesting to note that the high number of reports suggesting that manual therapy techniques are a contributor to wrmds do not explain the low percentage of wrist / hand wrmds recorded in this study. there is a possibility that the results suggesting that manual therapy was a contributor to wrmds among physiotherapists in this study were based on the stress to the spine due to prolonged standing while performing the task rather than the stress to the hands this study only involved physiotherapists from public hospitals and did not consider physiotherapists who worked in rural centers or private practices. issues may also be associated with the memory and ability to recall wrmds over the past 12 months. despite these limitations, the high response rate of the received questionnaires from the therapists is a strength of this study because earlier studies only had a response rate that ranged from 53% to 80%.7 this study concluded that the prevalence of work - related injuries among physiotherapists in malaysia is as high as those reported in other developed countries. the prevalence of wrmds is higher among females than males and higher for therapists working in the pediatric specialty. adequate preventive and appropriate management strategies are recommended to minimize work - related injuries in the physiotherapy practice. most of the previously published works from the western world and developed countries indicate that work - related musculoskeletal injuries among physiotherapists are very high and that it is a major health concern. this may be the first study that addresses the occupational injuries and health concerns of physiotherapists from a southeast asian perspective. because manual therapy practices among therapists are starting to emerge in malaysia and a large shortage of physiotherapists exists, this study attempts to expose the occupational health concerns and work - related challenges encountered by the therapists from a developing country such as malaysia. this current study identified that the rate of musculoskeletal injuries encountered by physiotherapists during their practice is far higher than previously reported rates from other parts of the world. the authors wish to thank all of the physiotherapists from kuala lumpur hospital, universiti kebangsaan malaysia medical centre and tuanku ampuan rahimah hospital for their participation in this study. | objectives : a cross - sectional study was conducted to measure the prevalence of work - related injuries among physiotherapists in malaysia and to explore the influence of factors such as gender, body mass index, years of work experience and clinical placement areas on the occurrence of work - related musculoskeletal disorders. methods : self - administered questionnaires adapted from the nordic musculoskeletal questionnaire were sent to 105 physiotherapists at three main public hospitals in kuala lumpur, malaysia. the questionnaire had 12 items that covered demographic information, areas of musculoskeletal problems and physiotherapy techniques that could contribute to work - related musculoskeletal disorders. the data obtained were analyzed using the statistical package for social science version 14 software. results : the overall prevalence of work - related injuries during the past 12 months was 71.6%. female therapists reported a significantly higher prevalence of work - related musculoskeletal disorders than the male therapists (73.0%, p 25 (2 = 9.0, p = 0.003) and the group with a bmi of 1825 (2 = 7.8, p = 0.006). manual therapy (58.6%) and lifting / transfer tasks (41.3%) were the two physiotherapy techniques that most often contributed to work - related musculoskeletal disorders. conclusion : work - related injuries are significantly higher among the physiotherapists in malaysia compared with many other countries. female therapists reported a higher incidence of work - related musculoskeletal disorders in this study, and work - related musculoskeletal disorders were more common among therapists working in the pediatric specialty. this study contributes to the understanding of work - related disorders among physiotherapists from a southeast asian perspective where the profession is in its development stage. |
spinal subdural hematoma (sdh) is a rare clinical entity that frequently occurs in the thoracic regions of the spine. this condition can be caused by major or minor trauma and iatrogenic causes such as ones resulting from spinal puncture performed for diagnostic- or anesthetic - related purposes2,5). it can also occur spontaneously in patients with coagulation abnormalities, underlying neoplasm, or arteriovenous malformation7). however, spontaneous cervical spinal sdhs occurring in the absence of these underlying conditions have been very rarely reported11,12). here we report a case of spontaneous non - traumatic acute sdh with normal hemostatic parameters presenting as neck pain and hemiparesis. magnetic resonance images (mri) was fully diagnostic and the patient was successfully treated with conservative management. we also present a discussion of the etiology, pathogenesis, and prognosis of this rare pathologic entity. a 48-year - old woman with neck pain and motor weakness was referred to our emergency room. the patient had no previous history of anticoagulant therapy or hematological coagulopathy, and was in good health before this episode. on physical examination, she was alert and fully oriented. no neurologic abnormalities were found in the cranial nerves and cerebellar system. however, sensory testing of right arm and leg demonstrated loss of touch along with pin - prick and vibration sensitivity. motor examination revealed hemiparesis on right side (grade iii on arm and grade i on leg). the patient 's coagulation profiles including platelet count, prothrombin time, and partial thromboplastin time were within normal ranges. the sagittal t1 and t2 weighted images revealed a dorsolateral subdural hematoma extending from c3 to c5 presenting as high signal in both sequences. the axial images showed left displacement of the spinal cord at this level (fig. there was no signal void area, indicating that there were no abnormal vessels in the lesion. based on these mr findings, the patient was diagnosed with an acute cervical spinal sdh in the right side at c3-c5 level. for differential diagnosis to distinguish this case from other conditions such as cervical epidural hematoma, a lumbar spinal puncture was performed. about 20cc of csf was drained and cerebrospinal fluid (csf) analysis revealed 190,000 red blood cells / mm and 267 white blood cells / mm (fig. 2). an urgent laminectomy and hematoma removal was planned, but the patient 's motor weakness gradually resolved during surgical preparation. at 5 hours after the symptom onset, her right side hemiparesis improved to grade iv. the next day, her muscle power on the right side had fully recovered and she was able to walk without any assistance. mr images obtained 10 days after admission revealed that the spinal sdh had completely resolved (fig. 3). the patient was in good health and free of neurological deficits during the 10 months follow - up period. acute spinal sdh is a rare pathological entity that is caused by compression of the spinal cord or cauda equina1). although there is no evidence of any underlying causes and this type of hematoma appears spontaneously, several authors have reported that hematological disorders, anticoagulant therapy, spinal puncture, or traumatic brain injury predispose individuals to developing spinal sdh3,5). acute spontaneous sdhs appearing in the absence these predisposing conditions are very rarely reported and are frequently located in the thoracic spine2). unlike the epidural space and intracranial anatomy of the dura, the spinal subdural space does not contain major blood vessels or bridging veins. instead, the vasculature consists of only a delicate network of vessels that run along the lateral margins of the dura. moreover, relatively larger and more vascularized epidural fatty tissues are present and the spinal dura does not adhere to bone. the causes and origin of this type of hemorrhage in the subdrual space are still undefined. rader.11) proposed that sudden and sharp increases in abdominal and thoracic pressure elevate intravascular pressure in the spinal subdural space to the extent that a momentary disparity between intravascular and extravascular pressure could result in the tearing of a spinal vessels. when cerebrospinal fluid (csf) pressure is lower than intravascular pressure, vessels rupture and result in subarachnoid hemo- rrhage. the same sudden increases in pressure can cause rupturing of small extra - arachnoidal vessels located on the inner surface of the dura, resulting in a subdural hemo- rrhage. several authors have described the presence of combined subdural and subarachnoid hemorrhages in patients9,13). this has led to the suggestion that subdural hemorrhage may originate in the more vascularized subarachnoid space and pass through the thin and delicate arachnoid membrane. conversely, the hematoma can originate in the subdural space and then pass through the arachnoid membrane, giving rise to a concurrent subarachnoid hemorrhage. there are questions as to whether this small delicate network of vessels can be the source of hemorrhage4,8). in the present case, we could n't confirm the presence of blood in both spaces because surgery was not performed. although the degree of symptoms varies from mild motor - sensory deficit to serious sphincter deficits, spinal sdh can rapidly compromise the spinal cord or cauda equina and result in paralysis or acute deterioration with severe neurologic deficits. mri is considered to be the best imaging technique for the diagnosis and follow - up of patients with spinal sdh6,10). this modality best depicts the extent of the hemorrhage and delineation from the epidural space. in commonly - used t2 weighted turbospinecho sequences, recently - developed hematomas are hyperintense to the spinal cord and iso - intense to csf. in t1 weighted sequences, the blood is mostly iso - intense to the spinal cord and only slightly brighter than csf. alteration of the spinal cord is less conspicuous than in epidural hemorrhage because the distribution of the blood is semicircular with subsequent compression instead of displacement. the majority of hematomas are located ventral to the spinal cord or circumferentially with a major ventral part. however, it is difficult to differentiate between a subdural and epidural hematoma on the spine in some cases. in such instances, lumbar tapping is a useful diagnostic tool to determine the exact location of the hematoma5). it is widely accepted that prompt surgical evacuation should be performed before permanent irreversible damage to the spinal cord however, we did not perform emergency surgical decompression due to the early progressive improvement of the patient.. however, the patients who exhibit only mild neurologic disturbances or early progressive improvement are likely to benefit from conservative treatment. | spontaneous cervical sdh with no underlying pathology is a very unusual condition. to the best of the authors ' knowledge, only two cases have been previously reported. a 48-year - old female patient was admitted to our emergency room due to severe neck pain following standing up position with rapid onset of hemiparesis. mri revealed a dorsolateral subdural hematoma from c3-c5 with cord compression. an emergency laminectomy was planned, but motor weakness gradually improved during surgical preparation. the patient showed substantial clinical improvement and complete recovery was confirmed after 7 days of conservative management without surgical treatment. to determine a differential diagnosis distinct from other conditions such as cervical epidural hematoma, a lumbar spinal puncture was performed. follow - up mri performed 10 days after admission revealed complete resolution of the hematoma. we report an extremely rare case of spontaneous cervical spinal subdural hematoma (sdh), present a review of relevant literature, and discuss the etiology, pathogenesis, and prognosis of this case. |
is often neglected despite its common occurrence in endemic areas.1,2 if detected at an early stage, fascioliasis can be treated successfully ; however, the disease in the advanced stages can cause liver fibrosis, hepatic cirrhosis, and sometimes death.3,4 therefore, early detection and treatment are important. however, owing to its nonspecific symptoms and observational findings, human fascioliasis is difficult to diagnose.57 some clinicians have limited experience with human fascioliasis in nonendemic countries. and this situation leads to misdiagnosis or delayed treatment. we herein report a case of fascioliasis in a traveler from jakarta, indonesia, who was diagnosed at an early stage based on a detailed history of the patient, eosinophilia, and the characteristic findings on computed tomography (ct), resulting in successful treatment without any sequelae. a 46-year - old japanese woman without a medical history expatriated to jakarta, indonesia, for 5 years. she lived in an upper - class residential area, in a clean environment. around the time she returned to japan, 1.5 months prior to her visit to our clinic, the patient started experiencing a dry cough, which subsided after several days. one month prior to her visit, she developed a fever of 40 c that persisted for several days accompanied by malaise, followed by fever and malaise every week. the patient had no history of consumption of raw seafood ; however, the patient had been eating organic raw vegetables on a daily basis, including watercress. at admission, her body temperature was 35 c, vital signs were stable, and the liver was enlarged and tender. she had eosinophilia (eosinophil count, 3,465 cells/l) and mild hepatic dysfunction (aspartate transaminase, 37 u / l ; alanine transaminase, 60 u / l ; -glutamyl transpeptidase, 70 u / l ; and alkaline phosphatase, 398 u / l). three of the stool specimens were negative, the latter for ova, cysts, and parasites, examined under a microscope. enhanced ct showed multiple microabscesses arranged in a tractlike pattern with ring enhancement in the right lobe of the liver (fig. endoscopic retrograde cholangiopancreatography did not show worms in the bile duct, and no ova were detected in the bile examined under a microscope. the diagnosis of fascioliasis was confirmed by demonstration of specific antibodies to fasciola sp. using the ouchterlony double - diffusion test (fig. 2).8 for treatment, 500 mg (10 mg / kg) triclabendazole (egaten, novartis pharma ag, switzerland) was orally administered two times at 12-h intervals. her eosinophil count decreased to 1,966/l in 1 month, 624/l in 2 months, and 273/l in 3 months, reaching the normal range. contrast - enhanced ct performed a year later showed almost complete disappearance of the multiple nodular lesions (fig. fascioliasis is a common trematodiasis distributed worldwide mainly in human - endemic areas and animal - endemic areas.9 however, in several nonendemic countries such as japan, most clinicians lack experience and knowledge about human fascioliasis. therefore, the disease can sometimes be excluded from differential diagnosis and overlooked.6,7 adult fasciola spp., the causative agent for fascioliasis, inhabit the bile duct of ruminants such as cattle and sheep, which then pass their ova in stool. the miracidium develops and invades a suitable snail (galba / fossaria group, genus radix and pseudosuccinea) as an intermediate host. in the snail, the cercaria are released from the snails and encyst as metacercaria on aquatic plants such as watercress.3,5 humans can accidentally become infected as the final host by eating raw aquatic plants or drinking water contaminated with metacercaria.1 therefore, the incidence of fascioliasis is considered scarce in urban areas but concentrated in rural areas, where these conditions are likely to be met.4 the present patient had lived in a clean environment in an urban area of jakarta. thus, the risk of becoming infected with fasciola spp. would have been considered low. however, she had routinely consumed organic raw vegetables grown at an organic farm that also raised cattle. therefore, contaminated organic raw vegetables were considered the source of infection. when ingested by humans, immature larvae invade the abdominal cavity via the small - intestinal wall 2 hours after ingestion, grow into juveniles, reach the liver within 6 days after excystment, and migrate into the liver through the hepatic capsule, causing the formation of linear cystic lesions.1 in the liver, juveniles migrate for 56 weeks. this is considered the invasive or acute phase1,10 and is distinctive on ct,11 as in the present case (fig., the patient might experience right hypochondrial pain, fever, nausea, vomiting, and cough, and eosinophilia is common.1 because the patient in the present case exhibited all these characteristics, she was considered to be in the invasive or acute phase. the biliary phase follows next, when juveniles migrate into the bile duct where they become sexually mature and deposit eggs at least 6 months from the beginning of the invasive or acute phase.1,10 it is possible to detect the eggs in both stool and bile. chronic - stage fascioliasis may be asymptomatic but can develop into irreversible liver fibrosis or liver cirrhosis as a result of recurrent cholangitis caused by worm - induced biliary obstruction.3 adult worms can also cause irreversible lesions by invading various organs other than the liver, such as the gastrointestinal tract, abdominal wall, lung, heart, brain, and eye (ectopic fascioliasis).3 because of the effects in the more advanced biliary phase, it is important to detect and treat fascioliasis early. while the presence of worms or ova provides a direct diagnosis of fascioliasis, a patient in the invasive or acute phase does not have worms in the bile duct or ova in the bile. therefore, even if worm or ova is negative, this result could not rule out the possibility of fascioliasis. if the clinician does not have knowledge of the lifecycle of fascioliasis, diagnosis is difficult during the invasive or acute phase, which can consequently lead to a misdiagnosis or delayed diagnosis.5 although fascioliasis during the invasive or acute phase can only be definitively diagnosed via serological testing, contrast - enhanced ct can sometimes significantly help, as in the present case. however, clinicians or radiologists who have limited experience with human fascioliasis often misdiagnose lesions observed on contract - enhanced ct as a malignant tumor, causing the patient to undergo liver biopsy or hepatectomy.6,7 therefore, it is very important that clinicians and radiologists outside endemic regions be aware of the typical contrast - enhanced ct findings of fascioliasis.6 triclabendazole is the first - line treatment for fascioliasis,1 with a cure rate of 78%100% at doses of 10 mg / kg.12 unlike other helminth infections, treatment with praziquantel is not effective. triclabendazole resistance concerned livestock in animal - endemic areas.1 fortunately, the symptoms of our patient disappeared rapidly and eosinophilia and ct findings were improved, and there was no triclabendazole in our case. the present case with fascioliasis was diagnosed and treated at an early stage of infection based on a detailed patient history, eosinophilia, and typical ct findings. fascioliasis is no longer a disease limited to human - endemic areas or animal - endemic areas. fascioliasis should be considered in the differential diagnosis of any patient who consumes raw vegetables and presents with gastrointestinal symptoms and eosinophilia, regardless of the patient s residence and living situation. the characteristic contrast - enhanced ct findings may provide a clue to the potential presence of fascioliasis, and treatment should be initiated immediately after a serological diagnosis. | a 46-year - old japanese female expatriate living in jakarta presented with intermittent fever lasting for a month. although she was considered at low risk of fasciola spp. infection because she lived in an upper - class residential area of the city, the patient presented with eosinophilia after consuming organic raw vegetables ; in addition, contrast - enhanced computed tomography detected microabscesses in a tractlike pattern in the liver. these findings led to an early diagnosis of fascioliasis, which was successfully treated without sequelae. in any patient with a history of consuming raw vegetables, fascioliasis should be suspected regardless of where the patient has lived. |
pure ligamentous flexion - distraction injury of the lumbar spine is a relatively rare clinical entity comprised of transverse disruption of the disc combined with tension splitting of posterior column. it has been first described by chance in 1948 as a result of flexion over a fulcrum positioned anterior to the spine. it is largely related to the use of lap belt restrains during motor vehicle accidents, when large forces are transmitted through the abdominal cavity often causing concomitant visceral injuries. neurologic deficit is rare as majority of cases involve lumbar levels, below the spinal cord. we report a 19-year - old female backseat passenger wearing a lapbelt who was involved in a head - on collision when the sport utility vehicle (suv) she rode in fell into gully. on admission she was paraplegic, with t12 sensory level and no motor and sensory function of s4-s5 (american spinal injury association (asia) a). plain x - ray [figure 1 ] and computerized axial tomography (cat) spine showed a flexion - distraction injury at the l3-l4 level, with a small fragment of the l4 vertebral body avulsed [figure 2 ]. after ruling out intra - abdominal injuries, patient was taken into surgery within 24 h after accident. immediately after skin incision further dissection of the subcutaneous tissue revealed that fascia was disrupted and within the interspinous space a neural structure with tangled nerve fibers was found [figure 3 ]. on closer inspection it was identified as a conus medullaris, which was wholly severed from the spinal cord. it was removed and laminectomy was performed to attempt dural repair. despite efforts, dura was not found as it probably recoiled into the upper portions of spinal canal as a result of injury. the pedicle screws were placed into l2-l5 vertebral bodies, with subsequent l3-l4 endplate preparation and implantation of an interbody spacer. autologous bone locally procured from facet joints mixed with synthetic bone substitute was used to fill in the interbody space. the postoperative course was complicated by formation of a large subcutaneous hematoma which developed despite placement of a wound drain. the wound was revised 3 days after initial surgery, hematoma was evacuated, and skin was closed in multiple layers. some 7 days later, patient developed a cerebrospinal fluid (csf) leak through the incision ; it was treated with a lumbar drainage (placed at l2 level) and prolonged bed rest. eventually the patient made a satisfactory recovery and 12 months after surgery was able to walk with a knee - ankle - foot orthosis. she had no motor function below knees, no sensation below l2, and no voluntary bladder control. x - ray taken in lateral decubitus position ct showing only modest malalignment and the naked facet sign, visible in all planes (arrows). chance type flexion - distraction injuries are rare, the incidence among entire series of thoracolumbar fractures ranges from 5 to 15%. they are a consequence of the hyperflexion of the spinal column around the fulcrum placed in front of the abdomen. typically this occurs as a result of injuries sustained while wearing the seat belt, especially the lap belt. the more anterior position of the axis of rotation, the greater distracting force acting on adjacent segments with a much lesser axial component. flexion - distraction injuries of the spine are often accompanied by serious visceral injuries which should be ruled out in each and every case. although this type of injury is considered highly unstable, neurological deficit is rare, and if present, it is usually caused by direct injury of lower thoracic to upper lumbar levels. in our case the deficit was a result of indirect injury : most likely the avulsion of the conus medullaris was caused by the sudden deceleration with subsequent distraction and eventual failure of ligamentous elements followed by substantial traction of the conus by the nerve roots. recent article by rade., provides an elegant illustration to support this hypothesis. it was found that during the straight leg raise (slr) test the conus descends by more than 2 mm. it is plausible to expect far greater dislocation with force acting upon nerve roots during abrupt deceleration in a restrained patient with both hips flexed. to our knowledge there have been only a handful of similar cases reported in literature, all among children and adolescents who sustained injuries in motor vehicle accidents. the authors of relevant articles and reviews emphasize the violent mechanism of injury and a significant risk of intra - abdominal visceral injuries. as the lap belts have been gradually phased out, the incidence of the type of injury described in this article is likely to decline. nevertheless both the practitioners and the motor vehicle users should be aware of potentially dreadful consequences of inadequate or improperly worn seatbelts. | pure ligamentous flexion - distraction injuries of the lumbar spine are relatively rare and even less commonly associated with neurologic compromise. they are largely related to the use of lap belt restrains during motor vehicle accidents. we report a 19-year - old female backseat passenger wearing a lap belt who was involved in a head - on collision. on admission she was paraplegic, with a t12 sensory level and no motor and sensory function of s4-s5 (american spinal injury association (asia) a). plain x - ray and computerized axial tomography (cat) spine showed a flexion - distraction injury at the l3-l4 level. during surgery in the interspinous space a conus medullaris was identified, which was completely severed from the spinal cord. the patient underwent a fusion procedure and made a good recovery. twelve months after surgery she was able to walk with a knee - ankle - foot orthosis, she has no motor function below knees, no sensation below l2, and no voluntary bladder control. although described type of injury is very rare, one should always have in mind devastating consequences of inadequate or improperly worn seatbelts. |
postmenopausal osteoporosis (pmop) is a highly prevalent disease, characterized by reduced bone mass, leading to increased bone fragility and fracture risk, caused by estrogen deficiency. a lot of recent reports provide evidence that the wnt/-catenin (canonical) signaling, one of the three known pathways of wnt signaling, may be implicated in pathogenesis of postmenopausal osteoporosis. the wnt/-catenin canonical pathway is modulated by a number of factors that include dickkopf (dkk-1) and sclerostin, which compete with the wnt/-catenin for binding to lrp5/6, disrupting (dkk-1) or antagonizing (sclerostin) lrp5/6 mediated wnt signaling. receptor activator of nf-b ligand (rankl) is highly expressed on the surface of bone marrow stromal cells (bmscs) and preosteoblasts. when rankl binds to rank, osteoclast osteoprotegerin (opg), produced by bmscs and osteoblasts, is a soluble decoy receptor to inhibit rank - rankl - mediated osteoclastogenesis [7, 8 ]. de toni. reported that opg expression is regulated by -catenin in colon cancer cells. recent evidence implicated that sclerostin, a major wnt/-catenin antagonist, stimulates expression of rankl [10, 11 ]. based on recent studies, we hypothesize that some relationships may exist between wnt/-catenin signaling and rankl / rank / opg system in pmop. to demonstrate the role of wnt/-catenin canonical pathway in postmenopausal osteoporosis, we evaluated the levels of serum -catenin, opg, rankl, sclerostin, and bone turnover markers in postmenopausal osteoporotic patients and compared them to those in postmenopausal nonosteoporotic women. in addition, we analyzed the relationships of -catenin with opg, rankl, the ratio of rankl / opg, sclerostin, and bone turnover markers. our cross - sectional study included 480 postmenopausal women with osteoporosis and 170 healthy postmenopausal women as a control group. according to the principle of statistics, the pmop patients were enrolled from 4 hospitals (2 in hubei province, 1 in jiangxi province, and 1 in jilin province in china) by advertisement recruiting during the period of december 2009 and march 2012 in china. eligible pmop participants were required to have a natural menopausal history of 210 years and a bmd t - score of < 2.5 at the lumbar spine using dual - energy x - ray absorptiometry (dxa). exclusion criteria were as follows : (1) treatment with calcitonin, bisphosphonates, raloxifene, estrogen, or estrogen / progestogens within 12 months, (2) coexistence of any other metabolic bone disease except for osteoporosis, (3) severe chronic disease, including malignancy, (4) medication that could affect bone metabolism, (5) previous radiation therapy, and (6) abnormal liver and kidney function tests. healthy postmenopausal women were enrolled from physical examination center in union hospital, tongji medical college, huazhong university of science and technology. after a medical examination, they were excluded from osteoporosis and other diseases affecting bone metabolism. the protocol was approved by the responsible clinical trial ethics committee. written informed consent was obtained from each participant. in all subjects, we measured height and weight and calculated body mass index (bmi) using the quetelet formula (weight in kilograms divided by the square of height in meters). bone mineral density (bmd) was measured for the anteroposterior lumbar spine (l1l4) by dual - energy x - ray absorptiometry (dxa) (lunar prodigy advance ; ge healthcare, madison, wi, usa). a control phantom was scanned every day, and all dxa measurements were performed by experienced operators in every hospital. osteoporosis was defined as a t - score of < 2.5 at the lumbar spine. venous blood samples were taken in the morning between 8:00 am and 9:00 am after an overnight fast. r / min within 30 minutes, and the serum was separated and stored at 80c prior to analysis. serum -catenin, rankl, opg, and sclerostin levels were measured by enzyme - linked immunosorbent assay (elisa, yanhui biotechnology co., ltd., the minimum detectable amount of human -catenin and opg kit was less than 1.0 pg / ml. serum -isomerized c - terminal crosslinking of type i collagen (ctx), intact n - terminal propeptide of type i collagen (pinp), n - mid fragment of osteocalcin (n - mid - ot), and 25-hydroxyvitamin d (25(oh)d) levels were measured using automated roche electrochemiluminescence system. serum levels of -catenin and other parameters between patients and controls were compared by independent - samples t - test. spearman 's coefficient of correlation was used for correlation between serum levels of -catenin and other parameters in both groups. multiple regression analysis was used to determine the influence of one independent variable after correcting for others. the main characteristics and laboratory data of the study population were listed in table 1. there was no statistically significant difference between patients and controls for age, weight, height, and bmi. ctx and pinp serum concentrations were higher in postmenopausal osteoporotic women than in nonosteoporotic postmenopausal women. there was no significant difference between patients and controls for serum estradiol levels, n - mid - ot levels, 25(oh)d levels, and opg levels. the differences for -catenin, sclerostin, and rankl levels between two groups were significant, with p < 0.001. we examined the correlations between serum -catenin levels and various parameters in patients and controls. as shown in table 2, serum -catenin levels were positively correlated with opg and negatively correlated with sclerostin, the ratio of rankl / opg, bmi, and bmd. no correlation between serum -catenin and age, estradiol, n - mid - ot, 25(oh)d, ctx, pinp, and rankl was found. -catenin was designated as the dependent variable, whereas age, bmi, bmd, estradiol, n - mid - ot, 25(oh)d, ctx, pinp, sclerostin, opg, and rankl were included as independent variables. in this analysis, bmd, pinp, sclerostin, and opg were found to be independent predictors of serum -catenin levels in pmop, after adjusting for age, bmi, estradiol, n - mid - ot, 25(oh)d, ctx, and rankl (table 3). scattered dots (figures 1, 2, 3, and 4) showed the correlations of serum -catenin levels with opg, rankl, the ratio of rankl / opg, and sclerostin, respectively. the adult skeleton undergoes continuous remodeling through tight coupling of opposing bone - resorbing osteoclasts and bone - forming osteoblasts. the contributing elements to the function of bone homeostasis are regulated hierarchically through a series of cell signals, cross - talk, and cascades, essentially focused on members of the tumour necrosis factor superfamily rankl and its receptors, rank and opg [1214 ]. during normal bone remodeling, rankl binds to the rank transmembrane receptor on osteoclast precursors and induces differentiation and activation. opg, also produced by bmscs and osteoblasts, is a soluble member of the tumor necrosis factor receptor family (tnfr family), inhibits the differentiation and fusion of the osteoclastic precursor cells, and blocks the activation of mature osteoclasts. thus, targeting the rankl / rank / opg system should produce potent effects on osteoclast differentiation and function. recent findings have shown that the wnt/-catenin canonical pathway in osteoblasts / stromal cells suppresses osteoclastogenesis through the upregulation of opg expression and the downregulation of rankl expression [16, 17 ]. the cross - sectional study confirms that -catenin is detectable in human serum, as observed very recently by gaudio. in patients with type 2 diabetes mellitus (t2 dm), and indicates that patients with pmop have -catenin serum levels lower than controls. -catenin, a pivotal signaling molecule of the wnt pathway, has been shown to be important in osteoblast differentiation, proliferation, and apoptosis. recent evidence has indicated that the wnt/-catenin pathway plays an important role in skeletal development and growth [21, 22 ], particularly in bone mass acquisition, remodeling, differentiation, and maintenance [23, 24 ]. the importance of the canonical pathway in bone biology has been emphasized by the identification of a link between bone mass and mutations in the lrp5 gene. loss - of - function mutations in lrp5 reduce the number of osteoblasts and cause osteoporosis [25, 26 ]. canonical wnts (wnt3a) bind to the receptor complex of frizzled and lrp5 or lrp6, inhibit gsk-3, and promote the accumulation of -catenin in osteoblasts. the accumulated -catenin translocates into the nucleus and together with tcf / lef induces the expression of opg to inhibit rank - rankl - mediated osteoclastogenesis. in our study, we detected higher rankl serum concentration and concomitantly similar opg serum concentration at the protein levels in pmop patients compared to controls. this suggested that there was a more seriously impaired balance between osteoblastic bone formation and osteoclastic bone resorption in patients with pmop. in our study, we found a significant negative correlation between -catenin and the ratio of rankl / opg. it suggested that some cross - links were present between wnt/-catenin signaling pathway and rankl / rank / opg system and provided evidence that increased rankl / opg expression was related to reduction of wnt/-catenin signaling activity in pmop. we also found a significant negative correlation between -catenin and sclerostin, which in fact agrees with a major contribution of sclerostin to the impairment of the wnt/-catenin signaling pathway in this setting. sclerostin, an osteocyte - derived and -secreted glycoprotein, has been shown to influence the activity of wnt signaling pathways. according to the current knowledge, sclerostin antagonizes the canonical wnt pathway by preventing the formation of the wnt - frizzled - lrp5 complex by competitively binding to lrp6 and lrp5, transmembrane proteins that together with frizzled receptors mediate the actions of wnts [4, 3033 ]. beyond that, sclerostin was found to interact with several other wnt pathway regulatory molecules such as secreted frizzled related protein 4 (sfrp4), casein kinase ii, and traf2- and nck - interacting kinase [3437 ]. both ctx and pinp significantly increased in pmop cohort, confirming that higher bone turnover took place in this population compared to controls. however, no association between the -catenin and ctx and the pinp was observed in both the pmop group and the control group.. showed that there were significant correlations of serum -catenin levels with age and serum sclerostin levels in t2 dm patients and with age in controls. our cohort in the study was limited to postmenopausal women with the age of 50.565.5 years old. in our pmop cohort, it is difficult to explain the negative correlation of serum -catenin levels with lumbar spine bmd. for the contradictory phenomenon another interesting thing was that we observed a negative correlation between serum -catenin and bmi. the result may be related to the role of wnt/-catenin signaling on inhibiting adipogenesis of mesenchymal precursors [38, 39 ]. in our control group, we found no associations of -catenin with opg and the ratio of rankl / opg. we hypothesized that the function communication between wnt/-catenin signaling and rankl / rank / opg system was not established in postmenopausal non - osteoporosis women. it was a cross - sectional design, and the causative nature of the associations between -catenin, rankl / opg, and other variables can not be established. we analyzed serum -catenin, rankl / opg levels which may not be sensitive enough to reflect their expression in bone cells. given the existence of multiple pathogeneses of pmop, the recognition of the role of wnt/-catenin signaling on bone metabolism has stimulated a number of studies on the effects of this signaling on treatment of pmop. however, the effects of wnt signaling on bone metabolism and the involved molecular mechanisms remain unclear. according to our findings, functional communication between rankl / rank / opg system and wnt/-catenin signaling pathway plays an important role in postmenopausal osteoporosis. targeting the wnt/-catenin signaling to change the ratio of rankl / opg to alter bone turnover | objective. to demonstrate the role of wnt/-catenin canonical pathway in postmenopausal osteoporosis by evaluating serum -catenin levels in patients with postmenopausal osteoporosis and analyzing their possible relationship with serum opg, rankl, the ratio of rankl / opg, sclerostin, and bone turnover markers. methods. 480 patients with postmenopausal osteoporosis and 170 healthy postmenopausal women were enrolled in the study. serum -catenin, opg, rankl, and sclerostin levels were measured by enzyme - linked immunosorbent assay. bone status was assessed by measuring bone mineral density and bone turnover markers. estradiol levels were also detected. results. serum -catenin levels were lower in postmenopausal osteoporotic women compared to nonosteoporotic postmenopausal women (26.26 14.81 versus 39.33 5.47 pg / ml, p < 0.001). serum -catenin was positively correlated with osteoprotegerin (r = 0.232, p < 0.001) and negatively correlated with the ratio of rankl / opg, body mass index, and sclerostin (r = 0.128, p = 0.005 ; r = 0.117, p = 0.010 ; r = 0.400, p < 0.001, resp.) in patients with postmenopausal osteoporosis. conclusion. the results indicate that lower serum -catenin and concomitantly higher ratio of rankl / opg may be involved in the pathogenesis of postmenopausal osteoporosis. functional communication between rankl / rank / opg system and wnt pathways plays an important role in postmenopausal osteoporosis. |
although pregnancy rate after complex urological procedures is increasing due to better understanding and management of underlying problems, the literature about delivery in patients with augmentation cystoplasty (ac) is still limited. we are reporting an interesting case of delivery in a primigravida with solitary functioning kidney and the history of ac with ureteric reimplantation. throughout pregnancy she had two episode of febrile urinary tract infection (uti). she was planned for cesarian section due to obstetric indications. despite altered pelvic anatomy, we successfully did the lower segment cesarian section. we reviewed the literature regarding pregnancy in patients with ac to find that what the treating urologist and gynecologist should know about these rare cases. a 23-year - old female presented to us with history of frequency, bedwetting, and recurrent fever for last 10 years. the only significant past history was anti - tubercular treatment for pulmonary koch 's 12 years back. ultrasonography (usg) and intravenous urography showed right gross hydro - ureteronephrosis (hdun) with small contracted left kidney with severe calcifications. micturition cysto - urethrogram (mcu) showed no vesico - urethral reflux, but was suggestive of thimble bladder. cystoscopy showed small contracted bladder and ureteral orifices could nt be identified due to edematous changes. bladder biopsy showed granulomatous changes. in view of solitary functioning right kidney with gross hdun, percutaneous nephrostomy (pcn) we did right nephrostogram, which showed gross hdun with smooth tapering at the right vesicoureteric junction [figure 1 ]. double j stent was removed after confirming normal mcu without leak [figure 2 ]. preoperative percutaneous nephrostogram, (a) scout film showing right pcn and left calcified (putty) kidney. (b) arrow is indicating smooth narrowing at the vesico - urethral junction post - operative micturating cystourethrogram (mcu) showing normal augmented bladder with dj stent in situ she became pregnant after 1 year of ac. at that time, she had no urinary complaint and her continence was normal. throughout pregnancy she had two episode of uti, treated with intravenous antibiotics. her antenatal usg showed symmetrical intrauterine growth retardation with polyhydroamnios. on routine antenatal investigation she found to have hypothyroidism, for that the thyronorm (75 g) was started. throughout the pregnancy she maintained the normal serum creatinine levels. at full term, the induction of labor was tried, but was unsuccessful. as the pregnancy was high risk in view of primigravida with solitary functioning kidney, hypothyroidism, and the history of previous urological reconstruction, the cesarian section was planned in the presence of urologist. bowel loop, which was used for augmentation, was visible on right side of bladder and was undisturbed [figure 4 ]. incision was given over lower uterine segment and a live female baby of 2 kg was delivered. the post - operative course was uneventful and the patient was discharged on day 3. at 3 month intraoperative photograph showing incised lower segment and undisturbed bladder intraoperative finding : forceps indicating the bowel loop, which was used previously for augmentation ac in females is usually performed for small contracted bladder due to variety of inflammatory conditions, neurogenic bladder, idiopathic urge incontinence, or eneuresis. the first pregnancy in a patient with ac for tubercular cystitis was published in 1955. till date important studies of delivery in patients with augmentation cystoplasty the goal in management of these patients is delivery of a healthy baby with preservation of renal function. the main problems encountered in these patients are uti, hdn, or deterioration in renal function. asymptomatic bacteriurea occurs in 50 - 100% patients with ac but only 4 - 43% has significant uti. in pregnancy with ac this risk obviously increases due to the altered anatomy, pressure effect, and urinary stasis. hill. found the presence of uti in 9 of 15 (60%) patients in there review. several authors have suggested the use of long - term prophylactic antibiotics throughout pregnancy especially in patients with reflux. some authors have also suggested the urethral catheterization, stenting or even pcn for the management of severe infections during pregnancy. taniguchi. described a case in which they required drip infusion of ritodrine hydrochloride from 21 week of pregnancy to prevent premature contraction due to pyelonephritis. entero - cystoplasty is fixed cranially by the mesentery, laterally by ureter, and caudally by the trigone and urethra. when the uterus enlarges, it pushes the mesentery to one side and reaches to the ventral abdominal wall. during cesarian section the patients with ac who also undergone the vesical neck reconstruction or artificial urinary sphincter are particularly at the risk of damaging continence mechanism. hill. have found the malfunction of artificial urinary sphincter after vaginal delivery in there patient. on the other hand, quenneville. found no adverse effect of vaginal delivery on continence mechanism in two patients with rectus fascial sling, when he kept the bladder empty at the time of delivery. several authors have recommended the cesarian section in patients with ac with bladder neck reconstruction, whether the vaginal delivery is not considered a contraindication in patients with ac alone. another problem in patients with ac is metabolic complications like hypokalemic hyperchloremic metabolic acidosis, hypocalcemia, hypomagnesemia, and vitamin b12 deficiency, but we have not encountered these problems in our patient. regular urinalysis and aggressive treatment of any urinary infection, if found should be done. it is important to monitor the renal function and to look for hydronephrosis on usg. if cesarian section is planned, it should be preferably done in the presence of urologist, who is well familiar with the anatomy of augmented bladder. | a young female with history of genitourinary tuberculosis with solitary functioning kidney became pregnant 1 year after augmentation cystoplasty (ac) with ureteric reimplantation. throughout pregnancy she had two episode of febrile urinary tract infection. her renal function remained normal. she was planned for cesarian section due to obstetric indications. despite altered pelvic anatomy, we successfully did the lower segment cesarian section. we reviewed the literature regarding pregnancy in patients with ac to find that what the treating urologist and gynecologist should know about these rare cases. various complications which should be anticipated and measures to prevent them are also discussed. |
lung cancer is the leading cause of cancer deaths in the united states among both men and women.1 among various types of cancers, small cell lung cancer (sclc) carries the worst outcomes, and accounts for 10%20% of all bronchogenic carcinomas in the united states.2,3 since its early description as the oat - cell carcinoma of the mediastinum, the fatality of sclc has not changed considerably,4 and early diagnosis and initiation of treatment for lung cancer remains a considerable problem plaguing medicine today. sclc continues to carry a grim prognosis, with median survival of approximately 24 months when untreated, and a 5-year survival rate in the range of 4%5% when treated.3,5 in view of the high mortality associated with this disease, it may be time to examine other ways to detect sclc in its earlier stages, which will likely result in more favorable outcomes. given the rapid doubling time, sclc may present as an acute syndrome that has a decent potential of being misdiagnosed as an infectious or inflammatory condition. whether this clinical scenario has a significant impact on patient outcomes is yet to be determined, but will probably delay the prompt administration of chemotherapy, which is shown to improve survival in all stages of sclc. a pubmed and google search was performed and 22 articles were found to be relevant to our topic and were therefore included in this review. various imaging modalities are currently ineffective for screening for sclc, including computed tomography (ct).6,7 therefore, alternative diagnostic methods are becoming essential in order to identify sclc at its earliest stages. chest radiograph was proven to be ineffective after several randomized trials demonstrate no significant effect.8 a number of randomized clinical trials are currently under way in europe to investigate lung cancer screening using low dose chest ct. chest x - rays, chest ct scans, bone scans, and magnetic resonance imaging (mri) of the brain have been proven effective in the staging workup of sclc. recently, positron emission tomography has been shown to improve staging accuracy, but none of these imaging modalities have played an effective role in screening for sclc. although modern chest ct scans are able to detect smaller solid nodules, these lesions lack specific radiographic features, which render this method incapable of differentiating the origin of the nodules.9 however, certain nodules escape identification by chest x - ray or ct scan secondary to factors such as the technique used or small size of the lesion.10 the challenge begins with cancers that do not manifest preneoplastic lesions, making their detection at an earlier stage (when therapeutic intervention may affect survival) a difficult, if not impossible, task. in a study comparing the cost effectiveness of ct screening to smoking cessation rates or combined approaches, the cost - effectiveness of chest ct screening was linked to smoking cessation rates.11 in a recently published european study, maisonneuve found that recalibrating the bach model to include nodule characteristic and size and investigating the presence of emphysema through a chest ct was a suitable way to select the high risk population for ct screening.12 the absence of characteristic changes in bronchial epithelium associated with sclc creates false negative findings when bronchoscopy is used.13 wistuba studied surgical specimens from patients with different types of primary lung cancers, and significant genetic damage was found in the majority of patients with sclc who had normal or mildly abnormal bronchial epithelium and preserved histologic examination.13,14 more recently, new means of screening for sclc are being investigated, such as autoantibody markers15 and multi - variable clinical scores.16 evidently, in cases of high - risk nodules, histopathologic diagnosis should be considered. a 66-year - old caucasian woman who was an exsmoker was being evaluated as an outpatient for exertional shortness of breath that had worsened over the course of several weeks. the performed imaging studies included normal chest radiography and thoracic ct with no evidence of any parenchymal or mediastinal lesions as interpreted by the radiologist (fig. three months after the onset of symptoms, the patient presented to the emergency department for worsening dyspnea, which had been occurring at rest for two weeks. it was associated with yellowish productive cough with no chills, fever, or weight loss. the physical examination was unremarkable with the exception of diffuse expiratory wheezing and labored breathing. a left multilobar opacity was present on the chest radiography, suggesting pneumonia. on the fourth day of hospitalization, thoracic ct was performed and revealed several abnormalities in addition to the tumorous lesion itself (fig. part of these findings could be secondary to obstructive pneumonia, so identifying an accurate area of cancerous lesion spreading is difficult. after discussions with the radiologist, we assumed that the left hilar mass was probably between 7 and 8 cm with associated lymphangitic spread. additionally, an obstructive endobronchial mass in the upper and lingular left lobes was visualized and biopsied using flexible bronchoscopy. findings from the pathological examination of the biopsied specimen were indicative of sclc. given the initial normal chest ct, the doubling time was probably too rapid to preclude early detection and, therefore earlier, more effective chemo - radiation therapy. given her acute presentation and negative initial chest ct scan, the admission diagnosis was presumed to be severe pneumonia and the patient was started on intravenous antibiotics and other supportive measures. this management approach delayed chemo - radiation therapy for about five days and the patient expired a few days later from multiorgan failure. a 66-year - old caucasian woman who was an exsmoker was being evaluated as an outpatient for exertional shortness of breath that had worsened over the course of several weeks. the performed imaging studies included normal chest radiography and thoracic ct with no evidence of any parenchymal or mediastinal lesions as interpreted by the radiologist (fig. three months after the onset of symptoms, the patient presented to the emergency department for worsening dyspnea, which had been occurring at rest for two weeks. it was associated with yellowish productive cough with no chills, fever, or weight loss. the physical examination was unremarkable with the exception of diffuse expiratory wheezing and labored breathing. a left multilobar opacity was present on the chest radiography, suggesting pneumonia. on the fourth day of hospitalization, thoracic ct was performed and revealed several abnormalities in addition to the tumorous lesion itself (fig. part of these findings could be secondary to obstructive pneumonia, so identifying an accurate area of cancerous lesion spreading is difficult. after discussions with the radiologist, we assumed that the left hilar mass was probably between 7 and 8 cm with associated lymphangitic spread. additionally, an obstructive endobronchial mass in the upper and lingular left lobes was visualized and biopsied using flexible bronchoscopy. findings from the pathological examination of the biopsied specimen were indicative of sclc. given the initial normal chest ct, the doubling time was probably too rapid to preclude early detection and, therefore earlier, more effective chemo - radiation therapy. given her acute presentation and negative initial chest ct scan, the admission diagnosis was presumed to be severe pneumonia and the patient was started on intravenous antibiotics and other supportive measures. this management approach delayed chemo - radiation therapy for about five days and the patient expired a few days later from multiorgan failure. therefore, the clinical presentation of these cancers is likely to differ from those experienced with more typical cancers and becomes more like an acute pulmonary manifestation, such as in an inflammatory or infectious disorder. doubling time is calculated by estimating the volume of the nodule or tumor in two different dimensions.17 the doubling time is usually calculated using the following equation : where ti = interval time, di = initial diameter, do = final diameter, vi = initial volume, vo = final volume. given the neuroendocrinological origin of sclc, it is considered the prototype of rapidly growing malignancies with doubling time in the range of 25 to 217 days according to several studies.10,1820 a described by wang, the doubling time of sclc ranges from 54132 days.9 in a study by son, the doubling time for sclc was 38 to 217 days and all patients were smokers.20 in addition, arai, kuroishi21 used serial chest x - ray films to study the doubling time of lung cancers in relation to prognosis and found that sclc mean doubling time was 86.3 days. sclc and large cell lung cancers represented most patients in the rapidly growing cancer group, which was defined as those having a doubling time of 109.6 days. it has been noted that the rate of growth in the size of the lung nodules is much faster for malignant versus benign nodules.22 many factors affect the rate of growth of these nodules, and hence should be taken into consideration when determining the frequency and type of imaging studies for followup. generally, among patients who have lung cancers with a rapid doubling time, sclc had the shortest doubling time, followed by squamous cell carcinoma and adenocarcinoma.9 eventually, the histological subtype of the tumor as well as the smoking status should be assessed in order to make suggestions concerning follow - up. in instances where sclc doubling time is exceptionally rapid, the clinical presentation can mimic other acute lung pathologies such as pneumonia and other acute interstitial and inflammatory lung diseases. among smokers who present with acute pulmonary manifestations, rapidly growing sclc can be an over - looked diagnosis. to date, screening for sclc is still an outreach. imaging techniques for detecting sclc at the curative or early stages seems to be ineffective and alternative methods are being investigated. | small cell lung cancer (sclc) is one of many types rapidly growing malignant diseases, such as burkitt s lymphoma and testicular germ cell cancers. at present, there is no reliable way to screen for sclc, and imaging modalities tend to be delayed in detecting this type of cancer. the clinical presentation of acutely and rapidly growing sclc can mimic those of pulmonary inflammatory or infectious disorders, and in some instances, this delays appropriate management and negatively affects patient outcome. |
a 40-year - old female patient was referred to ukurova university faculty of dentistry with the requisition of orthodontic treatment and odontalgia due to dental caries. medical anamnesis of the patient revealed that she had ts accompanying prolactinoma and osteoporosis (fig. the patient was also receiving care from the endocrinology, neurology, and physical and rehabilitation medicine departments at the university hospital. a general physical examination showed that the patient height was 140 cm and weight 40 kg with normal gait and vital signs. according to the patient 's medical history, it was revealed that she had undergone ear surgery due to otorrhea and recurrent otitis media at the age of 6. she had been diagnosed with ts at the age of 12 by endocrinologists due to the patient 's complaints of amenorrhea and shortness in stature. at the age of 16, the patient had been on medical treatment (dopamine agonist therapy) for a period of about 8 years for the tumor. she had responded to the medical treatment well without needing any surgical treatment. however, the last cranial magnetic resonance imaging (mri) of the patient showed a recurrence of the prolactinoma. she has been continuing to follow up at the neurology clinic and receiving medical treatment with a dopamine agonist for her microadenoma (fig. the patient 's general clinical symptoms were hirsutism, amenorrhea, congenital speech difficulties, partial hearing loss, sinusitis, headache, neck and throat pain, and dysphagia (fig. 3). she used calcium supplements, oral bisphosphonate, and vitamin d for osteoporosis ; ethinyl estradiol for increasing growth velocity ; cyproterone for hirsutism ; and lansoprazol for peptic ulcer. the intraoral examination and panoramic radiograph demonstrated that the patient had malocclusion, maxillary micrognatia, high and narrow palatal arch, retrusive mandible, bifid uvula, prevalent microdontia, hypoplastic first maxillary premolars, and short dental roots (figs. 4 and 5). we utilized the patient 's cbct (iluma cone - beam ct scanner, imtec imaging l.l.c., ardmore, ok, usa) scan, which was performed for orthodontic evaluation, and detected a number of craniofacial abnormalities specific to ts. the dental tomographic examination showed the crowding of teeth, especially in the maxillary anterior region, because of the maxillary narrowness. an elongated styloid process (length of 32.7 mm) was also detected on the right side (fig. 7). the patient was referred to the otolaryngology department of the university hospital for a detailed examination and assessment of speech problems. the temporal bone axial and coronal computed tomography (ct) reports noted that the tympanic membranes looked thick and retracted bilaterally ; on the left side, peripheral soft tissue density appeared at the external auditory canal. otoscopic examination showed that the right tympanic membrane was intact and calcified plaques were visible around the tympanic membrane. the left tympanic membrane was perforated, and there was an adhesion to the head of the stapes. according to endoscopic rhinoscopy, the nasal septum deviated to the left side ; there was also a slight polypoid degeneration in the middle nasal conchas and adenoid vegetation at the nasopharynx. there is evidence in the literature that genetic factors could contribute to dental and craniofacial abnormalities.8,16,17 patients with ts have a high palatal arch and cleft palate, sharpened canines and premolars, atypical cusps, root length alterations, and hypoplastic crown of premolars.6,8,17 " turner 's teeth " are described as permanent teeth, especially in the case of second mandibular premolars, that show hypoplasia in their crowns.18 our patient had hypoplastic maxillary first premolars, which could be described as turner 's teeth. some other specific abnormalities of ts, including imbalanced craniofacial skeletal growth with flattening of the basal skull, reduction of the transversal dimension of the maxilla, and retrusion of both jaws, has also been described.16 a high arched palate, a higher frequency of cleft palate occurrence, and malocclusion have been described.8,17 orthodontic anomalies of patients with ts were also reported by harju.19 they found that the occlusion of these patients seemed to be more abnormal than that of women without ts. it might be speculated that enamel genes on the x chromosome are involved in occlusal development.19,20 according to a hypothesis of gorlin, the absence of an x chromosome also has an effect on mandibular growth relative to maxillary development.21 intraoral examination and cbct investigation of our patient showed a number of craniofacial abnormalities specific to ts and an unexpected elongated styloid process specific to es. it was thought that some of the patient 's complaints, including headache, throat and neck pain, and dysphagia, might have been based on es. to the best of our knowledge, there has been no such case of turner syndrome accompanying es and prolactinoma reported in the literature. in conclusion, patients with ts carry various risks that make treatment more complicated ; thus advanced imaging techniques for proper treatment and follow - up would be extremely important. in the light of cbct examination, craniofacial abnormalities specific to ts and accompanying syndromes could be illustrated in detail. | turner syndrome (ts) is one of the most common chromosomal abnormalities, with an estimated frequency among female live births of 1/2,000 - 3,000. the syndrome is characterized by the partial or complete absence of one x chromosome (45,x karyotype). we reported a unique case of a 40-year - old woman with ts accompanying unexpected elongated styloid process specific to eagle syndrome (es) and followed up - prolactinoma. the present article is the first report to define the cone - beam computed tomographic (cbct) features of ts accompanying es. patients with ts carry various risks that make treatment more complicated ; thus advanced imaging techniques for proper treatment and follow - up are extremely important. in the light of cbct examination, craniofacial abnormalities specific to ts and accompanying syndromes such as the crowding of teeth especially in the maxillary anterior region caused by maxillary narrowness, micrognatic maxilla and mandible, relative mandibular retrusion, malocclusion, open - bite, and an elongated styloid process (length of 32.7 mm) on the right side were illustrated in detail. |
candida spp. is a common cause of device associated bloodstream infection in developed and developing countries. this disease has a tremendous attributable mortality in the order of 3040% according to available scientific literature. invasive candidiasis is generally associated with the formation of complex microbial communities, also known as biofilms over indwelling intravascular devices. biofilms are sessile communities consisting of microcolonies of yeast cells and an exopolymeric noncellular polysaccharide matrix secreted by the yeasts. candida albicans is the most common species of in the genus candida implicated in invasive candidiasis, in at least 5070% cases, although other species are also responsible. invasive candidiasis makes therapy very difficult, owing to factors like defective penetration of antifungal drugs through biofilms by forming a reaction - diffusion barrier and high cost of drugs available, like the echinocandins. moreover, antifungal drugs like amphotericin b have major adverse effects like nephrotoxicity and other ill - effects on health. intravascular catheters and other devices colonized with c. albicans can be removed, but this is not always feasible and antifungal treatment should be an adjunct to it. hence, low - cost safer alternatives are the need of the hour for treatment of device - associated invasive candidiasis. lipase enzyme expressed by c. albicans is one of the major virulence factors of the pathogen and its inhibition can be a strategy to abrogate infection by this pathogen. the present study was designed to evaluate the effect of filtrate of culture supernatant of staphylococcus epidermidis on the biofilm formation and lipase expression of c. albicans in vitro. this was a laboratory - based observational study, carried out in the department of microbiology, king george 's medical university (kgmu), lucknow, uttar pradesh, india. routine microbiological culture medium (5% sheep blood agar plate) was used to grow s. epidermidis isolates from different samples such as pus, blood, urine, and others. to isolate c. albicans from various clinical samples such as blood, pus, and urine, saboraud 's dextrose agar slant with emmon 's modification ten isolates each of c. albicans and s. epidermidis were randomly selected for the study. s. epidermidis isolates were identified by observing gram - positive cocci microscopically after performing gram - stain from the colonies on solid plates, positive catalase, and negative tube and slide coagulase tests and also a negative mannitol fermentation reaction. c. albicans isolates were identified by positive germ tube test and production of a single terminal chlamydospores on corn meal agar plate (dalmau slit inoculation technique) after aerobic incubation at 25c for 48 h. the microtiter plate model, as proposed by ramage., was employed for biofilm formation and its inhibition in vitro. at first, yeast isolates were grown in ypd broth (1% yeast extract, 2% peptone, 2% dextrose, w / v) overnight at 37c. s. epidermidis isolates were suspended in ypd broth (1 loopful of the colony in 2 ml broth) and centrifuged at 3000 rpm for 5 minutes. after that, the supernatant was filtered by passing it through the membrane filter of pore size 0.22 m (micro - por minigen syringe filter, genetix biotech asia, new delhi). then yeast cell turbidity was adjusted to 10 cells / ml in (a) ypd broth, (b) s. epidermidis culture filtrate. then 100 l of each set of suspension was dispensed in separate wells of a flat - bottomed 96-well polystyrene microtiter plate (nunclon a / s, kampstrupvej, denmark). sterile physiological (0.85%) saline was added in a well as a negative control. after incubating for 90 min at 37c, the wells were washed thrice with phosphate - buffered saline (pbs, ph 7.2) to remove non - adherent yeast cells and wells were reloaded with respective sterile liquid substrates. washing and reloading was repeated at intervals of 24 h and 48 h. after 48 h, wells were washed thrice with pbs and stained with 100 l of 1% safranine (weight / volume) in 95% ethanol for 1 min. after washing off excess stain with pbs, the wells were observed under inverted microscope under 200 magnification. subsequently their readings (optical densities) were also measured spectrophotometrically at a wavelength of 450 nm ultra violet light (imark microplate reader, bio - rad, usa). the first round of tests was carried out with c. albicans atcc 90028 strain and then with randomly selected clinical isolates. the test for inhibition of lipase was carried out by subculturing yeasts incubated overnight in (a) ypd and (b) culture filtrate on muhsin 's solid medium containing tween-80 and cacl2. a positive lipase activity was defined by a zone of haziness around yeast colonies on the medium. the toxic effects of the filtrate were studied by inoculating 100 l of the filtrate on hep-2 (human laryngeal epithelioma) cell line monolayer in small polystyrene vials, incubating it for 1 h at 37c, washing thrice with pbs, reloading the vials with 2 ml eagle 's minimum essential medium, reincubating at 37c, and periodic observation of the monolayer at 6 hourly intervals under an inverted microscope (40 magnification). this was a laboratory - based observational study, carried out in the department of microbiology, king george 's medical university (kgmu), lucknow, uttar pradesh, india. routine microbiological culture medium (5% sheep blood agar plate) was used to grow s. epidermidis isolates from different samples such as pus, blood, urine, and others. to isolate c. albicans from various clinical samples such as blood, pus, and urine, saboraud 's dextrose agar slant with emmon 's modification ten isolates each of c. albicans and s. epidermidis were randomly selected for the study. s. epidermidis isolates were identified by observing gram - positive cocci microscopically after performing gram - stain from the colonies on solid plates, positive catalase, and negative tube and slide coagulase tests and also a negative mannitol fermentation reaction. c. albicans isolates were identified by positive germ tube test and production of a single terminal chlamydospores on corn meal agar plate (dalmau slit inoculation technique) after aerobic incubation at 25c for 48 h. the microtiter plate model, as proposed by ramage., was employed for biofilm formation and its inhibition in vitro. at first, yeast isolates were grown in ypd broth (1% yeast extract, 2% peptone, 2% dextrose, w / v) overnight at 37c. s. epidermidis isolates were suspended in ypd broth (1 loopful of the colony in 2 ml broth) and centrifuged at 3000 rpm for 5 minutes. after that, the supernatant was filtered by passing it through the membrane filter of pore size 0.22 m (micro - por minigen syringe filter, genetix biotech asia, new delhi). then yeast cell turbidity was adjusted to 10 cells / ml in (a) ypd broth, (b) s. epidermidis culture filtrate. then 100 l of each set of suspension was dispensed in separate wells of a flat - bottomed 96-well polystyrene microtiter plate (nunclon a / s, kampstrupvej, denmark). sterile physiological (0.85%) saline was added in a well as a negative control. after incubating for 90 min at 37c, the wells were washed thrice with phosphate - buffered saline (pbs, ph 7.2) to remove non - adherent yeast cells and wells were reloaded with respective sterile liquid substrates. washing and reloading was repeated at intervals of 24 h and 48 h. after 48 h, wells were washed thrice with pbs and stained with 100 l of 1% safranine (weight / volume) in 95% ethanol for 1 min. after washing off excess stain with pbs, the wells were observed under inverted microscope under 200 magnification. subsequently their readings (optical densities) were also measured spectrophotometrically at a wavelength of 450 nm ultra violet light (imark microplate reader, bio - rad, usa). the first round of tests was carried out with c. albicans atcc 90028 strain and then with randomly selected clinical isolates. the test for inhibition of lipase was carried out by subculturing yeasts incubated overnight in (a) ypd and (b) culture filtrate on muhsin 's solid medium containing tween-80 and cacl2. a positive lipase activity was defined by a zone of haziness around yeast colonies on the medium. the toxic effects of the filtrate were studied by inoculating 100 l of the filtrate on hep-2 (human laryngeal epithelioma) cell line monolayer in small polystyrene vials, incubating it for 1 h at 37c, washing thrice with pbs, reloading the vials with 2 ml eagle 's minimum essential medium, reincubating at 37c, and periodic observation of the monolayer at 6 hourly intervals under an inverted microscope (40 magnification). as observed by both methods (microscopically and spectrophotometrically), biofilm formation in c. albicans was significantly reduced by crude culture filtrate of s. epidermidis, in vitro. the difference in mean values (optical density [od ] readings) of yeasts in ypd and the culture filtrate mean od of candida tropicalis in ypd and culture filtrate were 0.579 and 0.281, respectively (p < 0.05). optical density reading of c. albicans in ypd and culture filtrate lipase activity was found to be inhibited by culture filtrate of s. epidermidis. there was no zone of haziness around colonies subcultured from the culture filtrate in repeated experiments [figures 1 and 2 ]. haziness around candida albicans colonies subcultured from the culture filtrate of staphylococcus epidermidis no haziness around colonies subcultured from yeast extract - peptone - dextrose (without staphylococcus epidermidis filtrate) there was no observable change in morphology or cytopathic effect on of hep-2 cells inoculated with the culture filtrate after periodic observation for 2 days compared to control vial. thus, the crude filtrate was found to be nontoxic to host cells [figures 3 and 4 ]. hep-2 cell line incubated with staphylococcus epidermidis culture filtrate similar picture (here also no cytopathic effect) when hep-2 cell line is incubated with negative control (saline) invasive candidiasis is now regarded as the fourth most common cause of hospital - acquired bloodstream infection in the united states. very high incidence of nosocomial candidemia has also been reported from developing countries like brazil. in a study from north india, the incidence of candidemia was found to be about 45% among patients admitted in intensive care units. thus, the burden of this disease is considerable in both developed and developing countries. however, species other than c. albicans are also emerging as agents causing candidemia, and a report from north india indicated that c. tropicalis is the most common species associated with the condition. this disease entity is associated with the formation of complex microbial communities called biofilms over indwelling devices like intravascular catheters. formation of biofilms renders treatment difficult due to improper penetration of antifungal drugs through biofilms and slow growth of biofilm - associated cells. antifungal drugs available also have their own toxic effects which limit their routine use to treat this condition. for example, conventional amphotericin b is notorious for causing nephrotoxicity and hypokalemia and newer deoxycholate formulation is more expensive than the conventional one. hence, focus of researchers has shifted toward discovery of newer, low - cost, and safer alternatives in order to treat biofilm - associated candidemia. in this regard, it is worthy to mention that organochlorine derivatives (aspirochlorine) derived from aspergillus flavus have been shown to inhibit c. albicans growth in vitro. similar inhibition of candidal biofilm has been shown by pyocyanin and lipopolysaccharideof pseudomonas aeruginosa. in a mixed environment, the slime produced by s. epidermidis has been documented to facilitate adhesion of c. albicans to indwelling devices. conversely, c. albicans also shield the bacterium from the action of vancomycin. however, there is no study evaluating the effect of secreted substances by s. epidermidis broth culture on candidal biofilm formation and lipase expression. similar inhibition has been shown when yeasts were grown in culture filtrate of a. flavus. this filtrate was found to be nontoxic and hence can be precoated over the surface of indwelling devices to impair biofilm formation by c. albicans. further studies are required in this context to characterize the inhibitory substances in the crude filtrate and further check for host toxicity on animal systems. | background : candida spp. are fourth most common cause of bloodstream infection in developed countries and emerging agents of fungemia in developing countries, with considerable attributable mortality. candidemia is associated with the formation of complex, structured microbial communities called biofilms. biofilm formation makes treatment difficult due to improper drug penetration and factors like high cost and adverse effects of antifungal drugs available. hence, low - cost alternatives are urgently required to treat device - associated invasive candidiasis.objectives:to study the effect of culture filtrate of staphylococcus epidermidis on biofilm formation and lipase expression of candida albicans in vitro.materials and methods : yeast cells isolated from clinical samples were suspended to a turbidity of 106 in (a) yeast extract - peptone - dextrose (ypd) broth and (b) culture filtrate, and 100 l of each were dispensed in separate wells of microtiter plate. after repeated washing and reloading with respective liquid media, readings were taken spectrophotometrically. to check for lipase inhibition, yeasts were incubated overnight in ypd and filtrate and subcultured on media containing tween-80 and cacl2. positive lipase activity was denoted by haziness around colonies.results:mean reading of c. albicans in ypd broth was 0.579 while the same when yeasts were suspended in s. epidermidis culture filtrate was 0.281 (p < 0.05 by z - test of significance). lipase of c. albicans was inhibited by culture filtrate. filtrate was found to be nontoxic to human cell line.conclusions:culture filtrate of s. epidermidis can hence pave the way for development of new strategies to inhibit biofilm formation in device - associated candidemia. |
diabetes mellitus is a group of metabolic diseases characterized by elevated blood glucose levels resulting from defects in insulin secretion, insulin action, or both. the chronic hyperglycemia of diabetes is associated with long - term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels. one of the greatest factors in the development and progression of the complications of diabetes mellitus is hyperglycemia. hyperglycemia contributes to majority of diabetic complications by altering vascular cellular metabolism, vascular matrix molecules, and circulating lipoproteins. the oral glucose tolerance test (ogtt) measures the body 's ability to use glucose which is the main source of energy for body cells. treatment of diabetes involves use of drugs that reduce glucose levels, including insulin and oral antihyperglycemic drugs. although there is treatment for diabetes mellitus, most drugs in current use are seriously constrained by both their side effects and cost of treatment. due to these challenges, populations mainly in sub - saharan africa have resorted to cheaper and readily available alternative sources of treatment, such as use of medicinal plants or traditional medicines. the world health organization (who) estimates that 80% of the worlds ' populations use traditional medicine. the continued use of traditional medicines is linked to their low cost and a general belief that they have minimal side effects. zanthoxylum chalybeum which belongs to the family rutaceae is a traditional medicinal plant commonly used by some tribes in eastern uganda for treatment of malaria and diabetes. it is a deciduous spiny shrub that grows up to a height of 12 m, with a rounded but open crown. it has compound leaves comprising 3 to 5 pairs of shiny leaflets plus a terminal leaflet, with a strong citrus smell when crushed. stem bark decoctions or root bark decoctions are widely taken to treat diabetes, malaria, sickle cell disease, ulcers, and tumors. root bark decoctions are considered stronger than stem bark decoctions [6, 7 ]. however, although z. chalybeum is documented to be used in management of diabetes mellitus in traditional medicine, no scientific study has been done to validate its hypoglycemic activity. this study is the first to evaluate the antidiabetic activity of z. chalybeum using a laboratory rat model in order to provide scientific evidence to support its use by local communities in the management and treatment of diabetes. zanthoxylum chalybeum root bark was obtained from katakwi district in eastern uganda where it is abundant and locally called eusuk (ateso local language). the plant was authenticated by a plant taxonomist at natural chemotherapeutics research institute, ministry of health. after collection a voucher specimen was deposited at the national herbarium, makerere university, kampala. the root was washed, debarked, and dried in an air oven at 50c for 48 h. the bark was then pulverized into powder using a grinder. the powder was extracted by boiling in water for 30 minutes and allowing cooling to room temperature. the extract was then filtered and concentrated using an air oven at 50c to obtain the crude extract. the crude extract was reconstituted in distilled water and used to evaluate the hypoglycemic property in rats. twenty - four male and 24 female wistar albino rats (aged 1214 weeks, weighing 180240 g) were obtained from the college of veterinary medicine, animal resources and biosecurity (covab), makerere university, kampala, and used for evaluation of the antidiabetic property of z. chalybeum root bark extract in rats. the animals were allowed to acclimatize for 2 weeks, and then diabetes mellitus was chemically induced in the rats using freshly prepared solution of alloxan monohydrate in distilled water at a dose of 150 mg kg bw injected intraperitoneally. since alloxan caused fatal hypoglycemia due to massive insulin release by the pancreas, the rats were in addition orally given 20% (w / v) glucose solution (10 ml) after 6 h. they were further kept for 24 h on 5% (w / v) glucose solution to prevent hypoglycemia. rats which developed diabetes mellitus observed by glycosuria and hyperglycemia (i.e., blood glucose concentration > 250 mgdl) were selected for the subsequent experimental tests. this study was approved by graduate research committee, college of natural sciences, makerere university, kampala. animals were kept under standard laboratory conditions (25 3c, 12 h light / dark cycle) and had free access to food and clean tap water ad libitum for 28 days of experimental period. the procedures were of animal experiments in accordance with the organization for economic cooperation and development (oecd) guidelines for testing of chemicals. all the procedures were in accordance with the international council for laboratory animal science (iclas) and ethical guideline for researchers. six male and six female wistar albino rats were randomly allocated to each of the four groups. the groups were treated as follows : group i consisted of diabetic rats orally given water, food, and metformin by gavage (10 mg / kg bw) once daily for 28 days. group ii consisted of diabetic rats orally given water, food, and z. chalybeum extract by gavage (200 mg / kg bw) once daily for 28 days. group iii consisted of diabetic rats orally given water, food, and z. chalybeum extract by gavage (400 mg / kg bw) once daily for 28 days. group iv consisted of normal rats, orally given water and food with no treatment administered, that is, the normal control group. after 28 days of repeated treatment but before sacrifice, two male and two female wistar albino rats were randomly selected from each of the four groups and blood samples were collected from tail vein of the rats of control and treated groups after an overnight fast to obtain baseline blood glucose levels. subsequently, rats of both control and treated groups were orally given glucose (2 g / kg bw) by gavage. blood samples were collected from tail vein of the rats at intervals of 30 min up to 2 h for estimation of glucose concentrations using a glucometer. the animals were anesthetized and the pancreas was removed and fixed in a 10% solution of formaldehyde. the tissues were dehydrated because the reagents used at a later stage were immiscible with water. varying concentration of isopropyl alcohol, that is, 70%, 80%, 90%, 96%, and 100%, was used for the dehydration. the minimum time for dehydration between two different concentrations was 1 h. the fixed tissues were then cleared in xylene and embedded in paraffin wax. the sections (5 m) from each of the tissues were examined using a light microscope (40) after staining with hematoxylin and eosin dye. the glucose concentration results were expressed as mean standard error of the mean (sem). the mean and sem of the treatment groups were generated by use of the analysis of variance (anova) test. the significant difference between and within the treatment groups was considered significant at set p value < 0.05 using dunnett 's multiple comparison tests. oral glucose tolerance test was carried out 28 days after repeated doses of the aqueous root extract. there was a significant increase in blood sugar level after oral dosing within the same treatment group compared to treatment at 0 hr (figure 1). two hours following treatment with the extract, there was a significant reduction in blood glucose within the same treatment group compared to that after 0.5 h and 1 hr. however, there was no significant difference between the treatment group receiving 400 mg / kg bw and the control groups after two - hour treatment. the results further show that diabetic rats receiving z. chalybeum aqueous root extract (400 mg / kg bw) made some recovery and were able to regulate their sugar levels (figure 1). induction of diabetes with alloxan resulted in severe damage to the -cells of the islets of langerhans (figure 2(a)). a month after treatment with z. chalybeum (400 mg / kg bw), there was regeneration of the central -cells (figure 2(d)). the normal structure of the cells and the structure of the islets were restored. however the number of -cells was still low and yet the animals were able to maintain glucose levels near normal. this therefore suggests that the z. chalybeum extract also increases the sensitivity of the insulin receptors to the effects of insulin. diabetes mellitus causes disturbances in the uptake of glucose by cells as well as glucose metabolism. thus, alloxan - induced hyperglycemia is a very useful experimental way of studying and demonstrating the activity of new hypoglycemic agents. oral glucose tolerance tests were used to analyze blood glucose levels taken at different regular intervals after repeated treatments with z. chalybeum aqueous root extracts. results of the oral glucose tolerance test, using aqueous root extract of z. chalybeum (400 mg / kg bw), indicate significant decrease (p < 0.05) in blood glucose levels of the alloxan - induced diabetic rats (figure 1). this suggests that the aqueous root extracts of z. chalybeum enhance glucose utilization and hence improve glucose tolerance in diabetic rats. this also reaffirms that glucose tolerance test (gtt) is a suitable measure and indicator for the cells ability to use glucose, the body 's main source of energy. similarly, oral administration of aqueous root extract of z. chalybeum (400 mg / kg bw) had a significant glucose lowering effects in alloxan - induced diabetic rats (p < 0.05). other species in the genus zanthoxylum have been studied experimentally and reported to have significant antidiabetic activity. for example, various parts of z. zanthoxyloides including the roots, bark, and leaves have been used for medicinal purposes, including the treatment of diabetes mellitus. z. zanthoxyloides has been reported to significantly (p < 0.05) lower blood glucose in treated animals in comparison to nontreated groups. other species in the genus that are used traditionally to treat diabetes in india are z. armatum and z. nitidum [12, 13 ]. the beneficial effects of z. chalybeum treatment in diabetic rats in this study were likely due to improved insulin release and glucose uptake in remnant -cells. the induction of diabetes using alloxan resulted in severe damage of -cells of the islets of langerhans (figure 2(b)). however, after repeated treatment with z. chalybeum (400 mg / kg bw), for 28 days, there was regeneration of the central -cells (figure 2(d)). there was also a notable increase in the number of secretive -cells which are epithelial cells with ability to regenerate. the z. albeit the still low number of the -cells, the animals were able to maintain glucose levels close to the normal. this therefore also implies that the z. chalybeum extract increases the sensitivity of the insulin receptors to insulin. previous reports indicate that medicinal plants that possess hypoglycemic activity act through various mechanisms including improvement in the sensitivity of target cells to the effects of insulin, augmenting glucose - dependent insulin secretion, and stimulating the regeneration of -cells of islets of langerhans in pancreas of alloxan - induced diabetic rats. some of the medicinal plants seem to regulate enzymes of glycolysis, gluconeogenesis, and other pathways. active phytochemical compounds act through a variety of mechanisms ; however, in the present study, identification of the mechanism of action of the extract was not done ; thus the suggestions made are only hypothetical. earlier studies on phytochemical analysis of aqueous roots extract of z. chalybeum reported presence of the following compounds : tannins, reducing sugars, saponins, alkaloids, coumarin derivatives, flavonoides, steroid glycosides, triterpenes, and anthocyanosides. presence of flavonoids, steroids, terpenoids, and phenolic acids has been suggested by several authors to be responsible for antidiabetic activity. flavonoids have also been known to regenerate the damaged beta cells in alloxan - induced diabetic rats and act as insulin secretagogues. thus, the hypoglycemic activity of aqueous roots extract of z. chalybeum may be due to the presence of hypoglycemic flavonoids, terpenes, or saponins ; however, this also requires further investigation. the antihyperglycemic effect of z. chalybeum may be attributed to the potentiation of insulin from existing -cells of the islets of langerhans. the blood glucose lowering effect of z. chalybeum was compared with that of metformin, a standard drug which has been in use for many years for treatment of diabetes and acts by stimulating insulin secretion from pancreatic -cells. study has shown that oral administration of aqueous root bark extract of z. chalybeum to alloxan - induced diabetic rats improves their glucose tolerance, an important finding in the control of diabetes. this suggests that z. chalybeum is useful in protection and amelioration of diabetic complications through enhancement of regeneration of -cells of the islets of langerhans. further studies are needed to define the active agents present and their mode of activity. | background. medicinal plants offer cheaper and safer treatment options to current diabetic drugs. the present study evaluated the effect of aqueous root bark extract of zanthoxylum chalybeum on oral glucose tolerance and pancreas histopathology in alloxanized rats. method. diabetes was induced in rats by administration of alloxan monohydrate. root extract of z. chalybeum was administered to rats at 200 and 400 mg / kg bw daily for 28 days. blood glucose was measured by glucometer and pancreatic histopathology evaluated microscopically. results. initial increase was observed in blood glucose of the rats after oral administration of glucose from time zero. two hours after treatment with z. chalybeum, a significant reduction in blood glucose was observed within treatment groups (p < 0.05) compared to 0.5 hr and 1 hr. there was no significant difference between treatment group receiving 400mg / kg bw extract and the normal groups (p = 0.27), implying that the former group recovered and were able to regulate their blood sugar, possibly via uptake of glucose into cells. the reversal in pancreatic histopathology further supports the protective effect of z. chalybeum extract towards diabetic damage. conclusion. extract of z. chalybeum is effective in controlling blood glucose in diabetes and protecting pancreatic tissues from diabetic damage. |
disseminated intravascular coagulation (dic) is a life - threatening complication of several disease states including sepsis, cancer, obstetrical complications, trauma and aortic aneurysm, although dic associated with aortic aneurysm is rarely reported in korea. the definitive treatment for dic caused by an aortic dissecting aneurysm, involves lesion removal and the infusion of coagulation factors during the operation to minimize blood loss. we reported a case of dic caused by an aortic dissecting aneurysm, which was resolved by non - surgical therapy. a 55-year - old female patient was transferred from another hospital because of chest pain radiating to her back and thrombocytopenia. she had been diagnosed as having essential hypertension approximately 3 years earlier, but was not medicated. the chest pain developed upon exertion and radiated to her back the day prior to admission to a local hospital ; it persisted and became more severe after admission. on admission, her temperature was 36.8c, her pulse rate was 106, her respiration was 20, and her blood pressure was 190/100 mmhg. on physical examination, the patient appeared acutely ill, but her heart sounds were normal. urinalysis revealed many red blood cells and 05 white blood cells per high power field. chest radiographs revealed mediastinal widening, and a computer tomography (ct) scan of the thorax and abdomen revealed a dissecting aneurysm of the descending thoracic aorta, which descended inferiorly to the proximal abdominal aorta (figure 1). on the 2 day of admission, the patient reported an improvement in her chest pain, and she had a platelet count of 63,000/mm. on the 7 day, her platelet count was 377,000/mm, and her prothrombin and partial - thromboplastin times were normal. we followed up the aortic dissecting aneurysm by a thorax ct on the same day and disease progression was not observed. at 3 weeks after admission, her blood pressure and pulse rate had normalized and her chest pain had fully subsided. the dic profiles, including platelet count, prothrombin and partial - thromboplastin times, d - dimer, fdp, antithrombin iii, and fibrinogen, had normalized (table 1). outpatient follow - up by laboratory testing, thorax ct, and echocardiography have been performed at regular intervals over a period of 9 months, and the patient is doing well. disseminated intravascular coagulation (dic) is a rare complication of aortic dissecting aneurysm, but a well - recognized one. in the current series of patients with aortic aneurysm, 40% were found to have elevated levels of fibrinogen split products, but only 4% experienced significant bleeding and laboratory evidence of dic. the pathogenesis of coagulopathies, caused by aortic dissecting aneurysm, may be related to the release of endothelin, thromboplastin, and other attractants from the exposed subendothelial tissue and to the subsequent clot formation. dic implies the consumption of platelets, fibrinogen, and coagulation factors because of extensive in vivo coagulation. the mechanical destruction of platelets as the blood flows over the thrombus may also play a role there is no single laboratory test that can establish or rule out a diagnosis of dic. in clinical practice, a dic panel typically consists of a platelet count, a fibrinogen level, a prothrombin and a partial thromboplastin time, and a measurement of d - dimer (or fdp) level. tests for d - dimers may be helpful in the differentiation of dic from other conditions that are associated with a low platelet count or a prolonged clotting time. quantitations of selected coagulation inhibitors, including antithrombin iii and/or protein c, may provide useful prognostic information. a scoring system that uses simple laboratory tests has recently been published by the dic subcommittee of the international society on thrombosis and haemostasis. a score of 5 was deemed compatible with a diagnosis of dic, but the published scoring system has not yet been validated by prospective studies. in the present case, a low platelet count (< 50,000/mm, scoring 2), a moderately elevated fdp (20 g / ml, scoring 2), and a low fibrinogen level (< 1.0 g / l, scoring 1) were obtained, giving a total score of 5. on admission, the patient s d - dimer level was elevated to 2.0 g / ml and her anti - thrombin iii level depressed to 19.7 mg / dl, which is compatible with overt dic. when these two conditions coexist, they create a difficult clinical problem that requires optimal medical and surgical care. bleeding diathesis must be corrected before surgery in order to prevent massive intraoperative bleeding. in this respect, if bleeding is brisk or surgery is contemplated, cryoprecipitate, platelet concentrates, and fresh - frozen plasma should be given ; anti - thrombin iii concentrate and gabexate may also be effective in some cases. we recommended surgical intervention in the present case, but the patient refused, so we treated her using intravenous antihypertensive agents and antithrombin iii replacement. she was discharged without any complication, and has remained symptom - free and well during the 9 months since discharge. | disseminated intravascular coagulation (dic) is an acquired coagulation disorder that occurs when the normal hemostatic balance is disturbed, primarily by excessive thrombin formation. moreover, while dic is a rare complication of aortic dissecting aneurysm, it is also a well - recognized one. we reported a case of dic associated with aortic dissecting aneurysm in a 55-year - old woman who was transferred from another hospital because of chest pain radiating to her back and thrombocytopenia. laboratory findings showed dic with severe thrombocytopenia, and she was diagnosed as having an acute aortic dissection and dic. after medical treatment on the aortic dissecting aneurysm, her dic profile recovered. |
behet 's disease (bd) has been recognized as a systemic disorder of recurrent acute inflammation, characterized by the involvement of multiple organs and resulting in orogenital ulcers, uveitis, gastrointestinal ulcers, and skin lesions (1). its incidence is relatively high from eastern asia to the mediterranean and is estimated to affect approximately 1 - 10 out of 10,000 people in those areas, while in u.k. and north america, only 1 - 2 cases are found for every 1,000,000 people (2). while the etiology and pathogenesis of the syndrome are still obscure, it is thought that autoimmune mechanisms play a role because vasculitis is the primary pathological lesion, and circulating autoantibodies to human oral mucous membranes are found in approximately 50% of cases (3). however, involvement of the central nervous system, vessels, and intestines often leads to a poor prognosis. digestive manifestations in bd have been reported in up to 1 - 60% of cases, although this rate varies in different countries. the most frequent sites of gastrointestinal involvement are the ileocecal region with extension into the ascending colon (4). while esophageal involvement in bd is known to be very rare, anecdotal studies report esophageal involvement can cause serious complications such as strictures, bleeding, perforation, or fistulas (5, 6). despite a relatively high rate of intestinal involvement in korean bd patients (7), patterns of esophageal involvement in korean patients with bd remain unclear in terms of the prevalence and clinical characteristics. moreover, treatment response is controversial. some major issues have been examined for intestinal involvement in bd, but esophageal involvement has been not been sufficiently studied. accordingly, in this study, we aimed to evaluate the prevalence and clinical characteristics of esophageal involvement in korean bd patients. the medical records of 842 consecutive de novo bd patients cared at severance hospital, yonsei university college of medicine, seoul, korea between january 1990 and june 2006 were retrospectively analyzed. diagnosis of bd was based on a combination of clinical, enodoscopic, radiologic, and pathologic findings. to define bd two diagnostic criteria, suggested by the behet 's disease research committee of japan in 1987 (8) and the international study group for behet 's disease (isgbd) in 1990 (9), were applied. by using the classification of the research committee of japan, types were categorized into ' complete ', ' incomplete ', and ' suspicious '. the other criterion, proposed by the isgbd, was used as a supplement to certify the diagnosis of bd. demographic characteristics, endoscopic findings, clinical features, treatment responsiveness, and complications were evaluated. esophageal involvement in bd was confirmed when a discrete, punched - out ulcer, like those found in the intestines of similar patients, was detected during endoscopic examination and other causes, such as acid reflux - related, viral, or drug - induced esophagitis, were ruled out. biopsy specimens were obtained using standard - sized biopsy forceps from both the base and the margin of ulcerated lesions. for more accurate diagnosis of undetermined cases, serologic tests or quantitative polymerase chain reaction assays finally, these diagnostic processes were combined with the responses to specific therapy for esophageal involvement of bd and follow - up endoscopic findings. the activity index of bd was investigated at the timed of esophagogastroduodenoscopy (egd) due to upper - gastrointestinal symptoms. the clinical activity score was calculated by summing of each clinical manifestation present according to a previously proposed system (2). statistical analyses of the data were conducted using spss 12.0 computer software (chicago, il, u.s.a.) for comparison of bd activity with / without esophageal involvement. p value<0.05 were considered statistically significant and all p values correspond to two - sided significance tests. this study was approved by the institutional review board of severance hospital and informed consent was obtained from all patients before endoscopic examinations. among the 842 patients with bd, 129 (15.3%) patients experienced gastrointestinal symptoms ; all 129 underwent both egd and ileocolonoscopy. gastrointestinal symptoms or signs which led them to endoscopic examinations, were as follows : abdominal pain in 58 patients (45.0%), poor oral intake complicated with oral ulcer in 30 (23.2%), melena or hematochezia in 32 (24.8%), chest - pain in 4 (3.1%), dysphagia in 4 (3.1%), and hematemesis in one patient (0.8%). the clinical characteristics of the 129 patients are shown in table 1 and their endoscopic findings are listed in table 2. six patients were shown to have esophageal ulcerations suggestive of esophageal involvement in bd (fig. 1). the patients with esophageal involvement had a higher disease activity score numerically compared to those without esophageal involvement, which had no statistical significance (p=0.08). other esophageal diseases found through egd examinations include gastroesophageal reflux disease (5 patients), esophageal candidiasis (2 patients), hiatal hernia (2 patients), barrett 's esophagus (one patient), esophageal polyp (one patient), and esophageal varices (one patient). of the 129 patients, intestinal bd was found in 69 patients (53.5%) through colonoscopic examinations. esophageal ulcerations of most patients could be controlled by proton pump inhibitors / histamine-2 receptor blockers, colchicines, and/or 5-aminosalicylic acids. the median follow up period of the six patients was 39.5 months (14 - 78 months) after the diagnosis of esophageal ulceration. follow - up egd was performed on all the 6 patients. of them, in 3 patients, active ulceration previously noted was not found any more and the other 3 patients experienced the improvement of ulcers. moreover, no patients experienced serious complications, such as esophageal stricture, esophageal bleeding, or esophageal perforations, during follow - up. gastrointestinal manifestations have been known to lead to severe morbidity in bd patients and are commonly referred to as " intestinal behet 's syndrome ". the frequency of intestinal involvement in patients with bd was known to be 0 - 60%, with geographic differences (10). symptomatic intestinal involvement is rare in mediterranean patients with bd, but in east asian patients, including koreans, intestinal involvement is more common. shimizu. (11) reported that gastrointestinal symptoms are present in at least 50% of the patients with bd in japan. the intestinal lesions are usually resistant to medical treatment and recur frequently (25 - 78%) after surgical management, especially in people of western or chinese origin (10). the terminal ileum and the cecum are the most frequently involved parts of the gastrointestinal tract with esophageal involvement being very rare. the ulcerations can be single or multiple, shallow or deep, small or large, or clearly or unevenly marginated. esophageal lesions are most commonly located in the middle esophagus, although diffuse esophagitis and stenosis have also been reported (15). the histological aspects of esophageal ulceration in bd reportedly involve acute or chronic nonspecific infiltrates and granulation tissue and fibroblasts are typically seen at the base of the ulceration. in addition, serious complications such as stricture, bleeding, or perforations have been rarely described. since the first case of esophageal ulceration of this syndrome was presented by brodie and ochsner in 1973 (12), to date, less than 50 cases worldwide of behet 's patients with esophageal ulcers have been reported. however, the actual prevalence of esophageal involvement in bd is still uncertain and the proper management of this condition has yet to be established (13 - 15). thus, the purpose of this study was to investigate the prevalence of esophageal involvement in bd through a large cohort study in a single tertiary institution and to evaluate its clinical characteristics, especially in terms of therapeutic responsiveness and prognosis. our study investigated the largest population ever reported, and it was found that esophageal ulcerations were detected in only 6 of 129 bd patients (4.7%), which suggests that the prevalence of esophageal involvement in bd is quite low. moreover, such involvement may not be correlated with systemic disease activity, disease duration, disease activity, or any other aspect of bd. (16), conducted the first prospective study of the esophagus in patients with bd. this study involved 9 patients who underwent egd ; three patients were asymptomatic and 6 had upper gastrointestinal symptoms at the time of endoscopy. the prevalence of esophageal involvement in bd was low (11%) and non - specific, but was much higher than that of our data. however, this rate of esophageal involvement might be under- or overestimated because the case number was very small and endoscopic examination was not performed in all cases. in another anecdotal investigation, it was suggested that the esophageal abnormalities in bd were found rather frequently (14/21, 66.6%) and occur even in asymptomatic patients (13). however, in this series, gross endoscopic abnormality was detected in only one patient (4.8%), which yields a rate that is consistent with that of our study. because of a lack of randomized, controlled trials for treatment of various manifestations of bd, the standard treatment remains controversial. the usual initial management is combined drug therapies, involving drugs such as colchicines, non - steroidal anti - inflammatory drug (nsaids), glucocorticosteroids, immunosuppressive, or cytotoxic medications. furthermore, intestinal involvement is regarded as a serious manifestation of bd, like neurological symptoms or retinal vascultitis, and more potent drugs are usually chosen. in addition, several cases and trials reported the successful use of thalidomide, mesalazine, and anti - tnf-. however, at present, the appropriate treatment for esophageal involvement in bd remains controversial. corticosteroids represent the major therapeutic agent, but as stated by brodie and oshsner, corticosteroid therapy may cause esophageal perforation (12). in our series, the esophageal manifestations respond well to treatment with proton pump inhibitors, mesalazine, and/or colchicines. moreover, any serious complications, such as esophageal stricture, bleeding, or perforations, did not develop during follow - up. moreover, we did not perform egd for all the patients with bd, which may have lead to underestimation of the exact incidence of esophageal involvement in bd. regarding its treatment, further research is necessary to validate the appropriate treatment of esophageal involvement in bd through randomized prospective studies. in conclusion, our study demonstrated that upper gastrointestinal symptoms are common in bd patients, but esophageal involvement is very rare and serious complications may be negligible. moreover, the clinical characteristics of bd with esophageal involvement seem to be similar to those of bd without involvement. | while a significant amount of clinical information has been reported concerning intestinal involvement in behet 's disease (bd), esophageal involvement in bd has not yet been studied extensively. the aim of this study was to evaluate the prevalence of esophageal involvement in bd and its clinical characteristics. we retrospectively reviewed the medical records of 842 patients diagnosed with bd at a single tertiary institution in korea between january 1990 and june 2006. of the 842 patients with bd, 129 patients (15.3%) experienced upper gastrointestinal symptoms that required inspection through esophagogastroduodenoscopy. esophageal involvement was found in 6 (4.7%) of the 129 patients. the activity index of behet 's disease did not differ among patients with or without esophageal involvement. all patients with esophageal involvement responded well to medical treatment and no one experienced serious complications. the results of our study demonstrate that the prevalence of esophageal involvement in bd is very low and that most patients with such involvement face few complications and respond well to medical treatment. |
nevus sebaceous of jadassohn is a congenital hamartoma, which has been reported to be associated with secondary tumors such as syringocystadenoma papilliferum, trichoblastoma and basal cell carcinoma (bcc) [1, 2, 3, 4 ]. however, more than three multiple tumors arising from a nevus sebaceous of jadassohn is extremely rare. malignant transformation occurs in 1015% of nevus sebaceous in some series, although others suggest that this rate may be lower. studies indicate that the development of bcc or any other malignant neoplasm is very rare. multiple tumors arising from a nevus and/or a phacomatosis provide important clues to understand their histogenesis and their capacity to differentiate and proliferate. here we present a case of multiple tumors of syringocystadenoma papilliferum, sebaceoma, trichoblastoma and bcc that arose from a nevus sebaceous of jadassohn. a 67-year - old female presented with a tumor on the back of her head. the patient had recognized a nodule on the back of her head 37 years earlier. that nodule had gradually enlarged and had started bleeding 10 years earlier. physical examination revealed a 47 26 mm slightly elevated light brown plaque on the back of her head. a 16 13 mm erosive lesion and an 18 17 mm dome - shaped black node were recognized in that plaque (fig. dermoscopic analysis of the dome - shaped black node showed black homogenous areas, surface scales and arborizing vessels (fig. dermoscopic analysis of the erosive lesion showed glomerular vessels in pink homogenous areas (fig. histopathological examination of the black node showed tumor nests with a sheet - like appearance that extended downward into the deep dermis. melanin deposition was scattered irregularly and bleeding was also observed within the tumor nests (fig. insular tumor aggregations were recognized in the tumor nests and some of those aggregations contained melanin deposits (fig. immunohistochemistry showed a positive reaction for ber - ep4 in those tumor nests (fig. 2c). at the periphery of the black node, tumor nests also extended into the deep dermis and were composed of several different components (fig. 2d). in the upper portion, tumor nests with interlacing cords were noted. numerous structures resembling follicular germinative cells were recognized and some of those structures extruded from interlacing cords (fig. a diagnosis of trichoblastoma was made in this upper portion. in the lower portion, the tumor nests consisted of sebocytes, which had a foamy cytoplasm (fig. 2h). the epithelium of the tubules was lined by 23 layers displaying apocrine secretion of decapitation secretion and numerous plasma cells were noted in the stroma of the papillated structures (fig. a diagnosis of syringocystadenoma papilliferum was made at the erosive lesion. in the slightly elevated light brown plaque, a diagnosis of syringocystadenoma papilliferum, sebaceoma, trichoblastoma and bcc arising from a nevus sebaceous of jadassohn was made. the tumor was resected and there has been no relapse 1 year after the surgery. nevus sebaceous has the potential to generate different lineages of tumors that are not restricted to the sebaceous lineage [1, 4 ]. a secondary tumor from a nevus sebaceous may differentiate into follicular, sebaceous, apocrine and eccrine cells, and rarely, a secondary tumor can differentiate into muscle [6, 7 ]. the simultaneous occurrence of tumors from a nevus sebaceous is not rare, but the number of tumors is usually less than three. a previous study showed that one in 140 cases of nevus sebaceous simultaneously developed three tumors, and another previous study showed that one in 150 cases of nevus sebaceous simultaneously developed four tumors [8, 9 ]. in our case, four distinct tumors (syringocystadenoma papilliferum, sebaceoma, trichoblastoma and bcc) arose from a nevus sebaceous. those tumors reflect that nevus sebaceous has a pluripotency for apocrine, sebaceous and follicular differentiation. an intriguing fact is that this patient had a previous history of multiple tumors such as ovarian cystoma, submandibular gland tumor and thyroid cancer. in conclusion, we report a rare case of multiple tumors of syringocystadenoma papilliferum, sebaceoma, trichoblastoma and bcc arising from a nevus sebaceous of jadassohn. | nevus sebaceous is known by its association with one or more secondary tumors, but more than three multiple tumors arising from a nevus sebaceous is extremely rare. a 67-year - old female presented with a light brown plaque on the back of her head that contained a dome - shaped black node and an erosive lesion. histopathological examination showed atypical basaloid cells in the black node. at the periphery of that node, structures resembling follicular germs extruded from interlacing cords in the upper portion and tumor nests with sebocytes were in the lower portion. in the erosive lesion, papillated structures with an apocrine epithelium were observed. in the light brown plaque, enlargement of sebaceous lobules was noted. from those histopathological features, a diagnosis of syringocystadenoma papilliferum, sebaceoma, trichoblastoma and basal cell carcinoma arising from a nevus sebaceous was made. we discuss the rarity of multiple tumors arising from a nevus sebaceous. |
almost all marine fish and cephalopods can become infected with the third stage larva (l3) of a. simplex. the ingested a. simplex larvae by humans penetrate the stomach wall causing acute abdominal pain, nausea, and vomiting within a few hours. when the larvae invade the gastric or intestinal mucosa, inflammatory reaction often results in ulcer or eosinophilic granuloma. recently, it has become clear that anisakiasis is often associated with strong allergic reactions ranging from urticaria to anaphylactic shock [1 - 4 ]. reported that the prevalence of anisakiasis has remarkably increased throughout the world in the last 30 years. one of the main reasons for this increase is attributed to the preference for raw and slightly cooked food. this trend will bring about the rise of infectious diseases caused by parasitic infections, like a. simplex larvae, through marine fishery products. although anisakiasis might occur in any country where the people eat raw or undercooked fish or squids, the majority of cases have been diagnosed in korea, japan, spain, and some other countries because of their eating habits [6 - 9 ]. the prevalence of a. simplex infection has been reported to be different depending on the countries and areas. several studies have reported that more than 10% of gastrointestinal anisakiasis showed allergic symptoms related to fish consumption. the anisakid infection rate in spain was known to be around 0.43% in galicia area and 12.4% in the population of madrid. an epidemiological study in japan has shown that anisakiasis was more frequent in costal populations and among 20- to 50-year - old males. in addition, these patients were reported to engage in fishing industry or inhabit coastal areas. the high prevalence of a. simplex larval infection of fish and cephalopods has been reported in various fish species and squids [13 - 15 ]. favored fishes of the korean people, such as mackerels, cods, alaska pollacks, scabbard fish, and squids nevertheless, seroepidemiologic surveys among koreans have not been accomplished. to obtain data of seroprevalence against a. simplex allergens among koreans, we analyzed blood samples from residents in the southern parts of korea by elisa and western blot analysis using crude extract and excretory - secretory products (esp) from a. simplex l3. we prepared 498 sera from blood samples collected in 3 hospitals, each located in busan metropolitan city, masan city, and geoje city. they had no history of allergic symptoms, including allergic rhinitis, urticaria, atopic dermatitis, and asthma (total ige < 100 iu / ml). serum samples were obtained by clotting and centrifugation of the blood at room temperature and were stored at -70. the collection of sera for use in these studies was approved by the human subjects investigation committee of kosin university, busan, korea. a. simplex l3 larvae were collected manually from the viscera, flesh, and body cavities of naturally infected mackerels (scomber japonicus) and thoroughly washed with pbs. the crude extract of a. simplex l3 larvae was prepared from 300 larvae (about 1 g). larvae were frozen in liquid nitrogen and smashed by mortar. to extract proper amounts of proteins, the protein extraction solution was added and stored on ice for 30 min according to the manufacturer 's instructions (pro_prep, intron biotechnology, seoul, korea). the supernatant was collected after centrifugation at maximum speed for 10 min at 4. the amount of protein was measured by the bradford method. the collected larvae were incubated in pbs for 24 hr to remove salts and other foreign bodies. larvae were incubated in dmem (dulbecco 's modified eagle medium) with gentamycin (150 mg / ml) and vancomycin (10 mg / ml) at 0.5 ml / larva, 37 for 48 hr. the media were collected and centrifuged at 2,500 rpm for 20 min and concentrated with amicon stirred cells, cut off molecular weight less than 10,000 (millipore corp, massachusetts, usa). the crude extract and esp of a. simplex l3 larvae both of them were diluted for the elisa starter kit (koma biotech, seoul, korea) as 1 g / ml and 100 l of aliquots were added each well of 96 well elisa plate. antigens were incubated overnight at 4 and washed with tris - buffered saline with tween-20 (tbst) 3 times. blocking solution samples were incubated with 1:5 diluted patients ' sera and 1:500 diluted peroxidase conjugated goat anti - human ige (sigma aldrich, st. for color reactions, 100 l of tmb substrate solution was added and stopped the reactions when color reaction was enough to measure. absorbance was measured at 450 nm by elisa reader (emax, molecular devices, downingtown, pennsylvania, usa). a mean od value3sd of the negative control sera negative control sera were collected from 10 healthy persons aged 20 - 25 years who had no allergic history and the total ige level was less than 100 iu / ml. proteins were diluted with dissociation buffer (3:1) and boiled at 100 for 5 min. boiled proteins were run through 12% sds - polyacrylamide gel and transferred on a nitrocellulose membrane by mini - protean ii (bio - rad, richmond, california, usa) at 25 volt for 1.5 hr. transferred proteins were checked by ponceau staining and blocked with pbs containing 5% skim milk at room temperature for 1 hr. after 3 times washing with pbst for 10 min, membranes were incubated with diluted elisa positive patients ' sera (1:10) at 4 overnight. blotted membranes were washed 3 times with pbst for 10 min and incubated with horseradish peroxidase - labeled goat anti - human ige (1:500) (sigma) at room temperature for 1 hr. after 3 times washing with pbst for 10 min, color reaction was conducted by adding 4-chloro-1-naphtol (sigma) or dab substrate (pierce, rockford, illinois, usa) on the blotted membrane. we prepared 498 sera from blood samples collected in 3 hospitals, each located in busan metropolitan city, masan city, and geoje city. they had no history of allergic symptoms, including allergic rhinitis, urticaria, atopic dermatitis, and asthma (total ige < 100 iu / ml). serum samples were obtained by clotting and centrifugation of the blood at room temperature and were stored at -70. the collection of sera for use in these studies was approved by the human subjects investigation committee of kosin university, busan, korea. a. simplex l3 larvae were collected manually from the viscera, flesh, and body cavities of naturally infected mackerels (scomber japonicus) and thoroughly washed with pbs. the crude extract of a. simplex l3 larvae was prepared from 300 larvae (about 1 g). larvae were frozen in liquid nitrogen and smashed by mortar. to extract proper amounts of proteins, the protein extraction solution was added and stored on ice for 30 min according to the manufacturer 's instructions (pro_prep, intron biotechnology, seoul, korea). the supernatant was collected after centrifugation at maximum speed for 10 min at 4. the amount of protein was measured by the bradford method. the collected larvae were incubated in pbs for 24 hr to remove salts and other foreign bodies. larvae were incubated in dmem (dulbecco 's modified eagle medium) with gentamycin (150 mg / ml) and vancomycin (10 mg / ml) at 0.5 ml / larva, 37 for 48 hr. the media were collected and centrifuged at 2,500 rpm for 20 min and concentrated with amicon stirred cells, cut off molecular weight less than 10,000 (millipore corp, massachusetts, usa). both of them were diluted for the elisa starter kit (koma biotech, seoul, korea) as 1 g / ml and 100 l of aliquots were added each well of 96 well elisa plate. antigens were incubated overnight at 4 and washed with tris - buffered saline with tween-20 (tbst) 3 times. blocking solution samples were incubated with 1:5 diluted patients ' sera and 1:500 diluted peroxidase conjugated goat anti - human ige (sigma aldrich, st. louis, missouri, usa) as the secondary antibody. for color reactions, 100 l of tmb substrate solution was added and stopped the reactions when color reaction was enough to measure. absorbance was measured at 450 nm by elisa reader (emax, molecular devices, downingtown, pennsylvania, usa). a mean od value3sd of the negative control sera negative control sera were collected from 10 healthy persons aged 20 - 25 years who had no allergic history and the total ige level was less than 100 iu / ml. proteins were diluted with dissociation buffer (3:1) and boiled at 100 for 5 min. boiled proteins were run through 12% sds - polyacrylamide gel and transferred on a nitrocellulose membrane by mini - protean ii (bio - rad, richmond, california, usa) at 25 volt for 1.5 hr. transferred proteins were checked by ponceau staining and blocked with pbs containing 5% skim milk at room temperature for 1 hr. after 3 times washing with pbst for 10 min, membranes were incubated with diluted elisa positive patients ' sera (1:10) at 4 overnight. blotted membranes were washed 3 times with pbst for 10 min and incubated with horseradish peroxidase - labeled goat anti - human ige (1:500) (sigma) at room temperature for 1 hr. after 3 times washing with pbst for 10 min, color reaction was conducted by adding 4-chloro-1-naphtol (sigma) or dab substrate (pierce, rockford, illinois, usa) on the blotted membrane. the ages of patients were from teens to 98 years old. among them, 107 were in their twenties, and was the largest group. the numbers of thirties, forties, fifties, sixties, seventies, and eighties and older were 81, 71, 55, 55, 45, and 50, respectively (table 1). to analyze the pattern of antigens from crude extracts and esp of a. simplex l3 the optical density (od) by elisa of the 498 patients revealed 0.030.084 (meansd), and 0.010.108 for antigens from crude extract and esp, respectively. the seropositive rate was 5.0% with crude extract antigen while that with esp antigen was 6.6% (33 positives) by elisa among the 498 patients (by cut - off od value of 0.10). the od value of the positive group was 0.280.114 and 0.230.117, and that of negative group was 0.010.042 and 0.010.079 for antigens from crude extracts and esp, respectively. the specific serum ige level against a. simplex crude extracts and esp showed various distributions between the study populations. the serum samples from geoje city showed more extensive distribution and higher values than those from the other 2 cities (fig. western blotting carried out for elisa positive sera revealed a specific band of about 130 kda in 10 patients in both crude extract and esp (fig. 3). the mean od of elisa for 10 patients who showed the specific band in western blot was 0.280.221. the regional distribution of positive serum samples by western blotting showed that 8 positive patients were in geoje city, and 2 patients were in masan city. in our study, we analyzed the serum samples obtained from residents of busan metropolitan city, masan city, and geoje city by elisa and western blotting for the seroepidemiologic study of a. simplex l3 infection. the positive rates by elisa showed 5.0% and 6.6% for antigens from crude extracts and esp, respectively. western blot analysis of elisa positive sera revealed the specific band of about 130 kda in 10 patients in both crude extract and esp. this protein is considered similar to ani s 7 (139 kda) which was proposed by rodriguez., which was reported to be the typical antigen for a. simplex l3. our work provided the first data on the seroprevalence of anti - anisakis ige sensitization in korea. in spain, the prevalence of anisakiasis varied depending on location, such as 12.4% in madrid and 0.43% in galicia. valinas. suggested that the low positive infection rate (0.43%) of anisakis allergy in galicia, spain, was due to the difference between live or dead larvae. however, other reports set forth a counterargument that it can be different according to the habit of fish consumption, genetic background, and diagnostic methods the types of antigens and diagnostic methods, however, are the most important factors influencing seroepidemiological investigations. although the crude extracts and esp are frequently used for serologic diagnosis, esp were reported as the more potent and clinically important allergens for diagnosis [20 - 23 ]. however, cross reactions with other allergens, such as intestinal and blood - tissue nematodes, should be considered in regard to seropositive prevalence. many excretory - secretory and somatic anisakis - specific antigens have been reported, including ani s 1 (21 kda), ani s 2 (100 kda, paramyosin), ani s 3 (41 kda, tropomyosin) [28 - 29 ], ani s 4 (9 kda, cysteine protease inhibitor) [30 - 31 ], ani s 5 (15 kda protein homologous with the sxp / ral-2 family proteins), and ani s 6 (7 kda, serine protease inhibitor). recent studies also reported functionally unknown antigens, including ani s 8 (15 kda), ani s 9 (14 kda), and ani s 10 (21 kda protein with unknown function ; allergen nomenclature sub - committee ; http://www.allergen.org). ani s 7 (139 kda) was identified to have a 1096-amino acid fragment 7 (genbank : ef158010) and 19 repeats of a novel cx17 - 25 cx 9 - 22 cx8 cx6 tandem repeat motif. ani s 7 was reported probably the most important major excretory - secretory allergens since they were recognized by 100% of infected patients. in addition, anti - ani s 7 ige antibodies were reported that they were induced by live anisakis larvae peaked at about day 30 post - infection and then decreased slowly over the course of 2 months. our results also suggested that ani s 7 can be a potent serodiagnostic antigen in korean patients. in conclusion, we recognized that the seropositive rate of anisakiasis was 5.0% and 6.6% with elisa against crude extracts and esp, respectively. the specific 130 kda protein was confirmed by western blot analysis among elisa positive serum samples. this protein was similar with ani s 7 in molecular weight and can be a candidate for diagnosis of anisakiasis among koreans. | the present study was performed to estimate the seroprevalence of larval anisakis simplex infection among the residents health - examined in 3 hospitals in southern parts of korea. a total of 498 serum samples (1 serum per person) were collected in 3 hospitals in busan metropolitan city, masan city, and geoje city in gyeongsangnam - do (province) and were examined by ige - elisa and ige - western blotting with larval a. simplex crude extract and excretory - secretory products (esp). the prevalence of antibody positivity was 5.0% and 6.6% with elisa against crude extracts and esp, respectively. it was also revealed that infection occurred throughout all age groups and higher in females than in males. a specific protein band of 130 kda was detected from 10 patients with western blot analysis against crude extract and esp among those who showed positive results by elisa. our study showed for the first time the seroprevalence of anisakiasis in korea. the allergen of 130 kda can be a candidate for serologic diagnosis of anisakiasis. |
a petrous apex cholesterol granuloma (pacg) is a very common lesion of the petrous apex. (1) reported hearing loss as the most common symptom of petrous apex lesions, followed by vestibular dysfunction, headache, tinnitus, facial spasms, and diplopia. however, castillo. (2) reported headache and facial nerve weakness as the most common symptoms, and that most patients presented with more than one symptom. thus, there are no disease specific symptoms specific to pacg ; symptoms depend on the size and site of the pacg. here, we describe a case of pacg that manifested with a whirling - type vertigo and fluctuating hearing loss, initially mistaken for mnire 's disease. a 32-yr - old, previously healthy man visited the international clinic of asan medical center in may 2008, complaining of intermittent whirling - type vertigo. at that time, no obvious neurological deficit or nystagmus was found on examination. each vertigo attack lasted about 10 - 15 min, and the episodes occurred twice a month. in addition, he complained of right sided tinnitus, a sense of ear fullness, and had a fluctuating hearing disturbance. pure tone audiometry, speech audiometry, vestibular evoked myogenic potential (vemp), electrocochleography, and caloric testing, were planned, and a low - salt diet was recommended (3). bone and air conduction values on the right side were 12 db and 15 db, respectively, with up - sloping patterns. the cochlear summating potential to auditory nerve action potential ratio (sp / ap ratio) of the electrocochleogram was 0.43 - 0.56 (fig. 1a) (4). video caloric testing demonstrated a right unilateral weakness of 79% (fig. we diagnosed the patient with mnire 's disease of the right side and prescribed ginkgo biloba extract and betahistidine mesylate for 2 weeks. however, 4 days later the patient presented to our emergency room because of muscle twitching and numbness of the right side of the face. the patient was referred to a neurosurgeon. to rule out a central neurological lesion, temporal bone magnetic resonance imaging (mri) t1-weighted mri images showed a homogeneous, well - defined, high - intensity mass ; t2-weighted images of the same lesion were heterogeneous and of high - intensity, but the area was not enhanced after gadolinium administration (fig. these findings indicated the presence of a pacg (5). to evaluate the bony structures and assist in the plans for surgery, temporal bone computed tomography (ct) was performed. this showed a 3 cm isodense mass at the petrous apex, with thinning of the bony wall. after consideration of the location of the cyst and the patient 's hearing function as well as other possible complications such as contamination of the cranial fossa by cholesterol material during and after a middle fossa surgical approach, excision via an infralabyrinthine approach using a computer - aided image - guided surgical device (brainlab, heimstetten, germany) was performed (6 - 8). a preoperative contrast - enhanced three - dimensional t1-weighted mri was conducted to allow installation of the surgical navigation system. the patient 's head was fixed and three infrared emitting markers (red dots) and pointers (green dots) were placed (fig. the instrument recognized not only these markers, but also the surface image of the patient. paired - point and surface matching were performed to compare the actual position of the skull with the preoperative mri imaging data. the mastoid segment of the facial nerve, posterior semicircular canal, and the jugular bulb, were identified. using a diamond burr, the infralabyrinthine air cell tract was followed anteromedially along the long axis of the temporal bone toward the petrous apex. at the midpoint of the drilling process, a pointer was used to confirm the relationship between the location of the pacg and that of the drilling site (fig. postoperatively, the patient did not complain of vertigo, headache, or facial palsy. one month after surgery, the results of the pure tone audiogram returned to the normal range (fig. 5). a temporal bone ct scan revealed no aeration at the petrous apex and the patient had no vertigo. the patient continued to visit the outpatient clinic for four months after surgery, and remains well. classically, the pathogenesis of pacg has been described as being due to occlusion of the mastoid air cells. absorption of trapped air causes hemorrhage into the air cells and subsequent degradation of accumulated hemosiderin to cholesterol, which causes an inflammatory reaction and progressive granuloma formation. recently, a novel pathogenesis has been suggested ; hemorrhage from the exposed bone marrow of the hyper - pneumatized petrous apex is currently thought to be the etiology of pacg (9). treatment of a pacg is symptomatic or when there are signs of a growing lesion. a variety of surgical approaches are available to resect and drain the lesion, with stenting. to determine the optimal surgical approach, the patient 's hearing status, the tumor size and the anatomical relationship between the tumor location and the petrous apex bone, must be established. for a deaf patient however, an infralabyrinthine approach is better if the hearing is to be preserved ; however, this approach is limited to patients with high jugular bulbs. giddings recommended drainage for permanent aeration of a cholesterol - based cyst, and observed that total removal was unnecessary (10, 11). on the other hand, pacg can be accurately diagnosed by ct and mri if there are clinical manifestations. however, a pacg may show no definitive disease - specific symptoms or signs, because of the location and size of the pacg. therefore, the clinician must keep in mind the possibility of a petrous apex lesion in patients that have the clinical features of slowly aggravated endolymphatic hydrops. | a petrous apex cholesterol granuloma (pacg) is the most common lesion of the petrous apex mass. affected patients present with various symptoms such as hearing loss, vertigo, headache, tinnitus, facial spasms, and diplopia. we report the case of a 32-yr - old man with a pacg, who was first misdiagnosed with mnire 's disease. he was placed on a low - salt diet, and prescribed medication from another hospital, for several months, but the symptoms persisted and worsened. the patient presented to the emergency room complaining of left facial twitching and numbness. to rule out a central neurological lesion, temporal bone magnetic resonance imaging was carried out and a 2.5 cm mass with high signal intensity on t1- and t2-weighted imaging, without gadolinium enhancement, was found. because of the hearing and facial problems, we drained cholesterol - bearing material via an infralabyrinthine approach using a computer aided image - guided surgical device, the brainlab. after the operation, the vertigo and hearing loss were no longer present. it is likely that the patent 's mnire 's disease - like symptoms were due to the compression of the endolymphatic sac by a pacg. |
ussing chamber experiments tracheas and distal colon were removed from mice euthanized by ethically approved institutional procedures (c57bl/6, charles rivers, germany). tissues were put immediately into ice - cold buffer solution of the following composition (mmol / liter) : nacl 145, kci 3.8, d - glucose 5, mgci2 1, hepes 5, ca gluconate 1.3. after stripping the colonic mucosa and opening tracheas by a longitudinal cut, tissues were mounted into a micro ussing chamber with a circular aperture of 0.95 mm. mouse m-1 kidney cortex - collecting duct cells (kindly provided by c. korbmacher, physiologisches institut, universitt erlangen, germany) were grown to confluence on permeable supports and mounted into the ussing chamber (2). luminal and basolateral sides of the epithelium were perfused continuously at a rate of 5 ml / min. the bath solution containing (mmol / liter) nacl 145, kh2po4 0.4, k2hpo4 1.6, d - glucose 5, mgci2 1, hepes 5, and calcium gluconate 1.3, was heated to 37 c, and the ph was adjusted to 7.4. values for transepithelial voltages (vte) were referred to the serosal side of the epithelium. transepithelial resistance (rte) was determined by applying short (1 s) current pulses (l = 0.5 a) and after subtracting the resistance of the empty chamber, using ohm 's law (rte = vte/i). transepithelial resistances were 63 3.8 cm ; n = 12 (trachea), 31 2.1 ; n = 13 (colon), and 669 45 cm ; n = 38 (m1). crnas for enac subunits and cftr cdnas - encoding rat (flag - tagged or non - tagged) -enac (kindly provided by prof. 12) and the cl channels cftr were linearized in pbluescript with notl or mlul, and in vitro transcribed using t7, t3, or sp6 promotor and polymerase (promega). isolation and microinjection of oocytes have been described in detail elsewhere (2). the enac mutants s631a, t599a, r561x, y618a, and s633a were generated by pcr, and correct sequences were verified by sequencing. double electrode voltage clamp oocytes were injected with crna (10 ng, 47 nl double - distilled water). 2 - 4 days after injection, oocytes were impaled with two electrodes (clark instruments ltd, salisbury, uk), which had a resistance of < 1 m when filled with 2.7 mol / liter kci. using two bath electrodes and a virtual ground head stage, membrane currents were measured by voltage clamping (oocyte clamp amplifier, warner instruments llc, hamden ct) in intervals from -90 to + 30 mv, in steps of 10 mv, each 1 s. the bath was continuously perfused at a rate of 5 ml / min. chemiluminescence measurements oocytes were fixed for 60 min at room temperature with 3% paraformaldehyde (in tris - buffered saline (tbs), ph 8.0) and washed with tbs ; (mm) 50 tris, 138 nacl, 2.7 kcl, ph 8.0 at room temperature. then oocytes were incubated for 60 min in tbs with 1% (w / v) bovine serum albumin (bsa), another 60 min with 1 g / ml mouse monoclonal anti - flag m2 antibody in 1% bsa / tbs at 4 c (sigma - aldrich), washed at 4 c with 1% bsa / tbs and incubated with sheep anti - mouse igg peroxidase - linked whole antibody (amersham biosciences) diluted 1:20,000 in 1% bsa / tbs for 40 min at 4 c. afterward oocytes were washed for 60 min at 4 c in 1% bsa / tbs and finally in tbs (60 min, 4 c). oocytes were placed separately in 50 l of ecl plus western blotting detection reagents (amersham biosciences). after incubation for 5 min at room temperature, chemiluminescence was measured in a bioorbit 1250 luminometer (turku, finland), and an integration period of 1 s was allowed. oocytes were incubated for 60 min in nd96 solution (in mm : 96 nacl, 2.0 kcl, 1.8 cacl2, 1.0 mgcl2, 5.0 hepes, ph 7.4), fixed for 60 min with 3% paraformaldehyde (in tbs, ph 8.0) and washed in tbs. after embedding in optimum cutting temperature compound (sakura finetek europe, zoeterwoude, nl), oocytes were cut to 20-m slices with a cryostat (leica cm3050 s, wetzlar, germany). sections were put in either tbs or phosphate - buffered saline (pbs ; (mm) 137 nacl, 1.8 kh2po4, 10.3 na2hpo4, ph 7.4), incubated for 5 min in 0.1% (w / v) sds in pbs and washed two times with either tbs or pbs. sections were incubated for 60 min in tbs or pbs (5% bsa) and for 60 min at 37 c with the anti - flag m2 antibody diluted 1:50 in 2% bsa / tbs or a goat polyclonal casein kinase ii antibody (santa cruz biotechnology, heidelberg, germany) diluted 1:25 in 2% bsa / pbs. afterward sections were washed twice in pbs and incubated for 1 h with secondary antibodies (donkey anti - mouse igg - alexa fluor 488 conjugated and donkey anti - goat igg - alexa fluor 546 conjugated ; molecular probes, eugene, or) at a dilution of 1:1000 in 2% bsa / pbs. sections were washed two times with pbs for 5 min and covered with dakocytomation fluorescent mounting medium (dakocytomation, inc. images were obtained using a zeiss axiovert 200 m microscope with a 63 objective (carl zeiss, inc. materials and statistical analysis all compounds used were of highest available grade of purity. amiloride, 2-dimethylamino-4,5,6,7-tetrabromo-1h - benzimidazole (dmat), forskolin, ibmx (3-isobutyl-1-methylxanthine), heparin, okadaic acid, poly(e : y) peptide, and poly(k) were from sigma. l. pinna (department of biological chemistry, university of padua, italy). mnedd4 - 2-rnai (5-cca uuu guc cua uuu cac cuu cau u-3), xnedd4 - 2-rnai (5-gcg ugc cua uga aug gau u-3), and mck2-rnai (5-uug uca aga aga ucu agg gcc ucc g-3) were from invitrogen (paisley, uk). scrambled rna is a mixture of double - stranded rna sequences that has no match to any of the known xenopus mrna sequences. student 's t test was used for statistical analysis. a p value of < 0.05 was regarded as significant. original ussing chamber recordings of the transepithelial voltages vte detected in mouse trachea (a), mouse colon (c), and m1 cells (e). effects of amiloride (a, 10 m) and the ck2 inhibitor tbb (10 m). concentration - dependence of the effects of tbb on amiloride - sensitive transport in trachea (b), colon (d), and m1 cells (f). the asterisk () indicates significant effects of tbb (paired t - tests, number of experiments : 9 - 13 for each series). original ussing chamber recordings of the transepithelial voltages vte detected in mouse trachea (a), mouse colon (c), and m1 cells (e). effects of amiloride (a, 10 m) and the ck2 inhibitor tbb (10 m). concentration - dependence of the effects of tbb on amiloride - sensitive transport in trachea (b), colon (d), and m1 cells (f). the asterisk () indicates significant effects of tbb (paired t - tests, number of experiments : 9 - 13 for each series). a, current recording from a xenopus oocyte expressing -enac and effects of amiloride (10 m) and tbb (10 m). oocytes were voltage - clamped from -90 mv to + 10 mv in steps of 10 mv, and the resulting currents were recorded. b, summary of the effects of amiloride and tbb on whole cell conductance in enac - expressing oocytes. c, summary of the change of amiloride - sensitive conductance (gamil) after injection of water, the ck2 inhibitor poly(e : y) (50 m) and ck2 activator poly(k) (50 m), respectively. d, current recording of an enac - expressing oocyte and the effects of amiloride (a, 10 m) and okadaic acid (100 nm). e, summary of the effect of okadaic acid on the amiloride - sensitive whole cell conductance measured in oocytes. the number sign (#) indicates a significant difference for the effects of amiloride before and after incubation with tbb (paired t - test, 6 - 25 experiments for each series). a, current recording from a xenopus oocyte expressing -enac and effects of amiloride (10 m) and tbb (10 m). oocytes were voltage - clamped from -90 mv to + 10 mv in steps of 10 mv, and the resulting currents were recorded. b, summary of the effects of amiloride and tbb on whole cell conductance in enac - expressing oocytes. c, summary of the change of amiloride - sensitive conductance (gamil) after injection of water, the ck2 inhibitor poly(e : y) (50 m) and ck2 activator poly(k) (50 m), respectively. d, current recording of an enac - expressing oocyte and the effects of amiloride (a, 10 m) and okadaic acid (100 nm). e, summary of the effect of okadaic acid on the amiloride - sensitive whole cell conductance measured in oocytes. the number sign (#) indicates a significant difference for the effects of amiloride before and after incubation with tbb (paired t - test, 6 - 25 experiments for each series). ck2 blocker inhibits enac activity we examined the contribution of ck2 to the regulation of epithelial na channels in native epithelia from mouse trachea and colon. in ussing chamber experiments, we explored ck2-selective concentrations of the specific inhibitor tbb, which was shown to have no effect on over 30 other kinases. tbb selectivity depends on its ability to bind an unusual hydrophobic atp binding site that differs from the equivalent in conventional kinases. electrogenic na transport in mouse trachea was assessed by inhibition of enac with amiloride (10 m) (fig. tbb (10 m) reduced the transepithelial voltage vte and attenuated amiloride - sensitive short - circuit currents (isc - amil) in a dose - dependent manner (fig. similarly, tbb significantly blocked amiloride - sensitive na transport in mouse distal colon (fig. 1, c and d). this effect could be reproduced in cultured mouse m1-collecting duct cells, grown on permeable supports (fig. 1, e and f). these combined results suggest that endogenous epithelial na channels expressed in epithelial tissues are maintained in an active state by constitutively active ck2. to further confirm regulation of epithelial na channels by ck2, the three ()-enac subunits were expressed in xenopus oocytes and examined in double electrode voltage clamp experiments. as shown in the original recording in fig. 2a, the simultaneous expression of the three enac subunits produced a large current, which was inhibited by amiloride (a, 10 m). thus, tbb (10 m) also significantly reduced amiloride - sensitive whole cell currents and conductance (gamil), respectively (fig. another compound, dmat, has recently been shown to inhibit ck2 with higher inhibitory potency, but it has limited efficacy in vivo due to its rapid turnover.5 at 2 m, we did not observe inhibition of enac currents by dmat in xenopus oocytes ; however, 5 m reduced amiloride - sensitive enac conductance significantly from 31.9 6.8 to 25.7 4.1 s(n = 5). regulation of enac by ck2 was further validated using the structurally unrelated peptide inhibitor of ck2, poly(e : y) and conversely, by activating ck2 with polylysine (poly(k)) (16, 24). the peptides were injected into oocytes at final concentrations between 10 and 100 m, and amiloride - sensitive na conductances were compared before and 1 - 5 h after injection. during these few hours, enac conductance increased, almost doubling initial values in water - injected controls (fig. 2), this time - enhanced na conductance was nevertheless inhibited by poly(e : y) and was further activated through stimulation of ck2 by poly(k) (fig. ck2 is known to associate with protein phosphatase 2a (pp2a). to confirm phosphorylation - dependent activation of enac, ooctyes were exposed to okadaic acid at a pp2a - specific concentration (10 nm), which further increased amiloride - sensitive na transport (fig. thus enac appears to be stimulated by constitutively active ck2 with counter - regulation by pp2a - like inhibition. to further confirm this activation by ck2, injection of the nonspecific ck2 inhibitor heparin (10 m) figure 3.elimination of ck2 phosphorylation sites on enac inhibits channel activity. a, current recording from a xenopus oocyte expressing -enac and effects of amiloride (10m) and tbb (10m). oocytes were voltage - clamped from -90 mv to + 10 mv in steps of 10 mv, and the resulting currents were recorded. b, current recording from a xenopus oocyte expressing s631at559a - enac and effects of amiloride (10 m) and tbb (10 m). c and d, summaries of the effects of amiloride and tbb on gamil generated by -enac and s631at559a - enac. e, comparison of gamil produced by wt-()-, single mutants (s631a, t559a)-, and a doublemutant (s631at559a)-enac.f, summaries of gamil produced by dimeric wt-(,)- and mutant (s631a, t559)-enac channels. the asterisk () and number sign (#) indicate a significant difference (paired t - test, 13 - 25 experiments for each series). a, current recording from a xenopus oocyte expressing -enac and effects of amiloride (10m) and tbb (10m). oocytes were voltage - clamped from -90 mv to + 10 mv in steps of 10 mv, and the resulting currents were recorded. b, current recording from a xenopus oocyte expressing s631at559a - enac and effects of amiloride (10 m) and tbb (10 m). c and d, summaries of the effects of amiloride and tbb on gamil generated by -enac and s631at559a - enac. e, comparison of gamil produced by wt-()-, single mutants (s631a, t559a)-, and a doublemutant (s631at559a)-enac.f, summaries of gamil produced by dimeric wt-(,)- and mutant (s631a, t559)-enac channels. the asterisk () and number sign (#) indicate a significant difference (paired t - test, 13 - 25 experiments for each series). time course for gamil (a, c, e) and membrane expression of flag - enac (b, d, f). ooctyes were kept in nd97 or in nd97 containing tbb (10 m), or were injected with heparin (10 m), poly(e : y) (50 m), or equal amounts (30 nl) of water. the asterisk () indicates significant differences when compared with nd96 or water (unpaired t - tests, 6 - 13 experiments for each series). time course for gamil (a, c, e) and membrane expression of flag - enac (b, d, f). ooctyes were kept in nd97 or in nd97 containing tbb (10 m), or were injected with heparin (10 m), poly(e : y) (50 m), or equal amounts (30 nl) of water. the asterisk () indicates significant differences when compared with nd96 or water (unpaired t - tests, 6 - 13 experiments for each series). removal of ck2 sites from - and -enac inhibits amiloride - sensitive na conductance and renders it insensitive to tbb similar to endogenous na currents present in epithelial tissues (fig. 1), enac expressed exogenously in xenopus oocytes was inhibited by tbb in a dose - dependent manner (fig. enac contains two phosphorylation sites for ck2, in -enac (serine 631) and -enac (threonine 599). changing both ck2 sites to alanines (s631at599a) virtually eliminated na conductance and rendered residual conductance of the double mutant channel insensitive to tbb (fig. trimeric na channels carrying only one mutation in either -enac (s631a) or -enac (t599a), produced more measurable but still significantly attenuated na conductances when compared with wt - enac (fig. moreover, the remaining amiloride - sensitive na conductances formed by dimeric -enac and -enac channels were further reduced by s631a and t599a, respectively (fig. 3f). finally, coexpression of hck2 together with wt - enac increased gamil (65.9 9.0 s ; n = 7), when compared with injection of wt - enac alone (47.2 8.1 s ; n = 7). co - expression of hck2 did not augment gamil of s631at599a - enac (3.3 0.7 s ; n = 11 versus 3.4 0.5 s ; n = 11). taken together, these results demonstrate that phosphorylation of s631 and t599 is essential for enac. ck2 controls enac activity and membrane expression of -enac to examine whether ck2 phosphorylation controls membrane expression of enac, flag - tagged -enac was co - expressed with non - flagged -enac in xenopus oocytes. the appearance of flag - enac in the cell membrane was monitored by chemiluminescence, during an observation period of 40 - 48 h (fig. 4, b, d, and f and fig. 5a, see materials and methods). control injection with non - flagged enac did not produce a chemiluminescence different from background (data not shown). in parallel, amiloride - sensitive na conductance 4, a, c, and e). when flag-enac was expressed in control oocytes, both gamil and membrane expression continuously increased (fig. 4, a and b). in contrast, when oocytes were kept in 10 m tbb - containing buffer, amiloride - sensitive na conductance was completely abolished, and membrane expression was significantly reduced, albeit not completely. moreover, co - injection of -enac - crnas together with the ck2 inhibitors heparin (final concentration 10 m) or the peptide inhibitor poly(e : y) (final concentration 50 m) also inhibited na conductance along with membrane expression of -enac (fig. finally, m1 cells were grown on permeable supports, in the absence or presence of 10 m tbb. as observed in oocytes, tbb reduced amiloride - sensitive transport from 2.05 0.35 a / cm (n = 19) to 1.52 0.25 a / cm (n = 16). thus, ck2 phosphorylation differentially controls membrane expression of -enac and enac activity. of note, the inhibitory effect on conductance (3 - 5-fold) was significantly greater than on membrane expression (typically 2-fold). figure 5.ck2 is essential for enac activity and antagonizes the inhibitory effect of nedd4 - 2 on enac. a, summary of -enac membrane expression and gamil after 40 h. tbb inhibited membrane expression of -enac via single mutants (s631a,t559a) but not that of the double mutant (s631at559a). gamil was largely reduced for all mutants, and gamil produced by the double mutant was no longer inhibited by tbb. dashed lines indicate membrane expression and gamil of wt - enac. a mutation in the py motif (y618a) of -enac increased na conductance, and s631a no longer inhibited enac conductance. the grk2 mutant s633a inhibited enac, but not as a double mutant s631a / s633a. c, inhibition of xnedd4 - 2 expression by sirna - xnedd4 - 2 but not scrambled sirna. summary of enac whole cell conductances measured in the absence or presence of sirna - xnedd4 - 2 or scrambled sirna (see materials and methods). d, summary of the amiloride - sensitive short - circuit current and effects of tbb (10 m) in control m1 cells and m1 cell treated with scrambled rnai, mnedd4 - 2-rnai, and mck2-rnai (see materials and methods). the asterisk () indicates significant effects of tbb (paired t - tests). the number sign (#) indicates a significant difference compared with control (unpaired t - test, 6 - 24 experiments for each series). ck2 is essential for enac activity and antagonizes the inhibitory effect of nedd4 - 2 on enac. a, summary of -enac membrane expression and gamil after 40 h. tbb inhibited membrane expression of -enac via single mutants (s631a,t559a) but not that of the double mutant (s631at559a). gamil was largely reduced for all mutants, and gamil produced by the double mutant was no longer inhibited by tbb. dashed lines indicate membrane expression and gamil of wt - enac. a mutation in the py motif (y618a) of -enac increased na conductance, and s631a no longer inhibited enac conductance. the grk2 mutant s633a inhibited enac, but not as a double mutant s631a / s633a. c, inhibition of xnedd4 - 2 expression by sirna - xnedd4 - 2 but not scrambled sirna. summary of enac whole cell conductances measured in the absence or presence of sirna - xnedd4 - 2 or scrambled sirna (see materials and methods). d, summary of the amiloride - sensitive short - circuit current and effects of tbb (10 m) in control m1 cells and m1 cell treated with scrambled rnai, mnedd4 - 2-rnai, and mck2-rnai (see materials and methods). the asterisk () indicates significant effects of tbb (paired t - tests). the number sign (#) indicates a significant difference compared with control (unpaired t - test, 6 - 24 experiments for each series). ck2 is essential for enac activity, but has a small effect on membrane expression of -enac the contribution of both ck2 phosphorylation sites in -enac and -enac, to membrane expression of -enac was further examined by expressing single mutants (flags631a-enac or flagt599a - enac) or the double mutant (flags631at599a - enac) in oocytes. for the sake of simplicity, we only show a summary of the data obtained for membrane expression and enac conductance after 40 h of expression of those enac variants (relative to wild type). importantly, single mutations in -enac or -enac largely reduced the na conductance but had only a small effect on membrane expression. elimination of both ck2 sites in -enac and -enac also strongly reduced gamil, with a minor effect on membrane expression (fig. 5a). of note, tbb reduced -enac expression only when one ck2 site was intact. the nearby located grk2 phosphorylation site ser-633 was reported to control nedd4 - 2 binding to enac (9). we explored whether the same might hold true for ck2 phosphorylation sites. to this end, we compared whole cell conductances of several enac mutants to that of wt - enac. a mutation in the nedd4 - 2 binding site (y618a) increased enac whole cell conductance relative to the wild type and eliminated the inhibitory effect of s631a on enac conductance (fig. this strongly suggests that ck2 phosphorylation at ser-631 antagonizes the inhibitory action of nedd4 - 2 in enac (20) (fig. similarly, when we expressed the truncated r561x-enac channel, we found that amiloride - sensitive whole cell conductance was largely increased (64.5 5.9 s ; n = 14) when compared with -enac, and was now unaffected by 10 m tbb (64.1 6.1 s). interestingly, mutating the grk2 site also reduced enac conductance ; however, eliminating both ck2 and grk2 sites in -enac produced a channel with similar activity to that of wt - enac (fig. this suggests that the closely located phosphorylation sites of ck2 and grk2 (ser-631, ser-633) influence each other. the role of nedd4 - 2 on ck2 regulation of enac was further examined by sirna - knockdown of xnedd4 - 2, similar to a recent study (fig. two remarkable results were obtained, namely : (i) wt - enac was no longer inhibited by tbb. (ii) s631a-renac, which normally produced only small amiloride - sensitive na conductance, generated a whole cell conductance that was indistinguishable to that obtained for wt -renac (fig. the contribution of nedd4 - 2 to ck2 regulation of enac was further examined in mammalian cells. m1 cells were grown on permeable supports after treatment with rnai for mck2, mnedd4 - 2, or scrambled rnai, or as nontransfected control cells (fig. knockdown of ck2 and nedd4 - 2 was verified by western blotting (supplemental fig. isc - amil was enhanced in m1 cells following nedd4 - 2 knockdown, and inhibition by tbb was largely reduced (fig. 5d). in contrast, ck2 knockdown reduced isc - amil and also abolished the effect of tbb on isc - amil (fig. 5d). taken together, these results suggest that ck2 phosphorylation of enac antagonizes the inhibitory effects of nedd4 - 2. figure 6.ck2 is not essential for membrane expression of -enac and -enac. a, inhibition of membrane expression of flag - enac and flag - enac and gamil by 10 m tbb. b and c, tbb inhibited membrane expression of flag - enac and flag - enac in single mutants (s631a, t559a), but not in double mutants (s631at559a). gamil was largely reduced for all mutants, and gamil produced by the double mutants was no longer inhibited by tbb. dashed lines indicate membrane expression and gamil of wt - enac. the asterisk () indicates a significant effect of tbb (paired t - tests, 6 - 12 experiments for each series). ck2 is not essential for membrane expression of -enac and -enac. a, inhibition of membrane expression of flag - enac and flag - enac and gamil by 10 m tbb. b and c, tbb inhibited membrane expression of flag - enac and flag - enac in single mutants (s631a, t559a), but not in double mutants (s631at559a). gamil was largely reduced for all mutants, and gamil produced by the double mutants was no longer inhibited by tbb. the asterisk () indicates a significant effect of tbb (paired t - tests, 6 - 12 experiments for each series). ck2 controls membrane expression of -enac and -enac the activity of epithelial na channels largely depends on co - expression of both - and -subunits. membrane expression of -enac and -enac was monitored by injecting flag-enac or flag - enac, respectively, and na conductances were measured in parallel. na conductances generated by either flag-enac or flag - enac, respectively, were almost completely abolished when oocytes were exposed to 10 m tbb, while membrane expression of flag and flag were reduced by about 50% (fig. we further examined whether flag and flag behave in a similar fashion in the absence of -enac. dimeric channels formed by flag-enac or flag - enac produced small but detectable amiloride - sensitive whole cell conductances (0.74 0.2 s ; n = 8 and 0.2 0.1 s ; n = 7, respectively). interestingly expression of flag - enac and flag - enac in dimeric and trimeric channels was similar, but expression of both subunits in the dimeric channel was not affected by tbb (data not shown). this suggests a complex role of -enac for expression of -enac. to further assess the impact of ck2 phosphorylation on membrane expression of -enac and -enac in trimeric (flag-enac and flag - enac) channels, we expressed single mutants (s631a - flag-enac or t599a - flag - enac) and double mutants (s631a - flagt599a - enac or s631at599a - flag - enac) in the absence or presence of 10 m tbb. for all mutant combinations, na conductances were largely reduced in comparison to that of wt - enac, and residual conductances generated by the double mutants were insensitive to tbb (fig. in contrast, the membrane expression of -enac and -enac was not affected by any of the mutations individually, and membrane expression of the double mutants (s631a - flagt599a - enac ; s631at599a - flag - enac) was no longer inhibited by tbb (fig. these data suggest that when ck2 sites are present in -enac and -enac, they need to be phosphorylated to allow proper membrane expression of all three subunits. enac translocates ck2 to the cell membrane it has been reported that ck2 binds directly to enac (21). this suggests that ck2 may be translocated by enac to the cell membrane as found for cftr (24). oocytes expressing enac and non - injected control oocytes were immunolabeled, after embedding and cutting into 20-m sections. using an anti - flag antibody, -enac moreover, upon expression of flag-enac or flag - enac, - and -subunits could be immunolabeled in the oocyte membrane (fig. expression of the three subunits was similar in s631at559a - enac - injected oocytes (fig. however, while ck2 was detected in membranes of oocytes expressing wt - enac, no membrane staining of ck2 was detectable in s631at559a - enac - injected oocytes (fig. these results suggest that ck2 may be translocated to oocyte membranes by binding to enac - and -subunits. ck2 regulates ion channels ck2 is an essential, constitutively regulated multifunctional protein kinase whose functions are not fully understood (21). ck2 is believed to insert into protein complexes bringing its constitutive activity to many signaling complexes (p53, ion channels, actin capping at membranes, lipid flippases, etc). ck2 affects a number of membrane channels and pumps, among them is polycystin-2 (pc2), a divalent cation - selective channel. ck2 was shown to maintain the ca sensitivity of pc2. elimination of a ck2 site in pc2 reduced its ca sensitivity, without changing membrane expression (5). others reported the importance of phosphorylation of pc2 by ck2, for proper ciliary localization of the channel (13). ck2 also phosphorylates three critical serine residues within nephrocystin, another ciliary protein. ck2 phosphorylation is essential for co - localization of nephrocystin with the sorting protein pacs-1 at the base of cilia. inhibition of ck2 was shown to eliminate interaction of pacs-1 and nephrocystin and to induce defective nephrocystin targeting (19). ck2 may thus be relevant to the common autosomal form of polycystic kidney disease.. a, dic image of the oocyte membrane (left panels) and immunostaining of flag - enac in an enac - expressing (right upper panel) and a non - injected (right lower panel) oocyte. b, immunostaining of the three enac subunits (green) and ck2 (red) in wt - enac (left panel) and s631at559a - enac (right panel)-expressing oocytes. wt - enac translocates ck2 to the cell membrane. a, dic image of the oocyte membrane (left panels) and immunostaining of flag - enac in an enac - expressing (right upper panel) and a non - injected (right lower panel) oocyte. b, immunostaining of the three enac subunits (green) and ck2 (red) in wt - enac (left panel) and s631at559a - enac (right panel)-expressing oocytes. we found here that enac is regulated by ck2 in a related manner, i.e. ck2 regulates enac activity as well as membrane expression. lack of ck2 phosphorylation appears to reduce nedd4 - 2 binding to enac, thereby enhancing channel activity and membrane expression. notably, phosphorylation of enac by erk inhibits both channel activity and membrane expression, and both effects are mediated by nedd4 - 2 binding to enac (10). ck2 has also been described to influence other ion channels, namely by regulating the current amplitude of voltage - dependent ca channels (14), as well as the ca - dependent gating of small conductance ca - activated k channels (4). sk channels are organized in multiprotein complexes together with ck2 and pp2a, located in the postsynaptic pole of cochlear outer hair cells (4). furthermore, ck2 has recently been found to regulate cftr, the chloride channel that is defective in cystic fibrosis and, as in this study, ck2 becomes membrane - associated with cftr (24). once again both ck2 and pp2a co - localize with different domains of cftr (16, 24). importantly, ck2 binding may be disease relevant because phenylalanine 508 (f508), the common missing residue in most patients with cf, is critical in promoting the interaction with cftr. in both cftr - expressing xenopus oocytes and cell - attached mammalian cells, inhibition of ck2 induced prompt closure of cftr in less than 80 s (24). however, direct phosphorylation of cftr by ck2 at ser-511 close to f508 has not been confirmed by radiolabeling cells and direct phosphopeptide mapping, and the exact mechanism remains unknown. moreover, cftr expression determines the cellular localization of ck2, which in turn could affect other membrane conductances such as amiloride - sensitive na absorption. it is interesting to note that the effect of ck2 is not stimulatory of the function of all proteins studied to date. indeed, ck2 is inhibitory of another abc family member, abca1 ; thus, constitutively suppressing the lipid flippase activity of this cftr - related protein. c terminus of -enac, a target for kinase regulation of enac phosphorylation - dependent regulation of enac by ck2 is demonstrated in the present study by several agents, namely, heparin, a nonspecific inhibitor of ck2 directed against acidic motifs ; the peptide inhibitor of ck2 ; poly(e : y) ; and the specific ck2 inhibitors tbb and dmat. inhibition of protein phosphatases with okadaic acid further increased na conductance, indicating the importance of basal ck2-dependent phosphorylation for enac regulation (3). furthermore, elimination of ck2 phosphorylation sites (ser-6331) abrogated na conductance in xenopus oocytes. thus, together with results obtained in native epithelia and in epithelial cells, these data show that enac is compellingly regulated by ck2 (fig. 8). notably, ck1 was shown recently to control intracellular trafficking of enac (25). yet, in a previous report such regulation could not be detected in salivary duct cells (9). instead, activation of enac by the g protein - coupled receptor kinase 2 grk2 was found. because of phosphorylation of ser-633 by grk2 in the c terminus of -enac, the affinity of nedd4 - 2 binding to enac is apparently reduced, thus enhancing enac currents. according to the present data (fig. 5b), a similar scenario may hold true for ck2-dependent regulation of enac, because both phosphorylation sites for grk2 and ck2 are located in close proximity to one other. because ck2 classically engages in hierarchical phosphorylation with other kinases, phosphorylation through either grk2 or ck2 could control enac activity and membrane expression depending on the cell type and expression levels of the kinases involved. moreover, phosphorylation by ck2 is likely to affect regulation by grk2 and vice versa (fig. further experiments will be needed to unravel this complex regulatory network and should focus on the inhibitory phosphorylation by erk and its counteraction by activation through pka, grk2, and ck2, with nedd4 - 2 as the central control switch (1, 9, 20, 26). as suggested in a previous report and supported by the present data, acute and chronic regulation may be different, inasmuch as acute regulation is changing the open probability of the channel, while chronic regulation by nedd4 - 2 controls membrane expression (10). binding of the ubiquitin ligase nedd4 - 2 leads to ubiquitination of enac and subsequent degradation of the channel and/or channel inactivation. phosphorylation of enac at ser-631 reduces affinity of enac for nedd4 - 2, thereby maintaining membrane localization and enac activity. binding of the ubiquitin ligase nedd4 - 2 leads to ubiquitination of enac and subsequent degradation of the channel and/or channel inactivation. phosphorylation of enac at ser-631 reduces affinity of enac for nedd4 - 2, thereby maintaining membrane localization and enac activity. | ck2 is a ubiquitous, pleiotropic, and constitutively active ser / thr protein kinase that controls protein expression, cell signaling, and ion channel activity. phosphorylation sites for ck2 are located in the c terminus of both - and -subunits of the epithelial na+ channel (enac). we examined the role of ck2 on the regulation of both endogenous enac in native murine epithelia and in xenopus oocytes expressing renac. in ussing chamber experiments with mouse airways, colon, and cultured m1-collecting duct cells, amiloride - sensitive na+ transport was inhibited dose - dependently by the selective ck2 inhibitor 4,5,6,7-tetrabromobenzotriazole (tbb). in oocytes, enac currents were also inhibited by tbb and by the structurally unrelated inhibitors heparin and poly(e : y). expression of a trimeric channel lacking both ck2 sites (s631at599a) produced a largely attenuated amiloride - sensitive whole cell conductance and rendered the mutant channel insensitive to ck2. in xenopus oocytes, ck2 was translocated to the cell membrane upon expression of wt - enac but not of s631at599a - enac. phosphorylation by ck2 is essential for enac activation, and to a lesser degree, it also controls membrane expression of -enac. channels lacking the nedd4 - 2 binding motif in -enac (r561x, y618a) no longer required the ck2 site for channel activity and sirna - knockdown of nedd4 - 2 eliminated the effects of tbb. this implies a role for ck2 in inhibiting the nedd4 - 2 pathway. we propose that the c terminus of -enac is targeted by this essential, conserved pleiotropic kinase that directs its constitutive activity toward many cellular protein complexes. |
a major part of our cultural heritage has been recorded in various forms on paper for centuries and is therefore vulnerable to biodeterioration of its organic components through fungi (nol., 2001 ; corte., 2003 ; cappitelli and sorlini, 2005 ; michaelsen., 2006, 2009 ; rakotonirainy., 2007 ; zotti and ferroni, 2008 ; mesquita., 2009) and, in a minor way, bacteria (de paolis and lippi, 2008 ; jurado., 2010 ; michaelsen., fungal contamination is considered a major concern for libraries or archives full of paper - based books and documents. for the storage and maintenance of this often valuable material, it is crucial not only to control active fungal growth, i.e., hyphae, mycelium, or mould, but also to remove or reduce the amount of fungal ascospores and conidia. the structural nature of ascospores as progenitors of future growth allows the fungi to survive severe conditions, and ascospores are consequently harder to inactivate than the vegetative hyphae. in unfavorable conditions resting spores have low water content and their metabolism is inactivated but reversible (florian, 1993 ; deacon, 2005). hence, any treatment to conserve objects of cultural value should be directed toward the spores, as the vegetative hyphae are relatively easy to control by physical removal and through monitoring of the storage climate, in terms of temperature, relative humidity, and activity of water (nitterus, 2000). fungi are an increasing and dominant problem for archives and museums and thus the prevention of their growth and the development of appropriate treatment measures for contaminated objects are a challenge for restorers, curators, and scientists (sterflinger, 2010). a broad spectrum of chemical and non - chemical components has been used to sanitize microfungi attacking paper - made objects in an attempt to inhibit degradation (magaudda, 2004). in this study we chose three different conservation treatments for paper to compare their effectiveness over the short- and long - term, namely freeze - drying, gamma rays, and ethylene oxide fumigation. a commonly used strategy in paper conservation is freeze - drying, a method in which the water is frozen and then removed by sublimation, i.e., it goes from the solid phase to water vapor, bypassing the liquid phase. sublimation allows the water to be removed without the effects of water evaporative forces, which can cause dimensional changes. in addition, freeze - drying with dehydration can kill hydrated conidia and germinating conidia and it stops the growth of fungal mycelia and bacterial cells (sussman, 1966 ; mazur, 1968 ; florian, 2002). yet if the moisture content in the thawed materials remains high, resting dry conidia may be activated. freezing can also increase the porosity and thickness of organic materials and make them more hygroscopic. although it can not be considered a disinfecting treatment, freeze - drying is still the most effective method known for the physical, chemical, and biological stabilization of water - damaged archival and library materials, especially when large quantities are involved and time is of the essence (schmidt, 1985 ; mccleary, 1987 ; walsh, 1988 ; parker, 1989 ; florian, 1990). the application of gamma rays in conservation science dates back to the 1960s, when the radio - resistance of significant mould fungi from goods of cultural value was tested (belyakova, 1960). high - energy electromagnetic radiation is deeply penetrating and biocidal through the denaturation and cleavage of nucleic acids, which leads to a simultaneous and indiscriminate devitalisation of all living organisms (magaudda, 2004). gamma irradiation is successfully used to sterilize laboratory and hospital utensils and food, but it can have unwanted side effects when applied in paper conservation where high irradiation doses, which are often required in repeated doses, can result in cumulative depolymerization of the underlying cellulose in the paper. severe aging characteristics, such as lowered folding endurance and tear resistance, increased yellowing, and general embrittlement, have been reported in paper treated with gamma rays (butterfield, 1987 ; adamo., 1998), whereas more recent studies have suggested that the damage in terms of mechanical physical properties is not significant (adamo. the observed effects of gamma rays on fungi from paper have confirmed that radiation treatment of books and documents is effective, as there was no fungal growth detectable in cultivation studies (jrg., 1992 ; da silva., 2006). finally, ethylene oxide (eto) has been widely used for sterilization of objects of cultural heritage since 1933 (ballard and baer, 1986). ethylene oxide does not require activation energy, is used at room temperature, and expresses the high reactivity and diffusivity required for the inactivation of microorganisms (mendes., 2007). by adding alkyl groups to dna, rna, or proteins, eto prevents normal cellular metabolism and the ability to reproduce (rutala and weber, 1999). this ability to act as an alkylating agent was widely taken to indicate a carcinogenic potential, an assumption that has subsequently been proven for eto and some of its residuals (bolt, 1996 ; angerer., 1998), leading to a ban on eto for the practice of paper conservation in many countries. additionally, a study found paper material fumigated with eto to be more susceptible to microbial attack after fumigation (valentin, 1986). this phenomenon is not fully understood but it is claimed that ethylene glycol, formed as a by - product during fumigation, activates spores that contaminate the object in further storage (florian, 1993). in this study, the effects of these three conservation treatments on fungal spores and mycelium viability and activity were investigated by culture - dependent and -independent strategies. the main goals were : (a) to compare the efficacy of molecular versus cultivation techniques as models for the monitoring of conservation treatments, (b) to evaluate massive and aggressive disinfecting treatments on dna integrity, and (c) to use rrna analyses as a method of determining long - term viability of fungi. with this end, two paper grades were used for the in - vitro inoculation of spores from different fungal species to obtain infected paper samples. the physiological state of the fungal strains was monitored over the short- and long - term after the treatments to determine time - dependent effects of the treatments on fungal viability. these effects were examined by culturing and on a molecular level by generating dna - denaturing gradient gel electrophoresis (dna - dgge) profiles. as only metabolically active cells produce rna, we used the ability to retrieve fungal - specific rrna directly from the paper samples as an indicator of the viability of the respective fungi after the samples had been left for a year for eventual regrowth. this strategy is commonly used in bacterial ecology and is based on the fact that metabolically active species transcribe more rrna for ribosome synthesis than do inactive species (prosser, 2002). as rna is also highly unstable in the environment, the detection of rna in an environmental sample has been used as a strategy for detecting the most active microorganisms within a natural community (gonzalez., 2006). to our knowledge this is the first study combining molecular methods including rna detection of fungi with monitoring of conservation treatments for paper - made objects. fungal growth was induced in vitro by inoculating two types of paper with three fungal strains previously isolated : chaetomium globosum kunze, cladosporium cladosporioides (fres.) de vries, and trichoderma viride pers. paper a was a whatman paper type (whatman 1 chr category no. 3001917, not glued and not sized, 100% cotton linter, ph 6.57.0) consisting of pure cellulose with low ash content, and paper d was a mezzofino type paper with a high lignin content, namely a naturally aged rag paper produced by the istituto poligrafico dello stato in 1976 (gallo., 1999), no. 200953, consisting of bleached cellulose (45%), wood pulp treated with sulphites (25%), wood pulp from softwood (20%), glue (3%), aluminum sulfate (5%), and kaolin (2%), ph 4.5. each paper sample existed as a set of small discs (about 1 cm diameter) cut with a puncher (12.5 mm disc punch n. t5443, agar scientific ltd. fungal strains were grown on malt extract agar (2%) to obtain colonies with mature fruiting bodies or reproductive structures. conidia and ascospores were harvested with a sterile cotton swab and diluted in water with tween 80 (0.01%, sigma, italia) to obtain solutions with a standard concentration of about 5000 spores / ml. a further dilution was performed with a czapek broth, to obtain 50 spores l. the water vapor - sterilized paper samples were inoculated with 50 l of broth each to provide a physiological starter for germination. about 100% relative humidity (rh) was maintained with distilled water during fungal growth in double - bottom glass containers. samples were kept in a thermostatic cell at 27 c for 7 days (c. cladosporioides and t. viride), and 14 days (ch. chaetomium is a genus of filamentous fungi (phylum ascomycota, class sordariomycetes) encompassing species that typically possess densely setose, ovoid to pyriform ostiolate ascomata, clavate asci, and pigmented, one - celled ascospores (samson., 2000) several strains of trichoderma have been developed as biocontrol agents against fungal diseases of plants. the various mechanisms include antibiosis, parasitism, inducing host - plant resistance, and competition. the t. viride strain used in the production of paper samples was characterized by producing only conidia, and not the ascospores typical of its prefect state (hypocrea). cladosporium is a dematiaceous (pigmented) mould widely distributed in air and rotten organic material. the genus cladosporium is one of the largest genera of dematiaceous hyphomycetes, and is characterized by conidia in acropetal chains and davidiella teleomorphs. c. cladosporioides has conidiophora distinct from vegetative hypahe and conidia that are one - celled, thick - walled, and finely roughed, produced in branched chains (samson., 2000). one replica of each set was used as a control sample and not subjected to any treatment. the other replicas were treated with freeze - drying, gamma rays, or eto. viability of fungal spores was tested one month after treatment for all samples (t1) and one year later (t2) for the untreated control samples with the 25 points inoculum method. to avoid contamination from airborne fungi after the treatment, all samples were placed into sterile paper air - permeable envelopes before being treated and left therein until further testing. sample envelopes were stored in petri dishes in dark conditions at room temperature. gamma irradiation and eto fumigation are hazardous and consequently conducted by specialists and approved companies. the envelopes containing samples (a single envelope for each paper grade and each fungal inoculum) were positioned inside blocks of copying paper in order to simulate the treatment of a thicker volume and to avoid a direct heating of the samples on the warming grid of the freeze - dryer. a set of five paper leaves was interposed between each envelope and a 2-cm layer of paper leaves was used to isolate the envelopes from the heating plate. the treatment was conducted at the centro di legatoria e restauro frati e livi, s.r.l. (castelmaggiore, bologna, italy) in a 5-h cycle with warming temperatures of 50 c maximum. irradiation treatment was performed at the research centre of enea casaccia, rome, italy, by dr. inoculated paper samples underwent a gamma ray dose of 4756 gy / h with an exposure time of 1 h and 3 min (adamo., 2001). fumigation treatment was performed by spix italia s.r.l. in a vacuum cell device with a mixture of eto (10%) and co2 (90%) for 48 h at a temperature between 20 and 22 c. the treatment was verified with chemical (eo stripes n. 6615 eto chemical indicator, vwr international pbi srl) and biological (eo vials n. 3166 eto biological indicator, vwr international pbi srl) standard indicators (ballard and baer, 1986). sets of inoculated paper samples that developed fungal mycelia were air - dried in sterile polystyrene ventilated petri dishes at about 21 c and 50% rh for one week to stop active growth of fungal mycelia, but to keep conidia and ascospores alive. following natural dehydration the samples were sterilely introduced into paper envelopes and kept in petri dishes in the same manner as the treated samples. the treated and the untreated control samples were tested for viability of the fungal spores by means of the 25 points inoculum method : sets of inoculated and treated paper, three each, were washed three times in sterile water and divided into 25 sub - samples that were inoculated directly on solid nutritive agar (malt extract agar, 20 g l) distributed according to a geometric scheme (fig. 1). the development of the fungal mycelium in all or a fraction of the 25 inoculum points allows for a statistical comparison between different treatments and untreated samples (dix and webster, 1995, after adaptation on cellulosic materials). one - way anova was computed and followed by the fisher test according to massart. (1998) using xlstat statistical software (addinsoft, paris) to determine the significant differences between the treatments. the inocula on agar were performed directly after the treatments and 1 year later (long - term monitoring, t2). dna was extracted directly from the prepared small paper discs (about 1 cm in diameter) by using the fastdna spin kit for soil (mp biomedicals, solon, oh, usa). the protocol of the manufacturer was slightly modified, as described by michaelsen. (2006). to ensure the lysis of all cells and spores on the paper surface, the samples were pre - treated with lysozyme and proteinase k as described by schabereiter - gurtner. dna extraction was performed one month after treatment (short - term monitoring, t1) and again one year later (long - term monitoring, t2) to test the efficiency and long - term effects of the different treatments on the dna of the inoculated fungi. all pcr reactions were executed using pcr master mix (promega, mannheim, germany). fragments of about 500700 bp size corresponding to the its1its2 region of the ribosomal - dna internal transcribed spacer region were amplified with the primer pair its1 and its4 (white., 1990). for dgge analysis of the dna fragments, a nested pcr was performed with the pcr product of the first round as template dna using the primers its1gc with a 37-base gc clamp attached to the 5-end and its2 (white., 1990 ; (2006) and pcr products were visualized by electrophoresis in a 2% (w / v) agarose gel. denaturing gradient gel electrophoresis (dgge) was performed as previously described by muyzer. (1993) in 0.5 tae (20 mm tris, 10 mm acetate, 0.5 mm na2edta ; ph 7.8) with 8% (w / v) acrylamide gels containing a gradient of 3050% denaturants in a dgge 2401 system (c.b.s. gels were run at 60 c and 75 v for 14 h, stained with ethidium bromide, and visualized using a bio - rad gel doc imaging system (bio - rad laboratories pty., ltd, australia). rna was extracted from the paper samples using a combination of the method developed by griffiths. (2000) and the rneasy plant mini kit (qiagen, australia) with some changes to the manufacturer 's protocol. one disc per treatment was placed in a lysing matrix e tube (mp biomedicals, solon, oh, usa) and 600 l of buffer rlc from the rneasy plant mini kit was added. the tubes were processed on the fastprep beadbeater for 30 s at 5.5, followed by a centrifugation for 2 min at 13,000 rpm. the supernatant was then used with the rneasy kit following the protocol for fungi and plants from step 4 on, according to the manufacturer 's instruction. an on - column dnaase treatment was performed using the qiagen rnase - free dnase set (qiagen, australia). the final elution step was carried out twice with the provided water and stored at 80 c. rna extraction was performed one year after the treatment of samples (long - term monitoring, t2) to allow conclusions on the viability and actual growth of the inoculated and treated strains to be compared with the dna results. in all cases, pcr controls with the crude rna extracts rt - pcr was carried out using 2 l rna template for the initial denaturing step with 1 l of reverse primer 18sr (5-gatccttctgcaggttcacctac-3) for 5 min at 65 c. for dna synthesis, 1 l of amv reverse transcriptase (promega, australia), 0.5 l dntps (100 mm) and 2.5 l buffer omniscript were used in a 10-l reaction volume with molecular grade water (all qiagen, australia) for 60 min incubation at 37 c. the cdna was used to amplify approximately 250 bp products of the 18s rrna with primers ek555sr (5-gctgctggcaccagact-3) and 18s-300f gc, which includes a 39-bp gc clamp on the 5 end to be used in dgge analysis (moreno., 2010). the pcr reaction contained 2 l of cdna with 2.5 l amplitaqtm10 buffer, 1.5 l mgcl2 solution, 0.1 l amplitaqtm dna polymerase (all applied biosystems), 10 pmol of each primer, 0.5 l dntps (10 mm), and 1.25 l dmso in a 25-l volume. the pcr conditions used included an initial denaturation step at 95 c for 2 min, followed by 30 cycles of 95 c for 30 s, 61 c for 30 s, and 72 c for 30 s, and a final extension cycle of 10 min at 72 c. the gc - clamped pcr products were separated using dgge on 10% (wt / vol) polyacrylamide gels with a 3050% urea / formamide denaturing gradient using the dgge 2401 system (c.b.s. gels were run at 60 c and 55 v for 17 h, stained with ethidium bromide, and visualized using a bio - rad gel doc imaging system (bio - rad laboratories pty., ltd, australia). the development of fungal mycelium in the 25-spot grid enabled statistical comparisons between the treatments and the control samples and among the control samples as presented in fig. 1 and table 1, according to the analysis of variance (anova) method and fisher test. non - treated control samples showed good growth for all fungi with no statistical difference, but different inoculation results were obtained from paper samples subjected to freeze - drying, dependent on the species. c. cladosporioides appeared to be no longer cultivable and the recorded growth for t. viride was low and statistically different from both the control sample and ch. globosum and mixed inocula samples with respect to the control samples. both gamma irradiation and eto fumigation did suppress fungal growth of all single species and the mixed inocula over the short - term and showed full efficacy as disinfecting agents when evaluated with cultivation techniques (table 1). long - term monitoring showed differences in the growth of the singly inoculated fungal strains stored without treatment (t2). t. viride and c. cladosporioides were not able to grow after one year at room temperature. globosum did not behave in a statistically different manner compared to the sample one year before. these results can be explained by the higher resistance of chaetomium ascospores with respect to the conidia of trichoderma and cladosporium strains. ascospores are typically thick - walled and protected in a fruiting body, as in chaetomium spp. statistically significant differences (table 1) were also noted in a general favor of paper a, the whatman paper, for fungal growth after treatment. however, as the same effect was observed for the control samples of paper a, it indicates that cotton linter generally supported fungal growth better than rag paper d, the mezzofino paper made from cellulose and wood pulp, and the differences evaluated can be independent of the treatments. denaturing gradient gel electrophoresis is the technique most often used to study the structure of the microbial communities colonizing artworks (gonzalez and saiz - jimenez, 2004). furthermore, it has recently been implemented to monitor structural changes in microbial communities colonizing stone - works after conservation treatments (piar., 2010 ; hence, in this study we applied dgge for the first time to monitor paper samples that were inoculated with fungal spores and further subjected to different conservation treatments. this allowed monitoring of changes on the dna level of different fungal species in relation to the treatments, and determination of the treatments ' efficiency. to this end, the internal transcribed spacer region 1 (its1) was amplified from template dna extracted directly from the treated paper samples as well as from non - treated control samples and subsequently separated in dgge (michaelsen., 2006). interestingly, our results show only minor effects on the recovery of fungal dna from samples treated with freeze - drying or gamma rays, as minor differences between the dgge profiles of treated and non - treated control samples are mostly restricted to the mixed inocula samples (fig. 2). denaturing gradient gel electrophoresis fingerprints derived from untreated control samples one month after the treatment (t1) and one year later (t2) are nearly identical, as shown in fig dna from t. viride (lanes 5 and 6) was not detected in any sample when inoculated in a mixture with ch. however, it should be considered that t. viride was initially not competitive enough and was outgrown by the mycelium of the other two fungi, probably due to the antifungal effect of chaetomium (kanokmedhakul., 2002). the amount of fungal spores and mycelium from t. viride on the samples was below the detection limit of our pcr and dgge conditions. dgge bands representing c. globosum (lanes 3 and 4) were observed in both single and mixed inoculated control samples of both paper types, at both t1 and t2, whereas t. viride only showed clear bands when inoculated as a single strain at both time points monitored. unexpectedly, no dgge band could be obtained for c. cladosporioides inoculated as a single strain on paper d throughout the experiment, probably due to the difficulty of this species to grow as a single strain on the lignin containing / acidic paper. the conidia of this fungus deposited on paper d with the inoculum did not germinate but as conidia were possibly around they were probably below the detection limit of our pcr and dgge conditions. temperature has an effect on the growth and survival of fungi, as it dictates the rate of the reactions catalyzed by enzymes, but it is important to note that a temperature below which no growth occurs should not be understood as a lethal temperature for fungi (nitterus, 2000). reducing the temperature to subzero values, as happens in freeze - drying cycles, reduces the viability of many fungi and can finally exhaust even dormant spores if these temperatures are maintained over long periods after initially activating them (florian, 1993). as spores and hyphae in mature and active fungal colonies contain considerable amounts of water, the majority of them should be killed at temperatures below 0 c (nitterus, 2000). but our results show that, on the contrary, cultivation and molecular techniques both produced positive results for fungal growth or activity on samples subjected to freeze - drying, which are comparable with those of untreated samples. this explains also why freeze - drying is not considered a conservation treatment but an emergency mass - rescue procedure for flooding and other water - related incidents (florian, 1990, 2002). dgge fingerprints derived from samples treated with freeze - drying showed some differences compared to the control samples (fig. one month after the treatment (t1) the fingerprints derived from samples inoculated with the mixed culture differed from the control and between paper a, whatman, and paper d, mezzofino. the c. cladosporioides was represented by a strong band on paper a (t1, lane 1) but it was missing on paper d (t1, lane 2). globosum appeared as a strong band on paper d but was very faint on paper a, whereas t. viride was not detected in any mixed inoculated sample, as observed in the control samples. the bands representing each single fungus inoculated as pure culture did not show differences due to treatment. long - term effects due to the freeze - drying treatment were only observed for ch. globosum when inoculated as a single strain, showing an unspecific band - pattern (fig. furthermore, no dgge band corresponding to this fungus was detected when it was inoculated in a mixture with the other two fungi (fig. different functions of the paper or its components, such as cellulose, are affected by gamma irradiation. furthermore, its germicidal power on fungal microflora has been reported to be dose - dependent (magaudda., 2000 ; adamo., 2001). a dose of about 5 kgy, as applied in our experiment, was found to cause statistically significant variations on almost all paper properties, such as tensile strength, tearing resistance, and folding endurance, but also reduced the fungal population down to the blank relatively low irradiation doses of 23 kgy delivered comparable decontaminating power without the increasingly depolymerizing effects of gamma rays on cellulose. samples treated with gamma rays showed dgge patterns with numerous unspecific bands one month after treatment (fig. 2c, globosum, t. viride, and c. cladosporioides were still observed, but their identities were indecisive in some samples. these patterns indicate a mixed population of intact dna from surviving fungi and dna artifacts, as a consequence of dna fragmentation due to the irradiation, from affected fungi. 2c, t2), no more visible effect of gamma radiation on the dna of pure strains, compared to the control samples, was observed. this indicates that the surviving fungal population recovered with time and only the intact dna of this population was detected. for paper samples inoculated with mixed inocula, only the band for c. cladosporioides was shown to be weak on paper d (fig. the observation that eto can significantly reduce or erase the amount of amplifiable dna on small items has been reported before. ethylene oxide is successfully applied in forensics to remove dna contamination on items used both at crime scenes and in the forensics laboratory, without significantly affecting any needed downstream dna analysis when used to sterilize equipment (shaw. the gaseous eto is considered a radiomimetic agent, which means it is similar to ionizing radiation by virtue of its ability to induce the same biological end - points, such as gene mutations, mainly by alkylating and reacting with nucleophile centers, such as nitrogen and oxygen atoms in the dna bases (bolt, 1996 ; rutala and weber, 1999 ; chovanec. double - strand - breakages are induced, leading to fragmentation of the dna helix. our results could be explained by such a fragmentation of fungal dna through eto fumigation, resulting in pcr not delivering any evidence of fungal dna or rna on all samples submitted to fumigation. in accordance with this, a study on viability of fungal spores on paper after eto treatment indicated that fungal spores became non - cultivable and non - viable with eto inhibiting spore activity (rakotonirainy., 2003). (2008) did, we observed that eto fumigation was more effective than the application of gamma rays in reducing dna from the surface of samples. the superiority of eto as a sterilizing agent compared to gamma radiation was explained by chovanec. (2001) as a combination of differences in the nature of damage caused and the distribution of the target. ionizing radiation can react more locally and focused with free radicals following tracks along covalent bonds, whereas small molecules such as eto can diffuse quite freely in the cell nucleus, causing damage on a broader scale. although eto application is banned for use in conservation, it should still be recommended in certain cases where the careful restoration and conservation of valued material is assessed. the fumigation of paper with eto caused the loss of amplifiable fungal dna from all spore - inoculated samples tested in both single and mixed cultures. general eto inhibition properties on pcr performance were ruled out with pcr controls with dna extracts spiked with pure fungal dna as template (data not shown). as we confirmed the presence of dna on most samples, with the exception of those treated with eto, the next consequent step was to investigate if the fungi inoculated on the samples were still metabolically active. several methods are available to test fungal and microbial viability, such as the quantitation of cell 's atp content (rakotonirainy., 2003) or fluorescein diacetate (fda) and acridine - orange stainings that provide a qualitative observation of a microorganism 's active structures by means of an epifluorescent microscope, equipped with specific filters (pinzari., 2011). in this study we decided to use nucleic acids as markers of fungal viability. the existence of ribosomal - dna alone does not lead to assumptions about viability because rrna genes can persist in environmental dna pools for species that are metabolically inactive and functionally less important (ostle., thus, if responses of microbial communities to environmental perturbations are investigated, the rrna gene approach seems problematic, as rrna genes may be detected in dna pools for species whose growth or cellular activity has declined (anderson and parkin, 2007). to test the viability of the inoculated fungi, we targeted the fungal rrna directly, as it allows the detection of metabolically active and functionally important species, as they transcribe more rrna, a principle that is commonly exploited in bacterial ecology (prosser, 2002). determination of the viability of the causative agent is important in determining the active infection and in the evaluation of the efficacy of a particular treatment. in this study, rna was extracted from paper samples one year after the application of the respective treatments (t2), and subsequently transcribed into cdna for further dgge analyses. the dgge profiles generated with 18s primers from the fungal cdna are shown in fig. untreated control samples and those subjected to freeze - drying and gamma irradiation were all positive for amplification of fungal 18s cdna, whereas no positive rna extraction or pcr could be established for eto - treated samples. strong signals could be observed for all pure strains, with the exception of t. viride on paper d, in the control samples (fig. 3a). the pattern of single bands obtained for the pure inoculated strains indicated the presence of only one fungus on each paper sample. on samples inoculated with the mixed cultures, the dgge band corresponding to t. viride was not observable, a pattern that is congruent with the dna - generated gels. rna analyses revealed a minimal long - term impact of either gamma rays or freeze - drying treatments on the viability of the fungal spores, when inoculated as single strains (fig. all samples deliver clear bands with a pattern that matches the expectations and consistent for the different fungi. however, when fungi were inoculated as mixed cultures, no rna band at all or very faint signals were observed for paper d after freeze - drying and gamma ray treatments, respectively (fig. unfortunately, an amplification of the cdna with its primers, as used for the dgge analysis of dna, failed. its sequences are the most popular choice for species identification of fungi in environmental dna pools (white., 1990), but due to post - transcriptional processing of the main precursor rrna molecules containing 18s, its1, 5.8s, its2, and 28s rrna, the its spacer regions are spliced out to leave the rrna genes for ribosome synthesis (anderson and parkin, 2007). even if there is evidence that the its regions can be used for the detection of active fungi, as was done by anderson and parkin (2007), it is generally considered impossible to detect its sequences in rna pools (hibbett, 1992). a successful amplification of its rrna was performed by other groups from fungal isolate extracts and soil samples. we assumed, during the experimental work, that there was more rrna available in the soil samples themselves, as soils are active environments compared to the paper environment, which resembles starving conditions. while the present paper was under review, a paper by rajala. (2011) was published, which demonstrated that internal transcribed spacers (its) precursor rrna are a better marker of active fungi than small subunit rrna. this is because the its sequences, after being excised from the subunits turnover rapidly, and small and large subunit rrna particles are relatively stable in the environment. (2011) may be important for follow - up elaboration of the present work. our molecular results generally contradicted the cultivation results obtained in this study. by using culture - dependent techniques, samples subjected to freeze - drying showed growth in the case of t. viride, but is was recorded as low and statistically different from the control sample, whereas no statistical difference was found for c. globosum and the mixed inocula sample with respect to the control samples (see table 1). however, by using culture - independent techniques we could observe some differences on samples inoculated with the mixed culture samples one month after the freeze - drying treatment (t1) compared to the control samples (see fig. dgge bands representing each single fungus inoculated as pure culture did not show differences due to treatment, but a long - term effect was only observed for ch. globosum when it was inoculated as a single strain, showing an unspecific band - pattern. samples subjected to gamma irradiation and eto fumigation showed no fungal growth, either of all single species or the mixed inocula. therefore, the last two treatments indicated a full efficacy as disinfecting agents when evaluated with cultivation techniques (see table 1). however, when using culture - independent techniques, gamma rays were not able to eliminate fungal colonization of paper even if relatively high doses (5 kgy) were applied, but they could potentially reduce or slow down the growing process. dgge profiles obtained from dna extracted shortly after the irradiation process display a certain amount of fragmentation and unspecific bands that could be related to the effect of gamma radiation on the tertiary or secondary structure of the dna itself, but no long - term effect of gamma rays on dna or rna was observed, indicating the recovering of the surviving fungal fraction. gamma rays have been reported to inactivate fungi from and related to books (jrg., 1992 ; da silva., 2006 (2006) determined the efficiency of the radiation treatment on fungal spores by cultivation attempts only, and therefore described a higher degree of reduction than our dna- and rna - based studies. our cultivation results are in accordance with these observations, but are proven wrong when molecular data are included. only in the case of eto fumigation did both culture - dependent and -independent techniques indicate the total inactivation of the inoculated fungi. comparing dna and rna recovery, we could observe no differences in the case of samples inoculated with pure strains, being able to recover both dna and rna, which indicates that the fungi were indeed viable. however, differences in the rna recovery from samples treated with freeze - drying or gamma rays were observed when samples were inoculated with the mixed culture, especially on paper type d, indicating the inactivation of the fungi on this kind of paper and the higher sensitivity of rna- versus dna - based analyses for the detection of viability. the differences between culture and molecular results obtained in this study highlight the importance of molecular tools to complement conventional techniques, as they display higher sensitivity and bypass culturing methods. retrieval of fungal - specific rrna directly from paper can, in fact, be successfully used as an indicator for the viability of fungi after disinfecting treatments. information on the effects of mass treatments on the dna of paper - spoiling fungi is also provided. our results show, in fact, that the freeze - drying treatment displays only minimal effect on dna recovery over both the short- or long - term when compared to control samples, whereas the application of gamma rays shows a short - term slight reduction of dna recovery and dna fragmentation but no significant difference in long - term effect. when rna was used as an indicator for long - term fungal viability, differences in the rna recovery from samples treated with freeze - drying or gamma ray - treated samples were observed when samples were inoculated with the mixed culture, specially on paper type d. samples inoculated with the single strains showed no difference in the rna recovery compared to control samples. these results show the higher sensitivity of rna- over dna - based molecular analyses for the detection of viability of fungi on disinfected paper materials. considering our results, we can assume that gamma rays can be used to treat large amounts of paper simultaneously and without subsequent chemical hazard, but this can only be considered a decontamination treatment, a method to remove biodeteriogenous microorganisms to a controllable or blank level. this target of reducing and holding the biodeteriogens at under the danger threshold is crucial in paper conservation, and it can be achieved with gamma radiation, whereas freeze - drying can be applied only to stop heavy mould before further treatment. furthermore, our results point to ethylene oxide fumigation as the only effective and long - lasting treatment when the sterilization of a fungal infected paper object is needed. subsequent contamination is, however, possible, as ethylene oxide is not a chemical that remains as a preventive biocide on materials. it is worth emphasizing that no dna or rna could be recovered from paper samples fumigated with eto, a fact that is of particular importance for studies that address a molecular analysis of old contaminations or biological materials in cultural heritage characterization and diagnostics, including paleogenetic and archaeological molecular studies. ethylene oxide fumigation was used by museums and libraries in the recent past to treat insect or mould infestations, but it is often not traceable or not clearly mentioned in the reports that accompany the treated objects. what is needed is improved documentation of the conservation history of objects of cultural value, in order to account for treatments that can alter future studies and analysis and to avoid any impediments by past treatment based on eto. generally, any treatment that is applied to prevent the biodeterioration of paper documents or books has to be carefully assessed beforehand. the type of biological attack and the urgency of intervention have to be considered when choosing among treatments. economic factors such as cost of treatment, quantity of objects to be treated, and their historical or commercial value also have also an impact on the decision of conservators. | fungi are among the most degradative organisms inducing biodeterioration of paper - based items of cultural heritage. appropriate conservation measures and restoration treatments to deal with fungal infections include mechanical, chemical, and biological methods, which entail effects on the paper itself and health hazards for humans. three different conservation treatments, namely freeze - drying, gamma rays, and ethylene oxide fumigation, were compared and monitored to assess their short- (one month, t1) and long - term (one year, t2) effectiveness to inhibit fungal growth. after the inoculation with fungi possessing cellulose hydrolysis ability chaetomium globosum, trichoderma viride, and cladosporium cladosporioides as single strains or as a mixture, different quality paper samples were treated and screened for fungal viability by culture - dependent and -independent techniques.results derived from both strategies were contradictory. both gamma irradiation and eto fumigation showed full efficacy as disinfecting agents when evaluated with cultivation techniques. however, when using molecular analyses, the application of gamma rays showed a short - term reduction in dna recovery and dna fragmentation ; the latter phenomenon was also observed in a minor degree in samples treated with freeze - drying. when rna was used as an indicator of long - term fungal viability, differences in the rna recovery from samples treated with freeze - drying or gamma rays could be observed in samples inoculated with the mixed culture. only the treatment with ethylene oxide proved negative for both dna and rna recovery. therefore, dna fragmentation after an ethylene oxide treatment can hamper future paleogenetic and archaeological molecular studies on the objects. |
in 2016, tuberculosis (tb) is still a major cause of death and suffering worldwide. its control is a global public health issue and therefore needs to be conceived and carried out along with the basic principles of equity, human right to health and social protection. as marginalized people are always greatly exposed to health problems and often face difficulties in accessing care, social and economic determinants of ill health must be appropriately addressed together with adequate implementation of the specific interventions available today to combat those diseases that disproportionally affect the poorest. similarly to what observed for other infectious diseases, tb epidemiology is closely connected with social and economic conditions which makes tb prevention, care and control even more challenging. poverty reduction has been recognized as a global priority by the united nations and other international organizations since many decades, but it was only when the millennium development goals (mdgs) were first launched in 2000 that this issue was actually put in the spotlight. the mdgs, in short, called upon all governments to join forces to combat poverty and its consequences, including health conditions like tb, in order to foster development of societies and nations. in this context, community engagement played a pivotal role in reaching the most vulnerable groups, assessing their specific needs and promoting active mobilization of society. in this paper we will discuss the main aspects of tb epidemiology at global level as well as the threat of multidrug resistances. current achievements, future challenges, and prevention and control strategies in the context of the new sustainable development goals (sdgs) will also be examined. tuberculosis ranks alongside hiv / aids as the top infectious killer worldwide, with 9.6 million new incident cases and 1.5 million deaths estimated to have occurred in 2014. of note, more than two thirds of the global tb burden is reported in africa and asia, and in absolute terms india, indonesia and china account for the highest number of tb cases amounting to 43% of the global burden. the human immunodeficiency virus (hiv) is the strongest risk factor for tb, and tb is the first cause of death among people living with hiv (plhiv), even in an era of scale up of antiretroviral therapy (art), causing one third of all hiv - related deaths. in 2014, hiv - infected persons accounted for 1.2 million (12%) of the estimated 9.6 million people globally who developed tb. at the end of the same year, tb contributed to one third of the 1.2 million deaths from hiv / aids and hiv was responsible for 25% of the 1.5 million tb deaths. the majority (nearly three quarters) of these estimated hiv - associated tb cases and deaths are in the african region, with eastern and southern african countries carrying most of the global burden. art coverage among known hiv - infected tb patients in 2014 was 77%, which represents a further increase from previous years, but it is still far from the world health organization (who) recommendation that all hiv - positive people with tb should receive it. other co - morbidities such as those related to diabetes mellitus, smoking, and alcohol abuse are currently emerging in high, middle and low - income countries as potentially associated with a greater risk of progression from latent tb infection (ltbi) to active disease (two to three - fold higher in diabetics and smokers, for instance) as well as a less favorable treatment outcome. multidrug resistant tuberculosis (mdr - tb), defined as resistance to, at least, rifampin and isoniazid (the most commonly used and potent drugs of the currently recommended first - line therapeutic regimen), represents another important threat in the fight against the disease. the emergence and spread of drug resistances mostly derive from mismanagement of tb cases such as the use of inappropriate dosages, inappropriate regimens, limited availability of quality - assured pharmaceutical products, and little efforts to support patient s adherence. approximately 480,000 newly emerging cases of mdr - tb are estimated to occur every year. however, the proportion of cases detected and notified is only about a quarter of those estimated due to low implementation of drug susceptibility testing especially in resource - limited settings. the distribution of mdr - tb cases is geographically uneven (figure 1), with countries of the former soviet union reporting the highest percentages (up to 35%) and five countries (india, china, russia, pakistan and ukraine) accounting for as much as 60% of the global caseload in absolute terms. in september 2000 the united nations general assembly adopted an historical resolution, best known as the millennium declaration. this sent a strong signal to all countries and communities in an attempt to change their behaviors, socio - political choices and health responses based on the fundamental principles of freedom, equality, solidarity, tolerance, respect for nature and shared responsibility. three of them were devoted to health issues, and mdg 6 specifically addressed the three global epidemics of hiv / aids, malaria and tb. in particular, the tb - relevant target as defined in the mdg framework was to halt and begin to reverse the incidence by 2015. this target has been achieved both globally and in all six who regions, with 43 million lives estimated to have been saved between 2000 and 2014 (figure 2). through effective prevention, quality - assured diagnosis and proper treatment the burden of tb has therefore decreased. additionally, a 47% drop in tb mortality rates has been estimated since 1990, which means that the international goal of mortality reduction has almost been met. however, incidence is falling too slowly, with a 1.5% per year declining rate, which is far from sufficient to reach the future targets. importantly, while an average 86% cure rate has been attained at global level among new tb cases, some countries, like those in eastern europe, have documented much lower treatment success due to the high prevalence of mdr - tb. with respect to tb / hiv co - infection, assessment of achievements shows that significant progress has been made through the implementation of tb / hiv collaborative activities since 2005, with an estimated over 5 million lives saved up to now. if we look at the future challenges of tb control, five priorities for action can be delineated. first of all, around 3.6 million tb cases are estimated to be missing every year comparing with the estimated figure ; this means that they are not diagnosed and/or notified to public health systems. all such unknown cases may be diagnosed and treated but not reported, or simply never detected. this constitutes both an individual care and a transmission problem, as their outcomes may not be favorable and they may be responsible for continuous transmission thus representing a major public health concern. secondly, mdr - tb must be addressed as a crisis given its potential for hindering the achievement of current objectives. newly available rapid molecular diagnostics need to be expanded at the lowest possible care level and new medicines, such as bedaquiline and delamanid, added to current regimens to improve care of drug - resistant cases and prevent them from spreading further. third, the co - epidemic of tb / hiv requires an accelerated response based on a deeper and more comprehensive integration of services between often separately managed programs. the global fund and other key stakeholders have expressed concerns about the limited coverage of collaborative tb / hiv activities within grants of the global fund and other initiatives, and called for measures for improvement. this has led to the development of unified concept notes (i.e., proposals by countries applying for external financing) aimed to better address the dual epidemic through a more service - integrated approach. fourth, financial support to the fight against tb is pivotal to close the resource gap. about 8 billion us$ are annually required to cover the ordinary costs of case detection and treatment worldwide, but available funds, in the range of 6.4 billion us$, are not enough to address such needs (figure 3). last but not least, research needs to be intensified along the entire spectrum from basic towards the development pipelines and operational research, in order to successfully result in new diagnostics, drugs and eventually vaccines and the rapid uptake of such innovations. to this purpose, new investments from research institutions, governments and the corporate sector need to fill a gap that is estimated to be in the area of at least 1.3 billion us$ (figure 3). the future response to the challenges outlined above is included in a new set of seventeen sustainable development goals (sdgs) that were recently launched within the post-2015 global development agenda to come into effect on january 1, 2016 when they will replace the mdgs. one of the main differences between sdgs and mdgs lies in the fact that the former ones are universally applied to all countries instead of being focused on the developing world and depending largely upon aid funding. in addition, while the mdgs were limited to some well - defined categorical challenges to face, the sdgs are much broader in their reach, are integrated and indivisible, and rely not only on economic but also on environmental, moral and political considerations. among the sdgs, goal 3 (ensure healthy lives and promote well - being for all at all ages) covers a range of health - related issues such as maternal and child health, communicable and non - communicable diseases, and target 3.3 clearly calls for ending the major global epidemics (hiv, tb, malaria, neglected tropical diseases) by 2030. this is fully in line with what stated in may 2014 by the 67 world health assembly through a resolution that contains the new global who s end tb strategy and its ambitious targets. the vision behind the new strategy is a tb - free world, with no more people suffering from the disease or dying because of it. targets for 2035 include a 95% decline in tb mortality, a 90% reduction in tb incidence rate (to less than 10 cases per 100,000 population globally) and, to align with the quest for universal health coverage and social protection, the complete abolition of catastrophic expenditures for tb - affected people and families. broad poverty alleviation measures are therefore strongly expected to effectively overcome the impediments and cut down the barriers in access to healthcare. furthermore, some milestones have been introduced every five years along the pathway up to 2035 : in particular, coinciding with the sdg deadline of 2030, the number of tb deaths should be reduced by 90% and tb incidence rate should decline by 80%, compared to the baseline at 2015, by 2030. of note, there is an additional high - level target of zero catastrophic costs that should be achieved already in 2020. the new strategy stands on three pillars and is based on four cross - cutting principles defined as follows : i) government stewardship and accountability, with monitoring and evaluation ; ii) building a strong coalition with civil society and communities ; iii) protecting and promoting human rights, ethics and equity ; iv) adaptation of the strategy and targets at country level, with global collaboration (table 1). it refers to integrated, patient - centered care and prevention, and underlines the need to clearly identify the most vulnerable groups in a population in order to address their needs as a priority. active case finding along with drug susceptibility testing for resistance detection are among the core components of this pillar as are the concepts of treatment for all, including drug - resistant cases and children. special attention is also devoted to plhiv, given their high - risk condition, which claims for tailored interventions involving the provision of combined treatments for both diseases as well as the implementation of appropriate preventive measures. finally, preventive therapy to be administered to people at high risk is emphasized as a tool to prevent disease among the pool of latently infected people. the second pillar calls for bold policies and supportive systems to be based on a strong political commitment, greater financing, extended community engagement, active cooperation with non - governmental organizations as well as the civil society and public and private institutions. universal health coverage and social protection represent the cornerstones of the post-2015 broader agenda for effective health systems delivering the necessary services to people. although it is essential that tb diagnosis and treatment are provided free of charge to all people irrespective of their living area, other costs such as those related to travel to reach the healthcare facilities or those produced by the loss of working days must be taken into account to properly define the financial consequences of tb on affected patients. this pillar also emphasizes the broader policies that must be in place for any disease control effort to succeed. the third pillar underlines the importance of intensified research and innovation to make a real change in current tb trends. in order to achieve the ambitious targets of the end - tb strategy, the rate of decline of global tb incidence must increase to at least 10% per year by 2015. similar declining rates were reached in some european countries in the mid twentieth century, following the introduction of the first anti - tb drugs compounded with the significant improvement of general living conditions. the development of better diagnostics (including new point - of - care tests), safer, easier and shorter treatment regimens for active tb and ltbi, and effective pre- and post - exposure vaccines are critical to break the trajectory of the tb epidemic and further accelerate the reduction of tb incidence after 2025. without new effective tools it will not be possible to accelerate incidence decline at 17% per year, which is necessary to achieve the 2035 targets. new diagnostic tools are particularly awaited in order to improve the diagnosis of disease and latent infection, the rapid detection of drug - resistance, and for use in the pediatric population. several new diagnostics or diagnostic methods have been endorsed by the who since 2007 and many others are under investigation. whole - genome sequencing is being studied in an attempt to identify relevant mutations that could predict drug resistance : this approach may be potentially useful together with routine diagnostic tests in order to build up individualized therapeutic schemes in severe cases of mdr - tb and extensively drug - resistant tb (xdr - tb). a number of new molecules are currently in the early phases of the development pipeline although it will probably take about a decade to put them into the market, provided that their efficacy and safety will be demonstrated. after many years of financially limited research, two new drugs for mdr - tb (bedaquiline and delamanid) were approved between 2012 and 2013. these two drugs may save lives although they will probably yield a little impact of the global epidemiology of tb for which shorter and more effective regimens for both drug - susceptible disease and latent infection are necessary. vaccines may eventually be the most effective response to tb but the development of effective vaccines is considerably hindered by the complex biology of mycobacteria, whose nature is still partially undefined. bcg, which was first introduced in 1921, is the only currently available vaccine, mostly unsuitable for preventing infection and tb disease and therefore with limited use in specific contexts and for specific target populations (such as children in the first 2 - 3 years of life where it prevents wide dissemination of bacilli following exposure). looking beyond 2015, ending the tb epidemic is one of the best returns in investments. as recently highlighted in the economist (development : the economics of optimism ; issued on jan 24, 2015), reducing the deaths from tuberculosis would allow to gain an average 43 us dollars per dollar spent. lowering the burden of this disease therefore represents a high - impact and cost - effective poverty alleviation measure, more than many other health interventions, which is consistent with the close linkages existing between tb and impoverishment. although the mdg target of reversing trends in tb epidemiology has been met, the global burden remains enormous with several challenges that need to be properly addressed : we can and we definitely have to do more in tb control. global elimination in the next few decades | tuberculosis (tb) is a leading cause of morbidity and mortality worldwide, accounting for about 9.6 million new cases and 1.5 million deaths annually. the poorest and socially excluded groups carry the largest burden of disease, which makes it essential to properly address the social determinants of health through poverty reduction measures and targeted interventions on high - risk populations. the spread of multidrug - resistance tb requires special attention and highlights the need to foster research on tb diagnostics, new drugs and vaccines. although many advances have been made in the fight against tb over the last twenty years, a lot is still needed to achieve global elimination. the new end - tb strategy that was first launched in 2014 by the world health organization, is fully in line with the seventeen sustainable development goals that came into effect since january 2016 and sets ambitious goals for the post-2015 agenda. a 90% reduction in tb - related mortality and an 80% decline in tb incidence within 2030 as well as the abolition of catastrophic expenditures for tb - affected people are the main targets of this strategy. strong government commitment and adequate financing from all countries together with community engagement and appropriate investments in research are necessary in order to reach these objectives. |
studies evaluating the relationship between lipid parameters and bone mineral density (bmd) in healthy adults and those with metabolic syndrome have revealed inconsistent results. while most of the studies have been performed in women, there are a few studies in men and adolescents. since indians have differences in lipid profiles [higher prevalence of high triglycerides (tgs) and low high density lipoprotein cholesterol ] compared with other populations, we assessed the relationship between various lipid parameters with bmd at different sites in previously conducted cross - sectional population in healthy indian volunteers. this study was carried out as part of voluntary general health check - up of all members of resident welfare associations of four residential colonies, one each from north, south, east, and west delhi. the study included all participants > 20 years of age (2347 participants - male 39.4% ; female 60.6%) excluding those with infectious, hepatic, renal, neoplastic, gastrointestinal, dermatological and endocrine disorders, steroid intake or alcoholism and drugs affecting lipid parameters like statins, fibrates, diuretics, and beta - blockers. demographic, anthropometric, and clinical data were ascertained and a detailed physical examination conducted. body mass index (bmi) was calculated by weight in kilogram divided by square of height in meters. fasting blood samples were drawn for the estimation of serum 25-hydroxy vitamin d [25(oh) d ], intact parathyroid hormone (ipth), total and ionized calcium, inorganic phosphorus, alkaline phosphatase (alp), total cholesterol (tc), tgs, high - density lipoprotein cholesterol (hdl - c), and low - density lipoprotein cholesterol (ldl - c). biochemical parameters were carried out using automated analyzer (hitachi 902 fully automated biochemistry analyzer ; roche, manheim, germany) and commercial kits (roche, manheim, germany). measurements of plasma glucose were done by glucose oxidase - peroxidase method by trinder (clonital, italy). dyslipidemia was defined by tc > 240 mg / dl, serum tg > 150 mg / dl, hdl - c 160 mg / dl. all participants were divided according to age with cut - off of 50 years so that pre- and postmenopausal women can be separated. total population was divided and grouped for analysis into three groups male (n = 924), female 50 years (postmenopausal, n = 635). interquartile range for tc was 47.75, 34, and 51 mg / dl ; for tg was 59, 26.75, and 58 mg / dl ; for hdl was 8, 5, and 8 mg / dl ; for ldl 32, 14.75, and 39 mg / dl for three groups, respectively. the study was approved by the ethics committee of the institute of nuclear medicine and allied sciences and all participants gave written informed consent. the normal range for different biochemical parameters are as follows : serum total calcium (8.5 - 10.5 mg / dl), ionized calcium (1.12 - 1.32 mmol / l), inorganic phosphorus (2.5 - 4.5 mg / dl), alp were (females : 35 mg / dl), and ldl (50 years in both sexes as also defined by another study, while values higher than these were considered as normal bmd. instrument variation was determined regularly using a phantom supplied by the manufacturer and mean coefficient of variation was 20 years of age (male 39.4% ; female 60.6%). mean age and bmi were 49.1 18.2 years (range : 21 - 90 years) and 25.0 4.7 kg / m (range : 13.0 - 49.8) respectively. there were 788 (55.4%) premenopausal (50 years) and 635 (44.6%) postmenopausal women (> 50 years). male were older than females (54.0 16.7 vs. 45.9 18.5 years ; p 50 years) with quartiles of lipid parameters bmd at femoral neck decreased from second to highest quartiles of ldl - c, but no significant trend was noticed with tc, tg, and hdl - c. bmd at lumbar spine decreased significantly with quartiles of ldl - c and increased with tg. hdl - c had no effect on bmd at any site [supplementary table 2 ]. however, there was no correlation of any lipid parameters with bmd at any site in premenopausal women [table 3 ]. in multiple regression analysis, after adjusting for age, bmi, serum ionized calcium, alp,25(oh) d, and ipthlevels, ldl - c showed significant but weak negative correlation with bmd at femoral neck, total femur, and lumbar spine, but correlation with tg became nonsignificant [table 4 ]. bone mineral density (g / cm) in premenopausal women (females50 years) with quartiles of lipid parameters bmd at femoral neck decreased from second to highest quartiles of ldl - c, but no significant trend was noticed with tc, tg, and hdl - c. bmd at lumbar spine decreased significantly with quartiles of ldl - c and increased with tg. hdl - c had no effect on bmd at any site [supplementary table 2 ]. however, there was no correlation of any lipid parameters with bmd at any site in premenopausal women [table 3 ]. in multiple regression analysis, after adjusting for age, bmi, serum ionized calcium, alp,25(oh) d, and ipthlevels, ldl - c showed significant but weak negative correlation with bmd at femoral neck, total femur, and lumbar spine, but correlation with tg became nonsignificant [table 4 ]. bone mineral density (g / cm) in premenopausal women (females<50 years) with quartiles of lipid parameters total population was categorized in to subjects with normal bmd (1239 - 52.8%) and low bmd (1108 - 47.2%). in subjects with normal bone density, tc and ldl - c were significantly lower compared with subjects with low bone density in both sexes (men : tc-159 35 vs. 167 35 mg / dl, p = 0.001 ; ldl - c- 99 24 vs. 105 25 mg / dl, p < 0.0001 ; women : tc 153 32 vs. 174 38 mg / dl, p < 0.0001 ; ldl - c : 91 18 vs. 106 25, p < 0.0001). there was no significant difference in bmd at any site in any group when study population was categorized according to dyslipidemia (data not shown). in the present large population - based cross - sectional study, we found that femoral bmd was inversely correlated with total cholesterol and ldl - c in both men and women bmd at lumbar spine was negatively correlated with tc and ldl - c in men, and only with ldl - c in pre - menopausal women. similar to our results, a korean study also found a weak positive correlation of bmd with lipid profile (tc and ldl - c) after adjustment with age, bmi and age at menarche in pre- and postmenopausal women. in contrast, studies from usa (national health and nutrition examination survey - nhanes) and uk (framingham osteoporosis study - fos) reported no association of lipid parameters and bmd. both these studies (nhanes and fos) have only evaluated women and have not provided data separately for pre- and postmenopausal women. further, women with associated comorbidities, including alcohol and drug intake, were not excluded in these studies. many smaller studies have reported stronger but less significant correlation ; however, larger studies have found weaker but more significant correlation being large sample. there are very few studies which have evaluated the relationship between lipid parameters and bmd in men, mostly with small sample size. only two large community - based studies have reported the relationship between lipid parameters and bmd in men, but are not suitable for comparison because in one instance data for men was not separately reported, while in the other, bmd was measured only at the wrist. the nhanes - iii also reported a similar trend of a negative association between tc and ldl - c with bmd, though it became insignificant after adjusting for multiple variables. smaller studies among european men reported either absent or positive association of tc and ldl - c with bmd at femur and spine. it has been proposed that oxidized ldl increase receptor activator of nuclear factor kappa - b ligand expression on osteoblast and increase interaction with osteoclast which affect bone remodeling and may cause decrease in bmd. further, in animal models, the primary cholesterol metabolite, 27-hydroxycholesterol, interacts with estrogen and liver x - receptors, decreases osteoblast differentiation, and increases osteoclastogenesis, thereby resulting in increased bone resorption and decrease in bmd. bmd at femoral neck showed positive correlation with tg, which was lost when adjusted for various factors including age and bmi. a similar positive association of tg with bmd was reported in men and adolescents, which became insignificant when adjusted for body fat or markers of insulin resistance. however, other small studies have reported both absent and positive correlation of tg with bmd which persisted even after adjustment with body fat. obesity, weight, and bmi are positively correlated with bmd and tg is also positively correlated with obesity. hence, it is not surprising to find a positive correlation of tg with bmd, which gets neutralized when adjusted for bmi or fat mass. hdl - c was not correlated with bmd at any site in the present study, which was also reported previously. other studies in european men found a negative correlation of hdl - c with bmd at femur and spine, but the relationship was attenuated after adjustment for body fat content in one study. a similar finding was reported in a large population based study. on the contrary, a weak negative association of bmd at radius was observed with tc in one population - based study. serum tc and ldl - c had weak negative correlation with only total femur bmd. several studies, including large population - based studies, have also reported a negative association of tc and ldl - c with femur, lumbar spine, and radius. in contrast, a positive correlation of tc with hip bmd and total body bmd has also been reported, though in one of these studies, samples for lipid profiles were drawn in a nonfasting state. few studies have also shown no relationship between tc and ldl - c with bmd at any site. several of these studies are weakened by either small sample size, selection bias, or inclusion of subjects with comorbidities, as well as and consumption of medication known to affect bmd. bmd at femoral neck was positively related with tg, which became nonsignificant in multivariate regression analysis after adjustment with various factors. a similar positive association was reported in smaller studies as well large population - based studies. hdl - c revealed a positive correlation with lumbar spine bmd only in this group, which was maintained in multiple regression analysis. this relation was further confirmed by observation that hdl - c was higher in women with normal bone density compared with those women with low bone density. several large population based studies and smaller studies also reported a positive association between hdl - c and lumbar spine bmd. however, other studies reported either a negative association or no association of hdl - c and bmd. these differences have been explained by ethnic and racial differences, size of the study population, and inclusion of women on hormone replacement therapy. in the present study, bmd at femoral neck and lumbar spine decreased significantly with increasing quartiles of ldl - c, and this weak negative correlation was maintained in multiple regression analysis. large population based studies also found a negative association of tc and ldl - c with lumbar spine bmd and whole body mineral content, but not with femur in premenopausal women. after adjustment, no significant correlation was found between tg and bmd at any site. in contrast, a korean population - based study has reported a negative association of tg with bmd at total hip. hdl - c was not correlated with bmd at any site and a similar observation has been reported among chinese premenopausal women. however, another large population - based study found a positive relation between hdl and bmd at lumbar spine and femur. another limitation was absence of data on dietary habits, smoking, and physical activity, which can adversely affect both bmd and lipid parameters. the strength of our study was the large sample sizes from healthy indian population who were free from common morbidities and were not consuming any medication affecting bmd. there are very few studies which have evaluated the relationship between lipid parameters and bmd in men, mostly with small sample size. only two large community - based studies have reported the relationship between lipid parameters and bmd in men, but are not suitable for comparison because in one instance data for men was not separately reported, while in the other, bmd was measured only at the wrist. the nhanes - iii also reported a similar trend of a negative association between tc and ldl - c with bmd, though it became insignificant after adjusting for multiple variables. smaller studies among european men reported either absent or positive association of tc and ldl - c with bmd at femur and spine. it has been proposed that oxidized ldl increase receptor activator of nuclear factor kappa - b ligand expression on osteoblast and increase interaction with osteoclast which affect bone remodeling and may cause decrease in bmd. further, in animal models, the primary cholesterol metabolite, 27-hydroxycholesterol, interacts with estrogen and liver x - receptors, decreases osteoblast differentiation, and increases osteoclastogenesis, thereby resulting in increased bone resorption and decrease in bmd. bmd at femoral neck showed positive correlation with tg, which was lost when adjusted for various factors including age and bmi. a similar positive association of tg with bmd was reported in men and adolescents, which became insignificant when adjusted for body fat or markers of insulin resistance. however, other small studies have reported both absent and positive correlation of tg with bmd which persisted even after adjustment with body fat. obesity, weight, and bmi are positively correlated with bmd and tg is also positively correlated with obesity. hence, it is not surprising to find a positive correlation of tg with bmd, which gets neutralized when adjusted for bmi or fat mass. hdl - c was not correlated with bmd at any site in the present study, which was also reported previously. other studies in european men found a negative correlation of hdl - c with bmd at femur and spine, but the relationship was attenuated after adjustment for body fat content in one study. a similar finding was reported in a large population based study. on the contrary, a weak negative association of bmd at radius was observed with tc in one population - based study. serum tc and ldl - c had weak negative correlation with only total femur bmd. several studies, including large population - based studies, have also reported a negative association of tc and ldl - c with femur, lumbar spine, and radius. in contrast, a positive correlation of tc with hip bmd and total body bmd has also been reported, though in one of these studies, samples for lipid profiles were drawn in a nonfasting state. few studies have also shown no relationship between tc and ldl - c with bmd at any site. several of these studies are weakened by either small sample size, selection bias, or inclusion of subjects with comorbidities, as well as and consumption of medication known to affect bmd. bmd at femoral neck was positively related with tg, which became nonsignificant in multivariate regression analysis after adjustment with various factors. a similar positive association was reported in smaller studies as well large population - based studies. hdl - c revealed a positive correlation with lumbar spine bmd only in this group, which was maintained in multiple regression analysis. this relation was further confirmed by observation that hdl - c was higher in women with normal bone density compared with those women with low bone density. several large population based studies and smaller studies also reported a positive association between hdl - c and lumbar spine bmd. however, other studies reported either a negative association or no association of hdl - c and bmd. these differences have been explained by ethnic and racial differences, size of the study population, and inclusion of women on hormone replacement therapy. in the present study, bmd at femoral neck and lumbar spine decreased significantly with increasing quartiles of ldl - c, and this weak negative correlation was maintained in multiple regression analysis. large population based studies also found a negative association of tc and ldl - c with lumbar spine bmd and whole body mineral content, but not with femur in premenopausal women. after adjustment, no significant correlation was found between tg and bmd at any site. in contrast, a korean population - based study has reported a negative association of tg with bmd at total hip. however, this study was retrospective and suffers from selection bias. hdl - c was not correlated with bmd at any site and a similar observation has been reported among chinese premenopausal women. however, another large population - based study found a positive relation between hdl and bmd at lumbar spine and femur. another limitation was absence of data on dietary habits, smoking, and physical activity, which can adversely affect both bmd and lipid parameters. the strength of our study was the large sample sizes from healthy indian population who were free from common morbidities and were not consuming any medication affecting bmd. while we report a weak correlation between lipid parameters and bmd at various sites in men, pre- and premenopausal women, its clinical significance needs to be elucidated. | introduction : cardiovascular disease and osteoporosis share common risk factors including dyslipidemia. there are conflicting reports of differential relation of various lipid parameters on bone mineral density (bmd). hence, we studied the correlation between lipid parameters and bmd in healthy adult.materials and methods : a total of 2347 participants (male 39.4% ; female 60.6%) included in this cross - sectional study were divided according to sex and age. fasting blood samples were drawn for biochemical parameters. bmd at lumbar spine, femur, and forearm were measured by dual energy x - ray absorptiometry (dxa).results : in males, bmd at femur and lumbar spine decreased significantly with increasing quartiles of total cholesterol (tc) (p < 0.0001, and 0.004) and low - density lipoprotein cholesterol (ldl - c) (p = 0.001, and 0.01). in premenopausal women, bmd at femoral neck (p = 0.001) and lumbar spine (p = 0.029) showed declining trend with ldl - c (p = 0.007). in postmenopausal women, only bmd at total femur decreased significantly with tc (p = 0.024) and ldl - c (p = 0.036). all above findings were confirmed in correlation studies. in multiple regression analysis after adjusting for age, body mass index, ionized calcium, alkaline phosphatase, 25 hydroxy vitamin d, and parathyroid hormone levels correlation of bmd with tc and ldl - c persisted. tc, ldl - c was higher in subjects with low bone density compared those with normal bone density in both sexes.conclusions:tc and ldl - c had weak but significant negative correlation with bmd at femur and lumbar spine. |
orbital fat prolapse is a movable yellowish mass beneath the conjunctiva,1,2 typically located in the superotemporal quadrant.3 it is associated with localized weakness or thinning of tenon s capsule usually caused by aging or surgical trauma.1,2 males have been reported to be affected more likely than females.1,3,4 surgery is considered for irritative symptoms or for cosmetic reasons. a standard surgical method is the resection of herniated fat through conjunctival incision with or without connective tissue repair.19 alternative methods without resection have been reported, including suture - less repair using fibrin glue7 and conjunctival fixation to the sclera.10 among these methods, conjunctival fixation to the sclera, described by otaka and kyu,10 is a more simple and less invasive technique than the standard method. however, to our knowledge, there have been no reports describing the postoperative results in a large number of patients to date. in the study reported here, we retrospectively reviewed the outcome of this procedure, which involved some modifications to the original technique. twenty - three consecutive eyes of 19 patients who were treated surgically for orbital fat prolapse at the national hospital organization tokyo medical center, tokyo, japan, from august 2006 to january 2013 were retrospectively reviewed in this study. the study protocols followed the tenets of the declaration of helsinki and were approved by the local ethics committee of the tokyo medical center. at the first visit, comprehensive ophthalmic examinations were performed in all patients including measurement of visual acuity, intraocular pressure, slit - lamp biomicroscopy with fluorescein staining and indirect ophthalmoscopy. the diagnosis was made by slit - lamp examination of a typical yellowish, elevated mass beneath the conjunctiva. other orbital disorders such as dermolipoma, epibulbar dermoid, and lymphoid tumor were differentiated clinically by their appearance and mobility, which is a distinctive characteristic of orbital fat prolapse.1,2 the mobility was confirmed by repositioning the mass into the orbit with slight pressure (figure 1a, b). lacrimal gland prolapse was also differentiated by observing the normal appearance of the palpebral lobe of the gland under the upper eyelid. the exclusion criteria were recurrent cases after orbital fat surgery, history of any orbital disorders, and suspicious symptoms of other orbital diseases such as impaired ocular movement or exophthalmos. all cases were treated with the same surgical technique, involving conjunctival fixation to the sclera (figure 1c, d). efficacy and safety during the follow - up period were determined as the outcomes. after obtaining written informed consent for the surgical procedure, after rotating the eye to the opposite direction of the lesion using a traction suture, the prolapsed fat was slid posteriorly and repositioned into the orbit with gentle compression using a blunt instrument like a spatula. while the fat was kept repositioned by an assistant, conjunctiva in the quadrant of the fat prolapse was fixated to the sclera with 810 interrupted sutures of 10 - 0 nylon, in two rows located approximately 1214 mm posterior to the limbus measured with a caliper (figure 2). as the depth of the sutures was not recognized easily beneath the conjunctiva and tenon s capsule, the sutures were placed through the superficial layer of the sclera to prevent perforation. the distance between the two rows of sutures was not fixed precisely but was approximately 12 mm. these sutures were expected to form an embankment to keep the prolapsed fat at the posterior position, and were not to be removed after the treatment unless they caused any complaint such as irritation or pain. antibiotic and steroid eye drops were prescribed for 1 month postoperatively to prevent infection and to lessen inflammation. the patients were observed at 1 day, 1 week, and 1 month after surgery, and subsequently were examined every 26 months according to the condition of each patient. at each visit after the operation, the efficacy of the surgery was determined by slit - lamp examination. the age of the patients at operations was 64.910.4 years (average age standard deviation ; range : 4582 years). all the patients were male except for one female who had a unilateral lesion. among the 23 eyes, eight eyes (four patients) were bilateral cases and 15 eyes were unilateral cases.. none of them had any other ocular or orbital disorder that would exclude them from the study. the mean follow - up period was 19 months (range : 159 months). there were no intraoperative or postoperative complications, such as disorders of eye movements, decreased visual acuity, increased or decreased intraocular pressure, ocular recession, retinal detachment, or severe bleeding. no conjunctival folds or fibrosis were observed at the sutured site during the follow - up period. slit - lamp examination with fluorescein staining revealed no severe disturbance of the tear film either preoperatively or postoperatively. a 67-year - old male experienced recurrence at 36 months after the surgery. there was a large fat prolapse before the initial operation, which had been located adjacent to the limbus, and the recurrent prolapse presented with a similar appearance. reoperation was performed at 42 months with the standard surgical method of fat resection through conjunctival incision. although a standard surgical method involves resection of herniated fat through conjunctival incision, a less invasive method is desirable for this disorder. among several reports in the literature, conjunctival fixation to the sclera, introduced by otaka and kyu,10 appears to be the least invasive, consisting of very simple procedures and requiring no conjunctival incision. we used 10 - 0 nylon sutures, 1214 mm posterior to the limbus, whereas 6 - 0 vicryl sutures were located 810 mm posterior to the limbus in the original report.10 we selected 10 - 0 nylon for its thin, monofilament and nonabsorbable features instead of 6 - 0 vicryl, to reduce postsurgical irritation and inflammation. however, postsurgical inflammation was used to facilitate adhesion between the conjunctiva and the sclera, and was an essential element of this method.10 the lower inflammation and adhesion resulting from the 10 - 0 nylon sutures would have been a disadvantage. additionally, the thin suture threads of 10 - 0 nylon may not have had enough strength to form an embankment, long after the surgery. in order to overcome these disadvantages, as many as 810 sutures were located in two rows. the suture threads were placed more posteriorly than described in the original article10 to create the embankment as far as possible from the limbus to result in a better cosmetic outcome. consequently, the stitches were safely located 1214 mm from the limbus in all cases. this case had a large volume of fat prolapse which nearly reached the limbus. in cases with prominent prolapse, the pressure of the mass toward the embankment would easily overcome the adhesive strength of the sutures after the surgery. additional suture threads or stronger suture material such as vicryl might prevent recurrence in such cases, and could serve as a desirable supplementary procedure for re - treatment of recurrent cases using the same surgical method. the embankment in this procedure was primarily formed by the mechanical attachment of the conjunctiva, tenon s capsule, and the sclera. because 10 - 0 nylon monofilament suture loses its strength in several years, the long - lasting efficacy of the operation might have been provided by the adhesive scarring created by the apposition of conjunctiva and tenon s layer, as sniegowski suggested in a report of cases repaired with fibrin glue.7 they also reported that sufficient adhesion strength would be achieved within 1 week through apposition of the tissues. additionally, if the sutures were located around the break of tenon s capsule or intermuscular septa from which the prolapsed fat protrudes,6 the scar formation would reconstruct the break successfully. the long - term efficacy might have depended on whether the sutures maintained their strength until the adhesive scar was formed. recent progress in ocular imaging has enabled the detailed evaluation of the anterior segment and the ocular surface such as filtering blebs with the aid of optical coherence tomography.11 this technique may provide useful knowledge about the adhesive scarring process after this surgical procedure. in the present study, the efficacy of conjunctival fixation to the sclera proved to be effective to treat orbital fat prolapse. because the time limit for the sutures or adhesive scar to maintain the embankment was unknown, continuous observation was necessary to ensure its long - term efficacy. surgery for orbital fat prolapse by conjunctival fixation to the sclera may be a more simple and less invasive procedure than surgery using conjunctival incision and fat resection. | purposethe aim of the study described here was to report the outcomes of surgery for orbital fat prolapse by conjunctival fixation to the sclera.patients and methodstwenty - three consecutive eyes of 19 patients with orbital fat prolapse were retrospectively reviewed. all cases were treated with the same simple procedure without resection through conjunctival incision : fixation of conjunctiva to the sclera with interrupted sutures of 10 - 0 nylon in two rows located approximately 1214 mm posterior to the limbus. these sutures formed an embankment to keep the prolapsed fat posteriorly. postoperative results were determined by slit - lamp examination and recurrence of prolapse was defined as the presence of orbital fat anterior to the embankment.resultsduring the mean follow - up period of 19 months (range : 159 months), one case experienced recurrence which required further surgery. none of the other cases experienced recurrence, and there were no intraoperative or postoperative complications.conclusionconjunctival fixation to the sclera was a simple and effective surgical technique for orbital fat prolapse, with less invasion compared to the conventional method that requires conjunctival incision. |
despite refined roentgenographic, endoscopic and isotopic techniques, the diagnosis of pancreatic lesions still presents a great problem in many instances. recently, with rapid development of imaging diagnostic modalities such as ultrasonography and computed tomography, it has been possible to obtain information about the pancreas and to perform percutaneous fine needle aspiration biopsy of pancreatic cancer guided by these techniques. we wish to present our experience with percutaneous fine needle aspiration biopsy guided by ultrasonography in a total of 39 patients with pancreatic cancer and to evaluate the value of the technique for establishing a proved histologic diagnosis, avoiding operative intervention and facilitating treatment planning. thirty - nine patients with pancreatic cancer were studied from march 1984 to july 1987. the age range of the 39 patients was 29 to 72 years(mean 54 years). of the 39 patients, the pancreatic cancer was diagnosed by operation in 7 patients, three diagnostic methods (computed tomography, ultrasonography and ercp) in 5 patients, two diagnostic methods such as ultrasonography and computed tomography in 10 patients, ultrasonography and ercp in 6 patients and ultrasonography in 11 patients with clinical grounds. ultrasound investigations were performed in the fasting supine patients by means of a real - time scanning technique (aloka echo camera ls model ssd-280 with a real - time linear - array scanner having a frequency of 3.5mhz). the cancer of the pancreas was delineated, providing a three - dimensional presentation of its sonoanatomy. whenever possible, biopsy access routes representing the shortest distance between the skin and the target tumor were selected. the distance between the skin and the center of the target volume was measured on the ultrasonic display and transposed to the needle shaft with a sterile needle stop. the angle of the needle path (parallel to the ultrasonic beam) was indicated by the position of the transducer. biopsies were performed using commercially available chiba needles with a removable inner stylet and an outer diameter of 23 gauge. local anesthesia with 2% the needle and syringe were heparinized before use by wetting the whole inner surface of the syringe with a small amount of sodium heparin. during suspended respiration, a variation in resistance was felt in a significant number of patients when the needle entered the tumor mass. when the needle tip had been introduced into the target volume, the mandrel was withdrawn and a special suction device equipped with a 20ml plastic syringe flushed with heparin was attached. while the patient was asked to stop breathing, the needle was quickly moved back and forth several times while applying aspiration to the mass. suction should be discontinued before removal of the needle to prevent aspiration of neighboring tissue into the syringe or spillage of tumor cells along the needle track. the aspirated material was smeared on glass slides, immediately fixed in 95% alcohol and stained by the papanicolaou method, and collected on the filter paper in preparation for cell block. the results of cytology were reported as material insufficiency, or positive, suspicious or negative for cancer. the symptoms and signs of the patients with pancreatic cancer (39 patients) were abdominal pain (35 patients, 81.7%), weight loss (20 patients, 51.3%), anorexia (13 patients, 33.3%), back pain (13 patients, 33.3%), jaundice (10 patients, 25.6%), dyspepsia (7 patients, 17.9%), palpable mass (5 patients, 12.8%) and bowel habit change (2 patients, 5.1%) (table 2). of the 39 patients, aspirated material suitable for cytologic evaluation of smear preparations was obtained from 33 patients (84.6%). among the 33 patients, cytologic diagnosis was positive in 26 patients (78.8%), suspicious in 2 patients (6.1%) and negative in 5 patients (15.1%). there was mild abdominal pain only in one patient (2.6%) (table 3). all 39 patients were divided into three groups according to the size of the tumor such as group a (2.0 and < 4.0 cm, 22 patients), group b (4.0 and < 6.0 cm, 12 patients) and group c(6.0 cm, 5 patients). the aspirated material was insufficient for cytologic evaluation in 6 patients (4 patients in group a and 2 patients in group b). of the remaining 18 patients in group a, the cytologic diagnosis was positive in 15 patients and negative in 3 patients. of the remaining 10 patients in group b, the cytologic diagnosis was positive in 7 patients, suspicious in 2 patients and negative in 1 patient and of the 5 patients in group c, the cytologic diagnosis was positive in 4 patients and negative in 1 patient (table 4). the cancer of the pancreas was located in the head (21 patients), the body (9 patients), and the tail (6 patients) portions of the pancreas and diffusely in 3 patients. the aspirated material for cytologic evaluation was insufficient in 6 patients (head:4, body and tail : 1 and respectively). the cytologic diagnosis was positive in 26 patients (head:12, body:8, tail:4 and diffuse:2), suspicious in 2 patients (head and tail:1 each respectively) and negative in 5 patients (head:4 and diffuse:1) (table 5). the widespread acceptance of thin - needle (2123 gauge) aspiration biopsy for cytodiangosis of deep malignant tumors is due to the high yield of positive tissue samples as well as the low risk of serious complications. with recent improvements in localizing methods, the availability of thin needles for aspiration and the increased sophistication of the cytologic technique, the use of aspiration biopsies has been extended to that of mass lesions of the pancreas and other abdominal structures. aspiration biopsy offers an approach to pancreatic cancer which is distinct from histology or tissue biopsy. the smaller outer diameter of the aspiration needle results in a cross - sectional area 5 to 13 times less than that of tissue biopsy needles. it is this difference that permits uncomplicated passage of the fine needle through the liver, stomach or bowel on the way to the pancreas. the major technical problem is localization of the lesion and this has been achieved by biopsy during angiography with test injections of contrast material into appropriate arteries under fluoroscopic control, during ultrasonography using a special biopsy transducer, with computed tomography to image the position of the biopsy needle and following ercp. among them ultrasonography is well adapted to biopsy technique since the lesion can be quickly and accurately localized in any plane. success with the fine needle aspiration biopsy technique requires an experienced and skilled cytopathologist. also critical to a high yield is proper handling of the cytologic material. false - negative rates of 3% to 20% have been encountered, but false - positive results are virtually nonexistent. in this study, the diagnostic accuracy (84.9%) was slightly lower than that of other reports. there was no significant difference of diagnostic accuracy according to the size of the cancer. however, pancreatic cancer located in the body showed a higher rate of diagnostic accuracy than the other portions. lack of diagnostic cytologic material may be related to necrotic and/or scirrhous areas in the tumor. the reported falsepositive frozen section diagnoses have not occurred with fine needle aspiration. in this study, aspirated material for cytologic examination was insufficient in 6 patients (15.4%), especially in patients having a small cancer size (2.04.0 cm ; 4 patients and 4.06.0 cm ; 2 patients). goldstein performed fine needle biopsy of the pancreas in 4 dogs and on subsequent laparatomy found little or no evidence of the needle punctures. coel and niwayama performed biopsies on 16 pigs without clinically apparent complication, although histologic examination of one of the specimens demonstrated a small are of focal hemorrage. there were reports of complications such as intestinal bleeding, fatal necrotizing pancreatitis and hematoma. in this study, seeding of malignant cells along the needle track is a recognized risk of paracentesis and thoracentesis for malignant effusions in carcinoma of the breast and ovary. experimentally, engzell. were unable to demonstrate release of tumor cells after thin - needle aspiration of transplanted malignant lymph nodes in rabbits. reports of thousands of fine needle aspiration biopsies have generated a few reported cases of seeding of the skin at the aspiration site. in addition, the theoretical possibility of hematogenous metastases from cells dislodged during aspiration has not been supported by clinical studies. however, with fine needle aspiration biopsy, there has been no evidence of dissemination of cancer in a large series of patients with different tumor types of various organs. in patients of this study, there was no seeding of malignant cells during the follow - up periods until now. in conclusion, ultrasound - guided fine needle aspiration biopsy is considered to be a safe and useful method for histologic diagnosis of pancreatic cancer. | fine needle aspiration biopsy guided by ultrasonography was performed in 39 patients with pancreatic cancer to evaluate the value of the technique for establishing a proved histologic diagnosis. aspirated material suitable for cytologic evaluation of smear preparation was obtained from 33 patients(84.6%). among the 33 patients, cytologic diagnosis of pancreatic cancer was possible in 28 patients(84.9%). there was mild abdominal pain only in one patient(2.6%). in conclusion, percutaneous fine needle aspiration biopsy guided by ultrasonography proved to be a safe and useful method for histologic diagnosis of pancreatic cancer. |
the material most commonly used for fabricating removable partial and complete denture is polymethyl methacrylate. despite its popularity, the material although adequate in satisfying aesthetic demands is not ideal in fulfilling the mechanical requirements of such appliance. this material presents limitations particularly in flexural strength and impact strength.1 the fracture of acrylic resin denture is rather common occurrence and causes inconvenience. in addition to this, fracture has been attributed to porosity, presence of residual monomer, cracks, and poor adaptation of the removable prosthesis to the residual ridge. removable prosthesis is susceptible to high impact extra oral forces, such as being accidentally dropped. as a result, stress concentration is generated, and the denture base acrylic resin can initiate or propagate the existent cracks, thus by influencing failure rate.2 therefore, studies on morphology and micro structural behaviour, surface defects and fracture initiation sites may help to identify the cause of the fracture. to simulate experimentally, impact tests such as izod or charpy although, impact testing is influenced by the loading configuration, there is a good correlation between the charpy and izod impact measurements. this study is an effort to evaluate and compare the impact strength and fracture morphology of four commercially used heat cure denture acrylic resins. metal dies were prepared for mentioned dimension (65 mm 10 mm 3 mm) (figure 1a). the dies were coated with a thin layer of petroleum jelly and were invested in the lower half of the denture flask. while taking care, the one - half of the thickness was embedded in the dental plaster but in the base of the flask. this was allowed to set for half an hour, and a single layer of separating medium was applied. second pour was made in the dental plaster, and the flask was held in compression till the final set of dental plaster. the denture flask was then opened, and the preformed dies were retrieved from the dental plaster (figure 1b). lucitone 199 (p : m) 21 g:8 ml and dpi heat cure 21 g:10 ml ; trevalon (p : m) 21 g:9 ml and acrylyn - h 21 g:10 ml were manipulated according to manufacturer s instructions and material was packed into the mould in the dough stage. bench curing was done for 3 h and short curing cycle was followed for acrylization i.e. 74c for 2 h and 100c for 1 h. totally 120 test samples were trimmed by using acrylic trimmer. one hundred and twenty test samples were labelled on each end before testing as follows : total number of specimen : 120 specimenstotal number of group : 4 groupstotal number of specimens in each group : 30 specimensgroup d (dpi) was labelled as d1, d2, d3. d30group a (acrylyn - h) was labelled as a1, a2, a3 a30group l (lucitone 199) was labelled as l1, l2, l3 l30group t (trevalon) was labelled as t1, t2, t3. t30 total number of specimen : 120 specimens total number of group : 4 groups total number of specimens in each group : 30 specimens group d (dpi) was labelled as d1, d2, d3. d30 group a (acrylyn - h) was labelled as a1, a2, a3 a30 group l (lucitone 199) was labelled as l1, l2, l3 l30 group t (trevalon) was labelled as t1, t2, t3. t30 for impact strength, the samples were tested with pendulum impact tester (s.c. dey & co., calcutta, india) (figure 3a) using izod method. the specimens were clamped at one end vertically, and the swinging pendulum was used to break the unnotched specimens (figure 3b). the load at which the specimen fractures were noted and values obtained were tabulated for statistical analysis. the fracture surface of the specimen was evaluated by stereoscopic microscope (lawrence and mayo [london ] lm-52 - 1802 trinocular research microscope [n-400 m ]) to determine the type of fracture. the samples were examined with a scanning electron microscope (sem) (leo, japan) (figure 4) and photo micrographs of respective areas were obtained to verify fracture morphology. statistical methods used were one - way anova, scheffe s post hoc test, t - test, contingency co - efficient test. scanning electron microscope metal dies were prepared for mentioned dimension (65 mm 10 mm 3 mm) (figure 1a). the dies were coated with a thin layer of petroleum jelly and were invested in the lower half of the denture flask. while taking care, the one - half of the thickness was embedded in the dental plaster but in the base of the flask. this was allowed to set for half an hour, and a single layer of separating medium was applied. second pour was made in the dental plaster, and the flask was held in compression till the final set of dental plaster. the denture flask was then opened, and the preformed dies were retrieved from the dental plaster (figure 1b). lucitone 199 (p : m) 21 g:8 ml and dpi heat cure 21 g:10 ml ; trevalon (p : m) 21 g:9 ml and acrylyn - h 21 g:10 ml were manipulated according to manufacturer s instructions and material was packed into the mould in the dough stage. bench curing was done for 3 h and short curing cycle was followed for acrylization i.e. 74c for 2 h and 100c for 1 h. one hundred and twenty test samples were labelled on each end before testing as follows : total number of specimen : 120 specimenstotal number of group : 4 groupstotal number of specimens in each group : 30 specimensgroup d (dpi) was labelled as d1, d2, d3. d30group a (acrylyn - h) was labelled as a1, a2, a3 a30group l (lucitone 199) was labelled as l1, l2, l3 l30group t (trevalon) was labelled as t1, t2, t3. t30 total number of specimen : 120 specimens total number of group : 4 groups total number of specimens in each group : 30 specimens group d (dpi) was labelled as d1, d2, d3. d30 group a (acrylyn - h) was labelled as a1, a2, a3 a30 group l (lucitone 199) was labelled as l1, l2, l3 l30 group t (trevalon) was labelled as t1, t2, t3. for impact strength, the samples were tested with pendulum impact tester (s.c. dey & co., calcutta, india) (figure 3a) using izod method. the specimens were clamped at one end vertically, and the swinging pendulum was used to break the unnotched specimens (figure 3b). the load at which the specimen fractures were noted and values obtained were tabulated for statistical analysis. the fracture surface of the specimen was evaluated by stereoscopic microscope (lawrence and mayo [london ] lm-52 - 1802 trinocular research microscope [n-400 m ]) to determine the type of fracture. the samples were examined with a scanning electron microscope (sem) (leo, japan) (figure 4) and photo micrographs of respective areas were obtained to verify fracture morphology. statistical methods used were one - way anova, scheffe s post hoc test, t - test, contingency co - efficient test. scanning electron microscope the 120 specimens of heat cure denture base resins were tested for impact strength by using impact testing machine and fracture morphology was observed by using sem. the sem microscopy showed that brittle fractures exhibited well - defined, flat, compact and organized surface fractures whereas intermediate fractures presented disorganized and jagged surfaces(figures 5a and b,6a and b). impact strength of dpi is 0.1447 and standard deviation (sd) of 2.623 and mean impact strength of acrylyn - h is 0.1463 and sd of 3.429 and mean impact strength of lucitone 199 is 0.1973 and sd of 4.638 and mean impact strength of trevalon is 0.1727 and sd of 4.510. when the f values (12.339) using one - way anova are significant then individual mean is different from other so we must analyze using scheffe s post hoc tests, which showed significance (table 1). results of scheffe s post hoc tests for mean values on impact scores of different materials. the mean impact scores of dpi heat cure and acrylyn - h materials and results of independent samples by t - test were obtained. mean impact scores of dpi and acrylyn - h materials and results of independent samples t - test. the mean impact scores of trevalon and lucitone 199 materials and results of independent samples were analyzed by t - test. the sd of lucitone 199 was 0.046 and for trevalon was 0.045 ; the t value obtained was 2.088. mean impact scores of lucitone 199 and trevalon (fiber reinforced) materials and results of independent samples t - test. the occurrence of the type of fracture in each material used was as follows (21-brittle and 9-intermediate in dpi, 20-brittle, and 10-intermediate in acrylyn - h, 10-brittle and 20-intermediate in trevalon, 9-brittle and 21-intermediate in lucitone 199) percentage of occurrence was calculated in each material and cross tabulation of all materials and type of fractures was done using contingency coefficient test (cc). the value by cc test was 0.346 and was statistically significant (table 4). the fracture of acrylic resins is an unresolved problem in removable prosthodontics despite numerous attempts to determine the causes. modification of acrylic resins designed to improve the specific properties include plasticization co - polymerization cross - linking and reinforcement. an attempt has been done to study the influence of the cross linking agent on the mechanical properties of acrylic resin, and it has been found that an improvement in impact strength with the use of 10% cross - linking agent added to the monomer is possible, but the impact strength decreases progressively with higher concentrations. one such attempt led to the production of high impact resins that contain low molecular weight butadiene- styrene - b co - polymer the exact nature of this inclusion is regarded as manufacturers trade secret and requires extensive research in chemical engineering.2 - 5 this study investigated the impact strength properties and fractographic analysis of 4 heat - cure acrylic resins. to compare the performance of different products, various mechanical tests were carried out for the study ; impact strength is taken into consideration because of their influence on the selection of a denture base resin materials. the sample preparation followed here was similar to the one adopted by john.6 in this study, metal strips were preferred over wax patterns to avoid distortion in mould space and for ease of preparation and finishing. the preformed metal strips were directly invested in the dental plaster to form plaster moulds for fabrication of test samples.6 it was observed that the mean impact strength showed higher values when tested under dry conditions, and long curing cycle when compared to wet conditions and short curing cycle.7,8 the sample dimension of 65 10 3 mm was prepared as per the ada specifications no. there are basically two types of test, charpy and izod for evaluation of impact strength.9 - 13 depending upon the loading configuration, specimen dimensions and presence of notches and their geometry, these tests can result in different values.10 the izod impact test was used in this study. although there is a good co - relation between the two tests, the absolute values differ from each other. unnotched samples were cantilevered, and a swinging pendulum was used to break the specimens. the reduction in the swing of the pendulum or the energy absorbed by the material was measured. comparison of impact strength values among four acrylic resins showed dpi heat cure and acrylyn - h presented the lower values which are not re - in forced. lucitone 199 and trevalon presented higher values that are re - in forced ; this may be attributed to reinforcement of fibers in the resin. the mean impact strength values between dpi heat cure and acrylyn - h was not statistically significant and the values between lucitone 199 and trevalon were statistically significant. the values between acrylyn - h, lucitone 199 and trevalon were statistically significant. by the help of stereoscopic microscope, the fractures were classified into brittle fractures and intermediate fractures. acrylyn - h and dpi heat cure also exhibited more brittle fractures than intermediate fractures. analysis of fractured surfaces by sem revealed the micro structural behaviour of brittle and intermediate fractures. acrylyn - h and dpi heat cure showed well - defined crystallographic surface compared to lucitone 199 and trevalon. although irregularities could be seen in each acrylic resins fracture, a common finding was that a granular micro structure was clearly distinguishable demonstrating that acrylic resin fails by transgranular or transcrystalline fracture.14 the results from impact strength indicate that differences observed can be attributed to the composition of the acrylic resins. in this study, impact strength and fracture morphology was compared between four heat cure denture base resins. the mechanical behavior of a denture in services depends not only on the strength but also on the design and construction, but also on the effect of residual stresses and the conditions of loading. within the limitations of this study, it was concluded that, the impact strength of the acrylic resins is affected by the reinforcement of fibers.increased intermediate fractures increased impact strength.brittle fractures morphology showed fewer undercuts and clearer surface.intermediate fractures morphology showed more undercuts than clear surfaces. | background : the fracture of acrylic resin denture is rather common occurrence and causes inconvenience to the patients. this study was carried out to evaluate and compare the impact strength and fracture morphology of four different heat cure acrylic materials.materials and methods : acrylic resin specimens were prepared using preformed metal die of dimension 65 10 3 mm. the specimens were finished, polished and subjected to impact strength evaluation using impact testing machine. the loads at which the specimens fracture are recorded and subjected to statistical analysis. fracture surface analysis was done. macroscopic analysis was performed by visual inspection of the fractured surfaces using a stereoscopic microscope. about 5 mm sections of all the fragments were subjected to scanning electron microscopy for microscopic analysis to verify fracture morphology.results:mean values of the impact strength were compared by statistical methods. the impact strength data were subjected to variance homogeneity tests. fracture surface analysis data was analyzed by statistical methods. the mean impact strength of lucitone 199 was higher than acrylyn - h, dpi heat cure & trevalon.conclusion:within the limitations of this study, it was concluded that the impact strength of the acrylic resins is affected by the reinforcement of fibers. increased intermediate fractures increased impact strength. brittle fractures morphology showed fewer undercuts and clearer surface. intermediate fractures morphology showed more undercuts than clear surfaces. |
benign prostatic hyperplasia (bph) is among the most common ailments in older men. the prevalence of pathological bph is only 8% in the fourth decade of life ; however, 50% of the male population in the age range of 51 to 60 years is diagnosed with pathological bph. bph is a specific histopathologic entity characterized by the presence of nonmalignant, unregulated hyperplasia of the stromal and epithelial cells. clinically, bph may be associated with obstructive and irritative lower urinary tract symptoms (luts) secondary to the prostate enlargement and is associated with complications such as acute urinary retention, bladder stones, gross hematuria, and urinary tract infections. although bph is a highly prevalent disease, the cellular mechanism of bph involving the stromal and epithelial components of the prostate is not well understood. for over a century, there have been two known etiologic factors for the pathogenesis of bph : aging and testicular androgens. in addition, family history, race / ethnicity, cigarette smoking, hypertension, non - insulin - dependent diabetes mellitus (niddm), high insulin content, and central obesity have been reported to be risk factors for the development of bph. metabolic syndrome (ms) is a combination of several metabolic and physiological abnormalities in the individual, including increased waist circumference (wc), high blood pressure, fasting plasma glucose (fpg), low high - density lipoprotein cholesterol (hdl - c), and dyslipidemia. many reports have discussed the finding that diabetic and obese men have larger prostate glands than do men without these conditions [7 - 10 ]. also, recent reports have suggested an association between ms components and luts related to bph. however, ms as a risk factor for bph has been under debate despite the epidemiologic and pathophysiologic evidence. therefore, this study was conducted to evaluate the effect of ms components on the serum psa level and prostate volume (pv) in healthy men aged 40 to 70 years. from january 2005 to december 2010, 521 consecutive men (age range, 40 to 70 years) who underwent transrectal ultrasonography (trus) were enrolled in the study. the health screening data included blood pressure, body measurements (height, weight, wc, body mass index [bmi ]), biochemical analysis (complete blood cell count, serum glucose, total cholesterol, triglycerides (tg), hdl - c and low - density lipoprotein cholesterol [ldl - c ], fpg, serologic test, coagulation test, and tumor markers), stool and urine analysis, chest radiography, electrocardiography, and detailed clinical examination. all subjects were asked to complete a questionnaire designed to assess their detail medical history, particularly of systemic diseases such as diabetes mellitus (dm) and hypertension. among the 521 men, 101 men were excluded from this study owing to current medical treatment for bph, having a history of dm or cardiovascular disease, having a diagnosis of prostate cancer and previous prostate or urethral surgery, or having abnormal serum psa (4 ng / ml, n=15). in addition, patients with pyuria and bacteriuria on urinalysis were excluded from the study. ms was defined by using the criteria established by the national cholesterol education program - adult treatment panel iii - american heart association / national heart, lung, and blood institute (ncep atpiiiaha / nhlbi) statement published in 2005. ms was diagnosed when at least three of the following criteria were present : 1) wc of 90 cm, 2) tg levels of 150 mg / dl, 3) hdl - c levels of < 40 mg / dl, 4) systolic blood pressure (sbp) 130 mmhg and/or diastolic blood pressure (dbp) 85 mmhg and/or pharmacological treatment, and 5) fpg of 100 mg / dl. central obesity was defined as wc 90 cm in men or 80 cm in women by the modified atp iii guideline that the world health organization western pacific region and the international association for the study of obesity presented for asian populations in 2000. bmi was calculated as weight in kilograms divided by height in meters squared (kg / m). pv was measured according to the prostate ellipsoid formula, multiplying the largest anteroposterior (height, h), transverse (width, w), and cephalocaudal (length, l) prostate diameters by 0.524 (hwl/6) by using trus (aloka, prosound-5sv). pasw ver. 18.0 (ibm co., armonk, ny, usa) was used for all statistical analyses. the mean value and independent t - test were used to compare the pv and serum psa level in men with and without each metabolic component. linear regression analysis was performed to compare the relationship between serum psa level, pv, and clustering of ms components. from january 2005 to december 2010, 521 consecutive men (age range, 40 to 70 years) who underwent transrectal ultrasonography (trus) were enrolled in the study. the health screening data included blood pressure, body measurements (height, weight, wc, body mass index [bmi ]), biochemical analysis (complete blood cell count, serum glucose, total cholesterol, triglycerides (tg), hdl - c and low - density lipoprotein cholesterol [ldl - c ], fpg, serologic test, coagulation test, and tumor markers), stool and urine analysis, chest radiography, electrocardiography, and detailed clinical examination. all subjects were asked to complete a questionnaire designed to assess their detail medical history, particularly of systemic diseases such as diabetes mellitus (dm) and hypertension. among the 521 men, 101 men were excluded from this study owing to current medical treatment for bph, having a history of dm or cardiovascular disease, having a diagnosis of prostate cancer and previous prostate or urethral surgery, or having abnormal serum psa (4 ng / ml, n=15). in addition, patients with pyuria and bacteriuria on urinalysis were excluded from the study. ms was defined by using the criteria established by the national cholesterol education program - adult treatment panel iii - american heart association / national heart, lung, and blood institute (ncep atpiiiaha / nhlbi) statement published in 2005. ms was diagnosed when at least three of the following criteria were present : 1) wc of 90 cm, 2) tg levels of 150 mg / dl, 3) hdl - c levels of < 40 mg / dl, 4) systolic blood pressure (sbp) 130 mmhg and/or diastolic blood pressure (dbp) 85 mmhg and/or pharmacological treatment, and 5) fpg of 100 mg / dl. central obesity was defined as wc 90 cm in men or 80 cm in women by the modified atp iii guideline that the world health organization western pacific region and the international association for the study of obesity presented for asian populations in 2000. bmi was calculated as weight in kilograms divided by height in meters squared (kg / m). pv was measured according to the prostate ellipsoid formula, multiplying the largest anteroposterior (height, h), transverse (width, w), and cephalocaudal (length, l) prostate diameters by 0.524 (hwl/6) by using trus (aloka, prosound-5sv). (ibm co., armonk, ny, usa) was used for all statistical analyses. the mean value and independent t - test were used to compare the pv and serum psa level in men with and without each metabolic component. linear regression analysis was performed to compare the relationship between serum psa level, pv, and clustering of ms components. the mean pv and mean serum psa level in the patients were 26.88.2 ml and 1.00.7. there were no statistically significant differences between the two groups in mean age, height, bmi, or total cholesterol level. in the patients with ms, the weight (p<0.001), wc (p<0.001), sbp (p<0.001), dbp (p<0.001), tg (p<0.001), and fpg (p<0.001) were significantly higher, and the hdl - c (p<0.001) was significantly lower, than in the patients without ms. in addition, serum psa level and pv were significantly higher in the patients with ms than in the patients without ms, respectively (p<0.001, p<0.001). the differences in pv and serum psa level according to the presence of each metabolic component are shown in table 2. the patients with central obesity (p<0.0001, < 0.0001), high sbp (p=0.021, p=0.003), high dbp (p=0.001, p<0.0001), high tg (p=0.001, p<0.0001), low hdl - c (p=0.012, p<0.0001), and high fpg (p=0.0001, p<0.0001) had significantly higher serum psa levels and larger pvs than did the patients without these conditions. therefore, the presence of metabolic components in these patients was significantly related to higher serum psa level and larger pv. the frequencies of zero, one, two, three, four, and five ms components were 74 (17.6%), 114 (27.2%), 90 (21.4%), 88 (20.9%), 44 (10.5%), and 10 (2.4%), respectively. the relationship between pv, serum psa level, and the sum of ms components is shown in table 3. the serum psa level was increased similarly with the increasing sum of ms components (r=0.104, p<0.0001). every increase in an ms component resulted in an increase of approximately 0.17 ng / dl in the serum psa level. there was a significant contribution of ms components to pv also (r=0.139, p<0.0001), with an increase of approximately 2.28 ml in pv for every component increase in ms components. bph is a complex and highly prevalent disease in men older than 50 years and it may lead to aggravation in the quality of life of suffering men. previous studies have shown that bph is associated with several etiologic factors that may influence the prostate pathophysiology [12 - 14 ]. recently, many studies have also shown that components of ms (central obesity, hypertension, niddm, hyperlipidemia, low hdl - c, hypertriglyceridemia) are risk factors for bph development and that these components may play a part in bph pathogenesis [12 - 14 ]. hammarsten and hogstedt reported that men with ms had a significantly larger pv and faster annual bph growth rates than did men without ms. in particular, central obesity, niddm, hypertension, low hdl - c, and high fasting insulin level are risk factors in the development of bph [14 - 17 ].. demonstrated that pv was positively correlated with central obesity, as represented by wc, but that overall obesity measured by bmi was not correlated with pv. the present study illustrated that patients with high wc, high sbp / dbp, high tg, low hdl - c, and high fpg level had a significantly larger pv. the serum psa level test is a screening tool that is the most widely used marker for the early detection of prostate cancer and is used in the management of the disease after the diagnosis of bph. the serum psa level may be influenced by many factors, such as age, bmi, pv, and prostate disease. reported trends for a positive association between older age, diastolic bp, and the serum psa level, whereas bmi, hdl, and fbg correlated negatively with the serum psa level. we demonstrated that patients with central obesity, high sbp / dbp, high tg, low hdl - c, and high fpg had a significantly higher serum psa level than did patients without these conditions. although several investigators have observed a significant association between bph and ms or its components, others have opposing opinions. gupta. suggested that there were no significant relationships between bph and ms, weight, bmi, lipid level, or thyroid hormone status. jeong. suggested that ms was not associated with psa level in a screened population. among the risk factors for ms, bp, unfortunately, the relationships between pv, serum psa level, and components of ms have not been clearly established and there is no consensus on the basis of recent results. however, in the present study, most of the metabolic components were significantly related to larger pvs and a higher serum psa level. also, patients with more numerous ms components had increased serum psa levels and pv. in general, ms develops as a result of westernized diet and lifestyle, which are in turn are related with obesity and insulin resistance. adipose tissue secretes several substances (adipocytokines), and these substances can induce insulin resistance. hypertension was also associated with bph / luts in several animal models and epidemiologic studies. reported that spontaneously hypertensive rats develop bph - like features with aging in the absence of any inductive exogenous agents. similar to obesity, the relationship between bph and dyslipidemia has been documented in several epidemiologic studies. examined the data of 158 men and reported that individuals with a low hdl - c level had a larger pv and a higher annual bph growth rate than did individuals with a high hdl - c level. hyperinsulinemia contributes to activation of the sympathetic nerve system and leads to an increased level of catecholamine in the tissue. it may also contribute to ms development and increased smooth muscle tone of the prostate, leading to severe luts. these observations suggest that components of ms may interact to increase the risk of bph. as seen in the above mentioned studies, each study reported different results for the relationship between ms components and psa. these differences can be attributed to the different sample sizes and different selection process of the study subjects. also, there are several studies confirming a correlation between the serum psa level and pv. pv is strongly related to serum psa and age in men with symptomatic bph and in whom prostate cancer has been excluded. psa can also be used as an aid to estimate the degree of prostate enlargement. in summary, because patients with ms components have a larger pv than do patients without ms components, larger pv is related to a high serum psa level. this study reported a different result for the relationship between psa and ms components from the result of jeong.. our study was not community - based ; however, the subjects were healthy and were applicants for a routine health screening. therefore, the serum psa levels of our study subjects may not be significantly different from the serum psa levels of a community - based population. in addition, therefore, a prospective, community - based study including prostate biopsy is necessary to verify whether ms influences the accuracy of the serum psa level test and pv measurement. we demonstrated that ms components were associated with larger pv and a high serum psa level. each ms component could be an important factor in the development and management of bph. when treating patients with bph, the ms components of patients should be considered and should be managed. further studies are needed to confirm the relationship between ms components and bph and to explain the underlying mechanisms. | purposemetabolic syndrome (ms) plays a potential role in the etiology of benign prostatic hyperplasia (bph). recent studies have reported on an association between ms and bph. however, there has been no consensus on recent results. this study was conducted to evaluate the associations among prostate - specific antigen (psa), prostate volume (pv), and metabolic components in men who visited our health promotion center.materials and methodsduring the period from january 2005 to december 2010, 521 consecutive men (age range, 40 to 70 years) who underwent transrectal ultrasonography were enrolled in this retrospective study. the health screening program includes blood pressure, body measurements (height, weight, waist circumference, body mass index), biochemical analysis (serum glucose, total cholesterol, triglycerides, high - density and low - density lipoprotein cholesterol, fasting plasma glucose, tumor markers), stool and urine analysis, and a detailed clinical examination.resultsthe serum psa level and pv were significantly higher in patients with ms than in patients without ms, retrospectively (p<0.001, p<0.001). patients with more than one metabolic component were significantly more likely to have a larger pv and higher serum psa level. the serum psa level and pv were increased in a similar manner with the increasing sum of ms components (p<0.0001, p<0.0001).conclusionsthe ms components were associated with larger pv and higher serum psa level. therefore, each ms component could be an important factor in bph development and management. |
phospholamban (plb) is a 52 amino acid membrane protein that is located in the sarcoplasmic reticulum (sr) membrane of heart muscle cells. despite its small size, plb plays an important role in regulating heart muscle relaxation rate by interacting with sarcoplasmic reticulum ca atpase (serca). in the absence of plb, serca acts as a ca ion channel that transports ca ions from the cytosol into the sr and triggers the heart muscle relaxation process. plb interacts with serca and inhibits serca function by decreasing serca - ca affinity. the inhibition effect is removed when plb is phosphorylated at ser16 by protein kinase a (pka) or when the cytoplasmic ca ion concentration is elevated. plb inhibition and inhibition relief effects on serca regulate serca function, which as a result controls the heart muscle relaxation rate. plb has four domains : cytoplasmic domain (domain ia ; residues 116), loop region (residues 1722), domain ib (residues 2330), and the transmembrane domain (residues 3152). the bellflower model has been established with the cytoplasmic domain sticking out into the cytosol as an -helix or as an unstructured coil. recent studies have shown that the inconsistency of the cytoplasmic domain conformation results from dynamic changes. instead of adopting a fixed conformation in the membrane, it consists of a mixture of two or more conformation states. in vivo, plb forms a pentamer with the cytoplasmic domain as an -helix laying flat on the membrane surface. the flexibility of the cytoplasmic domain enables it to act as the regulatory region of plb function via shifting its conformation states with the interplay of serca, pka, and the membrane environment. domain ib is less well studied and located on the top of the hydrophobic region of the membrane as an -helix. epr studies have indicated that this segment of plb is much more dynamic than the cytoplasmic domain. two conformation states are identified in domain ib, which are folded and unfolded. recent studies have suggested that domain ib is also involved in regulating plb function. the transmembrane domain has abundant leucines and isoleucines forming a leu - zipper and the three cysteines forming disulfide bonds to help stabilize the plb pentamer. protein interaction studies suggest that one face of the transmembrane domain binds to serca, while the other face stabilizes the plb pentamer form by interacting between the monomers. in living cells, 78% of plb are in the pentamer form and the rest are in the monomer form. it is the monomer form that interacts with and inhibits serca, while the pentamer form acts only as an inactive storage form. when plb is phosphorylated, there is a slightly increase in pentameric form population. plb can adopt four different conformational states (t, t, r, and r). the low - energy level t state has the cytoplasmic domain forming an -helix on top of the membrane surface and domain ib folded. the high energy level r state has the cytoplasmic domain unfolded and extended into the cytosol, and domain ib is unfolded. the two conformation states (folded and unfolded) of domain ib are independent of the cytoplasmic domain. equilibrium is established between the t and r states with the t state as a dominant population in vivo. when phosphorylated, plb population shifts from the t state to an intermediate t state, which gives rise to the more disordered and extended r state. the t state has an extended cytoplasmic domain partially attached to the membrane surface. the intermediate r state has a completely unfolded cytoplasmic domain that is attached to the membrane surface. the conformational states are suggested to be well - related to the function of plb, that is, each state or each dominant state may correspond to a function such as inhibition. plb conformational studies are extremely important to relate structural investigations to the functional studies. interplay between plb, serca, and the membrane controls the regulation of plb on serca. it is believed that the membrane is not only acting as an environment to stabilize or localize serca and plb but is also involved actively in regulating function. plb mutants are divided into two types : gain - of - function mutants and loss - of - function mutants. gain - of - function mutants have a superinhibition effect on serca when compared with wild - type plb (wt - plb), whereas loss - of - function mutants have inhibitory function relief. mutation sites are not confined to certain domains according to their effects, which suggest that plb function is not controlled by certain sites on only one domain. several factors may be involved including : plb oligomerization states, structural conformations, and its interaction with the membrane and serca. this study is focused on the arg9cys (r9c)-mutated plb (r9c - plb) loss - of - function mutant. r9c - plb has been identified in individuals who have heart chamber enlargement, which can lead to heart failure in 5 to 10 years. structural conformations between wt - plb, ser16-phosphorylated plb (p - plb), and r9c - plb are compared for the first time with nmr spectroscopy. solid - state nmr spectroscopy is a robust method to study the structural and dynamic properties of membrane proteins. because of the large size, hydrophobicity, and sensitivity to the environment of membrane proteins, traditional structural biology methods are not appropriate to elucidate valid structural information. the local secondary structure can be investigated by analyzing the c nmr spectrum of a c = o group on a specific residue. static n nmr powder pattern spectra reveal information on the backbone dynamics of a specific residue. with mechanically aligned glass plate samples, membrane proteins are aligned in lipid bilayers with the bilayer normal oriented parallel to the static magnetic field.n labels at specific sites yield n chemical shift resonances to resolve the structural topology. the cytoplasmic domain, domain ib, and transmembrane domain are studied with solid - state nmr spectroscopy to elucidate the secondary structure, backbone dynamics, and topology changes of plb upon r9c mutation and phosphorylation. 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine (popc), 1,2-dioleoyl - sn - glycero-3-phosphocholine (dopc), and 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine (dope) were purchased from avanti polar lipids (alabaster, al). the phospholipids were dissolved in chloroform and stored at 20 c before use. trifluoroethanol (tfe) and n-[2-hydroxyethyl]piperazine - n-2-ethane sulfonic acid (hepes) were purchased from sigma - aldrich (st. louis, mo). ethylenediaminetetraacetic acid (edta) and sodium chloride (nacl) were purchased from fisher scientific (pittsburgh, pa). prephosphorylated fmoc - serine and fmoc amino acids were obtained from applied biosystems (carlsbad, ca). c = o labeled fmoc - alanine, c = o labeled fmoc - leucine, n labeled fmoc - alanine, and n labeled fmoc - leucine were purchased from isotec tm / sigma - aldrich (miamisburg, oh). all other peptide synthesis and hplc reagents and solvents were acquired from vwr (san dimas, ca). nmr glass sample cells were purchased from new era enterprise (vineland, nj). glass plates were purchased from superior marienfeld laboratory glassware (lauda - konigshofen, germany). specific c- or n - labeled wt - plb, p - plb, and r9c - plb were synthesized via fmoc - based solid - phase peptide synthesis on a cem solid - phase peptide synthesizer. n labels were placed at positions ala11, ala24, leu28, and leu42. to synthesize the specific labeled p - plb, we used prephosphorylated fmoc - serine at ser16. crude peptides were obtained after cleavage using a cleavage mixture. a general electric (ge) akta purifier hplc was utilized to purify peptides by reverse - phase chromatography on a c18 column. the purified peptides were lyophilized and characterized by matrix - assisted laser desorption ionization time - of - flight (maldi - tof) mass spectrometry. lyophilized specific labeled wt - plb, p - plb, and r9c - plb were dissolved in a minimal amount of tfe. popc (35 mg) was mixed with dissolved peptides (4 mol % with respect to lipid) in a 12 75 mm test tube. the solvent was removed by a steady stream of n2 gas and placed in a vacuum desiccator overnight. the lipid peptide mixture was rehydrated with 95 l of hepes buffer (5 mm edta, 20 mm nacl, and 30 mm hepes at ph 7.0). vesicles were formed in a warm water bath at 45 c for 30 min using a vortexer and bath sonication. the sample was transferred into a 4 mm nmr rotor for solid - state nmr experiments. lyophilized specifically labeled wt - plb, p - plb, and r9c - plb were dissolved in a minimal amount of tfe. next, plb (1 mol % with respect to the lipid) was mixed with 60 mg of dopc / dope (molar ratio 4:1). a thin layer of the mixture was applied to 3540 glass plates and dried in a vacuum desiccator overnight. h - depleted water (4 l) was added to the sample on each glass plate, and the glass plates were stacked on top of each other. the sample was rehydrated in a hydration chamber with saturated ammonium phosphate (humidity 93%) for 24 h at 42 c. the sample was then transferred to a rectangular glass sample cell and sealed for nmr measurements. all nmr spectra were acquired using a 500 mhz wb bruker avance solid - state nmr spectrometer. c and n nmr experiments using mlv samples were performed with a bruker 4 mm triple resonance cpmas probe. c cpmas experiments were conducted with a spinning speed at 5 khz at the magic angle. h 90 pulse length was set to 4.5 s ; contact time 3 ms, recycle delay 4 s. eight k scans were averaged, and 70 hz line broadening was used. static n nmr experiments using mlv samples were performed using a ramp - cp pulse sequence with h decoupling. h 90 pulse length was set to 4.7 s ; contact time was 1.5 ms, and recycle delay was 4 s. 120 k scans were averaged, and 300 hz line broadening was used. static n nmr experiments using mechanically aligned glass plate samples were performed with a flat coil h - x low - e nmr probe. the alignment of the samples was checked by observing the static p solid - state nmr spectra from the membrane phospholipids. a ramp - cp pulse sequence with h decoupling was used to acquire the n nmr spectra. the contact time was 1.5 ms, and the recycle delay was 4 s. 120 k scans were averaged, and 300 hz line broadening was used. r9c is a loss - of - function mutation of plb ; therefore, r9c - plb can not inhibit serca as efficiently as wt - plb. a few properties of these mutants can be investigated to relate to their function variation. plb affinity to serca, interactions with the lipid bilayer, oligomerization states, and conformations are the major factors. previous studies have indicated that wt - plb, p - plb, and a few type of plb mutants have similar apparent affinity to serca. wt - plb interacts actively with the lipid bilayers by increasing the dynamics of both the membrane surface and hydrophobic region. both p - plb and r9c - plb have significantly less interaction with the membrane. p - plb and r9c - plb as well as a few loss - of - function mutants have slightly increased pentamer form population, while some of the gain - of - function mutants have a dominant monomer form. structural conformations of wt - plb, p - plb, and r9c - plb are compared in this project. membrane protein topology, dynamics, and secondary structure are important aspects to investigate in structure biology. for the membrane protein plb studies, these investigations can give insights into its structure function relationship. veglia and coworkers have studied the conformational states of plb and the function of corresponding conformations. a linear correlation is established. it is suggested that the ordered folded t state relates to the inhibition function of plb, while the disordered and extended t state and r state relate to the inhibition relief. phosphorylation shifts plb conformation states from the dominant t state to the t state that are readily exchangeable with the r state. it is very likely that the t state with the cytoplasmic domain partially unfolded and dissociated from the membrane surface relieves inhibition by plb. the r9c - plb is a loss - of - function mutant that also relieves inhibition of serca similar to p - plb. conformation state shifting from the t state to the t state or the r state upon r9c mutation would be coupled to functionality loss and make the r9c - plb conformation studies extremely significant. c cpmas solid - state nmr spectroscopy provides secondary structure information by studying the c = o signal of a specific residue on a membrane protein.c cpmas nmr spectroscopy of membrane proteins yields well - resolved c = o nmr peaks in the region of 170 to 180 ppm. a c nmr signal with a chemical shift of 176 ppm corresponds to an -helix for alanine and leucine residues, while a peak at 173 ppm corresponds to an unstructured coil for an alanine residue. ala15 is located toward the end of the cytoplasmic domain and is the neighbor of a phosphorylation site (ser16), thus, it would be sensitive to changes induced by phosphorylation. secondary structure of the cytoplasmic domain of wt - plb, p - plb, and r9c - plb was examined by c cpmas nmr spectroscopy with a c = o label on ala15. wt - plb (figure 1a) has two peaks at 176.5 and 175.4 ppm, indicating that the cytoplasmic domain of wt - plb consists of two -helical structural conformations. both p - plb and r9c - plb (figure 1b, c) have peaks centered at 176.3 and 174.9 ppm, which are very close to the wt - plb signals, but the intensity of the 174.9 ppm signal is larger than the peak at 176.3 ppm. this suggests that phosphorylation and r9c mutation increases the population of one of the two -helical components. either phosphorylation or r9c mutation of plb does not induce significant secondary structure changes to the cytoplasmic domain but only involves a population shift. c cpmas solid - state nmr spectra of popc mlvs acquired at 25 c with c = o labeled on the ala15 residue in the cytoplasmic domain of wt - plb (a), p - plb (b), and r9c - plb(c). domain ib was investigated with a c = o label at ala24, which is located near the beginning of domain ib. figure 2 shows c nmr spectra of the domain ib of wt - plb, p - plb, and r9c - plb. the wt - plb sample (figure 2a) has a major peak at 175.8 ppm with a broad shoulder, while p - plb and r9c - plb (figure 2b, c) have two peaks with one centered at 173.2 ppm with a larger intensity and the other at 175.8 ppm. interestingly, upon phosphorylation or r9c mutation, a significant population of unstructured coil appears. the data indicate an unfolding or partially unfolding process in domain ib upon phosphorylation or r9c mutation. c cpmas solid - state nmr spectra of popc mlvs acquired at 25 c with c = o labeled on the ala24 residue in domain ib of wt - plb (a), p - plb (b), and r9c - plb (c). the transmembrane domain was probed with a c = o label placed at leu39. wt - plb and r9c - plb yield a single well - resolved peak at 176.3 and 176.7 ppm corresponding to a single -helical component. the data suggest that the transmembrane domain secondary structure is not affected by r9c mutation. previous studies on p - plb also indicate an undisturbed transmembrane domain upon phosphorylation. c cpmas solid - state nmr spectra of popc mlvs acquired at 25 c with c = o labeled on the leu39 residue in the transmembrane domain of wt - plb (a) and r9c - plb (b). n solid - state nmr spectroscopy with n labels placed on specific residues. n - labeled amide proteins in mlvs adopt random orientations, revealing a n powder pattern line shape. the chemical shift anisotropy (csa) width of the powder pattern spectra reflects the backbone dynamics of the specific labeled residue. figure 4 compares the n nmr spectra of wt - plb, p - plb, and r9c - plb with the n label on the ala11 residue of the cytoplasmic domain. n nmr spectra of all three samples consisted of a broad powder pattern line shape that is typical for unoriented solid - state n nmr membrane protein samples and an isotropic component at 121122 ppm. the csa widths of the broad components of wt - plb, p - plb, and r9c - plb are 171, 168, and 163 ppm. the n nmr data indicate that the cytoplasmic domain is a mixture of a dominant ordered component and a small mobile population. the data are consistent with wt - plb cytoplasmic domain studies that have an equilibrium between the t and r states, in which the t state is immobile and more populated and the r state is more dynamic. upon phosphorylation and r9c mutation, the intensity of the isotropic signal increases and suggests a population shift from the t state to the disordered t or r state. notably, the csa width of the immobile component of r9c - plb is reduced by 8 ppm when compared with wt - plb, while p - plb induces a 3 ppm csa width reduction. the n nmr data indicate a slightly increased motion of the immobile component of residue ala11 in the r9c - plb cytoplasmic domain. n solid - state nmr powder pattern spectra of popc mlvs acquired at 25 c with n labeled on the ala11 residue in the cytoplasmic domain of wt - plb (a), p - plb (b) and r9c - plb (c). backbone dynamics were investigated in domain ib with an n label on the ala24 residue. the n nmr spectra of all the wt - plb, p - plb and r9c - plb are shown in figure 5. the n nmr spectrum of wt - plb labeled on ala24 of domain ib yields a peak with an undistinguishable isotropic component and a broad powder pattern component. unlike the two distinct components observed in the cytoplasmic domain, the undistinguishable static n nmr peak of domain ib could rise from the fast exchange between a folded and unfolded state in microseconds. the exchange between the states in the cytoplasmic domain is on the n nmr spectroscopy time scale (milliseconds), and thus both states are observed. the data are consistent with the epr studies that show two states of domain ib due to the fast epr time scale. upon phosphorylation or r9c mutation, there is no significant change in the n nmr spectra of domain ib, indicating the fast exchange between folded and unfolded states. n solid - state nmr powder pattern spectra of popc mlvs acquired at 25 c with n labeled on the ala24 residue in the domain ib of wt - plb (a), p - plb (b), and r9c - plb (c). for comparison, figure 6 shows the n nmr spectra of wt - plb and r9c - plb with the n label placed on leu42 of the transmembrane domain. characteristic powder pattern line shape spectra with similar csa width (173 ppm) are observed for both wt - plb and r9c - plb. the broad single component n spectrum indicates that the transmembrane domain of both forms of plb is immobile in the membrane. the results are similar to that of the p - plb form according to previous studies. n solid - state nmr powder pattern spectra of popc mlvs acquired at 25 c with n labeled on the leu42 residue in the cytoplasmic domain of wt - plb (a) and r9c - plb (b). the structural topology of plb with respect to the membrane was studied using n solid - state nmr spectroscopy of plb incorporated into mechanically aligned glass plate samples. in these oriented samples, the direction of the bilayer normal is aligned parallel to the static magnetic field. an n nmr resonant peak near indicates that the labeled residue is located perpendicular with the bilayer normal, while a n nmr peak near // indicates the labeled residue is located parallel with the bilayer normal. figure 7 compares the n nmr spectra of wt - plb, p - plb, and r9c - plb with the n label on ala11 of the cytoplasmic domain. wt - plb (figure 7a) has a peak at 71 ppm, which is very close to, indicating that wt - plb cytoplasmic domain lies nearly flat on the surface of the lipid bilayer, which is consistent with previous wt - plb studies. p - plb and r9c - plb (figure 7b, c) reveal peaks at 92 and 86 ppm. the shift of the n nmr peak indicates a dynamic shift due to backbone motion, a change in cytoplasmic domain topology, or a secondary structure change. n cp solid - state nmr spectra of mechanically aligned dopc / dope (4:1 ratio) lipid bilayers acquired at 25 c with n labeled on the ala11 residue in the cytoplasmic domain of wt - plb (a), p - plb (b), and r9c - plb (c). if the 15 ppm downfield shift of r9c - plb is only due to the backbone dynamic increase, a similar csa width reduction can be expected when comparing the static n nmr spectra of n - ala11 wt - plb with those of r9c - plb in mlvs. mlvs are more hydrated than glass plate samples, so a csa width reduction of 15 ppm or larger can be expected. however, r9c - plb induced an 8 ppm decrease in csa width when compared with wt - plb. so, beside the backbone dynamic increase, topology and/or secondary structure change also contributes to the downfield shift of r9c - plb. no significant secondary structure change is observed at residue ala15 in the cytoplasmic domain when r9c is mutated. more likely, ala11 topology changes by shifting away from the membrane surface, as observed in previous p - plb studies. the hypothesis is compatible with the p nmr data revealing a significantly less disturbance of r9c - plb on the lipid bilayer surface when compared with wt - plb. nmr data further confirm the shift of population from a membrane - attached t state toward partially membrane detached and more disordered t state or r state upon phosphorylation or r9c mutation. aligned n nmr spectra of wt - plb, p - plb, and r9c - plb with n - label on the leu28 residue of the domain ib are shown in figure 8. ala24 is not used here because wt - plb shows no alignment (data not shown). it is not surprising because ala24 is at the beginning of domain ib, which is close to the loop region and the water lipid interface. it is very possible that it does not have a fixed orientation in the lipid bilayer. a n nmr resonant peak at 210 ppm is observed for the wt - plb (figure 8a). it is near // and the n nmr peak of wt - plb with a n label on the transmembrane domain. thus, domain ib of the wt - plb aligns nearly parallel with the membrane bilayer normal and the transmembrane domain. interestingly, because all samples were well - aligned with the bilayer normal parallel to the static magnetic field according to the p nmr data, upon phosphorylation or r9c mutation, the alignment of domain ib is lost with the appearance of a broad powder pattern line shape in addition to a 208 ppm peak (figure 8b, c). the spectra indicate a mixture of an unoriented component and an oriented component parallel to the bilayer normal. n cp solid - state nmr spectra of mechanically aligned dopc / dope (4:1 ratio) lipid bilayers acquired at 25 c with n labeled on the leu28 residue in the domain ib of wt - plb (a), p - plb (b), and r9c - plb (c). the unoriented broad component may be due to backbone dynamic changes, topology, and/or secondary structure changes. if it is not due to the secondary structure change, leu28 should adopt random orientations as an -helix in the lipid bilayer. a dramatic disturbance in the lipid acyl chain near the membrane surface however, h nmr spectra investigating lipid acyl chain dynamics do not show any effect upon r9c - plb or p - plb addition. unwinding is observed at ala24 in domain ib, suggesting a population shift from the dominant folded state toward an unfolded state. the transmembrane domain topology was investigated with the n label on the leu42 residue (figure 9). both wt - plb and r9c - plb (figure 9a, b) show a peak at 208 and 211 ppm, which is near //. the n nmr spectra indicate that the transmembrane domain of both types of plb aligns nearly parallel with the bilayer normal, which is also similar to p - plb according to previous studies. n cp solid - state nmr spectra of mechanically aligned dopc / dope (4:1 ratio) lipid bilayers acquired at 25 c with n labeled on the leu42 residue in the transmembrane domain of wt - plb (a) and r9c - plb (b). taking all of the data together, when r9c is mutated, the plb cytoplasmic domain undergoes a possible dissociation process from the membrane surface with increased population of fast backbone motion like the p - plb. if the t state is identified as dominated in p - plb, a more disordered cytoplasmic domain dissociated from the membrane matches perfectly with the conformation that is observed here for both p - plb and r9c - plb. thus, when phosphorylated or r9c - mutated, plb conformation probably shifts from a dominant t state to t state or even r state. studies of plb n27a superinhibition mutant concluded that there is no significant difference between the backbone dynamics of the wt - plb and n27a - plb for both the cytoplasmic domain and transmembrane domain. the data suggest that n27a - plb adopts a dominant ordered t state, similar to the wt - plb, and that this conformation retains or even elevates the inhibition function. both phosphorylation and r9c mutation appear to shift plb conformation from the t state to the t and/or r state. wt - plb cytoplasmic domain absorbed on the membrane surface could be partially stabilized by the three positively charged arg residues electrostatically interacting with the negatively charged bilayer surface. when arg9 is mutated into a neutral cys, the electrostatic interaction would be weakened, which may initiate the dissociation of the cytoplasmic domain from the membrane surface. the studies on the interplay between serca, plb, and the membrane indicate that both the transmembrane domain and domain ib interact with serca and that the membrane can bind to serca and activate it. loose binding of the transmembrane domain to serca is not crucial for inhibition but only to recruit serca. thus, it is not surprising to observe the undisturbed transmembrane domain by either phosphorylation or r9c mutation in this study. recent studies show that the interaction sites of serca with plb and the membrane may overlap. it is possible that the membrane and plb domain ib compete for binding to serca, with the plb inhibiting serca while the membrane activates serca. when phosphorylated or r9c - mutated, unwinding of domain ib may expose the interaction site with serca. the dissociation of plb cytoplasmic domain from the membrane surface may facilitate the lipid to bind to and activate serca, which could induce the inhibition relief process. the mechanism of serca inhibition relief by p - plb and r9c - plb and its mediation by the membrane requires further studies. on the basis of the solid - state nmr studies in this paper, the r9c mutation of plb induces the cytoplasmic domain to undergo a change in the population of the -helical component and partially dissociation from the membrane surface with increased backbone dynamics. domain ib of the wt - plb forms a mobile -helical component. when phosphorylated or r9c - mutated, the population shifts from a folded to an unfolded state in domain ib are observed. the data indicate that phosphorylation or r9c mutation induce a population shift of plb conformation from a dominant t state to a more extended and disordered conformation, which may correspond to a mixture of t state and r state. this conformation change correlates to the t state and r state with the plb inhibition function loss. | phospholamban (plb) is a membrane protein that regulates heart muscle relaxation rates via interactions with the sarcoplasmic reticulum ca2 + atpase (serca). when plb is phosphorylated or arg9cys (r9c) is mutated, inhibition of serca is relieved. 13c and 15n solid - state nmr spectroscopy is utilized to investigate conformational changes of plb upon phosphorylation and r9c mutation. 13c = o nmr spectra of the cytoplasmic domain reveal two -helical structural components with population changes upon phosphorylation and r9c mutation. the appearance of an unstructured component is observed on domain ib. 15n nmr spectra indicate an increase in backbone dynamics of the cytoplasmic domain. wild - type plb (wt - plb), ser16-phosphorylated plb (p - plb), and r9c - mutated plb (r9c - plb) all have a very dynamic domain ib, and the transmembrane domain has an immobile component. 15n nmr spectra indicate that the cytoplasmic domain of r9c - plb adopts an orientation similar to p - plb and shifts away from the membrane surface. domain ib (leu28) of p - plb and r9c - plb loses the alignment. the r9c - plb adopts a conformation similar to p - plb with a population shift to a more extended and disordered state. the nmr data suggest the more extended and disordered forms of plb may relate to inhibition relief. |
to describe a case of non - arteritic ischemic optic neuropathy (naion) secondary to acute primary - angle closure (apac). a 50-year - old woman with painful visual loss in the right eye was found to be in apac with a right afferent pupillary defect. diffuse optic disc edema in the right eye and a small cup - to - disc ratio in the left eye were evident. the patient was diagnosed with non - arteritic ischemic optic neuropathy (naion) secondary to apac, a rare clinical entity which can result in markedly decreased visual acuity. naion secondary to apac is a rare clinical entity that can result in severe vision loss. the prevalence of acute primary - angle closure (apac) is 0.04 to 0.6 per thousand in the united states in patients older than 40.1,2 the incidence of non - arteritic ischemic optic neuropathy (naion) is two to ten per 100,000 in the population older than 50.3,4 naion secondary to apac was described by sonty and schwartz in 1981.5 there have been four subsequent case reports (five eyes) describing naion secondary to apac.69 patients present with typical apac symptoms and signs, which include pain, headache, nausea and vomiting, conjunctival injection, corneal edema, elevated intraocular pressure (iop), closed angles, and a shallow anterior chamber. naion was present at the time of presentation for apac in four of six eyes and developed one week after apac in two eyes.59 a 50-year - old woman with a history of hypertension and dyslipidemia presented with redness, pain, decreased vision, headache, and nausea, for approximately 10 hours. the patient was taking metoprolol, 50 mg, at bedtime ; hydrochlorothiazide, 25 mg, daily ; amlodipine, 25 mg, daily ; lisinopril, 20 mg, daily for hypertension ; simvastatin, 40 mg, at bedtime for dyslipidemia ; and aspirin, 81 mg, daily for cardiovascular risk factors. best - corrected visual acuity (va) was hand motion at 4 feet right eye and 20/20 left eye (manifest refraction : plano + 0.75 135). her pupil was 5 mm in the right eye and 4 mm in the left, and she was noted to have a moderate relative afferent pupillary defect (rapd) in the right eye. the right eye had moderately severe ciliary injection, corneal edema, a shallow anterior chamber (closed peripherally, corneal thickness centrally), and a moderate nuclear sclerotic cataract. the left eye was quiet, had a shallow anterior segment (corneal thickness peripherally, corneal thickness centrally), and a mild nuclear sclerotic cataract. the view of the posterior segment in the right eye was obscured by the corneal edema. the left optic nerve head appeared normal with a cup - to - disc ratio of 0.1. gonioscopy demonstrated that her angle was closed 360 in the right eye and was shaffer grade ii (anterior trabecular meshwork) 360 in the left. the patient was treated for apac in the right eye with timolol, dorzolamide, and brimonidine drops every 30 minutes and acetazolamide 500 mg orally. one hour later, the iop was 34 mmhg and she was still experiencing severe pain and decreased vision. after instillation of pilocarpine 2%, a laser peripheral iridotomy (lpi) was performed in the right eye without complications and the anterior chamber deepened, the iop decreased to 14 mmhg, and the patient felt a marked improvement in pain. due to the right rapd, the patient was asked to return the following morning for a dilated fundus exam. upon the patient s return, she reported no pain in her right eye but noted no change in vision. va remained at hand motion at 4 feet with a moderate rapd and an iop of 12 mmhg. her lpi was patent, and her anterior chamber was shallow (corneal thickness peripherally), but significantly deeper than the previous day. the right optic nerve head had 360 edema and an area of subretinal fluid extending into the nasal aspect of macula from the optic disc temporally (figure 1a). optical coherence tomography (oct) demonstrated optic nerve edema and subretinal fluid extending to the macula in the right eye (figure 2a and b). a brain magnetic resonance imaging (mri) with contrast to rule out infiltrative or compressive causes of the optic nerve edema was within normal limits. the patient had no giant cell arteritis symptoms and no headaches prior to this acute event, and the headaches resolved completely after the lpi. the patient was counseled on the importance of stopping use of her antihypertensive medications in the evening and of improved blood pressure and dyslipidemia control. two days after presentation, the patient was started on oral prednisone taper (60 mg prednisone daily for 2 weeks, followed by 50 mg for 5 days and then 40 mg for 5 days, and then cutting down by 5 mg every 3 days to 5 mg, followed by 2.5 mg for 3 days and then 1 mg for 3 days) and instructed to follow - up in one month. the decision to use steroids in this patient was based on a retrospective study by hayreh and zimmerman, which demonstrated improvement in va and visual field in naion patients who presented within two weeks of onset.10 a larger improvement was seen in patients with poorer presenting va.10 one month later, va was count fingers at two feet in the right eye and 20/20 in the left. a humphrey visual field of the right eye demonstrated a cecocentral scotoma (figure 3). the optic nerve edema of the right eye resolved and the disc had diffuse atrophy (figure 1c). naion secondary to apac is a rare clinical entity that can result in severe vision loss. naion is caused by ischemia to the optic nerve head, mainly from the posterior ciliary artery and its branches.11 blood flow to the optic nerve head is determined by a ratio of perfusion pressure and resistance to flow.12 resistance to flow is determined by efficiency of autoregulation, vascular integrity, and the rheological properties of blood.12 these factors can be impaired in patients with diabetes, hypertension, and atherosclerosis.12 perfusion pressure is determined by the difference in mean arterial pressure (map) and iop.12 the map is reduced with relative nocturnal hypotension and antihypertensive medications.12 high iops during phacoemulsification may contribute to the increased incidence of naion after cataract surgery.1315 elevated iop has been identified as a cause of decreased blood flow to the optic nerve head in five cases (six eyes) previously reported by other authors and our case of naion after apac (table 1).59 taken together with our case, of all seven eyes with apac - associated naion, the duration of apac symptoms ranged from 1 day to 1 month. five of the seven eyes had naion at presentation, while the remainder developed naion one week after presentation for apac. five of seven eyes underwent lpi in the naion eye and contralateral eye.59 slavin and margulis described one patient who developed an naion one week after an apac attack in the left eye and refused an lpi in the right eye.8 one month later, the patient had an apac attack in the right eye and developed a right naion a week later.8 sequential naion has not been reported in the other apac - associated naion cases.57,9 one possible hypothesis for the 1 week delay in presentation with naion after presentation with apac in this patient is that the initial apac event may have started the cycle of naion, but it was not clinically significant for 1 week. hayreh described the cycle of naion as ischemia of axons resulting in axoplasmic flow stasis, which in turn cause axoplasmic accumulation and subsequent axonal swelling, which in turn compresses the capillaries, leading to more ischemia.12 including our patient, only two of the seven eyes (29%) with apac - associated naion attained acuity of 20/30 or better,59 which is worse than the 49% of naion patients who attained 20/30 or better in a study by hayreh.16 however, the low sample size of apac - associated naion patients is a barrier for meaningful comparisons with other naion patients. it is also possible that the 2 cases of the delayed onset naion after apac previously reported may be spontaneous naion. patients with apac can also develop mild optic nerve edema that is not associated with naion.17 tsai found a statistically significant difference in the retinal nerve fiber layer thickness (rnfl) of the apac eye and the contralateral eye measured by oct 1 week after the apac event.17 however, a statistically significant difference was not observed at the 4- and 12-week follow - ups.17 these patients can be differentiated from naion secondary to apac patients by the lack of subsequent optic nerve atrophy and preserved visual acuity and visual field. in addition to counseling patients about naion risk factors (eg, evening antihypertensive medications and control of hypertension, diabetes, and dyslipidemia), patients must be counseled on the importance of performing an lpi in the contralateral eye. in apac patients with an rapd, a large amount of optic nerve edema, persistent decreased visual acuity, and a small cup - to - disc ratio in the contralateral eye should raise clinical suspicion for naion. | purposeto describe a case of non - arteritic ischemic optic neuropathy (naion) secondary to acute primary - angle closure (apac).methodscase report.resultsa 50-year - old woman with painful visual loss in the right eye was found to be in apac with a right afferent pupillary defect. laser peripheral iridotomy relieved pain but did not improve vision. diffuse optic disc edema in the right eye and a small cup - to - disc ratio in the left eye were evident. magnetic resonance imaging was normal. the patient was diagnosed with non - arteritic ischemic optic neuropathy (naion) secondary to apac, a rare clinical entity which can result in markedly decreased visual acuity.conclusionnaion secondary to apac is a rare clinical entity that can result in severe vision loss. |
in the modern era of medicine, such huge mucinous ovarian tumors have become rare in the current medical practice, as most of the cases are diagnosed early during routine gynecological examinations or incidental finding on the ultrasound examination of the pelvis and abdomen. most of the patients who have large tumors present mainly with the pressure symptoms over the genitourinary system leading to urinary complaints and also pressure over respiratory system leading to respiratory embarrassment. the role of imaging modalities such as computed tomography (ct) scan and magnetic resonance imaging gives better idea about the extension of the tumor in the various quadrants of the abdomen and consistency of the tumor. management of ovarian cysts depends on the patient 's age, the size of the cyst, and its histopathological nature. four frozen section is very important to know the malignant variation of this tumor and that helps in the management of the patient. surgical expertise is required to prevent complications as in huge tumors the anatomical planes get distorted. here are four major categories of ovarian tumors : epithelial tumors (65 - 75%) serous or mucinous cystadenoma / carcinoma, clear cell carcinoma, and brenner tumor;germ cell tumors (15%) dysgerminoma, embryonal cell cancer, choriocarcinoma, and teratoma;sex - cord - stromal tumors (5 - 10%) granulosa cell tumor, thecoma, and fibroma;metastatic tumors (10%) uterine, stomach, colon, breast, and lymphoma. epithelial tumors (65 - 75%) serous or mucinous cystadenoma / carcinoma, clear cell carcinoma, and brenner tumor ; germ cell tumors (15%) dysgerminoma, embryonal cell cancer, choriocarcinoma, and teratoma ; sex - cord - stromal tumors (5 - 10%) granulosa cell tumor, thecoma, and fibroma ; metastatic tumors (10%) uterine, stomach, colon, breast, and lymphoma. extra - large benign and malignant cysts of the ovary are uncommon and involve diagnostic and management challenges, and determination of cancer antigen (ca)-125 can help to identify epithelial tumors of the ovary. the epithelium of the cysts is usually cylindrical and mono- or multi - stratified and cuboidal epithelium is due to the pressure inside the cyst. this report concerns an unsuspected giant mucinous ovarian cystadenocarcinoma in a 42-year - old woman with a huge abdominal enlargement. the main objective of this report is to call attention to ovarian epithelial cysts in the outpatient clinics and primary care services, contributing to a decrease in any underdiagnosis, misdiagnosis, and underreporting that might occur. a 42-year - old multiparous woman came with rapidly increasing severe distension of abdomen and pain in abdomen since 6 - 8 months. the patient was postmenopausal since 2 years and there was h / o cholecystectomy done 2 years back otherwise there was no other significant h / o any medical or surgical disorder in the past. on examination, her general condition was fair, thin built, and her weight was 50 kg. vitals were stable, pulse was 86 beats / min, and blood pressure was 120/80 mmhg., there was huge distension all over the abdomen and skin over the abdomen had thinned was and shiny. on palpation, the tumor was 36 weeks of uterine size, firm to hard in consistency, mobility restricted ; margins were ill defined, was arising from the lower pelvis, and extending till xiphisternum and both sides of iliac fossa [figure 1 ]. her ca 125 levels were 990 iu / ml. ultrasound examination was suggestive of cystic mass of size 31 cm 13.2 cm 35 cm cystic lesion, arising from right adnexa with internal septations and calcifications within. ct of abdomen + pelvis showed solid cystic mass lesion of size 21.9 cm 18.5 cm 12.3 cm extending from l2 to s1s2. preoperative picture of giant ovarian tumor on opening abdomen in situ, findings were evidence of huge left ovarian tumor [figures 24 ], cystic in nature, of 6 kg weight, and clamping of left ovarian pedicle done. ovarian mass excision done and sent for frozen section report was mucinous cystadenocarcinoma of the left ovary. the patient was operated for exploratory laparotomy with debulking of left ovarian mass with total abdominal hysterectomy (tah) with bilateral salpingo - oophorectomy with complete omentectomy. her final histopathology report was suggestive of mucinous cystadenocarcinoma of right and left ovary, with left ovary showing focally capsular breach and right ovary capsule intact. intraoperative picture of mucinous cystadenocarcinoma of ovary intraoperative picture of ovarian tumor gross specimen of mucinous cystadenocarcinoma of ovary the patient withstood the surgery well with no intraoperative or postoperative complications. on day 10 of surgery, in young people, the majority of ovarian cysts decreases in size or even disappears and therefore should be dealt with a careful expectant follow - up by ultrasonography. benign cysts of < 8 cm are conservatively managed, but cystectomy is indicated for cysts over 5 cm in postmenopausal women. giant cysts require resection because of compressive symptoms or risk of malignancy and their management invariably requires laparotomy to prevent perforation and spillage of the cyst fluid into the cavity. clinically, the differential diagnosis of large abdominal masses should include uterine enlargement (pregnancy and fibromyomatosis) ; pelvic endometriosis (pregnancy and abdominal cysts) ; abdominal pregnancy ; urinary retention (full bladder) ; intestinal tumors ; hydronephrotic kidney ; pelvic retroperitoneal tumor ; and accentuated obesity. women with abdominal - pelvic masses constitute a challenging condition in general practice because the clinical features and findings from physical examination are usually nonspecific. although tumor markers can be a useful tool for differential diagnosis of malignant cysts, some authors have described elevated levels of these markers in patients with benign tumors. major diagnostic difficulties are often posed if inner nodules are disclosed in these cystic cavities because this finding must be considered as indicative of a malignant tumor. in our present case study, the giant ovarian tumor was multilocular with diverse inner solid masses, and the histopathology evaluation characterized the diagnosis of cystadenocarcinoma. in our case, malignant tumors (anaplastic carcinoma, carcinosarcoma, fibrosarcoma, rhabdomyosarcoma, undifferentiated sarcoma), mixed nodules, and leiomyoma among others, were ruled out. based on cell origin, ovarian tumors are classified as germ cell tumors (undifferentiated and extraembryonic) ; stromal tumors (granulosa - theca, sertoli, and leydig cells) ; and epithelial tumors (cystadenoma, borderline cystadenoma, and cystadenocarcinoma). the interval between the symptoms ' onset and clinical presentation of ovarian cancer is a major concern and diagnostic challenge involving this malignancy. nevertheless, symptoms frequently develop insidiously and with intervals usually longer in the localized disease. noteworthy is that delay in ovarian cancer detection has a direct relationship with poor outcome, but some examples of longstanding localized evolution have been reported. despite uncommon presentation and unexpected diagnosis, the cystadenocarcinoma was removed intact, successfully without any spillage or spread. our present case study gives evidence of unsuspected malignant giant abdominal tumor in a woman. | giant cystadenocarcinomas of the ovary are rarely described. huge ovarian masses are mostly benign, but malignancy should be ruled out by investigations and clinical assessment. giant cysts require resection because of compressive symptoms or risk of malignancy and their management invariably requires laparotomy to prevent perforation and spillage of the cyst fluid into peritoneal cavity. here, we present a case of a 42-year - old female with severe and rapidly growing abdominal distension operated for exploratory laparotomy for cystic mass excision. on histology, mass was found to be metastatic mucinous cystadenocarcinoma with omental metastasis. the diagnostic and management challenges posed by this unexpected and unusual presentation of an ovarian cystadenocarcinoma are discussed. the main aim of this report is to draw attention to huge ovarian epithelial cysts with unsuspected presentation contributing to a decrease in any underdiagnosis, misdiagnosis, and mismanagement that might occur. |
retinal ganglion cells (rgcs) can be stimulated by electrical pulses so that action potentials are generated even when the responses of photoreceptors to visual stimuli are lost due to photoreceptor degeneration (1). a retinal implant using prosthetic electrical stimulation has been developed to provide alternative partial vision to blind people who lost their vision due to a retinal degenerative disease, such as retinitis pigmentosa (rp) or age - related macular degeneration (amd) (2). according to the results from clinical tests of retinal implants, patients with implants can perceive phosphene with simple spatial patterns, such as large objects and letters (3). spatial resolution may be improved to some degree by increasing the number of stimulation electrodes, which enables visual guidance of fine hand movements (4). still, the performance of retinal implants should be improved, as a recent clinical test in multiple human subjects reported inconsistent and variable results of phosphene perception when identical stimulation parameters were applied to different subjects (5). the observation of neural activities can provide valuable insights into the success of prosthetic electrical stimulation. we have recently shown that temporal patterns of visual input could be successfully decoded from population activities of rgcs evoked by a temporally patterned stimulation pulse train, in both normal (6) and photoreceptor - degenerated retinas (7). cottaris and elfar (8) demonstrated that the spatial origin, duration, and amplitude of each stimulation pulse could be identified with multisite cortical local field potential. prior to these studies, rgc neural activities had been investigated to provide preliminary information on the requirements for electrical stimulation, such as the threshold current level, the latency of evoked neural activities, and the effect of stimulation waveforms (9). it is essential to examine both temporal and spatial characteristics of rgc responses in order to develop the details of the stimulation methods ; i.e., the stimulation strategy, for a faithful delivery of spatiotemporal visual information to the brain. this is obvious in that the spatial and temporal aspects of external visual inputs should be properly encoded in the neural activities evoked by stimulation so that they can be transferred to and interpreted in the brain. previous studies were mainly focused on either the temporal (110) or spatial (11) aspects of rgc activity patterns, and the encoding of spatiotemporal temporal visual information by evoked rgc activities has not been analyzed in detail. in this study, our specific aim was to investigate whether spatiotemporal visual information can be decoded from the rgc network activity evoked by patterned electrical stimulation. based on an experimental setup of an in vitro model of retinal implants (11), we tested an amplitude - modulation scheme of biphasic current pulse trains. here, we assumed that spatiotemporal visual information would be translated to the neural activities by multiple amplitude - modulated electrical pulse trains, just as in the case of cochlear implants, in which the pulse amplitude is modulated by the amplitude of sound pressure. along with a thorough characterization of spatial spreading of stimulation current and temporal information encoding, we demonstrated that the multipixel spatiotemporal visual information can be accurately decoded from the population activities of rgcs that are stimulated by amplitude - modulated pulse trains. the rd1 (c3h / hej strain) mice (age : postnatal day 56) were used in this study. the methods for retinal tissue preparation and for the recording of rgc activities were previously reported (1) and are briefly described here. retinal patches from rd1 mice were prepared following the method of stett. (retinas were isolated, cut into patches of 3 3 mm, and mounted on a planar microelectrode array (mea ; multichannel systems gmbh, reutlingen, germany) so that the ganglion cell layer faced the mea. the mea contained 64 tin electrodes (circular shape ; diameter : 30 m, interelectrode spacing : 200 m, impedance : < 50 kat 1 khz) on a glass substrate in an 8 8 square - type grid layout. the waveforms from the electrodes were recorded with a sampling rate of 25 khz / channel (amplification gain : 1,200, bandwidth : 103,000 hz) and stored on a hard drive using data acquisition software (mc_rack ; multichannel systems gmbh). the data recorded by mc_rack were converted to text file by mc_datatool (multichannel systems gmbh). the recorded waveforms were futher analyzed to generate single unit spike trains by spike detection and spike sorting based on principal component analysis (12). amplitude - modulated current pulse trains were generated with a stimulus generator (stg 1004 ; multichannel systems gmbh) and applied to the retina via 2 or 4 channels of the mea at its vertices. the stimuli consisted of symmetric, charge - balanced biphasic pulses (anodic first, with no temporal separation between the anodic and cathodic phases). for the characterization of the modulation behavior of rgc responses by pulse amplitude, trains of 20 identical pulses were applied at 1-second interpulse intervals from one of the stimulating electrodes while the pulse amplitude was increased from 260 a (2, 5, 10, 20, 30, 40, 50, and 60 a). the pulse rate and pulse duration were fixed at 1 hz and 500 s, respectively. then, the amplitude - modulated pulse trains were applied to investigate whether the rgc responses can be made to encode spatiotemporal visual information. the procedure for the pulse amplitude modulations based on natural scenes is described below (fig. black - and - white movies of natural scenes were recorded by a camcorder (xacti hd1010 ; sanyo, osaka, japan) for 2 minutes at 60 frames / s with a 1,280 720 resolution. two or 4 pixels of the 16 9 resolution scene were randomly selected, and the amplitude of each pulse was modulated according to the time - series of the pixel intensity within the range of 120 a (1 a resolution). the pulse trains were simultaneously applied to the retina via 2 or 4 channels of the mea to present the electrical stimulation with spatial pattern, as shown in fig. 1. the pulse repetition rate varied from 210 hz (2, 4, 6, 8, and 10 hz). the pulse duration per phase varied from 200400 s (200, 300, and 400 s). pulse trains of which amplitudes were modulated according to the brightness of 2 (or 4) pixels were delivered to rgcs via the stimulation electrode on the mea. the visual information was reconstructed (i.e., decoded) from the evoked rgc activities. then, the goodness - of - fit between the original and decoded pulse amplitude time - series was calculated to evaluate the effectiveness of the stimulation. spike train decoding was used to estimate how accurately the spatiotemporal visual information was transferred to rgc neural activities by multichannel electrical stimulation. because each pulse train was applied to each stimulating electrode independently, it was possible to select rgcs whose evoked activities were effectively modulated by a specific pulse train (as in fig. thus, 2 and 4 groups of rgcs were used to decode visual spatiotemporal information with 2 and 4 pixels, respectively. the raw waveforms from each electrode were transformed into single unit spike trains, and then, the rgc spike trains were transformed to a firing rate time - series by counting the numbers of spikes in 50 ms bins. rhythmic bursting patterns of (a) spontaneous activity and (b) electrically evoked response (pulse amplitude : 20 a, pulse rate : 1 hz). as the arrows indicate, both spontaneous and electrically evoked activities show rhythms of bursts at similar frequencies. the response strength of the rgcs was measured by counting the number of poststimulus spikes within approximately 100 ms of the stimulus onset, which corresponds to the first peak in the psth shown in fig. 2c. rgc = retinal ganglion cell, psth = post - stimulus time histogram, isih = interspike interval histogram. the input to the decoder consisted of firing rates of multiple rgcs within a number of time bins. that is, the firing rates of multiple rgcs within a short temporal interval after the stimulus were used to decode the pulse amplitude time - series at every time bin, corresponding to the intensity time - series of multiple pixels. the input - output relationship can be described as follows : (1)s^(i)=p=1nj=0m-1{(rpi - jfpj) } here, (i) is the estimated value of the pulse amplitude of one electrode at the i time bin, and fp(j) is the j coefficient of the linear filter corresponding to the p rgc. rp(i) is the firing rate of the p unit at the i time bin of 50 ms duration. the decoder can be regarded as a temporal filter because it calculates the output from the past and present values of the input. when linear mapping is used, the decoding algorithm becomes an optimal linear finite impulse response (fir) filter (13). in this case, (.) is merely a unity function, and the coefficients fp(j) were obtained by the least squares method, as illustrated by warland. more generally, nonlinear regression algorithms, such as multilayer perceptron or support vector machine (svm), can be used for the input - output mapping of the decoding algorithm. the specific algorithm of the svm that we used is described in hoegaerts. the recordings were divided into 2 groups of equal size (i.e. 12 minutes recordings). the first one minute of the recording was used for the training of the decoding algorithms and the remaining one minute was used to test the trained decoding algorithms. to quantify the accuracy of the information decoded from rgc activity, similarities between the original and decoded pulse amplitude time - series were computed by correlation coefficient (cc). all experimental methods and animal care procedures were approved by the institutional animal care and use committee of chungbuk national university (approval number : cbnura-042 - 0902 - 1). the rd1 (c3h / hej strain) mice (age : postnatal day 56) were used in this study. the methods for retinal tissue preparation and for the recording of rgc activities were previously reported (1) and are briefly described here. retinal patches from rd1 mice were prepared following the method of stett. (retinas were isolated, cut into patches of 3 3 mm, and mounted on a planar microelectrode array (mea ; multichannel systems gmbh, reutlingen, germany) so that the ganglion cell layer faced the mea. the mea contained 64 tin electrodes (circular shape ; diameter : 30 m, interelectrode spacing : 200 m, impedance : < 50 kat 1 khz) on a glass substrate in an 8 8 square - type grid layout. the waveforms from the electrodes were recorded with a sampling rate of 25 khz / channel (amplification gain : 1,200, bandwidth : 103,000 hz) and stored on a hard drive using data acquisition software (mc_rack ; multichannel systems gmbh). the data recorded by mc_rack were converted to text file by mc_datatool (multichannel systems gmbh). the recorded waveforms were futher analyzed to generate single unit spike trains by spike detection and spike sorting based on principal component analysis (12). amplitude - modulated current pulse trains were generated with a stimulus generator (stg 1004 ; multichannel systems gmbh) and applied to the retina via 2 or 4 channels of the mea at its vertices. the stimuli consisted of symmetric, charge - balanced biphasic pulses (anodic first, with no temporal separation between the anodic and cathodic phases). for the characterization of the modulation behavior of rgc responses by pulse amplitude, trains of 20 identical pulses were applied at 1-second interpulse intervals from one of the stimulating electrodes while the pulse amplitude was increased from 260 a (2, 5, 10, 20, 30, 40, 50, and 60 a). the pulse rate and pulse duration were fixed at 1 hz and 500 s, respectively. then, the amplitude - modulated pulse trains were applied to investigate whether the rgc responses can be made to encode spatiotemporal visual information. the procedure for the pulse amplitude modulations based on natural scenes is described below (fig. black - and - white movies of natural scenes were recorded by a camcorder (xacti hd1010 ; sanyo, osaka, japan) for 2 minutes at 60 frames / s with a 1,280 720 resolution. two or 4 pixels of the 16 9 resolution scene were randomly selected, and the amplitude of each pulse was modulated according to the time - series of the pixel intensity within the range of 120 a (1 a resolution). the pulse trains were simultaneously applied to the retina via 2 or 4 channels of the mea to present the electrical stimulation with spatial pattern, as shown in fig. 1. the pulse repetition rate varied from 210 hz (2, 4, 6, 8, and 10 hz). the pulse duration per phase varied from 200400 s (200, 300, and 400 s). encoding and decoding of visual information. pulse trains of which amplitudes were modulated according to the brightness of 2 (or 4) pixels were delivered to rgcs via the stimulation electrode on the mea. the visual information was reconstructed (i.e., decoded) from the evoked rgc activities. then, the goodness - of - fit between the original and decoded pulse amplitude time - series was calculated to evaluate the effectiveness of the stimulation. spike train decoding was used to estimate how accurately the spatiotemporal visual information was transferred to rgc neural activities by multichannel electrical stimulation. because each pulse train was applied to each stimulating electrode independently, it was possible to select rgcs whose evoked activities were effectively modulated by a specific pulse train (as in fig. thus, 2 and 4 groups of rgcs were used to decode visual spatiotemporal information with 2 and 4 pixels, respectively. the overall procedure for spike train decoding the raw waveforms from each electrode were transformed into single unit spike trains, and then, the rgc spike trains were transformed to a firing rate time - series by counting the numbers of spikes in 50 ms bins. temporal patterns of the neural activities of rgcs. rhythmic bursting patterns of (a) spontaneous activity and (b) electrically evoked response (pulse amplitude : 20 a, pulse rate : 1 hz). as the arrows indicate, both spontaneous and electrically evoked activities show rhythms of bursts at similar frequencies. the response strength of the rgcs was measured by counting the number of poststimulus spikes within approximately 100 ms of the stimulus onset, which corresponds to the first peak in the psth shown in fig. 2c. rgc = retinal ganglion cell, psth = post - stimulus time histogram, isih = interspike interval histogram. the input to the decoder consisted of firing rates of multiple rgcs within a number of time bins. that is, the firing rates of multiple rgcs within a short temporal interval after the stimulus were used to decode the pulse amplitude time - series at every time bin, corresponding to the intensity time - series of multiple pixels. the input - output relationship can be described as follows : (1)s^(i)=p=1nj=0m-1{(rpi - jfpj) } here, (i) is the estimated value of the pulse amplitude of one electrode at the i time bin, and fp(j) is the j coefficient of the linear filter corresponding to the p rgc. rp(i) is the firing rate of the p unit at the i time bin of 50 ms duration. the decoder can be regarded as a temporal filter because it calculates the output from the past and present values of the input. when linear mapping is used, the decoding algorithm becomes an optimal linear finite impulse response (fir) filter (13). in this case, (.) is merely a unity function, and the coefficients fp(j) were obtained by the least squares method, as illustrated by warland. more generally, nonlinear regression algorithms, such as multilayer perceptron or support vector machine (svm), can be used for the input - output mapping of the decoding algorithm. the specific algorithm of the svm that we used is described in hoegaerts. the recordings were divided into 2 groups of equal size (i.e. 12 minutes recordings). the first one minute of the recording was used for the training of the decoding algorithms and the remaining one minute was used to test the trained decoding algorithms. to quantify the accuracy of the information decoded from rgc activity, similarities between the original and decoded pulse amplitude time - series were computed by correlation coefficient (cc). all experimental methods and animal care procedures were approved by the institutional animal care and use committee of chungbuk national university (approval number : cbnura-042 - 0902 - 1). in total, 1,185 single units of rgcs showing apparent spiking activities were identified from 27 retinal patches obtained from 27 mice. the average number of rgcs per patch was 43.89 5.66. among these, 894 rgcs (33.11 7.51 rgcs / patch, 75.44%) showed consistent and evident modulation of evoked responses according to pulse amplitude, as shown in fig. 2a and b show typical waveforms of spontaneous and electrically evoked activities of an rgc. the oscillatory bursts in the spontaneous activity produced the distinct shape of the interspike interval histogram (isih, fig. the first peak at 10 ms resulted from an interspike interval within each burst of spikes. rhythmic burst firing patterns were also observable in electrically evoked rgc activities, as indicated by the arrows with dotted lines in fig. the rgc responses were in the form of repetitive oscillatory bursts with strong phase - locking across different trials (fig. the phase - locked bursts of spikes were maintained up to 500700 ms (fig. 2b and c). in spite of the significant alteration in firing patterns of electrically evoked activities, the strength of the evoked activities could be effectively modulated by the amplitude of the stimulation pulses (fig. 2e), just as in the case of a normal retina (6). the number of poststimulus rgc spikes within 100 ms increased essentially monotonically when the pulse amplitude was increased to 20 a and were saturated thereafter. short latency responses elicited by direct activation of rgcs were also observed after artifact removal at 3 ms poststimulus., only a single short latency spike is evoked in response to a single current pulse, so that the decoding accuracy in our study is not depedent on the short latency response. thus, we did not included the short latency responses, which are severly contaminated by stimulation artifact and not essential for the decoding in this study (1617). the spatial patterns of evoked rgc activities were characterized by the relationship between the response strength and the distance between stimulation and recording electrodes (fig. the electrical stimulation was applied at one of the 2 stimulating electrodes in fig. 3a. as a typical example, for a 20 a amplitude biphasic pulse, the rgcs within 8001,000 m of the stimulation electrode fired 3 spikes (fig. 3a and b). we defined the rgcs, the firing rates of which could be effectively modulated by pulse amplitude, as well - modulated rgcs (fig. we tried to find the spatial range of effective stimulation from the distribution of the well - modulated rgcs (fig. the rgcs were clustered into 2 groups (group 1 and 2) according to the strength of response to the electrical stimulation. that is, if the stimulation at the stimulating electrode 1 (s1) elicited more spikes at a specific rgc than the stimulation at the stimulating electrode (s2), then the rgc was assigned to group 1. fig. it clearly indicates that the rgc activities were evoked and modulated more effectively by the stimulation site in closer proximity. 3d shows the ratio of well - modulated rgcs as a function of the distance from the stimulation electrode, which implies that the range of rgcs for effective encoding of information is almost linearly decreased as a function of the distance. stimulation pulse trains were applied independently to one of the 2 stimulation electrodes (pulse amplitude : 20 a). (b) response strength as a function of the distance between the stimulation and recording electrodes. (c) spatial profile of the well - modulated rgcs. the number of well - modulated rgcs displayed at the location of the recording electrodes. (d) the percentage of the electrodes with well - modulated rgcs as a function of the distance between the stimulation and recording electrodes. the percentages were calculated by the ratio between the number of all the electrodes and the number of electrodes where the well - modulated rgcs were observed. when 2 independent amplitude - modulated pulse trains were applied simultaneously at the 2 stimulating electrodes, both of the pulse amplitude time - series obtained from 2 pixels of natural scenes could be successfully decoded from the rgc spike trains (fig. 4). a nonlinear decoder using a svm resulted in more accurate decoding, although both linear and nonlinear methods yielded satisfactory decoding. the decoding accuracy was strongly dependent on the details of stimulation, especially on the pulse amplitude range. 4a and b show the comparison of the original and decoded pulse amplitude time - series when the range of the pulse amplitude was 120 a and 110 a, respectively (pulse rate : 8 hz, pulse duration : 300 s). we tested several stimulation parameters and found that the decoding accuracy was the highest for the pulse amplitude range of 120 a and the pulse duration of 300 s (fig. when the amplitude range was changed to 110 a while fixing the pulse rate and duration, the decoding accuracy significantly deteriorated (fig. 4c, cc was 0.5912 0.24 and 0.3827 0.23 at 120 and 110 a, respectively ; 17 retinal patches, p < 0.001). 4d, when the pulse rate was changed to 2 hz, the decoding accuracy was significantly lower than at 8 hz (cc was 0.6733 0.18 and 0.4177 0.23 at 8 and 2 hz, respectively ; 6 retinal patches, p < 0.001). the pulse amplitude time - series applied to 4 stimulating electrodes could also be successfully decoded from a group of rgcs (fig. more accurate decoding was possible by increasing the number of rgcs for the decoder input, but the increase of decoding accuracy was saturated at 10 cells (data not shown). although the distance between adjacent stimulating electrodes was as short as 1,216 m, 4 independent time - series were faithfully reconstructed (fig. the decoding accuracy was calculated for several different stimulation parameters, and it was the highest for the pulse amplitude range of 120 a and the pulse duration of 400 s (fig. when the amplitude range was changed to 110 a while fixing the pulse rate and duration, the decoding accuracy significantly decreased (fig. 5b, cc was 0.6679 0.10 and 0.4788 0.19 at 120 and 110 a, respectively ; 7 retinal patches, p < 0.001). the decoding accuracies of 2 channel and 4 channel stimulations were not compared directly since different retinal patches were used for the 2 channel and the 4 channel stimulation experiment. examples of original and decoded pulse amplitude time - series, obtained from 2 pixels of a natural scene. (a) pulse amplitude range : 120 a, pulse duration : 300 s, pulse rate : 8 hz. (b) pulse amplitude range : 110 a, pulse duration : 300 s, pulse rate : 8 hz. the location of stimulation electrode of the mea is described in each panel (open circle : stimulation electrode, closed circle : ground electrode). comparison of the decoding accuracies of (c) 2 different pulse amplitude ranges (obtained from 17 retinal patches, pulse rate : 8 hz, svm) and (d) 2 different pulse rates (obtained from 6 retinal patches, pulse amplitude range : 120 a, svm). the decoding accuracy significantly changed in both cases (t - test, p < 0.001). examples of original and decoded pulse amplitude time - series, obtained from 4 pixels of a natural scene. (a) pulse amplitude range : 120 a, pulse duration : 400 s, pulse rate : 8 hz. pulse amplitude time - series applied to 4 stimulating electrodes could also be successfully decoded from a group of rgcs, just as it was with 2-channel stimulation. four - pixel decoding accuracy was significantly different at 2 different pulse amplitude ranges (t - test, p < 0.001, obtained from 7 retinal patches, pulse rate : 8 hz, svm). in this study, we demonstrated that spatiotemporal visual information can be decoded from the rgc activities of a photoreceptor - degenerated retina evoked by simultaneous multichannel stimulation based on pulse amplitude modulation. in our recent studies on temporal information encoding by electrically stimulated rgcs, the pulse amplitude time - series could be successfully reconstructed from evoked rgc activities (67). this is supported by recent reports on human clinical trials of retinal implants, such as the report of greenwald. (18), where it was shown that the brightness of the phosphene elicited by electrical stimulation of the retina followed the current amplitude. thus, we inferred that pulse amplitude modulation might be an appropriate approach for delivering temporal visual information to the retina. cottaris and elfar (8) showed that specific features of electrical stimulation pulses, including spatial location of the stimulation site, could be identified from local field potential in v1. hence, it is natural to expect that spatial information of visual input might be effectively encoded by prosthetic electrical stimulation as well. our results have explicitly shown for the first time, to our knowledge, that multipixel spatiotemporal information can be decoded from the activities of the rgc network when evoked by a multichannel amplitude - modulated stimulation pulse train. these results support the plausibility of a pulse generation strategy based on amplitude modulation for successful encoding of spatial information in neural activities. the range of amplitude modulation was crucial, as it could be predicted from the characteristics of the rgc - evoked firing rate as a function of pulse amplitudes (fig. it is obvious that an excessively wide pulse amplitude range is not beneficial for high decoding accuracy, due to the firing rate modulation characteristics shown in fig. this resulted in strong dependence of the decoding accuracy on the range of amplitude modulation. when the pulse amplitude was modulated at 110 a, which is half of the optimum (120 a), the decoding accuracy considerably deteriorated, due to the insufficient changes in the firing rate that caused ineffective encoding of the intensity variation. considering the high level of spontaneous rgc firing in photoreceptor - degenerated retinas (19), it is also feasible that the level of electrically evoked activities was not differentiated from that of the spontaneous activities. the current spread to adjacent sites is another reason to limit the pulse amplitude range, and the spatial profile of the evoked rgc activities (fig. other parameters, such as pulse duration or pulse rate, also significantly affected the decoding accuracy. the optimization of the stimulation strategy should be performed in a multidimensional parameter space in real clinical applications of retinal implants, as demonstrated conceptually by eckmiller. the decoding - based strategy of our study can be utilized for this purpose as well. for successful perception of a geometrical pattern, multiple localized phosphenes should be generated, and their brightness should be controlled individually. this is expected to be achieved by selective activation of groups of rgcs in close proximity of each stimulating electrode. we tried to investigate whether such a spatially selective activation of groups of rgcs is possible by observing the evoked rgc activities as a function of the distance from the stimulation electrode. it is known that the spatial resolution that is achievable by long - latency rgc responses (i.e., by indirect activation) is limited in space by spatial extent of the ganglion cell dendritic field and is better than that of short - latency response (i.e., by direct activation) (21). however, this is for a case when the size of the stimulation electrode is sufficiently small. the spatial resolution deteriorates for a larger electrode due to the current spread. considering that the electrode size is considerably large and that the current amplitude is substantially above the threshold in our study, as well as in clinical trials conducted thus far, the spatial resolution is expected to be worse than the optimum determined by the ganglion cell dendritic field. the current spreading was indeed quite substantial, and the evoked activities were quite strong for the rgcs within 8001,000 m from the stimulating electrodes, as shown in fig. 3). less than 20% of the well - modulated rgcs were outside of 800 m. considering that 1 mm corresponds to a visual angle of 3.4 in human eyes (22), the spatial resolution achievable by our strategy is expected to be 23. it is accepted that a spatial resolution of 10 is required for perceiving large objects and movement direction (23) and thus, a spatial resolution of 23 is surely enough to be helpful in improving the quality of life of the blind. the decoding accuracy was strongly dependent on the pulse rate, as shown in fig. 4, where the pulse rate of 8 hz was significantly more effective than a rate of 2 hz. a higher pulse rate might be beneficial because it prevents abnormal rhythmic rgc responses due to the short inter - pulse intervals. however, the pulse rate should be determined carefully because excessively high pulse rate may suppress rather than activate the rgc activities (10). it is reported that short - width pulses can selectively evoke short - latency responses of rgcs and generate precise temporal spiking patterns by evoking only one spike with each pulse (10). a similar strategy has recently been implemented using optogenetic stimulation as well (24). however, this requires minute electrode size and short inter - electrode distance, which are not compatible with current retinal implant devices undergoing clinical trials, which are rather bulky. for example, the stimulating electrode size is as large 200 m (25). the size and inter - electrode spacing of these current devices are comparable to those in our study. thus, our in vitro model can provide a basis for the evaluation and optimization of a stimulation strategy for these current devices. in conclusion, we showed that multipixel spatiotemporal visual information could be decoded accurately from rgc network activity evoked by patterned electrical stimulation when the detailed parameters of the simulation pulse trains were carefully determined. our results suggest that useful visual function may be restored by amplitude modulation - based retinal stimulation. | retinal implants have been developed as a promising way to restore partial vision for the blind. the observation and analysis of neural activities can offer valuable insights for successful prosthetic electrical stimulation. retinal ganglion cell (rgc) activities have been investigated to provide knowledge on the requirements for electrical stimulation, such as threshold current and the effect of stimulation waveforms. to develop a detailed stimulation strategy for faithful delivery of spatiotemporal visual information to the brain, it is essential to examine both the temporal and spatial characteristics of rgc responses, whereas previous studies were mainly focused on one or the other. in this study, we investigate whether the spatiotemporal visual information can be decoded from the rgc network activity evoked by patterned electrical stimulation. along with a thorough characterization of spatial spreading of stimulation current and temporal information encoding, we demonstrated that multipixel spatiotemporal visual information can be accurately decoded from the population activities of rgcs stimulated by amplitude - modulated pulse trains. we also found that the details of stimulation, such as pulse amplitude range and pulse rate, were crucial for accurate decoding. overall, the results suggest that useful visual function may be restored by amplitude modulation - based retinal stimulation. |
the young mania rating scale (ymrs) is a diagnostic questionnaire commonly used to evaluate manic symptoms in patients with bipolar 1 disorder and to assess treatment efficacy in clinical trials. this rating scale comprises eleven items (elevated mood, increased motor activity energy, sexual interest, sleep, irritability, speech rate and amount, language thought disorder, content, disruptive aggressive behavior, appearance, and insight). the total ymrs score is calculated as the summation of each of the eleven individual item scores.1 asenapine is a tetracyclic antipsychotic with a unique pharmacologic profile indicated in several countries, including the united states and the european union, for use in adult patients with manic episodes in bipolar i disorder. acute efficacy was demonstrated in two 3-week trials in which asenapine monotherapy was significantly more effective than placebo at improving mania symptoms as assessed using the total ymrs score.2,3 the objective of this exploratory, post hoc analysis of the data pooled from the two positive trials was to identify the potential differential effects of asenapine or olanzapine on each of the eleven individual ymrs items, as indicators of the severity of specific bipolar mania symptom dimensions. data were pooled from two 3-week randomized, placebo - and olanzapine - controlled, double - blind, double - dummy, multicenter, parallel - group trials (a7501004 and a7501005).2,3 patients aged 18 years with a diagnosis of bipolar i disorder ; current manic or mixed episodes according to the mini international neuropsychiatric interview4 and diagnostic and statistical manual of mental disorders, fourth edition, text revision criteria5 ; and a ymrs total score 20 at screening and baseline were eligible. following a 7-day, single - blind placebo run - in phase, active treatment was initiated (day 1, n = 977) with asenapine 20 mg (10 mg twice daily [bid ], morning and evening ; n = 379), olanzapine 15 mg (once daily [qd ] ; n = 396), or placebo (n = 202). thereafter, treatment continued with flexible dosing (asenapine at 1020 mg daily [510 mg bid ] and olanzapine at 520 mg qd). patients remained in the research facility for 7 days of treatment and were discharged if clinically stable. a complete description of the design and eligibility criteria of both studies has been reported elsewhere.2,3 seven of the eleven individual ymrs items were scored on a 0 to 4 scale : appearance, insight, language thought disorder, increased motor activity energy, elevated mood, sleep, and sexual interest. the remaining four items were scored on a 0 to 8 scale : disruptive aggressive behavior, content, irritability, and speech rate and amount. the ymrs total score, with a range of 0 to 60, was a summation of each of the eleven individual scores, with the higher total ymrs scores refecting greater symptom severity. ymrs response rate was determined as the percentage of patients achieving 50% reduction from baseline in total ymrs score. an exploratory post hoc analysis of the change from baseline in the eleven individual ymrs items was performed. an analysis of covariance model adjusted by baseline value was used to test for differences in changes from baseline in ymrs scores between groups. pooled patient demographics and baseline characteristics are displayed in table 1. of the 977 treated patients, 680 (70%) treat population (treated patients with at least one post baseline ymrs assessment, n = 960) consisted of patients receiving asenapine 10 to 20 mg daily (n = 372), olanzapine 5 to 20 mg daily (n = 391), or placebo (n = 197). reductions from baseline to day 21 were significantly greater for asenapine and olanzapine compared with placebo for each of the eleven individual ymrs items (table 2). statistically significant reductions in ymrs total score were observed for both treatments compared with placebo, as reported previously.2,3 in six of the ymrs items, statistically significant improvements from baseline were observed as early as day 2 (mean change sd, locf) for asenapine (1020 mg daily) and olanzapine (520 mg daily) compared with placebo : disruptive aggressive behavior (0.3 1.15 ; 0.4 1.14 ; 0.1 1.16), content (0.4 1.22 ; 0.5 1.48 ; 0.2 0.90), irritability (0.5 1.41 ; 0.5 1.33 ; 0.3 1.15), elevated mood (0.3 0.75 ; 0.3 0.73 ; 0.1 0.63), sleep (0.4 0.88 ; 0.5 0.92 ; 0.2 0.74), and speech (0.6 1.23 ; 0.6 1.27 ; 0.1 0.96) (all comparisons p < 0.05 for asenapine or olanzapine versus placebo). no statistical difference was observed in the improvements seen with asenapine and olanzapine in the ymrs items of appearance, content, elevated mood, increased motor activity and insight. for the remaining items (language thought disorder, sleep, sexual interest, disruptive aggressive behavior, irritability, and speech) while both treatments were associated with significant improvement compared to placebo, olanzapine treatment was accompanied by a significantly greater improvement compared to asenapine (p < 0.05 with no multiplicity adjustment). following treatment with asenapine or olanzapine a greater response rate (50% improvement in total ymrs score) was observed compared to placebo (42.5%, 52.4% and 29.4% respectively ; p < 0.05 for asenapine or olanzapine versus placebo). pooled patient demographics and baseline characteristics are displayed in table 1. of the 977 treated patients, 680 (70%) treat population (treated patients with at least one post baseline ymrs assessment, n = 960) consisted of patients receiving asenapine 10 to 20 mg daily (n = 372), olanzapine 5 to 20 mg daily (n = 391), or placebo (n = 197). reductions from baseline to day 21 were significantly greater for asenapine and olanzapine compared with placebo for each of the eleven individual ymrs items (table 2). statistically significant reductions in ymrs total score were observed for both treatments compared with placebo, as reported previously.2,3 in six of the ymrs items, statistically significant improvements from baseline were observed as early as day 2 (mean change sd, locf) for asenapine (1020 mg daily) and olanzapine (520 mg daily) compared with placebo : disruptive aggressive behavior (0.3 1.15 ; 0.4 1.14 ; 0.1 1.16), content (0.4 1.22 ; 0.5 1.48 ; 0.2 0.90), irritability (0.5 1.41 ; 0.5 1.33 ; 0.3 1.15), elevated mood (0.3 0.75 ; 0.3 0.73 ; 0.1 0.63), sleep (0.4 0.88 ; 0.5 0.92 ; 0.2 0.74), and speech (0.6 1.23 ; 0.6 1.27 ; 0.1 0.96) (all comparisons p < 0.05 for asenapine or olanzapine versus placebo). no statistical difference was observed in the improvements seen with asenapine and olanzapine in the ymrs items of appearance, content, elevated mood, increased motor activity and insight. for the remaining items (language thought disorder, sleep, sexual interest, disruptive aggressive behavior, irritability, and speech) while both treatments were associated with significant improvement compared to placebo, olanzapine treatment was accompanied by a significantly greater improvement compared to asenapine (p < 0.05 with no multiplicity adjustment). following treatment with asenapine or olanzapine a greater response rate (50% improvement in total ymrs score) was observed compared to placebo (42.5%, 52.4% and 29.4% respectively ; p < 0.05 for asenapine or olanzapine versus placebo). reduction in manic symptoms over 21 days was associated with a broad - based improvement across all symptom domains, with no subset of symptoms predominating. for antipsychotics with sedative properties, such as asenapine or olanzapine, it might be suspected that efficacy in manic or mixed episodes would result mainly from effects limited to sleep disturbances and/or aggressive behavior. however, the post hoc analysis presented here suggests that manic and mixed symptom improvement with asenapine or olanzapine is broader and goes beyond effects of sedation, with the complete spectrum of ymrs items showing clinical benefit. in the present analysis, a subset of ymrs items including disruptive aggressive behavior, content, irritability, elevated mood, sleep, and speech showed significant differences in favor of asenapine or olanzapine, compared with placebo, as early as the second day of treatment. with both asenapine and olanzapine, improvement seen in the disruptive limitations of these analyses include that, as a post - hoc analysis, the hypotheses were not predefined and that the results were not corrected for multiplicity. clinicians can therefore expect clinically relevant benefits early in the course of treatment, at least in a subset of patients. in this context, it is worthwhile to mention that early improvement has been reported to be associated with an increased odds ratio of response or remission at the end of treatment.6 | backgroundan exploratory post hoc analysis was conducted to evaluate the potential differential effects over time of asenapine and olanzapine compared with placebo on the eleven individual items comprising the young mania rating scale (ymrs) in patients with manic or mixed episodes in bipolar i disorder.methodsdata were pooled from two 3-week randomized, controlled trials in which the eleven individual items comprising the ymrs were measured over 21 days. an analysis of covariance model adjusted by baseline value was used to test for differences in changes from baseline in ymrs scores between groups.resultseach of the eleven individual ymrs item scores was significantly reduced compared with placebo at day 21. after 2 days of treatment, asenapine and olanzapine were superior to placebo for six of the ymrs items : disruptive / aggressive behavior, content, irritability, elevated mood, sleep, and speech.conclusionreduction in manic symptoms over 21 days was associated with a broad - based improvement across all symptom domains with no subset of symptoms predominating. |
heterotrophic plate count (hpc), which is an aerobic anaerobic bacteria test in water, can be used for detection of all bacteria, but can not be used in fecal contamination test (1). they have various types, such as gram - negatives : proteus, enterobacter, aeromonas, citrobacter, pseudomonas, klebsiella, flavo bacterium, sratya, moraxella, alcaligenese and acinetobacter, and gram - positives : bacillus and micrococcus (2). heterotrophic bacteria are not indicators of pathogenic conditions but some of them like pseudomonas is opportunists and can cause some infections in skin and lung and the other type like aeromonas cause gastroenteritis (24). today identification of heterotrophic bacteria is a very helpful method in the quality assessment of drinking water in storage tanks and in water distribution network. the concentrations of heterotrophic bacteria in the drinking water may vary from less than 1cfu / ml to over 10000 cfu / ml, which depends on water temperature, residual of chlorine and the amount of absorbable organic material. in the distribution system, the maximum permissible level of heterotrophic bacteria is 500 cfu / ml (5). changes in bacterial populations in the distribution network affect the aesthetic quality of water by changing the taste, odor and color by creating a sticky and slimy layer. such bacterial populations can cause corrosion, deposition and decrease of water discharge, health effects by setting people at risk of pathogenicity (6), and corruption in main products such as food, water, medicines and cosmetics (7). with high concentrations of heterotrophic bacteria in the water, in addition, heterotrophic bacteria counts over 1,000 cfu / ml in water samples can cause low sensitivity in tube tests and the membrane filter. organisms like pseudomonas and flavo bacterium can prevent the growth of total coliform and prevent the observed gas production in lactose fermentation and with dispersed growth on the membrane filter, and interfere with coliform on m - endo medium, and therefore coliform detection could be hindered. increase in heterotrophic bacteria could be a sign of trouble in treatment, repair, installation or influence of microbial growth in the distribution system and presence of biofilm (9), and one reasons which may increase the risk of gastroenteritis (10). in some cases of water distribution system with 0.6 mg / l of residual chlorine, more than 500 heterotrophic bacteria cfu / ml have been isolated. this indicates that some certain bacteria may survive in high concentrations of free residual chlorine (11). in the presence of more than 500 cfu / ml heterotrophic bacteria, coliforms are suppressed of 1 to 10 cfu / ml in the water distribution systems (12). this study investigated the relationship between heterotrophic bacteria and coliform, fecal coliforms and fecal streptococci bacteria and their possible interactions in drinking water system (13). microorganisms are included as heterotrophic bacteria, coliform, fecal coliforms and fecal streptococci bacteria. the surface method was applied to test the heterotrophic bacteria and mpn method was used for coliform, fecal coliform and fecal streptococci bacterial test. because of high incidence of diarrheal diseases, taking samples was conducted in summer and early autumn. overall, 324 samples were taken from rural areas and 32 samples from urban area in aq qala city, golestan province in 2013. in order to get more accuracy in network water quality, 178 samples were taken from the beginning location of water network systems and 178 samples from the end points. coliform, fecal coliform, fecal steptococci, and heterotrophic bacteria over 500 cfu / ml were under test. the tubes were incubated in 0.5 35 c for 2448 h and then, from each positive tube inoculate (by loop) was added to brilliant green and ec broth media. then, the tubes with brilliant green medium were incubated at 0.5 35 c for 2448 h and the tubes containing an ec medium incubated in serology water bath at 0.5 44 c for 24 h. both media with gas were marked as positive. in order to determine fecal streptococci, 9-tube method was used. the tubes incubated at 0.5 35 c for 2448 h. the tubes with turbidity were considered as positive and inoculated in pse agar. after that, the plates were incubated at 0.5 35 c for 24 h. the plates having brown and black colonies and brown halo were indicators of fecal streptococci. surface method was used for heterotrophic bacteria (14) and the entire surface of plate with nutrient medium was covered by 0.1 ml of water sample. then, the plates were placed at room temperature and were incubated at 0.5 35 c for 48 h. at last, all colonies were counted and multiplied by 10 to obtain cfu / ml. in order to determine the significance relationships between variables chi - square and wilcoxon test were used and p - values less than 0.05 were considered statistically significant. all the available data were analyzed by a computer program (spss 18) (chicago, il, usa). microorganisms are included as heterotrophic bacteria, coliform, fecal coliforms and fecal streptococci bacteria. the surface method was applied to test the heterotrophic bacteria and mpn method was used for coliform, fecal coliform and fecal streptococci bacterial test. because of high incidence of diarrheal diseases, taking samples was conducted in summer and early autumn. overall, 324 samples were taken from rural areas and 32 samples from urban area in aq qala city, golestan province in 2013. in order to get more accuracy in network water quality, 178 samples were taken from the beginning location of water network systems and 178 samples from the end points. coliform, fecal coliform, fecal steptococci, and heterotrophic bacteria over 500 cfu / ml were under test. the tubes were incubated in 0.5 35 c for 2448 h and then, from each positive tube inoculate (by loop) was added to brilliant green and ec broth media. then, the tubes with brilliant green medium were incubated at 0.5 35 c for 2448 h and the tubes containing an ec medium incubated in serology water bath at 0.5 44 c for 24 h. both media with gas were marked as positive. in order to determine fecal streptococci, 9-tube method was used. the tubes incubated at 0.5 35 c for 2448 h. the tubes with turbidity were considered as positive and inoculated in pse agar. after that, the plates were incubated at 0.5 35 c for 24 h. the plates having brown and black colonies and brown halo were indicators of fecal streptococci. surface method was used for heterotrophic bacteria (14) and the entire surface of plate with nutrient medium was covered by 0.1 ml of water sample. then, the plates were placed at room temperature and were incubated at 0.5 35 c for 48 h. at last, all colonies were counted and multiplied by 10 to obtain cfu / ml. in order to determine the significance relationships between variables chi - square and wilcoxon test were used and p - values less than 0.05 were considered statistically significant. all the available data were analyzed by a computer program (spss 18) (chicago, il, usa). table 1 shows biological characteristics of water supply network samples in aq qala rural and urban areas. in this table, the average number of total coliform, fecal coliform, fecal streptococci, and heterotrophic bacteria in the water distribution networks in rural areas were 9.6, 0.76, 5.57 cfu/100 ml and 1042 cfu / ml, respectively. for urban areas, these parameters were 4.56, 0, 0.125 cfu/100 ml and 688 cfu / ml, respectively. our finding demonstrated that with increase of heterotrophic bacteria in rural area, the other microorganisms were increased remarkably. biological characteristics of water supply network samples in aq qala rural and urban areas, golestan province in 2013 in table 2 the frequency of microorganisms in rural and urban areas of aq qala is shown. frequency of all contaminations of rural and urban areas of aq qala, golestan province in 2013 table 3 shows the biological characteristics of samples from the beginning and the end parts of water supply networks in aq qala rural and urban areas. total coliforms, fecal coliform, fecal streptococci, and heterotrophic bacteria in the end points samples were more than the samples in beginning part. table 4 shows all contaminations in samples with heterotrophic bacteria 500 and with > 500 cfu / ml. the results showed that nearly, in 114 samples from all samples, the heterotrophic bacteria counts were over 500 cfu / ml, which fecal coliform, coliform, and fecal streptococci were 8, 32, and 20 cfu/100 ml, respectively. however, in 242 samples, the heterotrophic bacteria counts were less than 500 cfu / ml, and the fecal coliform, coliform, and fecal streptococci rates were 7, 23, and 11 cfu/100 ml, respectively. in the samples with high concentration of heterotrophic bacteria, the other types of bacteria also observed in high concentration. biological characteristics of samples from the beginning and the end parts of water supply networks in aq qala rural and urban areas, golestan province in 2013 all contaminations in samples with heterotrophic bacteria 500 and < 500 cfu / ml table 5 represents the correlation between heterotrophic bacteria with other variables in water supply networks. there is significant correlation between coliform and fecal streptococci bacteria with heterotrophic bacteria and there is low correlation between fecal coliform and heterotrophic bacteria. drinking water systems are the known locations for biofilms growing area, even if the environment is oligotrophic and contain disinfectants. the bacterial growth in the network systems has been observed in the water without residual chlorine at 25 c, where the system has received many biodegradable materials (16). often coliform bacteria can be detached from biofilm and contaminate the water (17). biofilms contain 34.9% carbon, 5.4% hydrogen and 5.4% nitrogen (18) that protects these bacteria against antimicrobial agents (11). if biofilm grows on surface of ferrous pipes, it causes iron corrosion and releases iron particles in water (19). shortage of nutrients in the pipe water can cause migration of bacteria toward biofilm that contain organic and inorganic substances. therefore, biofilms are an ideal habitant for the survival and growth (20). biofilm density is between 10 and 1.9 10 bacteria /cm and contains many bacteria such as pseudomonas, klebsiella and enterobacter without any salmonella and shigella. pathogens that survived in the water treatment can grow and produce biofilm colonies that can be released into water flow. the larger water network systems have fewer pathogens while smaller water network systems have a great numbers of gram - negative pathogens (22). these results suggest that the heterotrophic bacteria in high concentrations in water do not have any effect on determining the fecal coliform and pathogens in the mpn method and do not suppress any coliforms, fecal coliforms and fecal streptococci in the aq qala city water supply network. we suppose it is due to high concentrations of organic carbon and nutrients in these systems. presence of coliforms in clean drinking water without any cracks in water treatment is considered as a major problem in the water industry. coliforms in the distribution system may be proliferate and multiply in presence of organic carbon. organic and inorganic compounds in biofilms may provide more opportunities for bacteria to grow and release them to the liquid phase (24). in the distribution system, k. pneumonia in the presence of very low concentrations of organic carbon (00.1 mg / l), mg / l in water allows growing of the e. coli bacteria. in presence of biofilm, growth of k. pneumonia coliforms are able to grow in low levels of organic matters in the water distribution system and water treatment facility at 4.8 to 21.9 c. etec (one type of fecal coliforms) which can grow in low temperature and nutrition condition (18). soluble carbon concentration between 10 to 50 g / l, induces a significant growth of bacteria in the water network system (25). even, concentration less than 3 g / l can uptakes microorganisms (26). environmental conditions such as temperature above 15c, neutral ph and assimilable organic carbon (aoc) are ideal condition for coliforms to colonize (9). regrowth of coliform bacteria is related to some factors : filtration rate, temperature, disinfectant type, assimilable organic carbon, corrosion control and operational characteristics. failure to control of corrosion and annual flushing program may lead to appearance of coliform bacteria in the network system. in water systems with free chlorine, increase in temperature from 5 c to over 20 c, coliforms increase 18 times (27). heterotrophic bacteria (p. maltophilia) in concentrations of 10 to 10 ml will cause decrease in growth of coliforms (0.5 log) (28). fecal coliform in the end point of water supply system partly increased than the beginning point. despite environmental contaminants, fecal coliform has a slight increase, which may be due to higher sensitivity to the residual chlorine. except enterotoxigenic e. coli, other e. coli species are not able to regrow in the water network. in some cases, this bacterium attaches to the biofilm and multiply in the water distribution system (29). e. coli can be colonized in the absence of free residual chlorine in drinking water distribution systems at 20 c. however, colonization of e. coli was temporary (30). fecal coliform is very sensitive to disinfectants and their presence may be considered as a major deficiency in the water supply network systems and indicating the recent fecal contamination (26). in the distribution system with 7 10 cfu / ml of heterotrophic bacteria, there is no interfere in the ac test (the presence and absence of coliforms) (31). unexpectedly, the number of fecal coliform isolated from the gac water supply network is less than in nano filter network due to protozoan activity of bacteriophage (32). when biofilm is exposed to monochloramine or hypochlorous acid, fecal coliform can survive for at least 10 days, even at high levels of disinfectants (33). however, general agreement exists that in common water temperature and nutrients, the number of fecal coliform or pathogenic bacteria do not increase (34). in the end points of water distribution network, fecal streptococcus observed more than fecal coliform due to the high resistance of these microorganisms to the environmental conditions and the free residual chlorine. some samples with fecal streptococci and without fecal coliforms, fecal streptococci survival is longer in winter than other seasons in the water distribution network (35) and can tolerate moderately alkaline ph conditions and high sodium chloride (36). the number of heterotrophic bacteria at the end points of water distribution network is greater than the beginning point. at present, eradication of biofilms in the distribution system is not possible, but it is necessary to control and minimize. quality control of inlet water, including taking care of distribution system may be a good way to prevent the risk of infection. in addition, to avoid any contamination in the water supply, it is recommended to observe permanently the mechanisms that facilitate entering the pathogens to the distribution network, such as poor maintenance activities, long time water retaining and the availability of nutrients (33). there was no interaction between heterotrophic bacteria with other microorganisms in the aq qala city water distribution network, except fecal coliform bacteria, which may occur because of the high sensitivity of fecal coliform. this interaction could not be confirmed due to fecal coliforms existence in water supply distribution network in the short time. we suppose that high concentrations of organic carbon in water provide the necessary nutrient for all microorganisms in these water network systems. the average number of coliform bacteria, fecal coliforms, fecal streptococci and heterotrophic bacteria in the end point of water network systems is more than the beginning part. thus, except the fecal coliforms at the end of the network with partial increasing, all of the microorganisms show significant increase, which may indicate a secondary growth of coliform and heterotrophic microorganisms in the biofilm. biofilm serve as a protective layer for the other microorganisms such as fecal streptococci and fecal coliforms. ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors. | background : this study investigated the interaction between heterotrophic bacteria and coliform, fecal coliforms, fecal streptococci bacteria in water supply networks.methods:this study was conducted during 2013 on water supply distribution network in aq qala city, golestan province, northern iran and standard methods were applied for microbiological analysis. the surface method was applied to test the heterotrophic bacteria and mpn method was used for coliform, fecal coliform and fecal streptococci bacteria measurements.results:in 114 samples, heterotrophic bacteria count were over 500 cfu / ml, which the amount of fecal coliform, coliform, and fecal streptococci were 8, 32, and 20 cfu/100 ml, respectively. however, in the other 242 samples, with heterotrophic bacteria count being less than 500 cfu / ml, the amount of fecal coliform, coliform, and fecal streptococci was 7, 23, and 11 cfu/100ml, respectively. the relationship between heterotrophic bacteria, coliforms and fecal streptococci was highly significant (p<0.05). we observed the concentration of coliforms, fecal streptococci bacteria being high, whenever the concentration of heterotrophic bacteria in the water network systems was high.conclusion:interaction between heterotrophic bacteria and coliform, fecal coliforms, fecal streptococci bacteria in the aq qala city water supply networks was not notable. it can be due to high concentrations of organic carbon, bio - films and nutrients, which are necessary for growth, and survival of all microorganisms. |
people enjoy sports and leisure activities, but using muscles for long periods of time causes muscle soreness. delayed onset muscle soreness (doms) is pain and stiffness felt in muscles several hours after resistance movement or unaccustomed, strenuous exercise. the soreness is felt most strongly 24 to 48 hours after the exercise1, 2 and is thought to be caused by eccentric (lengthening) exercise. doms is thought to be caused by neuromuscular dysfunction, such as shortened muscle length or limited range of motion (rom) in terms of the physiological response to muscle injury. it has been attributed to the increased tension force and muscle lengthening from eccentric contraction rather than from concentric contraction3. in doms, injury of the muscle and connective tissue affects rom and serum creatine kinase (ck) levels3, 4. the activity of muscle enzymes in blood is a useful indicator to evaluate the stress exerted with exercise. the enzyme ck is assayed in blood tests as a marker5. in the study of child and jacobs., exercise was performed by 14 healthy subjects who repeated 3 sets of the exercise 10 times at 80% of maximum strength for 7 days. it was reported that the level of ck was highest at 4872 hours after eccentric exercise6. many treatments have been used for doms. with regard to physical and kinematic approaches of stretching7, sonophoresis with ultrasound8 has been used for doms. low - intensity ultrasound has been reported to be effective for reducing pain and swelling and for increasing the range of movement in damaged skeletal muscles9, 10. to date, many studies have analyzed the clinical application of ultrasound to infiltrate various drugs through the skin11. in a recent study, mixed local anesthetic and anti - inflammatory agents were used to reduce inflammation and pain12. in oriental medicine, bee venom (bv) is used as a local anesthetic and anti - inflammatory agent. injecting bv into tissue bv has the unique pharmacological function of stimulating the immune system as a biochemical foreign body13. the main ingredients of bv are various enzymes, peptides, and low - molecular - weight organic substances (non - peptide substances). the main peptides in bv are melittin, apamin, and adolapin, and they have powerful anti - inflammatory and analgesic functions13. local analgesic and anti - inflammatory factors are activated in lesions by injecting bv, as the level of cortisol in blood is elevated and the biosynthesis of prostaglandin is inhibited14. doyle15 reported that bv had both anti - inflammatory and analgesic actions. in those respects, the present study was aimed at verifying the effectiveness of using ultrasound with bv for treatment of doms. the subjects in this study were adult women who were 21 to 34 years old and worked in j hospitals. they were randomized into those who were treated with ultrasound and bee venom (the bvus group ; n = 10) (msd age 27.422.57 years, height 159.713.25 cm, weight 48.422.99 kg) and those who were only treated with normal ultrasound (the us group ; n = 10) (msd age 28.853.62 years, height 160.716.10 cm, weight 50.715.79 kg). none of the subjects performed any heavy exercise for their upper extremities or were on any medication for 1 month prior to the study. they had no skeletal and neuromuscular dysfunction, no upper extremity pain or open wounds, and no history of neurosurgery or psychiatric disorders, and had a normal response in an allergic reaction test to bv. this study was approved by the ethics committee of the eulji university graduate school of health science. for bv allergy testing, 0.05 ml of diluted bv (a ratio of bv : saline=1:1000) was injected intradermally into the forearm. after verification that the tested lesion resulted in a wheal with a diameter of less than 10 mm and erythema with a diameter of less than 26.5 mm after 10 to 15 minutes, subjects were cleared to participate in this study16. first, the maximum load for each subject was investigated for the elbow flexor using 1-repetition isometric maximum exercise. subjects then performed eccentric resistance exercises with 70% of the 1-repetition isometric maximum (e.g., maximum load 5 kg 7/10 = 3.5 kg). the exercises were performed 7 times with 10 repetitions, and the subjects took a break time for one minute per exercise17. if the muscle fatigue does not occur after repeating 1 bout, the exercise was repeated until the subjects could not exercise due to muscle fatigue. the subjects were excluded from all exercise and treatment for 24 hours after eccentric exercise of the biceps brachii muscle. ultrasound was applied to the belly of the biceps brachii muscle at a frequency of 1 mhz, intensity of 1.0 w / cm, and speed of 2.5 cm / s for 10 minutes in each session. as a coupling medium, ultrasound gel and diluted bee venom (0.001%) mixed at a ratio of 9:1 was used for the experimental group, and pure ultrasound gel was used for the control group. ten milliliters of coupling medium was used in both groups. for this research into the impact of the treatment on doms, the vas, rom (elbow flexion and extension), and ck level were measured as the assessment parameters just before exercise and 24, 48, and 72 hours after exercise. the data were analyzed by repeated measures anova to compare time following exercise in each group and the independent t - test to compare differences between pre- and post - measurement values in both groups. the vas was significantly different after the experiment compared with before the experiment in both the us and bvus groups (p0.05). there was no significant difference in ck level using the independent t - test (p>0.05) between the 2 groups up to 72 hours (table 1). elbow flexion was significantly better after the experiment than before the experiment in both the us and bvus groups (p<0.05), and there was a significant difference between the 2 groups at 48 and 72 hours using the independent t - test (p<0.05) (table 1). elbow extension was significantly better after the experiment than before the experiment in both the us and bvus groups (p<0.05), and there was a significant difference between groups at 24 and 48 hours using the independent t - test (p<0.05) (table 1). us, ultrasound ; bvus, bee venom ultrasound ; vas, visual analogue scale ; ck, creatine kinase. induced eccentric contraction is used in research simulating doms, because eccentric contraction in muscle is related to muscle rupture18. the reduction of locomotion caused by muscle flexibility training can last up to 4 days, and it can reach 80% of the normal level of function19. craig.20 reported that ultrasound significantly improved elbow flexion but had no impact on pain threshold and pain index in a study comparing elbow flexion and extension, mechanical pain threshold, and the pain index. in this study, the reduction in pain (vas) was significant (p<0.05) at 72 hours after the exercise in the us and bvus groups. in addition, the reduction in pain was more significant in the bvus group than in the us group due to absorption of bv. melittin and apamin are thought to reinforce immunity with powerful anti - inflammatory and analgesic actions based on a mechanism of infiltration through intercellular lesions. in the range of measured movements, the bvus treatment showed significant improvement in not only elbow flexion but also elbow extension. in contrast to the study by craig.20, bvus improved articular movement and reduced pain. potteiger21 reported that the level of ck significantly increased 24 hours after exercise and was a marker of muscle damage. the level of serum ck was significantly elevated after maximum eccentric exercise of the biceps followed by a break for 24 h. this result suggested that doms may have been induced in the biceps, and the level of serum ck when sampled at 72 h was about 8,000 previous studies with ultrasound reported that ultrasound treatment with piroxicam gel was effective for recovery from muscle injury induced by eccentric exercise22. hoogland23 reported that sonophoresis with ultrasound at a frequency of 1 mhz could penetrate soft tissue to about 9 to 50 mm and that a frequency of 3 mhz could penetrate to about 2 to 16.5 mm. based on hooglands23 study, we concluded that ultrasound at a frequency of 1 mhz in this study was effective enough for soft tissue when used with the bv coupling medium. in particular, the application of ultrasound with the bv coupling medium was effective for pain reduction (measured using the vas) and recovery of flexion and extension impairment caused by doms. in addition, bvus did not lower the serum ck level, but the ck level was lower at 72 hours after the exercise in the bvus group than in the normal us group. for recovery from doms, bvus in conjunction with other physical and kinematic interventions is an effective procedure for pain relief and improving the mobility of muscle. | [purpose ] the purpose of this study was to evaluate whether ultrasound alone or ultrasound with bee venom is effective in treating delayed onset muscle soreness of the biceps brachii muscle, using the visual analogue scale, range of motion test (flexion and extension), and serum creatine kinase level. [subjects ] twenty women participated in this study. [methods ] repeated eccentric contractions were used to induce delayed onset muscle soreness in the elbow flexor of the subjects. the subjects were randomized to be treated with ultrasound alone or ultrasound with bee venom. we evaluated the effects of treatments in the 2 groups. individual subjects were assessed using the visual analogue scale, range of motion test, and serum creatine kinase level. the assessment parameters were evaluated 4 times : before exercise and 24, 48, and 72 hours after exercise. [results ] the visual analogue scale scores were significantly different before and after the experiment in both the group treated with ultrasound and the group treated with ultrasound and bee venom. the difference in elbow flexion and extension before and after the experiment was significantly different in both groups. no significant difference was found in the serum creatine kinase levels before and after the experiment. [conclusion ] treatment with ultrasound and bee venom is effective for managing delayed onset muscle soreness. |
clinical teaching is a critical and indispensable section in medical education (1) that results in the evolution of knowledge, attitude and skills (2) ; i.e., the clinical competency of the students (3). clinical teaching consists of various fields, one of which is clinical rounds (4). bedside teaching is a patient - based teaching method in medical education that facilitates acquiring real knowledge, practical skills and professional attitudes (5). this field of learning includes every situation where learning takes place in the presence of the patient, regardless of the environment in which such education is presented (6). bedside teaching engages the training physicians with interaction with the patient beside their bed to extract a patient s records, represent the key features of clinical examination and discuss the best approach to diagnosing the disease and treating the patient (7). bedside teaching provides active learning in real situations, observing students skills, increasing their motivation, professional thinking, clinical integration, communicative skills, problem - solving skills, decision - making and moral skills along with improving the understanding of patients (1, 8). in addition, it transfers the human aspects of patient care to the medical students (5, 9) and helps the physician to view the patient as a real person and not merely a summary of the disease (4). this is why these qualities can not be developed effectively in classrooms (1, 10). in clinical medicine, 56% of the patients problems could be well recognized after a complete history taking, and this increases to 72% after physical examination. in some cases, in other words, performing a comprehensive physical examination could assist the individual physician to reach a diagnosis faster (1, 8, 11, and 12). previous studies indicate that real clinical teaching emphasizing the history and physical diagnosis over the last 17 years has decreased from 75 to 16%, and it is even less today (4, 10, and 13). in addition, clinical teaching has withdrawn from the bedside to hospital corridors, nursing stations and finally to the conference hall (5, 11, and 14). claim that only 48% of the trainees have stated that they have had bedside teaching during their study. these individuals are 100% sure that bedside learning is the most effective method for learning clinical skills (15). since the aim of clinical teaching is to provide opportunities for students to link theoretical information with practical realities, promoting its quality could result in training qualified students in different clinical domains (16, 17). unfortunately, no experimental evidence indicates that bedside teaching is the most effective strategy (6). since no official evaluation has been conducted on this important educational field in medical universities, no reliable information shows that bedside teaching objectives, including the increase of concept understanding and personal skills, have been achieved through this strategy. this study was planned and conducted with the aim of determining the quality of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad. the present study has been conducted with the general objective of determining bedside teaching quality in internal wards of qaem and imam reza educational hospitals in mashhad in 2014. the specific objectives of this study include : determining the quality of patient comfort during bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014determining the quality of targeted teaching in bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014determining the quality of group dynamics of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of patient comfort during bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of targeted teaching in bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of group dynamics of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 clinical teaching is a critical and indispensable section in medical education (1) that results in the evolution of knowledge, attitude and skills (2) ; i.e., the clinical competency of the students (3). clinical teaching consists of various fields, one of which is clinical rounds (4). bedside teaching is a patient - based teaching method in medical education that facilitates acquiring real knowledge, practical skills and professional attitudes (5). this field of learning includes every situation where learning takes place in the presence of the patient, regardless of the environment in which such education is presented (6). bedside teaching engages the training physicians with interaction with the patient beside their bed to extract a patient s records, represent the key features of clinical examination and discuss the best approach to diagnosing the disease and treating the patient (7). bedside teaching provides active learning in real situations, observing students skills, increasing their motivation, professional thinking, clinical integration, communicative skills, problem - solving skills, decision - making and moral skills along with improving the understanding of patients (1, 8). in addition, it transfers the human aspects of patient care to the medical students (5, 9) and helps the physician to view the patient as a real person and not merely a summary of the disease (4). this is why these qualities can not be developed effectively in classrooms (1, 10). in clinical medicine, 56% of the patients problems could be well recognized after a complete history taking, and this increases to 72% after physical examination. in some cases, in other words, performing a comprehensive physical examination could assist the individual physician to reach a diagnosis faster (1, 8, 11, and 12). previous studies indicate that real clinical teaching emphasizing the history and physical diagnosis over the last 17 years has decreased from 75 to 16%, and it is even less today (4, 10, and 13). in addition, clinical teaching has withdrawn from the bedside to hospital corridors, nursing stations and finally to the conference hall (5, 11, and 14). claim that only 48% of the trainees have stated that they have had bedside teaching during their study. these individuals are 100% sure that bedside learning is the most effective method for learning clinical skills (15). since the aim of clinical teaching is to provide opportunities for students to link theoretical information with practical realities, promoting its quality could result in training qualified students in different clinical domains (16, 17). unfortunately, no experimental evidence indicates that bedside teaching is the most effective strategy (6). since no official evaluation has been conducted on this important educational field in medical universities, no reliable information shows that bedside teaching objectives, including the increase of concept understanding and personal skills, have been achieved through this strategy. this study was planned and conducted with the aim of determining the quality of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad. the present study has been conducted with the general objective of determining bedside teaching quality in internal wards of qaem and imam reza educational hospitals in mashhad in 2014. the specific objectives of this study include : determining the quality of patient comfort during bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014determining the quality of targeted teaching in bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014determining the quality of group dynamics of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of patient comfort during bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of targeted teaching in bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 determining the quality of group dynamics of bedside teaching in the internal wards of qaem and imam reza educational hospitals in mashhad in 2014 the method employed in this research was a qualitative study to design tools and apply them to educational clinical rounds held in the internal wards of qaem and imam reza educational hospitals in mashhad in october 2014. these two hospitals were selected since they are currently regarded as general hospitals and among the largest centers of medical education and health in northeastern iran. training of general practitioners as well as specialty and subspecialty levels is mostly done in these two hospitals. sample size was calculated as at least 96 rounds using the cochran formula ; in total we investigated 131 educational rounds in both hospitals. 59 and 54 educational rounds were held in imam reza and qaem hospitals respectively. to supply the calculated sample size, then they were selected randomly and evaluated proportionally to the rank of the holding members of the faculty. to collect data, to do so, persian and english keywords of medical education, quality of education, clinical teaching, challenges and strategies in bedside teaching, along with new models in bedside and patient - based teaching. the above - mentioned keywords, in combination and separately, were then used as search terms in pubmed, google, scientific information database (sid), eric, web of knowledge, medline, science direct, scopus, magiran, google scholar, proquest, elsevier, and the iranian journal of medical education without considering the time limit. in total, the search yielded 138 articles published from 2003 to 2014. the author investigated the obtained texts based on the titles and abstracts. in this step, then the sections related to quality based on variables mentioned in these studies were collected. in the next step, the resulting items were investigated and discussed in numerous meetings with experts and professors, and the overlapping cases were cancelled or merged together. finally, the contents were classified into three domains : patient comfort, targeted - teaching and group dynamics, in accordance with the three - domain model of janicik and fletcher. the resulting checklist was presented to a number of medical education and clinical medicine specialists for content validity. the reliability of the checklist was verified using cronbach s alpha coefficient (0.74). the first part of the author s checklist consisted of demographic information including the name of the hospital, type of internal ward and the rank of the faculty member conducting the educational rounds. the second part of the checklist included 30 questions involving the factors of effective bedside teaching, including the domains of patient comfort (8 questions), targeted teaching (14 questions) and group dynamics (8 questions) (table 1). the students were asked to score the checklist questions from 0 to 10 (0= not done, 10= done perfectly). first, the researcher obtained permission from the department of higher education and the vice - chancellor for medical school. the subject of the study was presented to the heads of the internal wards of the mentioned hospitals, and permission was then obtained from the hospitals. in the next step, a list of the medical students in the clinical teaching course was provided ; those who were passing the internal ward course and the professors rounds were selected randomly. then the content of the checklist was given to the students to familiarize them with its content and data collection method. finally, the students filled out the checklists after attending the selected educational rounds in internal wards, maintaining the confidentiality of their answers. this study was conducted under the license of the research ethics committee of mashhad university of medical sciences. participation in this study was voluntary, and the names of the members of the faculty who held educational rounds were not entered on the data collection forms to maintain privacy and confidentiality of information. data description was conducted using descriptive statistical indices as frequency and mean along with standard deviation. to ensure the normal distribution of data, the kolmogorov - smirnov test (k s test or ks test) was first conducted. then, independent t - test, one - way anova and variance analysis were used, in which (p0.05). the average quality score of patient comfort in the internal wards of imam reza and qaem hospitals was 26.5 (15.4) and 31.1 (20.4) out of 80, respectively (table 3). the average score related to patient comfort in both groups was compared using independent t - test, and the difference was not significant (p>0.05). the average quality score of targeted teaching in bedside teaching in the internal wards of imam reza and qaem hospitals was 101.3 (25.7) and 110.7 (33.3) out of 140, respectively (table 3). the average score of targeted teaching in bedside teaching in both groups was compared using independent t - test, and the difference was not significant (p>0.05). the average quality score of group dynamics in bedside teaching in internal ward of imam reza and qaem hospitals was 35.7 (15.1) and 56.0 (21.6) out of 80 (table 3). the average score of group dynamics was compared in both groups using independent t - test, and the difference was significant (p0.05). however, it was significant in terms of group dynamics (p0.05). one - way anova was employed to compare the quality of bedside teaching in terms of the rank of faculty members. no significant difference was observed in terms of patient comfort and targeted teaching (p=0.235 and p=0.121, respectively). however, the quality was significantly different in terms of group dynamics considering the rank of faculty members (p=0.027). in sum, bedside teaching quality was not significantly different in the two hospitals in terms of the rank of faculty members (p=0.129). the quality of bedside teaching was significantly different only in terms of group dynamics among assistant to associate ranks of faculty members (p=0.049) in such a way that the average score obtained by assistant professors was higher for 10.7. in general, the results of the study showed that the quality of bedside teaching in internal wards of qaem and imam reza educational hospitals is not acceptable according to the indicators of patient comfort, targeted teaching and group dynamics. in the study by ziaee, the percentage of students consent from clinical teaching was 38.8%, which conformed to the results of this study. in the previous study, only a few items including the educational objectives being specified, the number of participating students and having a course plan were investigated (18). the results of the present study conform to the results of khorasani study in 2007, which had reported students attitudes toward the current situation of clinical teaching as negative. however, in that study, only limited aspects of clinical teaching were addressed, including the educational objectives being specified, the possibility of independent visits by the trainee, the possibility of prescribing medicine independently and criticizing and correcting the trainee s history (19). on the other hand, another study conducted in the iran university of medical sciences in 2004 showed that the quality of clinical teaching was regarded as relatively favorable, according to medical students. in that study, domains of scientific mastery of the professor, educational management and the quality of communication and consulting were considered. that study did not consider patient comfort as an independent item, and it conformed to our study only in the domains of targeted teaching and group dynamics (20). in his study in 2012, bagheriyan represented that the situation of clinical teaching is regarded as desirable, according to the students of anesthesiology and operating room in tabriz. in this study, the trainer, clarity of educational objectives, and the educational environment were shown to have the highest effect in the quality of clinical teaching (21). in the study by mardani in 2010, the situation of clinical teaching was reported from nursing students perspective. in this study, the evaluation method and proper manners of the trainer were regarded as strong points, and the lack of coordination of clinical teachings with theory was regarded as the weak point (22). it appears that the current study has included aspects that play more important roles in clinical teaching. possibly the strong point of the present study in comparison to others is its special attention to patient preferences as one of the factors affecting quality. in a study by lubetkin at cornell university, the quality of clinical teaching was regarded as desirable according to the professors and students, although the professors were more satisfied than the student (23). the results of that study did not conform to the results of our study. in the above - mentioned studies, items including educational equipment, mastery of professors of the subjects and the physical environment were considered. the results of the present study are devastating in terms of the quality related to patient comfort in bedside teaching, and these conform to the results of the study by dehghani. this category has been emphasized in numerous studies, and it is regarded as a high - quality component of clinical teaching (6, 2527). a study conducted in isfahan in 2006 showed that talking about mental and social issues satisfies only 40% of the patients, and nearly 60% of the patients were satisfied in terms of having emotional connection with their physicians. the patient s participation in the treatment process was not satisfying for 50% of the patients in terms of having treatment methods clearly explained, being included in understandable discussions, and feeling that their feedback was valued in terms of performing or not performing a treatment. 40% of the patients felt unsafe about scattered talks during clinical teaching and stated that they felt themselves to be regarded as educational tools (4). in lehmann s study of educational hospitals of the usa, it appears that not allowing the patient to discuss their emotional issues and social conditions, lack of participation and getting feedback from them in discussions, along with medical decisions and low emotional relations of the physician with the patient, have resulted in ignoring human aspects. however, in related studies, indicators such as good relations with the patient, patients participation in decisions, considering patients concerns, accelerating the solution of patients problems and answering their questions are considered to be measures of humanitarian behavior of the physician (13, 29). in 2012, a study by adibi in isfahan revealed that repeated examinations and visits, along with crowded and long clinical rounds in which information is presented to the patient in an unclear way, have resulted in patient dissatisfaction. in effect, patients feel that they are employed as educational tools (13). factors such as a high number of questions and examining individuals in clinical rounds and the main physician not being recognized, scattered and contradictory discussions in the patient s presence, along with the lack of presenting sufficient and understandable explanations of the disease and treatment measures, results in increasing the patient s sense of insecurity. in addition, it results in greater uncertainty about being treated by an individual who is not the main physician. the necessity of introducing the people present in clinical rounds and identifying the person in charge, along with explaining treatments and procedures clearly, are given serious emphasis (4, 30). these results conformed to the results of the present study. in our study, the following cases in the domain of patient comfort have been considered : coordination with the patient, stating educational objectives, introducing people, considering the patients concerns, minimizing possible patient misunderstandings at the end of teaching and the importance of patients preferences. in the section related to the quality of group dynamics in clinical teaching, the results of our study showed that this quality has been lower than average in the internal ward of imam reza hospital, and it was average in that of qaem hospital. the results of our study were similar to the results of bazzazi s study (31). it appears that to achieve desirable conditions, new ways of increasing students participation must be employed. in this regard, a more accurate evaluation of the current situation appears essential along with discovering weak and strong points. possibly one of the obstacles to achieving this goal is providing students an opportunity to judge professors capabilities. in their discussion of learning environments, kroenke and omori (2010) stated that during educational rounds, physicians are maybe anxious about their abilities in knowledge transmission. they claim that this fear may be more prevalent in young lecturers who do not have much clinical teaching experience. therefore, having friendly relations with students at the beginning of teaching would result in a more positive learning environment (10). in this regard, ramani in his 2013 study states that the professor should challenge the students minds without humiliating them, and he suggests that the professor should involve all learners in teaching process. this could be achieved by assuring that all students have an opportunity to answer questions (32). recommended that before visiting the first patient, it is preferable to set a time for discussion with students. by introducing all the team members from the professors to the residents and the medical students, they will have the opportunity to become familiar with each other. the professor should state his objectives and expectations for the students at the beginning as well (27). in our study, in the domain of quality related to the group dynamics for which an average score was obtained for both hospitals, the following issues were considered : team cooperation, freedom in presenting comments, creating an intimate relationship at the beginning of teaching, giving responsibility to students and creating a safe atmosphere for free discussion. in the section comparing clinical teaching quality in the internal wards of imam reza and qaem hospitals, considering the indicators related to patient comfort, targeted teaching and group dynamics, the results of our study showed that generally the quality of clinical teaching in both hospitals does not differ significantly. however, the score related to the quality of group dynamics in qaem hospital was better compared to that of imam reza hospital. the quality of clinical teaching according to the rank of the member of the faculty was not different for both hospitals. more dynamics were observed in teachers with the rank of professor compared to associate professors. to answer the present ambiguities in this regard, more research is required. in this study, we investigated the quality according to the three - domain model of janicik and fletcher, which includes patient comfort, targeted teaching and group dynamics. other studies conducted from the perspectives of nursing students and clinical professors have investigated the factors affecting clinical teaching from different points of view (22, 33, and 34). another study in gonabad revealed that the most effective factor in the quality of clinical teaching, according to both students and lecturers, has been the performance of instructors. collaborative working environment, educational equipment in clinical environment, amenities in the clinical setting and the ward setting being proportional to the number of students are domains considered in the mentioned study (33). in that study, collaborative working environment is regarded as the weakest factor. this finding conforms to the results of our study in terms of low collaborative working environment as one of the components of group dynamics. a 2010 study by mardani in ahwaz revealed that the interns and trainees views of clinical teaching is average and better than average. in this study, the strongest point of clinical teaching students awareness from clinical evaluation method at the beginning of practical course and proper behavioral treatment by the clinical instructor. in addition, the weakest points were related to the lack of cooperation of the ward with students and the lack of harmony between theoretical learning and clinical activities (22). in summary, the results of this study showed that the quality of clinical teaching in the internal wards of qaem and imam reza educational hospitals is weak in the domains related to patient comfort and group dynamics. improving and promoting the quality of bedside teaching requires continuous assessment of the current situation, recognizing strong points and correcting weak points, a process in which the comments and ideas of both professors and students could pave the way for later plans. it is suggested that mashhad university of medical sciences take some steps to improve the quality of clinical teaching, especially in domains related to the patient s comfort and trainee s participation through holding educational workshops and presenting new teaching methods, along with investigating possible problems. | background : bedside teaching is a patient - based teaching method in medical education. the present study has been conducted with the aim of investigating the quality of bedside teaching in the internal wards of qaem and imam reza educational hospitals.methods:this study follows a mixed qualitative - quantitative approach using checklists on educational clinical rounds in imam reza and qaem hospitals in mashhad. in the first stage consisting of qualitative study, the parts related to the quality of bedside teaching were recognized and a checklist was designed in three domains of patient comfort (8 questions), targeted teaching (14 questions) and group dynamics (8 questions), and its reliability and validity were verified. in the next step, data were collected and then analyzed using spss 16 software through statistical techniques of independent t - test, one - way anova and variance analysis.results:in total, 113 educational rounds were investigated in this study. among them, 59 (52.2%) and 54 (47.8%) educational rounds have been investigated in imam reza and qaem hospitals, respectively. the average total score of bedside teaching was 180.8 out of 300 in the internal wards of both imam reza and qaem hospitals.conclusion:the results of this study showed that generally the quality of bedside teaching in imam reza and qaem hospitals of mashhad is low according to the qualitative standards considered in this study. holding educational workshops along with more familiarity of the professors with effective bedside teaching strategies could be effective in improving the quality of educational rounds. |
colon cancer is the most common form of cancer worldwide and the second leading cause of cancer - related death in the western world. survival is related to stage, and the 5-year overall survival rate is approximately 60%. metachronous secondary tumors affect 2030% of colon cancer patients and are usually detected within 2 years after the resection of the primary tumor. however, there is still no consensus concerning the therapy and the duration of follow - up of patients who have had curative colonic resection. recently, peritonectomy combined with intraperitoneal chemotherapy was reported to improve the long - term survival of peritoneal carcinomatosis [4, 5, 6 ], but the role of intraperitoneal chemotherapy in addition to adjuvant chemotherapy and tumor resection remains undefined in patients with intra - abdominal recurrence of colon cancer. we herein report a case of ascending colon cancer with intraperitoneal recurrence 10 years after the resection of the primary lesion. treatment consisted of three resections against the intra - abdominal metastatic lesion and systemic and localized (intraperitoneal) chemotherapy, which resulted in no evidence of a fourth recurrence 12 months after the third recurrence. a 56-year - old japanese woman who had a history of ascending colon cancer underwent radical right hemicolectomy with d3 lymph node dissection at 43 years of age (fig. the pathological findings were pss (moderately differentiated, ly1, v1, infb) pn1, sh0, sp0, fstage iiia, cur a, and the case was classified as t3n1m0 (stage iiia) according to the tnm classification. after the surgery, the patient developed the symptoms of ileus two or three times within a year ; therefore, a synechotomy was performed at 44 years of age. however, at 53 years of age, 10 years after the primary tumor resection, the patient was admitted to the emergency room because of abdominal pain, and although the pain disappeared without special therapy, a 4-cm tumor in the lower left abdominal cavity was detected by computed tomography (ct) (fig. 2a), and tumor uptake on a positron emission tomography (pet) scan revealed a maximum standardized uptake value of 9.0 (fig. thus, the diagnosis was a recurrence of ascending colon cancer resected 10 years ago, and a recurrent tumor resection with partial small intestinal resection was performed. there were no additional tumors (either primary or recurrent lesions), and the intraoperative cytology of the peritoneal washing showed no evidence of malignancy. 3a) with infiltration into the fat tissue of the omentum and within the wall of the small intestine. immunohistochemically, adenocarcinoma cells were negative for cytokeratin (ck) 7 (clone ov - tl 12/30, dako, carpinteria, calif., usa) and strongly positive for ck 20 (clone ks20.8, dako), which is suggestive of colon origin (fig. 3b and c). characteristically, immunohistochemical expression of cd44 (clone f10 - 44 - 2, dako), a marker suggestive of colorectal cancer stem cells, labeled a few normal intestinal epithelial cells localized at the bottom of the crypt, which were assumed to be stem cells, and had a scattered distribution in cancer cell membranes (fig. 3d). vascular endothelial growth factor (vegf, clone a-20, santa cruz biotech, calif., usa) was expressed in endocrine cells in the crypt and diffusely expressed in cancer cells (fig. 3e). angiogenesis and lymphangiogenesis were detected by cd34 (clone nu-4a1, nichirei, tokyo, japan) and d2 - 40 (clone d2 - 40, dako), respectively (fig. 3f and g). after surgery, the patient received adjuvant chemotherapy consisting of tegafur uracil (300 mg / m) and calcium folinate (75 mg). ten months after the first recurrence, ct and pet revealed liver tumors (fig. 2c, d, e and f) ; therefore, a left lateral segmentectomy and s8 partial hepatectomy without any further adjuvant chemotherapy were performed. microscopic examination of the liver tumors showed moderately differentiated adenocarcinoma compatible with the first recurrence. then, 8 months after the second recurrence, ct and pet revealed a 3-cm tumor on the end of the ileum (fig. prior to surgery for this third recurrence, we treated the patient with 5 cycles of combination chemotherapy consisting of folinic acid, fluorouracil and irinotecan. tumor resection for the third recurrence including ileocolonic anastomosis, total hysterectomy, and bilateral salpingo - oophorectomy was performed. intraoperative chemotherapy with oxaliplatin (60 mg / m) and fluorouracil (300 mg / m) and intravenous injection of levofolinate (180 mg / m) was performed. microscopic examination of the tumor showed moderately differentiated adenocarcinoma compatible with the first and second recurrence. after surgery, the patient received adjuvant chemotherapy consisting of 5 cycles of combination chemotherapy with folinic acid, fluorouracil and oxaliplatin. the patient is well with no evidence of recurrence 12 months after the third recurrence. there is no consensus concerning the long - term follow - up of patients who have had colon cancer surgery. follow - up data suggest that the average disease - free survival time between primary tumor resection and recurrence is one and a half years. metachronous secondary tumors affect 2030% of patients and are usually detected during postoperative follow - up, with a higher incidence during the first 2 years. the present patient was disease - free for 10 years after the primary colon cancer surgery with no evidence of recurrence. there are a few reports of the development of metastic lesions a remarkably long time after the primary colon cancer surgery. thus, patient follow - up for more than 10 years after primary colon cancer surgery is desired. it is possible that micrometastases of the primary tumor can remain in a dormant state for a long period. the dormant micrometastases may escape dormancy by increasing their level of angiogenic activity through the disappearance of circulating angiogenesis inhibitors and/or a switch to the angiogenic phenotype by a subset of cells within the micrometastases. in our case, the cancer cells stained positive for vegf, and angiogenesis (as determined by cd34 staining) and lymphangiogenesis (as determined by d2 - 40 staining) occurred adjacent to vegf - positive tumor cell nests at the recurrent site. these results suggest that sufficient levels of nutrient supplementation may have increased cancer cell proliferation such that they escaped from the dormant state. vegf expressed in endocrine cells located in normal intestinal crypt but not in normal absorptive epithelial cells can be an internal control. high vegf expression in colon cancer cells is related to low overall survival and a short disease - free survival period. as in the present case, recently, it has become clear that tumors and hierarchically organized heterogeneous cells contain a cancer stem cell component. the cancer stem cells share many similarities with normal stem cells, such as self - renewing capacity and multilineage differentiation properties. cd44 is a transmembrane glycoprotein that participates in growth, survival, differentiation, and motility. cd44 is a robust marker and is of functional importance for colon cancer cells for cancer initiation. in our case, the scattered appearance of cd44-positive cells may suggest continuous tumor growth, metastasis, and cancer recurrence. at present, there is no published data that outlines the impact of the new systemic therapy regimens when given to patients with intra - abdominal recurrence of colon cancer. cytoreductive surgery treating macroscopically detectable tumor and intraperitoneal chemotherapy early after surgery to treat residual tumor measuring less than 1 mm has been proposed. this combined modality has already proved its superiority over standard treatment in patients with colon cancer in a randomized study the first and most commonly used agent for intraperitoneal chemotherapy in colon cancer is mitomycin c or fluorouracil [17, 18, 19 ]. however, mitomycin c is not an efficient treatment for metastatic colorectal cancer, and it seems logical to use intraperitoneal oxaliplatin in combination with systemic leucovorin. [17, 20 ] used oxaliplatin for intraperitoneal chemotherapy and reported survival rates of 48.554% at 5 years in patients with carcinomatosis of colorectal origin. in our case, we performed three surgical cytoreductions and administered intraperitoneal fluorouracil and oxaliplatin and systemic levofolinate. the patient is well with no evidence of recurrence more than 12 months after the third recurrence. our case suggests that colon cancer can metastasize many years after the initial resection and that aggressive local as well as systemic chemotherapy may be required for colon cancer management. | a 56-year - old japanese woman who underwent a curative resection of ascending colon cancer at 43 years of age was found to have a tumor in her lower left abdominal cavity by computed tomography at 53 years of age. the tumor in the omentum was resected and identified as an adenocarcinoma compatible with metastasis from the primary ascending colon cancer. although the patient received adjuvant chemotherapy with tegafur uracil and calcium folinate, liver metastasis was detected 9 months after the first recurrence. a segmentectomy and hepatectomy was performed, and histopathological findings indicated metastasis from the primary colon cancer. a third recurrence was detected in the right abdominal cavity 7 months after the second surgery. the patient received 5 cycles of combination chemotherapy consisting of folinic acid, fluorouracil and irinotecan before the third operation. the metastatic tumor resection together with intraperitoneal chemotherapy was performed, and histopathological findings indicated metastasis from the primary colon cancer. after the third surgery, the patient received adjuvant chemotherapy consisting of 5 cycles of folinic acid, fluorouracil and oxaliplatin. the patient is well with no evidence of recurrence 12 months after the third recurrence. this case suggests that colon cancer can be dormant for over 10 years and that long - term follow - up is required after curative resection. aggressive local as well as systemic chemotherapy may be required for the management of colon cancer recurrence. |
patient safety has drawn the attention of healthcare organizations and the public, especially after the 1999 institute of medicine report to err is human : building a safer health system, which estimated that as many as 98,000 deaths annually are due to medical errors in the united states.1 since that report, patient safety has come to the forefront of public health problems. the world health organization has estimated that millions of patients worldwide suffer disabling injuries or death every year due to unsafe medical care ; therefore, the world alliance for patient safety was launched in late 2004.2 recently, patient safety concerns have caught the attention of medical educators in postgraduate training and medical practice improvement programs.3 education, an essential tool for promoting safety in healthcare, mitigates the prevailing name - and - blame culture in healthcare organizations and reinforces teamwork. furthermore, education promotes the use of errors as learning opportunities.4 improvements in safety and quality in healthcare require the following changes : the delivery of patient - centered care ; effective communication with patients and their relatives ; the demonstration of an awareness of medical error occurrence, prevention, management and disclosure ; safety while working in interdisciplinary teams ; the upholding of medical ethics ; the use of evidence - based practices ; and finally, the demonstration of a familiarity with information technology and quality improvement strategies.7 throughout their education, future generations of physicians must be adequately taught, sufficiently trained, and satisfactorily assessed in this new dimension of patient care4. currently, most medical students receive insufficient training in these areas ; as a result, they are not armed with the skills that are required to fulfill their responsibilities.4,6,8, a growing body of evidence suggests that education on patient safety must begin during undergraduate medical education, preferably during clinical rotations or clerkships, when students are in direct contact with patients.6,13,14 a recent study of the experiences of trainees with patient safety showed that most students were involved with medical errors during clinical rotations. despite this exposure, the majority considered themselves insufficiently trained in safe practices, medical error recognition and disclosure at graduation.12 additionally, the students ' exposure to medical errors and adverse events negatively affected their attitudes and competencies.12 patient safety educational programs are generally offered during postgraduate training and medical practice improvement activities. the general educational strategy includes formal lectures and open discussions, which are enhanced by multifaceted training approaches.6 - 8,10, unfortunately, this approach is seldom incorporated into undergraduate medical education.13 - 17 moreover, few studies have focused exclusively on the patient safety programs that are provided during medical school clerkships.12, in addition to the adequate inclusion of patient safety concerns in medical school curricula, valid and reliable assessments of students ' learning outcomes must also be incorporated.3,4,6,7,16,17 the objective structured clinical examination,21 which uses standardized patients, is a powerful tool for the evaluation of patient safety competencies. there are several positive aspects of this tool, including professional development in a low - risk environment and the incorporation of skills and attitudes for handling medical error recognition and disclosure, patient - centered care, communication, and interpersonal skills and humanism issues.4,6,7,10,22 in addition, the objective structured clinical examination provides valuable feedback to both examinees and educators4,6,7,23 and offers reinforcement that is critical for promoting lasting changes in physician behavior.4,6,7,23 despite the above considerations, the objective structured clinical examination is seldom incorporated as an educational strategy to assess the development of skills and attitudes related to patient safety. the few relevant studies published on this topic have described excellent results from the use of patient safety - based objective structured clinical examinations during postgraduate training and medical practice.7,8,22, to our knowledge, no previous study has analyzed the use of the objective structured clinical examination to assess patient safety competencies in undergraduate medical training, particularly during the clerkship experience. the objective of the present study was to analyze fifth - year medical student performance in patient safety using an objective structured clinical examination scenario with standardized patients following the introduction of a one - time new patient safety curriculum. specifically, the assessment focused on medical error recognition and disclosure, the patient - physician relationship and humanism. this study was conducted at the so paulo university school of medicine, which admits 180 students annually to a six - year program in undergraduate medical education ranging from primary to tertiary care. the clerkship experience occurs during the final two years of undergraduate education and involves supervised, hands - on training in two university hospitals of increasing complexity : a 258-bed secondary hospital and a 1,200-bed tertiary hospital. students are divided into small groups and rotate among five main areas : pediatrics, internal medicine, obstetrics and gynecology, surgery, and preventive medicine. the internal medicine clerkship also corresponds to a 12-week program for fifth - year students, during which small groups of students participate in supervised clinical activities in two internal medicine wards and ambulatory clinics. this program includes simulations of medical scenarios that focus on effective interpersonal communication, invasive procedures, and resuscitation. student performance is evaluated using the following three complementary tools : a supervisor 's overall rating, written exams, and an objective structured clinical examination using a standardized patient. since the objective structured clinical examination was first introduced at our institution in 2002, this tool has been incorporated in almost all clerkship rotations. the present study enrolled 95 fifth - year medical students who were rotating through the internal medicine clerkship from july to december of 2007. this study was exempt from the need for review by the university of so paulo school of medicine institutional review board, but informed consent was obtained from every student. the patient safety educational program was first introduced into the clerkship training at our institution in 2007 and developed over time into two lectures per rotation. the program focuses on the following factors : medical error definition, epidemiology, and disclosure ; near misses ; adverse events ; patient - centered care ; the patient - physician relationship ; and ethics. this program has adopted the institute of medicine 's definitions of adverse events, near misses and medical errors. after attending lectures on these topics, students participate in two additional activities. they were invited to discuss real cases that they had witnessed or in which they had participated. after this discussion the anonymity of the patients and the health professionals involved in these situations was always maintained., students were evaluated using an objective structured clinical examination with five 10-minute stations that involved standardized patients. one station focused on patient safety and included three domains : medical error, patient - physician relationship attitudes, and humanistic behaviors. the other four stations included the following assessments : 1) a procedure station for the assessment of venipuncture and paracentesis skills ; 2) an orientation station to evaluate the student 's ability to teach the use of medication (e.g., insulin injection) ; 3) a clinical station that focused on physical examination skills ; and 4) a health promotion station at which students were asked to perform a health promotion consultation on a healthy patient. all the standardized patients received training in both the scenario that corresponded to their role as a patient and the detailed checklist that was used to assess the students. subsequently, the scenarios were pilot - tested by the researchers until the standardized patients accurately portrayed the scenarios, reliably scored the previously described domains and adequately completed the checklist. the patient safety checklist was constructed based on a literature review by a group of medical educators and patient safety researchers. it was pilot - tested for use in objective structured clinical examinations with a group of 45 fifth - year medical students (june 2007). the revised checklist contained 21 items grouped into three domains (medical error, patient - physician relationship, and humanistic behavior) (tables 2, 3, and 4). to summarize, the medical error domain consisted of eight questions on medical error recognition and related patient outcomes, medical error risk factors, human error theory, and medical error disclosure. the patient - physician relationship domain was composed of nine questions concerning expected behaviors and communication skills. medical student competency in both of the above domains was rated as follows : nonexistent = 0 points ; present but insufficient = 50 points ; and present and adequate = 100 points. medical student competency in the humanistic behavior domain was evaluated with five questions using a likert scale ranging from 1 (definitely disagree) to 5 (definitely agree) ; the corresponding scores were then converted to percentages. the standardized patients completed one checklist for each student during a 3-minute break between the exit of the previous student and the entrance of the next student. all students received the checklist results and detailed written feedback regarding their performance at each objective structured clinical examination station via e - mail. students were also strongly encouraged to discuss their performance with the objective structured clinical examination coordinators. at the end of the objective structured clinical examination, students were invited to anonymously complete a questionnaire to evaluate the patient safety program and the objective structured clinical examination assessment. all of the collected information was transferred to a database that had been created specifically for this program. independent double data entry and comparisons of the two databases were adopted to ensure internal validation. quantitative variables were described using the mean value, standard error (se) and minimum (min) and maximum (max) values. the objective structured clinical examination overall score, individual station scores, and patient safety domain scores were calculated and compared. this study was conducted at the so paulo university school of medicine, which admits 180 students annually to a six - year program in undergraduate medical education ranging from primary to tertiary care. the clerkship experience occurs during the final two years of undergraduate education and involves supervised, hands - on training in two university hospitals of increasing complexity : a 258-bed secondary hospital and a 1,200-bed tertiary hospital. students are divided into small groups and rotate among five main areas : pediatrics, internal medicine, obstetrics and gynecology, surgery, and preventive medicine. the internal medicine clerkship also corresponds to a 12-week program for fifth - year students, during which small groups of students participate in supervised clinical activities in two internal medicine wards and ambulatory clinics. this program includes simulations of medical scenarios that focus on effective interpersonal communication, invasive procedures, and resuscitation. student performance is evaluated using the following three complementary tools : a supervisor 's overall rating, written exams, and an objective structured clinical examination using a standardized patient. since the objective structured clinical examination was first introduced at our institution in 2002, this tool has been incorporated in almost all clerkship rotations. the present study enrolled 95 fifth - year medical students who were rotating through the internal medicine clerkship from july to december of 2007. this study was exempt from the need for review by the university of so paulo school of medicine institutional review board, but informed consent was obtained from every student. the patient safety educational program was first introduced into the clerkship training at our institution in 2007 and developed over time into two lectures per rotation. the program focuses on the following factors : medical error definition, epidemiology, and disclosure ; near misses ; adverse events ; patient - centered care ; the patient - physician relationship ; and ethics. this program has adopted the institute of medicine 's definitions of adverse events, near misses and medical errors. after attending lectures on these topics, they were invited to discuss real cases that they had witnessed or in which they had participated. the anonymity of the patients and the health professionals involved in these situations was always maintained., students were evaluated using an objective structured clinical examination with five 10-minute stations that involved standardized patients. one station focused on patient safety and included three domains : medical error, patient - physician relationship attitudes, and humanistic behaviors. the other four stations included the following assessments : 1) a procedure station for the assessment of venipuncture and paracentesis skills ; 2) an orientation station to evaluate the student 's ability to teach the use of medication (e.g., insulin injection) ; 3) a clinical station that focused on physical examination skills ; and 4) a health promotion station at which students were asked to perform a health promotion consultation on a healthy patient. all the standardized patients received training in both the scenario that corresponded to their role as a patient and the detailed checklist that was used to assess the students. subsequently, the scenarios were pilot - tested by the researchers until the standardized patients accurately portrayed the scenarios, reliably scored the previously described domains and adequately completed the checklist. the patient safety checklist was constructed based on a literature review by a group of medical educators and patient safety researchers. it was pilot - tested for use in objective structured clinical examinations with a group of 45 fifth - year medical students (june 2007). the revised checklist contained 21 items grouped into three domains (medical error, patient - physician relationship, and humanistic behavior) (tables 2, 3, and 4). to summarize, the medical error domain consisted of eight questions on medical error recognition and related patient outcomes, medical error risk factors, human error theory, and medical error disclosure. the patient - physician relationship domain was composed of nine questions concerning expected behaviors and communication skills. medical student competency in both of the above domains was rated as follows : nonexistent = 0 points ; present but insufficient = 50 points ; and present and adequate = 100 points. medical student competency in the humanistic behavior domain was evaluated with five questions using a likert scale ranging from 1 (definitely disagree) to 5 (definitely agree) ; the corresponding scores were then converted to percentages. the standardized patients completed one checklist for each student during a 3-minute break between the exit of the previous student and the entrance of the next student. all students received the checklist results and detailed written feedback regarding their performance at each objective structured clinical examination station via e - mail. students were also strongly encouraged to discuss their performance with the objective structured clinical examination coordinators. at the end of the objective structured clinical examination, students were invited to anonymously complete a questionnaire to evaluate the patient safety program and the objective structured clinical examination assessment. all of the collected information was transferred to a database that had been created specifically for this program. independent double data entry and comparisons of the two databases were adopted to ensure internal validation. quantitative variables were described using the mean value, standard error (se) and minimum (min) and maximum (max) values. the objective structured clinical examination overall score, individual station scores, and patient safety domain scores were calculated and compared. a total of 95 fifth - year medical students (63 males and 32 females) participated in the objective structured clinical examination. the overall objective structured clinical examination mean score was 85.890.66 (mean se). the mean grades for each station were as follows : orientation 90.570.67 ; patient safety 87.591.24 ; procedures 85.531.45 ; health promotion 85.121.41 ; and clinical exam skills 80.971.33 (tables 2, 3, and 4). the comparisons among all but one of these scores showed no significant difference ; the clinical exam skills station had a mean score that was statistically significant (p<0.05). another noteworthy finding was that student performance in the patient safety station had a high mean score that was surpassed only by the score in the orientation station. no significant differences were found between genders in either the global or specific station scores. despite the relatively few significant differences in the data, a detailed analysis of the patient safety station data revealed some intriguing results (tables 2, 3, and 4). when students ' patient safety performances were divided into the domains of medical error, patient - physician relationship, and humanistic behavior, the mean scores in the latter two domains were high, at approximately 90 points. conversely, the medical error domain resulted in the lowest mean score (77.962.21) compared to the other two domains (p<0.05). in the medical error domain, the best performance was obtained for the item explaining the medical error and its consequence to the patient or relative using comprehensible and colloquial language (94.771.75). regarding approaches to medical errors, students recognized the systemic chain of errors and the importance of avoiding behaviors related to blame and punishment (84.044.83). also, students performed well in the area of medical error identification by recognizing potential risk factors and preventive measures. nevertheless, performance related to apologizing and ensuring that the case will be reviewed resulted in the lowest mean scores in this domain (57.374.79 and 59.474.38, respectively). a strong and significant correlation was found between the medical error and humanistic behavior domain scores (table 5). the medical error domain scores also correlated significantly to the patient - physician relationship scores, albeit at a lower level. finally, a significant correlation was detected between the scores from the patient - physician relationship and the humanistic behavior domains. the patient safety curriculum and assessment were highly rated by students (94%), who recognized the importance of learning, discussing, assessing, and receiving feedback on medical error issues during the clerkship. the relationship between the experience of residents and medical error has drawn increasing attention in the medical literature in recent years.7 - 12,22,24 - 31 however, few studies have addressed this topic in undergraduate medical education.12 - 20 teaching hospitals and medical schools must better incorporate formal curricula on patient safety to provide students with the opportunity to discuss errors, practice related competencies and receive adequate assessment in this area.32 it is important to note that previous research has shown that the first experience of most physicians with medical error occurred during their undergraduate training.32 nevertheless, most medical students receive ambiguous messages on this topic. in a recent study, white and colleagues described the attitudes and experiences of 629 medical students and 509 postgraduate trainees about medical errors and disclosure to patients.12 despite only 35% of these students reporting previous education or training in medical error disclosure, as many as 79% reported involvement with cases involving medical errors, which were predominantly minor errors and near misses.12 martinez and lo, who analyzed the experience of 142 medical students with medical errors, found that 76% of students reported having witnessed a medical error, and 18% reported having committed a medical error during their clinical training.33 furthermore, many students disapproved of the senior doctors ' attitudes toward medical errors, which were manifested in the doctors ' attempts to hide the errors from patients and relatives.33 both findings reinforce the necessity to teach, train, and assess medical students in patient safety competencies and to provide them with adequate role models.12,33 to our knowledge, this is the first study to describe the performance of medical students in an objective structured clinical examination using standardized patients to teach patient safety during clerkships that included patient safety in the curriculum. the scarce data that have been published on patient safety in undergraduate medical training have predominantly assessed students ' experience with medical error using surveys and specific questionnaires that were anonymously completed by students before and after the incorporation of a patient safety program.12,14,15,17 - 20, these survey - based studies are limited by a dependence on students ' self - reporting of achievements rather than the application of observational methods that objectively evaluate student performance.17 moreover, few of these studies have focused exclusively on the clerkship opportunity, despite recognition of the clerkship as the ideal time for teaching and evaluating these skills.12, seiden and colleagues concluded that to teach safety in healthcare, students should be provided with the appropriate knowledge of medical error epidemiology and also be effectively trained in the systems approach to medical error, patient - centered care, interpersonal communication and professionalism skills.16 because assessment drives behavior, seiden highlighted the importance of the incorporation of multifaceted patient safety concerns in routine evaluations during clinical clerkships. although the ideal approach would be to assess how students handle medical error by observing them in actual clinical settings, ethical concerns prevent this approach.8 practical evaluations with standardized patients and the objective structured clinical examination are an excellent alternative for the evaluation of patient safety competencies because they are conducted in low - risk scenarios, incorporate issues related to communication, patient - centered care, empathy and medical error disclosure, and provide valuable feedback for examinees and medical educators.4,6,7,10,22,23 the use of standardized patients is extremely effective for evaluating the performance of healthcare professionals. this approach has been adopted for graduate physician assessment,7,8,22, but to our knowledge, no published data have yet described this experience during clerkships. in the present study, our students performed well at the patient safety station compared to the other scenarios. nevertheless, a detailed examination of the results raised intriguing concerns. it is important to note that students performed significantly poorer on all of the medical error issues compared to their performance in the humanistic behavior and patient - physician relationship domains. as previously noted, the only exception was the ability to disclose medical error using understandable language. moreover, apologizing and ensuring that the case would be reviewed were the issues with the lowest scores (less than 60%). concerning issues that were exclusively related to apologizing, most previous studies on medical error disclosure have exposed the presence of a critical gap between expected attitudes and the manner in which physicians actually behave.9, our results are in accordance with previous data from medical error disclosure simulations that describe doctors as seldom apologizing after a medical error occurs (47%) and almost never ensuring that the case will be reviewed to prevent future similar occurrences (8%).8 a survey focusing on residents ' changes in practice after their involvement with a medical error showed that only 54% of the residents reported that they had discussed the mistake with their attending physicians. only 25% of these residents reported that they had disclosed the error to patients, and only 21% reported that they had apologized.31 in addition, these authors demonstrated that accepting responsibility for the mistake and extensively discussing it with supervisors and patients were both predictors of constructive changes in practice on behalf of these residents.31 however, studies describing the experiences of patients (including physicians) who have suffered from a medical error have shown that only one third of them had been offered an apology or had perceived genuine interest in the situation by the hospital staff.37 this finding conflicts with the ethical standards and expectations of professional guidelines and accrediting organizations, all of which dictate that physicians have a responsibility to disclose medical errors to patients34 to enhance transparency in healthcare.12 the failure to provide truthful and compassionate explanations to patients and their relatives after medical errors occur decreases patient trust and satisfaction in healthcare.35,36 patients and families want to be told about any error that causes patient harm.34,38,39 furthermore, previous research on medical error and litigation has shown that the absence of a sincere apology and the lack of effective explanations were key factors related to legal claims.34 we observed that our students were highly rated on communication skills and expected humanistic behaviors. these two important competencies have been discussed, trained, and assessed using the objective structured clinical examination throughout the past ten years of our clerkship program. effective communication among health professionals, patients, and their relatives is essential for ensuring safety in healthcare.34,36,39 previous studies have shown that communication failure was an important contributing factor in almost 90% of adverse events and incidents.1 moreover, once an error occurs, effective communication is a fundamental component of the disclosure process.34 - 36, similar to previous researchers, we believe that interpersonal skills, empathy, and humanism can and should be included in the educational process and incorporated into multifaceted assessments to promote longer lasting change among trainees and physicians.6,7,34,35 the strong correlation between the scores in the humanistic behavior and medical error disclosure domains reinforces the crucial role of compassion and empathy in the externalization of medical errors to patients.8,34 - 36,39 this study has several important limitations. first, we analyzed the performance of students in one objective structured clinical examination station related to patient safety using only two different scenarios in a single school of medicine. therefore, our results are limited and can not be generalized to all medical students or patient safety competency acquisition in general. second, our patient safety checklist was constructed by a group of medical educators and members from a patient safety group that incorporated content described in previous research. a pilot study was conducted with preceptors and fifth - year medical students during a pilot objective structured clinical examination, and a final review of the tool was performed. nevertheless, this patient safety checklist is essentially a new instrument that may be limited by its lack of formal validation and reliability testing. furthermore, there is not always a correspondence between the behaviors observed in structured, practical evaluations using standardized patients and the behaviors that occur in real life. issues of inter - rater reliability must also be addressed in future studies to reduce inherent bias and subjectivity. in addition, this was a preliminary study in a newly emerging field ; we hope that our findings will contribute to future avenues for research. in conclusion, although several questions remain unanswered, we provided for the first time the results of a new curriculum and assessment in patient safety attitudes and competencies. the objective structured clinical examination is a useful tool for the evaluation of this new curriculum during medical school clerkships because it represents an opportunity to provide feedback and reinforce positive role - modeling. this approach also presents a unique opportunity to assess the complex interfaces among the dimensions of humanism, patient - centered care, the patient - physician relationship, and patient safety. we hope that the implementation of this interactive patient safety program and the related objective structured clinical examination assessment will transform the current unfortunate scenario characterized by a large proportion of young trainees who enter practice without adequate training in medical error recognition and disclosure. | introduction : patient safety is seldom assessed using objective evaluations during undergraduate medical education.objective:to evaluate the performance of fifth - year medical students using an objective structured clinical examination focused on patient safety after implementation of an interactive program based on adverse events recognition and disclosure.methods:in 2007, a patient safety program was implemented in the internal medicine clerkship of our hospital. the program focused on human error theory, epidemiology of incidents, adverse events, and disclosure. upon completion of the program, students completed an objective structured clinical examination with five stations and standardized patients. one station focused on patient safety issues, including medical error recognition / disclosure, the patient - physician relationship and humanism issues. a standardized checklist was completed by each standardized patient to assess the performance of each student. the student 's global performance at each station and performance in the domains of medical error, the patient - physician relationship and humanism were determined. the correlations between the student performances in these three domains were calculated.results:a total of 95 students participated in the objective structured clinical examination. the mean global score at the patient safety station was 87.591.24 points. students ' performance in the medical error domain was significantly lower than their performance on patient - physician relationship and humanistic issues. less than 60% of students (n = 54) offered the simulated patient an apology after a medical error occurred. a significant correlation was found between scores obtained in the medical error domains and scores related to both the patient - physician relationship and humanistic domains.conclusions:an objective structured clinical examination is a useful tool to evaluate patient safety competencies during the medical student clerkship. |
parent mentors have been used in interventions aimed at improving childhood asthma control, reducing malnutrition, and improving general parenting skills. they have also been used to provide support for families of children with chronic disabling conditions. new research has shown that using parents may be of greater benefit because of their ability to relate to the experiences of other parents. there are limited data describing the dynamics of the mentoring relationship when parent mentors are being used in an interventional rather than supportive context and how they are perceived by their mentees. this is an important gap given the heterogeneity in these types of interventions and the need to better contextualize outcomes between studies. there are few studies that have used a qualitative approach to assess the experience, behaviors, and activities of mentors [68 ]. one study found a strong role for affirmational and emotional support between mother mentors and their mentees and less emphasis on providing information or discussing specific medical information. increased self - efficacy and empowerment have been described in parent mentors, with one report describing the mentors ' job opportunities improving postmentoring. other investigators found that mentors are able to empathize with participants and provide affirmational support [9, 10 ]. the current report examines the experiences of parent mentors involved in a child obesity intervention study in a hispanic population using primarily a qualitative approach with complementary quantitative data. feeding and disciplinary behaviors are deeply rooted in culture and family and often intensely personal. furthermore, obesity in early childhood among the hispanic community is often not recognized as a problem. therefore, the challenges that parent mentors face in this context, despite coming from the same community, may be different from prior applications of the parent mentor model. mentors may be faced with challenging closely held family beliefs about the provision of food for comfort or suggest changes for the child that go against the normative behavior for the family 's social context. this project involved a clinical trial enrolling obese, 25-year - old children and their parents (dyads). the original study aimed to compare differences in parent - child dyads enrolled in the study that were assigned to either a parent mentor or a community health worker, both of whom delivered education and behavioral coaching over the course of 6 months as described previously. this paper aims to better understand the experiences and perceptions of parent mentors and how this translates to the experiences of their mentees. the overall study was designed as a randomized trial targeting hispanic, obese, 25-year - old children and their parents who were enrolled in a head start program (a federally - funded early childhood education program for low - income families) in the lower rio grande valley in south texas [11, 12 ]. there were 60 parent - child dyads enrolled in the study, and they were randomized 1 : 1 to receive teaching from either a parent mentor or a community health worker using a standardized curriculum. the parent mentors were all mothers who were identified by the head start staff as having positive personalities and potential leadership qualities from among the other parents enrolled in the head start program ; while fathers were potentially eligible to participate, none volunteered. the mentors otherwise had no prior mentoring experience and were selected based on a brief, unstructured interview. parent - child dyads in the parent mentor arm of the trial were paired with a trained parent mentor. parent mentors were trained in a one - day session using a bilingual handbook developed with input from the pilot study on positive deviance methods derived using local parental input, as previously described. the theoretical framework of positive deviance drove the development of the handbook and the overall intervention design. positive deviance is a complexity - theory based model which asserts that, even among those most at risk for a poor outcome, there are some individuals who find ways to succeed, and by learning from their success, we may be able to discover innovative and sustainable means of addressing the problem in the rest of the community at risk. parent mentors were instructed in active listening, reflective feedback, and peer coaching techniques as well as the positive deviance findings and local resources for parents of obese children. the parent mentors conducted a baseline home assessment with the parent and child and conducted follow - up phone calls at least once a month to encourage healthy habits and behaviors identified as foci for that particular parent. the parent mentors were given complete flexibility in terms of how often they contacted their mentees, what topics they chose to focus on, and how they interacted with the families. the parents were invited to participate in monthly community meetings facilitated by the parent mentor and focused on encouraging the behaviors previously identified as positively deviant and healthy habits identified as goals by the group. parent - child dyads randomized to the community health worker condition were also invited to attend community meetings on a monthly basis at head start centers. these meetings were facilitated by a local promotora, or community health worker, who used the eatplaygrow curriculum to teach about healthy habits. the eatplaygrow curriculum uses an interactive format to engage parents and children, and it has been piloted in multiple early childhood settings among diverse populations. parent - child dyads in the community health worker condition did not receive a home visit nor any follow - up phone calls. they were all hispanic mothers of at least one child 25 years of age who was at a healthy weight. the interviews were recorded using a digital voice recorder and then professionally transcribed with adrianna, bianca, and cynthia used as pseudonyms. we conducted the interviews in march 2015, three months into the overall study which began enrolling january 5, 2015. the interview guide was semistructured and flexible to allow for the interview to take its own direction. a junior staff person involved in the research project conducted the interviews. the basic outline for the interviews is available as a supplemental file (supplemental file 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/5769621). we also sought to understand whether the parent mentees participating in the study perceived the parent mentors differently than the more traditional community health worker. we administered a survey consisting of questions on perceptions of the parent mentors or community health worker, self - efficacy, and attitudes related to childhood obesity. likert - scale - based questions that prompted the parent to rate their self - confidence in addressing childhood obesity were also included. the parents ' self - efficacy was assessed using the general self - efficacy scale, a 10-item, likert - scale questionnaire. the general self - efficacy scale spanish version was used instead of being translated, as it has demonstrated to be valid and a reliable measure among the spanish speaking population in clinical and community settings. surveys were administered at the end of the study (after six months of intervention). this study was approved by the university of texas health science center at san antonio institutional review board. we used qualitative description to stay as close to the data as possible, in part given the limited prior research. given the pilot and exploratory nature of this study, we focused on describing the experiences, successes, barriers, and challenges faced by parent mentors. member checking was done with the mentors on the broad themes identified during a debriefing session two months after the end of the mentoring period. the interviews were read at least three times to get an overall sense for the flow and tone of each interview. each interview was separately coded, using the web - based dedoose application. after all interviews had been coded, each code was reviewed within each individual interview again ; some codes were modified in this second round. for the third round, codes were again reviewed, both within each interview and across interviews looking for commonalities and emergent themes. we used descriptive statistics to analyze the survey data administered to the parents enrolled in the study, the parent mentors, and the community health workers. data were collected using redcap and then transferred to spss 23.0, ibm (usa), for descriptive statistical analysis. adrianna was enrolled in a technical - college, her husband was unemployed, and they have one young child (age 25), and she was obese herself. bianca was unemployed, her husband is deceased, she has one young child (age 25) and two older preteen boys, and she was at normal weight. cynthia was employed part - time in hospice care and has four children with the youngest aged 25, her husband works part - time, and she was obese. four overarching themes emerged from the data : perceived outcomes of participation, parent mentor qualities, obstacles and resources to overcome, and building community. while the themes are discussed separately, we recognize that they are intertwined within each individual, and the dynamics between the mentor and participants is an evolving process. for example, the parent mentor qualities described clearly inform the obstacles they faced and the ways they approached them. their individual life experiences and qualities influence how they perceive the outcomes of participation and how they are each engaged in building community. each mentor described in some way how their participation in the study had affected their own life or habits. adrianna, who had the highest level of education of any of the mentors, seemed pleasantly surprised that she had learned from the process. bianca described concrete ways in which being a mentor had affected her.well, it 's changed my shopping experience for sure with my children. i myself have been changing with my children, so it 's helped me a lot. well, it 's changed my shopping experience for sure with my children. i myself have been changing with my children, so it 's helped me a lot. cynthia discussed how her mentoring had an effect on her as she described implementing what she was teaching in her own life. while she had a child who was at a normal weight, her awareness of the process and challenges of healthful eating seemed heightened. in discussing how the study had influenced the participants they were mentoring, the mentors discussed process change or the beginnings of change.when we 're talking in conversations they will actually open up and say, i fried this but you know what ? i 'm not doing that as often ' or you were right about some points in the meeting and we 're going to try this. ' when we 're talking in conversations they will actually open up and say, i fried this but you know what ? i 'm not doing that as often ' or you were right about some points in the meeting and we 're going to try this. ' interestingly, none of the mentors discussed weight or appearance of weight in their perceptions of success or effects of the study. also, they tied their own experience to the description of the mentee 's experience. being nonjudgmental was described explicitly by each mentor.it's just letting them know that i am just there to help them, i am available and i 'm not there to judge, most important because i think in the beginning, when somebody comes and tries to change or tell you that you 're making them feel they 're not doing something, they might be a little bit more defensive. it 's just letting them know that i am just there to help them, i am available and i 'm not there to judge, most important because i think in the beginning, when somebody comes and tries to change or tell you that you 're making them feel they 're not doing something, they might be a little bit more defensive. the tone and context of their descriptions implied that they were either reflecting on how some of the mentees in the study initially perceived their possible role or how they themselves might have perceived being mentored on their child 's health. it should be noted that this was despite significant resource limitations : one of the parent mentors (bianca) had no car and the other two had other significant commitments (adrianna in school and cynthia with work) in addition to being primary caregivers for their own children. adrianna described the intervention as something she owned rather than being a study subject.i mean, i do care about my parents. that we 're trying to do something positive and it 's not just a temporary get to know you and then goodbye thing. that we 're trying to do something positive and it 's not just a temporary get to know you and then goodbye thing. another common attribute was being encouraging. adrianna described being encouraged in contrast to what may be expected, even contrasting with what may be commonly said or heard at a health professional 's office.i'm able to break it down to a certain level where it 's just like a friend to friend talk, versus a medical doctor with big scientific words that as soon as you go home you have to look in a dictionary in order to understand what he just said i believe that there 's a difference and it feels, to me, that it might be a little bit better from coming from a parent mentor. i 'm able to break it down to a certain level where it 's just like a friend to friend talk, versus a medical doctor with big scientific words that as soon as you go home you have to look in a dictionary in order to understand what he just said i believe that there 's a difference and it feels, to me, that it might be a little bit better from coming from a parent mentor. when asked to discuss any obstacles or challenges, the parent mentors primarily discussed engagement with the parent mentees. the parent mentors were being potentially put outside of their usual social circles and asked to provide advice and coaching. another challenge was addressing the individual backgrounds, experience, and time constraints of their mentees. bianca had the least education of the mentors, and she vividly described being intimidated by her own perceived lack of expertise or knowledge.the first time that i met with them it 's like i was learning from them, but then again i have to teach them that this is a child and what portions to feed him and of course stay away from the sweets because they had a little issue with wanting cookies and ice cream. they 've been doing a lot better on that themselves before i even met them. i did n't know what to tell them to help them because they already knew. the first time that i met with them it 's like i was learning from them, but then again i have to teach them that this is a child and what portions to feed him and of course stay away from the sweets because they had a little issue with wanting cookies and ice cream. they 've been doing a lot better on that themselves before i even met them. i did n't know what to tell them to help them because they already knew. cynthia also described a unique challenge in which the mother who was her mentee was enrolled in the program (voluntarily) but, at the same time, really did not seem to want to engage. in accessing and using resources to mentor their parents, cynthia in particular was very careful about the resources she chose, mostly the booklet she was given during training and making sure her sources were reputable, perhaps reflecting her own experience working in hospice. somewhat in contrast and perhaps reflecting her higher educational background, adrianna described her use of independent resources that she sought out, primarily government, health, or education websites and scholarly journals. tied into the notion of ownership described above, adrianna described her approach to that challenge.we're busy, they 're busy and it 's challenging just to kind of meet in the middle. you will have some parents where they 'll really, really try and they 'll try and they 'll try but they 're just they 're busy and you 're just going to really have to put more effort and more work to meet, especially to meet the demands, meet your duties that you 're supposed to do within a certain time. we 're busy, they 're busy and it 's challenging just to kind of meet in the middle. you will have some parents where they 'll really, really try and they 'll try and they 'll try but they 're just they 're busy and you 're just going to really have to put more effort and more work to meet, especially to meet the demands, meet your duties that you 're supposed to do within a certain time. one of the unexpected results of the project was the connection between the parent mentors and their mentees described early on in the study. each mentor described sharing, openness, and friendliness around the study that seemed to transcend the structure of the context of a randomized controlled trial. cynthia 's description of her role was striking as a focal point for the beginnings of a community, describing openness and friendliness.everybody seems to be friendly with me. they seem open and some of them want to introduce me to some of their friends so that i get to all their friends about what they 're learning and i said, no, that 's where you come in. they seem open and some of them want to introduce me to some of their friends so that i get to all their friends about what they 're learning and i said, no, that 's where you come in. forty - six of the forty - eight participants who completed the overall study completed the end of study survey. the parent mentees rated the parent mentors on their abilities in the community meetings similarly to the paraprofessional community health worker (table 1). the only difference between the two groups was that the parent mentor group reported a higher ability to identify with the presenter (p = 0.04). finally, the lowest rated item was whether obesity was a significant problem in the community with an overall mean of 79, sd = 29. there was no difference in self - efficacy between the parent mentor group and the community health worker group with both having a high level of self - efficacy (median = 37, iqr = 5, p = 0.47 for differences between groups using mann - whitney u test). the mentees were divided into two groups based on self - efficacy score : those who fell above versus below the median (median = 37). we examined whether these levels of self - efficacy were associated with attitudes toward behavioral change. those scoring above the median on the self - efficacy scale reported feeling more confident finding resources (p = 0.05) and higher confidence in making healthier choices for their children (p = 0.06) though both parents reported mean confidence scores > 90 both above and below the median self - efficacy groups. interestingly, there was also a moderate correlation (0.42, p = 0.003, spearman 's coefficient) between self - efficacy scaled on a continuum and reported time spent with their parent mentor. parent mentors in this study on childhood obesity described an overall positive impact of participation on both themselves and perceived a positive impact for their mentees ; they described using a nonjudgmental and supportive approach and formed significant bonds over a relatively short period of time. this model of using parents as a primary intervention for behavior change in childhood obesity has theoretical appeal since there are multiple existing structures where this could be implemented (schools, head start, daycares, etc.). leveraging the people in the community to promote positive habits has significant appeal for a public health problem as large as childhood obesity. while the efficacy of these parent mentors in changing behaviors or reducing adiposity is outside the scope of this paper, the data discussed here support the feasibility and cultural acceptability of this model which is an important outcome of its own which has implications for similar studies. one of the clear themes that came across was the nonjudgmental attitude, openness, and even friendliness described in the dynamic between mentor and mentee. the literature describes some of these same principles on how to form a therapeutic alliance between doctors and patients around obesity ; these parent mentors were able to achieve such an alliance in a short amount of time. the parent mentors also described having high self - efficacy related to their experiences, which has been noted in another parent mentor study. the parent mentors also openly described some of the personality conflicts they encountered and the pressures of time ; they seemingly dealt with these conflicts and negotiated these pressures very effectively. given the fairly low level of support provided by study staff, this speaks to their character prior to the study rather than the training component. we conducted individual interviews with potential parent mentors after they were selected by their peers. in expanding this model of intervention, developing a more streamlined process for mentor selection, recruitment, and training parental beliefs and attitudes are a reflection of both the community and what was being taught by the peer mentors. the rating for believing there was a problem of childhood obesity was lower than expected and rated lower than any other question. this is consistent with a comment attributed to a parent by one of the parent mentors : none of the parent mentors ever mentioned trying to prove to parents that there was in fact a childhood obesity problem ; indeed the subtheme of being nonjudgmental may reflect an underlying belief in the community at large. recognizing that childhood obesity is a problem has been shown to be a barrier to modifying behaviors among hispanics in several studies ; however, having a mentor that does not focus on what parents have done wrong may be beneficial. a parent mentor from the community may also have the advantage of better identifying with the parent in helping them recognize the aspects of their life they can change. the peer mentors themselves mention several resources that they used for information : remind them, they can ask their pediatrician, they can ask a dietitian at school, they can ask a teacher and so it is possible that these reminders may have helped the parents feel that they can find resources themselves. it is well documented that hispanic mothers have miscued perceptions of feeding and the association of physical activity, nutrition, and health ; therefore, feeling confident that they can find reliable resources could significantly improve knowledge of feeding and obesity [20, 21 ]. parents who scored above the median in self - efficacy were more likely to feel confident in making healthier choices and feeling that their child could achieve a healthy weight. this reflects the general principle of self - efficacy : overcoming barriers even in the state of adversity. this is important as all of these parents are from low socioeconomic backgrounds and face barriers such as being able to afford healthy food and going against cultural practices. among caucasian white females in a health promotion intervention for weight loss, general self - efficacy scores among those with a higher bmi were found to be on average 31.5. a study using latina migrant farmworker mothers in a health promotion intervention reported a general self - efficacy score among the latina mothers to be 30.3. our population reported much higher scores at the end of the intervention, with a median of 37. no difference was found between the community health worker group and the parent mentor group. the internal consistency of the data is reinforced by the finding that those above the self - efficacy median even despite the overall high scores were more likely to report higher confidence in making a behavioral change such as making healthier choices, finding resources, and believing that their child can achieve a healthy weight. this is the first description to our knowledge of the experience of parent mentors working with other parents to reduce their child 's obesity. while there were some challenges to implementing a parent mentor model of behavior change for obesity, the parent mentors were able to overcome those challenges and had an overall very positive experience that has potential for replication and dissemination to address this significant public health problem. all of our mentors were females chosen by the staff and showing an interest in participating ; while the study could have included fathers as mentors, none volunteered or were chosen. in other studies, mothers have been shown to play an integral role in controlling nutrition in hispanic households, and it has been demonstrated that they are an important component in preventing obesity in their children [25, 26 ]. fathers were not excluded in this study and several did attend the education classes with their spouses. further research to examine roles of fathers and mothers in this type of intervention may be warranted. the two interventions of a community health worker versus a parent mentor also differed in not only the type of interaction and content but also the intensity of time. the parent mentors spent one - on - one time with their mentees either in person or over the phone providing individual support and advice. the lack of an attentional control group in this study is a limitation in making any comparison between the groups. | objective. parents mentoring other parents as a behavioral intervention for child obesity is novel with limited data describing the experience and dynamics of this approach. this study aimed to describe the experiences of parent mentors and the self - efficacy and attitudes of their mentees in the context of a clinical trial for childhood obesity. methods. the context for this study was a randomized clinical trial using either parent mentors or a community health worker engaging parents of obese children in behavioral change over six months. parent mentors were interviewed at the mid - point of the intervention using a semistructured questionnaire to elicit their perceptions and experiences during the process of mentoring. parent mentees completed a survey assessing their self - efficacy, perception of the parent mentor, and attitudes and beliefs related to their child 's weight. results. the qualitative analysis of parent mentor interviews indicated high commitment despite their nonprofessional status, facing challenges of engagement with fellow parents and attitudes of persistence and being nonjudgmental. the parent mentee ratings of parent mentors were overall very high and similar to the ratings of a community health worker (paraprofessional). conclusion. the data suggest that a parent mentor model of intervention for child obesity is an acceptable mode of approaching behavior change in the hispanic population around childhood obesity with potential for scalability if proven effective. |
malaria is a major health problem in the developing world today, with an estimated 300 - 500 million cases and more than one million deaths each year. artemisinin and its derivatives are rapidly acting antimalarial compounds effective against resistant strains of plasmodium falciparum malaria. this has lead to its increased use in public hospitals in developing countries. we herein report a case of sodium artesunate - induced diuresis in a patient with malaria. a 6-year - old boy presented with fever for five days and vomiting for two days. vomiting was 23 times per day and non - projectile, non - bilious, and contained ingested food particles. there was no dysuria, oliguria, abdominal pain, or bleeding manifestations. on examination, the patient was afebrile with a heart rate of 104/min, respiratory rate of 24/min, and blood pressure of 100/60 mm of hg. liver was soft and non - tender with a span of 6.5 cm in the midclavicular line. investigations revealed : hemoglobin 8.2 gm / dl, total leucocyte count 11,300 /mm (60% polymorphs, 40% lymphocytes) and platelet count 53000 /mm. liver function tests, renal function tests, serum calcium, and phosphorous were normal throughout the hospital stay. the arterial blood gas, urine microscopy, and chest x - ray were normal. widal test, dri - dot test for leptospira and dengue igm antibody test were negative. intravenous sodium artesunate (2.4 mg / kg), i.e., 38.4 mg was given at 0, 12, 24, and then once a day for 7 days. the patient did not receive intravenous fluids, diuretics, or vasodilators and continued normal diet and fluid intake. the serum electrolytes, urine output, and urinary electrolytes during the course of hospital admission are shown in table 1. on day 3 of artesunate treatment, the patient developed increased urine output along with increased excretion of sodium, potassium, and chloride. two days after stopping artesunate the urine output and urinary excretion of electrolytes started decreasing. patient became afebrile on day three and was discharged from the hospital on day 11 of admission. serial values of serum electrolytes, urine output, and urinary electrolytes in patient on artesunate treatment the artemisinin derivatives artemether, artesunate, and dihydroartemisinin are derived from the chinese medicinal herb, qinghao. sodium artesunate is a new antimalarial water soluble drug, which can be administered either by intravenous or intramuscular injection. it is more potent than quinine and rapidly reduces parasitemia and resolves clinical symptoms in resistant parasites strains. this action is due to increased nitric oxide synthesis and inhibitory effect on chloride transport across the cortical thick ascending loop of henle (talh) that suppresses tubuloglomerular feedback. nitric oxide causes diuresis and natriuresis by inhibiting the activity of the na, k - atpase and tubular sodium transport. the principal site of action of artesunate in kidneys is supposed to be talh, which has the highest concentration of the enzyme na, k - atpase per tubular length. the increase in the urinary excretion of sodium, chloride and potassium also suggests that this drug has a predominant effect on the proximal segments of the nephron i.e. proximal tubule and the talh. reduction in the reabsorption of sodium and an increase in the distal sodium delivery occurs with a consequent greater sodium reabsorption and potassium secretion in the collecting tubule. the diuretic effect of artesunate is a double - edged weapon that can modify the course of the acute renal failure in malaria. the increase urinary loss of water and electrolytes can worsen the renal failure in hypovolemic patients with pyrexia and tachypnea. however, the administration of artesunate can also be useful in the conversion of an oliguric renal failure to nonoliguric in patients with volume overload. in addition, rapid improvement in pulmonary function has been observed in a patient with respiratory distress syndrome due to the diuretic effect of artesunate. in conclusion, the diuretic effect of artesunate can modify the course of renal failure and respiratory distress syndrome, both of which complicate severe malaria. physicians should be aware of this effect of artesunate, so that it can be used judiciously and to the advantage of patients with severe malaria. | sodium artesunate is used in the treatment of malaria. we report a case of sodium artesunate induced diuresis and natriuresis in a patient with malaria. following artesunate administration there was polyuria accompanied by natriuresis that was reversed after discontinuation of artesunate treatment. the diuretic effect of artesunate can modify the course of renal failure in severe malaria. prescribers should be aware of this effect of artesunate, so that it can be used judiciously and to the advantage of patients with severe malaria. |
both the med associates and the coulbourn instruments systems can monitor multiple inputs and control multiple outputs. both can run multiple experiments concurrently with ease. both require a windows - based system and really should be run on a dedicated computer that is not burdened by being connected to a network. both offer a runtime screen that provides information about the status of the current experiment. both systems are designed to be used with proprietary interfacing equipment available from the manufacturer. the reader is urged to consult the manufacturers web sites (www.coulinst.com and www.med-associates.com) for detailed descriptions of the products. i should note that i have received timely and helpful assistance from both companies when it was needed. the user creates a text file consisting of statements written in medstate notation (easily learned by a pascal programmer, challenging for the user with no programming experience). this can be done using the editor provided with the system or with any editor or word processor capable of producing plain - text output. the program can contain as many as 32 distinct state sets, each acting as an independent mini - program. prior to use the program must be compiled, a process that will reveal coding errors that must be corrected before compilation can succeed. once a program is successfully compiled it is available to be run from a pull - down menu within a separate med associates program. medstate notation allows data to be stored in 26 variables (labelled a to z), each of which can take the form of either a single variable or a 1-dimensional array. these variables can store any data that the user specifies when the program is written. for example, one might contain the total number of times that a lever was pressed, another might contain a list of inter - response latencies, and a third might indicate the number of correct responses. although limiting the total number of variables to 26 might seem constraining, i have yet to find this too restrictive, especially because arrays are allowed. the data are written to disk as a plain text file ; several formats are available for the data file, and the one selected will probably be determined by whether or not the file will be imported into a spreadsheet. the programming window offers a picture of the front panel of the hardware that interfaces with the behavioral equipment. by pointing and clicking within this window the user can can specify the stimuli to be turned on during each state, the responses or time events that cause the state to terminate, and the target state(s) to be activated upon termination of the state. for example, to turn on the houselight for 10 s, the programmer would click on the houselight button to toggle it on, and specify that the state should end after 10 s. the program must be written via the graphic state 2 programming interface, and the program is saved in a proprietary format that requires it to be viewed via graphic state 2. because of this it is impossible to print the program for archival purposes. once the program is constructed, gs2 performs a check to ensure that it contains no states that are not called by at least one other state or that fail to call another state a check that helps to ensure that the program is at least likely to terminate. because the user writes no code, errors in programming syntax are far less likely than in med associates system. graphic state 2 consists of a single state set ; that is, only a single state can be active at any given time, as compared to medstate notation s 32 concurrent states. usually counters and timers are reset when a new state is entered, but the program allows them to retain their values across states or throughout an entire session. graphic state 2 logs all events while a session is being run, even if they have no effect on the program. if a state is ended by a response on lever 1, for example, the program will also log responses on lever 2 even if they do nothing. the data file that is created contains a record of every response or state change that occurred during the session, with a record of the time during the session when the event occurred and an indication of the cause of each state change. instead a record of the entire session is available at its conclusion for analysis by graphic state 2 s extensive library of analysis routines or for exporting as a text file. without question, the easier of the two systems to learn to program is graphic state 2. the point - and - click interface obviates the need to learn seemingly obscure programming language, and the fact that the program records everything that occurs during a session means that the user does not have to program variables to track relevant information. indeed, a student with no programming experience watched me write a simple program one morning, and by the afternoon had mastered graphic state 2 well enough to write a program to control a two - lever autoshaping procedure. in comparison, it is true that the commands are reasonably intuitive (e.g., on turns on a stimulus), but they must be learned and they must be entered correctly. furthermore, the user must specify and track all variables of interest ; the program will automatically record the values of variables a through z at the end of the session, but these values will be 0 unless the program contains code to update them (e.g., count responses in variable r, record number of trials in variable t, etc.). the availability of only one state set within a graphic state 2 program makes some tasks either very difficult or perhaps impossible to program, especially those tasks that involve independent timing of various stimuli. for example, consider the truly random control condition in a pavlovian conditioning study (rescorla, 1967). the investigator wants a tone to turn on for 5 s and a feeder to operate for 0.1 s ; the two should occur perhaps 20 times each during a 30-min session, but totally randomly with respect to each other. in medstate notation each could be controlled by its own state set, without any reference to or input from the other (as if each stimulus was controlled by its own 1960s - era tape timer). in gs2, because a stimulus can be turned on only by entry into a state, and because only a single state set is available, the two stimuli must of necessity be controlled together, and a complex program must be established consisting of states in which none is on, one is on, or both are on ; the durations of these states will be varied and complicated. i think that the only way to implement this program in gs2 would be to create a fixed schedule of the two stimuli that simulates the random occurrence of both, then to program this fixed schedule. a program in which the two stimuli occur truly randomly with respect to each other might well be impossible in gs2. gs2 creates a log of a session that contains every transition from one state to another (because stimuli are tied to states this corresponds to a record of stimulus presentations) and every response onset and offset (whether the responses cause state transitions or not). the result is a complete record of the session, available for export as a text file or for analysis within gs2 itself. because the record of the session contains all responses that were available to be monitored the researcher can ask questions after the fact that were not anticipated at the time the session was run. a large number of routines are provided by gs2 that allow extensive analysis of the data, including frequency of entry into each state, number of responses within each state, inter - response intervals, etc. unfortunately the explanations of these routines are far from intuitive, and the routines seem geared toward free - operant schedules. i run trial - based studies, often in runways or mazes, and the analyses have never been useful to me except as quick checks at the end of a session to ensure that data were recorded. i have relied on the exported raw data text files, which i then filter extensively and analyze with a spreadsheet program (e.g., excel or gnumeric). medstate notation s approach to data is far different : only the variables specified by the programmer are recorded ; there will be no record of the number of responses if the program does not contain a counter that is incremented with each occurrence of the response. medstate notation allows data to be saved in text files in a limited number of formats, some human friendly and some (supposedly) spreadsheet friendly. the text - files must be manipulated extensively with a spreadsheet before useful analysis can occur. med associates offer an additional program called mpc2xl to ease translation from their data files to a spreadsheet ; i have no experience with it. although both medstate notation and gs2 data files could be made far more user - friendly in terms of how data are formatted for subsequent use by spreadsheet programs, i find medstate notation s files easier to use. they are inherently smaller, because they contain only those data that i have requested. because i can specify counters, timers, and other variables in the program, the data file contains the information that i want with very little further analysis, for example, some of my behavioral routines allow rats to control the onset of their own pavlovian trials, and the number of trials initiated is a critical dependent variable. a variable within a medstate notation program can record this number as the session runs, display it on the screen, and save it in the data file. in gs2 this variable would correspond to the number of times a particular state is entered ; there is no way to display this, but instead an analysis must be run on the data at the conclusion of the session. the ability to create user - defined variables that track meaningful information, and to display these variables as they are updated during the session, make a medstate notation session far more informative than a session run in gs2. a well - designed medstate notation screen can provide all of the data of interest at the end of a session. gs2 sessions are likely to require post - session analysis in order to get even the most rudimentary of data. med associates medstate notation and coulbourn instruments graphic state 2 both offer the scientist useful tools for behavioral research. gs2 s strengths lie in its user - friendly programming interface and its many analytical routines. for the non - programming scientist medstate notation, with the availability of multiple concurrent state sets, offers a far more powerful programming environment. the time spent in mastering medstate notation i believe that the serious behavioral scientist is better served by the med associates product. | two of the most widely used programs for the control of behavioral experiments are med associates medstate notation and coulbourn instruments graphic state 2. the two systems vary considerably in their approach to programming and data recording, with graphic state 2 using a point - and - click interface that appeals to non - programmers while medstate notation requires the typing of programming code. graphic state 2 provides many data analysis routines, while medstate notation allows the user to embed simple data analysis within the behavioral protocol. graphic state 2 is simpler to use, but medstate notation is more versatile. |
this report describes the geographic disparity in all - cause, sex and disease chapter specific premature mortality in ontario, canada, at the regional, district health council and public health unit levels. analyses of geographic variations in population health status can help to guide policies that address the economic, social and environmental determinants of health, identify needs for health care services and assist health planners to target and prioritize health promotion and disease prevention programs within geographic areas. our objectives were to examine the extent of geographic disparity in premature mortality and to consider factors that may underlie variations in premature mortality across geographic areas. table 1 presents all - cause smrs for ontario planning regions, district health councils and public health units from 19921996. table 2 shows ontario average annual premature mortality rates per 100,000 by icd-9 chapter, total number of premature deaths and the percentage of total premature deaths attributable to each disease chapter from 19921996. information in this table is required to understand the impact of variations in disease chapter smrs on variation in total premature mortality. neoplasms and circulatory system diseases were the dominant causes of premature mortality among ontarians aged 074. these two disease chapters accounted for 64% and 69% of all premature deaths among males and females respectively. injuries and poisonings, respiratory system diseases, and digestive system diseases accounted for an additional 19% (males) and 15% (females). overall, the five leading disease chapters accounted for over 80% of all premature deaths. table 3 (see additional file 1) and table 4 (see additional file 2) present smrs for males and females by disease chapter at the public health unit level. all - cause standardized mortality ratios, ages 074, 19921996 data source : vital statistics records, office of the registrar general, ontario ministry of consumer and commercial relations. notes : all standardized mortality ratios are statistically different from 1.00 (p < 0.05) except the female huron smr ontario average annual mortality rates, number of deaths and percent distribution, ages 074, 19921996 data source : vital statistics records, office of the registrar general, ontario ministry of consumer and commercial relations notes :... figures not applicable the south west region had higher than expected all - cause premature mortality, with smrs of 1.07 (95% ci 1.07111.0714) for males and 1.06 (95% ci 1.0591.060) for females. high smrs of 1.12 (95% ci 1.1191.120) for males and 1.11 (95% ci 1.1061.108) for females in essex - kent - lambton (district health council) contributed to high regional smrs. at the public health unit level, chatham - kent had the highest all - cause smrs of 1.26 (95% ci 1.2551.259) for males and 1.29 (95% ci 1.2891.296) for females, which contrasted with other south west public health unit areas whose smrs ranged from 0.95 to 1.12. thomas also had substantially elevated all - cause smrs : 1.10 and 1.12 for males and 1.10 and 1.07 for females, respectively. chatham - kent had particularly high smrs for circulatory system diseases (1.44 for males and 1.50 for females), but smrs were also elevated for the other leading causes of premature mortality : neoplasms, injuries and poisonings, respiratory system diseases and digestive system diseases. endocrine, nutritional and metabolic diseases and immunity disorders smrs were substantially higher than expected in elgin - st. the central south region also had higher than expected all - cause premature mortality, with smrs of 1.06 (95% ci 1.05611.0564) for males and 1.07 (95% ci 1.06781.0683) for females. high smrs of 1.10 (95% ci 1.1031.104) for males and 1.12 (95% ci 1.1151.118) for females in grand river (district health council) contributed to high regional smrs. public health unit area smrs ranged from 1.03 (95% ci 1.0261.027) for males and 1.04 (95% ci 1.0391.040) for females in niagara to 1.13 (95% ci 1.1261.130) for males and 1.14 (95% ci 1.1401.146) for females in brant, demonstrating greater geographic disparity than at the regional and district health council levels. smrs were particularly high (greater than 1.20) for both males and females for circulatory system, respiratory system and digestive system diseases and for injuries and poisonings in males. haldimand - norfolk also had substantially elevated smrs for circulatory system diseases : 1.26 and 1.21 for males and females, respectively. the central west region demonstrated the lowest all - cause premature mortality with smrs of 0.85 (95% ci 0.85110.8513) for males and 0.89 (95% ci 0.89100.8913) for females. district health council area smrs ranged from 0.79 (95% ci 0.78960.7899) for males and 0.84 (95% ci 0.8440.845) for females in halton - peel to 0.96 (95% ci 0.95880.9595) for males and 0.97 (95% ci 0.9700.971) for females in waterloo region - wellington - dufferin. standardized mortality ratios at the public health unit level were similar to those at the district health council level. with the exception of neoplasms among females in halton, halton and peel public health unit smrs were among the lowest in the province for neoplasms, circulatory system diseases, injuries and poisonings, respiratory system diseases, and digestive system diseases. toronto had similar all - cause premature mortality to the province for males with an smr of 0.99 (95% ci 0.98870.9888) and lower than expected premature mortality for females with an smr of 0.92 (95% ci 0.91990.9201). large geographic disparities in all - cause premature mortality were observed in standardized mortality ratios at the public health unit level (i.e., east york, etobicoke, north york, scarborough, toronto city and york city). however, place of residence miscoding is a significant source of error in calculating vital statistics measures for public health units in toronto (personal communication, f. goettler, 2002) ; therefore, the data are not presented. with the exception of digestive system diseases among males, toronto smrs were lower than expected for all of the leading causes of premature deaths : neoplasms, circulatory system diseases, injuries and poisonings, respiratory system diseases, and digestive system diseases. however, infectious and parasitic disease smrs were substantially higher than expected in toronto among both sexes (especially among males). the central east region had lower than expected all - cause premature mortality with smrs of 0.92 (95% ci 0.91860.9188) for males and 0.96 (95% ci 0.96390.9643) for females. at the public health unit level, york 's smrs of 0.78 (95% ci 0.77760.7782) for males and 0.87 (95% ci 0.8700.871) for females contrasted with simcoe 's smrs of 1.05 (95% ci 1.0511.052) for males and 1.05 (95% ci 1.0451.047) for females, demonstrating greater geographic disparity in premature mortality at the small area level. york public health unit also demonstrated smrs among the lowest in the province for neoplasms, circulatory system diseases, injuries and poisonings, respiratory system diseases, and digestive system diseases. the east region all - cause premature mortality was similar to the province for males, with an smr of 1.01 (95% ci 1.0081.009), and higher than expected for females, with an smr of 1.05 (95% ci 1.04791.0484). however, geographic disparity in premature mortality at the public health unit level revealed smrs ranging from 0.88 (95% ci 0.8830.884) for males and 0.95 (95% ci 0.94650.9474) for females in ottawa - carleton to 1.17 (95% ci 1.1691.171) for males and 1.20 (95% ci 1.2031.207) for females in hastings - prince edward. all - cause smrs were also substantially higher than expected for leeds - grenville - lanark and eastern ontario public health unit areas. ottawa - carleton had low smrs for circulatory system diseases, injuries and poisonings, and respiratory system diseases for both males and females. conversely, hastings - prince edward and leeds - grenville - lanark had high smrs in each of the five leading disease chapters for both males and females. eastern ontario public health unit also had high smrs for both males and females for neoplasms, circulatory system diseases, injuries and poisonings, and respiratory system diseases. the north region had the highest smrs, indicating approximately 20% greater than expected all - cause premature mortality relative to the province ; 1.21 (95% ci 1.21051.2110) for males and 1.20 (95% ci 1.19561.1964) for females. all public health unit areas within the north region, except for muskoka - parry sound, had substantially higher than expected premature mortality, ranging from 14% to 32% higher among males and 17% to 31% higher among females. public health unit level all - cause smrs were highest for northwestern (1.28 for males and 1.27 for females) and porcupine (1.32 for males and 1.25 for females). all public health unit areas except muskoka - parry sound and thunder bay had high neoplasm smrs for both males and females. circulatory system smrs were also elevated for all public health unit areas for both males and females except muskoka - parry sound and northwestern. respiratory system smrs were elevated for all public health unit areas except thunder bay and northwestern. standardized mortality ratios for injuries and poisonings were substantially higher than expected in all public health units, ranging from 1.34 to 3.17 for males and 1.23 to 3.58 for females. because neoplasms and circulatory system diseases together account for approximately two - thirds of all deaths, high smrs in these disease categories account for most of the higher all - cause smrs in the north region the south west region had higher than expected all - cause premature mortality, with smrs of 1.07 (95% ci 1.07111.0714) for males and 1.06 (95% ci 1.0591.060) for females. high smrs of 1.12 (95% ci 1.1191.120) for males and 1.11 (95% ci 1.1061.108) for females in essex - kent - lambton (district health council) contributed to high regional smrs. at the public health unit level, chatham - kent had the highest all - cause smrs of 1.26 (95% ci 1.2551.259) for males and 1.29 (95% ci 1.2891.296) for females, which contrasted with other south west public health unit areas whose smrs ranged from 0.95 to 1.12. thomas also had substantially elevated all - cause smrs : 1.10 and 1.12 for males and 1.10 and 1.07 for females, respectively. chatham - kent had particularly high smrs for circulatory system diseases (1.44 for males and 1.50 for females), but smrs were also elevated for the other leading causes of premature mortality : neoplasms, injuries and poisonings, respiratory system diseases and digestive system diseases. endocrine, nutritional and metabolic diseases and immunity disorders smrs were substantially higher than expected in elgin - st. the central south region also had higher than expected all - cause premature mortality, with smrs of 1.06 (95% ci 1.05611.0564) for males and 1.07 (95% ci 1.06781.0683) for females. high smrs of 1.10 (95% ci 1.1031.104) for males and 1.12 (95% ci 1.1151.118) for females in grand river (district health council) contributed to high regional smrs. public health unit area smrs ranged from 1.03 (95% ci 1.0261.027) for males and 1.04 (95% ci 1.0391.040) for females in niagara to 1.13 (95% ci 1.1261.130) for males and 1.14 (95% ci 1.1401.146) for females in brant, demonstrating greater geographic disparity than at the regional and district health council levels. smrs were particularly high (greater than 1.20) for both males and females for circulatory system, respiratory system and digestive system diseases and for injuries and poisonings in males. haldimand - norfolk also had substantially elevated smrs for circulatory system diseases : 1.26 and 1.21 for males and females, respectively. the central west region demonstrated the lowest all - cause premature mortality with smrs of 0.85 (95% ci 0.85110.8513) for males and 0.89 (95% ci 0.89100.8913) for females. district health council area smrs ranged from 0.79 (95% ci 0.78960.7899) for males and 0.84 (95% ci 0.8440.845) for females in halton - peel to 0.96 (95% ci 0.95880.9595) for males and 0.97 (95% ci 0.9700.971) for females in waterloo region - wellington - dufferin. standardized mortality ratios at the public health unit level were similar to those at the district health council level. with the exception of neoplasms among females in halton, halton and peel public health unit smrs were among the lowest in the province for neoplasms, circulatory system diseases, injuries and poisonings, respiratory system diseases, and digestive system diseases. toronto had similar all - cause premature mortality to the province for males with an smr of 0.99 (95% ci 0.98870.9888) and lower than expected premature mortality for females with an smr of 0.92 (95% ci 0.91990.9201). large geographic disparities in all - cause premature mortality were observed in standardized mortality ratios at the public health unit level (i.e., east york, etobicoke, north york, scarborough, toronto city and york city). however, place of residence miscoding is a significant source of error in calculating vital statistics measures for public health units in toronto (personal communication, f. goettler, 2002) ; therefore, the data are not presented. with the exception of digestive system diseases among males, toronto smrs were lower than expected for all of the leading causes of premature deaths : neoplasms, circulatory system diseases, injuries and poisonings, respiratory system diseases, and digestive system diseases. however, infectious and parasitic disease smrs were substantially higher than expected in toronto among both sexes (especially among males). the central east region had lower than expected all - cause premature mortality with smrs of 0.92 (95% ci 0.91860.9188) for males and 0.96 (95% ci 0.96390.9643) for females. at the public health unit level, york 's smrs of 0.78 (95% ci 0.77760.7782) for males and 0.87 (95% ci 0.8700.871) for females contrasted with simcoe 's smrs of 1.05 (95% ci 1.0511.052) for males and 1.05 (95% ci 1.0451.047) for females, demonstrating greater geographic disparity in premature mortality at the small area level. york public health unit also demonstrated smrs among the lowest in the province for neoplasms, circulatory system diseases, injuries and poisonings, respiratory system diseases, and digestive system diseases. the east region all - cause premature mortality was similar to the province for males, with an smr of 1.01 (95% ci 1.0081.009), and higher than expected for females, with an smr of 1.05 (95% ci 1.04791.0484). however, geographic disparity in premature mortality at the public health unit level revealed smrs ranging from 0.88 (95% ci 0.8830.884) for males and 0.95 (95% ci 0.94650.9474) for females in ottawa - carleton to 1.17 (95% ci 1.1691.171) for males and 1.20 (95% ci 1.2031.207) for females in hastings - prince edward. all - cause smrs were also substantially higher than expected for leeds - grenville - lanark and eastern ontario public health unit areas. ottawa - carleton had low smrs for circulatory system diseases, injuries and poisonings, and respiratory system diseases for both males and females. conversely, hastings - prince edward and leeds - grenville - lanark had high smrs in each of the five leading disease chapters for both males and females. eastern ontario public health unit also had high smrs for both males and females for neoplasms, circulatory system diseases, injuries and poisonings, and respiratory system diseases. the north region had the highest smrs, indicating approximately 20% greater than expected all - cause premature mortality relative to the province ; 1.21 (95% ci 1.21051.2110) for males and 1.20 (95% ci 1.19561.1964) for females. all public health unit areas within the north region, except for muskoka - parry sound, had substantially higher than expected premature mortality, ranging from 14% to 32% higher among males and 17% to 31% higher among females. public health unit level all - cause smrs were highest for northwestern (1.28 for males and 1.27 for females) and porcupine (1.32 for males and 1.25 for females). all public health unit areas except muskoka - parry sound and thunder bay had high neoplasm smrs for both males and females. circulatory system smrs were also elevated for all public health unit areas for both males and females except muskoka - parry sound and northwestern. respiratory system smrs were elevated for all public health unit areas except thunder bay and northwestern. standardized mortality ratios for injuries and poisonings were substantially higher than expected in all public health units, ranging from 1.34 to 3.17 for males and 1.23 to 3.58 for females. because neoplasms and circulatory system diseases together account for approximately two - thirds of all deaths, high smrs in these disease categories account for most of the higher all - cause smrs in the north region there were significant disparities in the risk of premature mortality among geographic areas in ontario during the period 19921996. the north region demonstrated approximately 20% higher than expected all - cause premature mortality among both sexes relative to the province. some public health unit areas in the north had greater than 25% higher than expected premature mortality. south west, central south and east regions had higher than expected premature mortality at the regional level ; however, there was significant variation at the district health council and public health unit level. central west, toronto and central east all had lower than expected premature mortality. however, despite lower than expected all - cause premature mortality at the regional level, substantial disparities existed at the public health unit level. previous work has demonstrated geographical variation in other measures of mortality within ontario. from 19881992, life expectancy varied among public health units by as much as 6.0 years for males and 4.3 years for females. in 1996/97, age standardized premature (age 064) mortality rates ranged from 141 to 239 per 100,000 population among district health councils. the current work examined premature mortality at three geographic levels of population simultaneously and clearly identifies greater disparity in premature mortality at the small area level. geographic disparities in premature mortality undoubtedly reflect the underlying distribution of population health determinants such as health related behaviours, and social, economic and environmental influences. tobacco use as a risk factor has been suggested to account for the majority of cancer deaths. data from the 1996/97 national population health survey indicated that the prevalence of regular smoking was significantly higher than the provincial rate among all north region health areas, which may contribute to high neoplasm smrs revealed in the present study (table 5) (see additional file 3). however, due to the lag time required to develop cancer, this potential association assumes that a higher smoking rate is not a new phenomenon, but a characteristic of the resident population over time. health areas in the south west (essex), central south (brant, haldimand - norfolk), and east (hastings - prince edward and leeds - grenville - lanark) also demonstrated smoking prevalence rates which were greater than the provincial average, potentially leading to higher than expected neoplasm smrs. however, other important risk factors for neoplasm smrs must be considered, such as diet, occupation, family history and alcohol. furthermore, research in ontario has also shown significant associations between high neoplasm smrs for males and decreased environmental protection expenditures, which remained significant after controlling for conventional risk factors such as smoking. high circulatory system disease smrs were common in south west, central south, east and north region public health units. conditions or risk factors associated with circulatory system disease deaths include hypertension, diabetes, smoking, high body mass index, inactivity and high stress. data from the 1996/97 national population heath survey (table 5) (see additional file 3) and the 1990 ontario health survey indicate that prevalence rates for many of these conditions were higher than provincial averages in kent - lambton, eastern ontario and in northern ontario. high circulatory system disease smrs in the areas identified in the present study may be related to higher prevalence of these conditions or risk factors. high respiratory system disease smrs were common in the south west, east and north region public health units. data from the 1996/97 national population health survey indicated that the prevalence of regular smoking was significantly higher than the provincial rate among all north region public health units which may contribute to high respiratory system disease smrs. prevalence of regular smoking was also significantly higher in essex ; brant, haldimand - norfolk ; and hastings - prince edward and leeds - grenville - lanark. high respiratory system disease smrs may be related to higher prevalence of smoking in these areas. conventional risk factors such as smoking and low physical activity may be related to observed premature mortality for neoplasms, circulatory and respiratory system diseases. however, determinants of health also include factors such as social class, income and education. geographic distribution of these social and economic determinants of health may also influence premature mortality in ontario. for example, a comparative analysis of ten ontario regional municipalities examined a variety of indicators including work hierarchy and organization, unemployment, social networks, health choices and social rank. municipalities whose indicators were consistently lower than average (hamilton - wentworth, niagara, and sudbury) demonstrated high smrs in the current research. municipalities with determinants of health indicators consistently higher than average (halton and york) demonstrated low smrs in the current research. current results also indicated public health units with the highest smrs (north region) had higher than provincial rates of unemployment, economic families classified as low income, and residents aged 15 + with less than grade 9 education (table 5) (see additional file 3). conversely, public health units with the lowest smrs (halton, peel and york) had lower than provincial rates of unemployment, economic families classified as low income, and residents aged 15 + with less than grade 9 education. previous research in ontario has shown education to be an important variable in understanding premature mortality. this could be a direct effect of greater knowledge of potential health threats, or mediated through higher income, allowing better nutrition and housing. although figures on unemployment, education and income represent data from the 1996 census, it is important to note that social and economic determinants influence health over a lifetime. standardized mortality ratio ranges widened from region to district health council to public health unit (more so for disease chapter specific than all - cause smrs), indicating greater geographic variation in premature mortality at the public health unit level. although the wider range in standardized mortality ratios observed at the small area level may be attributable in part to smaller numbers of observed deaths resulting in less stable estimates, it is important to note that all standardized mortality ratios (except for huron females) were significantly different from 1.00. in most cases, standardized mortality ratios were based on very large numbers of expected and observed deaths leading to very narrow confidence intervals. although death certificate diagnoses and coding practices may lead to classification errors, virtually all deaths in ontario are registered to comply with reporting requirements (incomplete death registration has been noted in certain areas of ontario). even though all - cause mortality is not influenced by diagnosis coding errors, assuming complete death registration, systematic variation in underlying cause of death reporting practices among geographic areas could bias the results of disease chapter specific analyses. this research has examined the extent of geographic premature mortality disparity and considered various determinants of health which may underlie observed variation. knowledge of conventional risk factors such as smoking and physical activity coupled with other determinants of health such as environment, education and social hierarchy are all important to understand health inequalities. although no simple policy prescription is evident from the results, analyses of geographic variation in premature mortality help generate hypotheses for future research aimed at gaining a better understanding of health determinants. clearly, an appropriate policy response to premature mortality disparity in ontario would need to extend well beyond health care to address the wide range of determinants of health that are outside the reach of the health care system. mortality data including place of residence and international classification of diseases (icd)-9 chapter of the underlying cause of death for ontario residents aged 074 for years 19921996 were obtained from vital statistics records, office of the registrar general, ontario ministry of consumer and commercial relations. deaths for all ontario residents (regardless of where they occurred) were included in the tabulations ; deaths of non - ontario residents that occurred in ontario were excluded. population data for years 19921996 were obtained from statistics canada inter - censal population estimates. standardized mortality ratios (smrs) compare observed deaths in a local population to the deaths expected if the local population has been subject to provincial average death rates for each age group, sex and where applicable disease chapter. expected deaths were based on provincial age group, sex and disease chapter - specific mortality rates. to obtain expected deaths, provincial rates were applied to the seven regional planning areas, 16 district health council areas and 37 public health units in ontario, giving the expected number of deaths in the area if the age and sex - specific provincial rates were applied to these populations for each disease chapter. standardized mortality ratios were calculated as observed deaths divided by expected deaths by geographic level, sex and disease chapter. an smr value of one for a geographic area indicates the same mortality rate as for the whole of ontario after accounting for differences in the age distribution of the population between the geographic area and the ontario population. for example, an smr of 1.08 indicates that the region experienced mortality 8% higher than expected. similarly, an smr of 0.92 indicates that the region experienced mortality 8% lower than expected. aggregation of deaths over the five - year period 19921996 reduced the impact of year - to - year fluctuations. this was especially important when analyzing less populous geographic areas, as annual mortality rates calculated from small numbers of observed deaths can vary substantially from one year to the next. the smrs were based on very large numbers of expected and observed deaths leading to very narrow confidence intervals. although no single relative mortality measure is preferable for all purposes, premature mortality (death between birth and age 75) was selected to focus on potentially preventable deaths, given an ontario average life expectancy of 78.9 years in 1996. standardized mortality ratios were used to examine and focus on opportunities to reduce geographic disparity in premature mortality within the determinants of health framework. had the objective of the study been to examine and focus on opportunities to reduce premature mortality as a loss to society, potential years of life lost prior to age 75 may have been a better measure (i.e., deaths at earlier ages result in greater losses to society compared to deaths nearer to the cut - off age). ca and bg drafted the manuscript, participated in the design of the study and the interpretation of results. jh, sb, and je participated in the interpretation of results. all authors read and approved the final manuscript. this table presents standardized premature (ages 074) mortality ratios for males by disease chapter at the public health unit level, 19921996. this table presents standardized premature (ages 074) mortality ratios for females by disease chapter at the public health unit level, 19921996. demographic, social, economic and health characteristics for public health units and health areas, 1996. this table presents demographic, social and economic characteristics from the 1996 statistics canada census at the public health unit level and health characteristics from the 1996/97 national population health survey at the health area level. this work was completed during a studentship program, funded jointly by the centre for health economics and policy analysis and the faculty of health sciences, mcmaster university, hamilton, ontario, canada. | backgroundstandardized mortality ratios are used to identify geographic areas with higher or lower mortality than expected. this article examines geographic disparity in premature mortality in ontario, canada, at three geographic levels of population and considers factors that may underlie variations in premature mortality across geographic areas. all - cause, sex and disease chapter specific premature mortality were analyzed at the regional, district and public health unit level to determine the extent of geographic variation. standardized mortality ratios for persons aged 074 years were calculated to identify geographic areas with significantly higher or lower premature mortality than expected, using ontario death rates as the basis for the calculation of expected deaths in the local population. data are also presented from the household component of the 1996/97 national population health survey and from the 1996 statistics canada census.resultsresults showed approximately 20% higher than expected all - cause premature mortality for males and females in the north region. however, disparity in all - cause premature mortality in ontario was most pronounced at the public health unit level, ranging from 20% lower than expected to 30% higher than expected. premature mortality disparities were largely influenced by neoplasms, circulatory diseases, injuries and poisoning, respiratory diseases and digestive diseases, which accounted for more than 80% of all premature deaths. premature mortality disparities were also more pronounced for disease chapter specific mortality.conclusiongeographic disparities in premature mortality are clearly greater at the small area level. geographic disparities in premature mortality undoubtedly reflect the underlying distribution of population health determinants such as health related behaviours, social, economic and environmental influences. |
the causes of ckd are heterogeneous, ranging from infectious diseases and metabolic multisystemic disease to congenital and genetic disorders. this variety of possible etiologies makes it difficult to identify the mechanisms involved in its pathogenesis. in many cases the annual incidence of end - stage renal disease (esrd) in mexico is 346 cases per million people (pmp), with prevalence of 929 pmp. an estimated 8.5% of the mexican population have ckd, and close to 60,000 individuals are on dialysis. the glutathione s - transferases (gsts) are a group of enzymes that participate in the biotransformation and detoxification of xenobiotics and endogenous substances ; they catalyze the conjugation of glutathione with electrophilic xenobiotics. the gst superfamily is encoded by 16 genes, and at least seven different types of gst have been identified thus far. glutathione s - transferase mu 1 (gstm1) (online mendelian inheritance in man, omim 138350) and glutathione s - transferase theta 1 (gstt1) (omim 600436) are highly expressed in the human kidney, and they are located on chromosomes 1p13.3 and 22q11, respectively. null genotypes of both genes, homozygous deletions, lead to a lack of expression of their respective enzymes. therefore, the null polymorphisms of gstm1 and gstt1 result in a decreased antioxidant defense, high accumulation of reactive oxygen metabolites, and consequent loss of renal function. a few studies have demonstrated the association of genetic polymorphisms of gst with the development of ckd, mostly in diabetes, without any conclusive results. however, patients with esrd have total serum homocysteine three times higher than the general population. hyperhomocysteinemia is common among hemodialysis patients and may result from genetic defects in enzymes involved in homocysteine metabolism. recent studies have associated the methylenetetrahydrofolate reductase (mthfr) gene polymorphisms with the diabetic nephropathy, and hyperhomocysteinemia with microalbuminuria. therefore, the presence of the mthfr c677 t (rs1801133) polymorphism might influence the clinical course and progression of ckd. this study investigates the association between gstm1, gstt1, and mthfr polymorphisms in the context of esrd of unknown etiology in mexican patients. this was a case - control study designed to investigate the association of gst and mthfr polymorphisms in esrd patients. all patients and healthy subjects provided written informed consent to participate in this study, in adherence with the declaration of helsinki and the mexican regulations for health and research. this study was approved by the internal review board of the hospital civil fray antonio alcalde in guadalajara, jalisco, mexico. we recruited 110 patients with esrd of unknown etiology who were referred to the hospital civil of guadalajara fray antonio alcalde. esrd of unknown etiology was defined as esrd with a glomerular filtration rate of 0.05). the association between genotype combination and risk of esrd of unknown etiology was significant among gstm1/gstt1 null carriers (p = 0.01, or = 1.94, 95% ci = 1.14 - 3.32). gstm1 and gstt1 genotype and allele frequencies were also compared with other populations, confirming their genetic variability among populations [figure 1 ]. genotype and allele frequencies of mthfr c677 t, gstm1, and gstt1 polymorphisms in patients with esrd of unknown etiology and healthy participants glutathione s - transferase mu 1 worldwide allele frequencies (wild and null), including 10 populations in addition to our group ckd patients are exposed to oxidative stress as a consequence of an increase in reactive oxygen species or a decrease in antioxidant defense, which in turn leads to the progressive deterioration of kidney function. gst polymorphisms may influence responses to damage induced by oxidative stress, and therefore may be involved in the development and progression of ckd. gstm1 and gstt1 null polymorphisms are caused by the deletion of the gene, and have been studied the most. recent studies have demonstrated increased expression of gsts in epithelial cells of the proximal tubule during the early stage of diabetes, likely in response to oxidative stress triggered by hyperglycemia or other toxic effects of glucose. it has also been reported that the gstm1 + genotype is associated with better survival in elderly peritoneal dialysis patients in the chinese population. lin., reported that the gstm1 null genotype approximately doubled the risk for all - cause mortality among hemodialysis patients ; patients without gstm1 activity are more susceptible to oxidative stress and are at greater risk for death compared with those who possess gstm1 activity. in addition, lin., observed that, among maintenance hemodialysis patients, the gstm1 null genotype was associated with a significantly lower antioxidant capacity than the gstm+ genotype. the null / low polymorphisms of the gstm1 and gstt1 genes have been associated with the risk of developing esrd in north indian patients. in addition, singh., reported that patients with transplant therapy demonstrated an increasing trend toward carrying the gstm1 null genotype (51.3%) versus healthy controls (40.4%) with a risk of about 1.5-fold (p = 0.035), and patients with a variant genotype of gstm1 were at the higher risk of transplant rejection. the frequencies reported for homozygous gstm1 deletions are approximately 50% in caucasian, 21.7% in nigerian, 43% in french, and 58.3% in chinese populations. the gstt1 null polymorphism is present in 13 - 26% of asians and in 35%-52% of caucasians. the combined deletion frequency of both genes, gstm1 and gstt1, is 8% in malaysia, 6% in north america, and 8% in egypt. we compared the gstm1 and gstt1 frequencies obtained from this study with those reported elsewhere [figures 1 and 2 ]. the gstt1 comparison frequencies revealed that there are statistically significant differences among other populations (p < 0.0001), but not in mexico city (p = 0.05). the gstm1 frequencies in the western mexican population were statistically different from south - east asian (p = 0.004), northern european (p = 0.0025), caucasian (p = 0.032), and african populations (p < 0.0001). glutathione s - transferase theta 1 worldwide allele frequencies (wild and null), including 10 populations in addition to our group regarding the genetic distribution of gstt and gstm among esrd patients, there are a few studies reported mainly caused by diabetic nephropathy in asian population. the gstt null frequency is quite low in our experimental population compared with north indians (7% vs. 58.7%, p < 0.0001) ; however, the gstm null is more frequent in our group (60.6% vs. 46.7%, p = 0.02). although these differences may be explained by the genetic structure of our population, it is important to consider that the etiology of esrd may vary among these patient groups. it is also important to highlight the limitations of this study, which detected only the presence or absence of the gstm1 and gstt1 gene ; gene dosage effects could not be assessed. in addition, the enzyme activities of gst and mthfr were not determined, and markers of oxidative stress, such as serum vitamin c, malondialdehyde, carbonyl, and reduced glutathione concentrations, were not measured, neither were homocysteine levels. we observed an association between esrd of unknown etiology and the gstm1 null deletion, but not gstt1 or mthfr c677 t polymorphisms, in mexican individuals in an attempt to associate the clinical variables with the genetic variants. however, additional controlled studies involving enzymes other than gstm1 are required to elucidate whether genetic factors participate in the modulation of glomerular filtration rate. our results suggest that the presence of the gstm1 null genotype, alone or in combination with the gstt1 null genotype, might be associated with an increase in oxidative stress and susceptibility to esrd of unknown etiology in the mexican population, possibly because of the diminished expression of the gstm1 enzyme, which results in a reduced ability to defend against oxidative stress. changes in its function might contribute to the development of esrd through the increase of oxidative stress or the production of free radicals, or might be associated with other factors, such as pollution, nutritional status, smoking, or gene - environment interactions. additional studies are needed to understand the roles of these genes in the development of esrd and to confirm these results. in conclusion, the gstm1 null allele may be is a risk factor for esrd of unknown etiology in mexican individuals. | oxidative stress is increased in chronic kidney disease, owing to an imbalance between the oxidative and antioxidant pathways as well as a state of persistent hyperhomocysteinemia. the enzymes glutathione s - transferases (gsts) and methylenetetrahydrofolate reductase (mthfr) are implicated in the regulation of these pathways. this study investigates the association between polymorphisms in the glutathione s - transferase mu 1 (gstm1), glutathione s - transferase theta 1 (gstt1), and mthfr genes and end - stage renal disease (esrd) of unknown etiology in patients in mexico. a case - control study included 110 esrd patients and 125 healthy individuals. gstm1 and gstt1 genotypes were determined using the multiplex polymerase chain reaction (pcr). the mthfr c677 t polymorphism was studied using a pcr / restriction fragment length polymorphism method. in esrd patients, gstm1 and gstt1 null genotype frequencies were 61% and 7% respectively. gstm1 genotype frequencies differed significantly between groups, showing that homozygous deletion of the gstm1 gene was associated with susceptibility to esrd of unknown etiology (p = 0.007, odds ratios = 2.05, 95% confidence interval 1.21 - 3.45). the mthfr c677 t polymorphism genotype and allele distributions were similar in both groups (p > 0.05), and the ct genotype was the most common genotype in both groups (45.5% and 46.6%). our findings suggest that the gstm1 null polymorphism appears to be associated with the esrd of unknown etiology in patients in mexico. |
cutaneous manifestations of sweet 's syndrome (ss) are typically painful plaque - forming erythematous papules, while bullae are quite uncommon. we present a case of bullous variant of ss in acute myeloid leukaemia. in this case, a 75-year - old male with myelodysplastic syndrome first presented with herpes zoster virus infection - like bullae and erosive plaques on the left side of the face and neck. treatment with valacyclovir and antibiotics was effective only for the initial lesions, whereas the other bullae kept developing predominantly on the left side. histopathological study revealed epidermal bulla formation, pandermal neutrophilic infiltration, erythrocyte extravasation and subepidermal oedema, but no vasculitis. our case was unique in that bullous ss symptoms developed predominantly on one side of the cheek and neck where the herpes zoster infection occurred prior to ss. sweet 's syndrome (ss), also known as acute febrile neutrophilic dermatosis, is characterized by a rapid onset of fever, peripheral leucocytosis, and eruptions. although the precise aetiology is unknown, underlying malignancy, medication and infections have been postulated as a triggering factor of ss [1, 2 ]. cutaneous manifestations of ss are typically painful plaque - forming erythematous papules, while bullae are quite uncommon [2, 3, 4 ]. we report a case of bullous variant of ss, which occurred secondary to herpes virus infection of the left mandible in a patient with acute myeloid leukaemia. a 75-year - old male presented to our department with haemorrhagic and erosive plaques on the left side of the face and neck. five months earlier, he was diagnosed with myelodysplastic syndrome and treated with thalidomide at the department of haematology. about 2 weeks before his referral, his myelodysplastic syndrome turned into acute myeloid leukaemia, followed by abrupt development of blisters involving the left side of the oral mucosa and cheek (fig. 1). since herpes zoster virus infection and subsequent bacterial infection were suspected, valacyclovir and antibiotics were administered. the cutaneous blisters turned into crusts, although other erythematous plaques had newly developed on the left side of the cheek and neck and on the trunk. at the time of referral, the lesions coalesced, forming several large plaques covered with loose blisters (fig. 1, fig. body temperature was above 38c and c - reactive protein was 14.5 mg / dl (normal level : less than 0.2). histopathological study of a skin biopsy specimen from the anterior cervical region revealed epidermal bulla formation, pandermal neutrophilic infiltration, erythrocyte extravasation and subepidermal oedema, but no vasculitis (fig. microorganisms were not observed by ziehl - nielsen, grocott and pas staining of a biopsy specimen. repeated blood cultures, serum beta - d glucan, polymerase chain reaction of mycobacterium species of the skin lesion, quantiferon - tb2 g and cytomegalovirus antigenemia (c10, c11) were all negative. as systemic inflammation and renal and liver damage worsened, prednisone 40 mg / day (1 mg / kg) was initiated. the patient 's systemic and cutaneous condition improved rapidly, without scar formation after the use of steroid. bullous variant is a rare subtype of ss that presents predominantly with neutrophilic bullous lesions, but not with typical erythematous plaques [2, 4 ]. although one retrospective study showed that vesicles and pustules can sometimes be seen to a certain degree in half of ss cases, bullous variant is different from usual ss in that bullae are the dominant eruptions in bullous variant. in this case, important differential diagnosis was bullous pyoderma gangrenosum. however, good response to steroid therapy and healing without scar formation seem to favour the diagnosis of ss. cutaneous manifestations of ss are classified into the following five types : papules and nodules, plaques, bullae, ulcer and subcutaneous inflammation [1, 6 ]. caution has to be taken upon the diagnosis of bullae, ulcer and subcutaneous inflammation, since the ulcer type may mimic pyoderma gangrenosum and subcutaneous inflammation may mimic erythema nodosum. the possibility of bullous ss needs to be considered when blisters develop in febrile patients with haematological disorders. our case was unique in that herpes zoster virus infection seemed to trigger the development of bullous variant of ss. we think the preceding blisters were not caused by ss, but rather by the herpes zoster virus infection because the initial lesion subsided when ss progressed. interestingly, ss developed predominantly on one side of the cheek and neck where the herpes zoster infection earlier occurred. this tendency may be explained by the association between viral infection and development of ss. | aimcutaneous manifestations of sweet 's syndrome (ss) are typically painful plaque - forming erythematous papules, while bullae are quite uncommon. we present a case of bullous variant of ss in acute myeloid leukaemia. in this case, herpes infection of the left mandible had preceded the development of ss.case reporta 75-year - old male with myelodysplastic syndrome first presented with herpes zoster virus infection - like bullae and erosive plaques on the left side of the face and neck. treatment with valacyclovir and antibiotics was effective only for the initial lesions, whereas the other bullae kept developing predominantly on the left side. histopathological study revealed epidermal bulla formation, pandermal neutrophilic infiltration, erythrocyte extravasation and subepidermal oedema, but no vasculitis. the findings suggested the diagnosis of bullous variant of ss.discussionour case was unique in that bullous ss symptoms developed predominantly on one side of the cheek and neck where the herpes zoster infection occurred prior to ss. the tendency may explain the possible association between viral infection and development of ss. |
the paper by pagan., published in this issue, was originally reported as a brief abstract at the society for neuroscience in chicago in october 2015. the world s press and television news channels gave high coverage to this abstract, with some of the study patients shown worldwide in videos that focused on their purported clinical improvements. many neurology experts felt that the global media exposure which followed the initial announcement of the results was an object lesson on how not to report a small clinical trial that has no placebo control. while patients suffering from parkinson s disease (pd) and related alpha - synucleinopathies continue to feel an urgent need for better therapies, this news story inappropriately fed their hope with hype. following the global news story, the authors of this editorial were made aware that the off label use of nilotinib in pd escalated. it is currently conservatively estimated that > 200 pd patients across many countries are taking nilotinib. not only is nilotinib extremely expensive, but it carries a black box warning at its leukemia doses and its safety at low doses in pd and has only been studied in the 12 patients suffering from pd or dementia with lewy bodies (dlb) in the current trial. unfortunately some side effects were noted amongst the 12 patients within the 6 month study period. therefore, definitive proof is now required, one way or the other and as quickly as possible, as to whether nilotinib is safe in pd and dlb, and whether the hints of possible improvement are apparent in a larger group of patients when compared to placebo. nilotinib is a brain penetrant tyrosine kinase cabl inhibitor used for the treatment of chronic myeloid leukemia. it is approved for this condition at doses up to 1200 mg daily (eu) and up to 800 mg daily (usa) where it carries a black box warning for qt interval prolongation ; sudden cardiac death has been reported in patients taking nilotinib, and it can also cause myelosuppression. one patient of the twelve in the study had a serious cardiac event during the 6 month duration of the study, although the investigators felt it was partially related to an initial electrocardiogram (ecg) screening failure. these occur in a considerable number of leukemia patients taking nilotinib at the oncology doses [2, 3 ], and these can lead to fatal cardiac events. taken together, these facts indicate a study reporting on the safety of nilotinib in a small patient cohort during 6 months should be interpreted with caution. pagan and collaborators had selected a group of advanced and relatively frail pd and dlb patients for their trial. why this patient group was targeted is not clear, and indeed in the listing for this trial on the clinicaltrials.gov website it specifies that patients would be at hoehn and yahr stage < 2 as an inclusion criterion. selecting these later stage patients generated a large number of screen failures but, paradoxically, may have helpfully given a tougher test of the safety and tolerability issues surrounding the use of low dose nilotinib. preclinical studies had strongly suggested that cabl inhibition by nilotinib might interfere with pathogenic mechanisms that are relevant to pd and dlb and therefore could potentially modify the course of these diseases [5 16 ]. cabl is substantially activated (by phosphorylation) in brains of pd patients, as well as in mptp and in alpha - synuclein preclinical models of pd. cabl activation by phosphorylation is highly indicative of increased oxidative stress, and in dopaminergic neurons this is thought to contribute to the pathogenesis of pd. cabl inhibition by nilotinib has been shown both to protect against mptp damage, and to reduce intracellular levels of alpha - synuclein by autophagic degradation. also, given it is widely reported that parkin function is compromised in sporadic pd, the observation that nilotinib can also act as a parkin agonist by preventing its phosphorylation also represents yet another important putative mode of action. by inhibiting the phosphorylation of parkin, nilotinib may well offer protection against alpha - synuclein toxicity through a cytoprotective process that would be consistent with observations that overexpression of parkin protects against the effects of -synuclein - induced toxicity. in addition, there is evidence that some parallel parkin - independent benefits of cabl inhibition may also be clinically relevant. in 2012, and again in 2013, an international committee of pd experts discussed in great detail the preclinical evidence for taking nilotinib into a clinical trial in pd patients. at the time, it was questioned whether a low, and potentially safer, dose of nilotinib might be appropriate to test in pd. the conclusion was that, while the biological target (cabl inhibition) is highly relevant in pd, given potential safety issues with nilotinib it was not given top priority as a drug repurposing candidate. thus, the study reported in this issue became the first in - human testing of nilotinib in patients with alpha - synucleinopathies. the daily doses of 150 mg and 300 mg of nilotinib produced some adverse events, but helpfully opened the door on a lower dose range that might be appropriate to consider in the future definitive trial(s) of nilotinib. the choice of these doses was not arbitrary ; preclinical research would suggest efficacy in alpha - synuclein models at even lower doses than this, but nilotinib is only available in capsules of 150 mg minimum size and it is impractical to divide these. future pd trials involving nilotinib will have to determine if this is an optimal dose in pd and dlb. the value of measuring cerebrospinal fluid (csf) levels of the dopamine metabolite homovanillic acid (hva), as reported in this study, is unclear. prior studies suggest that hva levels in csf vary greatly between patients at similar disease stages and track poorly with disease progression. similarly, csf levels of s100b and neuron - specific enolase (nse), also used in the current study and which correlate with damage to neurons and glia, respectively, in stroke and in spinal & intracranial injuries, are not validated biomarkers for pd and dlb [20 22 ]. finally, the changes or lack of changes in alpha - synuclein measures in the study are difficult to interpret given our current understanding of the different molecular forms of this protein and their relation to disease progression. in short, substantial caution should be exercised when interpreting changes in csf levels of hva, s100b, nse and alpha - synuclein. ? given the publicity surrounding the trial when it was initially announced, it is important (as published in this issue) to have the full results of this trial made widely available to the scientific community. but it is impossible to extract definitive safety and valid efficacy signals from a small open - label unblinded study (lacking a placebo control) in pd and dlb. this is especially poignant as even a placebo effect can be very large over the time period used in this study, particularly in advanced pd, and can even induce biochemical changes in the dopamine system [25, 26 ]. perhaps the most important conclusions to be drawn from this study are that:1)a future double - blinded study is definitely warranted. given the importance of the next clinical study evaluating nilotinib, we encourage the involvement of leaders in pd clinical trial design and the use of well - established multi - site clinical study protocols. all raw data from any future study should be made widely available to the research and patient communities to allow detailed evaluation without any restrictions.2)the current study also helpfully gives us a first clue as to what dose(s) of nilotinib might offer a sensible balance between safety concerns and the search for efficacy. as future definitive trials are designed, there is a need further to consider the most appropriate dose and also to determine which pd patients are most likely to benefit from this type of therapeutic approach. a future double - blinded study is definitely warranted. given the importance of the next clinical study evaluating nilotinib, we encourage the involvement of leaders in pd clinical trial design and the use of well - established multi - site clinical study protocols. all raw data from any future study should be made widely available to the research and patient communities to allow detailed evaluation without any restrictions. the current study also helpfully gives us a first clue as to what dose(s) of nilotinib might offer a sensible balance between safety concerns and the search for efficacy. as future definitive trials are designed, there is a need further to consider the most appropriate dose and also to determine which pd patients are most likely to benefit from this type of therapeutic approach. in conclusion, the current paper by pagan. substantiates a new direction, addressing a molecular pathway not previously targeted in a clinical trial in this context, for potential disease modification in pd and dlb. however, this study is just a first step and a major concerted effort is needed to determine whether there is still hope that can match the hype for nilotinib in alpha - synucleinopathies. pb dr. brundin has received commercial support as a consultant from renovo neural, inc., roche, teva pharmaceutical industries, lundbeck a / s, abbvie inc, clearview healthcare, fcb health, ios press partners and capital technologies, inc. additionally he has received commercial support for grants / research from renovo neural inc, teva pharmaceutical industries and lundbeck a / s. | we discuss a report in the current issue on clinical and biochemical findings from a safety trial using the cabl tyrosine kinase inhibitor nilotinib (150 mg or 300 mg given daily for 24 weeks) in a small group of patients with either advanced parkinson s disease or dementia with lewy bodies. despite some side effects (one serious), the authors claim that nilotinib, which is normally used at much higher doses for treating leukemia, is safe and tolerated. furthermore, they report a possible benefit on motor and cognitive outcomes. we debate the safety of nilotinib and the reported efficacy signals. we emphasize that due to the small sample size, and lack of a control group, it is impossible to rule out a placebo effect. we briefly discuss a range of aspects surrounding the current and possible future use of this cabl inhibitor in patients with alpha - synucleinopathy, and what must now be done to obtain definitive information about its safety and efficacy in this population of patients. |
we analyzed the c - prm and the envelope genes of denv-3 isolates related to different clinical manifestations of dengue disease from minas gerais state, brazil, from 2002 through 2004. nine acute - phase serum samples from patients with df or dhf (previously identified by pcr as denv-3) were selected for this study (table). all serum samples were from patients living in the city of belo horizonte or neighboring cities (figure 1). df, dengue fever ; df(hm), dengue fever with hemorrhagic manifestations ; dhf, dengue hemorrhagic fever, according to world health organization definition ;, data not available. location of the city of belo horizonte and its boroughs pampulha, nordeste, and noroeste, minas gerais state, where samples of dengue virus serotype 3 were collected during 20022004. for viral isolation, 50 l of each serum sample was incubated with c6/36 cells, and at least 3 successive passages were conducted for each sample. microscopic examination of cells inoculated with serum from the patients showed a clearly visible cytopathic effect with changes in the monolayer such as syncytial cell formation and cytoplasmic vacuoles after the third passage (data not shown). supernatants of infected c6/36 cells showing typical cytopathic effect were used for viral rna extraction (qiaamp viral rna kit, qiagen, inc., rna was used as template in reverse transcription pcr (rt - pcr), as described (10). for determination of nucleotide sequences in the c - prm region from 9 virus samples,, madison, wi, usa), and 3 clones for each isolate were used in sequencing reactions. to determine the nucleotide sequence of the envelope gene from 4 isolates, we purified pcr amplicons (qiaquick gel extraction kit, qiagen) and directly used in sequencing reactions. each dna sample was sequenced at least 3 times in both orientations (megabace sequencer, ge healthcare, buckinghamshire, uk). the midpoint rooted phylogenetic trees were constructed by the neighbor - joining method with 1,000 bootstrap replicates using the tamura nei model implemented by the software mega 3.1 (arizona state university, phoenix, az, usa). sequence comparisons showed high degrees of identity among our isolates, and the paired identity at the nucleotide level ranged from 99.2% to 100% and from 99.7% to 99.9% regarding the c - prm region and the envelope gene, respectively. when sequences were compared with genotype 3 isolates, including isolates from latin america, the indian subcontinent, and east africa, the identity values ranged from 95.4% to 96.2% and from 94.0 to 95.6% in relation to the c - prm and envelope genes, respectively. when those were compared to genotype 1 sequences from the philippines and china, the nucleotide identities of c - prm region ranged from 98.4% to 99.4% ; when envelope sequences were analyzed, values from 99.1% to 99.5% nucleotide identity were observed. according to phylogenetic clustering with other denv strains (figure 2) both phylogenetic trees showed isolates from brazil grouped together in a well - supported distinct cluster of genotype 1 isolates. phylogenetic trees of established dengue virus serotype 3 (denv-3) and new sequences from minas gerais state, brazil, identified in this study. a) the tree is based on a 504-nt sequence alignment comprising the c - prm gene (nucleotides 137638). b) the tree is based on a 1,023-nt partial e nucleotide sequences (nucleotides 10222008). this tree was generated by neighbor - joining using the tamura nei model implemented by using mega3 software (www.megasoftware.net). numbers to the left of nodes represent bootstrap values (1,000 replicates) in support of grouping to the right various genomic regions of denv have been used for molecular phylogenetic analyses. as described (10,11), the c - prm junction and envelope genes have been used as the most sensitive method for virus detection because they harbor epidemiologically relevant sequence information. although consensus nucleotide sequences of denv isolated from different localities have provided some measure of genetic diversity, only a small number of studies use viruses isolated from the same location. moreover, phylogenetic studies indicated an association between specific genotype and the severity of the disease (8,12). by analyzing a conserved region of the denv genome (504 nt of c - prm gene) and a more variable region (1,023 nt of the envelope gene), we verified that the denv-3 isolates belong to genotype 1 (figure 2). other denv-3 isolates sampled from rio de janeiro, brazil, from 2001 to 2002 during an outbreak and also in latin america were assigned to genotype 3, which has been associated with dhf outbreaks (68,13). phylogenetic studies have shown that denv can move long distances between continents as well as short distances between neighboring countries (8,14). in this study, all denv-3 isolates, which were associated with df, dhf, and a fatal case, belonged to genotype 1. however, no consistent differences among the isolates in relation to c - prm or envelope sequences were found to be associated with distinct clinical outcomes nor did they form phylogenetically distinct groups. if the disease severity in denv-3infected patients does have a genetic basis, it can not be attributed to the c - prm or envelope gene. denv-3 genotype 1 in minas gerais state is more likely to be imported from asia because these isolates were closely related in the phylogenetic tree to the reference strain from the philippines and china (15). it is difficult to determine the precise route of importation of denv into minas gerais because the available data are limited. but one could speculate that the introduction of genotype 1 into minas gerais occurred in 2002, because this genotype had not been detected in that area before 2002. this study shows the emergence of a new denv-3 genotype not only in minas gerais, but in the entire subcontinent, which is a matter for prospective public health studies. | dengue serotype 3 viruses were isolated from patients in brazil from 2002 through 2004. on the basis of phylogenetic analyses, these isolates were assigned genotype 1. this genotype had never been reported in south america before. its appearance indicates a major risk factor for dengue epidemics and severe disease. |
a 37-year - old korean man was referred to our hospital for evaluation of a solitary nodule on his penile shaft. he had been aware of a slow - growing, asymptomatic nodule for the previous 10 years. more recently, however, he experienced pain, tenderness, increased size, and difficulties during sexual intercourse and erectile dysfunction. there was nothing notable in the patient 's past medical history or family history, and he was generally in good health. a physical examination revealed a 2 cm - sized, ovoid, movable lesion on the dorsum of the penile shaft. the results of a complete blood count, electrolyte battery, liver function, renal function, and urine tests were all within normal limits. magnetic resonance imaging (mri) revealed a 2 cm - sized ovoid mass abutting the right corpus cavernosum (fig. the mass located between the corpus cavernosum and buck 's fascia was removed completely (fig. the macroscopic appearance of the removed specimen was a 2 cm sized ovoid and bright reddish white mass such as a bean shape. after surgery, he had no pain, a normal sensation of the penile glans, and his erectile dysfunction had improved. at the present time, 26 months after surgery, the patient has experienced no signs of symptoms or recurrence. nf1 is much more uncommon in the genitourinary tract than in other systems, with the bladder being the most commonly involved organ of the genitourinary tract. only one patient with primary neurofibromas associated with penile neurofibromatosis has been described to date in korea. neurofibromatosis typically presents as genital masses or edema in males and clitoral hypertrophy, mimicking intersex condition, in females. although a penile neurofibroma involves continuity of the dorsal neurovascular bundle with the mass, the latter can be excised completely and safely, while preserving the dorsal bundle. although partial excision may be successful, complete resection is necessary not only for cosmetic and functional reasons but also to prevent recurrent problems and to prevent malignant degeneration. therefore, optimal timing of surgery is necessary to allow complete resection without organ impairment. in our patient, the mass did not involve the dorsal neurovascular bundle and could therefore be resected. difficulties in sexual intercourse may include an inability to penetrate due to the mass, despite experiencing normal sensation and erection, as well as erectile dysfunction, due to arterial stealing by the neurofibroma. a bilateral selective pudendal arteriogram showed that both arteries were hypertrophied and supplied the neurofibroma. thus, the erectile dysfunction in our patient was probably caused by a mass effect and psychological influence. the patient gradually became more reluctant to have sexual intercourse and expressed low levels of self - esteem. after surgery, all his symptoms improved and there was no evidence of recurrence. in patients with functional impairment of the penis, resection of the lesion is associated with good outcomes. | neurofibromas of the penis, although very rare, are often associated with neurofibromatosis type 1. primary solitary neurofibromas of the penis are extremely rare. we describe a 37-year - old man with a solitary neurofibroma in the dorsum of the penis. the patient reported difficulty with sexual intercourse owing to a penile mass and erectile dysfunction. after surgical excision of the neurofibroma, he had no pain and a normal sensation of the penile glans, and his erectile dysfunction improved. at the present time, 26 months after surgery, there has been no evidence of tumor recurrence. |
chloroquine has been in use for many years for the treatment of malaria and for the long term prophylaxis of inflammatory diseases such as rheumatoid arthritis, lupus erythematous, and sjogren s syndrome.1 for the latter indications chloroquine was previously prescribed in dosages of 400600 mg / day but has now been reduced to 250 mg / day.23 ocular toxicity associated with chloroquine has been extensively studied since its description in 1957 by cambaiaggi4 and in 1959 by hobbs.5 it can lead to corneal deposits, lens opacities, and maculopathy causing bull s eye lesion.67 chloroquine retinopathy (cr) is a severe form of retinal toxicity caused by long - term use of chloroquine with an incidence of 116%.89 retinal photoreceptors and retinal pigment epithelium have been postulated as the primary sites of involvement in chloroquine retinopathy.9 once established, cr is irreversible and may progress even after cessation of the drug.9 in this case report, we present the history and clinical findings of a patient with late onset ocular toxicity, 10 years after stopping chloroquine treatment. a 52-year - old female with sjogren s syndrome presented to us with diminution of vision in both eyes for 1 year. she was diagnosed 19 years prior to presentation and had been taking chloroquine (250 mg daily) for 7 years. the best corrected visual acuities using a snellen chart were 6/9, n6 and 6/6, n6 in the right and left eye respectively. she could identify 1 plate correctly in the right eye and 10 plates in the left eye using ishihara s isochromatic plates. indirect ophthalmoscopy of both eyes showed normal looking central fovea with a circular ring shaped area of depigmentation measuring approximately two disk diameters in size akin to a bull s eye appearance. fundus fluorescein angiography showed increased hyperfluorescence due to a window defect in the macular area corresponding to the area of depigmentation and reduced hyperfluorescence due to blockage of choroidal fluorescence in the central fovea [figures 1 and 2 ]. spectral domain optical coherence tomography of both eyes showed reduced central foveal thickness (right eye 108 and left eye 132). both eyes showed generalized loss of outer retinal layers in foveal and parafoveal areas while the left eye showed preservation of the inner segment outer segment (is- os) junction [figures 3a and b ]. multifocal electroretinogram showed reduced central and paracentral ring responses and reduced perifoveal ring responses in both eyes [figures 3c and d ]. based on the history and clinical findings, the patient was diagnosed with late onset chloroquine retinopathy. the patient was informed about the condition and a letter of recommendation addressed to her physician was sent documenting the importance of avoiding chloroquine. fundus fluorescein angiogram showing a ring of increased hyperfluorescence (window defect) surrounding a central area of reduced hyperfluorescence (blocked fluorescence) (a) and (b) optical coherence tomography showing generalized loss of outer retinal layers in parafoveal region in both eyes. (c) and (d) multifocal electroretinogram showing reduced perifoveal ring responses in both eyes in the present case, the patient had placed on chloroquine on a long - term basis over 10 years prior to presentation and had been under the care of an ophthalmologist thereafter. the onset of chloroquine retinopathy more than a decade after stopping chloroquine emphasizes the need for regular follow - up of patients with a history of long - term chloroquine usage. chloroquine is stored in tissues such as the liver and especially the pigmented uveal ocular tissue.10 urinary excretion of chloroquine continues years after cessation of chloroquine.10 these findings buttress the diagnosis of late onset chloroquine retinopathy in our patient. currently there is no definite treatment for chloroquine retinopathy ; hence, it is advisable to discontinue the drug as soon as the signs of toxicity are noted. according to the revised guidelines of the american academy of ophthalmology, each patient who may be placed on long - term chloroquine should undergo a baseline ophthalmic examination which should include both subjective (102 visual field testing) and objective tests (autofluorescence, multifocal electroretinogram, and spectral domain optical coherence tomography).2 these tests should be repeated annually if the patient falls in the high risk category for developing chloroquine retinopathy.2 the present case report reiterates the need for regular follow - up of patients who have been on chloroquine, even after discontinuation of the treatment. this is important both for the ophthalmologist and the physician to educate the patient about the delayed presentation of this disease entity. | chloroquine retinopathy is a known complication of long - term use of chloroquine. this retinopathy can appear even after usage of chloroquine has stopped. the present case report describes the history and clinical features of chloroquine retinopathy developing a decade after discontinuing the drug. |
lymphomas are among the most common cancers and the most common hematologic malignancy in adolescents and young adults (ayas, defined by the national cancer institute (nci) as individuals aged 1539 at diagnosis). in california, non - hodgkin 's lymphoma (nhl) represents approximately 7% of cancers diagnosed in ayas (unpublished data). mortality in aya nhl patients has been found to be higher than in younger children [3, 4 ], attributed in part to a lack of understanding of how best to treat nhl in ayas. recent attention has been given to investigating socioeconomic disparities in survival among cancer patient populations with diverse ethno - racial or socioeconomic backgrounds or with differential access to healthcare [510 ]. concurrently, nci as well as several other cancer advocacy agencies such as the lance armstrong foundation has begun to address the gaps in cancer research for ayas [1, 11 ]. this study sought to examine whether socioeconomic factors beyond race / ethnicity and treatment differences influence survival in ayas with nhl. the following research questions were investigated : does neighborhood - level socioeconomic status (nses) at diagnosis predict all - cause and lymphoma - specific mortality in ayas diagnosed with nhl, after adjustment for race / ethnicity, gender, insurance status at diagnosis, marital status, stage at diagnosis, nodality, and first - course treatment ? is there a linear trend between decreasing nses and shorter survival ? is the relationship between nses and mortality modified by race / ethnicity ? a retrospective case - only analysis was performed of nhl cases diagnosed in california between 1996 and 2005 among individuals aged 15 to 39 years old using the california cancer registry (ccr) (n = 3,762). the ccr, has been part of the nci 's surveillance, epidemiology and end results (seer) program since 1988 [1215 ] with annual patient follow - up. data were abstracted from medical and laboratory records, with tumor site and histology coded using the world health organization (who) criteria in the international classification of diseases for oncology (icd - o, 3rd edition). patient cases were selected according to icd - o-3 coding standards and based on histologic types for nodal and extranodal nhl : seer primary site codes 33041 and 33042. histologic types included burkitt 's (n = 228), diffuse large b - cell (n = 1,746), follicular (n = 480), lymphoblastic, (n = 201), anaplastic large cell (n = 148), and other types (n = 950). seventy - eight cases were identified only through death certificate, obituary, or the social security death index, and an additional two were lost to follow - up. the remaining cases were identified through hospitals, inpatient / outpatient centers, oncology treatment centers, laboratories, or private practitioners. recorded variables in the ccr include age at diagnosis, demographic information, histology, first - course therapy (radiation, chemotherapy, and surgery status), neighborhood ses (nses), vital status, treatment hospital type (pediatric or otherwise), and insurance status. for this analysis, health insurance status at diagnosis was categorized in one of the four following ways : (1) private insurance (including managed care, military and veterans administration, or other private) ; (2) government - funded insurance (including medicare, medicaid, or other state assistance programs) ; (3) no insurance ; or (4) unknown insurance status. individuals with government - provided insurance were not grouped with those who had private insurance because preliminary kaplan - meier analyses indicated that individuals with government - provided insurance had shorter survival than individuals without health insurance at diagnosis, corroborating previously published reports [1820 ]. the following four categories were used in the analysis : non - hispanic white (nhw), non - hispanic black (nhb), hispanic / latino (hl), and asian / pacific islander (api). the nses variable used in the ccr is a single index created from a principle component analysis of census block group - level measures of education, income, occupation, and an adjustment for cost of living, previously described. a subgroup analysis was performed on individuals aged 18 and over at diagnosis to examine the additional predictor of marital status at diagnosis, after all other variables were included in the model. cause of death was recorded according to the icd criteria in effect at the time of death, using icd-9 codes for deaths prior to 2000 and icd-10 codes for deaths in 2000 and later. hospital registrars contact cases annually and ccr staff review state death certificates on an annual basis to identify deceased patient cases. the last date of follow - up was the date of death or last date of contact. kaplan - meier curves were generated for age group, race / ethnicity, nses categories, and insurance status and were compared with the log - rank test. cox proportional hazard regression was performed to generate adjusted hazard ratios (hrs) for all - cause mortality (acm) and lymphoma - specific mortality (lsm) using sas 9.1 (sas institute, inc., cary, nc), controlling for age at diagnosis, race / ethnicity, histology, stage at diagnosis, nses, insurance status, gender, and diagnostic year. a total of 2,432 males and 1,330 females in california aged 1539 at diagnosis with nhl between 1996 and 2005 comprised the study group. table 1 presents distributions of demographic, clinical, and socioeconomic characteristics by race / ethnicity. there was significant variation in age, tumor staging, nodality, first - course chemotherapy, radiation, nses, and health insurance. the majority of the cases (all histologic types) were diagnosed at a late stage, but significantly more (p <.01) non - hispanic black (52%) than asian / pacific islander (34.7%) patients were diagnosed at a distant stage. figure 1 presents the frequencies of nses at diagnosis by race / ethnicity and shows that for nhws and apis, more individuals resided in higher ses areas, while for nhbs and hls, the opposite trend was true. for the remaining survival analyses, individuals with unknown race / ethnicity, stage, or chemotherapy status were excluded (n = 183). during the follow - up period through december 2005 the majority of deaths were due to lymphoma - related causes (n = 593 ; icd-9 codes : 2008, 2019, 2028, icd-10 codes : c819, c829, c833 - 5, c837, c844 - 5, c851, c859). the second most common cause of death was human immunodeficiency virus (hiv) disease resulting in nhl (n = 204 ; icd-9 code : 042, icd-10 codes : b21.0 - 3, b21.7 - 8) ; an additional 43 died due to hiv complications that led to noncancerous diseases (icd-10 code : b227). twenty - four others died of lymphoid leukemia (icd-9 code : 204.0, icd-10 codes : c91.0 - 5), and the remaining deaths were due to other causes (n = 217). table 2 displays the unadjusted and adjusted hazard ratios for both all - cause mortality (acm) and lymphoma - specific mortality (lsm) for this analysis. the estimated average hr increased by 2% (95% ci : 1.011.03) for every increasing year of age at diagnosis after adjustment for all other variables : gender, race / ethnicity, diagnostic year, histological subtype, nodality, stage at diagnosis, nses, insurance status at diagnosis, and first - course treatment with chemotherapy and/or radiation. in the univariate model, nhbs and hls appeared to have increased acm and lsm compared to nhws. however, after adjustment no significant differences in mortality remained between nhws and either nhbs or hls. conversely, adjustment increased rather than decreased the magnitude of difference in lsm for apis compared to nhws. compared to earlier stages, later stage at diagnosis appeared to have a slightly stronger effect on acm after adjustment (adj hr : 3.16, 95% ci : 2.633.81) and the adjusted hr for later stage at diagnosis remained high for lsm (adj hr : 3.14, 95% ci : 2.424.06). extranodal involvement appeared to increase risk of overall death, but only after adjustment (adj hr : 1.29, 95% ci : 1.111.50). for lsm, extranodal involvement appeared to have a protective effect (hr : 0.71, 95% ci : 0.590.86), but there was almost no effect after adjustment (adj hr : 0.99, 95% ci : 0.811.22). not having received chemotherapy as a first - course treatment appeared protective in the unadjusted acm analysis (hr : 0.75, 95% ci : 0.620.91), but in the full model conferred shorter acm (adj hr : 1.27, 95% ci : 1.021.57). on the contrary, not having first - course chemotherapy yielded protective lsm effects for both the unadjusted (hr : 0.24, 95% ci : 0.160.36) and adjusted hr (adj hr : 0.37, 95% ci 0.240.57). results were stratified by stage at diagnosis, and it appears that only in patients with distant - staged nhl was adjusted acm significantly higher than in patients that did not receive first - course chemotherapy (adj hr : 1.69, 95% ci 1.282.24). for patients who did not receive first - course chemotherapy, lsm was improved both in those with localized disease (adj hr : 0.16, 95% ci 0.070.38) and those with regional disease (adj hr : 0.11, 95% ci 0.010.76). not receiving first - course radiation therapy yielded significantly worse hazard ratios for the unadjusted acm (hr : 1.31, 95% ci 1.151.50) and lsm (hr : 1.43, 95% ci : 1.191.71) estimates, but did not have a significant effect after adjustment. the effects of decreasing nses on acm and lsm significantly worsened with every decreasing quintile both before and after adjustment, with the strongest effects evident at the lowest quintile (p <.05) (see figure 2 for overall unadjusted kaplan - meier survival curves). the adjusted hazard ratio for acm for those residing in the poorest ses quintile at diagnosis compared to the wealthiest was 1.40 (95% ci : 1.131.75). all - cause mortality was higher for individuals with government - provided insurance as compared to having no insurance (hr : 1.41, 95% ci : 1.071.87), but after adjustment the effect was only marginally significant (adj hr : 1.32, 95% ci : 1.001.75). for individuals with private insurance, unadjusted survival was longer (hr : 0.62, 95% ci : 0.470.82) compared to those without insurance, although the difference was not significant after adjustment. a subgroup analysis was conducted to examine whether marital status, as a means of social support, conferred longer survival among individuals aged 18 and over at diagnosis. after adjustment for the other demographic and clinical parameters in the full model, individuals who were married at diagnosis had 23% lower acm (adj hr : 0.67, 95% ci : 0.580.78) as compared to those who were single, separated, divorced, or widowed at diagnosis. there was no significant difference in lymphoma - specific survival for individuals who were married at diagnosis, as compared to other marital statuses (adj hr : 1.00, 95% ci : 0.831.20). overall survival analysis was next stratified by the four racial / ethnic groups : nhw, nhb, hl, and api (table 3). in the stratified analysis, a one - year difference in age conferred a significant survival effect only in nhws (adj hr : 1.02, 95% ci : 1.011.04) and hls (adj hr : 1.02, 95% ci : 1.011.04) ; however, the analyses may have been underpowered for nhbs and apis. later stage at diagnosis continued to be the strongest predictor of mortality across all age groups. extranodal involvement was a significant adverse risk factor only for nhws (adj hr : 1.51, 95% ci : 1.211.87). not receiving first - course chemotherapy was a significant adverse risk factor in nhbs (adj hr : 2.02, 95% ci : 1.093.71) and hls (adj hr : 1.71, 95% ci : 1.192.46), but interestingly, a significant protective factor in apis (adj hr : 0.25, 95% ci : 0.080.74). not receiving first - course radiation therapy was not a significant hazard for any of the racial / ethnic groups. after stratification by race / ethnicity, decreasing nses was associated with worse acm in nhws, with the middle (adj hr : 1.33, 95% ci : 1.011.75), low (adj hr : 1.62, 95% ci : 1.222.14), and lowest (adj hr : 2.25, 95% ci : 1.643.08) quintiles having significantly higher hazard of overall death than the highest. having private insurance imparted a protective effect only for hls (adj hr : 0.64, 95% ci : 0.430.95), and having government - provided insurance predicted worse acm in nhbs (adj hr : 5.97, 95% ci : 1.4125.24). this study is one of the first to examine the impact of socioeconomic status on survival in adolescents and young adults with non - hodgkin 's lymphoma. our analyses indicate that nses and treatment variables attenuate much of the racial / ethnic - specific differences in survival and that, after adjustment for demographic and clinical variables, both nhbs and hls tend to show similar survival patterns to nhws. asian / pacific islanders, however, showed significantly poorer lymphoma - specific survival than nhws. being married at diagnosis, a possible indicator of social support, as compared to being single, separated, widowed, or divorced, conferred strong protection against all - cause mortality, but not lymphoma - specific mortality. furthermore, when examined across racial / ethnic groups, a significant gradient in survival by nses was only evident in nhws. although not significant, there was a suggestion of lower survival in apis as nses decreased, but the low numbers of apis in the study likely contributed to wide confidence intervals. for hls, having private insurance contributed to better survival, but for nhb, having government - provided insurance was associated with worse survival. similar findings were reported in a cohort of elderly nhl patients (age at diagnosis 65) in a study examining the association of mortality risk and ses. over three times as many black patients resided in the lowest ses quartile than whites (p <.001). the authors found increasing hazard ratios by decreasing ses quartile after adjusting for race / ethnicity, sex, age, marital status, stage, comorbidity, and therapy, and for both all - cause and nhl - specific mortality. interestingly, individuals in the lowest ses quartile and diagnosed at stages i - ii had a higher adjusted hazard ratio (adj hr : 1.31, 95% ci : 1.191.44) than individuals diagnosed at stages iii - iv (adj hr : 1.22, 95% ci : 1.121.33). finally, after controlling for disparities in stage at diagnosis and treatment, the authors failed to find significant differences in all - cause or nhl - specific mortality between black and white patients. a brazilian study of hodgkin 's lymphoma patients also found higher mortality associated with lower ses that was unexplained by treatment regimen. a scandinavian study investigated ses influences on incidence and survival in adult nhl cases and found decreasing rates of both one- and five - year relative survival by decreasing level of education, dwelling size, and disposable income. however, two other studies of ses impacts on lymphoma survival failed to find a significant association. a hospital - based study in austria that investigated relapse - free survival (rfs) in a cohort of 218 hodgkin 's lymphoma patients (average age at diagnosis = 35.9 15.0 years) found that after adjustment for age survival rates actually decreased with corresponding increases in educational level and income. the authors commented that the findings seemed to be specific only to hodgkin 's patients and may partly be specific to austria 's equal - access healthcare system or to possible underlying immunological differences related to life - course exposures, such as epstein - barr virus positivity. a study on teenaged and young adult cancer patients (aged 1324) in england reported no gradient between survival and a composite measure of area - level poverty among nhl patients. the lack of an association may be partly due to the age range included in this study ; another study that examined nses impacts on survival in leukemia patients aged 039 only found a significant gradient in survival among 3039 year - olds. an investigation on survival in nhl patients in scotland and wales found 10% and 19% shorter survival in intermediate and most deprived areas, respectively,. the study used an area - level deprivation score based on four census variables (car ownership, male unemployment, overcrowding, and social class). race and ethnicity were not reported in the scottish study, perhaps because health research on racial / ethnic differences in survival is less widely conducted in the uk. if the study sample was fairly homogenously caucasian, the results would be consistent with our findings of an nses gradient in survival among non - hispanic whites. not finding an ses - mortality gradient in the non - white patients in our study raises several questions about the cancer experience in these populations. first, it is important to reiterate that for nhbs and hls, although the unadjusted hazard ratios for both acm and lsm were significantly higher than for nhws, adjustment for the other factors in the model including nses, stage at diagnosis, and first - course treatment attenuated these risks. as evident in figure 1, there were far more nhbs and hls residing in poorer nses areas, which often have lower access to resources. thus, it is possible the differences in survival by race / ethnicity commonly reported may be due more to confounding by later initiation of and poorer access to care, a phenomenon noted in hispanic populations. measuring access to care is quite difficult, particularly in a registry - based analysis, and further studies should be done to examine why a gradient exists for nhws but not other racial / ethnic groups. differential access to the most effective treatment regimens may still persist for lower ses groups. the lack of a consistent association between health insurance status and survival after adjustment was surprising, given the widely - documented increased vulnerability of patients lacking health insurance. finding higher all - cause mortality among those with government - provided insurance compared to those without insurance suggests that there may be important disparities in access to care among medicaid recipients. approximately double the percentage (17.8%) of those without health insurance compared to those with government - provided health insurance (9.4%) resided in the highest nses quintile at diagnosis. a paper analyzing the relationship of health insurance status and cancer outcomes across us demographic groups found striking disparities in cancer screening, stage at diagnosis, and survival for those uninsured or insured by medicare or medicaid. individuals in the general population aged 1824 were also found to have the highest probability of lacking insurance or being insufficiently insured. access to adequate healthcare coverage can affect cancer care, including challenges of covering premiums, deductibles, and co - payments, and difficulty remaining employed and eligible for insurance benefits [19, 29 ]. one study found an average increase of 13.1 weeks in wait time between diagnosis and initiation of treatment for non- or government - insured patients as compared to private health insurance, although no associations between wait times and ses, gender, age, race / ethnicity, or marital status were found. one of the strengths of this study is the use of ccr data with a large, heterogeneous, and population - based cohort with almost complete patient ascertainment and follow - up. registry - based explorations of factors that contribute to longer survival are important to help identify groups that are particularly vulnerable to premature cancer mortality. because social determinants affect health outcomes along several pathways, it is important to document the existence of persistent health disparities, particularly for understudied groups such as ayas. the limitations of this study include the estimation of ses based on the residence at diagnosis, which may not accurately capture some factors that contribute to healthy living environments and adequate medical care. transitioning through developmental life stages can make ayas a heterogeneous group ; some are dependent on parents and relatives while others provide for families of their own. as such, measuring ses as a one - time neighborhood composite variable may inadequately summarize an individual patient 's social and financial circumstances. the way that first course of treatment is measured in the registry is also somewhat limited ; it is not possible to know the type of therapy, the dose - intensity administered, and other factors related to the treatment regiment that may have significant influence on survival. furthermore, knowing a patient 's health insurance status only at diagnosis does not reveal whether the insurance was sufficient in covering the costs associated with cancer treatment, nor does it reveal whether there were any subsequent lapses in insurance coverage. these findings do not appear to be limited to the us ; however, comparability across borders may be limited due to differing national health systems. patient contact studies that address individual socioeconomic barriers to treatment and recovery and that can better guide potential interventions are warranted, as is the development and evaluation of programs specifically designed to meet the needs of the diverse and unique aya population. our study is one of the first to examine socioeconomic impacts on survival in ayas with nhl. we determined that as neighborhood ses at diagnosis increases, overall- and lymphoma - specific survival improves, after adjustment for demographic and treatment variables, and a linear trend persists. the impact of ses on mortality appeared to be independent of health insurance status at diagnosis. however, when stratified by race / ethnicity, the effects of nses on mortality were only significant in non - hispanic whites. | shorter survival has been associated with low socioeconomic status (ses) among elderly non - hodgkin 's lymphoma (nhl) patients ; however it remains unknown whether the same relationship holds for younger patients. we explored the california cancer registry (ccr), to investigate this relationship in adolescent and young adult (aya) nhl patients diagnosed from 1996 to 2005. a case - only survival analysis was conducted to examine demographic and clinical variables hypothesized to be related to survival. included in the final analysis were 3,489 incident nhl cases. in the multivariate analyses, all - cause mortality (acm) was higher in individuals who had later stage at diagnosis (p <.05) or did not receive first - course chemotherapy (p <.05). there was also a significant gradient decrease in survival, with higher acm at each decreasing quintile of ses (p <.001). overall results were similar for lymphoma - specific mortality. in the race / ethnicity stratified analyses, only non - hispanic whites (nhws) had a significant ses - acm trend (p <.001). reduced overall and lymphoma - specific survival was associated with lower ses in ayas with nhl, although a significant trend was only observed for nhws. |
the two - peptide lantibiotic haloduracin is composed of two post - translationally modified polycyclic peptides that synergistically act on gram - positive bacteria. we show here that hal inhibits the transglycosylation reaction catalyzed by pbp1b by binding in a 2:1 stoichiometry to its substrate lipid ii. hal and the mutant hal-e22q were not able to inhibit this step in peptidoglycan biosynthesis, but hal with its leader peptide still attached was a potent inhibitor. combined with previous findings, the data support a model in which a 1:2:2 lipid ii : hal:hal complex inhibits cell wall biosynthesis and mediates pore formation, resulting in loss of membrane potential and potassium efflux. |
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the endoplasmic reticulum (er) has diverse biological functions associated with cellular homeostasis and whole organism physiology. these include the biosynthesis, folding, posttranslational modification, assembly, and trafficking of membrane and secretory proteins. thus, the er provides quality control of those proteins, which ensure that only properly folded proteins are exported via the secretory pathway. unfolded or misfolded proteins leave the er to undergo ubiquitin - mediated proteasomal degradation, known as er - associated degradation (erad). the er is also tightly linked to cellular calcium homeostasis, since er luminal calcium binding proteins comprise the major intracellular calcium reservoir. the er is also a major cellular compartment of lipid biosynthesis including cholesterol, phospholipids, triglycerides, and sterols, for the assembly of very low density lipoprotein (vldl) in hepatocytes, and for lipid droplet formation in adipocytes. er homeostasis can not be maintained if physiological or pathological insults disturb protein - folding processes, resulting in er stress. a variety of stimuli can trigger er stress, including folding - defective mutations, disturbance of metabolic and calcium homeostasis, viral infection, inflammation, hypoxia, and oxidative stress. to resolve er stress, the er activates an elaborate adaptive response by triggering several signal transduction cascades collectively known as the unfolded protein response (upr). the upr was first discovered in yeast for a single upr signaling pathway based on the er transmembrane protein inositol - requiring enzyme 1p (ire1p). however, the mammalian upr is more diverse, consisting of at least three major signaling cascades from three er transmembrane proteins : ire1, which exists in and forms ; pkr - like er kinase (perk) ; and activating transcription factor 6 (atf6) (fig. it is generally believed that a primary short - term response to cope with er stress should be an acute decrease in rate of protein translation to prevent further accumulation of proteins in the lumen of the er. for the longer - term response, upr pathways rely on transcriptional mechanisms, typically inducing the expression of target genes encoding chaperones that increase er protein - folding fidelity, and proteins that are essential for the erad machinery. if the activation of these upr pathways is not enough to alleviate er stress, chronic upr initiates cellular apoptosis. under normal circumstances, these transmembrane er stress sensors and their signal transducers are inert due to their association with the major luminal er chaperone, immunoglobulin heavy chain bip (also known as glucose - regulated protein 78). during er stress, bip dissociates from the luminal domains of ire1, perk, and atf6 to bind to unfolded or misfolded er luminal proteins, which lead to the activation of the upr. in addition to the dissociation of bip from these three transmembrane proteins, there are other upr sensors and mechanisms proposed. for example, the luminal domain of ire1 itself may directly interact with unfolded or misfolded proteins ; the glycosylation of atf6 is profoundly decreased during er stress ; or thioredoxin - interacting protein (txnip) regulates er stress via protein disulfide isomerase (pdi) activation. ire1 is a single er transmembrane protein that contains an er luminal domain sensing unfolded or misfolded proteins, as well as a cytoplasmic domain with both intrinsic kinase activity and endoribonuclease (rnase) activity. in homeostatic conditions, kinase and rnase activities of ire1 are inactive due to its association with bip. upon a variety of stress conditions, free ire1 forms homodimers or oligomers, which increases autophosphorylation of the cytoplasmic domain this eventually triggers its specific rnase activity, which results in an unconventional splicing process that removes 26 nucleotides from the x - box binding protein 1 (xbp1) mrna. converting the unspliced form of xbp1 mrna (xbp1) into the shorter spliced form of xbp1 mrna (xbp1s), leads to the efficient translation of xbp1s protein. it acts as a basic leucine zipper (bzip) transcription factor to induce nuclear target genes encoding er chaperones, erad components, lipid and phospholipid biosynthetic enzymes, and inflammatory components. moreover, with its nonspecific rnase activity, ire1 promotes the degradation of er - localized mrnas, a process called a regulated ire1-dependent decay (ridd) of mrna, which contributes to the reduction of protein loading in the er. mrna substrates cleaved by ridd mechanism include genes involved in the lipid metabolism and specific micrornas that derepress translation of proapoptotic genes. ire1 also activates c - jun n - terminal kinase (jnk) and ib kinase (ikk) by recruiting the scaffold protein tumor necrosis factor receptor - associated factor 2 (traf2) and the apoptosis signal - regulating kinase 1 (ask1). jnk and ikk are two major kinases to induce ser phosphorylation of insulin receptor substrate 1. this results in inhibition of its tyr phosphorylation by insulin receptor, which leads to insulin resistance (ir). there are two mammalian homologs of yeast ire1p : ire1 is ubiquitously expressed in cells and tissues but expression of ire1 is limited to the epithelium of the gastrointestinal tract where it plays an important role in dextran sodium sulfate (dss)-induced colitis probably via contributing to efficient protein folding and secretion of mucin2, a major secretory protein in goblet cells. atf6 is an er transmembrane protein with a cytoplasmic transcription activator domain and an er luminal domain that senses protein - folding status. upon er stress, atf6 dissociates from bip and moves from the er to the golgi apparatus where it is cleaved by the mechanism of regulated intramembrane proteolysis (rip) performed by the two serine proteases, site 1 and site 2 proteases (s1p and s2p, respectively). the cleaved cytoplasmic domain is then translocated into the nucleus, where it acts as a bzip transcription factor to regulate expression of xbp1 and other genes involved in er chaperones, erad components, protein foldases, and c / ebp homologous protein (chop). chop also acts as a transcription factor that induces the expression of genes encoding the growth arrest and dna damage - inducible protein (gadd34), a regulatory subunit of protein phosphatase 1 (pp1), and er oxidoreductin 1 (ero1). it is of interest to note that the same mechanism of rip can cleave other atf6 homologs with relatively less known functions. these include camp - response element - binding protein h (crebh), camp - response element - binding protein 4 (creb4), old astrocyte specifically induced substance (oasis), and box - binding factor-2 human homolog on chromosome 7 (bbf2h7). under er stress, bip dissociation also allows perk homodimerization and subsequent autophosphorylation, activating its cytoplasmic kinase domain. activated perk is able to phosphorylate the subunit of eukaryotic initiation factor 2 (eif2) at ser 51, which inhibits the activity of the guanine nucleotide exchange factor eif2b. thus, eif2 phosphorylation rapidly decreases the initiation of mrna translation ; thereby, reducing the load of newly synthesized proteins in the er. paradoxically, the perk - eif2 pathway facilitates translation of atf4, which increases transcription of genes involved in amino acid synthesis and apoptosis, such as tribbles homolog 3 and chop. subsequently, chop is able to increase the expression of gadd34 that targets pp1 to eif2 for its dephosphorylation. thus, atf4 provides a negative - feedback loop to control perk - eif2 signaling cascades. it is now appreciated that both the atf6 and perk pathways trigger apoptosis by increasing chop expression. it is worth emphasizing that, in addition to its response to er stress, eif2 is also phosphorylated by three other kinases : double - stranded rna (dsrna)-activated protein kinase (pkr), general control non - derepressible kinase 2 (gcn2), and heme - regulated inhibitor kinase (hri), that are respectively activated by dsrna, amino acid deprivation, and iron / heme deprivation. because of its activation by various stresses, the eif2-atf4 pathway has been designated as the integrated stress response. recent evidence indicates that the er is associated with both the development of ir and its progression to type 2 diabetes mellitus. one of the mechanisms by which er stress contributes to the development of hepatic ir is the activation of transcription factors that regulate the expression of genes involved in gluconeogenesis. however, depending on which upr pathway is activated, gluconeogenesis could be enhanced or compromised. for example, activation of crebh, an atf6 homolog upon er stress primarily in the liver increases the expression of the gluconeogenic genes phosphoenolpyruvate carboxykinase (pepck) and glucose-6 phosphatase (g6pase), along with inflammatory markers such as c - reactive protein. by contrast, generating xbp1s by er stress - mediated ire1 pathway induces ubiquitin - mediated proteasomal degradation of forkhead box o1 (foxo1), resulting in reduced gluconeogenesis. hepatic ir by er stress may also be the result of increased lipogenesis, which leads to intracellular accumulation of diacylglycerol (dag) and ceramide. dag accumulation shows toxic effects for the hepatocytes and is pivotal for er stress to cause ir and hepatic steatosis. in this regard, exposure of hepg2 cells to hyperglycemia or the saturated fatty acid (fa) palmitate, induces er stress and activates the perk - eif2 pathway. this in turn activates the transcription factor sterol regulatory element binding protein (srebp)-1c to increase lipogenesis and lipid accumulation. this pathway may also contribute to er - stress - induced hepatic steatosis through the increased expression of hepatic vldl receptor, which promotes lipoprotein delivery to the liver. the mammalian target of rapamycin complex 1, a key regulator of srebp-1c expression, also activates er stress, which increases hepatic ir and lipid accumulation. an additional mechanism by which er stress activates srebp-1c processing includes rapid degradation of its upstream inhibitor insulin - induced gene 1 (insig1) via ridd mechanism. a previously dominant hypothesis that hepatic ir is secondary to er stress - mediated lipogenesis has been challenged by a relatively recent hypothesis that fat accumulation per se does not induce ir. the latter hypothesis is supported by the findings that the chop knockout (ko) mice showed normal glucose tolerance and insulin sensitivity despite marked obese phenotype. this discrepancy seems to be due to diminished inflammation in fat and liver tissues of chop ko mice, reinforcing the idea that ir is not induced by fat accumulation per se, but rather by inflammatory signaling cascades controlled by chop. by contrast, it has been shown that er stress - mediated perk activation may decrease insulin responsiveness through phosphorylation of foxo1 in hepatocytes. since insulin signaling reduces foxo1 activity via akt and promotes insulin responsiveness, it has been suggested that inhibition of perk might improve insulin signaling in the liver. fibroblast growth factor 21 (fgf21), an endocrine hormone predominantly secreted by the liver has a broad range of effects on metabolism. recent studies indicate that either er stress or autophagy deficiency induced fgf21 expression via the ire1-xbp1 or perk - atf4 pathway, respectively. moreover, diet - induced obesity led to decreased autophagic activity by downregulating autophagic gene expression, and to reduction in the activity of the proteasome in the liver. this was in turn associated with activation of chronic upr, hepatic ir, and increased glucose production. conversely, administration of recombinant fgf21 peptide or restoration of autophagic activity in the liver of obese mice alleviated tunicamycin - induced er stress and steatosis, and improved both hepatic insulin action and systemic glucose tolerance. as fgf21 exerts beneficial effects on lipid metabolism through counteracting er stress, it is possible that it may display antiobesity and antidiabetic activity. the nuclear hormone receptor liver receptor homolog-1 (lrh-1, also known as nr5a2) is expressed in tissues of endocrine origins including liver, pancreas, and intestine as well as reproductive tissue such as ovary. lrh-1 plays a pivotal role in pathways that appear quite distinct from er stress, including bile acid homeostasis, embryonic development and pluripotency of embryonic stem cells, and intestinal steroidogenesis. however, a potential link between lrh-1 and er stress resolution was raised by the observation that lrh-1 activation improves type 2 diabetes mellitus by alleviating nonalcoholic fatty liver disease (nafld). this was confirmed by our recent study showing that lrh-1 is necessary for resolution of acute er stress (fig. 2). this is dependent on a novel pathway in which er stress - dependent lrh-1 activation induces expression of polo - like kinase 3 (plk3) gene, which leads to increased phosphorylation of atf2. liver - specific lrh-1 ko (lrh-1), plk3 ko mice, and mice with decreased atf2 activity were all defective in er stress resolution upon tunicamycin challenge. interestingly, restoring plk3 expression in lrh-1 mice was sufficient to resolve er stress, demonstrating that plk3 is an important lrh-1 target gene in er stress resolution. as expected, treatment with an lrh-1 agonist protected primary hepatocytes against the toxic effects of strong upr activation. the molecular mechanisms for how er stress activates lrh-1 and how atf2 contributes to er stress resolution remain to be determined. at this stage, however, it is intriguing that the lrh-1-plk3-atf2 signaling pathway for er stress resolution seems to be independent of the three canonical upr pathways. more broadly, these studies suggest that targeting lrh-1 may provide a new therapeutic intervention in not only ir and type 2 diabetes mellitus, but also other human diseases caused by chronic er stress. several other members of the nuclear receptor superfamily affect er stress and upr pathways in liver, as well as in other tissues. increased hepatic gluconeogenesis is one of the hallmarks of ir and type 2 diabetes mellitus. particularly, glucocorticoid receptor (gr, also known as nr3c1) potently activated by endogenous glucocorticoids or a synthetic agonist dexamethasone is known to markedly increase hepatic gluconeogenesis by inducing the transcription of g6pase and pepck genes. earlier results have shown that the orphan nuclear receptor estrogen - related receptor (err, also known as nr3b3), a gluconeogenic transcription factor, is linked to er stress. this was supported by the observation that err can also induce expression of both atf6 and the truncated form of crebh that is activated upon er stress. the central role of err is reinforced by the observation that it is required for induction of crebh in response to the er stress inducer tunicamycin. it is likely that the antidiabetic effects of the err inverse agonist gsk5182 are due to inhibition of these er stress related responses as well as direct suppressive effects on gluconeogenic gene expression. a recent report has linked increased nafld in the livers of aged mice to downregulation of farnesoid x receptor (fxr, also known as nr1h4) signaling induced by er stress. the chemical chaperone tauroursodeoxycholic acid, a poor agonist ligand for fxr in vitro, significantly increased hepatic fxr expression and decreased lipogenic gene expression in aged mice. er stress inducers such as tunicamycin or thapsigargin also decreased expression of fxr and its downstream target genes, small heterodimer partner (shp, also known as nr0b2) and bile salt export pump (bsep, also known as abcb11). the decrease in fxr expression was attributed to er stress - mediated inhibition of hepatocyte nuclear factor 1 (hnf1) transactivation of the fxr promoter. decreased activity of peroxisome proliferator - activated receptor (ppar, also known as nr1c1) via gene ko or perturbation of its signaling by feeding high - fructose diet impairs mitochondrial fa oxidation and increases hepatic mitochondrial stress. decreased ppar activity was also linked to compromised expression of the sarco / endoplasmic reticulum calcium atpase (serca) and to induction of er stress and hepatic steatosis. in contrast, treatment of high - fructose diet - fed rats with the ppar agonist wy-14643 increased fa oxidation and strongly decreased both steatosis and markers of er stress. in another study of high - fat diet - fed mice, treatment with the selective ppar agonist fenofibrate completely reversed glucose intolerance and hepatic ir but did not decrease either hepatic steatosis or inflammatory signaling associated with jnk or ikk. this seems to be attributed to sequestration of dag, inhibitors of insulin signaling, into the lipid droplet / er compartment and away from the plasma membrane. overall, it is quite likely that increased hepatic fa oxidation contributes to the documented antidiabetic effects of fibrates in human clinical trials, and this may contribute to decreased er stress. er stress - activated indicator transgenic mice in which green fluorescent protein (gfp) expression is induced under er stress conditions were used to examine whether pioglitazone, a selective ppar (also known as nr1c3) agonist, improves er stress in vivo. in high - fat and high - sucrose diet - fed transgenic mice, gfp was clearly decreased as early as 4 weeks after initiation of pioglitazone treatment, prior to the improvement of ir. this study suggests that the antidiabetic effects of pioglitazone may be at least due to reducing er stress in the liver. thus, novel dual agonists for ppar/ showed antidiabetic effects associated with improving fatty liver, inflammation and er stress in metabolic tissues of leptin receptor (lepr)-deficient db / db mice. finally, liver x receptors (lxr and, also known as nr1h3 and nr1h2, respectively) agonist treatment has been reported to decrease saturated fa - induced er stress in primary hepatocytes by inducing expression of the remodeling enzyme lysophosphatidylcholine acyltransferase 3 (lpcat3), which modulates membrane phospholipid composition by increasing incorporation of unsaturated fa chains. although direct studies of lxr activation in vivo are complicated by its potent lipogenic effect, lpcat3 overexpression has been shown to be sufficient to ameliorate er stress and also improve glucose homeostasis in livers of both ob / ob and db / db mice. in this review, we have briefly described current understandings of how er stress activates upr pathways, and of how chronic er stress causes peripheral ir. in addition, we have described how nuclear receptors contribute to er stress resolution and er stress - mediated apoptosis in different metabolic tissues with an emphasis on the aspects of nuclear receptor lrh-1 in hepatic er stress resolution, which seems to be independent of canonical three branches of upr pathways. it would be of great interest to further investigate why the intact activation of each canonical upr signaling could not be sufficient for resolving tunicamycin - mediated er stress in lrh-1 mice. it would also be of interest to understand how lrh-1/plk3/atf2 pathway could integrate into canonical upr signaling pathways. lastly, it would be necessary to further understand the roles of nuclear receptor lrh-1 upon other kinds of physiologic or pathologic er stresses, which might provide a therapeutic strategy to overcome diverse metabolic diseases. | chronic endoplasmic reticulum (er) stress culminating in proteotoxicity contributes to the development of insulin resistance and progression to type 2 diabetes mellitus. pharmacologic interventions targeting several different nuclear receptors have emerged as potential treatments for insulin resistance. the mechanistic basis for these antidiabetic effects has primarily been attributed to multiple metabolic and inflammatory functions. here we review recent advances in our understanding of the association of er stress with insulin resistance and the role of nuclear receptors in promoting er stress resolution and improving insulin resistance in the liver. |
neonatal facial birth injuries are easily recognizable, but most under - reported form of birth injuries. they are often associated with face presentations, which occur in every 600 - 800 births at term gestation. face presentation usually presents as a deflexed head which becomes extended at pelvic inlet during labor. anything that delays or prevents flexion such as fetal anomalies, contracted pelvis, fetopelvic disproportion or cord around the neck can contribute to face presentation. the presenting part may feel soft and lumpy and the landmarks of the face are felt. the bridge of the nose is an important landmark and often the baby will suck on the examining finger due to rooting reflex. repeated per vaginal examination to assess the presenting part and the progress of labor may lead to bruises in the face as well as damage to the eyes. we describe one such potential case of preventable birth injury involving face, sustained by an unborn child with face presentation, in an institutional delivery. a 22-year - old primigravida was admitted at term with active labor in our tertiary care teaching hospital. per vaginal examination at admission as face presentation was quite uncommon, all our enthusiastic residents as well as the medical students posted in labor room wanted to have a feel of the face during labor. this resulted in repeated unofficial per vaginal examinations in spite of having strict guidelines in place. after delivery, baby was noted to have prominent ulcerations, bleeding and edema of the face [figure 1 ]. the infant also had injury to right eye with eyelid ulceration, swelling, subconjunctival hemorrhage and difficulty in breathing due to swollen tongue. respiratory distress settled within 2 days and the child was finally discharged after 7 days of hospitalization. facial ulcerations secondary to per vaginal examinations in face presentation iatrogenic neonatal facial injuries are rare, but are one of the most easily preventable neonatal injuries. teaching institutions should have strict guidelines in place in order to avoid iatrogenic birth injuries. | birth injuries involving face are easily recognizable, but are often under - reported. most of these injuries are associated with face presentation. we report an iatrogenic, but potentially preventable facial birth injury sustained by an unborn child in institutional setup. |
the cyclic mononucleotides camp and cgmp are potent signaling molecules that exert profound effects on cellular homeostasis. camp, which was originally identified by its ability to cause breakdown of glycogen in response to adrenaline, is produced by adenylyl cyclase, an enzyme first characterized in 1958. it took another decade to establish that cgmp and the enzyme responsible for its synthesis, guanylyl cyclase, are also present in mammalian tissues. in the rod and cone cells of the retina, cgmp plays a central role in the visual signal transduction cascade by opening ligand - gated ion channels in sensory neurons. cgmp is also produced in response to hormones, such as nitric oxide and atrial natriuretic peptide, which leads to the relaxation of vascular smooth muscle and is thus important for regulation of blood pressure. now, more than a decade after the first reported adenylyl cyclase structures, two atomic structures of a guanylyl cyclase catalytic core have been reported. the first describes a cyanobacterial guanylyl cyclase from synechocystis pcc6803 and the second a eukaryotic guanylyl cyclase from the green alga chlamydomonas reinhardtii. they provide a fresh look at the molecular basis for nucleotide selectivity and allosteric regulation in these important enzymes. mammalian adenylyl and guanylyl cyclases belong to the class iii nucleotide cyclase family. atomic structures of mammalian adenylyl cyclase [6 - 8 ] showed that the catalytic core of class iii cyclases consists of either a homodimer or a heterodimer of homologous domains that associate to form a wreath - like structure (figure 1, top left). one or two active sites are formed at the interface between the domains. in mammalian adenylyl cyclases, the catalytic core is composed of homologous domains (c1 and c2) that form only one functional active site. the non - functional active site is the binding site for the activator forskolin, a well - known hypotensive agent. comparison of the c. reinhardtii guanylyl cyclase catalytic core (green) with that of the c1:c2 domains of activated mammalian adenylyl cyclase (pdb i d : 1cju ; blue) bound to a nucleotide analog (shown as a stick model). the two active sites of the guanylyl cyclase are found in a cleft formed at the interface of two identical subunits (top left). the two subunits form a wreath - like structure, with active sites located at either end of the cleft (bottom left). the guanylyl cyclase structure is presumed to be in an inactive conformation : in the active state of the nucleotide cyclases, it is expected that helix 1 (labeled in the right - hand panel) moves into the active site and toward helix 4 of the other subunit of the dimer. the arrows in the right - hand panel indicate the conformational changes required to generate the active configuration. from winger. most mammalian adenylyl cyclases are large transmembrane proteins and subject to complex regulation, most importantly through direct interactions with heterotrimeric gtp - binding (g) proteins (figure 2, left - hand side). in contrast, mammalian guanylyl cyclases are modular receptor enzymes : they exist as homo- or heterodimers with one catalytic domain in each polypeptide chain, and their activity can be modulated directly by hormones or other factors that bind to their accessory domains (figure 2, right - hand side). mammalian adenylyl cyclases (left - hand side) are typically transmembrane proteins activated by heterotrimeric g proteins. mammalian guanylyl cyclases (right - hand side) are receptor enzymes directly activated by hormones, such as nitric oxide (no), or other factors that bind to its regulatory domains (for simplicity, the regulatory domains are not shown). not surprisingly, the catalytic core of both the synechocystis cya2 guanylyl cyclase (which has approximately 30% sequence identity to mammalian adenylyl cyclase and guanylyl cyclase isoforms) and the c. reinhardtii enzyme (which has 4050% sequence identity to mammalian guanylyl cyclases) adopt the same wreath - like structure that was described for adenylyl cyclase (figure 1). discrimination between gtp and atp among class iii nucleotide cyclases is critical for the fidelity of signaling by cgmp and camp. this specificity is determined, at least in part, by a pair of active - site residues (glu and cys) in guanylyl cyclases and their counterparts (lys and asp) in adenylyl cyclases. for example, when the guanylyl cyclase glu - cys pair is replaced with a lys - asp pair, the guanylyl cyclase is converted into a highly specific adenylyl cyclase. although the adenylyl cyclase lys - asp pair interacts directly with the purine base, it is not known for certain whether their equivalents in guanylyl cyclases contribute to specificity in the same manner. unfortunately, neither of the guanylyl cyclase structures contains a bound ligand to help define the mechanism of specificity in the active site. the c. reinhardtii enzyme contains the glu - cys pair typical of mammalian guanylyl cyclases at their expected topological positions, but covalent modification of the active - site cysteine (a consequence of the conditions necessary for crystallization) precludes further interpretation. interestingly, the bacterial cya2 guanylyl cyclase has a non - canonical glu - gly pair, with the critical glu side chain being supported by a third basic residue, conserved as arg or lys in guanylyl cyclases. unexpectedly, guanylyl cyclase activity is reduced and adenylyl cyclase activity increased when the glu - gly pair of cya2 is replaced by a glu - cys pair. thus, the specific features that help discriminate between atp and gtp in class iii cyclases are likely to be complex and not universally conserved. indeed, the relatively nonspecific mycobacterial rv1900c adenylyl cyclase contains a non - canonical asn - asp pair that seems to be completely dispensable for binding nucleotides. the molecular mechanism of mammalian adenylyl cyclase activation has been elusive because there is as yet no structure representing a physiologically relevant basal, inactive state of the catalytic core (a c1:c2 heterodimer in the absence of activating ligands). the structure of the c2 homodimer of adenylyl cyclase has been used as a surrogate, but its conformation is probably influenced by the presence of two molecules of forskolin, which is used to stabilize the dimer interface, and the fact that it lacks a c1 domain, which is required for catalytic activity. the guanylyl cyclase structures provide new images of basal conformations that may be more physiologically relevant. the catalytic core of the synechocystis guanylyl cyclase enzyme assumes a conformation most similar to catalytically competent states of adenylyl cyclases, although, surprisingly, it adopts a closed conformation that apparently needs to open before nucleotides can access the active site. this could represent a regulatory difference from the c. reinhardtii guanylyl cyclase and mammalian cyclases. the c. reinhardtii guanylyl cyclase adopts a conformation similar to that of the adenylyl cyclase c2 homodimer structure, in that the 1 helix, which binds to the - and -phosphates of the nucleotide (figure 1), is not properly aligned with the rest of the active site. the structure may therefore serve as a useful model for the inactive state of mammalian cyclases. however, the fact that crystals could not be obtained without covalent modification of cysteines leaves open the possibility that this structure still does not represent the true ground - state conformation. in 2005, tews. described the active and inactive structures of a ph - sensing mycobacterial class iii adenylyl cyclase. importantly, this structure included the regulatory domains of the enzyme. in the active state, the catalytic domains assume the familiar wreath - like dimer. in the inactive state, they adopt a dramatically different conformation in which they interact extensively with the regulatory domains. although this enzyme is more distantly related to mammalian adenylyl cyclases than the two guanylyl cyclases described here (approximately 23% identity), it highlights the fact that accessory domains, such as those found in the two guanylyl cyclase enzymes and in mammalian adenylyl cyclases, can have a dramatic impact on the conformation of the catalytic core in its inactive state. there is also evidence that the ground - state structures of some class iii nucleotide cyclases may consist simply of loosely associated, and therefore minimally active, catalytic domains. although the c. reinhardtii guanylyl cyclase enzyme was a homodimer under all conditions tested (j winger, personal communication), the cya2 catalytic core exists in an equilibrium between monomeric, dimeric and oligomeric forms. forskolin and g proteins are known to dramatically enhance the affinity of the independently expressed c1 and c2 domains of mammalian adenylyl cyclase, and the rv1900c adenylyl cyclase forms an asymmetric homodimer in its unliganded state, with an unusually open active - site cleft. obviously, more structures of class iii nucleotide cyclases are needed, especially of those that include regulatory domains. although interpretation of the molecular mechanism of activation for nucleotide cyclases is somewhat hindered by the conditions required to bring about their crystallization, it is also possible that each class iii enzyme has evolved a distinct basal conformation that can take optimal advantage of the regulatory inputs unique to that enzyme. | cyclic guanosine monophosphate (cgmp) is a critical second messenger that regulates cardiovascular function and vision in humans. two recent papers, including one in bmc structural biology, have revealed atomic structures of the enzymes that catalyze the synthesis of cgmp providing new clues about the molecular basis of substrate specificity and allosteric regulation in nucleotide cyclases. |
nanotechnology is being explored with the goal of improving nanomedicine for human health.1,2 there is an interest in the synthesis of nanomaterials because they exhibit unique physical, chemical, optical, electrical, magnetic, mechanical, thermal, dielectric, and biological properties, different from those of bulk materials, due to their distinct size- and shape - dependent characteristics.3 silver is a natural antimicrobial agent.4 therefore, silver nanoparticles (agnps) exhibit great potential as novel antimicrobial agents. besides their antimicrobial properties, agnps are also reported to exhibit anti - angiogenesis,5 anti - inflammatory,6 and anti - platelet7 activities. hence, agnps have diverse medical applications.810 although there are reports on the antimicrobial activity of free agnps and in combination with antibiotics,1116 very few reports have examined the effect of agnps on antibiotics belonging to different classes.17 agnps can be synthesized by chemical,18 physical,19,20 or biological routes involving plants8,17,21,22 or microorganisms.2328 biological synthesis is preferred nowadays because these methods are safe, cheap, eco - friendly, and do not involve any toxic substrate or by - product.29 unlike plants, microbial - mediated synthesis of nanoparticles is not affected by geographical and seasonal variations, avoiding inconsistent morphologies and properties. reports are available on agnp synthesis employing pathogenic and nonpathogenic bacterial cells and/or supernatant such as klebsiella pneumoniae, enterobacter spp., extracellular synthesis has advantages, since retrieval of nanoparticles is easy compared with intracellular synthesis where additional recovery steps are required. acinetobacter spp. is most commonly found in the environment including human skin microbiota30 and the mucus membrane of the upper respiratory tract.31 it is a versatile group of microbes with respect to nutrition, metabolism, and genetic organization.32acinetobacter spp. have been reported to exhibit metal resistance33 and plant growth - promoting properties such as phosphate and zinc solubilization,34 implying their ability to interact with metals rendering them nontoxic for their survival. so far there are only two preliminary reports which have included acinetobacter spp. for synthesis of agnps ; one being a. schindleri isolated from soil,35 and another is a. calcoaceticus w17.36 however, detailed reports on the synthesis of agnps by genus acinetobacter are not known. in view of this background, we proposed that acinetobacter isolated from wheat rhizosphere may produce novel and unique nanoparticles with respect to size, shape, and biological activities. we also investigated the effects of reaction parameters on the rate of synthesis and morphology of the agnps. synergistic effects of these agnps on activity of different antibiotics have been evaluated against pathogenic gram - negative and gram - positive bacteria. eighteen strains of acinetobacter spp., isolated from the wheat rhizosphere (pune, maharashtra, india), were used to screen the synthesis of agnps.34 these were already identified by 16s rrna sequencing (genbank eu221346, eu221386, eu221389, eu921458eu921472). the cultures were maintained on a luria bertani (himedia, mumbai, india) agar plate / slant at 4c and as glycerol stocks at 80c. for inoculum preparation, a loopful of the culture was inoculated into 250 ml sterile luria bertani broth and incubated at 30c/200 rpm for 24 hours. standard cultures for antibacterial assays were procured from microbial type culture collection (mtcc), chandigarh, india and national collection of industrial microorganisms (ncim), pune, india. these include gram - negative (enterobacter aerogenes ncim 2964, escherichia coli atcc 25922, pseudomonas aeruginosa ncim 5029, shigella sonnie mtcc 2957, salmonella typhimurium ncim 2509), and gram - positive (staphylococcus aureus mtcc 3160, streptococcus mutans mtcc 497) bacterial pathogens. acinetobacter baumannii aiims 7 (genbank eu779829) isolated and identified in our laboratory has been used.37 for screening, the cells of acinetobacter spp. (optical density [od]600 1 10 cfu / ml) were suspended into sterile milli - q water (ph 7) after repeated rinses to remove the traces of media and incubated at 30c/200 rpm for 72 hours. cell - free extract (cfe) used for synthesis was collected by passing the supernatant obtained after centrifugation through a 0.2 m syringe filter (pall corporation, port washington, ny, usa). three flasks, the first containing 1 mm agno3 (himedia) without cfe, the second containing only cfe, and the third containing cfe with 1 mm agno3, were incubated at 40c in static conditions. synthesis of agnps was visually observed for a color change over a period of 168 hours. reduction of ag ions was monitored by recording the uv - vis spectrum between 300 and 800 nm at regular intervals up to 168 hours on spectramax m5 multi - mode microplate reader (molecular devices llc, sunnyvale, ca, usa). after 168 hours of synthesis, the sample of agnps was centrifuged at 14,000 rpm for 30 minutes at room temperature. the pellet of agnps was suspended in 1 ml sterile milli - q water. the x - ray diffraction (xrd) data of dried thin films of agnps on a glass slide was recorded by d8 advanced brucker x - ray diffractometer with a cu k (1.5) source. a drop of agnp sample was dried on a glass slide for analysis under a scanning electron microscope (sem) (jsm-6360 ; jeol, tokyo, japan) at 20 kv accelerating voltage. samples for transmission electron microscopy (tem) and high - resolution tem (hr - tem) were prepared by drop - coating the agnps solution on a carbon - coated copper grid and drying under infrared radiation. the analyses were performed on fei tecnai g 12 biotwin (fei company, eindhoven, the netherlands) tem and jem-2100 (jeol) hr - tem. the presence of elemental silver was confirmed through energy - dispersive spectroscopy (eds) (jed-2300 ; jeol), equipment included with the hr - tem. the particle size of a 3 ml sample was estimated using a dynamic light scattering instrument (zetasizer nano-2590 ; malvern instruments ltd, worcestershire, uk) in a polystyrene cuvette. to obtain the optimized reaction parameters giving maximum synthesis of agnps, firstly, agno3 ranging from 0.5 to 5 mm (final concentration) after this, the optimum concentration of agno3 was added to the cfe and incubated for 168 hours at 4c80c for temperature optimization. besides uv - vis spectra, tem analyses were carried out to study morphology with varying salt concentrations and reaction temperatures. fourteen antibiotics, namely, amikacin, gentamicin, kanamycin, amoxicillin, ampicillin, ceftriaxone, vancomycin, ciprofloxacin, doxycycline, tetracycline, chloramphenicol (himedia, mumbai, india), trimethoprim (sigma - aldrich, st. louis, mo, usa), ceftazidime (glaxosmithkline pharmaceutical limited, nashik, india), and penicillin (alembic pharmaceutical limited, vadodara, india), were used for antibacterial assay. the disc - diffusion method was used to assay the antibacterial activity of the agnps, antibiotics, and combination of both against pathogenic bacteria on mueller - hinton (mh) (himedia) agar plates. in brief, a single colony of each test strain was grown overnight in mh broth on a rotary shaker (150 rpm) at 37c. the od was adjusted to 0.5 mcfarland (1 10 cfu / ml), and cultures were applied to the plates along with discs containing agnps (11,024 g / disc) and antibiotics (30 g / disc). to determine the synergistic effects, discs with 30 g of antibiotic were further impregnated with 5 l of freshly prepared agnps (15 g / disc). the minimum inhibitory concentration (mic) was determined by broth microdilution method given by the clinical laboratory standards institute (clsi) using 96-well microtiter plates. to each well, 5 l inoculum (5 10 cfu / ml) was added. to evaluate the effect of agnps on mics of antibiotics, a sub - inhibitory concentration of agnps was added to the wells containing different concentrations of antibiotics so that the final concentration of agnps in each well was 15 g. the microtiter plates were incubated at 37c for 20 hours, and results were recorded. the lowest concentration completely inhibiting the growth as detected by the unaided eye was reported as the mic. mic breakpoints of the clsi 200738 guidelines were used to define a resistant or susceptible strain. from the above assay, a 5 l aliquot was taken from the wells showing no visual growth after incubation and spotted onto mh agar plates. the lowest concentration showing no colony on the mh agar after 20 hours of incubation at 37c eighteen strains of acinetobacter spp., isolated from the wheat rhizosphere (pune, maharashtra, india), were used to screen the synthesis of agnps.34 these were already identified by 16s rrna sequencing (genbank eu221346, eu221386, eu221389, eu921458eu921472). the cultures were maintained on a luria bertani (himedia, mumbai, india) agar plate / slant at 4c and as glycerol stocks at 80c. for inoculum preparation, a loopful of the culture was inoculated into 250 ml sterile luria bertani broth and incubated at 30c/200 rpm for 24 hours. standard cultures for antibacterial assays were procured from microbial type culture collection (mtcc), chandigarh, india and national collection of industrial microorganisms (ncim), pune, india. these include gram - negative (enterobacter aerogenes ncim 2964, escherichia coli atcc 25922, pseudomonas aeruginosa ncim 5029, shigella sonnie mtcc 2957, salmonella typhimurium ncim 2509), and gram - positive (staphylococcus aureus mtcc 3160, streptococcus mutans mtcc 497) bacterial pathogens. acinetobacter baumannii aiims 7 (genbank eu779829) isolated and identified in our laboratory has been used.37 (optical density [od]600 1 10 cfu / ml) were suspended into sterile milli - q water (ph 7) after repeated rinses to remove the traces of media and incubated at 30c/200 rpm for 72 hours. cell - free extract (cfe) used for synthesis was collected by passing the supernatant obtained after centrifugation through a 0.2 m syringe filter (pall corporation, port washington, ny, usa). three flasks, the first containing 1 mm agno3 (himedia) without cfe, the second containing only cfe, and the third containing cfe with 1 mm agno3, were incubated at 40c in static conditions. synthesis of agnps was visually observed for a color change over a period of 168 hours. reduction of ag ions was monitored by recording the uv - vis spectrum between 300 and 800 nm at regular intervals up to 168 hours on spectramax m5 multi - mode microplate reader (molecular devices llc, sunnyvale, ca, usa). after 168 hours of synthesis, the sample of agnps was centrifuged at 14,000 rpm for 30 minutes at room temperature. the x - ray diffraction (xrd) data of dried thin films of agnps on a glass slide was recorded by d8 advanced brucker x - ray diffractometer with a cu k (1.5) source. a drop of agnp sample was dried on a glass slide for analysis under a scanning electron microscope (sem) (jsm-6360 ; jeol, tokyo, japan) at 20 kv accelerating voltage. samples for transmission electron microscopy (tem) and high - resolution tem (hr - tem) were prepared by drop - coating the agnps solution on a carbon - coated copper grid and drying under infrared radiation. the analyses were performed on fei tecnai g 12 biotwin (fei company, eindhoven, the netherlands) tem and jem-2100 (jeol) hr - tem. the presence of elemental silver was confirmed through energy - dispersive spectroscopy (eds) (jed-2300 ; jeol), equipment included with the hr - tem. the particle size of a 3 ml sample was estimated using a dynamic light scattering instrument (zetasizer nano-2590 ; malvern instruments ltd, worcestershire, uk) in a polystyrene cuvette. to obtain the optimized reaction parameters giving maximum synthesis of agnps, firstly, agno3 ranging from 0.5 to 5 mm (final concentration) was added to the cfe and incubated at 40c for up to 168 hours. after this, the optimum concentration of agno3 was added to the cfe and incubated for 168 hours at 4c80c for temperature optimization. besides uv - vis spectra, tem analyses were carried out to study morphology with varying salt concentrations and reaction temperatures. fourteen antibiotics, namely, amikacin, gentamicin, kanamycin, amoxicillin, ampicillin, ceftriaxone, vancomycin, ciprofloxacin, doxycycline, tetracycline, chloramphenicol (himedia, mumbai, india), trimethoprim (sigma - aldrich, st. louis, mo, usa), ceftazidime (glaxosmithkline pharmaceutical limited, nashik, india), and penicillin (alembic pharmaceutical limited, vadodara, india), were used for antibacterial assay. the disc - diffusion method was used to assay the antibacterial activity of the agnps, antibiotics, and combination of both against pathogenic bacteria on mueller - hinton (mh) (himedia) agar plates. in brief, a single colony of each test strain was grown overnight in mh broth on a rotary shaker (150 rpm) at 37c. the od was adjusted to 0.5 mcfarland (1 10 cfu / ml), and cultures were applied to the plates along with discs containing agnps (11,024 g / disc) and antibiotics (30 g / disc). to determine the synergistic effects, discs with 30 g of antibiotic were further impregnated with 5 l of freshly prepared agnps (15 g / disc). the minimum inhibitory concentration (mic) was determined by broth microdilution method given by the clinical laboratory standards institute (clsi) using 96-well microtiter plates. to each well, 5 l inoculum (5 10 cfu / ml) was added. to evaluate the effect of agnps on mics of antibiotics, a sub - inhibitory concentration of agnps was added to the wells containing different concentrations of antibiotics so that the final concentration of agnps in each well was 15 g. the microtiter plates were incubated at 37c for 20 hours, and results were recorded. the lowest concentration completely inhibiting the growth as detected by the unaided eye was reported as the mic. mic breakpoints of the clsi 200738 guidelines were used to define a resistant or susceptible strain. from the above assay, a 5 l aliquot was taken from the wells showing no visual growth after incubation and spotted onto mh agar plates. the lowest concentration showing no colony on the mh agar after 20 hours of incubation at 37c was recorded as the minimum bactericidal concentration. a study on the biosynthesis of agnps by cfe of 18 strains of acinetobacter spp. isolated from the wheat rhizosphere was carried out. only a. calcoaceticus lrvp54 gave a positive result for synthesis of agnps within 24 hours. this indicates that although these bacteria possess plant growth - promoting properties,34 only one has the ability to synthesize agnps. visual observation of cfe incubated with agno3 showed a color change from colorless to reddish brown, clearly indicating the formation of agnps (figure 1a). in the uv - vis spectrum, a single, strong, and broad peak was observed at 440 nm, confirming the synthesis of agnps using cfe (figure 1a). in metal nanoparticles such as silver, the electrons move freely due to the close proximity of the conduction and valence bands.39 the collective oscillation of electrons of agnps in resonance with the light wave gives rise to a unique surface plasmon resonance (spr) absorption band which is also the origin of the observed color.40,41 a peak located between 410 and 440 nm has been observed for agnps and is well documented for metal nanoparticles with sizes from 2 to 100 nm.41 according to mie s theory, only a single spr band is expected in the absorption spectra of spherical metal nanoparticles, whereas anisotropic particles could give rise to two or more spr bands, depending on the shape of the particles.42,43 in our case, a single spr peak was observed, which suggests that our agnps were spherical in shape and is consistent with the tem observations. with the progress of reaction time, more and more ag were reduced to ag, resulting in an increased concentration of agnps as indicated by increased od of the spr band (max = 440 nm) with time. the major reason for spr broadening is electron surface scattering, which may be enhanced for small aggregates.40 agnps formed in this process were stable, and the absorption spectra found to be unaltered even after 6 months at room temperature. peptides, proteins, and dna have been suggested to act as templates for reducing metal ions to form nanoparticles.23,4447 there are reports suggesting the involvement of enzymes such as nitrate reductase and phyochelatin synthase as reducing agents for agnps synthesis in bacteria and fungi.23,46,47 we observed that the nanoparticles were embedded in an organic matrix. it seems that these biomolecules are secreted by the cells in the cfe, which may act as reducing and stabilizing agents. the exact nature of the agnps can be deduced from the xrd spectrum of the sample. the xrd pattern (figure 1b) showed four peaks at 2 values of 38.1, 44.3, 64.4, and 77.2, corresponding to (111), (200), (220), and (311) respectively, in the whole spectrum of 2 values ranging from 20 to 80. a comparison of our xrd spectrum with the standard (jcpds file no 04 - 0783) confirmed the formation of crystalline agnps, in accordance with the study of kalimuthu.27 sem of the sample showed the formation of nanoparticles in dispersed and aggregated forms which were confirmed to be of silver by eds (figure 2a and b). eds analysis showed a peak at 3 kev, which is typical for the absorption of metallic silver nanocrystallites due to spr, thereby confirming the formation of agnps.14 additional peaks for copper were observed due to the copper grid used for eds analysis. tem analyses revealed the synthesis of polydisperse agnps of 1060 nm size from 1 mm agno3 after 168 hours. aggregates and dispersed nanoparticles were found to be embedded in an organic matrix (figure 2c). namasivayam employed a similar method for biosynthesis of agnps using cfe of lactobacillus acidophilus 01 where production of spherical agnps of 4560 nm was reported.24 dynamic light scattering data (figure 2c) showed that the majority of agnps synthesized with 1 mm agno3 were in the range of 1560 nm, along with some aggregates, which is well in agreement with sem and tem results. also hr - tem imaging distinctly showed the lattice fringes wherein d (the distance between two lattice fringes) was 0.23 nm (figure 2d), typically observed for the crystalline structure of the agnps.22 optimization studies revealed the significant effects of concentration of metal salt and reaction temperature on the rate of bioreduction of silver ions to agnps. morphological changes during variation of parameters were observed under tem. at a fixed temperature of 40c, variation in reaction kinetics was observed for the synthesis of nanoparticles by varying the agno3 concentration (figure 3a). maximum synthesis of nanoparticles occurred at 0.7 mm agno3 in the reaction mixture, followed by 0.5 mm agno3. highest concentration of 5 mm agno3 showed the least bioreduction of silver ions to nanoparticles. this can be explained on the basis of enzyme - substrate kinetics ; ie, the active site in the key biomolecule responsible for reduction is already saturated with the silver ions, and no site is available for excess ions to get reduced, hence there is no further increase in synthesis of agnps despite the addition of more salt. as compared with the uv - vis spectrum obtained for 1 mm agno3 after 168 hours, a blue shift from 440 to 430 nm was observed at 0.7 mm (figure 3b), which could be due to the variation in morphology of nanoparticles synthesized with different salt concentrations. at the optimized agno3 concentration of 0.7 mm, rate of synthesis was found to increase with an increase in reaction temperature up to 70c, which showed maximum synthesis (figure 4a), after which a decline in the synthesis was observed. a further blue shift in the spr peak to 420 nm was observed at 70c after 168 hours of incubation (figure 4b). it has been reported that small and homogeneous distribution of agnps causes the blue shift in uv - vis spectrum,48 parallel with our results as confirmed by uv - vis spectrum and tem images. tem analysis showed the formation of monodisperse spherical agnps of 812 nm size at 70c with 0.7 mm agno3 (figure 5). deviations from these optimized parameters resulted in an increase in size and polydispersity of agnps as observed under tem. despite a large number of reports on the synthesis of agnps using bacteria, only few reports are available on the optimization. one such study was reported by gurunathan, for e. coli - mediated agnp synthesis, where 5 mm agno3, 60c temperature, and ph 10 were reported to provide optimal conditions for the maximal synthesis of small sized nanoparticles.49 in the present study, the enhanced rate of synthesis of agnps at optimized conditions might be the direct result of the effect of substrate (silver ions) and temperature on a key biomolecule responsible for reduction present in the cfe of a. calcoaceticus lrvp54. it has been suggested that increase in reaction rate causes most silver ions to be consumed in the formation of nuclei and thus stops the secondary reduction process on the surface of the preformed nuclei, leading to the formation of smaller size particles.49 however, to have completely monodisperse nanoparticles through biological systems is still a big challenge. silver and its compounds are known for their antimicrobial properties and for the treatment of burns and chronic wounds.50,51 high surface area to volume ratio cause high bactericidal activity of agnps compared with bulk silver metal.52,53 here, individual and combined effects of agnps with 14 antibiotics belonging to seven classes were investigated against seven pathogenic bacteria using the disc- diffusion method. agnps exhibited antibacterial activity against p. aeruginosa and a. baumannii compared with s. aureus and s. mutans as observed by the zone of inhibition (table s1). similar results were obtained by fayaz.16 the reason for this is the structural difference in cell wall composition of gram - positive and gram - negative bacteria. gram - positive bacteria possess a thick layer of peptidoglycan (2080 nm), making it difficult for agnps to penetrate.16 wide variation has been found in the activities of antibiotics in the presence and absence of agnps, which is interpreted in terms of an increase in the area of zones of inhibition (table 1). aminoglycosides showed a minute increase in the range 0.00.8 fold, with the exception of gentamicin against a. baumannii and kanamycin against p. aeruginosa, where a 1.8-fold increase was seen. considerable enhancement in antibacterial activity was observed for amoxicillin in the presence of agnps against p. aeruginosa, where a 1.8-fold increase was observed. for penicillin, in the presence of agnps, a 3.0-fold increase against s. mutans was observed. vancomycin was found to have the highest overall synergistic activity in combination with agnps compared with all other antibiotics. for e. aerogenes, a 3.8-fold increase in inhibition zone was observed with the combination of vancomycin and agnps. in a similar study, ghosh reported the synergy using a 30 g / disc of agnps synthesized by dioscorea bulbifera tuber extract and 500 g of each antibiotic.17 our findings are comparable to their results even though we only used 30 g / disc of antibiotics and 15 g / disc of agnps. the broth dilution assay was performed to determine the mic. l against gram - negative bacteria, while for s. aureus and s. mutans, it was > 1,000 mg / l. therefore, to evaluate the synergistic effects of agnps on the respective mics of antibiotics, a sub - inhibitory concentration of 75 mg / l agnps was added to each well (15 g / well). mic breakpoints of the clsi guidelines38 were used for comparison of the obtained results (table 2). mic breakpoints are provided to define the resistance and susceptibility of pathogenic microorganisms depending on the extent of their antibiotic resistance. interpretation of the antimicrobial results as per these guidelines will provide a greater understanding of the efficacy of agnps and their synergistic effects on antibiotics against pathogenic microorganisms. e. coli atcc 25922, a control strain, was included in the study to check the accuracy of determined mics. out of seven bacteria, six exhibited resistance to one or more antibiotics belonging to the -lactam class (table 2). with the addition of agnps, only gram - negative bacteria were found to have a mic in the susceptible range. although a decrease in mic was observed, s. aureus and s. mutans still exhibited resistance towards -lactam antibiotics. significant synergistic effects of agnps were observed against a. baumannii, which showed resistance to seven of the ten antibiotics. on exposure to agnps in combination with the antibiotics, the mics were reduced significantly, and the bacteria were found to be susceptible to all of the antibiotics tested except cephalosporins, where no change was observed. although gram - positive bacteria showed resistance towards vancomycin, addition of agnps not only reduced the mic but also made s. mutans susceptible to the antibiotic treatment. all pathogens were already susceptible to the tetracycline class of antibiotics ; therefore, treatment with agnps was unnecessary, although a decrease in mics could be observed. the corresponding minimum bactericidal concentrations of these bacteria were also reduced on addition of agnps in combination with antibiotics (table s2). it is important to note that although there are large numbers of reports on mics of agnps against bacterial pathogens,5456 no study has been done to evaluate the synergistic potential of agnps with respect to the known clsi standards. however, in this study, we not only showed the synergistic effects of agnps on the activity of antibiotics, but also determined its significance using mic breakpoints given in the clsi guidelines. this shows that the administration of a small amount of agnps in combination with antibiotics can reduce the required dose of antibiotics by up to a 1000-fold to achieve the same effect besides combating the problem of multidrug resistance among pathogenic bacteria. antibacterial mechanisms of antibiotics are well - known.57 multiple mechanisms have been suggested to explain the antibacterial activity of agnps, such as release of silver ions from agnps,16 generation of reactive oxygen species, disruption of cellular morphology, inactivation of vital enzymes, dna condensation,11 and loss of dna replication.58 emerging resistance among bacteria renders the available antibiotics inefficient. moreover, simultaneous action of antibiotics and agnps will make it difficult for pathogenic bacteria to develop resistance, and hence, this combinational therapy can be further studied to develop new formulations of agnps in conjunction with antibiotics. a study on the biosynthesis of agnps by cfe of 18 strains of acinetobacter spp. isolated from the wheat rhizosphere was carried out. only a. calcoaceticus lrvp54 gave a positive result for synthesis of agnps within 24 hours. this indicates that although these bacteria possess plant growth - promoting properties,34 only one has the ability to synthesize agnps. visual observation of cfe incubated with agno3 showed a color change from colorless to reddish brown, clearly indicating the formation of agnps (figure 1a). in the uv - vis spectrum, a single, strong, and broad peak was observed at 440 nm, confirming the synthesis of agnps using cfe (figure 1a). in metal nanoparticles such as silver, the electrons move freely due to the close proximity of the conduction and valence bands.39 the collective oscillation of electrons of agnps in resonance with the light wave gives rise to a unique surface plasmon resonance (spr) absorption band which is also the origin of the observed color.40,41 a peak located between 410 and 440 nm has been observed for agnps and is well documented for metal nanoparticles with sizes from 2 to 100 nm.41 according to mie s theory, only a single spr band is expected in the absorption spectra of spherical metal nanoparticles, whereas anisotropic particles could give rise to two or more spr bands, depending on the shape of the particles.42,43 in our case, a single spr peak was observed, which suggests that our agnps were spherical in shape and is consistent with the tem observations. with the progress of reaction time, more and more ag were reduced to ag, resulting in an increased concentration of agnps as indicated by increased od of the spr band (max = 440 nm) with time. the major reason for spr broadening is electron surface scattering, which may be enhanced for small aggregates.40 agnps formed in this process were stable, and the absorption spectra found to be unaltered even after 6 months at room temperature. peptides, proteins, and dna have been suggested to act as templates for reducing metal ions to form nanoparticles.23,4447 there are reports suggesting the involvement of enzymes such as nitrate reductase and phyochelatin synthase as reducing agents for agnps synthesis in bacteria and fungi.23,46,47 we observed that the nanoparticles were embedded in an organic matrix. it seems that these biomolecules are secreted by the cells in the cfe, which may act as reducing and stabilizing agents. the exact nature of the agnps can be deduced from the xrd spectrum of the sample. the xrd pattern (figure 1b) showed four peaks at 2 values of 38.1, 44.3, 64.4, and 77.2, corresponding to (111), (200), (220), and (311) respectively, in the whole spectrum of 2 values ranging from 20 to 80. a comparison of our xrd spectrum with the standard (jcpds file no 04 - 0783) confirmed the formation of crystalline agnps, in accordance with the study of kalimuthu.27 sem of the sample showed the formation of nanoparticles in dispersed and aggregated forms which were confirmed to be of silver by eds (figure 2a and b). eds analysis showed a peak at 3 kev, which is typical for the absorption of metallic silver nanocrystallites due to spr, thereby confirming the formation of agnps.14 additional peaks for copper were observed due to the copper grid used for eds analysis. tem analyses revealed the synthesis of polydisperse agnps of 1060 nm size from 1 mm agno3 after 168 hours. aggregates and dispersed nanoparticles were found to be embedded in an organic matrix (figure 2c). namasivayam employed a similar method for biosynthesis of agnps using cfe of lactobacillus acidophilus 01 where production of spherical agnps of 4560 nm was reported.24 dynamic light scattering data (figure 2c) showed that the majority of agnps synthesized with 1 mm agno3 were in the range of 1560 nm, along with some aggregates, which is well in agreement with sem and tem results. also hr - tem imaging distinctly showed the lattice fringes wherein d (the distance between two lattice fringes) was 0.23 nm (figure 2d), typically observed for the crystalline structure of the agnps.22 optimization studies revealed the significant effects of concentration of metal salt and reaction temperature on the rate of bioreduction of silver ions to agnps. morphological changes during variation of parameters were observed under tem. at a fixed temperature of 40c, variation in reaction kinetics was observed for the synthesis of nanoparticles by varying the agno3 concentration (figure 3a). maximum synthesis of nanoparticles occurred at 0.7 mm agno3 in the reaction mixture, followed by 0.5 mm agno3. highest concentration of 5 mm agno3 showed the least bioreduction of silver ions to nanoparticles. this can be explained on the basis of enzyme - substrate kinetics ; ie, the active site in the key biomolecule responsible for reduction is already saturated with the silver ions, and no site is available for excess ions to get reduced, hence there is no further increase in synthesis of agnps despite the addition of more salt. as compared with the uv - vis spectrum obtained for 1 mm agno3 after 168 hours, a blue shift from 440 to 430 nm was observed at 0.7 mm (figure 3b), which could be due to the variation in morphology of nanoparticles synthesized with different salt concentrations. at the optimized agno3 concentration of 0.7 mm, rate of synthesis was found to increase with an increase in reaction temperature up to 70c, which showed maximum synthesis (figure 4a), after which a decline in the synthesis was observed. a further blue shift in the spr peak to 420 nm was observed at 70c after 168 hours of incubation (figure 4b). it has been reported that small and homogeneous distribution of agnps causes the blue shift in uv - vis spectrum,48 parallel with our results as confirmed by uv - vis spectrum and tem images. tem analysis showed the formation of monodisperse spherical agnps of 812 nm size at 70c with 0.7 mm agno3 (figure 5). deviations from these optimized parameters resulted in an increase in size and polydispersity of agnps as observed under tem. despite a large number of reports on the synthesis of agnps using bacteria, only few reports are available on the optimization. one such study was reported by gurunathan, for e. coli - mediated agnp synthesis, where 5 mm agno3, 60c temperature, and ph 10 were reported to provide optimal conditions for the maximal synthesis of small sized nanoparticles.49 in the present study, the enhanced rate of synthesis of agnps at optimized conditions might be the direct result of the effect of substrate (silver ions) and temperature on a key biomolecule responsible for reduction present in the cfe of a. calcoaceticus lrvp54. it has been suggested that increase in reaction rate causes most silver ions to be consumed in the formation of nuclei and thus stops the secondary reduction process on the surface of the preformed nuclei, leading to the formation of smaller size particles.49 however, to have completely monodisperse nanoparticles through biological systems is still a big challenge. silver and its compounds are known for their antimicrobial properties and for the treatment of burns and chronic wounds.50,51 high surface area to volume ratio cause high bactericidal activity of agnps compared with bulk silver metal.52,53 here, individual and combined effects of agnps with 14 antibiotics belonging to seven classes were investigated against seven pathogenic bacteria using the disc- diffusion method. agnps exhibited antibacterial activity against p. aeruginosa and a. baumannii compared with s. aureus and s. mutans as observed by the zone of inhibition (table s1). similar results were obtained by fayaz.16 the reason for this is the structural difference in cell wall composition of gram - positive and gram - negative bacteria. gram - positive bacteria possess a thick layer of peptidoglycan (2080 nm), making it difficult for agnps to penetrate.16 wide variation has been found in the activities of antibiotics in the presence and absence of agnps, which is interpreted in terms of an increase in the area of zones of inhibition (table 1). aminoglycosides showed a minute increase in the range 0.00.8 fold, with the exception of gentamicin against a. baumannii and kanamycin against p. aeruginosa, where a 1.8-fold increase was seen. considerable enhancement in antibacterial activity was observed for amoxicillin in the presence of agnps against p. aeruginosa, where a 1.8-fold increase was observed. for penicillin, in the presence of agnps, a 3.0-fold increase against s. mutans was observed. vancomycin was found to have the highest overall synergistic activity in combination with agnps compared with all other antibiotics. for e. aerogenes, a 3.8-fold increase in inhibition zone was observed with the combination of vancomycin and agnps. in a similar study, ghosh reported the synergy using a 30 g / disc of agnps synthesized by dioscorea bulbifera tuber extract and 500 g of each antibiotic.17 our findings are comparable to their results even though we only used 30 g / disc of antibiotics and 15 g / disc of agnps. the broth dilution assay was performed to determine the mic. for agnps, mic was in the range of 150600 mg / l against gram - negative bacteria, while for s. aureus and s. mutans, it was > 1,000 mg / l. therefore, to evaluate the synergistic effects of agnps on the respective mics of antibiotics, a sub - inhibitory concentration of 75 mg / l agnps was added to each well (15 g / well). mic breakpoints of the clsi guidelines38 were used for comparison of the obtained results (table 2). mic breakpoints are provided to define the resistance and susceptibility of pathogenic microorganisms depending on the extent of their antibiotic resistance. interpretation of the antimicrobial results as per these guidelines will provide a greater understanding of the efficacy of agnps and their synergistic effects on antibiotics against pathogenic microorganisms. e. coli atcc 25922, a control strain, was included in the study to check the accuracy of determined mics. out of seven bacteria, six exhibited resistance to one or more antibiotics belonging to the -lactam class (table 2). with the addition of agnps, only gram - negative bacteria were found to have a mic in the susceptible range. although a decrease in mic was observed, s. aureus and s. mutans still exhibited resistance towards -lactam antibiotics. significant synergistic effects of agnps were observed against a. baumannii, which showed resistance to seven of the ten antibiotics. on exposure to agnps in combination with the antibiotics, the mics were reduced significantly, and the bacteria were found to be susceptible to all of the antibiotics tested except cephalosporins, where no change was observed. although gram - positive bacteria showed resistance towards vancomycin, addition of agnps not only reduced the mic but also made s. mutans susceptible to the antibiotic treatment. all pathogens were already susceptible to the tetracycline class of antibiotics ; therefore, treatment with agnps was unnecessary, although a decrease in mics could be observed. the corresponding minimum bactericidal concentrations of these bacteria were also reduced on addition of agnps in combination with antibiotics (table s2). it is important to note that although there are large numbers of reports on mics of agnps against bacterial pathogens,5456 no study has been done to evaluate the synergistic potential of agnps with respect to the known clsi standards. however, in this study, we not only showed the synergistic effects of agnps on the activity of antibiotics, but also determined its significance using mic breakpoints given in the clsi guidelines. this shows that the administration of a small amount of agnps in combination with antibiotics can reduce the required dose of antibiotics by up to a 1000-fold to achieve the same effect besides combating the problem of multidrug resistance among pathogenic bacteria. antibacterial mechanisms of antibiotics are well - known.57 multiple mechanisms have been suggested to explain the antibacterial activity of agnps, such as release of silver ions from agnps,16 generation of reactive oxygen species, disruption of cellular morphology, inactivation of vital enzymes, dna condensation,11 and loss of dna replication.58 emerging resistance among bacteria renders the available antibiotics inefficient. moreover, simultaneous action of antibiotics and agnps will make it difficult for pathogenic bacteria to develop resistance, and hence, this combinational therapy can be further studied to develop new formulations of agnps in conjunction with antibiotics. this is the first report on biological synthesis of agnps using a. calcoaceticus lrvp54, an environmental isolate from the wheat rhizosphere. the efficiency of agnps to increase the susceptibility of drug - resistant bacteria measured as per mic breakpoints of the clsi standard we report here for the first time. further studies on the mechanistic aspect of biosynthesis of agnps, and their bactericidal activity are currently under investigation. zone of inhibition obtained with different concentrations of agnps against pathogenic microorganisms note : all experiments were repeated twice, and standard deviations were insignificant. mbc of antibiotics (g / ml) against pathogenic microorganisms in the presence and absence of agnps notes : all experiments were repeated twice, and the higher of the two values was recorded. columns a and b represent the mbc values of antibiotics and antibiotics in presence of agnps, respectively, for each pathogen. | backgroundthe development of nontoxic methods of synthesizing nanoparticles is a major step in nanotechnology to allow their application in nanomedicine. the present study aims to biosynthesize silver nanoparticles (agnps) using a cell - free extract of acinetobacter spp. and evaluate their antibacterial activity.methodseighteen strains of acinetobacter were screened for agnp synthesis. agnps were characterized using various techniques. reaction parameters were optimized, and their effect on the morphology of agnps was studied. the synergistic potential of agnps on 14 antibiotics against seven pathogens was determined by disc - diffusion, broth - microdilution, and minimum bactericidal concentration assays. the efficacy of agnps was evaluated as per the minimum inhibitory concentration (mic) breakpoints of the clinical and laboratory standards institute (clsi) guidelines.resultsonly a. calcoaceticus lrvp54 produced agnps within 24 hours. monodisperse spherical nanoparticles of 812 nm were obtained with 0.7 mm silver nitrate at 70c. during optimization, a blue - shift in ultraviolet - visible spectra was seen. x - ray diffraction data and lattice fringes (d = 0.23 nm) observed under high - resolution transmission electron microscope confirmed the crystallinity of agnps. these agnps were found to be more effective against gram - negative compared with gram - positive microorganisms. overall, agnps showed the highest synergy with vancomycin in the disc - diffusion assay. for enterobacter aerogenes, a 3.8-fold increase in inhibition zone area was observed after the addition of agnps with vancomycin. reduction in mic and minimum bactericidal concentration was observed on exposure of agnps with antibiotics. interestingly, multidrug - resistant a. baumannii was highly sensitized in the presence of agnps and became susceptible to antibiotics except cephalosporins. similarly, the vancomycin - resistant strain of streptococcus mutans was also found to be susceptible to antibiotic treatment when agnps were added. these biogenic agnps showed significant synergistic activity on the -lactam class of antibiotics.conclusionthis is the first report of synthesis of agnps using a. calcoaceticus lrvp54 and their significant synergistic activity with antibiotics resulting in increased susceptibility of multidrug - resistant bacteria evaluated as per mic breakpoints of the clsi standard. |
for decades, there are many interests and research focused on women 's health according to increasing of female 's life span. in particular, female patients who have problems such as menopause, hormonal and nutritional imbalance are continuously have risen as main health issues in women 's health.12345678 there have reported that women are suffering from the diseases after menopause by hormonal imbalance such as osteoporosis, obesity, diabetes, breast cancer, depression and dementia,91011 in addition, many senior ladies have been reported as mal - nutritional state depending on the elderly or lifestyles.1213 the new approaches using new types of insects - derived materials against chronic senior ladies ' disease following menopause have been tried to attenuate such a various problems for a recent decade, which is the use of a variety of medicinal and nutritional benefits from insect - derived materials.141516 historically, it has been a very long time for insects to be both used as a food and an alternative medicine in many asian countries,1718 however, scientific evaluation has not been well conducted yet on the medicinal and nutritional values for them. various peptides, extracts and compounds isolated from insects have reported that have epithelial proliferation and inflammatory response, and have been expected to have anti - metabolic diseases.192021 here, in this short review, it will be examined whether insect - derived materials can be a new promising agent for positively attenuation various health problem issues following menopause compared to a number of conventional agents. samia cynthia ricini (family saturniidae) has been introduced as chinese material medical in zhong - hua - bencao (state administration of traditional chinese medicine of people 's republic of china, 1999).22 gan.15, reported that treatment of the ethanol extraction from female moths samia cynthia ricini for 4 weeks showed increased keratinization of aged mice 's vagina epithelia, the weight of the ovary and amelioration of its degenerative process. also, it showed increased serum concentration of estradiol, progesterone concentration and bone mineral contents. since the declined estradiol and progesterone production disrupt the negative feedback of hypothalamic - pituitary - ovarian axis,152324 moreover, if the constant hormone levels are not well maintained, and fertility is affected also stress regulatory mechanisms of the body as well. in particular, samia cynthia recini has accelerating effect on estrogen on vaginal epithelial keratinization in rodent with no adverse effect.15 in this respect, samia cynthia recini can be a good candidate material for women following menopause. mealworms are the larvae of two species of darkling beetles of the tenebrionidae family, the yellow mealworm beetle (tenebrio molitor linnaeus), and the smaller and less common dark or mini mealworm beetle (tenebrio obscurus fabricius).25 mealworm beetles are indigenous to europe and are now distributed worldwide. mealworms are easy to breed and feed, and have a valuable protein profile.16 mealworms are a high quality feed. they contain large amounts of protein (45%-60% dry matter [dm ]) and lipid (30%-45% dm). they are relatively poor in ash (less than 5% dm), and like other insects they have a low calcium content and a very low calcium : phosphorus (ca : p) ratio. the exclusive feeding of mealworms caused ca deficiency and symptomatic metabolic bone disease.16 it must be noted that composition is highly variable and influenced by the diet. thus, they performed an experiment increasing the calcium content of mealworms (tenebrio molitor) to improve their nutritional value for bone mineralization of growing chicks.11 several dried waste materials from different origins were used as a substrate to grow tenebrio molitor l. nutrient / amino acid values differed depending on both larval size / weight and substrate. in addition, these larvae were experimentally used as a broiler feedstuff. afterwards, ramos - elorduy.25, also reported the nutritional value of tenbrio molitor that has the potential to be used as protein source for raising broilers. they may be easily reared on fresh oats, wheat bran or grain, with sliced potato, carrots, or apple as a moisture source. it might be expected to be a good source for anti - osteoporotic property and anti - malnutrition for elderly women. honeybee (apis mellifera) royal jelly (rj) has a long history in human medicine because of its health - protecting effects. rj contains rich proteins, carbohydrates, fats, free amino acids, vitamins and minerals.26 in particular, lipids in rj are useful as preventive and supportive medicines with functionalities that include potential inhibitors of cancer growth, immune system modulators, alternative therapies for menopause, skin - aging protectors, neurogenesis inducers.26 park.26, also reported that rj, in particular, caffeic acid phenethyl ester (cape) from honeybee propolis has been known to have capacity of attenuating osteoclastogenesis and bone resorption in ovariectomized (ovx) mice.27 according to the results, with cape treatment, the microarchitecture of the tibia metaphysis was significantly enhanced with a reduction of osteoclast formation as well bone mineral density (bmd). thus, cape is expected to be applied to the treatment of osteolytic condition including osteoporosis, arthritis, periodontal bone erosions and cancers - induced bone loss.27 as noted above, there have expected many kinds of insects are able to use for human health. however, still little thing is known their safe unique and alternative medicinal function when they used with food type. in general, since edible insects have very low toxicity, the use as alternative medicines or foods has been paid attention to especially women 's health. this is definitely potential and new area for many health problem issues to elderly women who is following menopause by chemical- or hormonal - based medicinal treatments. | human health problems due to long life are becoming major issues in society, and in particular greater interest collected on women 's health after menopause. many substances can be introduced to women 's health, however, materials from the substances have not shown all of the safety and efficacy properties that are not easily found. currently, it is known about the effects of the disease on the female insect - derived material that is capable of overcoming this problem significantly. when using the insect - derived material through the results of several studies suggest that it is possible to solve a hormonal imbalance and nutritional imbalance in the elderly. here, we 'd like to try to dissertate about the new trends for women 's health improvement using novel materials - derived from insects. |
before we work out what constitutes an assessment s value for a given cost in medical education, we must first outline the steps necessary to create an assessment, and then assign a cost to each step. in this study we undertook the first phase of this process : we sought to work out all the steps necessary to create written selected - response assessments. first, the lead author created an initial list of potential steps for developing written assessments. this was then distributed to the other three authors. these authors independently added further steps to the list. the lead author incorporated the contributions of these others and created a second draft. this process was repeated until consensus was achieved amongst the study s authors. next, the list was shared by means of an online questionnaire with 100 healthcare professionals with experience in medical education. the results of the authors and healthcare professionals thoughts and feedback on the steps, needed to create written assessment, are outlined below in full. we outlined the steps that are necessary to create written or web - based selected - response assessments. |
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between december 2010 and october 2011, 98 patients who were diagnosed with isolated acl ruptures underwent primary acl reconstruction using single bundle method with modified transtibial technique. informed consent was obtained from all patients prior to receiving 3d - ct and radiograph for analyzing the femoral tunnel position after the operation. the patient pool consisted of 81 men and 17 women with a mean age of 27.3 years (range, 15 to 60 years). 1c) by two orthopedic surgeons on two separate occasions at an interval of 2 weeks to assess the interobserver and intraobserver reliability. the quadrant method used to measure the postoperative femoral tunnel position by 3d - ct is as follows : 1) a true lateral view cutting at the middle of the intercondylar notch of the proximal femur by 3d - ct was attained through the picture of archiving and communication systems (pacs, marotech, seoul, korea) (fig. 2) the image was enclosed with a rectangular measurement frame that was either parallel or perpendicular to the blumensaat line on power point ver. 2007 (microsoft co., redmond, wa, usa) as shown in fig. t calculated the deep to shallow distances from the center of the femoral tunnel parallel to the blumensaat line, and h calculated the high to low distances perpendicular to the blumensaat line. in addition, the anatomical femoral attachment site of acl was shown on table 1 using the previous quadrant method. after thorough arthroscopic evaluations of the acl, a 3 cm longitudinal skin incision at 2 cm medial to the tibial tuberosity was made. the graft was cut in half and folded into half to produce a quadruple semitendinous graft. if the harvested graft was shorter than 24 cm, a quadruple semitendinosus / gracilis graft was prepared by harvesting both the semitendinosus tendon and the gracilis tendon. the intercondylar ridge of the lateral femoral condyle, and the bifurcate ridge that divides the am bundle and the posterolateral (pl) bundle were confirmed by removing the soft tissues at the femoral insertion of acl. a bony mark at the anatomic center of acl footprint (just posterior to the bifurcate ridge and center of anteroposterior [ap ] width of footprint) was made by passing a microfracture awl through the anteromedial portal as in fig. 2a. this center was deepened and widened to allow space for the guided pin through the tibial tunnel. since the anatomical femoral tunnel is prepared by the trans - am portal, the transtibial guide pin tends to be located more to the anterior and proximal position of anatomic center than anticipated. the ' triangular, funnel - shaped bony trough ' was employed to slip the eccentrically positioned guide into the anticipated anatomic center, which is part of our modified transtibial technique (fig. the starting point of the tibial edge / periphery was superior to the pes anserinus and the anterior margin of medial collateral ligament (mcl) (fig. the intra - articular site is positioned immediately lateral to the medial tibial spine and the extension of the inner margin of anterior horn of lateral meniscus. the reference point of tibial tunnel is a little posterior region of the acl footprint. the angle of the acl guide (linvatec, largo, fl, usa) was set at 47.5. the tibial tunnel was created using a reamer with diameter 1 mm smaller than the prepared acl graft. with a free hand technique, a guide pin was passed through the tibial tunnel towards the bony trough. generally, the guide pin was located distal and anterior to the anatomical center, and extension of the knee nudged the guide pin towards the proximal and posterior region of the anatomic center, but was being bent at the intra - articular orifice of the tibial tunnel (fig. femoral reaming was performed with knee flexion which reduces the chances of blowing out the posterior wall and increases the femoral tunnel length. when the reamer passes over the bending portion of the guide pin, the knee should be extended. after passing through the bending point after the graft passage, femoral fixation was done with endobutton and bio - cross pin (rigidfix system, mitek, johnson & johnson, norwood, ma, usa). the graft was tensioned and fixed to the tibia by placing the ha screw (bioscrew poly l - lactic acid, linvatec linvatec). we regularly performed follow - ups on the patients in the out - patient clinic and established a protocol for rehabilitation. partial weight bearing with crutches for 6 weeks was mandated for patients who underwent meniscal repairs. the treatment purpose of the patients was to gain a full range of motions at 2 to 6 weeks after surgery. running and side - cutting activities were allowed at 3 months, with a return to sports activities at 6 months after surgery. after thorough arthroscopic evaluations of the acl, a 3 cm longitudinal skin incision at 2 cm medial to the tibial tuberosity was made. the graft was cut in half and folded into half to produce a quadruple semitendinous graft. if the harvested graft was shorter than 24 cm, a quadruple semitendinosus / gracilis graft was prepared by harvesting both the semitendinosus tendon and the gracilis tendon. the intercondylar ridge of the lateral femoral condyle, and the bifurcate ridge that divides the am bundle and the posterolateral (pl) bundle were confirmed by removing the soft tissues at the femoral insertion of acl. a bony mark at the anatomic center of acl footprint (just posterior to the bifurcate ridge and center of anteroposterior [ap ] width of footprint) was made by passing a microfracture awl through the anteromedial portal as in fig. 2a. this center was deepened and widened to allow space for the guided pin through the tibial tunnel. since the anatomical femoral tunnel is prepared by the trans - am portal, the transtibial guide pin tends to be located more to the anterior and proximal position of anatomic center than anticipated. the ' triangular, funnel - shaped bony trough ' was employed to slip the eccentrically positioned guide into the anticipated anatomic center, which is part of our modified transtibial technique (fig. the starting point of the tibial edge / periphery was superior to the pes anserinus and the anterior margin of medial collateral ligament (mcl) (fig. 2c). the intra - articular site is positioned immediately lateral to the medial tibial spine and the extension of the inner margin of anterior horn of lateral meniscus. the reference point of tibial tunnel is a little posterior region of the acl footprint. the angle of the acl guide (linvatec, largo, fl, usa) was set at 47.5. the tibial tunnel was created using a reamer with diameter 1 mm smaller than the prepared acl graft. with a free hand technique, generally, the guide pin was located distal and anterior to the anatomical center, and extension of the knee nudged the guide pin towards the proximal and posterior region of the anatomic center, but was being bent at the intra - articular orifice of the tibial tunnel (fig. femoral reaming was performed with knee flexion which reduces the chances of blowing out the posterior wall and increases the femoral tunnel length. when the reamer passes over the bending portion of the guide pin, the knee should be extended. after passing through the bending point after the graft passage, femoral fixation was done with endobutton and bio - cross pin (rigidfix system, mitek, johnson & johnson, norwood, ma, usa). if femoral tunnel was short, the graft was press fitted with only endobutton. the graft was tensioned and fixed to the tibia by placing the ha screw (bioscrew poly l - lactic acid, linvatec linvatec). we regularly performed follow - ups on the patients in the out - patient clinic and established a protocol for rehabilitation. partial weight bearing with crutches for 6 weeks was mandated for patients who underwent meniscal repairs. the treatment purpose of the patients was to gain a full range of motions at 2 to 6 weeks after surgery. a perturbation training program began at 6 weeks after surgery. running and side - cutting activities were allowed at 3 months, with a return to sports activities at 6 months after surgery. the position of the femoral tunnel was measured using the quadrant method by 3d - ct,8) where t was 32.94% 5.16%, and h was 41.89% 5.58% (fig. these values were more distal and middle as compared with the previous data (mean t, 29.85% ; h, 33.2%) measured by quadrant method using cadaveric knees for validating anatomical acl footprints (p < 0.05).9,10) the interobserver intraclass correlation coefficients (iccs) were 0.766 and 0.793, whereas the intraobserver iccs were 0.875 and 0.893. the femoral tunnel angle on the ap view was 50.43 7.04, for which the interobserver iccs were 0.783 and 0.795, and the intraobserver iccs were 0.911 and 0.923. on the process of optimizing this modified transtibial technique, mishaps such as breakage of the bended wire occurred on 3 occasions as the reamer passed through the tibial tunnel, and a shallow tibial tunnel was formed on another occasion, which all occurred during the initial series. however, as the examiners became more experienced in executing this modified technique and when the safe zone of the bone stock was adjusted to the entry point of the tibia, these complications were avoided. the purpose of this study is to describe a single bundle acl reconstruction performed with modified transtibial technique for positioning the femoral tunnel to anatomical footprint. the femoral tunnel location was being evaluated with 3d - ct scan reconstructions with the quadrant method as described by bernard.,8) and the results of these scans were compared with those derived from arthroscopic identification. the results of this study showed that the t - value was 32.94% 5.16%, and the h - value was 41.89% 5.58%. abebe.11) and kaseta.12) reported that the femoral tunnels were placed " deeper or more proximal " and " higher or more anterior " than the native acl insertions of the femur when the transtibial technique was used, which could result in increased laxity of the graft. contrary to these results, the femoral tunnel in this study was located " shallower or more distal " and " lower or more posterior " than the mean value from multiple measurements of the anatomical acl footprints in cadaveric knees (t, 29.85% ; h, 33.2%) from previous studies that also used the quadrant method for calculation. the mean values of the femoral tunnel created by the authors were placed between the am and pl bundle position, which were calculated as described by kopf.13) and tsukada.10) rue.6) reported that a laterally - oriented transtibial drilling femoral tunnel effectively overlapped approximately half the am bundle and half the pl bundle. miller.14) proved that the intra - articular aperture of the femoral tunnel in all cases formed an ellipse, with the long and short axis averaging 13.9 1.6 mm and 9.4 0.8 mm in the trans - am method, and 12.1 0.8 mm and 10.6 0.6 mm respectively in the transtibial group. accordingly, if the center of the femoral tunnel is relatively " distal " and " posterior, " the ellipse - shaped tunnel would allow the incorporation of original portions of the am and pl bundles leading to a more anatomical reconstruction. hence, this modified transtibial technique has advantages over the traditional technique (t, 37.2% 5.5% ; h, 11.3% 6.6%) for being closer to its anatomical location.10,13) ferretti.15) reported the presence of a lateral intercondylar ridge in all patients who underwent acl reconstruction and the bifurcate ridge in 82% of 60 knees. van eck.16) described 88% and 48% of these landmarks, respectively for their patients at arthroscopy. however, to arthroscopically identify these landmarks on the medial wall of the lateral femoral condyle is difficult. therefore, postoperative 3d - ct scans were used to validate whether this modified technique can reproducibly be used for the placement of the anatomic femoral tunnel. our angle of the femoral tunnel in the coronal plane was on average 50.43 7.04, when viewed by ap radiography, and it was lower than the results (61.7 5.5, 58.8 8.3) from previous transtibial techniques, even when compared with the anteromedial portal technique (55.9 4.7, 50.9 8.3), which were measured by chang.17) and dargel.18) in addition, bedi.4) reported that the coronal angles of the oblique femoral tunnels from either the transtibial or the trans am portal techniques were 54.1 7.17 and 45.9 6.9, respectively. therefore, the rate of the posterior wall blowout was 50% with the latter technique. thus, not only is the modified transtibial technique more reproducible, but it is also safer when such complications of the am portal technique is taken into consideration. there was no significant posterior wall blowout in our case series. the entrance of the tibial tunnel is essential for the placement of the anatomic femoral tunnel when using the transtibial technique for acl reconstruction, especially if we want to prevent graft failures.19) recent studies have tried to accurately drill the tibial tunnel in relation to the native acl footprint of the femur, but without success.20,21) however, heming.7) reported the plausibility of the anatomical footprint using the transtibial technique, but commented that the starting point should be closer to the joint line, and the tibial tunnel length must be shortened for an accurately positioned footprint. arnold.22) attempted to produce anatomical tunnels by transtibial drilling just proximal to the pes anserinus and ventral to the mcl, but in their case, the guide pin was projected too high in the notch and missed the femoral insertion. in this study, the authors followed arnold 's method to avoid creating shallow tibial tunnels, and furthermore, used a new modified method to prevent over - projecting the guide pin in the notch into a non - anatomical position. there are some advantages of this modified transtibial technique in which the femoral tunnel is drilled appropriately through the tibial tunnel. first, it is similar to the conventional method and easily replicated by surgeons who are familiar with the transtibial technique. second, it is possible to create the femoral tunnel in an anatomic position through a relatively longer and deeper tibial tunnel with only a few additional and simple techniques ; triangular, funnel - shaped bony trough and by changing knee flexion angles during femoral tunneling. third, it permits early rehabilitation through firm graft fixation resulting from tunnel length - graft length matching. the limitations of the study may be that the authors used the mean values of the native acl insertions derived from multiple previous studies for comparison. although the methods used in this paper are the same as those in previous studies, mean variations in the experimental instruments do not reflect the differences that arise, and, are therefore, a crude averaging of values. in addition, the icc was lower for the interobserver correlation coefficient in two observers, which may reflect the difficulty of selecting the true lateral view on the magnetic resonance imaging sagittal image, and measures the center of the femoral tunnel (forming an ellipse interobservers). finally, there was a lack of full considerations for the length of the femoral tunnel and the characteristics of the tibial tunnel. further studies to overcome these limitations are required, and essential evaluations of clinical outcomes for modified transtibial technique are necessary to prove its superiority over the traditional techniques. in conclusions, our modified transtibial technique is anticipated to provide a true anatomical placement of the femoral tunnel during acl reconstruction than the previous traditional transtibial techniques. | backgroundthe purpose of this study is to report a modified transtibial technique to approach the center of anatomical femoral footprint in anterior cruciate ligament (acl) reconstruction and to investigate the accurate femoral tunnel position with 3-dimensional computed tomography (3d - ct) and radiography after reconstruction.methodsfrom december 2010 to october 2011, we evaluated 98 patients who underwent primary acl reconstruction using a modified transtibial technique to approach the center of anatomical femoral footprint in single bundle acl reconstruction with hamstring autograft. their femoral tunnel positions were investigated with 3d - ct and radiography postoperatively. femoral tunnel angle was measured on the postoperative anteroposterior (ap) radiograph and the center of the femoral tunnel aperture on the lateral femoral condyle was assessed with 3d - ct according to the quadrant method by two orthopedic surgeons.resultsaccording to the quadrant method with 3d - ct, the femoral tunnel was measured at a mean of 32.94% 5.16% from the proximal condylar surface (parallel to the blumensaat line) and 41.89% 5.58% from the notch roof (perpendicular to the blumensaat line) with good interobserver (intraclass correlation coefficients [icc ], 0.766 and 0.793, respectively) and intraobserver reliability (icc, 0.875 and 0.893, respectively). according to the radiographic measurement on the ap view, the femoral tunnel angles averaged 50.43 7.04 (icc, 0.783 and 0.911, respectively).conclusionsour modified transtibial technique is anticipated to provide more anatomical placement of the femoral tunnel during acl reconstruction than the former traditional transtibial techniques. |
enrofloxacin (efx) (1-cyclopropyl-7-(4-ethylpiperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydro quinoline-3-carboxylic acid) belongs to the group of synthetic 6-fluoroquinolones or 4-quinolones derived from the core structure of nalidixic acid. as a result of gradual changes to the basic molecule, antimicrobial properties were considerably increased and pharmacokinetics could be substantially improved, whereas the probability of adverse effects was reduced. coplanar carbonyl groups (c = o) at positions 3 and 4 of the core structure are generally required for antimicrobial activity of the fluoroquinolones. a fluorine atom, introduced at position 6, enhances the efficacy against gram - negatives and broadens the spectrum against gram - positive bacteria. efx is an amphoteric drug with pka1 = 5.94 corresponding to carboxyl group and pka2 = 8.70 corresponding to basic piperazinyl group and the isoelectric ph = 7.32. due to the presence of carboxylic acid and amine functional groups (basic), the molecule has amphoteric and zwitter ionic properties which make efx lipid soluble and enhance the ability to penetrate tissues, pus, and organic debris. the piperazine ring at position 7 further increases antimicrobial activity, especially against pseudomonas organisms. the presence of a -c2h5 group which is attached to the piperazine ring enhances tissue penetration and decreases central nervous system toxicity by reducing drug binding to gaba receptors in the brain. efx is a pale or light yellow colored crystalline substance with a high degree of purity. in water at ph 7, it is slightly soluble. however, as it contains acidic and basic groups (betaine structure), it can readily be brought into solution when the ph values are either alkaline or acidic. liquid formulations of baytril for parenteral administration contain freely soluble salts of efx in an aqueous solution. due to the high hydrolytic stability of the active ingredient, these solutions are very stable. the tablet formulations contain efx in its original betaine form-1 [1, 4, 5 ]. in veterinary medicine it is administered by subcutaneous injection to cattle and intramuscular injection to pigs and orally to cattle, pigs, turkeys, and chickens, for the treatment of infections of the respiratory and alimentary tract. from the literature survey it is evident that few methods were available for the determination of efx and its impurities in tablet dosage forms by using high performance liquid chromatography (hplc). garcia. developed a method for the simultaneous determination of efx and its primary metabolite ciprofloxacin in plasma by hplc with fluorescence detection. tyczkowska. developed high performance liquid chromatographic method for the simultaneous determination of efx and its primary metabolite ciprofloxacin in canine serum and prostatic tissue. developed simultaneous determination of benofloxacin, danofloxacin, enrofloxacin, and ofloxacin in chicken tissues by high performance liquid chromatography. idowu and peggins developed simple, rapid determination of efx and ciprofloxacin in bovine milk and plasma by hplc with fluorescence detection.. have developed methods for the determination of efx and its related impurities in drug substance by thin layer chromatography (tlc) and hplc methods. tlc method was developed for the determination of fluoroquinolonic acid and hplc method for the estimation of its two impurities, ciprofloxacin and desfluoro compound in both usp and ph. eur.. as per the literature review, no method was reported for the estimation of efx and its degradation products in finished dosage forms by using hplc. the present research work describes the simultaneous estimation of efx and its degradation products in tablet dosage forms using hplc. methanol was used as solvent for the development and validation of this method as it is often less expensive and less toxic than acetonitrile. the work gives a sensitive, specific, and stability - indicating method for the determination of impurities of efx in a single method by hplc rather than performing two analytical techniques of hplc and tlc. time required for the tlc analysis, man power, and solvent consumption for performing tlc analysis can be saved and finally supporting towards green environment by following health safety and environment guidelines. developed lc method was validated with respect to lod, loq, linearity, precision, accuracy, and robustness. forced degradation studies were carried out to verify the stability - indicating nature of the lc method. qualified standards (efx purity ~99.5%, decarboxylated impurity ~99.6%, ethylenediamine impurity ~95.6%, desfluoro impurity ~99.3%, ciprofloxacin impurity ~99.2%, chloro impurity ~99.9%, and fluoroquinolonic acid impurity ~98.9%) and samples of efx were obtained from local laboratories and were used without any further purification. hplc grade methanol (meoh purity ~99.7%) and acetonitrile (acn purity ~99.8%) were obtained from rankem (mumbai, india). orthophosphoric acid (purity ~85%) was received from qualigens fine chemicals (mumbai, india). the waters lc system (milford, ma, usa) equipped with a diode array detector was used for method development and forced degradation studies. waters lc consists of 2695 separation modules and 2996 pda detectors used for validation study. intermediate precision was carried out using waters 2695 separation modules with 2487 dual wavelength detectors. photolytic chamber was used for photolytic degradation and thermal degradation samples were kept at 80c for 5 days in an oven. the chromatographic separation was achieved on a kromasil c-18, 250 4.6 mm, 5 m column using mobile phase - a composed of 10 mm kh2po4 containing 0.1% of tea (v / v) (ph adjusted to 2.50 0.05 with orthophosphoric acid) and mobile phase - b was meoh. the mobile phase - a was filtered with 0.45 m nylon filter. gradient program used for chromatographic separation was shown in table 1. flow rate was set to 1.0 ml min with a column temperature of 35c. detection wavelength was carried out at 278 nm for ethylenediamine, desfluoro, ciprofloxacin, and chloro impurities and 254 nm for fluoroquinolonic acid and decarboxylated impurities. citrate buffer (ph 4.0) and meoh in the ratio of 50 : 50 were used as diluent for the preparation of standards and samples. citrate buffer was prepared by dissolving 2.0 g of citric acid and 0.5 g of potassium hydroxide in 1 liter of hplc grade water and adjusted ph of the resultant solution to 4.00 0.05 with dilute orthophosphoric acid. we accurately weighed and transferred 50 mg of efx working standard into a 100 ml volumetric flask. we added about 70 ml of diluent and sonicated it to dissolve with intermittent shaking. the resulting solution is diluted up to the mark with diluent and mixed well. we transferred 5 ml of efx standard stock solution into a 50 ml volumetric flask and diluted it up to the mark with the diluent. we further diluted 5 ml of this solution into 50 ml with the diluent mixed well. we accurately weighed and transferred a sample powder equivalent to 50 mg of efx into a 100 ml volumetric flask. we added about 70 ml of diluent and sonicated it for 60 min with intermittent shaking. we made up the volume of 100 ml volumetric flask with diluent and then filtered the solution through 0.45 m pvdf membrane filter. specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. the specificity of the developed method was established to prove the absence of interference from placebo peaks (excipients) which is part of required pharmaceutical preparation. degradation study was performed by subjecting the tablet powder to accelerated degradations such as acid, alkaline, oxidation, thermal, humidity, and photolytic conditions to evaluate the interference of degradation impurities. thermal degradation was performed by keeping the placebo and tablets in different petri dishes and then placed them in an oven at 60c for 3 days. humidity degradation was performed by placing the tablet and placebo powders in two separate petri dishes and kept in a humidity chamber at 90% rh, 25c for 7 days. photolytic study was carried out by placing the placebo and tablets in separate petri dishes in a photolytic chamber at 1.2 million lux hour 's illumination and 200-watt hours / square meter ultraviolet energy. acid, base, and oxidation degradations were performed by adding 1 ml of 5 n hcl, 1 ml of 5 n naoh, and 1 ml of 30% peroxide solution (h2o2), respectively, to the placebo and tablet powders at 70c for 1 hour. the detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. the quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. the quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices and is used particularly for the determination of impurities and/or degradation products. the limit of detection (lod) and limit of quantitation (loq) were important for the impurity tests and the assays of dosages containing low drug levels. the lod is generally quoted as the concentration yielding a signal - to - noise ratio of 2 : 1 or 3 : 1 and loq is quoted as the concentration yielding a signal - to - noise ratio of 10 : 1. the signal - to - noise ratio is determined by the following equation : (1)s = hh, where h = height of the peak corresponding to the component. h = absolute value of the largest noise fluctuation from the baseline of the chromatogram of a blank solution. lod and loq are also determined based on the standard deviation of the response and the slope. 10/s where is the standard deviation of the response and s is the slope of the calibration curve. the linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. linearity is the ability of the method to obtain results which are either directly or after mathematical transformation proportional to the concentration of the analyte within a given range. the linearity of response for efx and their related impurities were determined in the range from loq to 120%. the seven concentrations of each component were subjected to regression analysis by least - squares method to calculate correlation coefficient and calibration equation. the precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. precision is considered at two levels : repeatability (method precision), intermediate precision. repeatability expresses the precision under the same operating conditions over a short interval of time. intermediate precision expresses within - laboratories variations : different days, different analysts, different equipment, and so forth. the precision was expressed as the relative standard deviation (rsd) : (2)%rsd=(standard deviationaverage)100. precision and intermediate precision of the developed method were carried out by 6 determinations (preparations) of the test solution by injecting the impurities spiked solution and calculated the % rsd for each impurity. the accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted as either a conventional true value or an accepted reference value and the value found. was determined by applying the method to samples in which known amounts of analyte have been added. these should be analyzed against standard and blank solutions to ensure that no interference exists. the accuracy was calculated from the test results as a percentage of the analyte recovered by the assay. accuracy of the present method was carried out by injecting the impurities spiked solution at different concentration levels of loq, 100% and 120% to their specification limit, in triplicate determinations. the robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. robustness of the method indicates the reliability of an analysis to assess the system suitability parameters under the influence of small but deliberate variations in method parameters. it was performed by injecting the impurities spiked solution and the stressed degradation sample solutions by changing several parameters including different batch of the same column, flow rate, column temperature, and minor change in organic composition. the control sample solution and the standard solution containing efx were prepared as per the test procedure. the freshly prepared solutions and the solutions which were stored at room temperature up to 24 hours were injected at different time intervals. the % impurity obtained at initial was compared with the % impurity obtained at different time intervals. the main purpose of the current chromatographic method was to develop a lc method for the separation and quantification of known and unknown degradation products of efx in efx tablets at trace level. efx and its known impurities structures were shown in figure 1. from the structure of efx, it was observed that efx has pka1 = 5.94 corresponding to carboxyl group and pka2 = 8.70 corresponding to basic piperazinyl group. in spite of the fact that in reversed - phase separations, ph of selected buffer should have the ph 1.5 units from the pka values of the analytes, the selection of buffer with proper ph leads to ionization of analytes which results in the sharp and symmetric peak shapes and reproducible retention times (rt). the ph of the mobile phase was selected at lower side as the ph increases silica dissolves slowly and results in inconsistent retention times and results. kh2po4 has a wide range of pka values ; hence, initially we selected a buffer of 10 mm kh2po4 composed of 0.5% tea and set the ph of this solution to 3.00 0.05 using orthophosphoric acid. acn was used in the mobile phase along with hypersil bds c-18, 250 4.6 mm, 5 m column at a column temperature of 35c. acn was selected as solvent for initial method development trials as it produces sharp, symmetrical peaks with less column back pressure. tea was used in the mobile phase to reduce tailing factor for efx and its known impurities by reducing the silanol and sample interactions on the bonded surface of the hplc column. placebo interference was observed at the retention time of decarboxylated impurity and broad peak shape observed for chloro impurity. further trials were performed at a column temperature of 40c and 50c by using the same above chromatographic conditions ; however placebo peak was not separated from the decarboxylated impurity. hence, mobile phase was changed to 10 mm kh2po4 containing 0.1% of tea (v / v) (ph adjusted to 2.50 0.05 with orthophosphoric acid) and meoh was used as solvent with kromasil c-18, 250 4.6 mm, 5 m hplc column at a column temperature of 40c., decarboxylated impurity was well separated from placebo peak and the resolution between the cipro base impurity and efx peak is 1.6 only. column temperature was changed to 45c with slight change in gradient programme and remaining chromatographic conditions are unchanged. cipro base impurity was separated from the efx peak but placebo interference was observed again at the retention time of decarboxylated impurity. next trial run was carried out with slight change in linear gradient program at a column temperature of 40c by keeping remaining chromatographic conditions the same as previous run. further trial was performed with the changes in gradient program with a column temperature of 35c with all the chromatographic conditions the same as previous run. hence, this method was finalized for separation of all the known impurities of efx by using step gradient run. the screening studies were performed on a variety of columns to cover a wide range of stationary phase properties including carbon chain length, carbon loading, and surface area. each of the selected columns was screened with different mobile phase ratios, different column temperatures, and different type of organic solvents including meoh and acn. kromasil c-18, 250 4.6 mm, 5 m column was selected for the final method due to reproducible results and better peak shapes. in most of the trials, major impurities of efx are separated ; however resolution between cipro base impurity and efx is less and placebo peak interference with decarboxylated impurity is observed. the chromatograms of blank run are shown in figures 3 and 4, chromatograms of placebo are shown in figures 5 and 6, chromatograms of control sample (concentration ~0.5 mg ml) are shown in figures 7 and 8, and chromatograms of 1% impurity spiked samples are shown in figures 9 and 10. the elution order of the impurities in different chromatographic conditions was decarboxylated > ed analogue > desfluoro > cipro base > efx > chloro > fq acid. in presence of acn, ed analogue impurity was eluted first and desfluoro impurity was eluted next to the ed analogue impurity where as in presence of meoh, desfluoro impurity was eluted first and ed analogue impurity was eluted next. except for the above change, all the remaining impurities were eluted in the same order. spectra for all the known impurities and efx were measured from 200 to 395 nm for wavelength maxima. based on the spectra maxima, 278 nm was selected for identification and quantification of ethylenediamine impurity, desfluoro impurity, ciprofloxacin impurity, and chloro impurity, and 254 nm was selected for identification and quantification of fluoroquinolonic acid impurity and decarboxylated impurities. to study the temperature effect on resolution between the impurity peaks of efx, we injected the impurities spiked solution at different column temperatures. it was observed that at a column temperature of 35c, all the known degradation impurities were well separated when compared to the other column temperatures. the resolution between closely eluting cipro base impurity and efx was found to be not less than 2. the objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended use. the described hplc method has been extensively validated for its known degradation impurities and unknown impurities as per ich guidelines. after successful completion of method development [13, 15, 16 ], method validation [1733 ] was performed to ensure that the developed method was capable of giving reproducible and reliable results when used by different operators employed on the same equipment of the same lab or of different laboratories. stress testing needs to be performed to elucidate the inherent stability characteristics of the active drug substance and also to prove the stability - indicating capability of the method. the developed hplc method was validated to quantify the degradation impurities of efx in its tablet dosage form by determining the parameters including specificity, lod, loq, linearity, accuracy, precision, and robustness according to the ich guidelines. specificity of the developed method was performed by injecting the stressed degradation samples and the degradation impurities spiked solutions. the degradation study was carried out using the samples which include (i) tablet powder containing efx and (ii) placebo powder without active drug efx. efx was found to be stable in all the degradation conditions except in oxidation degradation where slight degradation was observed. purity angle value was less than the purity threshold for all peaks indicating all peaks are spectrally homogeneous. also spectral homogeneity of known impurities in degradation samples, found to be similar with those obtained for the individual impurities, suggests that no peak was being coeluted at the retention time of respective known impurities. the results indicate efx undergoes degradation in presence of oxidation condition to form major unknown impurity. the lod and loq were determined for efx and their impurities by injecting a series of solutions with known concentrations. we calculated the s / n ratio for these solutions and selected the concentration at which level s / n was about 3 for lod and the s / n ratio was about 10 for loq. s / n values of lod and loq for efx and their impurities were shown in table 7. the linear graphs were plotted between the peak areas versus concentration to obtain the calibration curve. the response obtained for all compounds was found to be linear from loq to 120% of standard concentration. the relative response factor for efx and all the impurities was determined against their respective standard and presented in table 7. the results demonstrate an excellent correlation between the peak area and concentration of all impurities. method precision was determined by injecting the impurities spiked solution of six determinations and the observed values of % rsd were shown in table 7. the % rsd for all compounds in impurities spiked solution for six determinations was not more than 1.9%. the intermediate precision of the method was studied by injecting the impurities spiked solution of six determinations and the values were shown in table 7. less difference between the two analysts shows that the developed method is precise and has good intermediate precision. the percentage recovery results for impurities of efx were varied from 91.2% to 106.7% at three different concentration levels and the results were shown in table 8. based on the % recovery data, we concluded that the developed method is capable of the estimation of its related substances and is adequate for routine analysis. in all the robust conditions (flow rate, column temperature, and organic composition change in mobile phase and columns) the resolution between two critical pairs (resolution between cipro base impurity and efx) was not less than 1.8. relative retention times (rrt) and resolution values for different robustness parameters were shown in tables 9 and 10. also the resolution between the remaining impurities from analytes was not significantly affected and elution pattern of the impurities remained unchanged. peak purity for all impurities also tested to observe no placebo peaks interference in all the robust conditions. the impurity percent difference was determined for control sample solutions and percent difference was determined for efx standard solution stored at room temperature in different time intervals up to 24 hours. all the impurities and standard solution were found to be stable up to 24 hours at room temperature. solution stability results of efx standard solution and impurities in control sample at room temperature were shown in table 11. a novel rp - hplc method was developed for the separation and quantification of efx and its related degradation impurities in its pharmaceutical dosage forms. unknown degradation impurity of 0.5% was formed from efx in oxidation degradation and no degradation peaks were observed in other stress conditions. all the known degradation impurities and the unknown degradation impurities were well separated from efx revealing the stability - indicating capability of the method. the developed method can be used for the quantification of related substances of efx in routine analysis. | the present work was the development of a simple, efficient, and reproducible stability - indicating reverse - phase high performance liquid chromatographic (rp - hplc) method for simultaneous determination enrofloxacin (efx) and its degradation products including ethylenediamine impurity, desfluoro impurity, ciprofloxacin impurity, chloro impurity, fluoroquinolonic acid impurity, and decarboxylated impurity in tablet dosage forms. the separation of efx and its degradation products in tablets was carried out on kromasil c-18 (250 4.6 mm, 5 m) column using 0.1% (v / v) tea in 10 mm kh2po4 (ph 2.5) buffer and methanol by linear gradient program. flow rate was 1.0 ml min1 with a column temperature of 35c and detection wavelength was carried out at 278 nm and 254 nm. the forced degradation studies were performed on efx tablets under acidic, basic, oxidation, thermal, humidity, and photolytic conditions. the degraded products were well resolved from the main active drug and also from known impurities within 65 minutes. the method was validated in terms of specificity, linearity, lod, loq, accuracy, precision, and robustness as per ich guidelines. the results obtained from the validation experiments prove that the developed method is a stability - indicating method and suitable for routine analysis. |
the ribosomal elongation cycle consists of three basic reactions, namely the binding of the new aminoacyl - trna (aa - trna) according to the codon presented at the decoding center (reaction 1 of figure 1a), the peptide - bond formation (reaction 2) and the translocation reaction (reaction 3). this cycle, described within the framework of the allosteric three - site model (1), is depicted in figure 1a. according to this model, there are at least two trnas on the ribosome during the elongation cycle : trnas in the a and p sites prior to translocation (pre state) and trnas in the p and e sites following translocation (post state) as seen in figure 1a. the allosteric three - site model also states that a - site occupation triggers the release of the deacylated trna from the e site (2,3). the linkage between a and e sites is also indicated by the fact that the activation energy is significantly larger with an occupied e site than with a free one, 120 and 80 kj / mol, respectively (4). however, the exact step of a site binding that triggers release of the e - site trna has not yet been identified. (i) the pre state (1b), where the a - trna is still bound in the form of the ternary complex and makes codon anticodon interaction with the a site codon of the mrna. (ii) the pre state (1c), where the aminoacyl moiety at the cca end of the a - trna is accommodated into the a site at the peptidyltransferase center (ptc) on the 50s subunit. (iii) finally, the pre state (2) arises following peptide - bond formation, such that the peptidyl moiety is now attached to the a - trna and the p - trna is now deacylated (uncharged). release of the e - trna could be triggered at any one of these steps. for example, (i) the antibiotic edeine, which binds in the e site on the 30s subunit induces translational misreading at the a site (3), (ii) weakening of the e - trna via mutations at the s7s11 interface, which binds the anticodon loop of an e - trna, induces dramatic selection problems such as misincorporation and readthrough (5), and (iii) loss of codon anticodon interaction at the e site provokes high - efficiency frameshifting (6,7). furthermore, allosteric effects between the a and e sites have been observed in the crystal structure of 70s ribosomes from thermus thermophilus at 4.5, where components of the e site were well ordered when an e - trna was present, whereas the elements were disordered when the a site instead of the e site was occupied (8). since the ptc is part of a and p sites and antibiotics that bind at the ptc on the 50s subunit have been shown to affect trna decoding at the a site (9), we rationalized that peptide - bond formation could also be regulated by the functional state of the ribosome, specifically the presence or absence of an e - trna. here we have determined the step of a - site occupation at which the e - trna is released from the ribosome, revealing that the e - trna is released before both ef - tu - dependent gtp hydrolysis and accommodation of the aa - trna into the a site. in addition, we show that the rate of transfer of peptidyl - moiety to puromycin is the same for pi and post states, revealing for the first time that peptide - bond formation is not regulated during protein synthesis. these results suggest that the occupation state of the e site influences only the 30s component of the a site, i.e. the decoding center, without effecting the 50s component of the a site, namely, the ptc. reassociated 70s ribosomes free of endogenous trnas and mrnas were prepared according to (10). heteropolymeric mrnas with two (mf - mrna) or three (mvf - mrna) unique codons in the middle were prepared with run - off transcription, according to (11). n - acetyl-[c]val - trna (ac[c]val - trna), [h]val - trna, ef - tu and ef - g were prepared as described previously (3). all complexes were prepared in polyamine buffer system which contained 20 mm hepes koh (ph 7.5 at 0c), 4.5 mm mgac2, 4 mm 2-mercaptoethanol, 150 mm nh4ac, 0.05 mm spermine and 2 mm spermidine. this buffer approximates physiological conditions, with respect to the concentrations of the essential ions using nh4 + instead of k. construction of the pi complexes. 70s ribosomes (0.3 m) were incubated with mrna (mf - mrna, or mfv - mrna, molar ratio mrna:70s = 7:1) plus acphe - trna or acval - trna (molar ratio 1.5:1), at 37c for 30 min in a volume of 1 ml. under these conditions, > 85% of the ribosomes carried a peptidyl - trna analog at the p site, and had free a and e sites. 70s ribosomes programmed with mf - mrna (described above) were incubated for 15 min at 37c with uncharged trnafmet (molar ratio to ribosomes 1.5:1), in order to prefill the p site. subsequently, acphe - trna was added (molar ratio 1.5:1) and incubated for an additional 30 min at 37c to allow non - enzymatic a - site binding. the puromycin reaction yielded no product indicating that trnafmet was at the p site and acphe - trna was bound at the a site. pre complexes (prepared as described above) were translocated in the presence of ef - g (molar ratio to 70s 0.2:1) and gtp (0.5 mm) during an incubation at 37c for 10 min. according to the puromycin reaction post state complexes carrying a trnafmet (labeled with p when indicated) in the e site and an ac[c]phe - trna in the p site were incubated with the ternary complex [h]val - trnagtpef - tu at 25c for 2 min (molar ratio 1.6:1). under these conditions, a peptide bond is formed and the peptidyl - trna is exclusively bound at the a site, as demonstrated with the puromycin reaction. in another series of experiments, we used gdpnp instead of gtp in order to freeze the ribosomal complex in the pre state prior to peptide - bond formation. for kinetics of the puromycin reaction, ribosomal complexes were freed of factors, excess of substrates and gtp, via centrifugation through a 10% sucrose cushion in the same buffer (beckman rotor tl-100, 45 000 g for 18 h, 4c). in each case, the ribosomal bound trnas were measured by nitrocellulose filtration, and the site location of the acylated trna was identified by the puromycin reaction. after formation of the ribosomal complexes, puromycin was added at the indicated concentrations (or if not indicated at 1 mm) and the reaction was carried out at various temperatures in a volume of 180 l. at various time intervals an aliquot of 20 l was withdrawn and the reaction stopped by adding an equal volume of 0.3 m sodium acetate (ph 5.5) saturated with mgso4. the incubation mixture was extracted with 1 ml ethyl acetate and the radioactivity in 800 l of the organic phase was measured after mixing with 5 ml scintillation liquid. background controls (minus puromycin) were subtracted. the puromycin product was expressed as percentage of the ribosomal complex formed before adding puromycin (measured by nitrocellulose filtration). the puromycin reaction was employed for two distinct purposes : (i) to measure the activity of peptidyl transferase via determination of kcat and km and (ii) to determine the amount of ribosomal complex that is puromycin reactive. in the first case the complete time course of the reaction was measured, while in the second only the extent of the reaction was required. if peptide - bond formation between the acyl residue of the trna at the p site and puromycin can be described as a first - order reaction, the following integrated rate law applies 1ln[c0c0p]=kobst, where c0 is the initial concentration of the trnaribosome complex reactive with puromycin (s) and kobs is the observed rate constant of peptide - bond formation. as shown in the results then the relationship between the concentration of puromycin and the apparent rate constant kobs is given by the equation 2kobs = kcatsks+s. where ks is the dissociation constant of the encounter complex between trnaribosomemrna complex and puromycin. the apparent rate constant kobs was measured for several functional ribosomal complexes at various temperatures, in a range of puromycin concentrations and with different donors. from the double reciprocal plot 1/kobs versus 1/[puromycin ], we calculated both kcat and ks (figure 3b). the apparent rate constants observed at various temperatures from 0 to 37c for both the pi and the post complexes were processed in an arrhenius plot according to 3lnkobs = lnaeart, where ea is the arrhenius activation energy which can be estimated from the slope of the resulting regression line in the graph ln / kobs versus 1/t and equals ea / r. the free enthalpy of activation h was calculated according to 4h=eart with r the gas constant and t the temperature in degree kelvin. the free energy of activation g is given by the eyring equation 5g=2.3rtlog([kcatks]nhrt), where h and n are the planck 's and avogadro 's constants, respectively, and ts by 6ts=hg, where s is the entropy of activation. after formation of the ribosomal complexes, puromycin was added at the indicated concentrations (or if not indicated at 1 mm) and the reaction was carried out at various temperatures in a volume of 180 l. at various time intervals an aliquot of 20 l was withdrawn and the reaction stopped by adding an equal volume of 0.3 m sodium acetate (ph 5.5) saturated with mgso4. the incubation mixture was extracted with 1 ml ethyl acetate and the radioactivity in 800 l of the organic phase was measured after mixing with 5 ml scintillation liquid. background controls (minus puromycin) were subtracted. the puromycin product was expressed as percentage of the ribosomal complex formed before adding puromycin (measured by nitrocellulose filtration). the puromycin reaction was employed for two distinct purposes : (i) to measure the activity of peptidyl transferase via determination of kcat and km and (ii) to determine the amount of ribosomal complex that is puromycin reactive. in the first case the complete time course of the reaction was measured, while in the second only the extent of the reaction was required. if peptide - bond formation between the acyl residue of the trna at the p site and puromycin can be described as a first - order reaction, the following integrated rate law applies 1ln[c0c0p]=kobst, where c0 is the initial concentration of the trnaribosome complex reactive with puromycin (s) and kobs is the observed rate constant of peptide - bond formation. as shown in the results then the relationship between the concentration of puromycin and the apparent rate constant kobs is given by the equation 2kobs = kcatsks+s. where ks is the dissociation constant of the encounter complex between trnaribosomemrna complex and puromycin. the apparent rate constant kobs was measured for several functional ribosomal complexes at various temperatures, in a range of puromycin concentrations and with different donors. from the double reciprocal plot 1/kobs versus 1/[puromycin ], we calculated both kcat and ks (figure 3b). the apparent rate constants observed at various temperatures from 0 to 37c for both the pi and the post complexes were processed in an arrhenius plot according to 3lnkobs = lnaeart, where ea is the arrhenius activation energy which can be estimated from the slope of the resulting regression line in the graph ln / kobs versus 1/t and equals ea / r. the free enthalpy of activation h was calculated according to 4h=eart with r the gas constant and t the temperature in degree kelvin. the free energy of activation g is given by the eyring equation 5g=2.3rtlog([kcatks]nhrt), where h and n are the planck 's and avogadro 's constants, respectively, and ts by 6ts=hg, where s is the entropy of activation. we determined kinetic and thermodynamic parameters of the puromycin reaction performed with three ribosomal complexes, (i) pi, (ii) post and (iii) a special pre complex (pre-2), where the ternary complex is frozen in the a site by the non - hydrolyzable gtp analog gdpnp (figure 1b). the pi state is similar to a 70s initiation complex (i for initiation) in that it contains an empty e site and carries an acphe - trna at the p site. the post complex is a standard complex of the elongation cycle, representing a posttranslocational ribosome state by having deacylated trnafmet at the e site and an acphe - trna, the peptidyl - trna mimic, at the p site. the occupancy of the p site in pi (acphe - trna) and pre-1 state (trnafmet) was 85% and binding of acphe - trna to the a site showed that 74% of ribosomes carried acphe - trna in the a site of the pre state ribosomes. following translocation (addition of ef - g and gtp), the occupancies of the trnas in the post complex remained the same, except that the trnafmet was located at the e site and the acphe - trna was at the p site. this demonstrates that the trna is stably bound at the e site following translocation under these buffer conditions. this contrasts with the loss of e - trna found to occur following translocation in standard buffers without polyamines as well as in polymix buffer (12). it should be noted that all complexes were isolated through a sucrose cushion via an 18 h centrifugation to remove elongation factors, unbound trnas and gtp (materials and methods), thus reiterating the stable binding of the trnafmet at the e site. this stability is consistent with the presence of e - trna in native polysomes following an isolation procedure of several hours (13). using these complexes we wanted to check whether the peptidyltransferase activity is regulated by the functional state of the ribosome. in order to do this the kinetics of the puromycin reaction were performed with pi and post complexes at different temperatures, ranging from 0 to 37c (figure 2). as expected, the rate of transfer of the acphe moiety of the p site bound trna to puromycin, forming acphe - puro, slows as the reaction temperature is decreased. furthermore, the data for pi and post complexes were almost identical at all the temperatures measured, providing the first hint that the peptidyltransferase activity is not regulated by the ribosome functional state, nor in this case the presence of an e - trna. if we assume that the equilibrium of puromycin binding to ribosomes is fast and the subsequent peptide - bond formation between the acyl residue of the p - trna and puromycin is slow and rate - limiting, then the kinetics of the puromycin reaction c+skscskcatp+c should follow a first - order law. in the scheme, the left half represents the binding reaction where c is the trnaribosome complex and s is puromycin, and the right half, peptide - bond formation with kcat as the rate - limiting step and p is the product acyl - puromycin. if the data from figure 2 are plotted according to the first - order rate law (materials and methods), then a linear relationship is observed in all cases (figure 3a), which verifies the rate law assumption. we measured the relationship between the puromycin (s) concentration and the apparent rate constant kobs according to equation 2 given in material and methods. the apparent rate constant kobs was measured for pi and post complexes at various temperatures in a range of puromycin concentrations. the data were processed in a double reciprocal plot of 1/kobs versus 1/[s ] and representative plots are given in figure 3b that were virtually identical for pi and post. the values of ks and kcat were estimated from the intercepts with x - axis and y - axis, respectively. kcat values are varied as a function of temperature (figure 3b), whereas ks values were kept constant and equal to 4 10 0.5 10 m. the ratio kcat / ks, which expresses the activity status of peptidyltransferase, was calculated for each complex at different temperature and the values are summarized in table 2. in addition, two controls were included : first, we checked whether the puromycin reaction of the pi state was affected by a preceding incubation with ef - ggtp, to enable a better comparison with the post state that is formed by incubating with ef - g and gtp. except for a spurious increase in the extent of reaction observed in some experiments, second, the donor of the puromycin reaction on the ribosome was changed : instead of acphe - trna, ac[c]val - trna was used for p site binding in the presence of mfv - mrna (pi state). the kinetics of the puromycin reaction was performed at 25c and the rate of reaction was shown to be independent of the species of donor occupying the p site (table 2). the observed rate constants (kobs) determined at various temperatures were used to determine the activation energy ea by applying the arrhenius equation (equation 3 in materials and methods), which enables the activation parameters of free energy g, of enthalpy h and entropy s to be calculated using equations 46 in materials and methods. the calculated values of g, s and ts are comparable with those reported previously [ref. (14) and erratum ], despite the fact that sievers. used a different substrate, namely, fmet - phe - trna instead of acphe - trna. next we analyzed whether the e - trna is released before ef - tu - dependent gtp hydrolysis. previously, we have shown that the a - site occupation with the ternary complex val - trnaef - tugtp induced an almost quantitative release of the e-[p]trna (3), i.e. the deacylated trna was released from the e site at some point during decoding and accommodation of the aa - trna at the a site, however, which of the exact three steps was not addressed. here we have tested whether or not e - trna can be released using a ternary complex [h]val - trnaef - tugdpnp. figure 4a (and table 1 pre-2) shows that the addition of this ternary complex with non - hydrolyzable gtp analogue gdpnp almost completely releases the e site. the a site was 60% occupied with [h]val - trnaef - tugdpnp, and the occupancy of the trnafmet dropped from 85% in the post state to 15% in the pre-2 state. ef - tu release and accommodation of the val - trna at the a site, these results clearly demonstrate that the e - trna is released after the decoding process but before gtp hydrolysis, ef - tu release and accommodation of an aa - trna at the a site. we reasoned that if this was really true, then the a site on the 50s should still be free for binding of puromycin since the cca end of the a site trna is still bound to ef - tu which is far from the ptc (15). figure 4a shows that indeed the p site bound ac[c]phe - trna reacted almost quantitatively with puromycin, whereas the a site bound ternary complex containing val - trna and gdpnp could not. this behavior stands in striking contrast to the analogous experiment in the presence of gtp, where both ac[c]val and [h]phe did not react with puromycin indicating dipeptide formation at the a site [figure 4a, right panel, from figure 6a in (3) for comparison ]. the puromycin reaction is extremely sensitive owing to the low - affinity (km of 0.2 mm) of this drug (16), such that an effect seen with the puromycin reaction might not occur when aa - trna is at the a site instead of puromycin (17). here, we use this highly sensitive method of puromycin reactions and do not find a change of kinetic parameters of the reaction under any of various conditions. therefore, it seems safe to conclude that peptide - bond formation does not in fact depend on the various functional states under observation. the observation that a ternary complex triggers e - trna release after the decoding step but before gtp hydrolysis and accommodation of the aa - trna into the a site provides an elegant explanation of the finding that pi and post states show the same activity concerning peptide - bond formation. this observation means that the a - site region of the peptidyltransferase is occupied by the acylated end of the a - trna only after the e - trna release, i.e. peptide - bond formation always occurs in the presence of an empty e site, either when the e site is free per se (pi state) or has become freed (post state plus ternary complex). we have found that a successful decoding event triggers e - trna release, but does not affect the active state of the ptc. an important implication of this finding is that the small subunit is a main player in the allosteric interactions between a and e site, supported by a wealth of data (3,57) without including the 50s region of the ptc, which is consistent with the large distance between the e site and ptc. in the future, it will be interesting to investigate how the e - trna is released in response to the binding of the tmrna complexed with smpb and ef - tugtp (18,19), which binds to ribosomes stalled on truncated mrnas that have no codon in the a site. (a) the elongation cycle in the frame of the allosteric three - site model. the binding of the new aa - trna at the decoding center (reaction 1), peptide - bond formation (reaction 2) and translocation reaction (reaction 3). the reciprocal linkage between a and e sites is indicated by a gap : an occupied e site induces a low - affinity a site and vice versa. a - site occupation induces the release of the e - trna [for review see (1) ]. kinetics of the puromycin reaction, at the following temperatures : 0, 10, 20, 25, 30 and 37c. the puromycin concentration was 1 mm and the product acphe - puromycin was expressed as a percentage of the preformed ribosomal complex (pi and post, squares and circles, respectively) as measured via nitrocellulose filtration. (a) first - order fitting of the data of figure 2 at the temperatures of 25 (circles), 30 (triangles) and 37c (squares) for pi and post complexes. the slope of the plots gives the apparent rate constant of product formation (kobs) at the specified puromycin concentration (1 mm). (b) double reciprocal plot of puromycin reactions at temperatures 25 (circles), 30 (triangles) and 37c (squares). the intercept on the y - axis gives 1/kcat, while the intercept on the x - axis gives 1/ks. (a) trna binding and puromycin reaction with ribosomal complexes in different functional states. the binding of [p]trnafmet (blue) to the p site and ac[c]phe - trna (red) to the a site (pre-1 complex) was followed by addition of ef - g and gtp to induce translocation (post). next, the ternary complex ef - tugdpnp[h]val - trna (green) was added and the new pre-2 complex was formed. in all cases the extent of binding of the respective trnas are indicated with upright bars of the corresponding color, and the accessibility of peptidyl- and aa - trnas to puromycin is indicated with hanging bars of the same color. all data are expressed with, i.e. pmoles of bound trna or peptidyl- or aminoacyl - puromycin formed per pmole ribosome. the corresponding data from the pre-2 state performed with gtp instead of gdpnp are taken from figure 6a in (3). the complex carried ac[c]phe - trna at the p site and [h]val - trna - ef - tu - gdpnp at the a site. the puromycin concentration was 1 mm and the product was expressed according to both isotopes, either ac[c]phe - puromycin (squares) or [h]val - puromycin (circles). occupation of trnas in various ribosome functional states using mfv - mrnaa all values are presented as (pmoles bound isotopes per pmol 70s ribosomes). all complexes were measured after isolation through sucrose cushion to remove unbound elongation factors, unbound trnas and gtp. the catalytic activity of peptidyltransferase at different temperatures for the three different functional ribosomal states ; pi, post and pre-2a the activity is expressed by the ratio kcat / ks, which were calculated from the double reciprocal plots like figure 3b. | the presence or absence of deacylated trna at the e site sharply influences the activation energy required for binding of a ternary complex to the ribosomal a site indicating the different conformations that the e - trna imparts on the ribosome. here we address two questions : (i) whether or not peptidyltransferase the essential catalytic activity of the large ribosomal subunit also depends on the occupancy state of the e site and (ii) at what stage the e - trna is released during an elongation cycle. kinetics of the puromycin reaction on various functional states of the ribosome indicate that the a - site substrate of the peptidyltransferase center, puromycin, requires the same activation energy for peptide - bond formation under all conditions tested. we further demonstrate that deacylated trna is released from the e site by binding a ternary complex aminoacyl - trnaef - tugdpnp to the a site. this observation indicates that the e - trna is released after the decoding step but before both gtp hydrolysis by ef - tu and accommodation of the a - trna. collectively these results reveal that the reciprocal linkage between the e and a sites affects the decoding center on the 30s subunit, but does not influence the rate of peptide - bond formation at the active center of the 50s subunit. |
electro convulsive therapy (ect) is an effective treatment modality in the management of depression, mania, bipolar affective disorder (bad), schizophrenia as well as a number of psychiatric disorders since 1930s. complications of ect like subdural hematoma (sdh), intracerebral hemorrhage (ich), have been reported in the literature sparsely. we report a case of chronic sdh in a 38-year - old lady resulting from ect for management of bad. a 38-year - old lady who was a known case of bad for last fifteen years presented with increasingly agitated behaviour along with intense mood fluctuations for last six months. she had a total of 12 sessions over a period of six weeks when she developed frontal headache and frequent vomiting with increasing frequency for last three weeks. there was additional history of altered sensorium, agitation, slurring of speech (dysphasia), and weakness of the right side of the body without loss of consciousness or seizure. on examination by the neurosurgeon and performance of computed tomography (ct) scan of the brain, a left temporo - parietal subdural hematoma with midline shift was revealed [figures 1 and 2 ]. computed tomography scan of the brain showing post electro convulsive therapy subdural hematoma ct scan of the brain showing post ect subdural hematoma owing to the similarity of symptoms with ect, the diagnosis of chronic subdural hematoma following ect is difficult in clinical practice. ect is a well accepted treatment modality for severe mental illness in which a short application of electrical stimulus is used to produce a generalized motor seizure. the generalized seizure lasts several minutes and includes a short 10 - 15 seconds tonic phase followed by a more prolonged clonic phase, lasting for 30 - 60 seconds. most patients scheduled to undergo ect are receiving tricyclic antidepressants (tcas), monoamine, selective serotonine reuptake inhibitors (ssris), lithium carbonate, or a combination of these drugs. the newer drugs such as trazodone and bupropion have lesser complications.[24 ] lithium carbonate prolongs the action of neuromuscular blockade. therefore, pre - ect workup should include a complete medical and neurologic evaluation of the patients. ect can be used safely in elderly patients and in persons with cardiac pacemakers or implantable cardioverter - difibrillators. generalised autonomic nervous system stimulation causes an initial bradycardia and occasional asystole, followed by a more prominent sympathetic response of hypertension and tachycardia. occasionally cardiac dysrrhythmia, myocardial ischaemia, infarction, or neurologic vascular events may be precipitated. first, the medical complications that can be substantially reduced by the use of appropriately trained staffs, best equipments, and best methods of administration of therapy. death due to ect is mostly from cardiovascular and hemodynamic complications and occur most frequently in patients with already compromised cardiovascular profile. the adverse effects of ect includes laryngospasm, circulatory insufficiency, headache, emergence agitation, tooth damage, vertebral compression fractures, status epilepticus, peripheral nerve palsy, skin burns, and prolonged apnea. common adverse effects associated with ect are headache, nausea, fatigue, confusion, and delirium, shortly after the seizure while the patient is recovering from anesthesia. marked confusion may occur in up to 10% of patients within 30 minutes of the seizure and can be treated with barbiturates and benzodiazepines. delirium is usually most pronounced in after the first few treatments and in patients who received bilateral ect or those who have co - existing neurological disorders. high risk group of patients for ect include those with recent myocardial infarction and/or ischemia, uncontrolled blood pressure, intracranial sol, and previous spinal injuries. memory loss is most often reported by patients who have experienced little improvement after ect. few cases of neurological complications such as ich and sdh were not found to increase or deteriorate following ect thereby establishing the safety of this procedure in patients with such neurological complications. although it is difficult to establish the diagnosis of intracranial hemorrhage as iatrogenic, but any change in mental status, worsening of symptoms, or development of focal neurological abnormality in patients undergoing ect should be treated with utmost urgency and investigated with a ct scan of brain to rule out intracranial complications. this case report and other publications suggest that one can not rely only on clinical symptoms when symptoms worsen after ect, although ect is a safe procedure. therefore, it is important to investigate those patients who are having neurological signs and symptoms following ect and a ct or magnetic resonance imaging (mri) scan should be performed appropriately. anticipation of complications along with their early diagnosis and treatment are the most important factors in delivering a comprehensive ect. | subdural hematoma is a rare but serious complication following electroconvulsive therapy (ect), a frequently used treatment modality in the management of various psychiatric morbidities including bipolar affective disorder (bad). there are very few reports of intracranial bleeding following ect in the literature. a 38-year - old female, known case of bad for last fifteen years receiving ect, presented with the symptoms of dysphasia, headache, left sided paresis, and sudden deterioration of sensorium. computed tomography (ct) scan of the brain was suggestive of left - sided fronto parietal chronic subdural hematoma with midline shift that was drained successfully. |
even in the earliest explorations of eukaryotic ultrastructure it was clear that the nucleus, the defining organelle of eukaryotes, can and does differ widely in size and morphology between different species. these nuclei do, however, seem to share a number of certain features in the electron microscope : every nucleus is bounded by a double - membraned nuclear envelope (ne), perforated by nuclear pore complexes (npcs) and often underlayered on the nuclear periphery by a dense lamina. electron dense bodies such as the nucleolus are commonly found in the nuclear interior ; and even the earliest light microscopic studies recognized that chromatin, the nucleus packaged dna, seemed to come in less and more densely packaged forms, termed euchromatin and heterochromatin respectively. though all these features are clearly present in the model metazoan organisms, such as vertebrate, fly, and nematode cells, stark differences emerge when we look at another favorite group of organisms, the fungi. the model yeast saccharomyces lacks both a lamina and obvious heterochromatin, and the npcs seem far smaller than those in metazoa. thus, metazoan organisms generally disassemble their ne and npcs completely at the onset of mitosis to allow the spindle to assemble and segregate the chromosomes. a new ne and the npcs reassemble around the daughter chromosomes at the end of mitosis. by contrast, both the ne and npcs remain intact throughout the entire cell cycle of yeast, with the spindle forming inside the unbroken nucleus (see refs. 8 and 9 for reviews). yeast and metazoa are actually rather close cousins from the perspective of the entire eukaryotic evolutionary tree, both sharing the opisthokont branch or supergroup (fig. 1). however, many unicellular parasitic protozoa are highly divergent eukaryotes, and many sources have suggested that their nuclear organization is markedly different, albeit lacking much in the way of a molecular characterization. thus, our normal opisthokont - centric view, focusing on those cells and organisms most closely related to our selves, i.e., animals and fungi, may be inadequate in attempting to understand these economically important and human - impacting disease organisms. further, as many protists are major components of the biosphere, their overall impact on the environment is immense. here we use one such divergent organism, trypanosoma brucei, as a perspective on this issue. remarkably, recent advances have revealed that many of the underlying structures and systems in nuclei seem far more conserved than appearances suggested from the rather divergent transcriptional mechanisms in trypanosomes, but that there are crucial differences. these advances allow us not only to view the whole tree of eukaryotic evolution anew, but shed new light on how our own cells are organized and function, with therapeutic implications not only for these divergent parasites but also for ourselves. (a) cartoon of structures at, and associated with, the nuclear envelope. shown are the nuclear pore complex / karyopherins (red), sun / kash (linc complex) proteins (blue) and known components of a nuclear lamina (lamins, green ; nup-1, yellow). (b) the evolutionry distributions of the nuclear pore complex / karyopherins, sun / kash proteins and known nuclear lamina proteins are shown colorized and overlaid upon a schematic phylogeny of the eukaryotes, emphasizing the five contemporary - recognized supergroups. feca, first eukaryotic common ancestor ; leca, last eukaryotic common ancestor ; kap, karyopherin ; sar + ccth, stramenopiles alveolates, and rhizaria + cryptomonads, centrohelids, telonemids and haptophytes. trypanosomes are members of the euglenozoa, a group of organisms that belongs to the excavata supergroup (fig. 1). they most likely branched very early during eukaryotic evolution, which in part may explain some of their more unusual biology. species include the photosynthetic euglenids, which populate most fresh water environments, free living phagotrophs, such as bodo saltans, and plant and animal parasites, the phytomonads and trypanosomatids, respectively. trypanosoma brucei has emerged as the model organism for this group, on account of both its importance as the causative agent of human african trypanosomiasis, and from technical advances that include robust rnai systems, excellent cytology and, most recently, forward genetics based on whole genome rnai screening. accordingly, our understanding of transcription and chromatin organization is comparatively advanced for t. brucei, although major gaps remain in our knowledge. as far as we know, all trypanosomatids exploit primarily polycistronic transcription, where large (tens) numbers of open reading frames are transcribed in tandem. there is little evidence for functional association between orfs in individual polycistronic transcription units (ptus), although evidence suggests that the position within the ptus is associated with mrna copy number. the nascent rnas that are produced from the ptus are processed by a combination of trans - splicing and polyadenylation to yield mature mrnas that, bar an unusual 5 cap structure, are organizationally indistinguishable from higher eukaryotes. however, this mode of transcription does mean that much of the genome is constitutively transcribed, and mrna abundance is primarily controlled at the post - transcriptional level, in sharp contrast to metazoan and yeast model systems, where the transcription of each gene is usually highly regulated by a dedicated promoter. notable exceptions to this mechanism are the loci encoding the superabundant surface antigens, the variant surface glycoprotein (vsg) and procyclin ; these molecules are expressed at very high levels, and rather remarkably, their transcription is driven by rna polymerase i, rather than the more usual rna pol ii which is responsible for transcribing the majority of ptus. both vsg and procyclin are developmentally regulated, with the former expressed exclusively in mammalian infective forms and the latter in insect vector stages. while procyclin orfs are embedded within the body of the chromosomes and consist of small loci of a handful of orfs, vsg transcription is substantially more complex. vsg is the molecular basis for antigenic variation, the primary immune evasion mechanism in these organisms. the sequential expression of distinct vsgs, in a semistochastic manner, serves to allow escape from immune destruction at the population level, and which appears to rely on monoallelic expression, i.e., a single vsg expressed in any one cell at any one time. key to this is the restriction of transcriptionally active vsg orfs to subtelomeric expression sites (es), where the vsg is the most telomere - proximal orf in a pol i - driven ptu. selection of a single vsg is achieved by repression of most vsg ess within heterochromatin, and the translocation of the active expression site into a specialized region of the nucleus called the expression site body (esb). it is likely that the positioning of vsg ess at the nuclear periphery, by virtue of their subtelomeric location, places the inactive ess within the heterochromatin. both telomere suppression and vsg - specific repression mechanisms appear to operate (see below). further, the procyclin locus, which is also transcribed by pol i, is also subjected to control via positioning within the nucleus ; when active, i.e., in the insect, the locus resides peripherally to the nucleolus. while definitive data are lacking, all evidence points to a peripheral nuclear position for the locus in the mammalian infective stages, where it is (relatively) inactive. while polycistronic transcription is certainly not unique to trypanosomes, the high proportion of the genome transcribed by this mechanism is highly unusual. however, taken with the presence of pol i - mediated transcription of protein - coding genes, exploitation of 4d positioning of loci encoding super - abundant surface antigens whose expression is modulated in a life cycle dependent manner and the divergent evolutionary position of trypanosomes suggests the presence of unusual mechanisms at the nuclear periphery. although it had been suggested from in silico analysis that the nuclear transport system of trypanosomes had little in common with that of opisthokonts, a combination of proteomics, structural studies, and comparative genomics has revealed that the npc and associated karyopherin transport factors are an ancient feature, and that the overall configuration of this organelle and the transport mechanism and diversity of pathways were established in the last eukaryotic common ancestor (termed leca). by contrast the lamina and associated macromolecules appear to be more divergent. the basic npc architecture, in terms of division into a structural scaffold, consisting predominantly of protocoatomer - related proteins, together with a gating system comprising about ten disordered fg repeat - containing nucleoporins, is conserved across the eukaryotes. further, the karyopherin family is likewise ancient and, perhaps more remarkably, based on an ability to reconstruct the majority of karyopherin clades as being widely represented across the eukaryotes, the level of diversity and specificity in nucleocytoplasmic transport is both well conserved across the eukaryotic lineage and a feature of the leca. what these studies imply is that the plural mechanisms of nucleocytoplasmic transport, which are intimately wrapped up with mrna processing and translocation into the cytoplasm and hence gene expression, are extremely ancient. minor differences in the compositions between mammalian, yeast, plant and protozoan npcs may signal some diversification / adaptation of mechanism that reflects the differing transcriptional systems in place in these various lineages, but at a level that is quite detailed and one that is not immediately obvious or accessible from a study of the proteins comprising the npc and in silico approaches alone. in part, this is due to our rather limited grasp of the precise functions of many individual nucleoporins themselves, and it may well be that some functionality is redundant, such that the presence of a specific subunit from one organism has little or no clear impact on functionality ; this is certainly an area where more investigation is warranted. we have evidence for a nucleoporin, tbnup92, that appears functionally similar to the mitotic spindle - associated tpr nuclear basket proteins, despite an absence of obvious sequence - based homology and also an fg - repeat nucleoporin, tbnup53b, potentially involved in transcriptional control and hence analogous to the nup98 proto - oncogene from drosophila, albeit again with no obvious primary structural similarity (holden, j, mpr and mcf, unpublished data, and refs. it is unclear if these functional similarities in the absence of evidence for sequence relatedness are a result of convergent evolution or a failure in search algorithms to detect such similarity. in sharp contrast to the npc / karyopherin system, there is good evidence for significant divergence in the nuclear lamina. while a nuclear lamina has been clearly detectable by ultrastructural analysis in various eukaryotes, and might be expected, in the sense of a basic requirement for structural support of the nucleus (though yeast does manage without apparent support), a knowledge of the molecular basis was restricted to metazoa, where the lamin proteins have been well characterized. despite extensive searches of the genomes of yeasts and many other organisms, no evidence for lamin orthologs had been forthcoming, leading to the suggestion that lamins represent a metazoan innovation. first, through a serendipitous route, a clear lamin ortholog was identified for dictyostelium discoideum, which even includes a c - terminal prenylation signal, heptad repeat signatures and a potential cdk-1 phosphorylation site consensus ; orthologs are also present in additional amoebozoa genomes. with lamins restricted to metazoa, lineage - specific innovation is the obvious explanation, but the new data suggest that in fact lamins arose several hundred million years earlier, at least as early as the origin of the unikont lineage, which includes the opisthokonta and amoebozoa. clearly then, the absence from fungi can now be interpreted as a secondary loss, and has profound implications for our views on gene expression mechanisms and lifestyles of rather early eukaryotes close to the radiation of the modern supergroups. this study also provides a salient lesson as well identification of divergent proteins and detecting an ortholog is often challenging ; the tools available have especial difficulty with coiled coil proteins. this topic is discussed further in a recent extra view article in these pages to which the interested reader is referred. in the remaining supergroups, the bikonts, there is so far only one example of a lamina - associated protein for which convincing functional evidence is available. this is the trypanosome nup-1 protein, which actually has structural similarities with and fulfills many of the reported functions of lamins. nup-1 forms a fenestrated, apparently fibrillar, network on the inner face of the nucleus, and is implicated in functions including maintaining the structural integrity of the nucleus, positioning of npcs and telomeres, and mediating telomeric silencing. interactome analysis also indicates a physical association between the npc and nup-1 (obado s, mcf, mpr and chait bt, unpublished). in trypanosomes, the role in telomere positioning is particularly critical as monoallelic vsg expression is dependent on heterochomatinization of subtelomeric vsg expression sites, and indeed there is good evidence that this process is disrupted by knockdown of nup-1. while the parallels between nup-1 and unikont lamins are striking, there are some critical differences. first, there is no obvious sequence relatedness and the amino acid repeats in t. brucei are 144 residues, significantly larger than the heptad repeats of the lamins. second, nup-1 is a huge protein of 407 kda, compared with ~60 kda for lamins, which means that nup-1 can reach across a substantial proportion of the trypanosome nuclear volume, and hence may indicate a rather distinct structural organization to the lamins (fig. 2). however, this may in part be specific to trypanosomes as the orthologs in leishmania are considerably smaller, albeit still predicted to contain over 2,000 residues. hence, while there is no evidence for any relationship or common ancestry between nup-1 and the lamins, we remain agnostic on this issue. blue ovals indicate the relative sizes of a fibroblast and trypanosome nucleus, at 10 m and 1.5 m in diameter. the molecular weight of the unikont lamins is approximately 60 kda while nup-1 is 450 kda ; these are indicated as white bars close to the indicator lines for each nucleus. the inference is that the structural arrangement of the lamina in these systems is potentially very different. also, as mitosis is closed in trypanosomes, the nup-1 network does not break down as does the mammalian lamin network, but rather it is maintained throughout mitosis, where it may participate in chromosomal segregation (holden j and mcf, unpublished data). the absence of a massive re - assortment of the nuclear envelope during mitosis is quite significant in terms of maintaining epigenetic marks and the mechanisms by which specific dna sequences are incorporated into heterochromatin, as in metazoa several lamin - dependent mechanisms that deliver loci to heterochromatin occur as a component of mitosis and reassembly of the nuclear envelope at late anaphase, which clearly can not be the case for trypanosomes. hence it is unclear just how many of the proteins that control entry, exit and maintenance of heterochromatin are shared across the eukaryotes. however, it seems to us a key point that peripheral organization of developmentally - regulated chromatin (kept inactivated as heterochromatin until needed) at a nuclear lamina composed of coiled - coil proteins is a shared feature of representative unikont and bikont organisms. linc complexes are comprised of trimers of sun and kash domain proteins ; these trimers then interact within the lumenal space of the nuclear envelope. these trans - membrane domain proteins are responsible for providing a physical tether for the lamina to the nuclear envelope, as well as a connection between the nuclear lamina and the cytoskeleton. significantly, sun and/or kash domain proteins with the same architecture as those present at the nuclear envelope of metazoa are found in all eukaryotic lineages (including excavata lineages such as the diplomonads and heterolobosids), with the exception of the trypanosomatids (fig. by contrast, sun - like proteins, have different architecture to classical sun proteins (fig. 3) and their function is likely distinct from the classical sun proteins of the linc complex, and are found in trypanosomatids as well as elsewhere. this is perhaps consistent with the restriction of the nup-1/nup-2 lamina to trypanosoma and perhaps argues against convergent evolution from a common lamin / sun / kash system. if this is the result of loss from a leca possessing a lamin - based system, or reflects differential evolution of the lamina in the trypanosomatid and unikont lineages remains to be determined. it is also possible that, as trypanosomes likely branched early from the eukaryotic lineage, that evolution of the lamin / sun / kash system postdates their speciation event. however, the discovery of the d. discoideum lamin is a further salient lesson in relying too heavily on in silico data alone, if any more are really needed at this juncture, and resolution of this issue will require deeper experimental probing. in summary, the nuclear periphery of the trypanosomes appears to be a mixture of highly conserved elements, together with other elements that may be lineage specific, or are at the very least, extremely divergent (fig. 1). the values supporting the separation into two main subfamilies are : approximate likelihood ratio test calculated with phyml 3.0/non - parametric bootstrap calculated in raxml 7.2.6/posterior probabilities from phylobayes 3.3b. the right panel depicts the domain structures of sun - domain containing proteins of the representative species used in the phylogenetic analyses. given the evidence that nup-1 is involved in the positioning of telomeres, npcs and also the repression of both the vsg and procyclin loci, it is important to consider how the t. brucei lamina impacts singular, mono - telomeric vsg expression and organizes chromatin in a broader context. it seems unlikely that a lamina itself would directly control allelic exclusion, but tethering could certainly be important for maintaining the silencing of all but one of the telomeric vsg genes. for example, a single telomeric vsg may " untether " as part of the process that allows transcriptional activation. a swap in tethering could then allow transcription to switch from one telomeric vsg to another, a process known as in situ switching. it is worth considering what is known about the sub - nuclear context of the active and silent telomeric vsgs in this regard. silent vsgs are thought to be associated with the nuclear periphery while the active vsg is found in the esb. however, no esb - specific factor has been described, and cells depleted for the telomeric - repeat dna binding protein, rap1, that is required for full telomeric vsg silencing, form multiple esbs. this then provides support for the idea that rna pol i recruitment and esb formation are consequences of activation. thus, current observations are consistent with a tethering - untethering model as outlined above, but it remains to be seen whether activation and untethering, if it happens, occur in a specific order or if they occur concomitantly. significantly here, no increase in the number of esbs was observed following nup-1 knockdown, suggesting a distinct mechanism to rap1. there is increasing evidence that basal telomeric silencing in t. brucei and vsg gene silencing are mechanistically distinct. the first evidence in support of this distinction came from studies on telomeres themselves, which could be removed, disrupting basal telomeric silencing, but without disrupting vsg silencing. a second piece of evidence derives from studies on the histone deacetylase, silent information regulator 2 related protein 1 (sir2rp1), which is required to maintain basal telomeric silencing in many organisms but had no major impact on vsg silencing. interestingly, studies on nup-1 suggest that it is only vsg silencing that is dependent on the intact nuclear lamina. it remains possible that basal telomeric silencing is dependent upon another component of the nuclear lamina but characterization of these other components will be required to determine whether this is indeed the case. the lamina component, nup-1, is now known to play a role in vsg silencing, and other observations are consistent with the idea that the t. brucei nuclear lamina is important for organizing and reducing the mobility of many subtelomeric vsg genes within the confines of the nuclear space. it will be important to identify lamina - associating sequences and lamina - associated domains in t. brucei and structural or regulatory rnas should probably also be considered in possible models of vsg expression control. ultimately, any model that seeks to explain mono - telomeric and switchable vsg transcription, a system that requires communication among telomeric genes, will likely require the identification of new key regulators and will also need to deal with issues relating to cause and effect. whatever comes next, we suspect that further characterization of the t. brucei lamina will make an important contribution to improving our understanding of this complex process. the structure and segregation of the t. brucei nuclear genome, suggests the possibility of differential interactions with the nuclear lamina, with implications for recombination and gene expression. the 32 mbp haploid nuclear genome of t. brucei is distributed across more than 120 linear chromosomes, divided into three classes : 11 pairs of megabase chromosomes (mbc) of at least a megabase in size, one to five intermediate sized chromosomes (ic), and more than 100 minichromosomes (mc) of 50 to 150 kbp. the mbcs contain all of the housekeeping genes, as well as about 15 telomere - proximal vsg ess. the mcs are highly repetitive in nature and constructed around a palindromic arrangement of a characteristic 177 bp direct repeat ; many also contain vsg genes, providing a library of distinct variants. the intermediate chromosomes also carry vsg genes, though in contrast to the mcs, these are present in ess. in addition to the linear chromosomes, the t. brucei nucleus contains an unknown number of circular extra - chromosomal dnas termed nlaiii repeat (nr)-elements of up to 400 kbp in size. the nr - elements contribute approximately 6% of the total dna content of the nucleus, but have no known function. in common with the mcs, these different classes of nuclear dna exhibit significant variation in segregation during mitosis and their subsequent organization in the interphase nucleus. the mbcs are segregated by a spindle - kinetochore interaction via microtubules peripheral to the main central spindle, so enabling the telomeric regions of the mbcs to maintain their association with the nuclear lamina during mitosis and remain within the established heterochromatin throughout the cell cycle, although to date components of the trypanosome kinetochore remain uncharacterized. this arrangement may serve to prevent interaction with the active vsg es, which localizes to the extranucleolar expression site body, the site of rna polymerase i driven expression of the active vsg, and which replicates later during the cell cycle. by contrast the mcs and nr - elements segregate along the central spindle and rapidly migrate to the poles of the daughter nuclei, after which the mcs maintain an asymmetric distribution during interphase, and the nr - elements disperse throughout the interphase nucleus. compared with the mbcs, the interaction of the mcs with the nuclear lamina therefore appears episodic, with the association breaking down during mitosis as they migrate along the central spindle, and then becoming re - established upon completion of mitosis, as indicated by a persistent asymmetric distribution during interphase. the vsg genes on the mcs are not in ess meaning that they can not be expressed from these loci, so any potential loss of their heterochromatin state during mitosis will presumably not lead to expression, negating a need for this level of repression. instead, periodic dissociation from the nuclear lamina may allow for recombination between mcs, potentially serving to generate further vsg variants. establishment of putative mc - lamina interactions may also allow recombination with silent vsg ess also present in the peripheral heterochromatin, but likely also prevents disruption of the active vsg during interphase on account of the differential positioning and kinetics of replication of the esb. in contrast to the mbcs and mcs, there is no evidence that the nr - elements interact with the nuclear lamina. indeed, their circular structure and consequent lack of telomeres may preclude any such interaction, resulting in their dispersal during interphase. our understanding of the evolution of the ne has advanced considerably in the past five years, with 2012 being a particularly good year for moving forward in our understanding of the evolutionary biology of the lamina. the old paradigm of lamins as metazoa specific is now clearly obsolete, and there is robust evidence for a lamina in at least one branch of the excavata. many gaps remain, and we currently lack definitive information on the compositions of the lamina of plants, chromalveolates, stramenopiles and several excavata lineages, despite the clear evidence for heterochromatin in these taxa. in the case of plasmodium, a member of the chromalveolates, a role for telomeric positioning in antigenic variation and with gross similarities to african trypanosomes is well known, but the absence of a molecular definition of a lamina is a significant gap in comprehending the virulence mechanisms of a major pathogen. much novel biology and many surprises likely await the investigations of the lamina in these organisms. the rather unusual minichromosomes, which contribute to antigenic variation, are an important aspect of the biology of trypanosomes, and may have had a strong influence of the evolution of the nuclear lamina in these taxa, potentially to avoid promiscuous recombination, reactivation and other defects that could disrupt antigenic variation. however, genome structure alone can not explain the high divergence between lamin - based and nup-1-based lamina as, for example, minichromosomes are absent from t. cruzi, a related trypanosomatid that has a clear nup-1 ortholog. in terms of defining the functions of the trypanosomatid nup-1 lamina, a pre - requisite for deeper comprehension of how related the mechanisms of organization of chromatin truly are in trypanosomes and unikonts, it will be essential to probe the interactions of these proteins in greater detail. this will necessitate interrogation of how trypanosome lamina components operate in the context of specific dna sequences, determination of the nature and distribution of lamina - targeting sequences (if such exist), how the nup-1 lamina organizes chromatin - modifying activities, and how this system integrates with the nuclear envelope and spindle. answering these questions is most likely a long journey, but the identification of nup-1 as a lamina component has finally provided a doorway through which we can begin that journey. lastly, we can ask : why do eukaryotes seem to share, as a most fundamental mechanism of genetic control, developmental regulation by packaging of huge blocks of chromatin at the nuclear periphery, a periphery organized in many cases by an extensive coiled - coil protein network ? such developmental heterochromatin in higher eukaryotes is used to store the genetic information needed to build tissues. cohorts of tissue - specific genes are packaged away in lineages where expression is not needed, or perhaps more critically, would be detrimental if allowed to occur ; the dynamic range of heterochromatin suppression is very much greater than simple promoter - based inactivation. hemoglobin genes are thus normally inactive in the nuclear peripheral heterochromatin of all but hemopoetic cells while cell adhesion molecules that specify cell type and position are similarly inactivated during metazoan embryogenesis. but leca was undoubtedly unicellular, and therefore these sophisticated multicellular organism - related processes can not explain the need for such tight regulation. likewise, the parasitic lifestyle of trypanosomes necessitates massive remodeling to accommodate drastically different insect and vertebrate host environments, but again leca predates multicellularity, and hence such parasitic lifestyles. however, the inference that leca possessed such a developmental regulation mechanism indicates that it could undergo significant changes in its lifestyle. the primordial environment leca faced was potentially much more unstable and chaotic than most organisms face nowadays, as it is our extensive biosphere that contributes to stabilizing earth s modern climates. it is also possible that the specific location that leca found itself in had specific challenges that necessitated periodic modulations of gene expression patterns, for example salinity or seasonal changes. we speculate that perhaps leca evolved to change its form and function to adapt to these shifting environments. an ability to store great amounts of genetic information to be used upon need could have been the path to the eukaryotic colonization of diverse land and sea environments, and the basis for the multicellularity required for the complex lifeforms that enabled such colonization. | temporal and spatial organization of the nucleus is critical for the control of transcription, mrna processing and the assembly of ribosomes. this includes the occupancy of specific territories by mammalian chromosomes, the presence of subnuclear compartments such as the nucleolus and cajal bodies and the division of chromatin between active and inactive states. these latter are commonly associated with the location of dna within euchromatin and heterochromatin respectively ; critically these distinctions arise through modifications to chromatin - associated proteins, including histones, as well as the preferential localization of heterochromatin at the nuclear periphery. most research on nuclear organization has focused on metazoa and fungi ; however, recent technical advances have made more divergent eukaryotes accessible to study, with some surprising results. for example, the organization of heterochromatin is mediated in metazoan nuclei in large part by lamins, the prototypical intermediate filament proteins. despite the presence of heterochromatin, detected both biochemically and by em in most eukaryotic organisms, until this year lamins were thought to be restricted to metazoan taxa, and the proteins comprising the lamina in other lineages were unknown. recent work indicates the presence of lamin orthologs in amoeba, while trypanosomatids possess a large coiled - coil protein, nup-1, that performs functions analogous to lamins. these data indicate that the presence of a nuclear lamina is substantially more widespread than previously thought, with major implications for the evolution of eukaryotic gene expression mechanisms. we discuss these and other recent findings on the organization of nuclei in diverse organisms, and the implications of these findings for the evolutionary origin of eukaryotes. |
adults with bd spend approximately 9% of their time in manic or hypomanic episodes, whereas they spend 32% of the time in depressive episodes. children and adolescents with bd clearly experience significant depressive symptoms as well as depressive episodes. however, the phenomenon of depression is less studied in pediatric bd. in a phenomenological study of 438 children and adolescents with bipolar spectrum disorders, suicidal thoughts and behaviors were common as well, with 76% having past suicidal ideation, and 31% having made a prior suicide attempt. it is not clear if children with bd arc commonly misdiagnosed with unipolar depression, but this phenomenon is common in adults. it should also be noted that irritability is commonly a presenting symptom of depression, rather than only mania, in children. thus, the dsm - iv allows for the predominant mood to be irritability or dysphoria for children to meet criteria for a depressive episode. irritability is a common symptom in children with bd, even outside a clearly established manic episode. therefore, it is possible that a certain portion of irritability in children with bd stems from a more depressive etiology. this is important to remember, in that, much as adults with bd are often misdiagnosed with unipolar depression, children with bd should not have their symptoms of irritability misdiagnosed as mania if they are truly stemming from depression. finally, mixed episodes occur frequently in pediatric bd. in adults, these episodes have been thought to be more difficult to treat than pure mood episodes, and also carry the highest risk of suicide attempts. similarly, in a pediatric bd cohort, mixed episodes were one predictor of suicide attempt. thus, depressive symptoms may also occur within the context of mania symptoms in children, and therefore such children should also be carefully assessed for potential mixed episodes. there are several reasons why such depressive episodes and symptoms in children with bd may be missed by clinicians. foremost, manic symptoms usually are what bring the child into the office, including symptoms of high energy, impulsivity, recklessness, sleeplessness, hypersexuality, and irritability and anger. children presenting with more subtle symptoms of anhedonia and fatigue are not always as obvious in presentation to parents or teachers as those presenting with such manic symptoms. irritability, as mentioned above, is also often mistaken as mania and not recognized as a symptom of depression. finally, in younger children such depressive episodes are not as common as for adolescents with bd, but depressive symptoms may often intermingle with manic symptoms, and thus be underidentified. clinicians may benefit from carefully eliciting depressive symptomatology in any child with bd, and recognizing any type of suicidal ideation, even passive, as a red flag for a serious depressive episode. nonetheless, due to the morbidity and mortality of depression in youth with bd, it is necessary to treat these children. one may look to the treatment of bipolar depression in adults for some guideposts, as this topic has been more studied in adults. there exist many treatment options for patients with bipolar depression. while antidepressants have historically been the first line of treatment for bipolar depression, concern over the propensity for antidepressants to cause manic switching or cycle acceleration has led to questioning of this approach. it is becoming clear that overall, the addition of antidepressants to mood stabilizers for adult bipolar depression offers no greater benefit than placebo, and up to 44% of adults with bd have experienced a switch into mania or a mixed episode with an antidepressant trial. thus, several expert consensuses have recommended nonanti depressant medications as first - line treatment for adults with bipolar depression, including lithium, lamotriginc, olanzapine - fluoxetine combination, and quetiapine. other options showing some efficacy in controlled trials include divalproex, olanzapine, and pramipexole. despite these adult data, it is still important to remember that children are distinct neurodevelopmentally, and so may not respond as adults do to psychotropic medications, both in positive and negative ways. indeed, it appears that youth, particularly pcripubertal children, may be more susceptible to deleterious effects of selective serotonin reuptake inhibitors (ssris) than adults. in an analysis of an hmo database of 87 920 patients aged 5 to 29 years old, children 10 to 14 years old were at the highest risk of switching from a diagnosis of mod to bd after being prescribed an ssri. however, despite case reports of ssri - induced mania in depressed children, one study found no evidence retrospectively that antidepressant exposure in depressed children led to higher rates of mania than children without such exposure. it is possible that bipolar youth are more susceptible to aim. in a retrospective chart review, 42 children with bd who were prescribed ssris were seven times more likely to improve in depressive symptoms than children with bd who were not prescribed any other medication, but three times more likely to experience a subsequent manic episode. furthermore, in a retrospective study of 54 children with bd, 50% had a manic episode within 1 month of starting an ssri, and 25% had new - onset suicidal ideation. thus, it is possible that bipolar youth may account for some of the concerns regarding ssris and suicidally that eventually led to the black - box warning on all antidepressants mandated by the us fda. thus, alternative treatments need to be considered in children and adolescents with bd, perhaps even more so than for adults. while no placebo - controlled studies have yet been reported in pediatric bipolar depression, two prospective treatment studies in adolescents with bipolar depression chang and colleagues studied the effectiveness and tolerability of lamotrigine as mono- or adjunctive therapy in an 8-weck open - label study of 20 adolescents with bd experiencing a depressive episode. the authors reported statistically significant improvement in depressive symptoms, as measured by the children 's depression rating scale - revised version (cdrs - r). 32 sixty - three percent of subjects were classified as responders, with at least a 50% decrease in cdrs - r score between baseline and end point, and 47% were considered remitters by virtue of a score of 24 or less on the cdrs - r and a clinician global impression - severity rating of not ill or mildly ill. additionally, 84% of subjects showed much or very much improvement by the end of the study by the clinical global impression - improvement scale. despite the historical risk of serious rash with lamotrigine, particularly in children, no serious rashes occurred in this study. patel and colleagues conducted a 6-week open - label study of lithium monotherapy in 27 depressed adolescents with bipolar i disorder. forty - eight percent of subjects were considered responders by a 50% reduction in cdrs - r score from baseline to end point. commonly reported side effects were headaches (74%) and nausea / vomiting (67%). thus, while promising, these studies point to the need for larger, placebo - controlled studies of these and other agents (eg, quetiapine, bupropion) in youth with bipolar depression. another agent that might be studied in this regard is omega-3 fatty acid supplements, given some mild efficacy in preventing adult bipolar depression, and in treating adult bipolar depression. even when youth with bd are not in full depressive episodes, it is becoming clear that they often experience subsyndromal depressive symptoms as well as mixed states. birmaher and colleagues studied 263 children and adolescents with bd i, ii, and not otherwise specified (nos) over 2 to 3 years. subjects were symptomatic 60% of the time, but only in full syndromal depressed or manic episodes 22% of the time. children with bd changed between mania and depression an average of 16 times per year, with 34.1 % shifting polarity more than 20 times per year. because manic symptoms are often the first targeted symptoms in youth with bd, these patients often experience residual or emergent depressive symptoms, even with treatment. an older report found 3 out of 6 (50%) of manic children placed on lithium to have a significant worsening of depressive symptoms. in a more recent study of 100 manic adolescents treated prospectively with lithium, mean ham - d scores decreased overall from 12.63 to 6.74 over 4 weeks. it is not reported how many subjects had significant residual depressive symptoms at the end of the study. however, having depressive symptoms at the start of the study did not predict whether or not the subject responded to lithium by mania criteria. in another open study, kowatch and colleagues reported on the naturalistic prospective treatment of 35 manic children and adolescents who had previously been treated with 6 weeks of monotherapy with either lithium, valproate, or carbamazepine. two subjects (5.7%) had a depressive episode despite treatment with two concurrent mood stabilizers (lithium, divalproex, or carbamazepine). of 90 children and adolescents with bd treated openly with divalproex and lithium combination therapy, none continued to have significant depressive symptoms requiring discontinuation from the study. more recent large placebo - controlled studies offer some insight into the natural course and treatment of depressive symptoms in youth with bd. for example, olanzapine was compared with placebo in a 3-week study of 1 58 youth with acute manic or mixed episodes. eight percent of subjects on olanzapine and 14% of subjects on placebo switched to a full depressive episode by the end of the study. this was not a significant difference between groups, and the change in depressive symptomatology, while not reported, was also not different between groups. thus, it would appear that at least for the acute treatment of depressive symptoms in the context of pediatric manic or mixed episodes, olanzapine is not effective. similarly, in a study of divalproex versus placebo in 150 youth (10 to 17 years old) with manic or mixed episodes, there was no difference in the amount of change in cdrs - r scores between groups. in a study of 116 similarly diagnosed youth, oxcarbazepine resulted in a 7.9point decrease in cdrs - r score, versus 6.4 for placebo, a nonsignificant decrease. thus, as yet it does not appear that these commonly used treatments for children with bd result in effective changes in depressive symptoms. due to the predominance of subsyndromal depressive symptoms in bipolar youth, it would be important to conduct studies specifically examining treatment of these symptoms versus placebo. in a questionnaire of adults with bd, depressed mood was the most common presenting symptom before the onset of a full mood pisode. furthermore, 50% to 66% of adults report onset of symptoms before the age of 18 years, with 28% occurring before 13 years. geller report 20% to 49% of children with mdd experience a full manic episode by adulthood. a positive family history of bd would seem to further elevate the risk of future mania in a depressed child ; however, the exact risk in these children is not known. given that many if not most of these children will not ever experience mania, careful diagnosis and biological markers for predication would be essential. unfortunately, at this time there are no clear biological markers that do predict such likelihood, despite recent advances in neuroimaging and genetics research. in the future, markers such as decreased amygdalar volume, increased limbic activity, and the short allele of the serotonin transporter gene, may all be combined to calculate relative risk of bd development. proposed clinical clues of first episode bipolar depression include an acute onset, psychosis, prominent irritability and labile mood, and poor or brief hypomanic reactions to antidepressants. furthermore, features of atypical depression, including hypersomnia, hyperphagia, and other neurovegetative symptoms, may indicate risk for future manic episodes. despite the uncertainty of actual bd risk, early interventions in youth with depression and family histories of bd are beginning to be studied. geller and colleagues performed the first such study in 30 prepubertal children, all with m.dd and family histories of mood disorder. forty percent had a parent with bd, 40% had a more distant relative with bd, and 20% had a family history of unipolar depression only. subjects were randomized to lithium or placebo, and after 6 weeks no differences were found between the two groups in improvement in depressive symptoms. the final clinical global assessment of severity scores in both groups did improve from baseline, but remained below 60, indicating continuing clinical problems. as there was a significant distribution of subjects who responded well and subjects who responded poorly, some subjects may have had unique factors associated with response, but whether family history was a factor is unknown. nonetheless, lithium may have limited efficacy in youth with depression at high risk for bd. in another early intervention study, chang and colleagues investigated the effectiveness of open divalproex in 24 bipolar offspring with mood and/or disruptive behavioral disorders. none of the subjects, aged 7 to 17, had bipolar i or ii disorder, but all had at least some mild affective symptoms as manifested by a minimum score of 12 on the young mania rating scale (ymrs) or hamilton rating scale for depression (ham - d). of these subjects, 21 % (5) were diagnosed with mdd, and 8% (2) with dys thymia. subjects were tapered off of any current medications, and then begun on divalproex monotherapy, eventually reaching a mean final dose of 821 mg / day (serum level = 79.0+/-26.8 ug / ml). after 12 weeks, 78% of subjects were considered responders, having general improvement in mood and functioning, with the majority showing improvement by week 3. depressive symptoms appeared to resolve especially rapidly, with mean ham - d scores achieving the end point mean by week 1. this rate of response may have been due to placebo response, receiving support from an academic institution, being in a study, and having regular visits to a physician. however, this placebo response would have been carried throughout the 12 weeks of the study. of note, 6 out of 7 (86%) of subjects with mdd or dysthymia were considered responders. again, caution should be applied to these results given the small sample size and lack of a control arm. despite these promising findings regarding divalproex, findling and colleagues found divalproex to be no more effective than placebo in preventing worsening of mood symptoms in youth with cyclothymia or bipolar disorder not otherwise specified who were bipolar offspring. in this study, 56 subjects 5 to 17 years old were randomized to divalproex or placebo and assessed over an acute 8week period, and then followed monthly for up to 5 years, until clinical intervention was needed for mood symptoms. there was no difference between the treatment arms in time to discontinuation from the study. however, both groups did show significant improvement in depressive and manic symptoms over time. notably, divalproex was superior to placebo in time to discontinuation in a subset of patients who had three or more firstor second - degree relatives with an emotional and/or behavioral problem. it should also be noted that subjects in this study differed from those in the study by chang and colleagues in that they had not had a past full depressive episode, and they were required to have a past significant 4-hour period of elation, indicating that they may have had less symptoms of depression. nonetheless, there was no difference between divalproex and placebo for efficacy regarding depressive symptoms. ouetiapine would seem a good candidate for use in first - episode bipolar depression, given its efficacy in adult bipolar depression. delbello and colleagues conducted a 12-week study of open quetiapine for bipolar offspring with mood disorders (mean age = 14.7 years), that were considered subsyndromal to full bd (no subjects had a history of mania). 11 (55%) had bd - nos.3 had bipolar ii disorder, 3 (11%) had dysthymia, 2 cyclothymia, and 1 mdd. thus, almost all subjects had a bipolar spectrum disorder, and as such these subjects were farther along the progression line for bd than the previously discussed studies involving valproate. quetiapine was begun at 100 mg / day, then increased every day up to 400 mg / day, with flexible dosing thereafter to achieve 300 - 600 mg / day based on clinical need. 15 subjects completed the study, and final mean dose was 460 mg / day. response was considered a 1 or a 2 on the cgi - bp (much or moderate improvement in bipolar symptoms). after 1 week there were 4 responders (25%), which grew to 81 % by week 12. of note, the one subject with mdd was a nonresponder, but all three subjects with dysthymia were responders. ymrs score decreased from 18.1 to 8.7, and mean cdrs - r score decreased from 38.2 to 27.7. therefore, quetiapine may have clinical utility in this population, but larger controlled studies are needed to clarify its role in first episode bipolar depression in youth. it should be remembered that these studies did not address prevention of mania, as longer longitudinal studies are needed to address that issue. agents such as lithium, divalproex, and quetiapine may be efficacious in decreasing depressive symptoms in these children at risk for bd, but is unclear if they are more effective than placebo or actually prevent or delay mania. in none of these studies, however, did these agents precipitate mania, so in this sense they may prove to be safer than antidepressants in this population. as for other bipolar depressed states, these and other agents deserve further study in this population. psychotherapy may prove to be an effective adjunctive or alternate treatment in depressed youth with or at risk for bd, as it may be more targeted than medications, without the potential for physical or behavioral adverse effects. various psychotherapeutic approaches, including cognitive - behavioral therapy (cbt), dialectical - behavioral therapy (dbt) and family therapy, are beginning to demonstrate efficacy in pediatric bd. in an open study of dbt in 10 adolescents with bd, depressive symptoms and suicidal ideations and behaviors decreased significantly over 1 year. in a small controlled study of cbt for adolescents with bd, significant decreases in parent and child reported depressive symptoms were reported in the cbt condition. however, compared with control bd youth who did not receive cbt, there were no differences in post - treatment depression scores by clinician assessment. these individual therapy approaches show promise and should be studied in larger, controlled studies. a recent study of family - focused therapy (fft) in 58 adolescents with bd found that fft was more effective than enhanced care (ec), a series of psychoeducational sessions. subjects receiving fft recovered faster from their baseline depressive symptoms than did subjects receiving ec. while fft did not more effectively prevent relapse of depressive episodes, subjects receiving fft spent fewer weeks in depression than subjects in ec. a modification of fft is now being studied for bipolar offspring with mdd or bd - nos. therefore, depression in the context of bd in youth may be particularly responsive to psychotherapeutic interventions, potentially more so than mania. common themes of these interventions are psych oeducation, behavioral and cognitive interventions, including reducing stress and improving coping strategies, and mood regulation techniques. future studies incorporating larger samples, with youth with bipolar depression, depressive symptoms, or unipolar depression at risk for bd would be highly illuminating to the field. as the existence of bd in youth is becoming less controversial, clinicians and researchers are now able to concentrate on understanding the full spectrum of symptoms experienced by these patients, and researchers are able to concentrate on all aspects of the illness, including depressive symptomatology. it is clear that while mania is impairing in this condition, depressive symptoms may ultimately be just as if not more damaging, particularly leading to suicidal thoughts and behaviors. recognition of depressive episodes in bipolar youth and in youth at high risk for bd is essential for the purposes of early intervention and prevention of progression of the disorder. meanwhile, it is becoming clear that ssris have dangerous potential in this population, while certain mood stabilizers, antipsychotics, and psychotherapics may be better alternatives. treatment options are slowly growing and future research will allow clinicians to more confidently identify and treat depression in the context of pediatric bd. | there has been great public and academic interest in the diagnosis and treatment of bipolar disorders (bd) in children and adolescents over the past decade, originally in the us, but now extending internationally. much of the interest in pediatric bd has focused on the unique manifestation of mania in younger populations. depression is often overlooked, both as a topic, and as a clinical reality, in these children. while it is becoming clear that adults with bd spend the majority of their symptomatic time in depressive rather than manic episodes, less is known about the pediatric experience of bipolar depression. however, children and adolescents with bd clearly do experience significant depressive symptoms as well as depressive episodes, and therefore early recognition and treatment is necessary. this review addresses what is known about the prevalence, presentation, and treatment of depressive symptoms and episodes in youth with bd, and includes a discussion about the recognition and treatment of bipolar depressive episodes that occur before the first manic episode. |
type 1 diabetes mellitus (t1 dm) is a well - established risk factor of accelerated cardiovascular disease, and, on the other hand, cardiovascular disease is the major cause of mortality in this group of patients. the relative risk for coronary heart mortality in t1 dm patients is seven times higher than in matched counterparts without the disease and two times higher compared to risk of t2 dm patients [1, 2 ]. lately, it has been postulated that the main reason is not only chronic hyperglycaemia or other traditional risk factors, but frequent hypo- and hyperglycaemia episodes that accompany the disease daily course, and a new cardiovascular risk factor excessive glycaemic variability has been postulated. blood glucose instability may contribute, perhaps even more than elevated hba1c, to the development of diabetes complications [3, 4 ]. these extreme glucose fluctuations lead to endothelial dysfunction and accelerated atherosclerosis, independently of average glucose concentrations, probably in mechanism of oxidative stress occurrence [5, 6 ]. alterations in endothelial function precede the development of morphological changes and contribute to atherosclerotic lesion development and progression. it is now clear that vascular disease begins in childhood and progresses silently until complications, such as stroke or myocardial infarction, later appear. t1 dm in childhood is recognized as a high - risk factor for premature atherosclerosis [810 ]. appreciation of the role of the vascular endothelium throughout the atherosclerotic disease process has led to the development of a range of invasive and noninvasive techniques which permit evaluation of different aspects of its function. at present, several noninvasive imaging techniques used in children offer an opportunity to study the relationship of surrogate markers to the atherosclerosis development. the use of these techniques may help to identify high - risk individuals in preclinical phase who may benefit from active therapy to prevent clinical disease. endothelial function, that is, the vasodilator response to increased blood flow (flow - mediated vasodilatation, fmd), can now be accomplished using high - resolution ultrasound. fmd is an early endothelial dysfunction marker, used to detect minimal endothelial changes in children and adolescents. t1 dm poses a challenge to the goal of maintaining adequate glucose variability for years. from the 1990s, a new device has been introduced to the market to support patients in achieving this goal. real - time continuous glucose monitoring system (rt - cgm) continuously measures the glucose level in interstitial fluid of subcutaneous tissue. it provides the ability to track blood glucose trends and, thanks to warning alarms, prevents dramatic glucose variability before these changes become problematic. the potential clinical benefit of this technology as a tool to assist with optimization of glucose control has been demonstrated in several recent clinical studies, reviewed in. the studies demonstrate that strict t1 dm control helps not only in hba1c and glycaemic variability reduction, but also in diminishing oxidative stress level and dyslipidemia. both of these are predominant in children and adolescents with t1 dm, playing a key role in endothelial dysfunction and leading to early atherosclerosis development [1517 ]. some recent data indicate that even up to thirty percent of pediatric t1 dm patients have impaired endothelial function, independently associated with a1c and related to endothelial nitric oxide synthase t(-786)c polymorphism to some extent. noteworthily, there are no studies so far assessing possible influence of the rt - cgm device use on the cardiovascular system neither in t1 dm adults nor in children. we hypothesized that use of rt - cgm via reduction of glycaemic variability might influence improvement of endothelial function. therefore, we aimed firstly to assess brachial artery flow - mediated dilatation before and after short - term use of real - time continuous glucose monitoring in adolescents with t1 dm. secondly, we aimed to define a possible group of young patients who would benefit most from the use of this method. the entire study involved a total of forty adolescents (21 girls) at the age of 1118 years mean 14.6 2.1, with the diagnosis of t1dm who had been treated by continuous subcutaneous insulin infusion (csii). mean disease duration was 7.4 3.7 years and mean hba1c level at the baseline was 9.35 1.53%. primary eligibility requirements were diabetes duration above 1 year, exogenous insulin requirement at least 0.5 j / kg / day, and insulin pump therapy for at least 0.5 years. patients were excluded from the study if they presented other diseases that could affect the outcome (additional autoimmune disease, hypertension, hyperlipidemia, obesity, and early microangiopathy) as well as having education difficulties and presenting a lack of cooperation. we enrolled patients who have never used real - time continuous glucose monitoring system before but expressed their willingness to participate in the study. all of the patients used either a sensor augmented insulin pump (paradigm 722, medtronic minimed, northridge, ca) or guardian rt (medtronic, northridge, ca) device when the original pump (paradigm 715, medtronic or accucheck spirit, roche) did not have a sensor option. the study was based on one - month continuous glucose sensors use combined with technical training and proper education of the patients and their caregivers concerning insulin therapy, diet, and physical activity. there was also the written instruction of operating system given to all of the patients. children and their parents were able to contact the diabetology team if any problem occurred. every patient was supplied with 4 - 5 sensors, changed every 6 - 7 days, to provide the 4-week trial. after the first and last sensor usage, the obtained data were uploaded to the computer using the carelink pro (medtronic). clinical issues were analyzed and discussed together with diabetologist. during the study, patients were told to live normal life adjusting the insulin therapy based on sensor records, confirmed with smbg measurements, by themselves or prior telephone consultations. next, we performed the analysis of computer uploaded glucose variability parameters that included mean blood glucose, standard deviation (sd) for the mean glucose, and area under the curve (auc) for glucose level > 140 mg / dl and 7.5%, mean 9.88 1.22%). decrease of hba1c by at least 0.5% after 3-month follow - up was the criterion to divide the study group into improved group versus not improved group. the ultrasound measurement procedure was conducted between 8.00 and 10.00 am after a fasting period of 812 hours. examinations of the brachial and carotid arteries were performed with hewlett packard sonos 4500 apparatus, using a 7.5 mhz linear transducer. ultrasound examination of the right brachial arteries was performed in longitudinal sections 210 cm above the elbow, according to guidelines. the principle is to induce vasodilatation in the proximal (brachial) artery by postischemic (forearm) enhanced flow. all lumen diameter measurements were scanned at end diastole by use of the r - wave of the electrocardiogram. first scans were taken at rest and second scans during reactive hyperemia. increased flow was induced by deflating a pneumatic tourniquet placed on the right forearm, inflated to the pressure about 50 mmhg above the patient 's resting systolic blood pressure for 4.5 min. flow - mediated dilatation (fmd) was derived from the percentage change of the brachial artery diameter after ischemia of the forearm from baseline. nitric oxide - dependent fmd of the brachial artery was assessed at baseline (before rt - cgm sensor placement) and after one month of sensors usage. the study was approved by the bioethics committee, medical university of biaystok, poland. the caregivers gave a written consent, whereas the children expressed a spoken consent before examination. the statistical analysis was performed using the statistica 9.0 software (krakow, statsoft). to determine the differences between the study groups for variables with normal distributions, the student t - test was applied. the student t - test for paired variables was used to compare the variables within the respective groups at baseline, after one month (glycaemic variability parameters), and after 3 months (hba1c values). since in this study the variables our analysis of metabolic control in the study group was based on hba1c level, which has significantly decreased in entire study group after three months from 9.35 1.5% to 8.81 1.8% (p 140 mg / dl decreased from 41.23 using the first sensor to 21.22 using the last sensor (p 7.5%. all glycaemic variability parameters, apart from auc 7.5% and 140 mg / dl before the study (r = 0.35, p = 0.026). fmd after the study significantly correlated inversely with hba1c after the study (r = 0.78, p 140 mg / dl for the whole study (r = 0.34, p = 0.27). no associations were observed between subclinical marker of atherosclerosis and total cholesterol, ldl - cholesterol, and triglycerides or between disease duration and onset either. the present study is the first to our knowledge to show that use of real - time cgm can be helpful in reduction of glycaemic variability in order to improve endothelial function in young t1 dm patients without any symptoms of clinically present cardiovascular disease. among the whole study group, we have observed significant endothelial function improvement, mainly in those patients who have also improved their hba1c level. in those patients, the initiation of rt - cgm caused both a rapid reduction of glucose variability and improvement in vascular function. glucose variability affects quality of life and can contribute to pathogenesis of diabetic complications [4, 20 ]. continuous subcutaneous insulin infusion (csii) and multiple daily insulin injections (mdi), established therapies for type 1 diabetes, are thought to prevent hyperglycaemia and deleterious glucose fluctuations. patients using insulin pumps present with lower glycaemic variability, better glycaemic control, and treatment satisfaction compared with those using mdi [21, 22 ]. rt - cgm can be considered as one step further in achieving a safe way to target near normoglycaemia. although there is still a debate as to whether rt - cgm can improve glycaemic control and improve quality of life, increasing amount of data of observational and rct studies in the pediatric age groups found improvement in metabolic control with use of this device. most of the rct studies, including pediatric t1 dm, have demonstrated that the frequency of the cgm use was significantly associated with the effect of lowering hba1c levels. real - time cgm has been shown to lead to sustained reduction in mage and hypoglycaemia ; nevertheless, benefits from sensor augmented pumps relate to sensor use frequency [13, 14 ]. our results are in agreement with these studies, as we observed significant improvement in hba1c in almost 70% of patients and significant improvement in glucose variability parameters. recently, the close relationship between increased glucose variability and endothelial dysfunction has been demonstrated in several studies in both type 1 and type 2 diabetic patients [5, 23, 24 ]. noteworthily, in the study of obese adults with or without metabolic syndrome and type 2 diabetes, results of cgm revealed the close connection between glycaemic variability and endothelial function. interestingly, it appeared that glycaemic variability may be elevated even in nondiabetic, despite being obese, subjects and is also independently correlated with endothelial function. furthermore, one of australian studies demonstrated that children with t1 dm following continuous subcutaneous insulin infusion (csii) initiation have early improvements in vascular function, blood pressure, and metabolic control associated with reduced glucose variability. in our study, all of our patients have already used csii. moreover, we have gone a step further and used real - time glucose monitoring to give the patients the possibility of positive feedback and immediate reaction. in the discussed studies, patients were using cgm blindly, and the results were analyzed retrospectively, whereas in our study patients were given a possibility for dynamic modulation of the displayed glycaemic variability. they could react immediately by changing the insulin dosage, diet, physical activity, and lifestyle. in the abovementioned study, the effects were unfortunately not sustained after twelve months, and fmd and glucose variability returned to baseline levels, with deterioration of metabolic control. poor adherence to csii - related tasks, such as insulin bolusing for meals, is frequently seen in adolescents. in our study, we have discovered that using real - time cgm may help to improve not only glucose variability parameters, but endothelial function as well, in patients already treated with csii. in some contrast to our results, where only hypoglycaemia, but not glucose variability, during continuous glucose monitoring related to impaired vascular endothelial function in children with t1 dm. however, this was not interventional study, like our study was. in our group, we found that auc for hypoglycaemia and minimal glycaemia value in the whole study group did not change significantly after one - month glucose sensor use. interestingly, however, in the group with initial optimal glycemic control, where the highest improvement in endothelial function was observed, we noticed higher minimal glucose value during last week of sensor use compared to the first sensor use and insignificantly decreased auc for hypoglycaemia. it has been speculated that oxidative stress is the link between glucose variability and vascular dysfunction. both in vitro studies and animal models have demonstrated evidence of vascular inflammation and endothelial cell apoptosis following fluctuations in blood glucose levels, via the production of reactive oxygen species [27, 28 ]. oscillating blood glucose may have much worse effect on endothelial function and oxidative stress than stable hyperglycaemia in diabetic as well as nondiabetic subjects. still, the gold - standard method to measure glucose variability in research and clinical practice was not established. using just glycated hemoglobin (hba1c) as a glycaemic variability parameter has a number of limitations : it presents average glucose levels from the last three months, while the research shows that avoiding hypo- and hyperglycaemia episodes may be even more important. in some patients, the superiority of glycaemic variability was presented in recent studies, which recommend that clinicians should focus first on limiting glucose variability, before attempting to reduce median blood glucose, that is, hba1c. standard deviation (sd), an index of the dispersion of data around mean blood glucose, was by design viewed as the simplest approach for the evaluation of glucose variability, beyond the simple determination of mean blood glucose. a clear consensus on the gold - standard method to measure glucose variability in clinical practice and research is still lacking, although a number of indicators have been proposed. in our study, we have chosen generally accepted methods of the quality of blood glucose control and variability. these include the area under the curve (auc) and the percentage of time inside, above, and below the blood glucose target. we have intentionally avoided the use of newly introduced parameters, such as continuous overlapping net glycaemic action (conga) or mean amplitude of glycaemic excursions (mage) due to reports showing the limitations of those methods and to the fact that those have not gained widespread use in clinical practice [24, 31 ]. the impact on endothelial function seems to be more effective in the patients who present better adherence. our study has confirmed the correlation of fmd increase with metabolic control improvement. in our study, we named responders to cgm these patients who were able to improve the hba1c level at least by 0.5% after three months of follow - up. almost 70% of our adolescents achieved this result, and what is more, responders achieved greater improvement in endothelial function as fmd increased in this group significantly more than in nonresponders. patients who improved their metabolic control presented greater decrease of parameters of glucose variability the average glucose, sd for the mean glucose, auc > 140 mg / dl, and maximal glucose level. fmd showed similar trend ; greater increase was observed in patients with more significant hba1c improvement. therefore, our study demonstrated that responders to rt - cgm benefit most in endothelial function improvement. surprisingly, we observed the greatest increase in fmd in those with optimal glycaemic control from the onset. while hba1c in this subgroup decreased even more from 7.25% to 6.7%, changes in glycaemic variability parameters did not achieve statistical significance, except for the already abovementioned decrease in auc for hypoglycaemia. we can speculate that it is due to their already initial high adaptation to diabetes - related tasks and educational potential and abilities to view and use real - time data to make insulin, nutrition, and lifestyle modifications. however, the mechanism is still unknown and requires further research. our data provide important information on the role glycaemic variability may play in influencing cardiovascular risk even in children with t1 dm and the possibility to undertake deliberate action to improve endothelial function in this group of high - risk patients. we are aware that there are certain limitations of our study implicating a careful interpretation of the conclusions. metabolic control and endothelial function improvement might be caused by extra training and more intensive education schedule that was given to the patients and their caregivers rather than the effect of cgm itself. an additional limitation is that the improvement of a1c after 3 months was directly attributed to the 4 weeks of cgm use ; there is no proof from the data that these two are directly related. our group numbers were quite small, with only 8 patients in the < 7.5% group. notwithstanding, the aim of the study was rather to demonstrate the additional benefits of rt - cgm device use in children and adolescents than to prove the superiority of cgm over smbg in improving metabolic control and endothelial function. rt - cgm can be considered as an additional, educational tool which offers type 1 diabetic adolescent the quick reaction to decrease glycaemic variability parameters in short - time observation. whether such approach may allow for the improvement in endothelial function and further influence to reduce the risk of future cardiovascular disease remains to be elucidated. the best outcome was demonstrated in initially well - controlled patients (hba1c < 7.5%), but also in rt - cgm responders, that significantly improved hba1c and glycaemic fluctuations in one - month system use. | children with type 1 diabetes (t1 dm) are the high - risk group of accelerated atherosclerosis. real - time continuous glucose monitoring (rt - cgm) provides possibilities for the detection of glycaemic variability, newly recognized cardiovascular risk factor. the aim of the study was to assess the usefulness of rt - cgm as an educational tool to find and reduce glycaemic variability in order to improve endothelial function in t1 dm adolescents. forty patients aged 14.6 years were recruited. the study was based on one - month cgm sensors use. parameters of glycaemic variability were analyzed during first and last sensor use, together with brachial artery flow - mediated dilatation (fmd) to assess endothelial function. in the whole group, fmd improvement was found (10.9% to 16.6%, p < 0.005), together with decrease in all studied glycaemic variability parameters. in patients with hba1c improvement compared to the group without hba1c improvement, we found greater increase of fmd (12% to 19%, p < 0.005 versus 8.2% to 11.3%, p = 0.080) and greater improvement of glucose variability. rt - cgm can be considered as an additional tool that offers t1 dm adolescents the quick reaction to decrease glycaemic variability in short time observation. whether such approach might influence improvement in endothelial function and reduction of the risk of future cardiovascular disease remains to be elucidated. |
despite modern reperfusion strategies have been well accepted around the world, acute myocardial infarction (ami) still remains a leading cause of death worldwide. this suggests that ami patients still need more understanding of potential pathophysiology for recurrent events of treatment especially in the early post - acs period. so far, the systemic inflammatory response after ami has been well described and may play an important role in series of events after ami. both circulating inflammatory markers, such as interleukin- (il-) 6 and high sensitive c reaction protein (hs - crp), as well as circulating inflammatory cells, including leukocytes and inflammatory monocytes, are elevated acutely after an ami event in a temporal pattern that corresponds to elevated event rates and is predictive of recurrent events. anti - inflammation strategy is a good option of improving treatment for patients after ami. anti - inflammatory cytokines such as il-10 are involved in the events of early ami. the ratio of il-18/il-10 is found as an indicator for prognosis of ami [2, 3 ]. il-37 is a recently found anti - inflammatory cytokine in the il-1 ligand family and proved as a fundamental inhibitor of innate immunity. il-37 was elevated in some inflammatory diseases such as inflammatory bowel disease, atopic dermatitis, rheumatoid arthritis, and systemic lupus erythematosus [58 ], indicating il-37 may have potential protective effect on inflammatory diseases. il-37 is found to be increased in patients with acute coronary syndrome but not investigated in patients after percutaneous coronary intervention (pci). additionally, il-37 is normally expressed at low levels in peripheral blood mononuclear cells (pbmcs), mainly monocytes, and dendritic cells (dcs), which is rapidly upregulated in the inflammatory context after ami. il-37 effectively suppresses the activation of macrophage and dcs, and therefore il-37 may conversely inhibit the production of inflammatory cytokines in pbmcs and dcs after ami. given that il-37 may be associated with the development of atherosclerosis, we hypothesize that il-37 may play a potential role in the inflammation response including plasma and leukocytes in ami patients after pci. so il-37, as a new anti - inflammatory cytokine, may suppress immune responses and inflammation. understanding of il-37 expression helps us further to explore that role and effect of il-37 in ami situation. since it is also reported that a complex of the il-37 and il-18-binding protein reduces il-18 activity, we wanted to explore (1) the expression of plasma and leukocytic il-37 in early period after ami ; (2) the possible relationship between plasma il-37, il-18, and il-18bp ; and (3) the possible inhibitory effect of il-37 on icam-1 in leukocytes. -actin antibody was purchased from santa cruz biotechnology (dallas, texas, usa), and hrp - conjugated rabbit anti - human igg was purchased from jackson immunoresearch (west grove, pa, usa) ; icam-1 and il-18 antibody were purchased from abcam (cambridge, ma, usa). il-37 was purchased from adipogen ag, liestal, switzerland, il-18 from mbl, nagoya, japan, il-18bp from raybiotech, norcross ga, usa, and hs - crp from elisa biotech (shanghai, china). from october, 2013, to april, 2014, a total of 112 cases of healthy volunteers (56) and patients (56) with astemi agreed to participate in this test. stemi was defined as chest pain suggestive of myocardial ischemia for at least 30 minutes before hospital admission and the electrocardiogram (ecg) with new st - segment elevation in 2 or more contiguous leads of 0.2 mv or more in leads v2 to v3 and/or 0.1 mv or more in other leads. the exclusion criteria were as follows : (1) patients presenting with stemi after 12 hours from symptom onset ; (2) patients presenting with vasospastic angina (as determined by the resolution of st - segment elevation and relief of symptoms after an iv administration of nitroglycerin) ; (3) patients over 75 years old ; (4) recent (250 seconds during the procedure. administration of glycoprotein iib / iiia inhibitors will be based on the physicians ' discretion. the goals of the procedure are to achieve optimal angiographic efficacy of pci at the infarct - related artery and minimize the risk of procedure - related complications. a full range of commercially available guiding catheters, balloon catheters, and guide wires will be readily available. all patients included in this trial will be treated according to the current american college of cardiology (acc)/american heart association (aha) guidelines regarding poststenting management, which specify treatment with at least 100 mg of aspirin daily and 75 mg clopidogrel daily for at least 12 months after pci. angiotensin converting enzyme inhibitors and -blockers will be administrated after pci if no limitation exists. blood collection from consented healthy volunteers and patients was approved by the human ethics committee of first affiliated hospital of shantou university medical college. an approximate volume of 3 ml peripheral venous blood was collected from all patients at different time point (12 h, 24 h, and 48 h) after pci procedure into the procoagulation tube and ethylenediaminetetra acetic acid (edta)-k2 anticoagulation tube separately. plasma was isolated from blood sample in procoagulation tube after centrifugation at the speed of 3,000 rpm, 15 min. leukocyte was isolated from blood sample in edta - k2 tube using erythrocyte lysis buffer (qiagen, hilden, germany) and then collected after centrifugation at the speed of 2000 r / min, 10 min. membranes were blocked for 1 h at room temperature with 5% dry milk in tpbs (pbs containing 0.1% tween 20) and then incubated with the appropriate primary antibodies (icam-1 antibody was diluted into 1 : 1000, il-37 1 : 500, and -blocker 1 : 1000) overnight at 4c. after washing with tpbs, membranes were incubated with horseradish peroxidase- (hrp-) linked secondary antibodies (1 : 5000 dilution with tpbs containing 5% dry milk) at room temperature for 1 h. bands were developed using ecl and exposed on x - ray films. plasma hs - crp, il-18, il-18bp, and il-37 were quantified by elisa kits. plasma hs - crp was just detected at 12 h point, while il-18, il-18bp, and il-37 were detected at 12 h, 24 h, and 48 h point. absorbance of standards and samples was determined spectrophotometrically at 450 nm using a microplate reader (khb labsystem wallscan k3, thermo scientific, finland). analysis of variance (anova) was performed, and differences were considered significant when p < 0.05, as verified by fisher post hoc test. baseline clinical characteristics on the basis of age, gender, and leukocyte count between healthy volunteers and patients with stemi are presented in table 1. most patients have 2 or more cardiovascular risk factors, while few patients have single parameter (2 have only hypertension, 2 only diabetes, 4 only hypercholesterolemia, and 4 only smoking history). most patients have one or two diseased arteries, while 3 cases have 3 diseased ones. and most patients were occluded in lad or rca, while only 3 cases were infarcted in lcx. plasma hs - crp was elevated and indicated high inflammation situation after ami with pci. we tested plasma il-37, il-18, and il-18bp expression at different time points (12 h, 24 h, and 48 h after pci) in patients with stemi with elisa kit (table 2). both il-37, il-18, and il-18bp expression were balanced but decreased at all time points in patients if compared to those in healthy volunteers (p < 0.05). we then test il-37 and icam-1 expression in leukocytes to check cellular inflammation and anti - inflammation after pci. leukocyte is an important cell involved in inflammatory response after ami and expressed il-37 under inflammation. we found that both cellular il-37 and icam-1 protein were highest expressed at 12 h point but significantly decreased at 48 h point (figure 1, p < 0.05). in this study, we demonstrated the expression of plasma il-37 and leukocytic il-37 in stemi patients after pci in early period. we found that plasma il-37 did not increase and leukocytic il-37 went down while plasma hs - crp is high which indicates high inflammation response in the first 2 days after pci. plasma il-18, which was found to be inhibited by il-37, was also not increased under this situation. it is proved that inflammation is involved in atherosclerostic plaque formation and rupture, coronary thrombosis, and myocardial necrosis and repair after myocardial infarction [1315 ]. anti - inflammation strategy may be good for myocardial prognosis, but some anti - inflammatory cytokines such as il-10 reduced in acs patients, reflecting the imbalance in systemic cytokine response following an acs [2, 16 ]. il-37 is already proved as an anti - inflammatory cytokine and reported to be elevated in acs patients, but we found that it decreased in patients after pci in our study. hereby, we found that systematic plasma il-37 and leukocytic il-37 decreased in the early period. in vivo expression of human il-37 in mice reduces local and systemic inflammation in cona - induced hepatitis and lps challenge. therefore, il-37 reduction may fail to inhibit systematic inflammation in patients with astemi after pci. not only hs - crp but also il-18 had been proved to be good indicators for prognosis for patients [1820 ]. il-18 is enhanced in stemi situation [2123 ], and the expression change of il-18 is not reported before in patients after pci. we found that il-18 is decreased in the patients with pci compared to that in healthy volunteers. a complex of the il-37 and il-18-binding protein reduces il-18 activity ; but plasma il-37, il-18 and il-18bp were increased in patients with acs. we wanted to test whether there is any relationship between changes of il-18 and il-37 in stemi after pci. the synchronous reduction of systematic il-37 and il-18 could not reveal the inhibitory effect of il-37 on il-18. the reduction of systematic il-18 after pci is not due to enhanced anti - inflammatory response. the ratio of il-18/il-10 was found to be an independent predictor of adverse events in patients with acs [2, 3 ]. to decrease il-18 and the potential predictor effect of the ratio of il-18/il-37 on adverse events could be explored in the future. and to change the imbalance between inflammation and anti - inflammation is still meaningful. although inflammatory markers such as crp predict future cardiovascular events in acs patients, when all inflammatory mediators are taken into account in a prospective analysis of risk, markers reflecting anti - inflammatory mechanisms may be better prognostic markers. furthermore, elevated level of plasma il-37, il-18, and il-18bp had no correlation with the severity of the coronary artery stenosis, and decreased level of those was not related with that in our study. leukocyte is an important inflammatory cell in stemi patients and is involved in myocardial necrosis and repair after stemi. circulating monocytes could express high level of proinflammatory cytokines, tnf - alpha, and il-6, as well as anti - inflammatory cytokine il-10. icam-1 induces the interaction between leukocytes and endothelial cells, which is involved in the myocardial remodeling. we isolated circulating leukocytes and found reduction of both icam-1 and il-37 in early period, indicating a balance of inflammatory and anti - inflammatory response in circulating leukocytes. we can suggest that icam-1 expression decreased because of il-37, which is better to be confirmed by isolated leukocyte culture. different from the systematic imbalance, the changes of il-37 and icam-1 indicated a balance of inflammation and anti - inflammation on leukocytes. leukocytes include several types including neutrophils, lymphocytes, and monocytes, so we can not acutely tell which subtype has only or more il-37 expression. we think neutrophils, most percentage of leukocytes, may be a potential good target to explore il-37 expression and change in the future. in this study, we found that it is lower in the stemi than that in the healthy, which may be because anti - inflammatory response is inhibited [2, 16 ]. compared to other studies about anti - inflammatory cytokines, we can suggest that this is due to the inhibitory effect of anti - inflammatory response in acs. enhancing anti - inflammatory effect in stemi may help to repair myocardial damage [27, 28 ]. how to increase il-37 expression may be helpful and it is interesting to investigate that in future study. furthermore, sample size is not too much in our study ; we can investigate expression difference in different subgroup if we have more samples. in conclusion, our study firstly demonstrates that systematic il-37 expression was decreased in stemi with pci situation and on decline in leukocytes after pci. these suggest that il-37 does not play an important role in the systematic inflammatory response but may be involved in leukocytic inflammation in astemi after pci. | objective. acute st - segment elevation myocardial infarction (astemi) is accompanied by increased expression of inflammation and decreased expression of anti - inflammation. il-37 was found to be involved in the atherosclerosis - related diseases and increased in acute coronary syndrome. however, the level of il-37 in blood plasma and leukocytes from patients with astemi after percutaneous coronary intervention (pci) has not been explored. methods. we collected peripheral venous blood from consented patients at 12 h, 24 h, and 48 h after pci and healthy volunteers. plasma il-37, il-18, il-18-binding protein (bp), and high sensitive c reaction protein (hs - crp) were quantified by elisa and leukocytic il-37 and icam-1 by immunoblotting. results. plasma il-37, il-18, and il-18 bp expression decreased compared to those in healthy volunteers while hs - crp level was high. both leukocytic il-37 and icam-1 were highest expressed at 12 h point but significantly decreased at 48 h point. conclusion. these findings suggest l-37 does not play an important role in the systematic inflammatory response but may be involved in leukocytic inflammation in astemi after pci. |
myotonic muscular dystrophy type 1 (dm1) is an autosomal dominant, multisystem degenerative disorder due to unstable expansion of ctg trinucleotide repeats. in addition to the clinical features of muscle wasting, weakness and myotonia, the patients with dm1 often have involvement of heart, eye, brain, endocrine and other organ systems, which impact the function, quality of life and survival in these patients. cardiac involvement in the form of arrhythmias and/or conduction defects is frequent, occurring in 5065% of patients with dm1. primary cardiomyopathy in dm1 presenting as congestive heart failure is rare ; however, subclinical systolic dysfunction has been reported with a prevalence of 028% in different studies using echocardiographic or magnetic resonance imaging. the frequency of cardiac conduction defects and tachy / bradyarrhythmias correlates significantly with increasing age, male gender, number of ctg repeats and neuromuscular disability. in contrast, there is no consistent association of such risk factors with structural myocardial involvement and systolic dysfunction. in the present study, we describe the nature and prevalence of various structural myocardial abnormalities as noted on echocardiographic imaging in adult patients with dm1, and attempt to define factors associated with the abnormal echo findings. we retrospectively reviewed the clinical, electrocardiographic (ekg) and echo findings in adult patients with dm1. the information was obtained from the mda (muscular dystrophy association) clinic and database of the echocardiography laboratory at our institute. the diagnosis of dm1 was based on characteristic clinical phenotype, positive family history consistent with autosomal dominant transmission, electromyographic findings, and/or expansion of ctg repeats on genetic testing. adult patients (> 18 years), in whom echocardiogram results were available, were included in the study. patients with known underlying cardiac disease (ischemic, congenital or valvular) were excluded. the medical records of the patients were reviewed for the demographics, history of cardiac symptoms, duration of illness, proximal (shoulder abduction, hip flexion) and distal (grip, ankle dorsiflexion) muscle strength in extremities [grade 05 of medical research council (mrc) scale ], presence of congenital myotonic dystrophy, ekg findings, and number of ctg repeats if the results of genetic testing were known. echo reports and video recordings (when available) were reviewed by a cardiologist (ka). various parameters assessed on echocardiogram included left ventricular ejection fraction (lvef), chamber enlargement, valvular dysfunction, wall motion abnormality and doppler findings (e / a and e deceleration time pulse wave recording from left ventricular inflow, tricuspid regurgitation maximum velocity on continuous wave doppler). lvef was calculated as : end diastolic volume - end systolic volume, divided by end diastolic volume. patients with abnormal vs. normal echo were compared for age, gender, cardiac symptoms, ekg findings of prolonged pr interval, left anterior fascicular block and qtc interval, presence of congenital myotonic dystrophy (cmd), severe distal weakness (mrc 200 ms826left anterior fascicular block752qtc ms414.630.1414.027.8415.534.7proximal weakness19118severe distal weakness (mrc < 4)1376congenital myotonic dystrophy651p=0.017 all 5 patients with systolic dysfunction (lvef 3550%) were men, aged 2752 years, who had dm1 for 6 to 27 years. the systolic dysfunction was mild in 2 (lvef 50%) and moderate (lvef 35%, 37% and 40%) in 3 patients. proximal muscle weakness was present in 4 patients, severe distal weakness in 3 and history of tachyarrhythmia in another 2 patients (table 2). any association between the subclinical systolic dysfunction and demographic or other risk factors, or ctg repeat length could not be determined due to the small number of patients. table 2characteristics of adult myotonic dystrophy type 1 patients with systolic dysfunction.n.lvef (%) age (years)duration illness (years)other echo abnormalityekgweakness proximal / distal < 41504011nonenormaly / y1637349nonea fibn / n19502727apical hypokinesialafby / n23355227noneavb1y / y2840476left atrial dilatationafl avb1y / ycongenital myotonic dystrophy. lvef, left ventricular ejection fraction ; echo, echocardiogram ; ekg, electrocardiogram ; avb1, first degree atrioventricular block ; lafb, left anterior fascicular block ; afl, atrial flutter ; a fib, atrial fibrillation. congenital myotonic dystrophy. lvef, left ventricular ejection fraction ; echo, echocardiogram ; ekg, electrocardiogram ; avb1, first degree atrioventricular block ; lafb, left anterior fascicular block ; afl, atrial flutter ; a fib, atrial fibrillation. among the 6 patients (2 female) with cmd, an abnormal echo was observed only in one patient (27 years - male) in the form of apical wall hypokinesia and lvef 50%. the observed frequency of abnormal echo in the congenital type was not different from the adult type of dm1 (table 1). the significant findings in our study were frequent echocardiographic abnormalities in adult patients with dm1, with overall significant correlation with increasing age, but the subclinical systolic dysfunction (reduced lvef) was observed even at younger (< 40 year) age. we noted echocardiographic abnormalities in over one third of the adult patients with dm1, including findings of reduced lvef in half of these patients. other echo abnormalities included diastolic dysfunction, left atrial dilatation, mitral valve prolapse, regional wall motion abnormality and left ventricular hypertrophy. the pattern and prevalence of various abnormalities were similar to those observed by previous investigators. such wide range of abnormalities appear to be commensurate with the reported pathological changes in dm1, of widespread patchy fibrosis and fatty degeneration affecting myocardium in all chambers. the overall echocardiographic abnormalities in our study correlated significantly with older age ; however, 2 of the 5 patients with reduced lvef were aged 27 and 34 years. in contrast, previous studies in a large cohort have documented significant association of systolic dysfunction in dm1 with increasing age, noting that it almost always occurred after the age of 40 years. it is possible that mechanisms other than age related changes may also underlie the left ventricular dysfunction in dm1 patients. among other clinical factors evaluated to determine the dm1 patients at risk for left ventricular dysfunction, significantly higher incidence has been reported in male patients, but no consistent association has been observed with number of ctg repeats, duration of illness or neuromuscular disability. of note, all patients with reduced lvef in our study were male ; however, a specific relationship with various clinical and demographic factors could not be determined due to the small number of cases. we observed mild diastolic dysfunction in 4 patients, and this was an isolated finding in 2 of these patients. several studies, using conventional or tissue doppler imaging have documented diastolic dysfunction in dm1 patients with normal ejection fraction, and hence these findings have been considered an early indicator of left ventricular dysfunction. it has been proposed that the myocardial degenerative process as well as poor relaxation due to myotonia may contribute to the diastolic dysfunction. an interesting observation was paradoxical movement of the interventricular septum in a patient who also had left bundle branch block (lbbb). in other cardiac conditions, lbbb is reported to be associated with paradoxical septal movement and may have significant hemodynamic consequences. however, paradoxical septal motion in the setting of lbbb has not been described previously in patients with dm1. among other ekg changes, we noted a trend, albeit not significant, for association of prolonged pr interval with abnormal echo. previous investigators have shown significant correlation of prolonged pr interval and/or prolonged qrs with reduced lvef and regional wall motion abnormality in patients with dm1. on the other hand, kaminski. observed that an abnormal echo in dm1 patients was a predictor of high risk for cardiac arrhythmias and conduction defects. similarly to other investigators, we also found an interdependence of ekg and echo abnormalities in patients with dm1, such that the abnormality in either one could prompt further evaluation to determine the full range of cardiac involvement. the findings of subclinical left ventricular dysfunction in dm1 patients in our study thus ranged from reduced lvef to diastolic dysfunction and regional wall motion abnormality similar to the previous studies. our study did not include a longitudinal follow up, therefore long term clinical impact of these echo changes could not be determined. long term follow up of dm1 patients in a few studies has revealed increasing frequency of left ventricular dysfunction, and association of echo abnormalities with increased mortality and sudden death. congenital myotonic dystrophy is considered the severest phenotype of dm1, with high mortality in infancy and childhood, and only about 50% of patients survive to adulthood. in addition to cardiac conduction defects, obstructive cardiomyopathy and systolic dysfunction have been reported. in our study, abnormal echo (apical hypokinesia and lvef 50%) was noted in only 1 out of 6 patients with cmd. the incidence is similar to the adult type of dm1, and patients with cmd who survive to adulthood do not seem to have any increased vulnerability to myocardial involvement. the major limitation of our study is the retrospective review of a relatively small number of patients, and genetic testing not being available in all patients. therefore a definitive relationship of clinical features, ekg changes and ctg repeat length with structural myocardial involvement could not be assessed. in conclusion, we found a wide range of echocardiographic abnormalities in over one third of adult patients with dm1. subclinical systolic dysfunction on echo was present even at younger age (< 40 years), although myocardial involvement was more frequent with increasing age. the frequency of abnormal echo in adult patients with cmd was similar to that observed in patients with the classical type of dm1. our findings support the need for investigation of structural myocardial involvement in adult patients with dm1. | myotonic dystrophy type 1 (dm1) is the commonest muscular dystrophy in adults, affecting multiple organs in addition to skeletal muscles. cardiac conduction system abnormalities are well recognized as an important component of dm1 phenotype ; however, primary structural myocardial abnormalities, which may predispose these patients to congestive heart failure, are not as well characterized. we reviewed the retrospective analysis of the clinical and echocardiographic findings in adult patients with dm1. among 27 patients (16 male ; age 1961 years) with dm1, the echocardiogram (echao) was abnormal in 10 (37%) including one of 6 patients (16%) with congenital myotonic dystrophy. reduced left ventricular ejection fraction (lvef 50%) was noted in 5, diastolic dysfunction in 4, left atrial dilatation in 3, left ventricular hypertrophy in 2, apical hypokinesia in 1 and mitral valve prolapse in 3 patients. one patient had paradoxical septal movement in the setting of left bundle branch block. echocardiographic abnormalities significantly correlated with older age ; however, patients with systolic dysfunction on echocardiogram ranged in age from 27 to 52 years including 2 patients aged 27 and 34 years. we can conclude that echocardiographic abnormalities are frequent in adult patients with dm1. the incidence is similar in the classical and congenital type of dm1. overall, echocardiographic abnormalities in dm1 correlate with increasing age ; however, reduced lvef is observed even at young age. cardiac assessment and monitoring in adult patients with dm1 should include evaluation for primary myocardial involvement. |
the early diagnosis of 21-hydroxylase deficiency (21-ohd) is mandatory for both correct sexual assignment and early treatment to prevent adrenal crisis. in term infants, 17-hydroxyprogesterone (17-ohp) in a filter paper - dried blood spot serum levels of 17-ohp are very sensitive markers for biochemical diagnosis of 21-ohd ; however, in premature infants no marker for biochemical diagnosis of 21-ohd has been found. in preterm infants, 17-ohp is not useful because of interference by delta 5 steroids from the fetal adrenal cortex. the serum levels of 17-ohp can be high in unaffected preterm infants, precluding the biochemical diagnosis of 21-ohd (1,2,3,4). we present here a case of a female infant with 21-ohd, born at 31 wk of gestation. analysis of urinary steroid profiles by gas chromatography / mass spectrometry in selected ion monitoring (gcms - sim) showed a high pregnanetriolone (ptl) level, which was strongly suggestive of 21-ohd. this is the first report which presents the usefulness of urinary ptl by gcms - sim for the biochemical diagnosis of 21-ohd in preterm infants. a female japanese infant was born to unrelated parents, as the first child of trichorionic, triamniotic triplets. she was delivered at 31 wk of gestation by an emergency cesarean section because of preterm labor ; the birth weight was 1,464 g and the apgar score was 6 and 8 at 1 and 5 min, respectively. mechanical ventilation and medical treatment were required for respiratory distress syndrome, patent ductus arteriosus, and heart failure. phototherapy was given from 3 through 6 and from 8 through 11 d of age for hyperbilirubinemia. at 5 d of age, we found mild clitoromegaly as well as pigmentation of the tongue and gingiva. meq / l), hyperkalemia (7.7 meq / l), disseminated intravascular coagulopathy (dic), pulmonary hemorrhage, and subependymal hemorrhage. a provisional diagnosis of 21-ohd was made, prompting us to do endocrinological studies : 17-ohp > 93.5 (elisa 7 prime extractive method in filter paper - dried blood spot) and 718.3 ng / ml (ria after high performance liquid chromatography extraction in serum) ; plasma acth 690 pg / ml ; plasma renin activity 77.4 ng / ml / h ; serum 21-deoxycortisol (21-dof) 368 ng / ml ; serum androstenedione 292 ng / ml ; and serum testosterone 3,169 ng / dl. ultrasonography found bilaterally enlarged adrenal glands. urine steroid profile analysis was performed according to the method previously reported (5). in brief, the method consisted of enzymatic hydrolysis of 0.52 ml of urine sample, extraction, derivative formation, purification, gcms - sim analysis and quantification. the urinary steroid profiles at 8 d of age showed a high ptl level (0.80 mg / g creatinine ; table 1table 1 urinary steroid profile by gcms - sim at 8 d of age). this high level of ptl strongly suggested 21-ohd when correlated with the cut - off level in term infants. we analyzed cyp21a2 gene on the patient and her parents with genomic dna extracted from their peripheral blood leukocytes by pcr direct sequencing (6). informed consent for gene analaysis was obtained from her parents. the patient had compound heterozygosity, one allele having a cluster mutation in exon 6 (i236k, v237e, m239k) and the other having a large deletion including cyp21a2. the biochemical diagnosis of 21-ohd was made by the finding of a high urinary ptl level that had been determined via gcms - sim in this preterm infant. the level of ptl was compatible with 21-ohd previously reported in term infants (7). the diagnosis of 21-ohd is difficult because : preterm infants with 21-ohd may have subtle ambiguous genitalia due to prematurity of the labia majora pudenda ; preterm infants without 21-ohd often show hyponatremia and hyperkalemia, which are common laboratory findings in 21-ohd ; although gene analysis of cyp21a2 is a good test for the diagnosis of 21-ohd, it is time consuming and expensive (8) ; most importantly, a marker for the biochemical diagnosis of 21-ohd has not been found. in preterm infants, 17-ohp is not useful because of the interference of delta 5 steroids from the fetal adrenal cortex. an unaffected preterm infant may have high serum levels of 17-ohp (1,2,3,4). reported 17-ohp levels had a statistically significant negative correlation with gestational age and birth weight (9). in newborn mass - screening for 21-ohd, a modification of the cut - off limits of 17-ohp in a filter paper - dried blood spot has been reported ; this modification has been found to have slightly increased specificity in preterm infants. allen. reported weight - adjusted criteria for 17-ohp to reduce the false - positive rate of 21-ohd in preterm infants (10). their weight - adjusted criteria, however, do not factor in small for gestational age infants. reported cut - off limits allowing for the equivalent age of gestation at blood sampling (11). this method may prove to be useful for screening, but can not provide a definitive diagnosis. urinary steroid profiles, especially ptl, by gcms - sim can be useful for the biochemical diagnosis of 21-ohd in preterm infants because : they can detect a full spectrum of steroid hormone metabolites (fig. urinary steroid profiles obtained by gcms - sim can detect all urinary metabolites shown here. an : androsterone 11 ha : 11-hydroxyandrosterone) ; the sample collection is noninvasive and a random sample is appropriate ; reference values of urinary steroids in term newborn infants by gcms - sim have been established (5) ; precursor / product ratio assessments can be measured, which may increase both the sensitivity and specificity of the diagnosis (7). the literature contains reports that describe urinary steroid profiles by gcms - sim for the biochemical diagnosis of 21-ohd in newborn infants. via urinary steroid profiles with gcms - sim, malunowicz. studied 161 newborns and infants, with clinical suspicion of congenital adrenal hyperplasia (cah). in eight cases (two full - term and six preterm infants), a false - positive diagnosis of cah reported three diagnostic ratios, measured by gcms - sim : 17-hydroxypregnanolone 100/cortisol metabolites (cm) ; pregnanetriol (pt) 100/cm ; and ptl 100/cm having no overlap between 21-ohd and normal infants (7). urinary steroid profiles obtained by gcms - sim can detect all urinary metabolites shown here. an : androsterone 11 ha : 11-hydroxyandrosterone among several metabolites of 17-ohp, urinary ptl may be the best biochemical marker for the diagnosis of 21-ohd in term infants. ptl exhibited no overlap between the 21-ohd cases and the control when we compared several urinary steroid metabolites by means of gcms - sim (fig. 2fig. 2 urine pregnanetriolone (ptl) in the patient, 21-ohd, and control. ptl levels measured by gcms - sim in term control infants (number=29, 614 d of age), term infants with 21-ohd (number=28, 714 d of age), and our patient (8 d of age). the level of ptl in our patient was beyond that of control infants.). other metabolites, such as pt and androsterone, exhibited overlap between the control and 21-ohd (data not shown). the mechanisms which explain why ptl is superior to pt, in the early biochemical diagnosis of 21-ohd, despite the fact that both are specific metabolites of 21-ohd (fig. one possible reason for the disparity may be because of a relatively greater inactivity of the enzymes metabolizing 17-ohp to pt than 21-dof to ptl in newborns. urine pregnanetriolone (ptl) in the patient, 21-ohd, and control. ptl levels measured by gcms - sim in term control infants (number=29, 614 d of age), term infants with 21-ohd (number=28, 714 d of age), and our patient (8 d of age). in preterm infants, urinary ptl with gcms - sim may have good sensitivity and specificity for the biochemical diagnosis of 21-ohd. our study suggests that urinary ptl with gcms - sim may have the ability to make a biochemical diagnosis of 21-ohd (true positive). reported urinary ptl levels obtained by gcms - sim were helpful in diagnosing two very low birth weight infants as having transient high 17-ohp (43.6 and 27.7 ng / ml at 35 and 10 d of age, respectively) and transient high 21-dof (6.0 and 4.2 ng / ml at 35 and 16 d of age, respectively) (false positive) (13). finally, we propose that the measurement of urinary ptl by gcms - sim is a useful biochemical marker for the diagnosis of 21-ohd even in preterm infants. we recommend all neonatologists and pediatricians to measure urinary ptl by gcms - sim in any premature infants who have high 17-ohp with filter paper - dried blood spot in newborn screening. additional premature infant case studies are indicated to establish and confirm the usefulness of urinary ptl by gcms - sim. | the biochemical diagnosis of 21-hydroxylase deficiency (21-ohd) is difficult in preterm infants. to date, no marker for the biochemical diagnosis of 21-ohd has been found. seventeen -hydroxyprogesterone (17-ohp), is not useful because of interference by delta 5 steroids from the fetal adrenal cortex. a 5-d - old female infant, born at 31 wk of gestation, was suspected of having 21-ohd based on physical findings (mild clitoromegaly, pigmentation of the tongue and gingiva) as well as laboratory data (17-ohp > 93.5 ng / ml by elisa 7 prime extractive method in filter paper - dried blood spot and 718.3 ng / ml by ria after high performance liquid chromatography extraction in serum ; plasma acth 690 pg / ml ; and serum testosterone 3,169 ng / dl). we examined her urinary steroid profiles by gas chromatography / mass spectrometry in selected ion monitoring (gcms - sim) at 8 d of age. the pregnanetriolone (ptl) level was noticeably high (0.80 mg / g creatinine), which was strongly suggestive of 21-ohd. gene analysis of cyp21a2 showed compound heterozygosity, one allele having a cluster mutation in exon 6 and the other having a large deletion including cyp21a2, confirming the diagnosis of 21-ohd. this case suggested that, in preterm infants, urinary ptl by gcms - sim can be useful for the biochemical diagnosis of 21-ohd. |
a definite diagnosis of alzheimer 's disease (ad) can be accomplished only post - mortem by histopathology of the autopsied brain so as to reveal amyloid beta protein (a) in neuritic plaques and intraneuronal neurofibrillary tangles [1, 2 ]. in past years several alterations in peripheral cells and biological fluids of ad patients have been proposed as potential antemortem ad biomarkers [36 ]. however, none have met the criteria established for an ideal biomarker [7, 8 ], capable of assessing whether a mild cognitive impairment (mci) might reveal early stages of disease or should rather be linked to normal aging. in fact, advances in our knowledge of ad have shown that symptoms usually develop after a long preclinical pathogenetic process, making early detection of ad in asymptomatic subjects of great interest. indeed, the assessment of presymptomatic subject cohorts regardless of genetic predisposition [10, 11 ], including relatives of ad patients, could be highly useful in determining the effectiveness of pharmacological intervention in slowing ad onset and/or cognitive decline in ad patients. clinical criteria for ad diagnosis have recently been revised and the terms possible and probable ad have been redefined by including some biomarkers for the pathophysiological process in support of ad diagnosis. however, their use has been limited to research settings due to the fact that these procedures are not easily available to all patients. the combination of multiple parameters [5, 13 ] obtained through neuropsychological testing and conventional and functional imaging [14, 15 ], together with the relatively low accuracy attainable in discriminating ad from other dementia, requires elaborate, serial, and costly procedures, available only in the best academic centers and for a limited number of patients. therefore, there is a great need of reliable and low - cost peripheral markers to identify persons with incipient ad and complement clinical ad outcomes in a large number of subjects. the national institute on aging and alzheimer 's association international workgroup recently reviewed the state - of - the - art in this field to develop recommendations to determine which factors best predict the risk of progression from a combination of csf t - tau and a42/p - tau ratio has shown to be useful for defining mci patients at high risk of developing ad, yielding a positive predictive value of 81%. the various constituents of blood, including plasma, platelets, and cellular fractions, are now being systematically explored as a pool of putative peripheral biomarkers for ad for the ease of sampling and repeatability of measures [15, 19 ]. in this context, disease - specific autoantibody profiles, increased glutaminyl cyclase expression, and angiopoietin-1 levels have also been described in human sera. on the whole, all these findings need verification in further studies [23, 24 ] ; nevertheless they lend further support to the hypothesis that ad patients suffer from a systemic metabolic dysfunction that, in addition to the brain, also affects peripheral tissues. fibroblasts have also often been employed as an in vitro model for neurological diseases and, particularly, for ad [4, 25, 27 ]. for instance, ad fibroblasts, unlike their normal counterpart, display a variety of alterations related to gm1 ganglioside catabolism, the function of k+ channel and ca++ homeostasis, erk1/2 levels in response to bradykinin, cholesterol ester cycle, and, eventually, dysregulated proteolysis [3236 ]. recently, a fibroblast biomarker profile was proposed to identify accurately ad patients for therapeutic intervention. a very attractive approach could be that of using patient fibroblasts reprogrammed to neurons and exhibiting ad phenotype as a model. herein, we report that early prediction and diagnosis of ad might be improved by measuring typical proteolytic alterations that we formerly found in cultured ad fibroblasts. these alterations involve transketolase (tk), a thiamine - dependent enzyme that undergoes limited degradation by the enhanced activities of cysteine proteinases released upon cell extraction [32, 33 ], to yield isoforms with unusually high alkaline pi (tk - alkaline bands, tk - ab). in this paper, for the first time subjects from 36 families of probable late - onset ad patients were analyzed for the frequency and intensity of the tk - ab signal, and for the apoe genetic asset. then, statistic analyses were carried out to correlate tk - ab positivity and the presence of apoe 4 allele. remarkably, asymptomatic first - degree relatives were recruited and identified as subjects at greater risk of developing ad, independently of their apoe genetic asset. these subjects are often overlooked in most studies focused on detection of disease biomarkers without considering that ad relatives would probably be the most cooperative and interested individuals for investigation with noninvasive analyses. healthy subjects, patients with other non - ad neuropathologies, and early - onset ad patients were also analyzed. our results reveal tk - ab as a steady indicator of cultured ad fibroblasts, that is, associated significantly with both late- and early - onset forms of ad regardless of age - of - onset or stage of disease. it could also be of prognostic value, helpful in assessing ad risk in single individuals and applicable to asymptomatic relatives of ad patients and apoe-4 carriers. bace1 is the key rate - limiting enzyme for the production of the a peptide and its activity has been found to increase under starvation in vitro and after energy inhibition in app transgenic mice. the amount of active bace1 increased significantly in extracts of cultured fibroblasts positive to the tk - ab test, supporting the usefulness of cultured fibroblasts as an excellent in vitro model for the study of the pathogenetic process of ad. individuals who entered the study have been enrolled in the geriatric unit of prato hospital (italy) and included (a) elderly healthy subjects (controls, n = 29, mean age sd = 66.8 11.1, age range : 5086) ; (b) patients with a clinical diagnosis of probable late - onset or sporadic ad (n = 36, mean age sd = 70.8 7.3, age range : 6186) ; (c) asymptomatic first - degree relatives of ad patients (n = 38, mean age sd = 46.7 10.9, age range : 3268) ; and (d) neurological non - ad patients (n = 12, mean age sd = 67.2 10.5, age range : 5084) including 4 patients with vascular dementia, 3 patients with frontotemporal dementia, 2 patients with parkinson 's disease, 1 patient with severe neurosis, 1 patient with intermittent ataxia, and 1 patient with olivopontocerebellar ataxia. clinical diagnosis of probable ad was made according to the criteria established by the diagnostic and statistical manual of mental disorders (4th edition, dsm iv), the national institute of neurological and communicative disorders and stroke, and the alzheimer 's disease and related disorders association (nincdsadrda) and reevaluated according to the nia - alzheimer 's association workgroups on diagnostic guidelines for ad. medical examinations by neurologists with the aid of neuropsychological and laboratory tests and sometimes computed tomography or magnetic resonance of the brain were performed. the mini - mental state examination (mmse) and global deterioration scale (gds) informed written consent to use blood and dermal cells for analysis was obtained from the subjects or, when necessary, from their legal guardians under local institutional review board supervision and approval. further, we analyzed fibroblasts (kindly provided by s. sorbi, department of neurology, university of florence) from early - onset ad patients (n = 5, mean age sd = 48.0 6.9, age range : 3855) carrying mutations of app (val717ile, 1 case), presenilin 1 (ps1) (met146leu, 2 cases), and presenilin 2 (ps2) (met239val, 2 cases). skin biopsies were obtained from the forearm of subjects by a 2 mm punch, under local anesthesia with 2% xylocaine ; healing was usually complete within a week. tissue fragments were placed in culture and fibroblasts were then propagated under culture conditions favoring tk - ab expression. briefly, cells were seeded using a medium at ph 7.8 and maintained for 2 weeks without medium changes. then fibroblasts were harvested and lysed by sonication and cell extracts were separated by isoelectric focusing (ief) as previously reported. separated proteins were transferred to a nitrocellulose membrane and then probed with rabbit polyclonal igg raised against human tk. louis, mo) or ecl procedure (amersham biosciences) [25, 33 ] and quantified by densitometric analysis with the aid of imagej software (see section 3 and legend in figure 1). peripheral blood mononuclear cells were isolated by centrifugation at 1,700 g on lymphoprep (eurobio, les ulis cedex b, france). genomic dna was then extracted by using the nucleospin tissue kit (macherey - nagel gmbh & co. kg, dren, germany). dna was amplified by pcr in a dna thermal cycler (geneamp pcr system 2700, applied biosystems, nj) as reported by hixson and vernier. fibroblasts from two healthy aging control subjects (cont 1 and cont 2), from six probable ad patients (ad1 - 6), and from two asymptomatic first - grade ad relatives (rel 1 and rel 2) were analyzed. cells were seeded and maintained in parallel under conditions favoring tk - ab production (see above), called positive, and under normal conditions (with normal medium ph and detached at confluence), called negative. harvested fibroblasts were extracted with ripa buffer (r0278, sigma, 0.1 ml/1,000,000 cells) and protein concentration was assessed by the bicinchoninic acid protein determination kit (sigma). for bace1 determination, aliquots (20 g) of total cell extracts were separated on 412% nupage bis - tris gels with mops - sds running buffer (novex, invitrogen, carlsbad, ca) and blots were probed with anti - bace1 antibody (nb120 - 10716, novus biologicals, littleton, co). after incubation with hrp - conjugated secondary antibodies, specific bands corresponding to bace1 proteins were detected by the ecl procedure (amersham, freiburg, germany) and quantified by densitometric analysis with the aid of imagej software. anti - gapdh antibodies (cell signaling, beverly, ma) were used for detection of the housekeeping protein. tk - ab expression and apoe genotype frequencies in different groups of subjects were compared by means of fisher 's exact test. the analysis of variance test (anova) was used to establish whether the degree of ad severity or patient age differed significantly between tk - ab - positive and -negative cases. sensitivity, specificity, accuracy, and predictive values (pv) of tk - ab expression were reported together with 95% confidence intervals (ci). altered tk - isoforms (tk - ab) have frequently been observed in extracts of cultured fibroblasts from ad patients and proposed as potential indicators of disease (see section 1 and [25, 32, 34 ]). the conditions for optimal expression and detection of tk - ab following ief separation have been reported elsewhere [32, 47 ] ; however, for the sake of clarity, a panel of the typical and of the most representative altered tk profiles as well as the densitometric criteria used to evaluate results of the analysis were reported (figures 1(a) and 1(b), resp.). the protein band migrating at ph 8.4 was the major tk isoform in control samples, together with at least three minor and less basic bands. instead, tk - ab exhibited a more alkaline pi and migrated within a ph range of 8.59.4. the presence of tk - ab was usually assessed by visual inspection ; however, to decide on uncertain cases a densitometric analysis of immunostained bands was employed. intensity values of tk - ab (ph 8.5) were calculated and divided by those of the normal tk band (ph = 8.4) ; the border between negative and positive samples was arbitrarily set at a ratio of 0.4. of note, a strong positive tk - ab signal (ph 8.5) as observed for many of the ad patients was usually accompanied by a decrease in size and staining of the major tk band (ph = 8.4). tk profiles were analyzed in fibroblasts derived from 5 distinct groups of individuals and results are reported in figure 2 (for details see legend and section 2). the number of tk - ab - positive cases was markedly high among probable ad patients while both healthy controls and neurological non - ad patients (neurol. controls) were all tk - ab negative. based on these findings, tk - ab could be validated as a potential peripheral ad signature, showing a sensitivity = 69.4% (ci = 51.983.6), a specificity = 100% (ci = 91.4100), and an accuracy = 85.7% (ci = 75.992.6). the negative or positive predictive value (pv) was 78.8% (ci = 65.388.9) or 100% (ci = 86.3100), respectively. tk - ab - positive and -negative cases did not differ significantly with regard to age - at - onset (analysis of variance : p = 0.76) or disease severity as determined by gds and mmse clinical tests (p = 0.20 and p = 0.26, resp.). notably, early - onset ad patients (dominant ad) carrying app, ps1, and ps2 mutations were all tk - ab positive. nearly half (47.4%) of asymptomatic first - degree ad relatives (mean age 1), in fibroblast extracts which contemporarily yielded a positive tk - ab signal. we propose that the tk - ab test may be used as a peripheral indicator of disease in fibroblasts from ad patients in this study. the sensitivity of this test (as opposed to specificity, accuracy, and predictive values) was not extraordinarily high but it should be remembered that these individuals were diagnosed as probable ad patients only by standard clinical criteria. there are, however, some aspects concerning the significance and applications of tk analysis that deserve further comments. first, the tk - ab signature seems to be independent of age - of - onset, severity, and form of ad. such a steady expression from the early to the late clinical stages of disease is a distinctive feature of tk - ab as compared to other peripheral ad indicators. in fact, even a peptides, the most sensitive and direct hallmark of ad, increase in plasma of rare dominant ad patients but not in most common late - onset ad patients who actually show a decline in a-42 levels [9, 55 ]. secondly, tk - ab could be detected even in the absence of clinical signs such as in (i) approximately half of the subjects younger than 50 who, irrespective of 4 frequency, have had an ad patient in the family and run a major risk of developing ad later in life and (ii) in asymptomatic 4/4 carriers who have a high probability of developing ad with age. the identification of the tk - ab signature in combination with genetic profile in relatives of ad patients but it might also be worth for subjects with mci or less clearly determined cognitive deficits could provide early recognition of at - risk subjects and allow targeted intervention to delay neurodegeneration. we can not say that all asymptomatic individuals with a single apoe 4 gene dose and positive for tk - ab will necessarily develop the disease at advanced age but, at least, it could be possible to identify those subjects who should enter a follow - up study. thirdly, our results suggest that mechanisms underlying tk - ab production and bace1 activation might be related. in addition, the fact that the amount of active bace1 increased significantly in extracts of cultured tk - ab - positive fibroblasts reinforces the specificity of our test. on the whole, this study supports the usefulness of cultured fibroblasts as an excellent in vitro model for the study of the pathogenetic process of ad and for preliminary tests of toxicity and efficacy of agents capable of reestablishing the control of intracellular proteolysis, including bace1 activation. | there is great interest in developing reliable biomarkers to support antemortem diagnosis of late - 's disease (ad). early prediction and diagnosis of ad might be improved by the detection of a proteolytic dysfunction in extracts from cultured ad fibroblasts, producing altered isoelectrophoretic forms of the enzyme transketolase (tk - alkaline bands). the tk profile and apolipoprotein e (apoe) genotype were examined in fibroblasts from 36 clinically diagnosed probable late - onset sporadic ad patients and 38 of their asymptomatic relatives, 29 elderly healthy individuals, 12 neurological non - ad patients, and 5 early - onset ad patients. tk alterations occurred in (i) several probable ad patients regardless of age - of - onset and severity of disease ; (ii) all early - onset ad patients and apoe 4/4 carriers ; and (iii) nearly half of asymptomatic ad relatives. normal subjects and non - ad patients were all negative. notably, culture conditions promoting tk alterations were also effective in increasing active bace1 levels. overall, the tk assay might represent a low - cost laboratory tool useful for supporting ad differential diagnosis and identifying asymptomatic subjects who are at greater risk of ad and who should enter a follow - up study. moreover, the cultured fibroblasts were confirmed as a useful in vitro model for further studies on the pathogenetic process of ad. |
one hundred and forty type 2 diabetic patients aged 2967 years recruited from the iranian diabetes society and gabric diabetes education association, both located in tehran, participated in the study. to be eligible, patients needed to be free of any clinical disease including gastrointestinal, cardiovascular, and other endocrinological disorders (i.e., absence of specific symptoms and history of the diseases) ; a nonsmoker ; nonpregnant ; and not using insulin or any dietary supplements at least 3 months prior to the study. this study was approved by the ethics committees of the national nutrition and food technology research institute (nnftri), shahid beheshti university of medical sciences, and tehran university of medical sciences (tums). this study was a part of a larger controlled randomized clinical trial that has previously been described (11). briefly, after a 2-week run - in period, all subjects were instructed to consume a 250-ml bottle of vitamin d fortified persian yogurt drink (doogh), containing 170 mg calcium and 500 iu vitamin d/250 ml per bottle, twice a day with their meals (i.e., lunch and dinner). two bottles of doogh were replaced for one exchange of dairy product (one glass of milk or yogurt) in the participants ' diets so that the total energy intake was not affected by the intervention. every two weeks, all subjects were visited for assessment of their compliance and provision to them of a new package. blood pressure measurements as well as anthropometric, dietary, physical activity, and biochemical evaluations were performed for all of the subjects before and after the intervention. finally, all participants were divided in three groups according to vdr fok - i variants, i.e., ff, ff, and ff. responses to the intervention were compared among the fok - i genotypic groups. to ascribe changes of the variables to vitamin d intake, the intervention was performed during fall and winter, when dermal vitamin d synthesis was negligible. moreover, a control group (n = 50) receiving equal amount of plain doogh was also included in the main study. as the results of the comparisons between fortified and plain groups have previously been reported (3,4), data are not presented here. weight was measured in light clothing without shoes using a digital scale (seca 808 ; seca, hamburg, germany). bmi was calculated as weight in kilograms divided by the square of height in meters. body fat mass was evaluated by bioelectrical impedance analysis (quadscan 4000 system ; bodystat, beaconsfield, u.k.). dietary intakes were assessed by three 24-h recalls on 3 days (including 2 working days and 1 weekend day) at the beginning and the end of the intervention period. trained dietitians completed all questionnaires through face - to - face interviews with the participants using a multipass approach. for better this method has been used in a national survey on household food consumption patterns in iran (12). blood samples collected from all participants after 1214 h fasting by phlebotomy were divided in two tubes either with edta for genotyping and hba1c determination or without the anticoagulant for serum chemical analyses as previously described (11). fasting serum glucose (fsg) and insulin concentrations as well as serum lipid profile and albumin - to - creatinine ratio (acr) were determined as previously described (3). insulin resistance was evaluated by quantitative insulin check index (quicki) (13). serum 25(oh)d3 concentrations were measured by high - performance liquid chromatography method (14). in this study, vitamin d status was defined based on serum 25(oh)d3 concentration as deficiency, 27.5 nmol / l but 50 nmol / l. according to the institute of medicine, these cutoffs were set based on the fact that the vitamin d requirements of at least 97.5% of the population can be met with circulating 25(oh)d concentrations of 50 nmol / l (15). serum intact parathyroid hormone (ipth) was measured by enzyme immunoassay (eia) (drg, marburg, germany) with the aid of a semiautomatic plate reader (statfax 3200 ; awareness technology, palm city, fl). systemic inflammatory biomarkers including high - sensitivity c - reactive protein (hscrp) and serum amyloid a (saa) were determined as previously described (4). serum endothelial biomarkers including endothelin 1, e - selectin, and matrix metalloproteinase (mmp)-9 were measured as reported earlier (3). peripheral blood mononuclear cells were separated using ficoll density gradient and cultured in rpmi-1640 (16). after 24 h incubation, cell culture supernatants were used for cytokine measurements using enzyme - linked immunosorbent assay (4). dna was extracted from anticoagulated blood samples using genet bio dna isolation kit (primeprep, chungnam, south korea) according to the manufacturer s protocol. the genotypes of vdr gene polymorphisms were determined by pcr amplification and enzymatic digestion of the products with fok - i restriction enzyme. the forward and reverse primers (5-agc tgg ccc tgg cac tga ctc tgc tct-3 vs. 5-atg gaa aca cct tgc ttc ttc tcc ctc-3) for amplification of the fok - i vdr polymorphism were the same as those used elsewhere (17). pcr was performed with a gradient palm - cycler (corbett research, sydney, australia) for 30 cycles and at 58c annealing temperature. dna was digested with fok - i enzyme (fermentas ; thermo scientific, burlington, ontario, canada), and the products were analyzed by electrophoresis on a 1.5% agarose gel containing ethidium bromide and visualized in a gel documentation system (uvidoc ; uvitec, cambridge, u.k.). genotype frequencies of fok - i were tested for hardy - weinberg equilibrium using test. for trend was done to compare the proportions of vitamin d status among the groups. the statistical difference in genotype distribution among the groups was assessed by. two - factor repeated - measures anova was used to evaluated time - group interactions, with time and group as factors. in case of a significant time group interaction, between - group comparison of changes was done using anova followed by tukey post hoc analysis and with polynomial contrast analysis for trend when indicated. categorical and continuous variables are expressed as n (%) and means sd, respectively. in this study, all statistical analyses were done using spss (version 14 ; spss, chicago, il). this study was a part of a larger controlled randomized clinical trial that has previously been described (11). briefly, after a 2-week run - in period, all subjects were instructed to consume a 250-ml bottle of vitamin d fortified persian yogurt drink (doogh), containing 170 mg calcium and 500 iu vitamin d/250 ml per bottle, twice a day with their meals (i.e., lunch and dinner). two bottles of doogh were replaced for one exchange of dairy product (one glass of milk or yogurt) in the participants ' diets so that the total energy intake was not affected by the intervention. every two weeks, all subjects were visited for assessment of their compliance and provision to them of a new package. blood pressure measurements as well as anthropometric, dietary, physical activity, and biochemical evaluations were performed for all of the subjects before and after the intervention. finally, all participants were divided in three groups according to vdr fok - i variants, i.e., ff, ff, and ff. responses to the intervention were compared among the fok - i genotypic groups. to ascribe changes of the variables to vitamin d intake, the intervention was performed during fall and winter, when dermal vitamin d synthesis was negligible. moreover, a control group (n = 50) receiving equal amount of plain doogh was also included in the main study. as the results of the comparisons between fortified and plain groups have previously been reported (3,4), data are not presented here. weight was measured in light clothing without shoes using a digital scale (seca 808 ; seca, hamburg, germany). bmi was calculated as weight in kilograms divided by the square of height in meters. body fat mass was evaluated by bioelectrical impedance analysis (quadscan 4000 system ; bodystat, beaconsfield, u.k.). dietary intakes were assessed by three 24-h recalls on 3 days (including 2 working days and 1 weekend day) at the beginning and the end of the intervention period. trained dietitians completed all questionnaires through face - to - face interviews with the participants using a multipass approach. for better this method has been used in a national survey on household food consumption patterns in iran (12). blood samples collected from all participants after 1214 h fasting by phlebotomy were divided in two tubes either with edta for genotyping and hba1c determination or without the anticoagulant for serum chemical analyses as previously described (11). fasting serum glucose (fsg) and insulin concentrations as well as serum lipid profile and albumin - to - creatinine ratio (acr) were determined as previously described (3). insulin resistance was evaluated by quantitative insulin check index (quicki) (13). serum 25(oh)d3 concentrations were measured by high - performance liquid chromatography method (14). in this study, vitamin d status was defined based on serum 25(oh)d3 concentration as deficiency, 27.5 nmol / l but 50 nmol / l. according to the institute of medicine, these cutoffs were set based on the fact that the vitamin d requirements of at least 97.5% of the population can be met with circulating 25(oh)d concentrations of 50 nmol / l (15). serum intact parathyroid hormone (ipth) was measured by enzyme immunoassay (eia) (drg, marburg, germany) with the aid of a semiautomatic plate reader (statfax 3200 ; awareness technology, palm city, fl). systemic inflammatory biomarkers including high - sensitivity c - reactive protein (hscrp) and serum amyloid a (saa) were determined as previously described (4). serum endothelial biomarkers including endothelin 1, e - selectin, and matrix metalloproteinase (mmp)-9 were measured as reported earlier (3). peripheral blood mononuclear cells were separated using ficoll density gradient and cultured in rpmi-1640 (16). after 24 h incubation, cell culture supernatants were used for cytokine measurements using enzyme - linked immunosorbent assay (4). dna was extracted from anticoagulated blood samples using genet bio dna isolation kit (primeprep, chungnam, south korea) according to the manufacturer s protocol. the genotypes of vdr gene polymorphisms were determined by pcr amplification and enzymatic digestion of the products with fok - i restriction enzyme. the forward and reverse primers (5-agc tgg ccc tgg cac tga ctc tgc tct-3 vs. 5-atg gaa aca cct tgc ttc ttc tcc ctc-3) for amplification of the fok - i vdr polymorphism were the same as those used elsewhere (17). pcr was performed with a gradient palm - cycler (corbett research, sydney, australia) for 30 cycles and at 58c annealing temperature. dna was digested with fok - i enzyme (fermentas ; thermo scientific, burlington, ontario, canada), and the products were analyzed by electrophoresis on a 1.5% agarose gel containing ethidium bromide and visualized in a gel documentation system (uvidoc ; uvitec, cambridge, u.k.). blood samples collected from all participants after 1214 h fasting by phlebotomy were divided in two tubes either with edta for genotyping and hba1c determination or without the anticoagulant for serum chemical analyses as previously described (11). fasting serum glucose (fsg) and insulin concentrations as well as serum lipid profile and albumin - to - creatinine ratio (acr) were determined as previously described (3). insulin resistance was evaluated by quantitative insulin check index (quicki) (13). serum 25(oh)d3 concentrations were measured by high - performance liquid chromatography method (14). in this study, vitamin d status was defined based on serum 25(oh)d3 concentration as deficiency, 27.5 nmol / l but 50 nmol / l. according to the institute of medicine, these cutoffs were set based on the fact that the vitamin d requirements of at least 97.5% of the population can be met with circulating 25(oh)d concentrations of 50 nmol / l (15). serum intact parathyroid hormone (ipth) was measured by enzyme immunoassay (eia) (drg, marburg, germany) with the aid of a semiautomatic plate reader (statfax 3200 ; awareness technology, palm city, fl). systemic inflammatory biomarkers including high - sensitivity c - reactive protein (hscrp) and serum amyloid a (saa) were determined as previously described (4). serum endothelial biomarkers including endothelin 1, e - selectin, and matrix metalloproteinase (mmp)-9 were measured as reported earlier (3). peripheral blood mononuclear cells were separated using ficoll density gradient and cultured in rpmi-1640 (16). after 24 h incubation, cell culture supernatants were used for cytokine measurements using enzyme - linked immunosorbent assay (4). dna was extracted from anticoagulated blood samples using genet bio dna isolation kit (primeprep, chungnam, south korea) according to the manufacturer s protocol. the genotypes of vdr gene polymorphisms were determined by pcr amplification and enzymatic digestion of the products with fok - i restriction enzyme. the forward and reverse primers (5-agc tgg ccc tgg cac tga ctc tgc tct-3 vs. 5-atg gaa aca cct tgc ttc ttc tcc ctc-3) for amplification of the fok - i vdr polymorphism were the same as those used elsewhere (17). pcr was performed with a gradient palm - cycler (corbett research, sydney, australia) for 30 cycles and at 58c annealing temperature. dna was digested with fok - i enzyme (fermentas ; thermo scientific, burlington, ontario, canada), and the products were analyzed by electrophoresis on a 1.5% agarose gel containing ethidium bromide and visualized in a gel documentation system (uvidoc ; uvitec, cambridge, u.k.). genotype frequencies of fok - i were tested for hardy - weinberg equilibrium using test. for trend was done to compare the proportions of vitamin d status among the groups. the statistical difference in genotype distribution among the groups was assessed by. two - factor repeated - measures anova was used to evaluated time - group interactions, with time and group as factors. in case of a significant time group interaction, between - group comparison of changes was done using anova followed by tukey post hoc analysis and with polynomial contrast analysis for trend when indicated. categorical and continuous variables are expressed as n (%) and means sd, respectively. in this study, all statistical analyses were done using spss (version 14 ; spss, chicago, il). in our study population, the occurrence of vdr variants was in hardy - weinberg equilibrium (p = 0.056), with highest frequency of ff genotype, followed by ff and ff genotypes, respectively. personal characteristics among the genotypic groups, including age (p = 0.248), sex (p = 0.600), and duration of sun exposure in a day (p = 0.230), did not show any significant difference. in the whole - study population, the supplementation resulted in a considerable decrease in the occurrence of vitamin d insufficiency / deficiency and a remarkable increase in vitamin d sufficiency (from 26 to 92%). however, 4.4 and 14.9% of the subjects still remained vitamin d deficient and insufficient, respectively. reanalysis of data among fok - i variant subgroups revealed no significant trend of distribution of vitamin d deficiency through fok - i genotypes (= 0.24 ; ptrend = 0.620) at baseline. however, after intervention, trend test was significant (= 5.3 ; p for trend = 0.020) showing that 50% of the initially vitamin d deficient subjects in the ff genotype still remained deficient after 12 weeks intervention (table 1). there was no significant difference in any of the initial variables among the genotypic groups except for fsg, which showed a significant difference (p = 0.011). post hoc tukey test revealed that the ff group had significantly lower fsg compared with ff (p = 0.034) and ff (p = 0.019). however, this difference was not accompanied by a significant difference of other glycemic variables such as hba1c or quicki. vitamin d status among the fok - i genotypic subgroups before and after the intervention improvement of vitamin d status was accompanied by amelioration of glycemic, lipidemic, and inflammatory markers in the whole - study population as previously described (3). repeated - measures anova revealed a significant time effect (i.e., within - subject difference over time) for 25(oh)d (p < 0.001), ipth (p < 0.001), fat mass (p < 0.001), waist circumference (p = 0.002), systolic blood pressure (sbp) (p = 0.003), diastolic blood pressure (dbp) (p = 0.001), fsg (p < 0.001), hba1c (p < 0.001), quicki (p < 0.001), total cholesterol (p < 0.001), triglycerides (p < 0.001), hdl (p < 0.001), saa (p = 0.013), e - selectin (p = 0.012), endothelin-1 (p = 0.001), interleukin (il)-2 (p = 0.006), il-4 (p < 0.001), and tumor necrosis factor (tnf)- (p = 0.044). the time effect was insignificant for -interferon (ifn-) (p = 0.058) (table 2). however, time intervention effect was significant only for 25(oh)d (p = 0.031), hdl (p = 0.011), hscrp (p < 0.001), il-4 (p = 0.018), and il-6 (p = 0.011). when changes of the variables were compared using anova followed by tukey post hoc test, a significant difference in 25(oh)d was observed between ff and ff (p = 0.023), but the differences between ff and ff (p = 0.573) or ff and ff (p = 0.23) were not statistically significant. as for hscrp, ff differed significantly with both ff (p < 0.001) and ff (p = 0.010), but there was no significant difference between ff and ff (p = 0.833). the proinflammatory cytokine il-6 also showed a significant difference between ff and ff (p = 0.038) as well as between ff and ff (p = 0.048). again, here, ff and ff did not differ significantly (p = 0.990). table 3 demonstrates the results of anova of the changes of the variables among the three vdr fok - i genotypes. comparisons of the variables among the fok - i genotypic groups with type 2 diabetes before and after 12 weeks intervention comparison of the changes of the variables among the fok - i genotypic groups with type 2 diabetes our data show a significant difference in the diabetic host response to supplemental intake of vitamin d according to fok - i vdr variants. specifically, the significantly lowest serum 25(oh)d response to daily intake of vitamin d fortified doogh in the ff group is considerable. the occurrence of vdr genotypes could, therefore, be pivotal in the outcome of any vitamin d intervention in a healthy as well as a patient population. some fortification experiences failed to protect the desirable proportion of the study population from vitamin d deficiency (18), which might be, at least partly, due to the genetic variations. the influence of vdr genotypes on absorption of both calcium and vitamin d has already been reported (19). in a pilot study on the efficacy of vitamin d plus calcium supplementation in 92 u.s. adult subjects, after 6 months supplementation with 800 iu vitamin d and 2,000 mg calcium, 25(oh)d reached the desirable levels in only half of the subjects (20). similarly, supplementation with 800 iu / day vitamin d during the winter resulted in normalization of circulating 25(oh)d in 80% of the postmenopausal women (21). although no genetic analyses were performed in these studies, genetic variations of vdr among the study populations could be one explanation. in support of this notion, in subjects with low bone density who are heterozygous for allele f, compared with ff homozygotes, the inverse association between vdr fok - i ff genotype and lower vitamin d status has also been observed among the subjects with multiple sclerosis (23) and -thalassemia (24). it is noteworthy that despite different 25(oh)d responses, changes of ipth did not differ significantly among the groups. contrary to our finding, in an observational study on 95 postmenopausal women, fok - i variants were related to both secretion and degradation of parathyroid hormone (25). notwithstanding, our data showed that a parathyroid hormone suppressing effect of intake of 1,000 iu / day vitamin d could be similar among fok - i vdr genotypic groups. the ameliorating effect of vitamin d intake on glycemic status of diabetic subjects was observed in our previous study (3). in the current study, lower 25(oh)d response to vitamin d intake in the ff group had no remarkable effect on either fsg or quicki. this might be due to the compensatory increased formation of the active vitamin d metabolite, 1,25(oh)d2 from 25(oh)d by 1--hydroxylase, which is also expressed in pancreatic -cells (26). although vdr fok - i polymorphism has been shown to explain some 29% of the variability in insulin sensitivity, it has no influence on -cell function in healthy caucasians (10). the association of vdr genotypes with glycemic status has been the focus of several studies. polymorphisms of vdr bsm - i have been linked to development of both diabetes and coronary artery disease (cad) in 293 subjects at high risk of cad (27). in another study, while the distribution of apa - i, bsm - i, and taq - i variants did not differ between diabetic and nondiabetic subjects, bsm - i polymorphism was associated with insulin resistance only in nondiabetic caucasians (28). high prevalence of vitamin d deficiency and its inverse association with apa - i aa genotype and fok - i f allele in caribbean subjects with type 2 diabetes has been described previously (29). as previously reported (3), daily consumption of vitamin d fortified doogh resulted in a small but significant increase in serum hdl. however, only in homozygotic subgroups (i.e., ff and ff) were the changes of hdl concentrations statistically significant. contrary to our findings of increased serum hdl in vdr fok - i homozygotic groups ff and ff, in a study on 276 nondiabetic overweight men (bmi 28.06 kg / m) aged 2565 years, subjects with ff and ff genotypes, compared with the ff variant, had lower hdl concentrations and higher fasting serum insulin (30). one of the reasons for this inconsistency in observations could be the interactive effects of other genes with vdr, including hla (31). cardiovascular disease (cvd), including cad, stroke, and peripheral vascular disease, is well - known as the major cause of morbidity and mortality in diabetic subjects (32). however, some studies have shown that intensive treatment of hyperglycemia would lead to only 16 and 25% risk reduction of myocardial infarction and microvascular disease, respectively (33). therefore, normalization of other risk factors, including inflammatory status, has been proposed to prevent cvd in diabetes (34). the association of inflammation with diabetes and its long - term complications has previously been documented (35). in the current study, although hscrp decreased in all groups, it did so more in ff than in ff and ff. similarly, the decrease of cellular il-6 in ff was in order of magnitude compared with ff and ff, which was accompanied by a significant increase in il-4. these changes suggest the vitamin d induced immune deviance toward t - helper 2, which was more prominent in ff than in ff or ff. considering the predisposing effect of inflammation in long - term diabetes morbidities, notably cvd (36), our findings may indicate different protective effects of vitamin d intake among diabetic subjects carrying various vdr fok - i genotypes. improvement of endothelial (3) as well as systemic (4,37) inflammatory biomarkers after vitamin d intake in the subjects with type 2 diabetes has already been reported. the subsided inflammatory response, however, was not accompanied by a significant change in urinary albumin excretion in either fok - i genotypic group. the possible synergistic protective effect of both suppressed systemic / endothelial inflammation and raised serum hdl after improvement of vitamin d status in diabetic subjects and their interaction with vdr fok - i variants could be the focus of further studies. as vitamin d deficiency adversely affects both glycemic and inflammatory status, it can intuitively be regarded as a predisposing factor for long - term diabetes complications, including atherosclerosis (32). in the current study, 74% of the subjects initially had vitamin d deficiency / insufficiency, similar to 73% occurrence of hypovitaminosis d observed in another study (38). however, not all vdr genotypic groups responded similarly to increased vitamin d intake. actually, the ff variant showed the lowest response in terms of increments of 25(oh)d and certain inflammatory biomarkers. vdr ff genotype has also been proposed as a contributing factor in aggressive breast cancer, which is less responsive to vitamin d therapy (39). the possible role of vdr variants in diabetes still remains to be clarified by further studies. low responders who may have an increased need for vitamin d. a nutrigenetic approach may, therefore, be needed to protect diabetic subjects from vitamin d deficiency. polymorphisms of other vdr genotypes, i.e., taq - i, apa - i, bsm - i, and cdx-2, and their possible interactions with fok - i variants were not evaluated. extension of the changes observed after 12 weeks intervention to longer periods of time and, above all, their possible protective effect against long - term diabetes complications require well - designed longitudinal controlled studies. regarding the small amount of 25(oh)d variation explained by genetic determinants, a mendelian randomization approach could be beneficial to reveal a causal relationship in a large population (40). as hypovitaminosis d may negatively affect glycemic status (38), and probably the related consequences, the very high prevalence of vitamin d insufficiency / deficiency among the participants of the current study is likely to have a major impact on the results. also, evaluation of a vast array of variables further allowed us to detect the positive effects of vitamin d supplementation. to the best of our knowledge, this is the first report of the interactive effects of vdr fok - i polymorphisms and vitamin d intake on diverse aspects of diabetes host response, including glycemic, lipidemic, and inflammatory as well as immune responses. our findings indicated that those with vdr fok - i ff genotype may be regarded as low responders to vitamin d intake in terms of circulating 25(oh)d and certain inflammatory biomarkers. the prevalence of different vdr fok - i variants among diabetic subjects could explain, at least in part, some discrepancies observed in the effect of vitamin d on various aspects of diabetes host response. | objectiveinterpopulation as well as interindividual variations in response to vitamin d intake commonly observed in subjects with type 2 diabetes may be related to genetic makeup. one of the candidate genes potentially responsible for this diversity is vitamin d receptor (vdr). this study aimed to investigate the interactive effect of vdr fok - i polymorphism and vitamin d intake on diverse aspects of diabetic host response.research design and methodsglycemic status, lipid profiles, inflammatory biomarkers, and vdr fok - i genotypes were determined in diabetic subjects (n = 140) who participated in a randomized controlled trial. participants consumed two 250-ml bottles per day of yogurt drink (doogh) fortified with 500 iu vitamin d/250 ml for 12 weeks.resultsmean serum 25(oh)d increased by ~30 nmol / l (p < 0.001). the time intervention effect was significant for 25(oh)d (p = 0.030), hdl (p = 0.011), high - sensitivity c - reactive protein (hscrp) (p < 0.001), interleukin (il)-4 (p = 0.008), and il-6 (p = 0.017) among the genotypic groups. the alleles were defined as f or f depending on the absence or presence of the restriction site, respectively. the least increment in 25(oh)d was in ff (23.0 3.8 nmol / l) compared with ff (31.2 3.4 nmol / l) and ff (35.6 2.7 nmol / l) (p for trend = 0.009), but only the difference between ff and ff was significant (p = 0.023). ff group had the largest decrement of both hscrp and il-6 compared with ff (p < 0.001 and p = 0.038) and ff (p = 0.010 and p = 0.048), respectively.conclusionswe concluded that those of vdr ff genotype may be regarded as low responders to vitamin d intake in terms of response of circulating 25(oh)d and certain inflammatory biomarkers. a nutrigenetic approach may, therefore, be needed to protect diabetic patients from vitamin d deficiency. |
the human immunodeficiency virus type 1 (hiv-1), a small diploid rna retrovirus with a genome size of approximately 10,000 nucleotide bases, was isolated for the first time about three decades ago and recognized as the etiologic agent of the acquired immunodeficiency syndrome (aids). early study soon demonstrated that hiv-1 displayed remarkable inter- and intra - host genetic heterogeneity, as well as specific evolutionary patterns within the infected host. first, the low - fidelity of the reverse transcriptase characterized by an error rate of 3.410 mutations per nucleotide per cycleresulting in the introduction of one nucleotide substitution every second to third newly synthesized genome. second, the rapid viral generation time defined as the time from release of a virion until it infects another cell and causes the release of a new generation of viral particles estimated in 1.5 - 2.6 days with a production of ~1010 new virions each day. third, the elevated recombination rate with a number of crossovers between two rna templates estimated to range from three to nine per genome per round of replication. these factors make hiv-1 one of the fastest evolving organisms known and the heterogeneous viral swarm that infects most hiv-1seropositive individuals fits the definition of a measurably evolving population, i.e. a population from which molecular sequences taken at different points in time will display a statistically significant number of genetic differences. in other words, hiv-1 evolution in vivo is an observable microevolutionary process that can be investigated through the analysis of viral sequences sampled longitudinally (heterochronous sequences) from an infected host. hiv-1 intra - host evolutionary and population dynamics have been under extensive investigation, especially for their potential role in pathogenic processes, and because they constitute a major challenge for the development of an effective vaccine. what are the intra - host ecological factors shaping viral diversity over time and what is the potential relationship between viral evolution and pathogenesis are still presently debated. in the next sections, the current knowledge on hiv-1 intra - host evolutionary patterns in the absence of antiretroviral treatment (art) will briefly be reviewed with special emphasis on results obtained through the development and application of phylogenetic techniques. i will show how phylogenetic analysis has been able to shed light on the complex interplay between viral evolution and immune selection that can ultimately result in disease progression. hiv-1 genetic variability at the nucleotide level can reach up to 5% within an infected subject and provides the viral population with the ability to adapt rapidly to changes in its environment. the infection is typically characterized by three phases acute infection, clinical latency and progression to aids during which viral genetic divergence and diversity display consistent patterns that have been associated with disease progression. hiv-1 intra - host diversity and divergence over time can be defined according to specific phylogenetic criteria. given a data set of hiv-1 sequences sampled longitudinally from an infected patient, diversity is the average pairwise genetic (nucleotide or amino acid) distance within the sequences sampled at a given time point, while divergence is their average genetic distance from the most recent common ancestor (mrca), i.e. the root of the viral genealogy (phylogeny). when an individual becomes infected with hiv-1, a relatively homogenous population of the virus is initially harbored because transmission is usually associated with a significant population bottleneck. a new infection can be established by just one single virion, although the number of variants transmitted seem to be dependent on the transmission route and bottlenecks associated with sexual transmission may be greater than with vertical transmission (e.g. mother to child or injecting drug use). it has been shown that in men who have sex with men (msm) the minimum number of transmitted viruses can range from 2 to 10 with subsequent viral recombination leading to rapid and extensive genetic shuffling among viral lineages. a combined analysis also revealed a significantly higher frequency of multivariant transmission in msm than in heterosexuals. the severity of transmission bottlenecks could be the result of anatomical barriers (e.g. mucosal barrier in heterosexual transmissions) and by immune selection mediated by early cytotoxic t cell responses. however, in an animal model, infection with a highly heterogeneous siv viral swarm of cd8 depleted rhesus macaques, where immune selection is expected to be weak or inexistent, low frequency siv variants characterized by specific amino acid motif in gp120 were still transmitted more efficiently than higher frequency ones in six different primates. overall, it is important to keep in mind that these studies used, by necessity, indirect methods to quantify the amount of transmitted variants, because it is obviously very difficult to measure hiv-1 genetic diversity at the exact moment of transmission. indeed, it has rightly been observed that homogeneity of the viral population during primary infection does not necessarily imply homogeneity of the transmitted variants. the clinical latency phase of hiv-1 infection is characterized by a progressive increase in both viral divergence and diversity, while during the last phase, when the immune system collapses and progression to aids begins, viral divergence stabilizes and viral diversity declines. this observation has been explained as a consequence of cd4 t cell depletion, which likely results in less effective selection pressure on the virus, as well as significant decrease in target cells capable of sustaining viral replication. phylogenetic studies have clearly shown that, in the absence of art, the central paradigm governing hiv-1 intra - host evolutionary dynamics following primary infection is the turnover of genetic variants over time through sequential population bottlenecks. hiv-1 intra - host genealogies of heterochronous sequences display strong temporal structure, where sequences from the same sampling time tend to cluster together and are the direct ancestors of sequences from the following time point. a typical example, using a data set of hiv-1 env gp120 v1v3 sequences from a pediatric patient infected via mother to child transmission, is given by the maximum likelihood (ml) tree in figure 1a. increasing branch lengths from root to the latest sampled tips also reveal the increase in both viral diversity and divergence over time. the key point is that topology and branch length patterns of hiv-1 intra - host genealogies are the result of specific phylodynamic processes, defined as the interaction between evolutionary (i.e. mutation, genetic drift, selection) and ecological (population dynamics and environmental stochasticity) factors. in particular, the striking similarity between hiv-1 intra - host and influenza a inter - host genealogies, both characterized by strong temporal structure, seem to indicate that hiv-1 population bottlenecks may be driven by continual immune selection. studies based on pairwise sequence comparisons of synonymous (dn) and nonsynonymous (ds) substitutions have suggested that neutral genetic drift is the dominant force shaping the intra - host evolution of the virus. however, such methods have the tendency to underestimate the strength of selection. analyses of dn / ds ratios using an ml framework that takes into account the phylogenetic tree relating the sequences under study, have shown that both purifying and positive selection play a substantial role in the continuous emergence in vivo of new variants and the ability of the virus to evade immune response. hiv intra - host adaptation rate in the env gene has, indeed, been estimated to the astounding value of one adaptive fixation event every ~2.5 months. moreover, the comparison of hiv-1 replication rate of virus genomes isolated at early times following infection with that of later viruses has demonstrated an increase of fitness during chronic infection, and escapes from both cd8 + t - cell responses and neutralizing antibodies the degree of temporal structure exhibited by hiv-1 genealogies of longitudinally sampled sequences can be affected by additional factors that can alter the ideal ladder - like topology. the ml tree in figure 1b is an example obtained by analyzing gag p24 viral sequences from a subject carrying the hla class i allele b5701, which is associated with slower disease progression. by applying a nonparametric test for population structure, it has been shown that a complete within host viral population turnover may take up to 22 months. therefore, the intermix of viral sequences at 70 and 106 days post infection shown in figure 1b is expected and typical of genealogies inferred from longitudinal samples with relatively short time intervals. other deviations, however, can provide critical insights on factors driving hiv-1 intra - host population dynamics. for example, the clustering of sequences sampled at more distant time points, like the one indicated by the arrows in figure 1b, within a monophyletic clade of earlier sequences may indicate the activation (or re - activation) of archival genomes, possibly from a viral reservoir. peripheral blood mononuclear cells have been noted to harbor small latent reservoirs of infectious virus, which seem to provide one of the mechanisms for lifelong persistence of hiv-1 under art. although phylogeny - based criteria have been proposed to detect reservoirs where viral replication is restricted, a full characterization of hiv-1 reservoirs is still lacking. from a phylogenetic perspective, a limiting factor is that topological differences among genealogies can intuitively be assessed by visual inspection (figure 1a and b), but are actually difficult to quantify. a new statistic, temporal clustering (tc), has recently been developed to provide a quantitative measure of the degree of topological temporal structure in a serially sampled genealogy. tc values represent the expected deviation of an observed genealogy from the null hypothesis of no temporal structure (tc=0) and can be used to compare trees of different size. the tree with perfect temporal structure in figure 1a has by definition tc=1, while the tree in figure 1b has tc=0.72. in future studies, comparison of hiv-1 intra - host genealogies from patients sampled under different conditions could provide important insight on the dynamic of viral reservoirs. in addition, the ability to quantify and compare topological deviations from perfect temporal (ladder - like) structure among different trees may shed light on qualitative and quantitative differences in the evolutionary and ecological processes shaping each phylogeny. strong evidence for the importance of selection on hiv-1 intra - host evolution is also provided by studies on the total viral population size within an infected host, estimated on the order of 10 individual viruses. according to the general prediction of evolutionary theory, mutations in small size populations are produced rarely and their fixation will essentially depend on random genetic drift. on the other hand, in populations large enough that can be considered for any practical purpose of infinite size, mutations are produced more frequently and their fixation will eventually be decided by the deterministic action of natural selection. it has been suggested that it would be appropriate to consider the hiv-1 population within an infected host as if its size were infinite and, therefore, evolutionary driven by selection. on the other hand, several studies have calculated that hiv-1 intra - host effective population size (ne), corresponding to the size of a hypothetical neutral idealized population (i.e. described by the standard wright - fisher model), is several orders of magnitude smaller (10 - 10) than the total population size. such estimates would be more consistent with an evolutionary process driven by random genetic drift, despite the strong evidence of selection provided by dn / ds analyses (see previous section). first, ne estimates in these studies were derived from methods implicitly assuming neutrality, making it impossible to assess whether low values reflect true stochastic processes or continual selective sweeps. second, hiv-1 populations do not evolve under panmixia (one single population), but rather with some metapopulation structure (i.e. division in sub - populations) that causes a reduction in ne. indeed, several studies have shown the existence of diverse hiv-1 subpopulations infecting different body tissues, for example semen, breast milk, genital tract, and brain. the term compartments has been introduced to indicate tissues or cell types were viral replication is ongoing but viral gene - flow (in and out) is restricted because of anatomical barriers. compartmentalization is shown, phylogenetically, by the existence of supported monophyletic clades including sequences isolated from a specific tissue, as displayed in figure 2. the ml tree was inferred from hiv-1 sequences isolated from necropsy tissues of an infected subject who died with widespread atherosclerosis. additional studies have also demonstrated that different brain regions harbor distinct viral populations characterized by specific evolutionary dynamics that may be associated with pathogenic processes like the onset of hiv-1 associated dementia. estimates of hiv-1 intra - host evolutionary rate range from 10 to 10 nucleotide substitutions per site per year depending on specific patients and gene region analyzed. several studies have shown an inverse relationship between rate of viral evolution and disease progression, but contrary evidence has also been presented. a sophisticated molecular clock analysis of gp120 c2v5 sequences from nine subjects followed from seroconversion up to 11 years found that hiv-1 disease progression seems to be predicted by synonymous substitution rates, which are expected to be selectively neutral and, therefore, proportional to the underlying viral replication rate. specifically, lower substitution rates were found in patients with slow disease progression. the authors speculated that such lower rates might reflect lower levels of persistent immune activation, which in turn impose significant constraints on viral generation times and viral evolution. t cell activation is the strongest predictor of progression to aids, and can determine the rate and continuity of viral replication. in the attempt to unify all these observations, lee. in 2008 developed an hiv-1 intra - host evolution model that simulated the effects of mutation and fitness of sequence variants. the model indicated that the decrease of diversity and stabilization of divergence in the last phase of the infection are the result of a decrease in the proportion of offspring that are mutants as the distance from the founder strain increases, rather than of an increase in viral fitness. in other words, hiv-1 intra - host evolutionary rate is expected to decrease towards the end of the infection in correlation with the rate of cd4 t cell decline because of the progressive disappearance of target cells that can sustain viral replication. phylogenetic and molecular clock analysis of empirical data (15 patients followed for 3 - 12 years) supported the prediction of the model. interestingly, a small proportion of hiv-1 infected patients (500 cells / mm for more than 10 years without any therapy. most ltnps carry the hlab 5701 allele and their ability to control the infection have intensively been studied for its obvious clinical implications. from the standpoint of virus evolution, little work has been done since intra - host genetic variation is often minimal and difficult to evaluate given the undetectable vl. however, some subjects expressing hla - b5701 do show low levels of viremia (usually ranging between 1000 and 10,000 copies / ml) and can still remain clinically and/or immunologically stable for years without therapy. cd8 + t cell responses in these patients target several epitopes in the gag p24 viral gene, and an interesting association between hiv-1 intra - host evolutionary rate and immune response has recently been found. heterochronous p24 sequence data were employed to estimate with a bayesian molecular clock method the posterior distribution of viral evolutionary rates within subjects classified as either high - risk progressor (hrp) or low - risk progressor (lrp) on the basis of their cd4 t cell count in early (10 - 13 weeks) infection. lower evolutionary rates were found in lrps who also exhibited higher polyfunctional responses than hrps in terms of higher proportion of cd8 t cells producing a combination of interferon-, inteleukin-2, mip-1 and perforin when induced by wild - type hla - b5701-restricted epitopes. further investigation of the interplay between specific cd8 t cell responses and intra - host viral rate of evolution in these subjects may help defining correlates of protection, one of the greatest challenges in hiv-1 vaccine design. due to super - infection and reverse transcriptase switches between alternative genomic templates, that are an integral part of hiv lifecycle, hiv-1 recombination rate per replication is likely exceeding the mutation rate. the impact of recombination on viral genetic diversity is evident at the molecular epidemiological scale. the majority of hiv-1 strains circulating worldwide cluster within a large group called m (for main) that includes, besides ten phylogenetically distinct subtypes and two subsubtypes, several inter - subtype recombinants, known as circulating recombinant forms (crfs). recombination can affect hiv-1 evolutionary dynamics by providing a mechanism even more efficient than fast mutation rate for the virus to escape from the accumulation of deleterious mutations or to jump between adaptive peaks. it might accelerate progression to aids and can foster the emergence of viral variants able to evade immune pressure or with increased resistance to drugs. analysis of hiv-1 sequences amplified from different tissues, sampled post mortem from patients with a number of aidsrelated malignancies, have shown that recombination is common and more extensive in tissues with abnormal histopathology compared to normal tissues. as shown in figure 3, intra - host recombination could be detected, in principle, by moving a sliding window along a multiple sequence alignment and calculating the bootstrap support for the phylogenetic clustering of a query sequence, the potential recombinant, with different reference sequences in different genomic regions. this analysis is an example of bootscanning ; a method specifically developed to investigate recombination in hiv. recombinant sequences can not be clustered correctly within one single phylogenetic tree but can be displayed within network - like graphs able to represent simultaneously different tree topologies (figure 3). using algorithms that do not explicitly model recombination can bias molecular clock as well as population genetic estimates. unfortunately, this is not the case for most of the work discussed in the previous sections, where authors have chosen either to ignore recombination or the common approach to identify recombinants and exclude them from the analysis. first, because recombination is so frequent and plays such a central role, inferences on the evolutionary dynamics of the virus after exclusion of recombinants may be meaningless or, at the very least, incomplete. second, despite the plethora of methods developed to detect recombination, each algorithm has limitations resulting, in general, in an under estimation of recombination, especially in data sets of closely related sequences. for example, while inter - subtype recombination is relatively straightforward to detect, bootscanning performs very poorly with hiv-1 sequences from individuals infected by the same subtype. network - based methods have been more successful and have shown that the number of recombinant sequences detected by classic phylogenetic analysis can lead to a significant under estimation of the actual number of recombinants within an intra - host data set. however, it is clear that novel techniques and additional studies are still needed to assess the full impact of recombination on hiv-1 intra - host evolutionary and population dynamics. this review briefly highlights some of the major findings that have contributed to our current understanding of hiv-1 intra - host phylodynamics. there is general agreement on deterministic selection processes as the main driving force of viral evolution within the infected host. the study of the temporal structure of genealogies inferred from heterochronous sequences is one of the key techniques that can be used to assess the impact of continual immune selection on viral population turnover and a promising tool to investigate further the activation or re - activation of viral reservoirs. in addition, the recent application of high - resolution deep sequencing to study hiv-1 population complexity and structure (biodiversity) within an infected host can allow the characterization of low frequency variants responsible for significant changes in the viral evolutionary landscape that would be impossible to detect by conventional methods. on the other hand, a thorough understanding of early evolutionary events at the moment of transmission and during primary infection, as well as a full account of the role played by recombination is still lacking and among the future challenges of hiv molecular evolutionary research. | the intra - host evolutionary and population dynamics of the human immunodeficiency virus type 1 (hiv-1), the cause of the acquired immunodeficiency syndrome, have been the focus of one of the most extensive study efforts in the field of molecular evolution over the past three decades. as hiv-1 is among the fastest mutating organisms known, viral sequence data sampled over time from infected patients can provide, through phylogenetic analysis, significant insights about the tempo and mode of evolutionary processes shaped by complex interaction with the host milieu. five main aspects are discussed : the patterns of hiv-1 intra - host diversity and divergence over time in relation to different phases of disease progression ; the impact of selection on the temporal structure of hiv-1 intra - host genealogies inferred from longitudinally sampled viral sequences ; hiv-1 intra - host sub - population structure ; the potential relationship between viral evolutionary rate and disease progression and the central evolutionary role played by recombination occurring in super - infected cells. |
bleeding complications following percutaneous coronary intervention (pci) are associated with increased short and longterm morbidity and mortality.1, 2, 3, 4, 5 oral anticoagulant (oac) use prior to coronary stenting is a significant predictor of postprocedure bleeding events.6, 7 previous studies estimated that the frequency of chronic oac use among patients undergoing pci is between 3% and 7%.7, 8, 9, 10, 11 many of these analyses, however, examined selected patient populations, such as those admitted with acute myocardial infarction (mi) or atrial fibrillation, and preceded the market approval of non vitamin k antagonist oacs (noacs).12 as such, the contemporary rate of chronic oac use among all patients undergoing pci is presently unknown. furthermore, recent use of oac therapy among ambulatory patients has increased,13, 14 corresponding with the growing use of noacs, which are now prescribed as frequently as vitamin k antagonists (vkas).14, 15, 16, 17 compared with vkas, noacs are associated with reduced thromboembolic events and severe bleeding in patients with atrial fibrillation.18 nevertheless, there is a lack of data assessing management practices and outcomes among patients undergoing coronary stenting on noac therapy.19 with this in mind, we sought to determine contemporary practice patterns and temporal trends in use of oac therapies among all patients undergoing coronary stenting, using data from a large integrated health care system between 2009 and 2014. in addition, we examined the association of chronic oac therapy with short and longterm outcomes and whether these relationships were modified by use of noacs in addition to traditional vkas. partners healthcare is an integrated health care system that currently includes 8 massachusetts hospitals, 21 community health centers, and a network of independent ambulatory practices with > 500 affiliated primary care doctors. the pci cohort was derived from the partners longterm outcomes database, an ongoing, institutionally sponsored registry of patients undergoing pci within the partners healthcare system.20 all pcis in which patients presenting to 1 of 2 partners healthcare academic medical centers (brigham and women 's hospital and massachusetts general hospital) between june 2009 and september 2014 were included. if > 1 pci was performed in the same patient during the same hospitalization, only the first pci was included in the analysis. the present project was reviewed and approved with a waiver of informed consent from the institutional review board at partners healthcare. clinical and procedural characteristics analyzed for all pcis were abstracted from institutional registry data, based on the data collection form from the national cardiovascular data registry 's (ncdr) cathpci registry. a full description of the data elements of the cathpci registry are available online (http://cvquality.acc.org/~/media/qii/ncdr/data%20collection%20forms/cathpci%20registry_datacollectionform.ashx).21 to determine chronic anticoagulant status, we developed an algorithm that was applied to the entire electronic medical record, including admission notes, consultation notes, nursing notes, outpatient notes, catheterization reports, pharmacy notes, discharge summaries, and medication prescriptions, to identify the presence of oac use within the 30 days preceding the pci. the search terms included the medications warfarin, coumadin, dabigatran, pradaxa, rivaroxaban, xarelto, apixaban, and eliquis. the algorithm was designed to maximize sensitivity over specificity to provide a screen of the study population for patients taking oac therapy prior to pci. to remove falsepositive records, all electronic medical records of identified oac patients and n.b.) to confirm documented oac use within 30 days of the admission. of patients identified as taking oac therapy, 70.6% exclusion on manual review primarily resulted from the algorithm incorrectly identifying patients for whom phrases reflecting nonuse of oacs were found in unstructured data (eg, patient not on warfarin in a clinic visit note). additional data for oac patients were also manually collected and included : type and dose of anticoagulant at admission, type and dose of antiplatelet at admission, aspirin status and dose at admission, indication for anticoagulant, use and duration of bridging agent prior to pci, type and dose of antiplatelet at discharge, aspirin status and dose at discharge, type and dose of anticoagulant at discharge, discontinuation of anticoagulant after pci, and reason for discontinuation of anticoagulant after pci. the primary outcome measures were inhospital major bleeding, inhospital mortality, and longterm mortality. inhospital major bleeding was based on the ncdr version 4 definition,22 which included any of the following occurring before hospital discharge : arterial accesssite bleeding (defined as external bleeding at the access site or a hematoma > 10 cm for femoral access, > 5 cm for brachial access, or > 2 cm for radial access) ; retroperitoneal, gastrointestinal, or genitourinary bleeding ; intracranial hemorrhage ; cardiac tamponade ; postprocedure hemoglobin decrease of 3 g / dl in patients with a preprocedure hemoglobin level 16 g / dl ; or postprocedure non bypass surgery related blood transfusion for patients with a preprocedure hemoglobin level 8 g / dl. longterm mortality was assessed through the national death index and subsequent linkage with the institutional registry, as described previously.23 secondary outcome measures included accesssite bleeding (defined as external bleeding at the access site, hematoma at the access site, or retroperitoneal bleeding), non accesssite bleeding (defined as gastrointestinal or genitourinary bleeding, intracranial hemorrhage, or cardiac tamponade), red blood cell transfusion, postprocedure mi (new occurrence of a biomarkerpositive mi after pci), inhospital stent thrombosis (defined as a subsequent pci performed during the same index hospitalization as the related pci for the indication of stent thrombosis), cerebrovascular accident or stroke (defined as loss of neurological function caused by an ischemic or hemorrhagic event with residual symptoms lasting at least 24 hours after onset or leading to death), and postprocedure length of stay in days. in addition, readmissions to hospitals only within the partners healthcare system were identified for all pci patients within 30 and 90 days of discharge. clinical and procedural characteristics with dichotomous and categorical variables were reported as counts and percentages, and continuous variables were reported as means with standard deviations. betweengroup differences were assessed using fisher exact or chisquare tests for binary and categorical variables and student t tests or wilcoxon rank sum tests for continuous variables. meier methods were used to estimate the cumulative incidence of longterm mortality following pci stratified by use of oacs at admission, and logrank tests were used to compare the curves. predicted risks of inhospital bleeding and inhospital mortality were calculated for each group using previously validated risk models developed within the ncdr cathpci registry.22, 24 logistic regression models were created to measure the association of oac status at admission with inhospital outcomes. cox proportional hazards models were created to assess the influence of oac use at admission on longterm mortality following pci. for all outcome models, we adjusted by the propensity score predicting treatment with an oac. the propensity score was based on the following variables : sociodemographic variables (age, sex, ethnicity, and body mass index), comorbidities (tobacco use, diabetes mellitus, hypertension, dyslipidemia, prior mi, prior coronary artery bypass grafting, prior valve surgery, prior pci, family history of coronary artery disease, prior heart failure, chronic lung disease, cerebrovascular disease, peripheral vascular disease, renal failure, and anemia), clinical presentation (pci urgency, acute coronary syndrome, history of angina, history of heart failure symptoms, cardiogenic shock, cardiac arrest, ejection fraction, and need for mechanical support), and procedural characteristics (lesion complexity, lesion location, stent thrombosis, preprocedure thrombolysis in mi flow, postprocedure thrombolysis in mi flow, arterial access site, and multivessel disease). this propensity score was used as a covariate for adjustment and had a c statistic of 0.772. because 31.8% of patients were missing data on ejection fraction, these data were imputed by stratifying the population based on history of heart failure, prior mi, preprocedure cardiogenic shock, and the presence of stsegment elevation mi, as done previously.24 similar analyses were performed to examine the association between noac versus vka use and outcomes, limited to patients who were receiving oacs. these models were adjusted by the propensity score for receiving treatment with a vka, based on the variables listed above, which had a c statistic of 0.783. partners healthcare is an integrated health care system that currently includes 8 massachusetts hospitals, 21 community health centers, and a network of independent ambulatory practices with > 500 affiliated primary care doctors. the pci cohort was derived from the partners longterm outcomes database, an ongoing, institutionally sponsored registry of patients undergoing pci within the partners healthcare system.20 all pcis in which patients presenting to 1 of 2 partners healthcare academic medical centers (brigham and women 's hospital and massachusetts general hospital) between june 2009 and september 2014 were included. if > 1 pci was performed in the same patient during the same hospitalization, only the first pci was included in the analysis. the present project was reviewed and approved with a waiver of informed consent from the institutional review board at partners healthcare. clinical and procedural characteristics analyzed for all pcis were abstracted from institutional registry data, based on the data collection form from the national cardiovascular data registry 's (ncdr) cathpci registry. a full description of the data elements of the cathpci registry are available online (http://cvquality.acc.org/~/media/qii/ncdr/data%20collection%20forms/cathpci%20registry_datacollectionform.ashx).21 to determine chronic anticoagulant status, we developed an algorithm that was applied to the entire electronic medical record, including admission notes, consultation notes, nursing notes, outpatient notes, catheterization reports, pharmacy notes, discharge summaries, and medication prescriptions, to identify the presence of oac use within the 30 days preceding the pci. the search terms included the medications warfarin, coumadin, dabigatran, pradaxa, rivaroxaban, xarelto, apixaban, and eliquis. the algorithm was designed to maximize sensitivity over specificity to provide a screen of the study population for patients taking oac therapy prior to pci. to remove falsepositive records, all electronic medical records of identified oac patients and n.b.) to confirm documented oac use within 30 days of the admission. of patients identified as taking oac therapy, 70.6% exclusion on manual review primarily resulted from the algorithm incorrectly identifying patients for whom phrases reflecting nonuse of oacs were found in unstructured data (eg, patient not on warfarin in a clinic visit note). additional data for oac patients were also manually collected and included : type and dose of anticoagulant at admission, type and dose of antiplatelet at admission, aspirin status and dose at admission, indication for anticoagulant, use and duration of bridging agent prior to pci, type and dose of antiplatelet at discharge, aspirin status and dose at discharge, type and dose of anticoagulant at discharge, discontinuation of anticoagulant after pci, and reason for discontinuation of anticoagulant after pci. the primary outcome measures were inhospital major bleeding, inhospital mortality, and longterm mortality. inhospital major bleeding was based on the ncdr version 4 definition,22 which included any of the following occurring before hospital discharge : arterial accesssite bleeding (defined as external bleeding at the access site or a hematoma > 10 cm for femoral access, > 5 cm for brachial access, or > 2 cm for radial access) ; retroperitoneal, gastrointestinal, or genitourinary bleeding ; intracranial hemorrhage ; cardiac tamponade ; postprocedure hemoglobin decrease of 3 g / dl in patients with a preprocedure hemoglobin level 16 g / dl ; or postprocedure non bypass surgery related blood transfusion for patients with a preprocedure hemoglobin level 8 longterm mortality was assessed through the national death index and subsequent linkage with the institutional registry, as described previously.23 secondary outcome measures included accesssite bleeding (defined as external bleeding at the access site, hematoma at the access site, or retroperitoneal bleeding), non accesssite bleeding (defined as gastrointestinal or genitourinary bleeding, intracranial hemorrhage, or cardiac tamponade), red blood cell transfusion, postprocedure mi (new occurrence of a biomarkerpositive mi after pci), inhospital stent thrombosis (defined as a subsequent pci performed during the same index hospitalization as the related pci for the indication of stent thrombosis), cerebrovascular accident or stroke (defined as loss of neurological function caused by an ischemic or hemorrhagic event with residual symptoms lasting at least 24 hours after onset or leading to death), and postprocedure length of stay in days. in addition, readmissions to hospitals only within the partners healthcare system were identified for all pci patients within 30 and 90 days of discharge. clinical and procedural characteristics with dichotomous and categorical variables were reported as counts and percentages, and continuous variables were reported as means with standard deviations. betweengroup differences were assessed using fisher exact or chisquare tests for binary and categorical variables and student t tests or wilcoxon rank sum tests for continuous variables. meier methods were used to estimate the cumulative incidence of longterm mortality following pci stratified by use of oacs at admission, and logrank tests were used to compare the curves. predicted risks of inhospital bleeding and inhospital mortality were calculated for each group using previously validated risk models developed within the ncdr cathpci registry.22, 24 logistic regression models were created to measure the association of oac status at admission with inhospital outcomes. cox proportional hazards models were created to assess the influence of oac use at admission on longterm mortality following pci. for all outcome models, the propensity score was based on the following variables : sociodemographic variables (age, sex, ethnicity, and body mass index), comorbidities (tobacco use, diabetes mellitus, hypertension, dyslipidemia, prior mi, prior coronary artery bypass grafting, prior valve surgery, prior pci, family history of coronary artery disease, prior heart failure, chronic lung disease, cerebrovascular disease, peripheral vascular disease, renal failure, and anemia), clinical presentation (pci urgency, acute coronary syndrome, history of angina, history of heart failure symptoms, cardiogenic shock, cardiac arrest, ejection fraction, and need for mechanical support), and procedural characteristics (lesion complexity, lesion location, stent thrombosis, preprocedure thrombolysis in mi flow, postprocedure thrombolysis in mi flow, arterial access site, and multivessel disease). this propensity score was used as a covariate for adjustment and had a c statistic of 0.772. because 31.8% of patients were missing data on ejection fraction, these data were imputed by stratifying the population based on history of heart failure, prior mi, preprocedure cardiogenic shock, and the presence of stsegment elevation mi, as done previously.24 similar analyses were performed to examine the association between noac versus vka use and outcomes, limited to patients who were receiving oacs. these models were adjusted by the propensity score for receiving treatment with a vka, based on the variables listed above, which had a c statistic of 0.783. from june 2009 through september 2014, a total of 9566 pcis met the inclusion criteria. of these procedures, 837 (8.8%) were performed in patients with oac use within 30 days of admission. noacs were used in 66 (7.9%) of these pcis, with vkas used in the remainder. during the study period, overall oac use remained stable (8.1% of pcis in 2009, 9.0% in 2014 ; p=0.11 for trend), whereas noacs comprised an increasing proportion of all oac use among pcis (0% in 2009, 17.6% of oacs in 2014 ; p0.05 for all) (table s4). from june 2009 through september 2014, a total of 9566 pcis met the inclusion criteria. of these procedures, 837 (8.8%) were performed in patients with oac use within 30 days of admission. noacs were used in 66 (7.9%) of these pcis, with vkas used in the remainder. during the study period, overall oac use remained stable (8.1% of pcis in 2009, 9.0% in 2014 ; p=0.11 for trend), whereas noacs comprised an increasing proportion of all oac use among pcis (0% in 2009, 17.6% of oacs in 2014 ; p0.05 for all) (table s4). among contemporary patients undergoing pci, 1 in 11 patients was on chronic oac therapy. from 2009 through 2014, there was no significant change in the number of patients treated with oacs undergoing pci ; however, there was an increase in the use of nonvka agents among these patients. in addition, triple therapy after coronary stenting was used with high frequency throughout the study period. patients on chronic oac therapy had greater adjusted risks of inhospital bleeding, need for readmission, and longterm death compared with those not on oac therapy. our analysis found that 9% of all patients undergoing coronary stenting are currently treated with chronic oac therapy, comprising a large proportion of the pci population. atrial arrhythmias were the main reason for treatment, yet pci patients also had a variety of other indications for chronic anticoagulation. consistent with previous analyses in ambulatory patients,14, 15 we noted a brisk increase in use of noacs during the study period, starting at the time of market approval of the first noac in 2010 ; however, we did not observe any major change in the overall number of patients being treated with oacs. this finding contrasts with results from a recent analysis of ambulatory patients with atrial fibrillation on oac therapy that demonstrated a near doubling in patient visits since 2009.14 our results likely differ due to the expanded use of noacs in patients at lower risk of stroke,17 many of whom have fewer comorbidities and thus lower likelihood of undergoing pci. pci patients were discharged on triple therapy (oac plus a p2y12 inhibitor and aspirin) at a rate of 85%, which remained constant during the study period. this preference for triple therapy persisted among treating clinicians despite presentation of the woest (what is the optimal antiplatelet and anticoagulant therapy in patients with oral anticoagulation and coronary stenting) trial results in august 2012. the woest trial randomized stented patients with an indication for oac to treatment with a vka plus clopidogrel with or without aspirin and found no difference in outcomes at 1 year between treatment strategies.25 this study was further supported by similar findings in large registry analyses26, 27 and resulted in the inclusion of restricted oac plus p2y12 inhibitor use following pci in the most recent consensus document for the management of atrial fibrillation patients following coronary stenting.19 nevertheless, it appears from our analysis that these data have yet to influence actual clinician practice patterns within the studied health care system, at least at the time of discharge. we did not assess whether changes in antithrombotic regimens may have occurred later in followup. despite continued efforts among coronary interventionalists to improve treatment practices to reduce postpci bleeding,28, 29 1 in 10 patients on chronic oac therapy had an inhospital major bleeding event following coronary stenting. conversely, chronic oac use prior to pci did not reduce rates of postprocedure ischemic events, similar to a prior study among patients with acute mi.7 bleeding events primarily consisted of non accesssite bleeds, suggesting that current pci vascular bleeding avoidance strategies, such as increased use of radial access29, 30 and arterial closure devices,28, 31 may not alone mitigate the bleeding risk associated with oac use. it is noteworthy that oac patients had greater predicted risk of bleeding regardless of oac treatment, suggesting that the bleeding hazard in these patients is not solely related to use of these agents but also due to a greater proportion of clinical characteristics associated with postpci bleeding. patients taking oacs at the time of pci had decreased survival compared with those not on anticoagulants, with a 36% greater adjusted risk of longterm mortality. although causality can not be established from our analysis, this association may be related to the longterm consequences of increased bleeding events32, 33 or mediated by inherent patient factors, such as a higher prevalence of frailty among those on oac therapy.34 in addition, use of triple therapy at discharge may be associated with risk of decreased longterm survival.25 at minimum, chronic treatment with an oac represents a strong marker of risk for future morbidity and mortality following pci. use of hospital resources was also greater among oactreated patients following pci, including longer durations of stay and more need for transfusions. in addition, the frequency of readmission was marked, with 22% of chronically anticoagulated patients requiring rehospitalization within our health care system within 90 days of pci. of importance, at discharge, the majority of these patients were on triple therapy, which increases the risk of outofhospital adverse events necessitating medical attention, particularly risk of bleeding.26 consequently, it is important to recognize that oac patients have a substantial influence on hospitalrelated costs after coronary stenting. demographics and cardiovascular risk factors were comparable between those receiving noacs and vkas and differ from those of ambulatory patients on noacs, who are often younger and have fewer comorbidities.17 in addition, patients undergoing pci on noacs versus vkas did not vary in procedural characteristics, including sites of access, types of parenteral anticoagulants administered, and use of triple therapy at discharge. although the point estimates comparing short and longterm outcomes following pci between treatment strategies were not statistically different, the study was underpowered to draw definitive conclusions. the results of this analysis must be considered in the context of the study design. the analysis used a computerized method of searching electronic health records to identify patients on oac therapy. although all patients identified as being on oac therapy were manually reviewed to confirm treatment, this analysis could have missed patients whose treatment was poorly documented or who had treatment terms not included in the search algorithm. in addition, data were not available for time in the therapeutic range among patients treated with vkas, including prior to the procedure, and for events occurring after discharge, such as discontinuation of oac therapy or the occurrence of outofhospital bleeding events. as such, rates of events could have been under or overestimated ; however, we expected misclassification of events to be similar between oac and nonoac patients, thus biasing the comparison of the treatment strategies to the null. this study was conducted using pci data from 2 academic tertiary care medical centers from a single integrated health care system and may not be generalizable to other settings. readmission data were available only for patients returning to a hospital within the partners healthcare system. patients were primarily treated with clopidogrel and received pci via femoral arterial access, whereas more potent antiplatelets and greater use of transradial arterial access may have differential effects on outcomes. last, we do not know whether the associations observed between oac treatment and adverse events following pci are causal in nature, and unmeasured factors could confound the relationships observed. patients on oac therapy had greater adjusted risks of inhospital bleeding, need for readmission, and longterm mortality following pci. as such, use of oacs at the time of pci is an important prognostic marker. efforts are needed to specifically reduce the occurrence of adverse events following pci in this population. the work was funded, in part, by a spark award from the corrigan minehan heart center at the massachusetts general hospital. vitamin k antagonist oral anticoagulant versus vitamin k antagonist use table s2. presentation and procedural characteristics of percutaneous coronary interventions by non unadjusted and adjusted risks of outcomes with vitamin k antagonist oral anticoagulant use figure s1. temporal changes in use of antithrombotic agents at admission and discharge among patients on chronic oac therapy undergoing percutaneous coronary intervention during the study period. | backgroundcontemporary rates of oral anticoagulant (oac) therapy and associated outcomes among patients undergoing percutaneous coronary intervention (pci) have been poorly described.methods and resultsusing data from an integrated health care system from 2009 to 2014, we identified patients on oacs within 30 days of pci. outcomes included inhospital bleeding and mortality. of 9566 pcis, 837 patients (8.8%) were on oacs, and of these, 7.9% used non vitamin k antagonist agents. oac use remained stable during the study (8.1% in 2009, 9.0% in 2014 ; p=0.11), whereas use of non vitamin k antagonist agents in those on oacs increased (0% in 2009, 16% in 2014 ; p<0.01). following pci, oactreated patients had higher crude rates of major bleeding (11% versus 6.5% ; p<0.01), accesssite bleeding (2.3% versus 1.3% ; p=0.017), and non accesssite bleeding (8.2% versus 5.2% ; p<0.01) but similar crude rates of inhospital stent thrombosis (0.4% versus 0.3% ; p=0.85), myocardial infarction (2.5% versus 3.0% ; p=0.40), and stroke (0.48% versus 0.52% ; p=0.88). in addition, prior to adjustment, oactreated patients had longer hospitalizations (3.95.5 versus 2.84.6 days ; p<0.01), more transfusions (7.2% versus 4.2% ; p<0.01), and higher 90day readmission rates (22.1% versus 13.1% ; p<0.01). in adjusted models, oac use was associated with increased risks of inhospital bleeding (odds ratio 1.50 ; p<0.01), 90day readmission (odds ratio 1.40 ; p<0.01), and longterm mortality (hazard ratio 1.36 ; p<0.01).conclusionschronic oac therapy is frequent among contemporary patients undergoing pci. after adjustment for potential confounders, oactreated patients experienced greater inhospital bleeding, more readmissions, and decreased longterm survival following pci. efforts are needed to reduce the occurrence of adverse events in this population. |
the various nail signs in psoriasis are the presence of coarse irregular pits, splinter hemorrhages, subungual hyperkeratosis, oil drop sign, onycholysis etc. these changes are detected on gross examination. by then considerable damage to the nail unit has usually occurred and normal functions are hampered affecting the day to day life of the individual. in this study, we used dermoscope as a tool to detect signs of psoriasis in the nails and to find if any signs can help in early detection of nail affection. the study was conducted in the department of dermatology, venereology and leprology at k.e.m hospital, mumbai from september 2013 to november 2014. this study was approved by the institutional ethics committee of k.e.m hospital in the month of august 2013. patients who only had subtle nail findings, not easily discernible by naked eye, were taken. detailed history including the age, sex, duration of the disease and details of any past topical or systemic treatment was noted. examination of the nail was done using a contact dermoscope (heine 's delta 20) and a video dermoscope. oily medium such as liquid paraffin or gel was used for interface in case of a contact dermoscope. the nails were examined by the video dermoscope on a white light, polarized light. the parts of nail that were examined were the nail plate, proximal and lateral nail fold, cuticle and the subungual region. the findings were appreciated well through a hand held dermoscope with an oily medium at the interface or using a video dermoscope with a polarized light. the statistical analysis was done using the fishers exact test with the mid - p method. twenty - two patients did not show any findings [table 1 ]. out of the 46 patients who had findings, the mean age was 38.36 years with a range of 13 to 66 years. mean average duration of the disease is 2.69 years with duration ranging from 2 months to 10 years. frequency of patients showing findings of the various findings, irregular pits were the most common. it was seen in 18 out of 46 patients in the study population [table 2 ] and in 2 persons in the control group (p < 0.0001) [figure 1 ]. onycholysis, most commonly presenting as distal lateral onycholysis, was present in 10 patients and in 1 in the control group (p < 0.001) [figures 2 and 3 ]. an oil drop sign was seen in two patients and none in the control group. frequency of dermoscopy findings coarse irregular pits of various sizes and shapes (40x) onycholysis occurring at the distal lateral border onycholysis appearing as reddish orange discoloration through a video dermoscope (40x) subclinically, the following findings of interest were noted : dilated globose vessels at the onychodermal band were a consistent finding seen in nine patients and were statistically significant. stout, globose, dilated, red to maroon - appearing nail bed vessels arranged longitudinally at the onychodermal band were surrounded by a prominent halo [figures 4 and 5 ]. splinter hemorrhages, manifesting as purplish stain were seen in five patients (p = 0.057) [figure 6 ]. in 16 patients, rather than fusiform dilatation, dilated globose vessels at the onychodermal band (20x) through a contact dermoscope dilated vessels with a prominent halo as seen through a non - contact dermoscope in a polarized light (40x) splinter hemorrhages appearing as purple - colored streaks (40x) the dermoscopic features described in literature for nail psoriasis are pitting, onycholysis, salmon spot, oil spot, splinter hemorrhages and subungual hyperkeratosis. in our patients, the most common findings seen in nails were small irregular pits over the nail plate. they were seen as indentations over the nail plate and are irregular, both in size and shape. the keratinization of the stratum corneum of the proximal nail matrix is disrupted leading to formation of parakeratotic cells. these columns of loose parakeratotic cells fall off leading to deep and coarse pits later. the separation of nail plate from the nail bed allows air to enter inside giving a white appearance. the nail appears whitish in color as compared to the pale pink appearance of the normal nail. there is a reddish orange- to brownish - colored band separating the area of onycholysis with the normal nail. in traumatic onycholysis, the line of detachment of the plate from the bed is regular and smooth, and is surrounded by a normally pale pink bed. nine patients showed bright red to dusky colored dilated vessels arranged parallel over the onychodermal band of the nail plate. they can be appreciated well with either a video dermoscopeor a hand held dermoscope. in our study it was seen to be more common in psoriatic patients as compared to general population. in 2012, piraccini., described dilatation and tortuosity of the nail bed vessels in psoriatic patients which were appreciated by examining the hyponychium. however, it was not appreciated in our study due to the pigmentation of the skin in the indian population. in 2008, iorizzo., showed regular red dots in the hyponychium corresponding to the dilated tortuous vessels in the nail beds of patients with psoriasis. they compared this finding with hyponychium of normal persons and with patients of lichen planus. it was seen that the vessels were less tortuous and reduced in number in patients of lichen planus. moreover, they showed that capillary density is positively correlated with the severity of the condition. this study also showed that quantitative analysis of the capillaries correlates with response to treatment. in this study, following 3 months of topical treatment with calcipotriol, a reduction in the number of capillaries was found in the patients. similar to onycholysis, this area shows a red brown- colored margin separating it from the normal pink nail. in our study, recent splinter hemorrhages were purple in color, whereas long - standing splinter hemorrhages were darker and appeared blackish in color. they were seen as marks that run longitudinally in the direction of nail growth, and are caused by the successive incorporation of blood in the ventral nail plate. in 16 patients, nail bed capillaries were visible through the nail plate as bright red streaks. dermoscopy is a noninvasive, quickly applied and inexpensive test that may aid diagnosis of nail psoriasis in inconclusive cases. subclinical lesions of nail psoriasis can be appreciated well with the help of a dermoscope and appropriate treatment can be instituted. dermoscope is a handy non invasive instrument used in dermatology.use in onychopathies has been established for subungual melanoma.psoriasis of nails can be reliably diagnosed at present by histopathology. | background : onychopathies constitute one of the major challenges faced by a dermatologist in terms of its early detection and diagnosis. utility of dermoscope as a tool for detection is increasing by the day and its use in onychopathies needs to be explored.aims:to study the dermoscopic features of nails in patients of chronic plaque psoriasis.materials and methods : in a cross - sectional study, a total of 68 patients with chronic plaque psoriasis were recruited. dermoscopy of nail plate was conducted and were compared with equal number of age and sex matched healthy volunteers.results:forty-six patients showed dermoscopic findings. twenty - two patients did not show any dermoscopic findings. coarse pits (18/46, p < 0.0001), onycholysis (10/46, p < 0.001), oil drop sign (2/46, p = 0.12) and splinter hemorrhages (5/46, p = 0.05) were seen. in addition certain findings of interest were stout, globose, dilated, pink- to red - colored nail bed vessels arranged longitudinally at the onychodermal band surrounded by a prominent halo (9/46, p = 0.01). in contrast, splinter hemorrhages appeared as streaks and were purple in color.conclusion:in a psoriasis patient, dermoscope can be a useful tool to detect early nail involvement in psoriasis and aid in differentiating it from other disorders of nails. |
progress toward an effective treatment to prevent alzheimer s dementia (ad), or to retard its progression, has proven to be exceedingly elusive. in the last year alone, three promising drugs have failed to show clinical efficacy (bapineuzumab, latrepirdine, and semagacestat). although current strategies directed at reducing or eliminating the production of amyloid plaques, or at eliminating plaques already produced, may finally lead to a solution, the time may have come to examine alternative approaches. in 1982, ball proposed that the herpes simplex type 1 virus (hsv-1) might be involved in the pathogenesis of ad and degenerative lesions of the normal aged human brain.1 ball noted that hsv-1 was well known to establish a lifelong presence in the trigeminal nucleus, and that the trigeminal nucleus has projections into the mesial temporal (limbic) areas of the brain that are affected in ad and in herpes encephalitis. a reactivated virus might therefore have access downstream to manifest as herpes labialis (cold sores), and upstream into the limbic regions of the brain. in recent years, several researchers have made progress in developing what might be called a strong circumstantial case for ball s hypothesis. among these is the laboratory of dr itzhaki who has studied hsv-1 and ad for nearly 20 years. in a recent review article she summarizes some of the findings of her group and others,2 noting that 1) hsv-1 dna has been found in a high (90%) percentage of the brains of elderly people, including the brains of ad patients ; 2) that hsv-1 can persist in an active or inactive state throughout a host subject s lifetime ; 3) that intrathecal antibodies to hsv-1 have been identified in the cerebral spinal fluid (csf) of ad patients and elderly normal controls, indicating that the virus had at some point become active;3 4) that hsv-1 infection is associated with inflammation, and several mediators of inflammation have been identified that are common to both hsv-1 infection and ad;4,5 5) that hsv-1 may contribute directly to the formation of amyloid plaques and neurofibrillary tangles, the histologic hallmarks of ad;69 6) that hsv-1 infection leads to accumulation of cholesterol in infected cells, which has been found to be associated with the formation of amyloid;10,11 7) that acute hsv- 1 cerebral infection (encephalitis) affects the temporal and frontal cortices of the brain, but not the occipital lobes, which are many of the same areas affected in ad;12 8) that the long term sequelae of hsv-1 encephalitis include memory loss,12 a clinical hallmark of ad ; and 9) that hsv-1 confers a high risk of ad in patients who carry the apoe-4 allele, a well - established genetic risk factor for ad.13 interestingly, apoe-4 is also a risk factor for development of herpes labialis, a peripheral manifestation of hsv-1 reactivation.14 these and other findings demonstrate that hsv-1 is present in relevant cerebral regions and capable of causing the amyloid plaques, neurofibrillary tangles, inflammatory changes, and memory loss characteristic of ad. although they do not constitute proof of causation, they are consistent with the hypothesis that hsv-1 may be responsible for at least some cases of ad. however, if the virus is present in such a high percentage of the general population, the question must arise, why is ad primarily a disease of the elderly ? why do many elderly develop the disease, but a majority do not ? or to put it another way, what are the changes that occur in later age in some people that are conducive to the reactivation of the virus and the subsequent development of ad ? two factors will be considered in the text below : the lysine to arginine ratio in the brain, and activity of the immune system. it has been known since 1968 that hsv-1 requires arginine for replication,15 and that lysine inhibits hsv-1 replication by competing with arginine.16 these findings led to the use of lysine as a treatment for the common condition known as herpes labialis which, as noted above, is known to be caused by hsv-1. seven randomized, double - blind, placebo - controlled studies have examined the effectiveness of lysine in preventing outbreaks of herpes labialis and reducing the severity of outbreaks that do occur. six of these studies found lysine to be effective in preventing or decreasing outbreaks, and only two found that lysine reduced the severity of out - breaks. the first study, conducted in 1978, employed a dosage of 500 mg / day of lysine and found it to be ineffective.17 a 1984 study specified 1,000 mg of lysine, given once a day, and also measured serum lysine levels. lysine was found to be effective in reducing outbreaks when serum lysine concentration was greater than 165 nmol / ml, but not when it was less than this.18 another study, also published in 1984, found that a dosage of 1,248 mg / day was effective in reducing outbreak frequency, but that 624 mg / day was ineffective.19 a 1987 study studied lysine 1,000 mg three times a day, and found it to be effective in reducing both the frequency and severity of attacks.20 it seems beyond question, then, that lysine in sufficient concentrations relative to arginine suppresses reactivation of hsv-1 in vivo, at least in so far as it is manifested peripherally as herpes labialis. it has been shown to be effective in reducing frequency of herpes labialis attacks, and possibly the severity of attacks, when given in dosages of at least 1,500 mg / day in divided doses. lysine is an essential amino acid, and the most highly conserved of all amino acids. brain barrier by a basic amino acid carrier, and competes with arginine for transport. there are very few reports about this transporter, although there is evidence that the cat-1 transporter is responsible for the movement of lysine and arginine across the blood brain barrier.21 from the point of view of the current discussion, this is a pivotal point : the relative concentrations of lysine and arginine will depend on their transport across the blood brain barrier, as well as their rates of efflux from the central nervous system. lysine, unlike arginine, as an essential amino acid, can not be made within the central nervous system. if lysine is not consumed in the diet in adequate quantities relative to arginine, or if it is not transported across the blood brain barrier in adequate quantities relative to arginine, conditions may evolve in the central nervous system that are favorable for the reactivation of hsv-1. it is known, however, that csf levels of amino acids are not constant, and may change with disease states. for example, csf amino acid levels, including lysine and arginine, vary in patients with parkinson s disease relative to normal controls.22 it is known that older age is associated with dietary changes, and these may reduce the amount of lysine available to be transported. many factors have been proposed to explain this shift, but in general elders consume fewer calories with advancing age, and dietary choices move away from protein and energy dense foods such as meats, and towards foods that are less protein and energy dense, such as grains.23 this is a change, in general, from foods that have a favorable lysine to arginine ratio to foods that do not. as a result of diminished total caloric intake, and a change to foods with less lysine relative to arginine, it is likely that over time an environment more favorable to reactivation of hsv-1 evolves. there are, then, at least two factors in older age that might shift the lysine to arginine ratio in favor of arginine, and thereby favor the reactivation of hsv-1 : first, the activity of the basic amino acid transporter and second, the quantity of lysine acquired through the diet. since 2005 evidence has been accumulating that points to cd8 + t cells in maintaining hsv-1 latency.24,25 immunosenescence, or the decline of the immune system with age, appears to be related to involution of the thymus over time, with subsequent imbalance in t cell subtypes. there are no published reports specifically addressing the question of immunosenescence and hsv-1 reactivation in humans, although interestingly it has been reported that in mice the immune response to herpes simplex encephalitis is modulated by vitamin e.26 vitamin e, of course, has long been the object of scientific interest in the search for a means to retard the progression of ad. at present there is little that can be done to prevent or slow the process of immunosenescence. however, this does not necessarily apply to the lysine to arginine ratio in the csf. the question arises, given that lysine is effective in suppressing hsv-1 activation as manifested by herpes labialis, would it also suppress hsv-1 activation in the temporal and frontal cortices leading to ad ? there are no data describing the prevalence of ad in older people who have taken at least 1,500 mg / day of lysine continuously over a period of years. however, given that it is known that diets rich in lysine and poor in arginine suppress hsv-1 replication, it would be instructive to know whether such diets are associated with lower prevalences of ad. it emphasizes grains, but also emphasizes fruits, vegetables, cheese, yoghurt, and fish, all foods high in lysine and low in arginine.27 perhaps more than any other food, weekly consumption of fish is associated with a lower risk of ad.28 this is generally attributed to omega-3 fatty acids in fish ; however, it is also true that fish have a high lysine to arginine ratio. a study published in 2001 described what may be the lowest incidence rates of ad ever found, in the northern indian rural community of ballabgarh : 4.7 per 1,000 person - years, compared with 17.5 per 1,000 person - years in the monongahela valley, pennsylvania.29 the authors do not speculate on why this may be so. the residents of ballabgarh are farmers, remain physically active into older age, and obesity is essentially unknown. they also eat a diet proportionally high in dairy products, which have a very high lysine to arginine ratio. conversely, unlike fish and dairy products, tofu has a high arginine to lysine ratio. it was recently reported that high tofu intake among elderly sudanese and javanese, a population much like that of ballabgarh, was unexpectedly associated with an increased risk of cognitive impairment.30 these observations are consistent with the hypothesis that if hsv-1 causes some cases of ad, a diet high in lysine relative to arginine may help prevent this from occurring by preventing reactivation of the latent virus in vulnerable regions of the brain. to summarize, ad is a disease process, not a natural result of the aging process ; hsv-1 is present in 70%90% of the brains of older adults ; hsv-1 has been identified in the tissue of patients with ad at autopsy ; acute hsv-1 encephalitis affects many of the same regions of the brain that are affected by ad, and leads to long term memory loss ; hsv-1 is known to reside in a latent form in the trigeminal nucleus, which projects to areas of the brain known to be affected by ad ; there are indicators of an inflammatory process associated with ad, suggesting the possibility of an infectious etiology, possibly as a result of immunosenescence ; activated hsv-1 virus is associated with formation of plaques and tangles, which are histologic hallmarks of ad ; it is also associated with elevated cholesterol levels, also considered a risk factor for ad. finally, there are indications that diets high in lysine and low in arginine may be associated with lower prevalences of ad. this leads to the following hypothesis : hsv-1, latent in brain, becomes activated when in older age the ratio of lysine to arginine in the csf favors arginine, providing a medium conducive to viral reactivation, and the process of immunosenescence releases the virus from immune system surveillance. active hsv-1 then in turn causes ad. this process may be prevented or attenuated by increasing lysine, either in the diet, or as a supplement, or both. studies of lysine treatment of herpes labialis suggest that supplements of 1,500 mg twice a day or more are effective for this purpose. however, as noted above, the time may have come for thinking outside of the box in our approach to this terrible disease. the hypothesis as described above is highly testable, although it would require a very large study over many years to reach a definitive conclusion. some would argue that it would be preferable to test the efficacy of the antivirals, rather than lysine supplementation. it can not be argued, however, that the risk to patients posed by treatment with lysine is negligible, and the potential benefit enormous : a safe, inexpensive approach to the prevention or attenuation of ad. | there is a growing body of evidence that implicates the herpes simplex type 1 virus (hsv-1) in the development of alzheimer s dementia (ad). hsv-1 has been found to be present in the cerebrum of the great majority of older adults, and in many of the same areas of the brain that are affected by ad. when active, the virus may contribute to the formation of the neuro - fibrillary tangles and amyloid plaques characteristic of ad. like ad, hsv-1 encephalitis may cause long term memory loss. hsv-1 replication is suppressed in lysine - rich / arginine poor environments, and population studies suggest that diets high in lysine and low in arginine may be associated with lower rates of ad. there are no prospective studies of the efficacy of lysine supplementation to prevent or reduce the incidence of ad. supplementation with adequate doses of lysine could prevent the development of ad. |
in the united states, stroke is the number one cause of chronic disability and the fourth leading cause of death, with approximately 7 million adults affected. annually there are approximately 800,000 strokes in the us, of which 87% are ischemic strokes, 10% are primary hemorrhages, and 3% are subarachnoid hemorrhages. cerebral ischemic stroke is caused by an occlusion of a cerebral blood vessel, typically by a thrombus, which causes a decrease in cerebral blood flow and thus limits the supply of oxygen and nutrients globally (in global ischemia) or to certain regions of the brain (in focal brain ischemia). this absence of blood flow in a brain region causes neuronal death in addition to damaging the vascular tree ; the vascular tree is usually made more fragile during the ischemic period and damaged during reperfusion. time is an important parameter in the evolution of brain injury. in 2006, saver. have estimated the impact of stroke on the brain tissue to be immense ; the brain may lose up to 120 million neurons, 830 billion synapses and 714 km of myelinated fibers for each hour after stroke onset. ischemic stroke seems to accelerate aging of the brain at a rate of 3.6 years each time when the symptoms are not treated. therefore, the clinical goal of acute stroke treatment is to reduce brain damage by limiting the time of ischemia through thrombectomy (mechanical endovascular approach) or thrombolytic therapy, which consists of in lysing the blood clot in order to restore cerebral blood flow. recombinant tissue plasminogen activator (rtpa) is currently the only thrombolytic molecule administered during acute cerebral infarction that provides a clinical benefit in terms of survival and neurological outcome. the rtpa administration must be within the first 4 hours 30 minutes after stroke onset to maintain the beneficial effects without substantially raising the side effects / risk [5, 6 ], which limits its use. based on the organization of emergency care, only 5% of stroke patients are eligible for this therapy in this narrow time window, which leaves the remaining 95% of patients without any beneficial treatment available. the major risk of rtpa is the extension of the damage due to potential bleeding. the need for drug development to prevent the neuronal loss has driven research on neuroprotective agents that aim to save viable neurons located in the ischemic penumbra area. however, all of the proposed neuroprotective treatments specifically targeting neurons that showed promise on the bench have failed in clinical trials. in 2000, the neurovascular unit (nvu) was proposed as a physiological unit composed by neurons, astrocytes, and endothelial cells ; there is a growing interest in studying the changes of the nvu after stroke. in addition to cell death, ischemic stroke is characterized by changes in the properties of the blood - brain barrier (bbb) with physical disruption of the tight junctions contributing to aggravation of cerebral edema and consequently neuronal death. the new strategy for drug development is to have molecules with a broader spectrum targeting not just the neurons but the nvu as a whole entity. in the present paper, we will focus on some molecular and cellular mechanisms of astrocytes and endothelial cells. we will look specifically at : (1) the ways astrocytes and endothelial cells work in concert in stroke pathophysiology such as bbb disruption and edema formation, (2) how they could be affected after rtpa treatment, and (3) new drug developments in the future. several groups have proposed the nvu as a physiological unit composed of not only endothelial cells, astrocytes, and neurons but also pericytes, smooth muscle cells, and the interacting circulating peripheral immune cells [1012 ]. the term gliovascular emphasizes the importance of the interactions between astrocytes and cerebral blood vessels within the nvu, which are critical in cerebral blood flow regulation, brain energy metabolism, and also the maintenance of the bbb properties. the bbb is located in the endothelial cells of brain vessels, with the presence of tight junctions and adherens junctions between the cells (figure 1) that prevent paracellular diffusion and act as a unit to regulate ions and other molecules between peripheral blood flow and brain parenchyma. tight junctions are composed of several protein families : trans - membrane proteins (claudins and occludins), cytoplasmic proteins, and zona occludens proteins. adherens junctions are formed by proteins such as platelet - endothelial cell adhesion molecule (pecam) and vascular endothelial - cadherin, which contribute to the close physical contact between endothelial cells and facilitate the formation of tight junctions. the brain endothelial cells of the bbb also present specific transport proteins located on the luminal and abluminal membranes for nutrients, ions, and toxins to cross the endothelial layer between the blood stream and brain [13, 16 ]. for example, energy molecules are transported by specific solute carriers such as glucose transporter 1 (glut 1) and monocarboxylate transporters 1 and 2 (mct1, mct2). large molecular weight solutes (e.g., large proteins and peptides) are able to cross the bbb and enter the intact cns via endocytotic mechanisms called receptor - mediated transcytosis, such as with insulin, or adsorptive - mediated transcytosis, exemplified by albumin. on the other hand, transport can also be achieved by the atp - binding protein (abc) family, which consumes atp to effectively transport a wide range of lipid - soluble compounds from the brain endothelium. in the bbb examples of abc transporters for efflux transport are p - glycoprotein (p - gp), multidrug resistance - associated protein (mrp), and breast cancer resistance protein (bcrp). these efflux transporters are understood as gatekeepers of the brain because they keep tight control over which substances are allowed to enter the cns through the endothelial cell barrier (figure 1). endothelial cells also present a metabolic barrier of the bbb, which functions to inactivate molecules capable of penetrating cerebral endothelial cells. quite recently it has been proposed that the primary barrier of the bbb may extend to the basal lamina, thus preventing the entry of immune cells into the parenchyma under normal brain conditions. historically the brain was thought to be an immune cell deficient organ, and the bbb was thought to prevent passage of any immune cells into the brain. however, peripheral immune cells from the blood have been observed to enter and be present in the brain at multiple time points during embryonic development and in normal physiological conditions in adults. therefore, the theory of the cns as an immune - independent organ has recently started to be reexamined and revised. engelhardt and collaborators elegantly compare the perivascular space as a castle moat with perivascular antigen presenting cells floating as guards, confined by the inner and outer wall, which is the basement membrane of the astrocytic endfeet and the endothelial cell, respectively. endothelial cells and other cells, such as the astrocytes, may also contribute to the tight regulation of the movement of immune cells between the peripheral blood stream and the brain. however, the exact mechanisms by which peripheral cells enter the brain are still a matter of discussion. moreover, rather than the bbb being a rigid wall, it provides a dynamic interface between the brain and the rest of the body. as mentioned previously, the presence and the maintenance of these barrier properties are important for brain homeostasis and for neuronal functioning. in fact, disruption of tight junctions leads to bbb disruption and extravasation of blood components and water, which contribute to vasogenic edema formation. cerebral edema has been traditionally divided into 2 major classes : cytotoxic and vasogenic for cerebrovascular diseases and other brain pathologies. cytotoxic edema is defined by intracellular accumulation of water coming from the extracellular space without bbb disruption. vasogenic edema appears after bbb disruption, leading to a diffusion of proteins from the blood to the tissue followed by water accumulation in the extracellular space. however, this division alone does not explain fully the diversity and the complexity of the edema process in brain ischemia as well as in the other brain injuries and disorders. based on several recent advances in the understanding of the molecular mechanisms of edema formation and bbb properties, a third subtype of edematous processes was named ionic edema and described as a continuum between the cytotoxic to vasogenic edema in the cerebrovascular diseases [19, 20 ]. in fact, cytotoxic, or anoxic, edema occurs within the first few minutes after cerebral blood flow stoppage and is characterized as swelling of the astrocytes and neuronal dendrites [20, 21 ]. the cellular swelling within the first 10 minutes is a result of oxygen and glucose deprivation followed by a slow rise in extracellular [k ]. the absence of oxygen and energy nutrients induces a disruption of the cellular ionic gradients and leads to entry of ions into cells. cytotoxic / anoxic edema may evolve quickly to become ionic edema because the absence of oxygen and nutrients further alters the energy balance in endothelial cells and the ionic gradients, including transcapillary flux of na in these cells [19, 23 ]. the endothelial cells also require a large amount of atp production, characterized by the high density of mitochondria, which are important for the regular homeostatic bbb functions such as maintenance of ionic gradients and membrane transporters [24, 25 ]. reperfusion induces overpressure accompanied by shear stress on the nonperfused vascular tree that results in early transient leakage of the bbb [26, 27 ]. this leakage results in further entry of water through the endothelial cells resulting in brain swelling within 30 minutes after reperfusion [26, 27 ] and additional bbb permeability [27, 28 ]. this early opening of the bbb has also been described clinically in humans and is frequently associated with hemorrhagic transformation. early reperfusion probably mitigates the bbb alterations, but if it is delayed, reperfusion will exacerbate the amount of endothelial injury [3032 ]. the final step is the development of vasogenic edema, in which there is disruption of cerebrovascular endothelial tight junctions leading to increased permeability to albumin and other plasma proteins. another contributing factor of brain edema formation in addition to tight junction disruption is brain endothelial transcytosis. bbb disruption is usually coupled with the inflammatory response and activation of matrix metalloproteinases (mmp) [34, 35 ]. in fact, vasogenic edema development is aggravated by mmp-9, which degrades basal lamina, the connection between astrocytic endfeet and endothelial cells. in the clinic, diffusion - weighted imaging (dwi) and t2-weighted imaging (t2wi) magnetic resonance imaging (mri) modalities t2 values represent water content and apparent diffusion coefficient (adc) values derived from dwi images represent water mobility in the tissue [20, 37 ]. adc values decrease rapidly after stroke onset, indicating restricting water movement, and are interpreted as evidence of ionic edema with the characteristic swelling of the brain cells causing a decrease in extracellular space as proposed in our classification mentioned before. t2 values increase at later time points, which are associated with vasogenic edema [20, 39 ]. however, the cellular and molecular mechanisms involved in edema resolution are not well understood in stroke and other brain diseases. the healing of the endothelial cells with stabilization of the tight junctions may be a critical step to limit the entry of blood components into the brain. thus, stabilizing the nvu may be an essential component of controlling edema formation and bbb breakdown after stroke. postischemic bbb disruption has been commonly believed to be biphasic, but recent work suggests that the bbb disruption may be continuous for up to 5 weeks after ischemia in rats. bbb leakage was demonstrated using gadolinium and magnetic resonance imaging (mri) at 25 min ; 2, 4, 6, 12, 18, 24, 36, 48, and 72 hours ; and 1, 2, 3, 4, and 5 weeks after ischemia. similarly, albumin leakage through the bbb, especially in the hippocampus, has also been observed in spontaneously hypertensive stroke prone rats long term. although these data do not completely rule out the possibility of a biphasic pattern in the opening of the bbb, the long - term leakage of the bbb is important to note from the standpoint of postischemic edema because this disruption could account for a prolonged vasogenic edema. as part of the nvu the astrocyte endfeet in contact to the blood vessels the recent knowledge on the transporters and channels in this astrocyte subdomain gives new perspectives on the understanding of astrocyte swelling. in fact, aquaporin 4 (aqp4), a member of the family of 13 water channel proteins, is proposed to have an important role in edema formation [20, 45 ]. aqp4 is the most abundant water channel in the brain, in part due to its high concentration on astrocytic endfeet which are in contact with all the cerebral blood vessels [46, 47 ]. more recently, aqp1 has also been described in a subpopulation of astrocytes within the nonhuman primate but not in rodents, suggesting interspecies differences and a possible role in brain water homeostasis. aqp1 has also been reported to be present in peripheral endothelia and primary rat brain endothelial cell cultures. interestingly, dolman and collaborators observed that mrna aqp1 levels were lower in cultured brain endothelial cells when cocultured with astrocytes, suggesting an inhibition effect of the astrocytes on the aqp expression in endothelia. in fact, there are publications reporting a low level of aqp in endothelial cells in vivo, although aqp is more abundant in astrocytes [49, 5154 ]. currently, aqp4 is considered as a key player in the edema process by its location on the astrocyte endfeet [20, 55 ]. interestingly, aqp4 is also organized in the astrocyte endfeet membrane in a larger geometric structure known as an orthogonal array of particles (oaps), which has been described with freeze - fracture techniques and electron microscopy studies (figure 2). oaps are present in all astrocyte endfeet in contact with the blood vessels as well as the glia limitans. oaps are formed with two isoforms of aqp4 : long (aqp4-m1) and short splice variants (aqp4-m23). the ratio of aqp4-m1 to aqp4-m23 determines the size of these oaps in contact with the basal lamina of brain vessels (figure 1). experiments in oocytes showed that the aqp4-m23 isoform stabilizes the oap structure [57, 58 ]. however, the exact functional roles of the oaps remain unknown in normal and pathological conditions. recently, aqp4-m1 mrna and protein were found to increase quickly after stroke onset, while aqp4-m23 remained the same. the increase of aqp4-m1 early after ischemia could favor a shift toward m1 in the m1/m23 balance, which is known to favor small size oaps. in accordance with this work, previous studies have shown that early disorganization of oaps on the astrocyte endfeet after global cerebral ischemia preceded astrocyte swelling. although a direct effect of the modification in the ratio of aqp4-m1 to aqp4-m23 on water permeability has not yet been directly investigated in vivo, in a preconditioning model, a strong increase in aqp4 expression and increase of aqp4-m1 were correlated with reduced edema and less water in the tissue, suggesting increased water diffusibility which resulted in the removal of excess liquid from the brain tissue. interestingly, it was recently proposed that the assemblage of 4 aquaporin molecules forms a central pore, through which water, ions, and gases may flow depending on the aqp subtype. for example, the central pore is permeable for o2, co2, and possibly nitric oxide for aqp1, 4, and 5 [56, 60 ]. thus, the disruption of the oaps may also affect the diffusion of ions and gas through the central pore. due to its location in the astrocyte endfeet in contact with the blood vessels, aqp4 has been proposed to be linked with bbb integrity [52, 61, 62 ] and cell adhesion. in the epithelial cells of the eye lens aqp0 is present in the oaps and participates in epithelial cells linkage ; however it does not facilitate water flux. in this case, the presence of aqp4 in the astrocyte endfeet membrane was dependent on the presence of proteins in the basal lamina such as agrin, -dystroglycan, and laminin [65, 66 ] in addition to syntrophin and dystrophin protein complexes [67, 68 ]. the connection of aqp4 to proteins in the basal lamina may explain the ability of astrocytes to maintain the integrity of the blood - brain barrier, suggesting a possible role for aqp4 as a structural molecule within the perivascular space. however, reports using aqp4 knock out (aqp4-ko) mice show contradicting results regarding the modifications in the bbb structure suggesting that aqp4 may not be integral to the bbb structure [61, 69 ]. similarly in our sirna silencing studies, bbb permeability was not significantly changed at distance from the site of injection after injection of sirna against aqp4, even though aqp4 expression was decreased. we also showed that the upregulation of aqp4 in a preconditioning model did not prevent the early opening of the bbb after stroke. heparan sulfate proteoglycan is a large family of proteins with agrin and perlecan, involved in the basal lamina composition located between the astrocyte endfeet and endothelial cells [54, 70 ]. agrin and dystroglycan seem to play an integral role in the maintenance of astrocyte polarity by the interaction with aqp4 in the astrocyte endfeet. specifically, agrin ko mice showed a significantly decreased density of oap in the astrocyte endfeet when compared to wildype but overall immunoreactivity of aqp4 did not differ significantly. dysfunctions in the basal lamina are related to increase of the bbb disruption, promoting edema formation. in fact, a family of endopeptidases, matrix metalloproteinases (mmps), has been shown to degrade the proteins of the basal lamina and contribute to vasogenic cerebral edema. in the human brain, mmps are usually very low in concentration under nonpathological conditions. however, after injuries such as ischemic stroke, certain mmps such as mmp-2, -3, and -7 and especially mmp-9 have been shown to be upregulated in the brain (reviewed in). this layer between astrocytes and endothelial cells is a potential future target for the nvu protection. recently, dr. bix and collaborators have shown that administration of perlecan domain v, which is the c - terminal fragment, administered 24 hours after ischemic stroke has beneficial effects by interacting with integrins. perlecan domain v increased expression of vascular endothelial growth factor (vegf), thus promoting angiogenesis, and interestingly did not lead to increased bbb permeability even though vegf is known to increase bbb permeability after ischemia. perlecan has also been shown to modulate postischemic astrogliosis through interaction with dystroglycans and integrins in the astrocytes. astrocytic aqp4 is not only linked with the matrix proteins but also with several other channels present in higher concentration in the astrocyte endfeet such as potassium inner rectifying channel 4.1 (kir4.1), connexins (cx), and also chloride channel 2 (cic-2) [76, 77 ]. colocalization of aqp4 and kir4.1 suggests that aqp4 may have a role in potassium homeostasis by facilitating water diffusion along the potassium gradient and aqp4-ko mice display a delay in potassium reuptake during electrical activity. the decrease of aqp4 expression using sirna showed an associative decrease of connexin 43 (cx43), a protein involved in gap junction formation, and a decrease of cic-2, involved in the regulatory volume decrease function of the astrocytes. interestingly, gap junctions and aqp4 are morphologically closely associated with the astrocyte endfeet. the gap junctions in the astrocyte contribute to the formation of a complex network named the astroglial network. intercellular and intracellular communication that facilitate the movement of second messengers, amino acids, nucleotides, energy metabolites, and small peptides [7982 ] in astrocyte processes occur through gap junctions, which are made up of a family of channel proteins called connexins [83, 84 ]. in astrocytes, however, it is also important to note that cx43, along with cx37, cx40 [86, 87 ], and cx45, is also expressed in brain endothelial cells. the protein level of cx40 and cx45 was shown to increase in cerebral arteries, but no change in protein or mrna was observed for brain endothelial cx43 and cx37 after a model of brain injury causing cerebral vascular dysfunction. the effect of astrocytic cx43 upregulation or downregulation after ischemia still remains controversial and there is no consensus as to what provides beneficial effects. however, in humans, there are reports that show that cx43 protein levels were increased in the penumbra. and because cx43 and cx30 knockouts have been observed to be more edema prone, it is possible that the increase in cx43 after ischemia may be a physiological response to decrease edema. the induction of cx43 may be facilitating water flow throughout the astrocyte network to diversify and dissipate the accumulation of fluid from just one region. from these data we hypothesize that gap junction proteins, specifically cx43 on astrocytes, are working with aqp4. evidence for this also comes from a significant decrease of cx43 observed in mouse astrocyte cell cultures after administration of small interference rna against aqp4. although direct functional data are still lacking, one possibility is that aqp4 and cx43 is working together to direct water flow between astrocytes and could be controlling astrocytic swelling. the role of aqp4 in cerebral edema formation and resolution has been studied in several models. however the precise role of aqp4 remains unclear and depends on the pathological model used [92, 93 ]. indeed, the absence of aqp4 was shown to prevent the formation of edema in a permanent ischemia model in aqp4-ko mice. similarly, edema formation is prevented in -syntrophin knockout mice at 24 h after stroke. this decrease of brain swelling was correlated with the loss of the perivascular aqp4 domain in -syntrophin - ko mice.. however, the absence of aqp4 in aqp4-ko mice also prevents water clearance in an experiment of intrastriatal infusion of a saline solution, showing that aqp4 is critical for water removal from tissue. conversely, in a preconditioning stroke model, a higher induction of aqp4 was correlated with edema reduction. however, this reduction of edema may be referring to vasogenic edema, in which case, aqp4 is said to aid in edema resolution by actively pumping out water from the cerebral tissue to peripheral blood. the redistribution of the water in the astrocyte compartment through the astrocyte network would also be possible for the csf compartments. this hypothesis is supported by a publication showing an increase of aqp4 in ependymal cells in the border of the ventricles in a traumatic brain injury model. to summarize, the exact mechanism causing decreased edema formation is not yet fully understood, but aqp4 and the astrocyte network with the gap - junction proteins may certainly be contributing. osmotic gradients can also play an important role, and recently, high aqp4 expression was observed in hypersaline treatment after stroke correlating with decreased edema formation at 48 hours. as discussed in the introduction, recombinant tissue plasminogen activator (rtpa) is currently the only thrombolytic molecule fda approved for treatment of acute ischemic stroke. the dysfunction of the bbb after ischemia could cause problems for the therapeutic function of rtpa. this protease targets fibrin - bound plasminogens and converts them into plasmins, which then cut the fibrin clot and lyse it. intravenously infused at a dose of 0.9 mg / kg over one hour, rtpa provides increased survival and better neurological outcomes. to be beneficial for the patient, rtpa must be administered within the first 4 h 30 min after stroke onset [5, 6 ]. despite the organization of emergency care, in fact, late administration of rtpa translated to a higher risk of bleeding and extension of the lesion. higher doses of rtpa do not bind only the fibrin clot but also activate the circulating plasminogen activator (tpa). this activation contributes to a generalized fibrinolysis and fibrinogenolysis, which is suspected to be a cause of bleeding. but the mechanisms of the hemorrhagic transformation after rtpa treatment seem to be more complex than can be accounted for by the affinity of rtpa for fibrin alone. in fact, the enhanced fibrin specificity of tenecteplase and reteplase, two rtpa derivatives, resulted in no significant difference in terms of cerebral hemorrhage [98, 99 ]. interestingly, the comparison with myocardial infarction shows a low incidence of cerebral hemorrhage after rtpa administration suggesting a direct link between bleeding and the ischemic pathophysiology. clinical studies showed that 80% of bleeding after cerebral thrombolysis occur preferentially in the ischemic territory. to have a better understanding of the aversive effects of rtpa it is well known that endogenous tpa is present in the blood stream, endothelial cells, neurons, and microglial cells. in the brain parenchyma, tpa activity was found to be pleiotropic and associated with synaptic plasticity and cell death [102104 ]. in fact, tpa interacts with several neuronal proteins such as n - methyl - d - aspartate (nmda) receptors, one subtype of glutamatergic receptors, low - density lipoprotein - receptor - related protein (lrp), and annexin - ii [101, 105, 106 ]. tpa is synthesized in neurons, stored in presynaptic vesicles, and released following depolarization in synergy with the neurotransmitters. in the synaptic cleft, tpa binds and cleaves the nr1 subunit of nmda receptors that causes an amplification of calcium influx in postsynaptic neurons and an increase of the glutamatergic response in physiological conditions. however, this physiological response becomes excitotoxic after ischemia and is magnified after rtpa injection [101, 107, 108 ]. the injection of antibodies against the nr1-subunit prevented these proexcitotoxic effects of endogenous tpa and reduced brain infarction and bbb leakage after stroke. these data suggest that the nmda receptor may be a protective drug target for the nvu after stroke and may provide a potential extension of the rtpa therapeutic window. the presence of rtpa in the brain parenchyma has been explained by its passage through the bbb in several in vitro models with different proposed mechanisms.rtpa diffuses into the brain parenchyma through an already opened bbb as a consequence of the ischemic process. as we discussed previously, the kinetics of the bbb opening is complex in the early stages after stroke and it is difficult to observe this with clinical imaging. interestingly, in vitro endothelial monolayer cultured with astrocytes enables us to observe the ability of rtpa to cross the intact bbb, which is increased under oxygen - glucose deprivation (ogd). therefore, as rtpa potentially diffuses through an open or closed bbb in early time points after stroke onset, it may aggravate neuronal cell death as described previously.rtpa could cross the bbb by degrading the endothelium via its own proteolytic activity, but it is not a requirement in the intact bbb. the ability of rtpa to cross the intact bbb at a thrombolytic dose suggests that this protease may interact first with the endothelial cells before the bbb breakdown. in fact, rtpa promotes breakdown of the bbb by stimulating the synthesis activity of mmp-9 [113116 ] and other mmp isoforms exacerbating the degradation of the basal lamina and subsequent vasogenic edema formation and hemorrhage. the thrombolytic products could exacerbate the proposed mechanism.finally, lrp potentially contributes in trans - endothelial transport of the exogenous rtpa [106, 119, 120 ] and then activates the astrocytic mmp-9 and nuclear factor nf-b, which promotes the expression of inducible nitric oxide synthase (inos). rtpa diffuses into the brain parenchyma through an already opened bbb as a consequence of the ischemic process. as we discussed previously, the kinetics of the bbb opening is complex in the early stages after stroke and it is difficult to observe this with clinical imaging. interestingly, in vitro endothelial monolayer cultured with astrocytes enables us to observe the ability of rtpa to cross the intact bbb, which is increased under oxygen - glucose deprivation (ogd). therefore, as rtpa potentially diffuses through an open or closed bbb in early time points after stroke onset, it may aggravate neuronal cell death as described previously. rtpa could cross the bbb by degrading the endothelium via its own proteolytic activity, but it is not a requirement in the intact bbb. the ability of rtpa to cross the intact bbb at a thrombolytic dose suggests that this protease may interact first with the endothelial cells before the bbb breakdown. in fact, rtpa promotes breakdown of the bbb by stimulating the synthesis activity of mmp-9 [113116 ] and other mmp isoforms exacerbating the degradation of the basal lamina and subsequent vasogenic edema formation and hemorrhage. finally, lrp potentially contributes in trans - endothelial transport of the exogenous rtpa [106, 119, 120 ] and then activates the astrocytic mmp-9 and nuclear factor nf-b, which promotes the expression of inducible nitric oxide synthase (inos). together, yepes and collaborators have proposed the following potential cellular and molecular events to explain the toxicity of the rtpa and tpa on the nvu.circulating endogenous tpa and rtpa cross the bbb (intact or damaged endothelial layer) and increase mmp-9 activity in the basal lamina soon after stroke onset which compromises the nvu integrity and makes it fragile. then tpa and rtpa bind to the astrocytic lrp, inducing the loss of the extracellular domain of lrp [122, 123 ] in the basal lamina, and release the intracellular domain of lrp in the astrocytic cytoplasm to activate nf-b. this nf-b activation increases inos and mmp9 expression and overall function in the whole nvu, causing separation of astrocytic endfeet from the basal lamina. however, it is tempting to speculate that this cascade, which involves the perivascular cells of the nvu, would be an accelerated pathological process resulting from the use of rtpa. it is possible that rtpa and tpa may also affect the phenotype of the astrocyte endfeet by changes in the level of expression of key proteins such as aqp4 and also cx43. circulating endogenous tpa and rtpa cross the bbb (intact or damaged endothelial layer) and increase mmp-9 activity in the basal lamina soon after stroke onset which compromises the nvu integrity and makes it fragile. then tpa and rtpa bind to the astrocytic lrp, inducing the loss of the extracellular domain of lrp [122, 123 ] in the basal lamina, and release the intracellular domain of lrp in the astrocytic cytoplasm to activate nf-b. this nf-b activation increases inos and mmp9 expression and overall function in the whole nvu, causing separation of astrocytic endfeet from the basal lamina. however, it is tempting to speculate that this cascade, which involves the perivascular cells of the nvu, would be an accelerated pathological process resulting from the use of rtpa. it is possible that rtpa and tpa may also affect the phenotype of the astrocyte endfeet by changes in the level of expression of key proteins such as aqp4 and also cx43. the bbb is definitely not a barrier to rtpa in stroke but the bbb does become a serious barrier to the effective usage of this drug in clinic due to the neurotoxic effects and the risk of hemorrhagic transformation. interestingly, tpa may be endogenously synthesized by the central nervous system in neurons and endothelial cells. however, tpa and rtpa have effects on the endothelial cells, astrocytes, and neurons and possibly other glial cell types such as oligodendrocytes and microglia. in order to prevent the aversive effects of rtpa while maintaining the benefits of early reperfusion, several new therapeutic strategies have been examined to prevent the interaction of rtpa with the nmda receptor within the nvu. in fact, nmda receptors are expressed not only in neurons but also in oligodendrocytes and endothelial cells [125, 126 ]. one of these strategies uses an lrp antagonist (rap) to minimize the binding of rtpa with lrp in the endothelial cells. a second strategy uses the atd - nr1 antibody to block rtpa binding of the nr1 subunit on neuronal nmda receptors. the last one uses a mutation of the rtpa to decrease its adverse effects on the nervous tissue. an example of a natural drug, desmoteplase, the vampire bat desmodus rotundus salivary plasminogen activator (dspa), is a thrombolytic agent under development. it shows little neurotoxicity and has the ability to interact with the bbb endothelium through the same receptor (lrp) as that of tpa [127, 128 ]. unfortunately, the clinical trial of dias-2 (desmoteplase in acute ischemic stroke) showed no benefit of the desmoteplase versus placebo. although the outcome of this clinical trial was disappointing, promising alternatives pathways are being investigated. in fact, gleevec, a fda approved drug for treatment of chronic myelogenous leukemia, was recently proposed to prevent the complications associated with rtpa treatment. gleevec inhibits the activation of platelet - derived growth factor alpha receptor (pdgfr). it was shown that tpa increases bbb permeability through the indirect activation of perivascular astrocytic pdgfr. mmp inhibition is a good strategy based on reports of easy monitoring of mmp blood levels, defining them as potential biomarkers of brain damage [131, 132 ]. but because endogenous mmps are also key mediators in stroke recovery by contributing to inflammatory and remodeling responses, pharmacological targeting must be accurately applied for acute stroke phases so ; their beneficial effects are not compromised [133, 134 ]. despite efforts to understand the complex link between bbb integrity and the hemorrhage risk, a better definition and understanding of nvu kinetics and the mechanisms underlying their dysfunction is still needed to better define eligibility criteria for rtpa treatment. thus, alternative approaches other than mmp inhibition as mentioned before in some recent developments will offer interesting treatment strategies after stroke. given the small number of patients eligible for thrombolysis, many pharmaceutical compounds have been developed to limit the progression of brain injury by targeting different mechanisms leading to neuronal death. despite promising protective effects observed in preclinical studies, no compound to date has demonstrated benefit against stroke - induced neuronal death after facing the rigorous wall of clinical trials. as mentioned in section 1, research on brain diseases has focused on neuronal damage, as it was thought to be the major cause of cognitive deficits. however, ischemic stroke is a complex brain disease characterized by sudden onset of disabilities related to brain damage with a vascular origin. because the development of many neuroprotective molecules for treatment over the last twenty years has been unsuccessful, researchers have switched gears towards investigating the natural endogenous neuroprotection of ischemic tolerance. the purpose of the ischemic tolerance preconditioning is to induce endogenous defense mechanisms prior to the ischemic event that will attenuate the eventual consequences of ischemia. this resistance to ischemic damage can be achieved experimentally by several stimuli including ischemic preconditioning. in fact, a short duration of coronary occlusion is unable to cause myocyte necrosis. however, when carried out before a prolonged occlusion, a short occlusion significantly reduced the final infarct volume of the myocardium. this initial nonharmful ischemic insult triggered endogenous mechanisms that made the organ more resistant to the next attack for up to two periods of ischemic tolerance. the first period of ischemic tolerance resulted from posttranscriptional responses and began minutes after preconditioning. the second, longer period, began 24 hours after preconditioning and lasted up to 7 days with maximal protection found at 3 days. as with the cardiac preconditioning, ischemic tolerance in the brain also has delayed mechanisms leading to neuroprotection the induction of ischemic tolerance likely depends on the coordinated responses at the genomic, molecular, cellular, and tissue levels [141143 ], which suggests the importance of the interactions between the astrocyte and endothelial cells in the nvu. regarding neurovascular events in stroke pathophysiology, there has been a growing interest in vascular approaches to the preconditioning mechanisms. protective effects of preconditioning were observed in vivo, demonstrating that endothelium function is preserved by improving cerebral blood flow during reperfusion in areas surrounding the lesion, and that bbb integrity is maintained with a reduction in edema formation. the induced protection was again correlated not only with a decreased expression of mmp-9 but also with a reduced neutrophil adhesion to endothelial cells through a decreased expression of icam-1 [147, 148 ]. these results were confirmed by in vitro studies that report a protective effect via preservation of bbb integrity, by both a decreased expression of the inflammatory molecules icam-1 and vcam-1 [149, 150 ] and maintenance of tight junction structure. moreover, preconditioning also facilitates the increase of aqp4 expression at early time - points after stroke onset, which is associated with a decrease of the edema formation. a recent study also reported the protective role of glial tissue preconditioning in severe stroke. these recent observations suggest that future drug development must focus on drugs affecting the entire nvu instead of one cell type as was proposed in the 1990s with the development of calcium channel and nmda inhibitors. recently, some compounds like edaravone, an antioxidant, showed benefits in preclinical and clinical studies by protection of the nvu [152, 153 ]. but further trials are needed to confirm these promising preliminary results. preventive neuroprotection also involves management of risk factors, which is supported by studies showing that physical exercise or lipid - lowering treatment reduces the occurrence and severity of stroke [156158 ]. in this context, the involvement of pharmacological agents that are activators of nuclear receptors like peroxisome proliferator - activated receptors (ppars) could be a promising study. present in three isoforms,, /, and, these receptors exhibit pleiotropic activity in the sense that they can activate or repress the transcription of many genes involved in lipid and carbohydrate metabolism in addition to inflammation [159, 160 ]. activation of the ppars has long - term effects lasting from hours to days, which correspond to an activation of gene transcription (named transactivation) as has been seen in lipid and carbohydrate metabolism. however, activation of the ppars induce a cellular response within minutes to hours and this corresponds to an inhibition of gene transcription named transrepression. the latter mechanism does not require binding to dna, but rather protein - protein interaction involving other transcription factors like nf-b of stat-3 and ap-1, to inhibit their activity as reported for inflammatory genes. independent of its lipid - lowering activity, ppar- activation was found to be neuroprotective in several in vivo studies carried out in mice subjected to transient ischemia with preventive or curative treatments by agonists such as fenofibrate, wy-14643, and resveratrol (a polyphenol present in grapes) [164166 ]. the observed protection is the result of an anti - inflammatory mechanism, which decreases the expression of adhesion molecules, icam-1 and vcam-1, in brain endothelial cells. however, a study using a bbb in vitro model combining endothelial cells with glial cells from wild - type or ppar- knockout mice has demonstrated not only that the observed protection against ogd - induced hyperpermeability was dependent on this nuclear receptor activation but also that the ligand targeted specifically the endothelial cells without modulation of the classical ppar- target genes associated with inflammation or metabolism. moreover, protective effects of ppar- were not only reported through similar mechanisms but also via an inhibition of nfb and tnf- pathways [169, 170 ] and macrophages / microglial cells activation, thus preventing cytokine production. one study also suggests that ppar- agonists could inhibit excitotoxicity - induced neuronal death. this enzyme catalyzes the conversion of hmg - coa (3-hydroxy-3-methylglutaryl coenzyme a) to mevalonate, a precursor of cholesterol. as lipid lowering agents statins also exert pleiotropic effects at the vascular level. in addition to protection against excitotoxicity in cultured neurons, statins have demonstrated preservation of bbb endothelial cells ' integrity against glutamate excitotoxic challenge in vitro. the effects of statins may involve nuclear receptors, through an increase in both expression and activity of ppar- [177179 ]. more recently, brain endothelial ppar- activation has proven to be protective against ischemia - induced cell death through inhibition of the mir-15a microrna, thus strengthening the therapeutic concept based on activation of ppars for the treatment of stroke - related microvascular dysfunction. the c - jun n - terminal kinases (jnks) belong to the mitogen activated protein kinase (mapk) family ; the two other members being p38 and erk [181, 182 ]. the isoforms jnk1 and jnk2 are ubiquitously distributed, while jnk3 is primarily expressed in the heart, brain, endocrine pancreas and testis. jnks are activated by phosphorylation, which is catalyzed by upstream kinases mkk 4 and 7 [182184 ]. however, the activation of jnks plays several roles ranging from regulation of cell survival and apoptosis to cell proliferation [183, 185187 ]. they are activated under pathological conditions both in the brain [188, 189 ] and in the periphery [190, 191 ]. in fact, jnk phosphorylation initially decreases after stroke and then starts to increase at 1.5 hours with a maximum at 9 hours after onset. phosphorylation of c - jun, a jnk substrate, follows the same temporal pattern, peaking at 8 hours post - stroke [192, 193 ]. the development of the peptide named djnki, a competitive inhibitor of the jnk signaling pathway, has been shown to reduce lesion volume of mice with transient mcao by 90% even when induced 6 hours after injury. this lesion volume decrease was accompanied by behavior improvements as well, suggesting an increase of the therapeutic time window almost 2 times longer than tpa. this positive outcome was also observed in a more severe model with a permanent occlusion model. moreover, djnki has been shown to be compatible for treatment of ischemic stroke even in the presence of rtpa and was shown to decrease lesion volume. djnki also improved neurobehavior scores and decreased hemispheric swelling after a model of intra - cerebral hemorrhage. thus, djnki could possibly attenuate the highly probable side effect of hemorrhagic transformation caused by rtpa. interestingly, in this model of intracerebral hemorrhage, djnki administration significantly increased aqp4 expression 48 hours after injury. this increase in aqp4 expression negatively correlated with decreased hemispheric swelling, thus pointing towards a possible role of djnki controlling edema as well. in fact, activation of the jnk pathway is present not only in the neurons but also in glial cells and brain endothelial cells. such activation in nonneuronal cells may negatively impact neuronal cell death and function. in the context of broad effects of this drug, benakis. showed that djnki-1, injected peripherally, is able to modulate some nonneuronal inflammatory processes. as discussed previously, the development of a drug targeting several cells such as in the nvu may help to move towards success in the clinic. in summary, the data found in the literature suggest that the failure of agents in protecting the brain against stroke may come from the fact that each developed compound targeted only one mechanism and one cell type of stroke pathophysiology. ischemic preconditioning appears to be an attractive experimental strategy that would identify endogenous mechanisms of protection and regeneration. recent evidence of such protective mechanisms supports a complex action on cells of the nvu, underlining the importance of the interactions between endothelial cells and astrocytes in the pathophysiology after stroke. as our knowledge of the nvu increases, molecules with pleiotropic activity will become increasing useful in the development of post - ischemic treatments in the clinics. | the neurovascular / gliovascular unit has recently gained increased attention in cerebral ischemic research, especially regarding the cellular and molecular changes that occur in astrocytes and endothelial cells. in this paper we summarize the recent knowledge of these changes in association with edema formation, interactions with the basal lamina, and blood - brain barrier dysfunctions. we also review the involvement of astrocytes and endothelial cells with recombinant tissue plasminogen activator, which is the only fda - approved thrombolytic drug after stroke. however, it has a narrow therapeutic time window and serious clinical side effects. lastly, we provide alternative therapeutic targets for future ischemia drug developments such as peroxisome proliferator- activated receptors and inhibitors of the c - jun n - terminal kinase pathway. targeting the neurovascular unit to protect the blood - brain barrier instead of a classical neuron - centric approach in the development of neuroprotective drugs may result in improved clinical outcomes after stroke. |
alcohol is regularly consumed by 20% of french adults, and more than 6.4 million french people consume it daily.1 alcohol is responsible for 33,000 deaths per year in france,2 and comorbidity with psychiatric disorders is important.3 alcohol abuse and dependence lead to somatic, psychological, family, social, professional, and financial disruption.4,5 dependent patients have a special link with the substance, with an inability to refrain from it (according to diagnostic and statistical manual of mental disorders, 4th edition [dsm - iv ] criteria).6 therapeutic strategies for alcohol dependence are based on acute treatment (withdrawal) and long - term rehabilitation.7 after withdrawal, relapse prevention is the major challenge for the treatment of this disease. usually, two approaches are combined : psychotherapy and pharmacotherapy.7 recently, new psychotherapeutic approaches developed to improve cognitive impairments described in mental illnesses (eg, schizophrenia or bipolar disorder) have been used in the treatment of addictions.8 the functioning of the brain of an addicted patient may differ from that of a nonaddict.9 a dependent subject no longer has full control of their external environment. this lack of control led to the observation that substance - related stimuli (ie, the word alcohol for an alcohol - dependent patient) induced strong activation compared with non - substance - related stimuli. this activation results in an attentional bias (ab) in which the attentional system is not able to filter correctly the external information. ab can also be defined as a phenomenon whereby attentional channeling is directed toward personally valued stimuli, despite an individual s efforts to ignore them.10,11 thus, ab would be strongly associated with relapse, and in particular with craving.12 development and an increase in craving are influenced by the excess of attention to substance cues.13 thus, craving and ab are highly correlated.10,1416 the evaluation of ab at the end of hospitalization is evident : several authors have shown that this can predict the likelihood of relapse and therefore the effectiveness of the treatment.10 indeed, cox observed that after 4 weeks of hospitalization, patients who had a greater ab had an increased risk of relapse compared with those who had a lower ab. the most used tool to show ab in the case of psychological disorders is a modified stroop task.11 the classic stroop task18 is used in clinical practice to highlight attentional deficits. in this task, participants are instructed to name the color of the ink used to write a word, eg, red, when the word door is written in red. in 1935, stroop showed in one of the conditions of his study that participants required more time to name the color when the word itself was an incongruent color (for example, blue written in red) than when it was a congruent color (blue written in blue). this task measures the participant s ability to ignore irrelevant stimuli (the meaning of the word) and to focus on the relevant stimulus (the color of the word). the more the stimulus is semantically close to the color name, the more ignoring it is difficult.19 the emotional stroop task is a modification of the original to highlight a lack of attention related to an emotional concept (for review, see williams).11 in an emotional stroop task, stroop interference is achieved by words emotionally relevant for the participant. for patients with anxiety disorders, the time to name the color of the word is longer when the word is related to the concept of anxiety than when the word is neutral. in patients with post - traumatic stress disorder, this effect is especially marked when the selected words refer to the traumatic event. this interference would be a cue that the patient filters information in an inappropriate way. in this case, the concept linked to the disorder is active and present in memory automatically, and too much attention is allocated to this information.20 the result is then a longer processing time to treat another feature of the stimulus the color. in the alcohol stroop test (ast ; recently reviewed),10 stimuli are alcohol - related words. an ab towards alcohol is noticeable when participants require more time to name the color of an alcohol - related word than for a neutral word. cox showed that patients at admission to inpatient treatment have a greater ab than a group of nonabusers, who themselves have a greater ab than patients in discharge after 4 weeks of treatment. our study was designed to evaluate the efficacy of hospital care on the modification of ab. for this, we compared the ab score in alcohol - dependent admission to inpatient treatment in the french version of the ast. we expected to observe results similar to those of previous studies:17 patients at admission should have a greater ab than nonabusers, whose ab should be greater than patients at discharge. the classic stroop effect, measuring the general capacity of attention, should not be significantly different between these three groups, indicating that the reduction in attention bias is not due to an increase in attentional capacities. cox used a within - subjects design, which could be a problematic method because the second measure of ab could just reflect a learning process. we chose to use a between - subjects design, also indicating that the reduction in ab is not due to a learning process. the test group was composed of alcohol - dependent participants who were recruited from the university hospital gabriel - montpied in clermont - ferrand, and more specifically the ward devoted to the treatment of addictive disorders. all subjects were evaluated and diagnosed as dependent by a senior addiction specialist with extensive clinical experience (fp), using dsm - iv criteria. one subgroup of the test group, the admission group, was composed of patients included during the 4 days after their admission, and another subgroup, the discharge group, was composed of patients included immediately prior to discharge, approximately 4 weeks after their admission. the control group consisted of healthy volunteers who had never presented addictive, neurological, or psychiatric disorders. all participants gave written informed consent to participate in this study, as required by the local ethical committee. finally, all patients in the test group had undergone standard oxazepam treatment (see table 1). stimuli consisted of four neutral words : robe (dress), pont (bridge), train, and studio ; four words semantically related to the notion of alcohol : alcool (alcohol), ricard, vin (wine), and whisky ; and four color names : rouge (red), jaune (yellow), bleu (blue), and vert (green). stimuli were individually presented in lowercase letters in courier new font, 72-point bold, centered on the screen. on average, a word covered a visual angle of 3 broad and height 0.9. there were three different blocks : one for the neutral words, one for the words for colors, and one for words related to alcohol. the color words were always presented in an incongruent way (eg, red appeared only in green, blue, or yellow). in addition, an analysis of the objective frequencies of the words from the french lexical database lexique 321 showed no significant difference between alcohol - related words with the exception of the word ricard, which was not contained in this base, and neutral words (t = 0.96, p = 0.38), and color words and neutral words (t = 0.4, p = 0.71). the experiment was conducted in a quiet room for nonabuser participants and in patients hospital rooms. following the reading of consent the presentation of stimuli and recording of response times were managed and measured to the nearest millisecond by the dmdx program.22 the response times were recorded by a microphone on the pc. the choice of a verbal response was preferred to the choice of a manual response. manual response offers worse stroop interference,23 and it remains less natural, because it requires a learning phase. they were instructed to concentrate on the fixation cross (+) for 500 ms before it was replaced by the word. they were to name the color of the word, and they were reminded to respond as quickly as possible, and without error. as recommended,24 stimuli were presented in blocks to avoid the cognitive influence of the item alcohol being carried over to the following item. in the first, the words were replaced by a series of xs (eg, xxxxx). this step allowed detection of possible color - perception problems or problems related to poor microphone detection. the second practice phase was the presentation of neutral words, not used in the rest of the study (voiture [car ], fauteuil [chair ], route [road ], tlvision [television ], and chaussure [shoe ]) (in total, ten items). finally, the three blocks were presented in random order between participants, with a pause of a few seconds between each block. in total, except for practice items, each participant saw 72 items. the average response time given by item for each block of correct answers was analyzed. responses less than 300 ms and more than 1500 ms were excluded from analyses (less than 1% of data). the tests were first made on the words semantically related to alcohol, compared with neutral words, then on the color words compared with neutral words, using spss 19 (ibm, armonk, ny, usa). a shapiro - wilk test on the two principal variables (alcohol stroop interference and classical stroop interference) indicated that these two variables were normally distributed (p = 0.37 for alcohol stroop interference and p = 0.097 for classical stroop interference). two types of analysis were undertaken, with a repeated - measures plan of 2 3 with type of stimulus (color words or alcohol - related words vs neutral words) and the status of the participant (nonabusers vs admitted patient vs discharged patients). we conducted contrast analysis to assess the intensity of the stroop effect (classic and alcohol) in different groups of patients and controls. in addition, we undertook comparisons for each type of participant with a bonferroni adjustment on this data. the test group was composed of alcohol - dependent participants who were recruited from the university hospital gabriel - montpied in clermont - ferrand, and more specifically the ward devoted to the treatment of addictive disorders. all subjects were evaluated and diagnosed as dependent by a senior addiction specialist with extensive clinical experience (fp), using dsm - iv criteria. one subgroup of the test group, the admission group, was composed of patients included during the 4 days after their admission, and another subgroup, the discharge group, was composed of patients included immediately prior to discharge, approximately 4 weeks after their admission. the control group consisted of healthy volunteers who had never presented addictive, neurological, or psychiatric disorders. all participants gave written informed consent to participate in this study, as required by the local ethical committee. finally, all patients in the test group had undergone standard oxazepam treatment (see table 1). stimuli consisted of four neutral words : robe (dress), pont (bridge), train, and studio ; four words semantically related to the notion of alcohol : alcool (alcohol), ricard, vin (red), jaune (yellow), bleu (blue), and vert (green). stimuli were individually presented in lowercase letters in courier new font, 72-point bold, centered on the screen. on average, a word covered a visual angle of 3 broad and height 0.9. there were three different blocks : one for the neutral words, one for the words for colors, and one for words related to alcohol. the color words were always presented in an incongruent way (eg, red appeared only in green, blue, or yellow). in addition, an analysis of the objective frequencies of the words from the french lexical database lexique 321 showed no significant difference between alcohol - related words with the exception of the word ricard, which was not contained in this base, and neutral words (t = 0.96, p = 0.38), and color words and neutral words (t = 0.4, p = 0.71). the experiment was conducted in a quiet room for nonabuser participants and in patients hospital rooms. following the reading of consent the presentation of stimuli and recording of response times were managed and measured to the nearest millisecond by the dmdx program.22 the response times were recorded by a microphone on the pc. the choice of a verbal response was preferred to the choice of a manual response. manual response offers worse stroop interference,23 and it remains less natural, because it requires a learning phase. they were instructed to concentrate on the fixation cross (+) for 500 ms before it was replaced by the word. they were to name the color of the word, and they were reminded to respond as quickly as possible, and without error. as recommended,24 stimuli were presented in blocks to avoid the cognitive influence of the item in the first, the words were replaced by a series of xs (eg, xxxxx). this step allowed detection of possible color - perception problems or problems related to poor microphone detection. the second practice phase was the presentation of neutral words, not used in the rest of the study (voiture [car ], fauteuil [chair ], route [road ], tlvision [television ], and chaussure [shoe ]) (in total, ten items). finally, the three blocks were presented in random order between participants, with a pause of a few seconds between each block. in total, except for practice items, each participant saw 72 items. the average response time given by item for each block of correct answers was analyzed. responses less than 300 ms and more than 1500 ms were excluded from analyses (less than 1% of data). the tests were first made on the words semantically related to alcohol, compared with neutral words, then on the color words compared with neutral words, using spss 19 (ibm, armonk, ny, usa). a shapiro - wilk test on the two principal variables (alcohol stroop interference and classical stroop interference) indicated that these two variables were normally distributed (p = 0.37 for alcohol stroop interference and p = 0.097 for classical stroop interference). two types of analysis were undertaken, with a repeated - measures plan of 2 3 with type of stimulus (color words or alcohol - related words vs neutral words) and the status of the participant (nonabusers vs admitted patient vs discharged patients). we conducted contrast analysis to assess the intensity of the stroop effect (classic and alcohol) in different groups of patients and controls. in addition, we undertook comparisons for each type of participant with a bonferroni adjustment on this data. nineteen comprised the admission group, and 23 comprised the discharge group. sixteen participants made up the control group. there was no difference between groups on the basis of age or sex (fs < 1). this response - time analysis revealed an effect of type of stimulus (f = 5.752, p < 0.05) and an effect of status of the participant (f = 4.25, p < 0.05). the interaction between these two factors was not significant (f < 1) (figure 1). to analyze these specific results this showed a trend effect of status of the participant (t = 1.714, p = 0.092), indicating that patients on admission had an alcohol stroop effect greater than the nonabusers, who themselves had a higher effect than discharged patients. orthogonal contrasts centered on this linear contrast were not significant (t = 1.174, p = 0.246). planned comparisons showed that patients in admission had a tendency towards an alcohol stroop effect (34 ms, f = 3.283, p = 0.075) while nonabusers and discharged patients did not have this alcohol stroop effect (respectively, 23 ms, f = 1.252, p = 0.268 and 12 ms, f = 1.552, p = 0.218). the mixed model on error - proportions analysis did not show any effect of type of stimulus or status of the participant, and these two factors had no interactions. the response - time analysis revealed a type of stimulus effect (f = 43.53, p < 0.001), indicating that response times to the color words were generally greater than for neutral words. the analysis also revealed an effect of status of the participant (f = 6.164, p < 0.05), indicating that the response times were different from nonabusers, patients at admission, or those at discharge. the interaction between these two factors was not significant (f = 1.343, p = 0.269). this did not show any effect of status of the participant (t = 1.393, p = 0.169) on the stroop effect, indicating that there was no difference between the participants on the classic stroop effect. planned comparisons showed a classic stroop effect for all participants : 127 ms at admission (f = 25.096, p < 0.001), 92 ms for nonabusers (f[1,55 = 11.178, p < 0.001 ], and 76 ms for discharged patients (f = 9.497, p < 0.01). we found no effect of type of stimulus on error proportions (f < 1), but status of the participant had an effect on error proportions (f = 3.723, p < 0.05), and there was interaction between these two factors (f = 3.134, p = 0.05). planned comparisons showed that only patients on admission had a difference in error proportions (f = 5.63, p < 0.05). nineteen comprised the admission group, and 23 comprised the discharge group. sixteen participants made up the control group. there was no difference between groups on the basis of age or sex (fs < 1). this response - time analysis revealed an effect of type of stimulus (f = 5.752, p < 0.05) and an effect of status of the participant (f = 4.25, p < 0.05). the interaction between these two factors was not significant (f < 1) (figure 1). to analyze these specific results this showed a trend effect of status of the participant (t = 1.714, p = 0.092), indicating that patients on admission had an alcohol stroop effect greater than the nonabusers, who themselves had a higher effect than discharged patients. orthogonal contrasts centered on this linear contrast were not significant (t = 1.174, p = 0.246). planned comparisons showed that patients in admission had a tendency towards an alcohol stroop effect (34 ms, f = 3.283, p = 0.075) while nonabusers and discharged patients did not have this alcohol stroop effect (respectively, 23 ms, f = 1.252, p = 0.268 and 12 ms, f = 1.552, p = 0.218). the mixed model on error - proportions analysis did not show any effect of type of stimulus or status of the participant, and these two factors had no interactions. the response - time analysis revealed a type of stimulus effect (f = 43.53, p < 0.001), indicating that response times to the color words were generally greater than for neutral words. the analysis also revealed an effect of status of the participant (f = 6.164, p < 0.05), indicating that the response times were different from nonabusers, patients at admission, or those at discharge. the interaction between these two factors was not significant (f = 1.343, p = 0.269). this did not show any effect of status of the participant (t = 1.393, p = 0.169) on the stroop effect, indicating that there was no difference between the participants on the classic stroop effect. planned comparisons showed a classic stroop effect for all participants : 127 ms at admission (f = 25.096, p < 0.001), 92 ms for nonabusers (f[1,55 = 11.178, p < 0.001 ], and 76 ms for discharged patients (f = 9.497, p < 0.01). we found no effect of type of stimulus on error proportions (f < 1), but status of the participant had an effect on error proportions (f = 3.723, p < 0.05), and there was interaction between these two factors (f = 3.134, p = 0.05). planned comparisons showed that only patients on admission had a difference in error proportions (f = 5.63, p < 0.05). our results on ab measured with a french version of the ast showed a trend difference between our three groups of participants : ab was greater in patients on admission, whose ab was greater than that of nonabusers, who had a greater ab than that observed in discharged patients. these results suggest an improvement in cognitive performances against related - substance cues in the treatment period. in spite of low statistical power, these results are consistent with those reported in the literature.17 while we observed a decrease in the classic stroop effect on response times, it did not differ significantly between the different types of participant. this seems to indicate that the observed decrease in ab was not completely due to a better attentional process, nor to an effect of the attentional process of the oxazepam treatment, which is in accordance with the literature.17 our results show that hospital care leads to reduced ab in alcohol - dependent patients. our results had the same pattern as those of cox,17 who used a within - subjects design. a between - subjects design could be problematic for drawing conclusions, because it is difficult to be sure that there was no other factor of difference between the two groups. our results, viewed together with previous results, seem to indicate that diminution of ab was not due to a training bias and that the difference observed between patients at admission and at discharge could be observed with the two types of design. williams have shown that the subjective frequency of items has an influence on the stroop task. the more frequently a word is encountered by an individual, the more it creates interference. the difference observed between our patients at admission, when they were confronted with many alcohol - related items, and our nonabusers could be explained by this phenomenon, but this seems unlikely, because we also observed a difference between patients at admission and at discharge, patients who could estimate that the subjective frequency of items used was comparable. cox used the staff in the same unit of addiction treatment as their patients, a group control, to avoid this bias. these authors continued to observe a difference between patients and the control group concerning ab. this suggests that the emotional valence of a word is more important in the emotional stroop task than the subjective frequency. the ab reduction in patients between admission and discharge is probably due to a decline in the emotional importance of alcohol - related words. this decrease between patients at admission and at discharge is probably an indication of the efficacy of the treatment on this dimension. indeed, the use of psychotropic drugs (ie, oxazepam) can have an effect on the emotional processing of stimuli.25 thus, the decrease in ab for discharged patients may be due to decreased emotional perception. moreover, the cognitive - care services probably encourage the patient to establish a functioning inhibiting process from alcohol - related cues. indeed, patients are advised to participate in discussion groups, for psychological education and relapse prevention, and to engage in individual behavioral and cognitive therapy. we must nevertheless be careful with these results, due to limitations on use of the stroop task to explore ab. this ab could be a problem of initial orientation of spatial attention or a disengagement bias. phaf and kan26 showed recently in a review that ab can occur in short stimulus - onset asynchrony (between 50 and 200 ms), which favors an initial orientation spatial bias, but also an ab with long stimulus - onset asynchrony, reflecting a disengagement bias. unfortunately, the stroop task can not distinguish between these two types of process.27 the diminution observed could be attributed to either explanation. it is difficult to determine which process is enhanced by hospital care : initial spatial orientation or the disengagement process. moreover, the emotional stroop effect could reflect the fact that emotional stimuli are less readily suppressed or filtered in normal populations, with negative consequences for primary task performance.28 indeed, studies on the emotional stroop task showed an effect with emotional words in a normal population.29 this effect exists in reading, lexical decision, and color naming.30 these tasks are all slower with emotional words, and this delay is immune to task - irrelevant variation and to changes in the relative salience of the words and the colors. further, the delay was absent when emotional and neutral words appeared in a single block, suggesting that the so - called emotional stroop effect could reflect a stimulus - driven generic slowdown effect rather than a selective - attention mechanism, such as that associated with the classic stroop effect. the ast used and developed in this study could indicate the efficacy of the general concept of ab in the hospital care of a patient, even if it can not highlight exactly the process involved in this diminution. at a clinical level, this study and the observation of ab in a patient population underline the importance of this process in pathology. as mentioned previously, ab is positively correlated with craving and relapse.17 these data support the importance of ab in the maintenance of the disorder. the alcohol attention - control training program developed in a nonaddicted population shows that participants in this program reduce their alcohol consumption and their ab during the following 3 months.31 this type of reducing ab this study, associated with the previous results of a study of ab17 with the ast, shows a diminution of ab for hospitalized patients. treatment that works on this ab will probably benefit the patient, and more specifically be associated with a diminution of relapse. the use of other measures of ab could be considered to explore in more specific detail which process exactly is enhanced during treatment. an answer to this question could allow us to design a more specific rehabilitation program, and to be more efficient in the hospital care of these patients. | background and objectives : previous studies in alcohol - dependent patients have shown an attentional bias (ab) under related substance cues, which can lead to relapse. this ab can be evaluated by the alcohol stroop test (ast). the ast is a modified stroop task in which participants have to name the color of an alcohol - related word or a neutral word. ab is the response - time difference between these two types of words. the goal of the current study was to examine modification of ab during specialized hospitalization for alcohol dependence, with the suppression of a training bias that could be present in within - subject design.methods:individuals with alcohol - dependence disorders (diagnostic and statistical manual of mental disorders, 4th edition) and admitted for withdrawal in the addiction unit of the university hospital of clermont - ferrand (test group, n = 42) and persons with no alcohol or psychiatric disorder (control group, n = 16), recruited among colleagues and friends of the staff, performed the ast. a subgroup of the test group performed the ast in admission (admission group, n = 19), and another subgroup undertook the test immediately before discharge (discharge group, n = 23).results : results showed an ab only for patients seen at admission (f[1,55 ] = 3.283, p = 0.075). moreover, we observed that the ab in the admission group (mean = 34 ms, standard deviation [sd ] = 70.06) was greater than the ab in the control group (mean = 23 ms, sd = 93.42), itself greater than the ab in the discharge group (mean = 12 ms, sd = 93.55) (t[55 ] = 1.71 ; p = 0.09).conclusion : although the results are preliminary, the present study provides evidence for changes in the ab during alcohol - addiction treatment and for the value of these methods to diminish ab during detoxification. |
recently, tumor - infiltrating lymphocytes (tils) have been strongly associated with the anti - tumor immunity and have shown prognostic and predictive significance in a range of types of solid neoplasms [1 - 4 ]. triple - negative breast cancers (tnbcs) show the poorest prognosis among all breast cancer subtypes ; there are no known targeted treatments for this subgroup of patients. on the other hand, previous studies have indicated that a large number of tils are associated with high response rates to neoadjuvant chemotherapy (nac) along with good survival rates in patients with tnbc. moreover therefore, gaining a better understanding of the mechanism whereby lymphoid cells infiltrate tumors and interact with malignant cells might facilitate the development of more efficient immunotherapy. tertiary lymphoid structures (tlss) are one of the major sources of tils. tlss are ectopic, vascularized lymphoid formations that can be found in inflamed or tumoral tissues. in breast cancers, tlss can be observed mainly in areas that surround the tumor and adjacent normal terminal ductal lobular units. the mechanism whereby tlss are formed is not known, but the roles for high endothelial venules (hevs) and the chemokine cxcl13 have been suggested. the hevs are specialized blood vessels with plump and cuboidal endothelial cells, which express the peripheral node addressin (pnad) and facilitate the extravasation of lymphocytes from the bloodstream and formation of tlss. many studies have reported positive correlations between the tls components, particularly hev, and favorable clinical outcomes in various types of cancer, such as cutaneous melanoma, non - small cell lung cancer, colorectal carcinoma, and breast carcinoma [7,11 - 13 ]. gu - trantien. reported that the infiltration of cxcl13-producing cd4 follicular helper t cells and a cxcl13 gene expression signature are related to a longer disease - free survival in breast cancer patients. a previous study recently suggested that increased cxcl13 gene expression is predictive of a more frequent pathologic complete response (pcr) and a good prognosis in tnbc patients treated with nac. despite the clinical significance of tlss and tils, they are difficult to assess in situ. the term tls can only be used when the structure exhibits all of the following features : distinct and adjacent t and b cell compartments, fibroblastic reticular cells, hevs, activation - induced cytidine deaminase enzyme expression, and follicular dendritic cells. on the other hand, in clinical practice, it can be difficult to distinguish simple lymphoid aggregations from true tlss using histopathological assessments alone. meca79, which is an epitope binding to cd62l and induces tls formation, can be used to evaluate tls, but there has been no trial using meca79 in tumor tissues. in addition, measurements of tils do not allow for the differentiation of various subpopulations, and there has been no consistent evidence regarding which til subpopulation(s) play the dominant role in anti - tumor immunity and are associated with good clinical outcomes. some papers have reported that the presence of cd8-positive cytotoxic t cells is the most important predictive and prognostic factor in breast cancer, whereas others have underscored the significance of b cells. therefore, this study evaluated the amount of tlss in prenac core needle biopsies of tnbc by calculating the meca79-positive hev density, measuring the til subpopulations by immunohistochemistry (ihc), and using a digital computer analyzer, and assessed their relationship with cxcl13 mrna expression and the clinical outcomes. a total of 108 patients, who were diagnosed with primary tnbc, received anthracycline and taxane - based nac, and were treated surgically between 2010 and 2012 at the asan medical center, were enrolled in this study. all patients received anthracycline and taxane - based regimens, which included four cycles of 60 mg / m adriamycin and 600 mg / m cyclophosphamide followed by four cycles of 75 mg / m docetaxel. the occurrence of pcr was defined as the absence of a residual invasive carcinoma in the breast and regional lymph nodes (ypt0/tis, n0). approval of the study protocol was granted by the institutional review board of the asan medical center (approval number : 2013 - 0866). the histological parameters were evaluated based on hematoxylin and eosin (h&e)stained slides and pathology reports. the clinicopathological parameters evaluated in each case included the patients age at diagnosis, sex, tumor size, histological subtype, histological grade, pathologic tumor (pt) stage, lymphovascular invasion, lymph node metastasis, overall tumor stage, the presence or absence of recurrence, the most recent follow - up date, and the survival status. h&e - stained slides of the pre - nac biopsy and post - nac surgery samples of all 108 tumors were reviewed for a histological diagnosis, subtype and grade, tumor size, ypt stage, ypn stage, lymphovascular invasion, and responsiveness to chemotherapy. the histological type was defined based on the 2012 world health organization classification criteria, and the histological grade was assessed using the modified bloom - richardson classification. the miller - payne grade and residual cancer burden (rcb) were also assessed for their response to chemotherapy. pre - nac biopsy slides were analyzed histopathologically to determine the level of tils (this is defined as the mean percentage of stroma of an invasive carcinoma infiltrated by lymphocytes and plasma cells in 10% increments ; 1% or 5% criteria were used if less than 10% of stroma was infiltrated by tils ; all available full sections were evaluated), the tlss in the adjacent tissues, including carcinoma in situ components (none, little, moderate, or abundant), and the presence or absence of a germinal center in tlss. lymphoid aggregation with vessels that exhibited hev - like features (plump, cuboidal endothelial cells) with or without germinal centers was considered to be a tls. among the 108 cases, formalin - fixed paraffin - embedded (ffpe) tissue samples were available for 55 cases, which were analyzed by ihc and mrna expression. the ffpe tissue sections were stained using an automatic immunohistochemical staining device (benchmark xt, ventana medical systems, tucson, az). the antibodies to cd3 (1:50, novocastra laboratories, newcastle upon tyne, uk), cd8 (1:400, dako, glostrup, denmark), cd20 (1:500, novocastra laboratories), and meca79 (1:200, santa cruz biotechnology, dallas, tx ; recognizes sulfate - dependent carbohydrate epitopes of pnad expressed in endothelial cells of hevs), were used. the immunostained slides were scanned using a digital microscopy scanner (pannoramic 250 flash, 3dhistech ltd., the entire tumor area was selected in whole slides. in each case, the images were then scanned using computer viewer software (pannoramic viewer 1.15.2, 3dhistech ltd.) and the number of cd3-, cd8-, and cd20-positive lymphocytes were counted in the tumor area using the nuclearquant module. the positive cell densities were calculated by dividing the immuno - positive cell numbers by the tumor area. the levels of the cxcl13 mrna transcripts in the ffpe tissue samples were evaluated by digital transcript counting (ncounter gx human immunology v2 kit) assay (nanostring technologies, seattle, wa). the total rna (100 ng) was assayed on a counter digital analyzer (nanostring technologies) according to the manufacturer s instructions. the data were normalized by scaling with the geometric mean of the built - in control gene probes for each sample. wilcoxon signed - rank tests, kruskal - wallis tests, mann - whitney u tests, fisher exact tests, logistic regression, spearman s correlations, and binary logistic regression were used where appropriate. a threshold for statistical significance a total of 108 patients, who were diagnosed with primary tnbc, received anthracycline and taxane - based nac, and were treated surgically between 2010 and 2012 at the asan medical center, were enrolled in this study. all patients received anthracycline and taxane - based regimens, which included four cycles of 60 mg / m adriamycin and 600 mg / m cyclophosphamide followed by four cycles of 75 mg / m docetaxel. the occurrence of pcr was defined as the absence of a residual invasive carcinoma in the breast and regional lymph nodes (ypt0/tis, n0). approval of the study protocol was granted by the institutional review board of the asan medical center (approval number : 2013 - 0866). the histological parameters were evaluated based on hematoxylin and eosin (h&e)stained slides and pathology reports. the clinicopathological parameters evaluated in each case included the patients age at diagnosis, sex, tumor size, histological subtype, histological grade, pathologic tumor (pt) stage, lymphovascular invasion, lymph node metastasis, overall tumor stage, the presence or absence of recurrence, the most recent follow - up date, and the survival status. h&e - stained slides of the pre - nac biopsy and post - nac surgery samples of all 108 tumors were reviewed for a histological diagnosis, subtype and grade, tumor size, ypt stage, ypn stage, lymphovascular invasion, and responsiveness to chemotherapy. the histological type was defined based on the 2012 world health organization classification criteria, and the histological grade was assessed using the modified bloom - richardson classification. the miller - payne grade and residual cancer burden (rcb) were also assessed for their response to chemotherapy. pre - nac biopsy slides were analyzed histopathologically to determine the level of tils (this is defined as the mean percentage of stroma of an invasive carcinoma infiltrated by lymphocytes and plasma cells in 10% increments ; 1% or 5% criteria were used if less than 10% of stroma was infiltrated by tils ; all available full sections were evaluated), the tlss in the adjacent tissues, including carcinoma in situ components (none, little, moderate, or abundant), and the presence or absence of a germinal center in tlss. lymphoid aggregation with vessels that exhibited hev - like features (plump, cuboidal endothelial cells) with or without germinal centers was considered to be a tls. among the 108 cases, formalin - fixed paraffin - embedded (ffpe) tissue samples were available for 55 cases, which were analyzed by ihc and mrna expression. the ffpe tissue sections were stained using an automatic immunohistochemical staining device (benchmark xt, ventana medical systems, tucson, az). the antibodies to cd3 (1:50, novocastra laboratories, newcastle upon tyne, uk), cd8 (1:400, dako, glostrup, denmark), cd20 (1:500, novocastra laboratories), and meca79 (1:200, santa cruz biotechnology, dallas, tx ; recognizes sulfate - dependent carbohydrate epitopes of pnad expressed in endothelial cells of hevs), were used. the immunostained slides were scanned using a digital microscopy scanner (pannoramic 250 flash, 3dhistech ltd., the entire tumor area was selected in whole slides. in each case, the images were then scanned using computer viewer software (pannoramic viewer 1.15.2, 3dhistech ltd.) and the number of cd3-, cd8-, and cd20-positive lymphocytes were counted in the tumor area using the nuclearquant module. the positive cell densities were calculated by dividing the immuno - positive cell numbers by the tumor area. the levels of the cxcl13 mrna transcripts in the ffpe tissue samples were evaluated by digital transcript counting (ncounter gx human immunology v2 kit) assay (nanostring technologies, seattle, wa). the total rna (100 ng) was assayed on a counter digital analyzer (nanostring technologies) according to the manufacturer s instructions. the data were normalized by scaling with the geometric mean of the built - in control gene probes for each sample. wilcoxon signed - rank tests, kruskal - wallis tests, mann - whitney u tests, fisher exact tests, logistic regression, spearman s correlations, and binary logistic regression were used where appropriate. a threshold for statistical significance the patients ages ranged from 23 to 70 years (median, 42 years) ; all patients were women. among the 108 cases analyzed, eight were ct1 tumors, 58 were ct2 tumors, 37 were ct3 tumors, and five were ct4 tumors. for the nodal status, there were six cn0 tumors, 48 cn1 tumors, 23 cn2 tumors, and 31 cn3 tumors. the median patient follow - up duration was 34.9 months (range, 12.0 to 55.8 months). table 1 summarizes the clinicopathological characteristics of the tumors without and with available ffpe blocks. no significant differences were observed between the two groups regarding the patients age, clinical t or n stage, histological grade, tils, tlss, pcr rate, miller - payne grade, or rcb class. based on assessments of the h&e - stained sections, uniand multi - variate analyses were performed to assess the associations between pcr, til, and other established clinicopathological parameters (table 2). among the 108 tumors, univariate logistic regression analysis revealed a lower ct stage and higher til and tls levels in the pre - nac biopsy to be significantly associated with pcr. multivariate analysis, including the ct stage, til, and tls, showed that only til (hazard ratio [hr ], 1.038 ; 95% confidence interval [ci ], 1.012 to 1.065 ; p=0.004) was an independent predictor of pcr. in univariate disease - free survival analysis, the ct and til were closely associated with a longer disease - free survival rate (table 3). on the other hand, multivariate analysis, including the ct stage, cn stage, til, and tls, showed that only til (hr, 0.975 ; 95% ci, 0.954 to 0.997 ; p=0.028) was a robust independent prognostic factor for the disease - free survival. ihc and gene expression analyses were carried out in 55 cases with available ffpe tissue blocks. the median of the tissue areas in each slide was 69.46 mm (range, 23.47 to 175.33 mm), whereas the median of the tumor areas was 20.92 mm (range, 1.23 to 83.15 mm). 1). finally, the mean cd3-, cd8-, and cd20-positive cell densities in the tumor areas of each slide were 357.2412.2/mm, 402.2382.4/mm, and 246.8343.5/mm, respectively (fig. the til and tls levels were correlated based on the h&e - stained slides, the densities of hev and til subpopulations, and expression levels of the cxcl13 mrna transcripts using the nanostring method with the pathological responses to nac (supplementary table 1). the tls levels were correlated significantly with the til levels, cd3-, cd8-, and cd20-positive cell densities, the hev density, and expression levels of the cxcl13 mrna transcripts. the miller - payne grade was significantly correlated with the tls level, cd20-positive cell density, hev density, and expression levels of cxcl13 mrna transcripts. finally, the rcb class was correlated significantly with cxcl13 expression. based on an analysis of the ihc slides, 17 patients (30.1%) with available ffpe tissue blocks achieved pcr ; none of these patients showed a recurrence during a median follow - up period of 31.4 months (range, 21.1 to 53.0 months). among the patients without pcr (n=38), approximately half had disease recurrence (range of recurrence - free survival, 1.5 to 19.7 months). cd20-positive cell density (hr, 1.004 ; 95% ci, 1.000 to 1.007 ; p=0.037), hev density (hr, 20.325 ; 95% ci, 1.455 to 283.857 ; p=0.025), and the levels of the cxcl13 mrna transcripts (hr, 1.706 ; 95% ci, 1.079 to 2.696 ; p=0.022) were correlated significantly with pcr. in survival analysis, the cd8-positive cell density and levels of the cxcl13 mrna transcripts were closely associated with the disease - free survival (table 4). the patients ages ranged from 23 to 70 years (median, 42 years) ; all patients were women. among the 108 cases analyzed, eight were ct1 tumors, 58 were ct2 tumors, 37 were ct3 tumors, and five were ct4 tumors. for the nodal status, there were six cn0 tumors, 48 cn1 tumors, 23 cn2 tumors, and 31 cn3 tumors. the median patient follow - up duration was 34.9 months (range, 12.0 to 55.8 months). table 1 summarizes the clinicopathological characteristics of the tumors without and with available ffpe blocks. no significant differences were observed between the two groups regarding the patients age, clinical t or n stage, histological grade, tils, tlss, pcr rate, miller - payne grade, or rcb class. based on assessments of the h&e - stained sections, uniand multi - variate analyses were performed to assess the associations between pcr, til, and other established clinicopathological parameters (table 2). among the 108 tumors, univariate logistic regression analysis revealed a lower ct stage and higher til and tls levels in the pre - nac biopsy to be significantly associated with pcr. multivariate analysis, including the ct stage, til, and tls, showed that only til (hazard ratio [hr ], 1.038 ; 95% confidence interval [ci ], 1.012 to 1.065 ; p=0.004) was an independent predictor of pcr. in univariate disease - free survival analysis, the ct and til were closely associated with a longer disease - free survival rate (table 3). on the other hand, multivariate analysis, including the ct stage, cn stage, til, and tls, showed that only til (hr, 0.975 ; 95% ci, 0.954 to 0.997 ; p=0.028) was a robust independent prognostic factor for the disease - free survival. ihc and gene expression analyses were carried out in 55 cases with available ffpe tissue blocks. the median of the tissue areas in each slide was 69.46 mm (range, 23.47 to 175.33 mm), whereas the median of the tumor areas was 20.92 mm (range, 1.23 to 83.15 mm). 1). finally, the mean cd3-, cd8-, and cd20-positive cell densities in the tumor areas of each slide were 357.2412.2/mm, 402.2382.4/mm, and 246.8343.5/mm, respectively (fig. the til and tls levels were correlated based on the h&e - stained slides, the densities of hev and til subpopulations, and expression levels of the cxcl13 mrna transcripts using the nanostring method with the pathological responses to nac (supplementary table 1). the tls levels were correlated significantly with the til levels, cd3-, cd8-, and cd20-positive cell densities, the hev density, and expression levels of the cxcl13 mrna transcripts. the miller - payne grade was significantly correlated with the tls level, cd20-positive cell density, hev density, and expression levels of cxcl13 mrna transcripts. based on an analysis of the ihc slides, 17 patients (30.1%) with available ffpe tissue blocks achieved pcr ; none of these patients showed a recurrence during a median follow - up period of 31.4 months (range, 21.1 to 53.0 months). among the patients without pcr (n=38), approximately half had disease recurrence (range of recurrence - free survival, 1.5 to 19.7 months). cd20-positive cell density (hr, 1.004 ; 95% ci, 1.000 to 1.007 ; p=0.037), hev density (hr, 20.325 ; 95% ci, 1.455 to 283.857 ; p=0.025), and the levels of the cxcl13 mrna transcripts (hr, 1.706 ; 95% ci, 1.079 to 2.696 ; p=0.022) were correlated significantly with pcr. in survival analysis, the cd8-positive cell density and levels of the cxcl13 mrna transcripts were closely associated with the disease - free survival (table 4). to the best of the authors knowledge, the current study is the first to identify and measure the tlss in biopsy specimens in an nac setting, and evaluate their predictive value. several reports have indicated a prognostic value for the presence of tlss in cancers [7,11 - 14 ]. on the other hand, no studies have assessed this in the context of nac, which is performed frequently in the treatment of breast cancer. in the present study, both higher levels of tlss represented by meca79-positive hev densities and higher levels of tils in the h&e slides were found to be predictors of pcr. in previous studies, currently, measurements of tils are generally performed based on visual assessments of the h&e - stained sections, which is an inexpensive, simple, and versatile method that is often easier to perform than complementary methods, such as ihc. in h&e - stained tissues, however, it can be difficult to distinguish lymphoid aggregates from true tlss and it is particularly challenging to quantify the amount of tlss. in addition, there have been trials in which dendritic cell lysosomal associated membrane protein (dc - lamp)positive dendritic cells were counted as surrogate markers for tlss. this represents a quantitative and objective method compared to simple visual assessments, even though counting a large number of small dendritic cells by microscopy is quite demanding. in addition, the tlss show a wide spatial heterogeneity in tumor tissues, and may therefore, be underrepresented when measured in small core needle biopsy specimens or with tissue microarrays. this can be problematic for breast cancers because many breast cancer patients only receive neoadjuvant systemic therapy after a needle biopsy, which represents the only tissue reference available for determining the drug regimens and assessing responses. previously, the expression of immune - related genes in pre - nac biopsies obtained from tnbc patients was investigated and the expression of the cxcl13 mrna transcripts, which encodes a potent b cell - attracting chemokine that contributes to tls formation in inflammatory and cancerous settings, was found to be a predictor of pcr, which indicates a better prognosis in tnbc patients. on the other hand, it can be easy to under or overestimate gene expression analyses of each biopsy specimen because the proportion of tumor versus normal tissues in a biopsy specimen is not considered. furthermore, gene expression analysis is expensive and can be cumbersome to perform in daily pathology practice. despite these difficulties, the present study obtained some important findings based on meca79 ihc staining. the number of meca79-positive hevs were counted in each slide and the hev density was calculated. this quantitative method is easier than counting dc - lamp because of the small number of hevs. the hev density was positively correlated with the amount of tlss measured by assessing the h&e slides, and a greater hev density was not only a predictor of pcr, but was also associated with a better disease - free survival. hevs contribute to til infiltration, and consequently to anti - tumor immunity, so cancer immuntherapy approaches have been proposed, in which the endothelial cell properties are modified to increase immune cell infiltration. hopefully, the discovery of a method to increase hev in the peritumoral areas will be beneficial for the development of treatment strategies for breast cancer, along with many other cancer types. finally, the densities of the til subpopulations were measured based on ihc staining for cd3, cd8, and cd20, along with a digital microscopic scanner and automated analysis software. several studies used ihc to evaluate the til subpopulations in breast cancer [7,20,25 - 27 ]. on the other hand, most of those studies used semiquantitative methods for the measurements because fully quantitative methods are often difficult and time - consuming due to the large number of tils. using automated computer software, quantified the cell densities were more accurately and the inter - observer bias was avoided. a higher cd20-positive cell density was found to be a predictor of pcr, whereas a higher cd8-positive cell density was associated with a better disease - free survival. cd8-positive cytotoxic t lymphocytes exhibit antitumor effects via direct tumor cell lysis and interferon release, which are strongly associated with better therapeutic responses and enhanced patient survival. in contrast, the effects of b cells on the tumors remain the subject of debate. some studies have reported that b - cell infiltration is associated with a good prognosis. the possible mechanisms whereby b cells might mediate improved anti - tumor responses have been suggested, such as the generation of anti - tumor antibodies, the secretion of proimmunogenic cytokines and chemokines, antigen presentation to t cells, and direct cytotoxicity via granzyme b production. nevertheless, more study will be needed to explore how the immune components interact in response to tumors. the density of meca79-positive hev is an easy to assess and accurate surrogate marker of tls in needle biopsies of tnbc. | purposethe tertiary lymphoid structure (tls) is an important source of tumor - infiltrating lymphocytes (tils), which have a strong prognostic and predictive value in triple - negative breast cancer (tnbc). a previous study reported that the levels of cxcl13 mrna expression were associated with tlss, but measuring the gene expression is challenging in routine practice. therefore, this study evaluated the meca79-positive high endothelial venule (hev) densities and their association with the histopathologically assessed tlss in biopsy samples. in addition, the relationship of tlss with the cxcl13 transcript levels and clinical outcomes were examined.materials and methodsa total of 108 tnbc patients treated with neoadjuvant chemotherapy (nac) were studied. the amounts of tils and tlss were measured histopathologically using hematoxylin and eosin stained slides. the hev densities and til subpopulations were measured by immunohistochemistry for meca79, cd3, cd8, and cd20. cxcl13mrna expression levels using a nanostring assay (nanostring technologies).resultsthe mean number of hevs in pre - nac biopsies was 12 (range, 0 to 72). the amounts of tils and tlss, hev density, and cxcl13 expression showed robust correlations with each other. a lower pre - nac clinical t stage, higher til and tls levels, a higher hev density, cd20-positive cell density, and cxcl13 expression were significant predictors of a pathologic complete response (pcr). higher cd8-positive cell density and levels of cxcl13 expression were significantly associated with a better disease - free survival rate.conclusionmeca79-positive hev density in pre - nac biopsies is an objective and quantitative surrogate marker of tls and might be a valuable tool for predicting pcr of tnbc in routine pathology practice. |
the ability to communicate effectively is one of the most essential skills to have as a scientist. not only is the exchange of ideas critical for the advancement of research and human health, but also communicating major discoveries to the public is essential to the future of biomedical research funding. although the internet has opened the door to new ways of communicating scientific ideas and results, cutting - edge research is still mostly presented in the form of scientific articles published in journals with a pay - per - view or subscription service. ibiology (www.ibiology.org) provides a free and open online platform for communicating the practice of science. the site, which is split into ibioseminars, ibiomagazine, and ibioeducation sections, holds a growing collection of more than 275 videos by scientists about research and topics related to science. the project is funded by the national science foundation, the national institute of general medical sciences, and the howard hughes medical institute and is supported by the american society for cell biology and the university of california at san francisco. seminars are one of the most important forms of communication within the scientific community. in addition to allowing scientists to convey their findings to one another, seminars usually lay out the thought process behind a series of experiments, set them in the context of the field, and describe the steps taken to reach the conclusions presented. often seminars present years of experiments and results, making them an effective way to learn about an area of science. as new technologies are rapidly being developed and applied in novel ways, seminars are useful for keeping up with the latest cutting - edge techniques. scientists also develop fresh ideas after attending seminars, resulting in new experimental directions or interest in a new area of research. despite their importance, accessibility to seminars by scientists who are leaders in their field is limited, as speakers can travel to only a small number of institutions or conferences each year. in 2006, ron vale, of the university of california at san francisco, realized that the internet could be used to make cutting - edge research seminars accessible to anyone with an internet connection. to give a face and personality to the scientist, he used green - screen technology to allow viewers to see both the slides and the speaker simultaneously. with some seed money from the carnegie foundation and the howard hughes medical institute, the first videos, called ibioseminars, were created with seminars by joseph derisi (university of california at san francisco), julie theriot (stanford university), baldomero olivera (university of utah), and martin raff (medical research council). since then, ibioseminars has produced more than 80 seminars covering topics ranging from ecology to chemical biology, allowing a worldwide audience to hear talks from leading researchers. two years after launching ibioseminars, ron vale developed another series of videos, called ibiomagazine, aimed at offering more insight into what it really means to be a scientist, with videos about famous discoveries, professional development, education, outreach, and scientific careers. nobel laureate tom cech quietly did an early experiment to show that rna can catalyze splicing because he thought that the experiment might not work and did not want it to fail in front of his lab (cech, 2010). ron vale discovered kinesin after a slam - dunk control experiment did not work (vale, 2010). cynthia kenyon could not convince a graduate student to work on aging, so she published her first paper on the subject with several rotation students (kenyon, 2010). for nobel laureate martin chalfie, a decision to only clone the coding sequence of green fluorescent protein instead of using an existing plasmid with an extra gene sequence was instrumental in seeing green fluorescence in escherichia coli (chalfie, 2011). these are a few of the many behind - the - scenes ibiomagazine stories about major discoveries that demonstrate that trusting one 's intuition, overcoming significant roadblocks, and allowing for uncertainty and guesswork are key aspects of major scientific discoveries. research articles typically present the end result and the culmination of years worth of work, leaving out some of the realities of the scientific process. the ibiomagazine videos allow aspiring scientists to learn these important lessons directly from the authors of these studies. ibiomagazine videos include other types of advice for young scientists. in his talk, bruce alberts describes the lessons he learned from his failures and advises aspiring scientists to study their failures carefully and learn from them, pointing out that successful people never make the same type of mistake twice (alberts, 2010). lydia villa - komaroff talks about how persistence through tough or discouraging times is one of the most important attributes for a scientist (villa - komaroff, 2011). enrique de la cruz emphasizes the need to use time wisely and have a weekly plan that is constantly being revised and evaluated to make sure one is spending time as productively as possible (de la cruz, 2014). ibiomagazine talks also provide a window into the unexpected career paths of successful scientists. alfredo quiones - hinojosa, a neurosurgeon at johns hopkins university, came to the united states from mexico as an undocumented immigrant when he was a teenager and worked on a farm before going to college (quiones - hinojosa, 2011). erich jarvis, a neuroscientist at duke university, chose science over a career as a professional dancer (jarvis, 2012). yixian zheng, a cell biologist at the carnegie institution for science, grew up during the cultural revolution in china and was an aspiring novelist before realizing that she enjoyed biology (zheng, 2011). these life stories demonstrate that scientists came from a variety of backgrounds and took a wide range of paths before they became the successful scientists they are today. each of these videos offers unique insights into the experiences of scientists and provides important lessons for young scientists or students thinking about a career in science. by identifying common themes the importance of persistence, trusting one 's instinct, learning from failure we can also start to identify characteristics that successful scientists share and that aspiring scientists can learn from. in 2013, ibioeducation was added to the ibioseminars and ibiomagazine series, and all were merged into one site named ibiology. ibioeducation is a place for educators to find video resources and accompanying assessments related to biology education. over the past several decades, there has been an intense push in biology education to shift away from teaching through lecture and toward helping students develop critical thinking and analytical skills through active learning (american association for the advancement of science, 2011). study after study has shown that incorporating active learning activities into a classroom increases student learning gains and results in higher grades and lower dropout rates than lecture - based classes (hake, 1998 ; handelsman., 2004 ; minner., 2010 ; freeman, 2014). although ibiology videos are online lectures, can they be used in active learning approaches to promote skill development and facilitate student learning about the process of science ? at the university of california at los angeles, utpal banerjee and ira clark have used an ibioseminar video in their research deconstruction model, in which students learn to analyze data and think critically about experiments presented in a scientific seminar. in this model, instructors and students spend each class session analyzing one segment of a seminar, and students learn about the fundamental concepts underlying the experimental approaches, analyze data, and think critically about the research. at the end of this course, students are able to discuss the experiments critically and apply what they learned to other seminars, as well as to hypothetical research scenarios (clark., 2009). ibioseminar videos have also been used in flipped classroom models, including one developed in a collaboration between ibiology and jon scholey at the university of california at davis. in this model, students watch the ibioseminars for homework, which gives them the content needed for the next class period. class time can then be spent discussing a scientific question related to the seminar, analyzing data from the experiments presented in the video, and critically examining conclusions made from the research. other instructors have also used this flipped classroom approach to increase problem - based learning and small - group work during class time, activities that have been demonstrated to improve student learning (springer., 1999 ; allen and tanner, 2003). humanizing the scientific process or providing historical relevance can help provide context when students are learning about topics or concepts (chamany. ibiology talks about science discoveries, as mentioned in the preceding section, often give insights into the failures, roadblocks, and serendipitous paths that are encountered in the quest for new knowledge. other talks give historical insights into issues surrounding research on recombinant dna, stem cells, and the teaching of evolution, giving a societal context to these concepts. ibiology talks can also be combined with corresponding primary literature, giving a face and personality to the scientist behind the research. the scientific teaching series features videos addressing problems in how biology has been taught traditionally and providing tools for educators to introduce active learning into their classroom. this series incorporates perspectives and experiences from multiple instructors, has classroom footage of active learning in action, and highlights evidence from the literature that supports active learning approaches. how can resources such as ibiology harness the power of the web in other ways to increase exposure to scientific ideas and thinking ? recently, ibiology has been using a tool called google hangouts on air to visually connect people from around the world with thought leaders in the scientific community. google hangouts on air provides the ability to host a live question and answer session (q&a) by creating a video broadcast in which viewers can submit questions and vote on questions submitted by other viewers. to date, seven google hangouts on air have been hosted by ibiology about research, academia, industry, education, and policy, featuring leaders in the scientific community such as bruce alberts, bonnie bassler, and lydia villa - komaroff. prominent speakers such as gregory petsko, keith yamamoto, and jon lorsch had q&as focused on current biomedical workforce issues. the typical model for these q&as involved recording an ibiomagazine video before the q&a, freeing time during the google hangouts on air broadcast to field questions from the audience. soon, ibiology plans to build a forum on which scientists can share their expertise and experiences and connect with one another. with the web, conversations about science need not be limited to institutions or conferences and can include those outside of the usual scientific circles. now we can create a global conversation with a worldwide network of people about research, education, and issues in the scientific community, helping to make sure science flourishes well into the future. | the internet hosts an abundance of science video resources aimed at communicating scientific knowledge, including webinars, massive open online courses, and ted talks. although these videos are efficient at disseminating information for diverse types of users, they often do not demonstrate the process of doing science, the excitement of scientific discovery, or how new scientific knowledge is developed. ibiology (www.ibiology.org), a project that creates open - access science videos about biology research and science - related topics, seeks to fill this need by producing videos by science leaders that make their ideas, stories, and experiences available to anyone with an internet connection. |
type 2 diabetes mellitus (t2 dm) is a metabolic disease that affects over 340 million people worldwide. the dysfunction and degeneration of pancreatic -cells are caused amongst others by the formation and deposition of extracellular amyloid plaques [14 ]. however, its main amyloidogenic component human islet amyloid polypeptide (hiapp), also named amylin, was extracted and sequenced 85 years later [7, 8 ]. therefore, t2 dm belongs to the protein misfolding diseases, also known as proteopathies, which are associated with abnormal accumulation of insoluble fibrillar protein aggregates in tissues and organs. although distinct proteins are involved in the formation of those deposits in different diseases such as alzheimer 's disease, parkinson 's disease, huntington 's disease and t2 dm, amyloids feature a common morphology with cross--sheets as secondary structure [912 ]. some peptides, including peptide hormones, show a tendency to aggregation due to their small size, lack of secondary structure as well as their appearance at high local concentrations [13, 14 ]. during aggregation, specific species such as monomers, oligomers and fibrils can be observed at different stages. in the past it was believed that the fibrillar deposits are the toxic species and are responsible for the pathological phenotype of the disease because they are found in post mortem organs or tissues. nowadays, there is much evidence that the aggregation process itself or even the intermediate species are cytotoxic, whereas the final fibrillar aggregates and inclusions, respectively, may even have protective functions [11, 1517 ]. however, fibrils are the best studied species due to their low solubility and high stability. the in vitro formed fibrils consist of several proto - fibrils which are twisted around each other and feature intermolecular -sheets perpendicular to the fibril axis. iapp is a 37 amino - acid residues long peptide hormone, which is coproduced and cosecreted along with insulin through the secretory pathway in -cell in a ratio of 1 : 100, but can increase to 1 : 20 in case of t2 dm. during protein translation, hiapp is processed and modified (figure 1). the 20 amino - acid residue long signal peptide, which is located at the n - terminus, guides the protein from the endoplasmic reticulum (er) to the trans - golgi network. during that transport. arrived at the trans - golgi network, the signal peptide is cleaved resulting in a 67 amino - acid residue long proiapp, which is further processed by prohormone convertase (pc) enzymes. first, the 16 c - terminal amino acids are cleaved by pc1/3 in the trans - golgi network. next, in the secretory vesicles, pc2 cleaves the 11 n - terminal amino acids. finally, carboxypeptidase e (cpe) catalyzes the cleavage of the two c - terminal basic amino acids and activates the peptidyl amidating monooxygenase (pam) complex which operates the cleavage of glycine at position 38 and the amidation of tyrosine at position 37 [1, 1922 ]. contradictory to the concept of folding funnels, the monomeric hiapp is intrinsically disordered and thus features numerous flexible and random coil conformations [2325 ] with a transient amphipathic helix in the n - terminal region [26, 27 ]. it has been suggested that aggregated iapp is folded into a double -hairpin with three -strands between residues 12 and 37. however, an alternative atomistic structural model of a single -hairpin has been obtained from three independent studies based on nuclear magnetic resonance (nmr), electron paramagnetic resonance (epr) and x - ray diffraction approaches [3032 ], but they differ in the details of the monomeric folding and the packing of the peptide within the fibrils. principally, the polymorphic nature of amyloid fibrils provides the possibility that different monomeric conformations and various sets of interresidue interactions within the fibrils can coexist. interestingly, amyloid formation of iapp does not occur in every mammalian species, although its primary structure is well conserved through evolution. for example, pathological deposition of iapp amyloids can not be found in the islets of langerhans of rodents. the main reason for this is proline mutations (acting as -sheet breaker) in the most fibrillogenic iapp2029 region. similar to its monomeric structure, the effect and exact physiological functions of hiapp are contradictory and still under debate. due to its cosecretion with insulin, it may act as a hormone regulating the glucose homeostasis. despite the difficulty to distinguish between its physiological and pathophysiological effects, first, it acts as an auto- or paracrine molecule in the islets of langerhans regulating the secretion of insulin and glucagon. however, the results are ambiguous, ranging from stimulation via no effect through to inhibition [3442 ]. this phenomenon might be explained by the fact that monomeric hiapp as an intrinsically disordered protein (idp) features conformational diversity and thus interacts with its target differently depending on its conformation. second, hiapp functions as a hormone for the central nervous system. as an anorectic moreover, inhibitory effects of hiapp on gastric emptying and bone resorption have been reported. the clear link between islet amyloid deposition and reduction of the -cell mass gives rise to the pathophysiological effects of hiapp. an increased expression level and aggregation of hiapp have been reported to cause dysfunctions and death of -cells at different subcellular levels. an inductive effect of hiapp on the unfolding protein response (upr) within the er compartment has been detected when the expression level of hiapp is upregulated. however, contrary findings have also been reported [45, 46 ]. therefore, the er stress and its role in iapp toxicity are controversial and are still an open question. additionally, accumulation of polyubiquitinated proteins and autophagosomes have been found in -cells from t2 dm patients at autopsy, indicating dysfunction of the two major intracellular protein degradation systems ubiquitin - proteasome system (ups) and autophagy [4851 ]. there is also evidence that hiapp contributes to islet inflammation by being internalized in macrophages in its aggregated state in order to activate the nlrp3 inflammasome and thus the production of the pathogenic cytokine il-1 [5254 ]. an additional major pathophysiological effect of hiapp is its interaction with membranes. in vitro studies show that hiapp fibrillation is membrane - mediated, especially in the presence of anionic lipids [23, 5559 ]. exogenous exposure of pancreatic -cells (ins-1) to hiapp via culture medium causes dysfunction of mitochondria and finally death of cells [60, 61 ]. there is much evidence that hiapp can escape from the secretory pathway by attacking the vesicle membrane [6267 ]. thus, the hiapp - mediated cytotoxicity is proposed to be initiated by intracellular oligomerization and fibrillation and presumably caused by disrupting membrane integrity of different cellular compartments, but its mechanism is still under debate. three general, but not exclusive theories of membrane disruption by hiapp have been developed (figure 2). (1) the nonspecific model, where the membrane integrity is disturbed by fibril growth on the membrane. in a detergent - like mechanism hiapp fibrillation causes large - scale defects in the lipid bilayer, resulting in membrane thinning and fragmentation, accompanied by increased membrane conductance [6873 ]. (2) binding of monomeric hiapp to the membrane facilitates the structural transition of hiapp from a disordered structure into a partially -helical conformation, followed by oligomerization. the hydrophobic and membrane permeable on - pathway oligomers represent the toxic species in this case [17, 26, 74 ]. (3) electrophysiological measurements and small molecule selectivity support the pore theory, where hiapp forms ion channel - like pores (barrel stave) within the membrane, leading to deficient ion homeostasis [7579 ]. however, the relationship between the formation of islet amyloid and the onset of t2 dm is still largely unknown. the question if hiapp aggregation is a cause for -cell dysfunction and destruction or just a consequence remains unanswered. in secretory vesicles, hiapp exists at high concentrations (mm range). in vitro at those concentrations, hiapp would rapidly aggregate. this leads to the suggestion that hiapp has to be stabilized to prevent rapid aggregation in vivo. it has been shown that insulin, but not proinsulin, is able to inhibit hiapp fibril formation in vitro by forming heteromolecular complexes. therefore, deficient insulin processing would prevent this protective interaction and lead to hiapp aggregation [8082 ]. moreover, age - related changes of environmental factors (ph, salt concentration, chemical modifications, and changes in lipid composition) and protein homeostasis could also lead to destabilization of monomeric hiapp [80, 83, 84 ]. however, the exact factors that are responsible for hiapp aggregation are still poorly understood. in vitro, hiapp fibrillogenesis has been thoroughly studied by varying salt [85, 86 ], ph and temperature. on the other hand, hiapp transgenic mice have been developed to study the consequence of hiapp aggregation in vivo. however, less attention has been paid to studies considering the heterogeneity of cellular membrane systems and the highly crowded milieu encountered in cells. for example, it has been shown that hiapp cytotoxicity highly depends on the location of the peptide. there is an enormous difference if pancreatic -cells are exposed exogenously and endogenously to hiapp, respectively. here, we compare the amyloidogenic properties of hiapp in bulk solution and in the presence of various membrane systems which have been found to drastically modulate fibril formation. an interaction between extracellular islet amyloid fibrils and -cell membrane has already been reported in 1973. since then, the membrane disruption hypothesis for cytotoxicity has become the most studied for hiapp and is thus one major focus of this review, with a special focus on the structural changes that occur in hiapp upon membrane binding and aggregation as investigated in our laboratory. moreover, the effects of crowding agents and osmolytes, both important constituents of cellular environments, are discussed. the aggregation kinetics depends on the monomer concentration as well as on the presence of aggregation nuclei and is often too fast to be resolved by spectroscopic methods. one strategy to decelerate the fibrillation time, which is often needed for time - dependent studies, is directly derived from nature. after in vivo synthesis, hiapp is stored in the -cell granules of the pancreas at a ph of approximately 5.5, and, when in need, released into the extracellular compartment at a ph of 7.4. they also showed that these differences in kinetics are directly linked to changes in the conformational behavior of the peptide. one explanation is the protonation of his18, resulting in repulsive interactions between the peptides. in addition, hiapp is often stored in or pretreated with hexafluoroisopropanol (hfip) in order to dissolve any form of aggregates and to keep the peptide in its denatured monomeric conformation. lowering the ph value to 5.5 and pretreatment with hfip allowed the study of the conformation of monomeric hiapp by using far - uv cd, ftir, and nmr spectroscopy. a typical cd spectrum of monomeric hiapp exhibits a minimum at ~201 nm along with a shoulder around 220 nm, indicating a predominantly disordered structure (~40%) of hiapp in its initial conformation (figure 3(a)), which is in good agreement with ftir data showing an amide - i band maximum around 1645 cm. the predominant random coil conformation of native hiapp is also in agreement with literature data [24, 25 ]. corresponding results of molecular dynamics (md) simulations revealed an essentially random - coiled conformation of hiapp in solution, although transient -helices were observed as well. 2d - nmr spectroscopy data was also employed to elucidate the monomeric structure of hiapp and the role of specific amino acids. solution nmr data [59, 92, 93 ] also suggest that the monomeric states of hiapp transiently sample helical states and show a lack of stable secondary structures. thus, the helical state of hiapp seems to be a low - lying excited state conformer. relating to the hiapp self - association in the bulk phase, time - lapse nmr data strongly suggested that the n - terminal region of hiapp is involved in the initial step of aggregation, followed by transient -helical intermediate structures. this is consistent with observations that the presence of low percentages of the helix - inducing solvent hfip strongly catalyzes the aggregation of hiapp. in another recent md simulation study on monomeric hiapp, singh. highlighted the interconversion of hiapp between an -helix and a -hairpin as an important activating process that could be the initial step of the nucleation process. the extrinsic fluorescent dye thioflavin t (tht) has been established as a standard tool to follow the fibrillation process of amyloidogenic peptides. tht displays enhanced fluorescence upon noncovalent binding of mature amyloid fibrils where it binds to -sheet rich areas, probably in a channel binding mode [95, 96 ]. as an example, figure 3(b) shows data using the tht assay for 100 m hiapp in acetate buffer, ph 5.5 at 10c. a lag phase for the first ~100 h followed by a slow exponential growth phase with the fibril formation completed after ~400 h was detected. hiapp oligomers appeared nearly exclusively between the time points of 0 and 100 h which is the lag phase of the aggregation process. the mean height standard deviation of the oligomers detected at 0 h was 0.7 0.2 nm. exponential growth was observed subsequently in the tht assay where hiapp proto - fibrils are formed, as revealed by afm. these species exhibited a mean height of 3.9 1.0 nm at 150 h of incubation time. after longer aggregation times up to 28 days, higher - ordered m - long fibrillar structures were detected showing a mean height of 6.4 1.8 nm. only few oligomeric structures with a mean height of ~0.9 nm remained in the solution. the interaction of hiapp with lipid membranes has been considered to be a main reason for the cytotoxicity of hiapp. hence, the properties of hiapp, while interacting with lipid membranes of different composition, have been extensively studied. in the past, attenuated total reflectance fourier - transform infrared (atr - ftir) spectroscopy has been established in our laboratory as a promising tool to investigate the time - dependent secondary structural changes of hiapp aggregation in the presence of lipid bilayers. in figure 4, the aggregation propensity of hiapp in the presence of a neutral, zwitterionic dopc and an anionic dopc / dopg 7 : 3 w / w lipid bilayers was evaluated by atr - ftir spectroscopy. comparison of the time evolution of the amide - i band shows that the presence of dopc / dopg strongly favoured the peak shift of the amide - i band from 1644 cm to 1627 cm, indicating a decrease in unordered conformations and a concomitant increase in intermolecular -sheet structures. a broad peak at 16161619 cm appeared during the aggregation process, reflecting the formation of intermolecular -sheets with strong hydrogen bonding. conversely, no significant aggregation could be observed in the presence of the pure zwitterionic dopc membrane within 30 h. only a small shoulder appeared after 20 h in the intermolecular -sheet region around ~1625 cm, indicating the formation of less ordered, probably oligomeric, aggregate structures after long incubation times. in many amyloidogenic proteins, small oligomers have been found to form metastable intermediates such as small oligomers which are transiently formed and rapidly converted to amyloid fibrils. by performing sedimentation velocity experiments, vaiana. showed that only < 1% of the total population of hiapp form low - weight oligomers before fibrillation, indicating that once oligomeric aggregation - prone species are formed, they are rapidly utilized in the formation of -rich fibrils. the observation of an essentially all - or - nothing process of hiapp aggregation can also be found in the atr - ftir data (figure 4(d)), which reveals an isosbestic kind of point between the initial and the final aggregate state, indicating the absence of a large amount of intermediate oligomeric species. complementary results have been obtained using infrared reflection absorption spectroscopy (irras) on lipid monolayers. no significant aggregation of hiapp was observed in the presence of a neutral, zwitterionic popc lipid monolayer, whereas results for negatively charged popg monolayers revealed significant fibrillation. thus, the aggregation process of hiapp is considerably enhanced in the presence of lipid bilayers with a negatively charged head group. additional x - ray reflectivity (xrr) and afm studies confirmed that the accelerative effect is initiated by an electrostatic interaction between the positively charged n - terminal amino acid residues of hiapp and the negatively charged lipid head groups. based on the data obtained a multiple step fibrillation mechanism for hiapp was proposed : first, hiapp inserts into the membrane through its n - terminus via electrostatic interaction resulting in a conformational transition from a predominantly random coil structure to an -helical conformation. knight and coworkers showed that membrane binding of iapp is a cooperative process leading to the formation of membrane - bound and heterogeneous -helical aggregates. the structural conversion of the monomeric iapp from predominantly disordered to -helical and the subsequent formation of heterogeneous -helical aggregates upon membrane binding hold true for both amyloidogenic hiapp and nonamyloidogenic ratiapp. thereafter, rapid conversion to a -sheet conformation of hiapp takes place, followed by formation of ordered fibrillar structures. in addition, membrane binding causes a reduced dimensionality and thus an increased local peptide concentration which finally promote the growth of hiapp fibrils. in a recent md study by jia. it has been found that the quick adsorption of hiapp monomers to the lipid bilayer surface is mediated by strong electrostatic interactions of the positively charged residues k1 and r11 with the negatively charged lipid head groups. a stable helix through residues 722 realizes a parallel binding of hiapp to the lipid bilayer surface via electrostatic and h - bonding interactions. this is in agreement with the observation that the fragment hiapp2029 features a lower affinity to membrane and without any preference for anionic lipids. the facts that hiapp119, the nonamyloidogenic fragment, and ratiapp at high concentrations are able to cause disruption in anionic lipids as well suggest that amyloid formation is not a necessary condition for membrane damage [74, 102 ]. this is in line with kinetic leakage studies with hiapp wildtype and mutants by cao. these results would fit to the model of a biphasic kinetics of membrane disruption by hiapp with distinct fibril - independent and fibril - dependent phases as shown by dye leakage experiments [68, 104 ]. interestingly, his18 plays an important role in the orientation of the peptide on the membrane and its protonation, as found in -cell granules, might modulate the membrane disruption effect of hiapp [103, 105 ]. however, the exchange of histidine to arginine at position 18 and the lower lipid affinity of ratiapp at ph 7.4 compared to hiapp suggest that the electrostatic contribution is not the only factor controlling the membrane binding behavior. a conformational change in the -helix induced by the difference between the membrane - binding domains of hiapp and ratiapp might define their membrane affinity. upon formation, the mature hiapp fibrils show evidence to detach from the lipid membrane into the bulk solution or they remain adsorbed at the lipid interface. via direct fluorescence microscopic observation, domanov and kinnunen showed that hiapp fibrillation on the surface of supported lipid bilayers induces deformation, vesiculation and tubulation of the membrane. in addition, hiapp fibrils have been observed to be coated by lipid membranes derived from the vesicles and tubes. complementary, sasahara. performed fluorescence recovery after photobleaching (frap) measurements and reported on a significant fluidity decrease of model lipid bilayers upon binding of soluble hiapp, suggesting morphological and functional perturbation caused by the hiapp - membrane interaction. they play a key role in many biological processes, such as modulating a broad range of signalling cascades [108110 ]. therefore, hiapp - membrane studies were extended to neutral and anionic heterogeneous membrane systems displaying a coexistence of liquid - ordered (lo) and liquid - disordered (ld) phase. in our laboratory, a neutral dopc / dppc / cholesterol (1 : 2 : 1) and an anionic dopc / dopg / dppc / dppg / chol (15 : 10 : 40 : 10 : 25) lipid raft mixture, both exhibiting lo and ld phase coexistence were used [111, 112 ]. confocal fluorescence microscopy of giant unilamellar vesicles (guvs) of those lipid mixtures showed that hiapp rapidly and preferentially partitions into the liquid - disordered (ld) domains of the neutral model raft membrane, that is, the domains containing less cholesterol. with time, hiapp was observed to induce permeabilization of the membrane and disintegration of the guvs. however, colocalization of hiapp and the fluid lipid domain was still detectable, indicating an incorporation of lipids into the hiapp aggregates. after ~72 h of incubation no intact guvs were detectable anymore [17, 111 ]. the same systems were investigated by time - lapse tapping mode afm to yield structural data on a nanometer scale. the results indicated a rapid permeabilizing effect of hiapp on the zwitterionic lipid raft membrane (dopc / dppc / chol, 1 : 2 : 1), accompanied by disruption of the lateral organization of the lipid bilayer within minutes after peptide addition. this degrading effect of hiapp to the heterogeneous membrane seemed to occur through an unspecific, detergent - like mechanism. corresponding studies carried out on the anionic lipid raft membrane (dopc / dopg / dppc / dppg / chol, 15 : 10 : 40 : 10 : 25) showed an even more accelerated kinetics than for the aggregation in the presence of the homogeneous anionic lipid membrane. complementary atr - ftir studies revealed a slower aggregation kinetics of hiapp in the presence of the neutral heterogeneous membrane compared to the scenario with 30% anionic membrane. taken together, these data clearly demonstrate that the hiapp - membrane interaction is more pronounced at anionic membranes since a stronger adsorption of hiapp to both the homo- and heterogeneous anionic membrane compared to the neutral membrane systems was observed. the electrostatic interaction between the positively charged n - terminal amino acid residues of hiapp and the negatively charged lipid head groups were found to be a dominating effect causing the peptide - membrane interaction. however, whereas hiapp does not seem to aggregate substantially in the presence of the homogeneous zwitterionic membranes, significant aggregation at heterogeneous zwitterionic bilayers takes place as well, as also found for other raft containing membrane systems. the rapid initial adsorption of hiapp at defect states, such as the rim of the coexisting lo and ld lipid domains, may be the reason for an increased local peptide concentration at the heterogeneous membrane which leads to an enhanced fibrillation even in the absence of charged head groups. next to the lipid bilayer 's lateral organization, also the constituting lipid components influence the aggregation propensity of hiapp. for example, phosphatidylethanolamine (pe), a lipid with an intrinsic negative curvature, has been shown to hamper the fibril - independent phase of membrane disruption, but enhances the membrane leakage correlated with the growth of fibrils on the membrane surface via a detergent - like mechanism. cholesterol has been found to effectively modulate hiapp fibrillation as well [115, 116 ]. model lipid bilayers consisting of few components are often insufficient in order to represent the scenario in vivo. therefore, our laboratory extracted cell membrane lipids from a pancreatic -cell line of rat (ins-1e) to be able to study the hiapp - membrane interaction in a more natural lipid environment. mass spectrometry analysis of the extracted lipids revealed phosphatidylcholine (pc) as the major head group component of the lipid mixture and a ratio of 2.5% negatively charged lipids. the atr - ftir spectroscopy data show that hiapp adsorbs readily at the membrane and shows an increasing amide - i band intensity at around 1623 cm indicating intermolecular -sheet formation already 1 h after the measurement was started (figure 5). these findings are similar to those obtained for hiapp aggregation and fibrillation at the homo- and heterogeneous anionic lipid membranes. however, a much stronger adsorption of hiapp to the biological model membrane was detected (figures 5(b) and 5(f)). this could be explained by a higher roughness of the biological membrane with a higher concentration of membrane defects which could foster the interaction of hiapp with the membrane surface. fluorescence microscopy measurements on giant unilamellar vesicles (guvs) of the extracted biological lipids from the -cell membrane supported these findings. to detect the permeabilization of the vesicles upon hiapp - induced disintegration of the lipid membrane, a leakage test was employed. the lipids were labelled by addition of n - rh - dhpe (n-(lissamine rhodamine b sulfonyl)-1,2-dihexadecanoyl - sn - glycero-3-phospho - ethanolamine triethylammonium salt). for visualization of hiapp, the peptide was c - terminally labelled with bodipy - fl and a 5 m solution of hiapp - k - bodipy - fl was added to the guvs. fluorescence microscopy images of the interaction are depicted in figure 6. at first (t = 0 min) the guvs are shown before hiapp was added, to visualize the atto647 fluorophore containing buffer (blue channel) within the n - rh - dhpe labelled guvs (red channel). already after 5 min, hiapp - k - bodipy - fl (green channel) could be mainly detected at the lipid membrane of the vesicles and led within the next minutes to permeabilization and leakage of the membrane. however, colocalization of hiapp and the biological membrane was still detectable even after t = 40 min. at this time point, disintegration of the guvs was observed, indicating incorporation of lipids into the growing hiapp aggregates. taken together, the rapid permeabilization and disintegration of guvs observed and induced by soluble hiapp confirm a fibril - independent mechanism of membrane disruption. subsequent guv disintegration and lipid incorporation into the hiapp aggregates give evidence for a second fibril - growth dependent mechanism of membrane disruption, which is in agreement with literature data [68, 75 ]. one reason for the fibril - independent mechanism could be the hiapp - insertion induced formation of negative curvature within lipid bilayers as smith. concluded from studies using bicelles. hiapp- and pe - induced curvature effects may be expected to feature different geometries and energetics, however. the intrinsic negative curvature of pe containing membranes hampers a deep insertion of the peptide into the membrane and favors a shallow binding of amyloid fibers onto the membrane. hence, insertion of the peptide into the membrane and thus the fibril - independent induced membrane disruption seem to sensitively depend on the geometry and curvature elastic stress of the membrane. upon fibril growth, further curvature might be induced by the twist of the -sheets of the hiapp fibrils. in addition, lipid extraction from the lipid bilayers has been observed during fibril growth which might be a second reason for the fibril - dependent membrane damage. the pore model for the fibril - independent membrane leakage has been put forward by last and miranker. using the amphipathic peptides magainin 2 and ratiapp, they pointed out that initial binding of the peptide in the intermediate region between the head group and acyl chains of the bilayer, expands the head group region relative to the acyl region of the membrane. this results in a thinning of the acyl chain region and thus the formation of an internal surface tension within the bilayer due to the nonideal packing of the acyl chains. recent imaging total internal reflection - fluorescence correlation spectroscopy (itir - fcs) studies suggest a carpet model and showed that below the critical concentration for peptide aggregation and upon binding to the plasma membrane of living cells, monomeric hiapp increases the fluidity of the plasma membrane by carpeting the plasma membrane and forming microdomains. such dynamic microdomains, presumably consisting of peptide - lipid complexes, are able to extract lipids in a hiapp - concentration dependent manner [121, 122 ]. in vivo, stability and function of proteins are tightly controlled by a protein control network, including chaperone - mediated folding and degradation of misfolded proteins via proteasome and autophagy. its deficiency results in accumulation of misfolded proteins and causes protein misfolding diseases including t2 dm. the mechanism of hiapp - induced cytotoxicity is not yet fully understood, but it seems to cause dysfunctions at different cellular and subcellular levels including production of ros, er - stress, defects in ups and autophagy, increased production of proinflammatory cytokines, and in particular permeabilization of plasma and mitochondria membranes. the hypothesis of intermediate species such as oligomers gaining toxic functions and causing degeneration of functional cells is common [912 ]. however, such oligomers are metastable, probably polydisperse in nature, and thus ill - defined. using the pancreatic -cell line ins-1e and a wst-1 cell proliferation assay, the results of the proliferation assay clearly show a correlation between the hiapp cytotoxicity and the aggregation time. within the lag phase of hiapp aggregation, the cells showed only small survival rates between 3.5 and 10% where predominantly oligomeric hiapp species have been found by afm experiments. proceeding of the growth reaction led to an increase in tht intensity correlated with a significant decrease in cytotoxicity. samples taken within this exponential growth phase contained mainly proto - fibrils as detected by afm and showed an 8085% survival rate of ins-1e cells. mature hiapp fibrils, as found in the saturation phase of hiapp aggregation and confirmed by afm, exhibited with 90% cell viability the lowest comparative cytotoxicity. as a control, samples at different time points after incubation with ratiapp were taken. at same concentration, the nonamyloidogenic ratiapp did not show cytotoxicity within the whole incubation period (figure 3(d)). however, using the mtt assay (loss of mitochondrial activity) and elevated peptide concentrations or another cell line, ratiapp has been reported to be also cytotoxic [61, 123 ]. these results are in agreement with the biphasic mechanism of iapp - induced membrane disruption found in vitro. soluble hiapp and even ratiapp, either monomeric or oligomeric, binds to the membrane surface by electrostatic and hydrophobic interactions. once initial binding is achieved, further peptides are recruited and growth of hiapp fibrils is promoted. both iapp binding (fibril - independent) and fibril growth of hiapp lead to membrane disruption and finally cell death. mature fibrils were shown to be least cytotoxic, which is consistent with in vitro data showing that preformed fibrils do not cause membrane disruption. however, the hiapp cytotoxicity can not be simply scaled - down to a membrane - disruption phenomenon. using nontoxic and nonamyloidogenic iapp mutants, cao. demonstrated that there is no one - to - one relationship between disruption of model membranes and induction of cellular toxicity. obviously, the typical extracellular amyloid deposits found in t2 dm play a minor role in cytotoxicity. in contrast, small and structurally ill - defined oligomers might play a more prominent role in the development of t2 dm. for example, recent data illustrate that hiapp cytotoxicity is correlated with mitochondrial dysfunction upon abnormal intracellular release of toxic hiapp oligomers from -granules [61, 66 ]. protein aggregation and fibrillation occur naturally in a densely crowded, viscous, and heterogeneous solvent, namely, the cytoplasm, which is filled up to a volume of 40% by differently sized biomolecules such as proteins, nucleic acids, osmolytes, and salts. the consequential reduced accessible volume, also known as the macromolecular crowding effect, is predicted to have a significant impact on the equilibria and kinetics of biochemical processes by limiting the conformational sampling space to maximize the overall available volume. in other words, macromolecules restrict the available molecular space by their mutual impenetrability and repulsive interactions (termed as excluded volume effect) and thus entropically stabilize proteins by favoring the more compact conformations of the protein. the excluded volume effect strongly depends on the relative sizes and shapes of the test molecule and the background macromolecules. the more comparable the crowded molecule and the cosolutes are, the more the excluded volume effect dominates. however, the excluded volume effect is accompanied by an increase of viscosity, reduced dynamics, and changes in solvent polarity and water activity, which in total describes the macromolecular crowding effect. since in - cell techniques are rare and limited, inert and water soluble synthetic polymer crowding agents such as ficoll, peg or dextran have been used to mimic macromolecular crowding conditions and to study the effect of steric repulsion on equilibria of different cellular processes, such as protein folding and aggregation. ficoll is a copolymer of sucrose and epichlorohydrin and behaves as compact and rigid spheres due to its high branching and cross - linking. in contrast, dextran is a polymer of d - glucopyranose with less branching and thus features high flexibility and linearity typical for a quasi - random coil. due to their hydrophilicity, inertness, and neutrality, both polymers have been established as well - suited macromolecular crowding agents to mimic biological fluids, whether intra- or extracellular. despite its high water - solubility, polyethylene glycol (peg), a linear polymer of ethylene glycol, is a less promising macromolecular cosolute, since evidence of attractive interactions between peg and hydrophobic side chains on the protein surface has been found [127129 ]. more recently, the macromolecular crowding effect includes also the consideration of nonspecific (soft) interactions between solute and cosolutes, which are of enthalpic nature and can have stabilizing and destabilizing effects [130135 ]. therefore, in order to mimic the in - cell scenario with more biologically relevant cosolutes, proteins such as bsa and lysozyme were introduced as proteinaceous crowding agents because they feature chemically heterogeneous surfaces, providing chemical interactions such as hydrophobic and electrostatic interactions as well as hydrogen bonding. bsa has a molecular mass of 66.4 kda and is negatively charged at physiological conditions, whereas the 14.3 kda lysozyme has a positive net charge. many studies reported on the stabilizing effect of in vitro macromolecular crowding on protein folding which ranges from modest to strong, whereas cellular crowding was shown to only weakly shift protein folding equilibria towards the folded state [136138 ] or even to destabilize the native state, such as of the surface antigen vlse. over the last decades, it was shown that the aggregation and fibrillation reaction of several idps, including -synuclein [141145 ], a, apolipoprotein c - ii, prion protein (prp) [148150 ], and copper - zinc superoxide dismutase (sod1), are accelerated by synthetic and protein crowders. in order to understand the accelerative effect of macromolecular crowding on the protein fibrillation reaction, several major steps within the aggregation pathway which might be affected, have to be considered : (1) the structural collapse of the monomer producing aggregation - prone partially folded species, (2) formation of dimers and oligomers inducing the nucleation process, and (3) the growth and elongation of fibrils. the excluded volume effect is expected to favor the first two steps due to structural compaction upon formation, whereas the viscosity of such crowded solutions might decelerate the diffusion - limited process of fibril elongation. by studying the morphology of fibrils from the idps described above and formed under macromolecular crowding conditions, increased amounts of fibrils featuring shorter lengths have been reported. this indicates that the excluded volume effect favors the steps of structural transformation within the monomer and the oligomer formation, which dominate over the viscosity effect of macromolecular crowding. however, the net effect of macromolecular crowding on protein fibrillation might be different for different idps. since hiapp fibrillation occurs naturally in crowded biological fluids, both intra- and extracellular, our laboratory recently analyzed the influence of macromolecular crowding on the aggregation properties of hiapp by using different polymeric (dextran 70, ficoll 70) and protein (bsa, lysozyme) crowding agents (figure 7). in contrast to previous studies on idps, we found suppressive effects of macromolecular crowding on the hiapp fibril formation [151, 152 ]. first, by applying fluorescence correlation spectroscopy (fcs) we found that the mobility of monomeric iapp was highly restricted by interaction with the cosolute and increased viscosity under such macromolecular crowding conditions. 20% of ratiapp was found to bind to the polymeric crowding agents ficoll and dextran, whereas in solution with bsa nearly 50% of ratiapp and with lysozyme nearly 90% of ratiapp were bound to the protein crowder (figure 7(b)). by applying tht fluorescence spectroscopy (figures 7(c) and 7(d)) and afm, however, for all concentrations of ficoll and dextran the aggregation kinetics of hiapp remained nearly unchanged, except for higher amount of dextran (3040 wt%), resulting in a slightly extended elongation phase. in contrast, the protein crowders caused a prolonged lag phase and an extended elongation phase in case of bsa and a complete inhibition in the presence of 10 wt% lysozyme. additional data gained by atr - ftir spectroscopy showed that the structural transition from partially -helical and disordered in the monomeric state to the formation of intermolecular -sheets in the aggregated state remained unchanged under macromolecular crowding conditions. the well - known accelerative effect of lipid environment on hiapp fibrillation was not affected by macromolecular crowding. taken together, these observations confirmed that the in vitro hiapp aggregation pathway from monomeric species to fibril formation via nucleation is competing with stabilization of early - stage hiapp species under macromolecular crowding conditions by nonspecific binding between structurally disordered iapp monomers and crowding agents and increased viscosity. this study showed that the cellular crowding might not only be an effect of excluded volume, at least for hiapp, but also strongly involves nonspecific interactions (enthalpic contribution). compared to the other idps studied under macromolecular crowding conditions, hiapp is the smallest peptide with only 37 amino acid residues and thus is rather insensitive to the excluded volume effect. the cavities confined by the cosolutes might be still too large in order to favor the structural transition and nucleation of hiapp as expected from the excluded volume effect. in a second study, we compared the modulation effect of the macromolecular crowding agent ficoll 70 and its monomeric equivalent sucrose on the aggregation reaction of hiapp. we found that both have approximately the same suppressive effect on the aggregation kinetics and the amount of fibrils formed confirming that ficoll behaves osmolyte - like and that the steric excluded volume effect does not have a major effect on hiapp aggregation. reported on inhibitory effects of ficoll and promoting effects of dextran as crowding agents on the fibrillation reaction of a, a comparable peptide in size and involved in alzheimer 's disease, under nonagitating conditions. these results revealed that the chemical nature and the increased viscosity of a crowding solution can determine the macromolecular crowding effect and thus dominate over the steric effect of excluded volume. the probability of the aggregated state vanishes upon decreasing the system size suggesting that the finite size of biological cells or their compartments may be playing a key role in hampering intracellular aggregation of highly amyloidogenic peptides, whereas aggregation occurs more frequently in lower crowded environments, such as the extracellular space. recently, huang. reported on the stabilization of soluble and neurotoxic -oligomers of the recombinant human prion protein (prp) under macromolecular crowding conditions. in contrast, we found that the globular, early - stage hiapp species stabilized under macromolecular crowding conditions were not toxic when exposed exogenously to the pancreatic -cell line ins-1e, indicating that those species are formed off - pathway (figure 7(e)). mostly, they inhibit protein aggregation and subsequent fibrillation by direct and hydrophobic interaction due to their planar structure and hydrophobic nature. thereby, they bind to partially folded regions within the monomer, lead to formation of nontoxic off - pathway oligomers, or destabilize the fibrillar states upon binding to mature fibrils. however, for the development of such pharmacological chaperones, it is also crucial to consider the effect of macromolecular crowding on its inhibition efficiency since the biological fluid itself has a significant regulative effect on protein aggregation and fibrillation in vivo. therefore, we characterized and compared the effect of an orcein - related small molecule, o4, which has been shown to reduce the cytotoxicity of a by stimulating its aggregation, on the hiapp fibrillation in the absence and presence of crowding agents. in vitro, we have shown that o4 is an efficient dose - dependent inhibitor of hiapp aggregation by redirecting the aggregation pathway towards off - pathway globular species, thereby reducing the cytotoxicity of hiapp. further, o4 is able to interact with hiapp fibrils at a mature stage, causing disassembly of fibrils into smaller, less stable structures. a less effective inhibitory effect of o4 was found in the presence of ficoll 70, whereas in the presence of its monomeric unit sucrose, the dose - dependent inhibitory effect of o4 is similar to that in the diluted buffer, indicating that macromolecular crowding does modulate the efficiency of o4. taken together, those studies clearly demonstrate that the biological fluid itself might be an active contributor regulating the amyloidogenic propensity of hiapp in vivo. an increase of water content and thus a decrease of macromolecular crowding, as found in the extracellular space or induced by a loss of the health status of the cell, might be a reason for the onset of hiapp aggregation in vivo. in addition, the data highlights the importance and need to develop in - cell methods in order to get insight into the mechanism of hiapp fibrillation in living cells where the influence of biomolecular solvation, viscosity, excluded volume and complex membrane systems act in concert. the recently developed optical super - resolution techniques such as stimulated emission depletion (sted) and stochastic optical reconstruction microscopy (storm) might be promising tools to spatiotemporally resolve structural and morphological properties of hiapp inside living cells. used direct storm to successfully show the fibrillar structure of in situ formed a aggregates. apart from macromolecules such as proteins, nucleic acids and lipids, the cellular cytoplasm contains additional small organic molecules, also known as osmolytes or chemical chaperones. they function as responders to equilibrate cellular osmotic pressure and thus to maintain protein stability and functionality [157159 ]. depending on how they affect the folding equilibrium of a protein, the osmolytes can be divided into two classes, namely denaturants and protecting (or compatible) osmolytes. denaturants such as urea and guanidinium chloride favor the unfolded states of proteins by forming intermolecular hydrogen bonds with the peptide backbone and polar side chains, which are more favored than intramolecular hydrogen bonds required for the formation of secondary and tertiary structures. in contrast, protecting osmolytes including carbohydrates (e.g., trehalose), polyoles (e.g., glycerine, sorbitol, inositol), amino acids (e.g., glycine, proline, and taurine), and methylamines (e.g., tmao, glycine - betaine) stabilize protein 's native structure by favoring interaction with the solvent water and excluding themselves from the protein surface. as a consequence, this preferential exclusion effect (also termed as osmophobic effect) shifts the folding equilibrium towards more compact structures with less solvent accessible surface area (sasa) [160162 ]. in nature, denaturants and protecting osmolytes such as urea and tmao often act in a certain ratio in concert to be able to regulate the protein stability under varying osmotic and other environmental conditions. it is still poorly understood how osmolytes affect the stability of aggregation - prone proteins and thus their aggregation propensity. in literature, the rarely investigated osmolyte effect ranges from aggregation - inhibiting to aggregation - inducing. at a closer inspection, chemically or mutation - induced protein destabilization leads to partial unfolding and thus formation of aggregation - competent species that can be ameliorated in the presence of osmolytes such as sorbitol, glycine - betaine and trehalose [163165 ]. in contrast, fibrillation of natively unfolded proteins such as idps has been reported to be enhanced and accelerated in the presence of osmolytes such as tmao, glycerine or glycine - betaine [166169 ]. the latter observation indicates stabilization of compact structures, probably aggregation - competent nuclei, whose free energy is decreased due to the osmophobic effect. recently, our laboratory investigated the effect of tmao, glycine - betaine, proline, and urea on the fibrillation reaction of hiapp [152, 170 ]. tht fluorescence and atr - ftir spectroscopy data revealed a tmao- and glycine - betaine - induced stabilization of hiapp proto - fibrils causing retardation of fibril elongation, whereas afm images showed that the morphology of the mature fibrils were not affected by those osmolytes. despite the similar outcome, the mode of action of tmao and glycine - betaine differed from each other. the prolongation effect of tmao was concentration - dependent, whereas the same effect of glycine - betaine was concentration - independent. in case of the denaturant urea, a concentration - dependent prolongation of the lag phase has been observed, indicating stabilization of hiapp in an aggregation - incompetent state and retardation of iapp nuclei formation. interestingly, that effect was fully compensated by adding tmao in a molar ratio of 2 : 1 urea : tmao, which could not be found for glycine - betaine indicating direct interaction between tmao and urea via hydrogen bonding. for the natural amino acid proline, we found a weak concentration - dependent retardation of the elongation phase as well as a dose - dependent decrease of the amount of hiapp fibrils formed. afm measurements revealed shortening of hiapp fibrils and formation of globular, amorphous aggregates apart from the fibrillar assemblies in the presence of proline (figure 8). this suggests that proline diverts the amyloidogenesis of hiapp into an alternative aggregation pathway where shorter and smaller, fibrillar and nonfibrillar species are formed. the observation of disfavoring fibril formation is consistent with the hypothesis of proline being excluded from the protein 's surface. as a response to the preferential exclusion effect, hiapp might form nonfibrillar aggregates in order to minimize the exposed surface area. studies of auton and bolen determined tmao as the most effective protecting osmolyte followed by glycine - betaine and proline when the transfer free energy values of peptide backbone units from 1 m osmolyte to water were considered [171, 172 ]. tmao and glycine - betaine stabilize hiapp in its proto - fibrillar state, whereas proline shifts the equilibrium of hiapp aggregation towards formation of amorphous assemblies. however, the results also imply that additional interactions between the peptide and the osmolyte are involved apart from the preferential exclusion effect in order to subtly modulate the osmolyte effect of hiapp aggregation. evidence can be found also in the literature by comparing the tmao effect on different idps. the fibrillation reaction of tau, -synuclein, and a has been reported to be accelerated in the presence of tmao, whereas tmao induces the formation of amorphous aggregates in case of the glutamine - rich idp huntingtin exon 1. interestingly, despite a 25% identity and 50% similarity between a and hiapp in their primary structure and a cross - aggregation ability with hiapp [60, 174 ], tmao acts differently on their aggregation reaction. taken together, the results clearly show that osmolytes do not only modulate the stability, but also the aggregation propensity of idps inside cells. however, the modulation outcome highly depends on the structural and chemical properties of the monomeric species. to conclude, we have shown that by studying specific steps in the aggregation and fibrillation process of hiapp, such reductionist biophysical approach can yield useful information on the very complex behavior of hiapp at the molecular level, which might also contribute valuable insights into the mechanisms by which the amyloidogenic peptide may induce cell toxicity. however, as no simple relationship between the disruption and iapp - induced leakage of membranes and cellular toxicity has been found, additional factors may seem to play a role as well. moreover, there must be factors operating in vivo that attenuate the otherwise strong amyloidogenic propensity and membrane disruptive characteristics of hiapp found in vitro. acidic conditions, divalent ions (ca, zn [85, 104 ]) and interaction with insulin [18, 113, 176 ] have been reported to strongly reduce hiapp 's amyloidogenesis and membrane disrupting propensity. in addition, we demonstrated in this review that ubiquitous effects in cells such as macromolecular crowding and the presence of osmolytes have significant regulative and even suppressive effects on hiapp aggregation. however, in future studies more complex and physiologically relevant models including in - cell studies are needed in order to uncover all mechanistic aspects of hiapp 's cellular toxicity. particularly, a molecular understanding how obesity and aging correlate with the onset of hiapp aggregation in vivo has to gain more attention. such studies are essential for the development of therapeutic strategies to prevent the age - related and metabolic disease t2 dm. | type 2 diabetes mellitus (t2 dm) is an age - related and metabolic disease. its development is hallmarked, among others, by the dysfunction and degeneration of -cells of the pancreatic islets of langerhans. the major pathological characteristic thereby is the formation of extracellular amyloid deposits consisting of the islet amyloid polypeptide (iapp). the process of human iapp (hiapp) self - association, and the intermediate structures formed as well as the interaction of hiapp with membrane systems seem to be, at least to a major extent, responsible for the cytotoxicity. here we present a summary and comparison of the amyloidogenic propensities of hiapp in bulk solution and in the presence of various neutral and charged lipid bilayer systems as well as biological membranes. we also discuss the cellular effects of macromolecular crowding and osmolytes on the aggregation pathway of hiapp. understanding the influence of different cellular factors on hiapp aggregation will provide more insight into the onset of t2 dm and help to develop novel therapeutic strategies. |
female icr mice 89 weeks old were purchased from taconic (hudson, ny) or charles river laboratories (wilmington, ma). all of the mice were housed with a 12-h light/12-h dark cycle and fed standard laboratory chow and water ad libitum. the mice were fasted overnight (9:00 p.m. to 8:00 a.m.) prior to study. protocols for animal use were reviewed and approved by the institutional animal care and use committee of the joslin diabetes center and were in accordance with national institutes of health guidelines. briefly, total rna was isolated from mouse tibialis anterior muscle using the rneasy fibrous tissue mini kit (74704 ; qiagen). rna was reverse transcribed to cdna using the accuscript high fidelity 1st strand cdna synthesis kit (200820 ; stratagene). the long form of tbc1d1 (ak122445) was amplified by pcr with the phusion hot start high fidelity polymerase (new england biolabs) using gene - specific primers : forward : 5-ggaaaatagtagcacgttttcctg-3 ; reverse : 5-ctctgcctctgacttccatctctcttg-3. the pcr product was gel purified with the qiaquick gel extraction kit (28706 ; qiagen) and ligated to the blunt ii topo cloning vector (k280020 ; invitrogen). the ligation product was transformed into top10 chemically competent escherichia coli (c404003 ; invitrogen). single clones were cultured and plasmid dna was extracted with the qiaprep spin miniprep kit (27106 ; qiagen) and sequenced in both the forward and reverse directions (brigham and women 's sequencing center, harvard medical school). a hemagglutinin tag was added to the nh2-terminus and xhoi sites to both termini of tbc1d1 by pcr cloning (phusion) using the following composite primers : forward : 5-tttctcgaggccaccatgtacccgtacgacgtgcccgactacgcg-3 ; reverse : 5-aaactcgagctctgcctctgacttccatctctcttg-3. the pcr product was gel purified, ligated to the blunt ii topo cloning vector, and transformed into top10 e. coli. the hemagglutinin - tbc1d1 open reading frame was cut from the blunt ii topo vector with xhoi and ligated into the xhoi site of the pcaggs mammalian expression vector. true clones were identified by ampicillin selection of transformed e. coli (top10) and sequencing purified plasmid. this pcaggs expression vector drives a target gene under the cytomegalovirus immediate - early enhancer chicken -actin hybrid (cag) promoter and has been demonstrated to have high activity in skeletal muscle (8). five tbc1d1 mutant constructs were generated : 1) tbc1d1 mutated to ala at four phosphorylation motifs (ser, thr, thr, and ser), rendering these sites incapable of being phosphorylated (4p) ; 2) tbc1d1 mutated to trp at arg (r125w) ; 3) tbc1d1 mutated to lys at arg within its rab - gap domain to eliminate tbc1d1 gap activity (r / k) ; 4) tbc1d1 double mutant containing both the 4p and r / k mutations ; and 5) tbc1d1 double mutant containing both the r125w and r / k mutations. tbc1d1 cdna constructs were sequenced to confirm accuracy, using the high - throughput dna sequencing service at brigham and women 's hospital. plasmid dna was amplified in e. coli top10 cells (invitrogen), extracted using an endotoxin - free plasmid mega kit (qiagen), and suspended in saline at 4 g/l. tbc1d1 plasmids (100 g) were injected into mouse tibialis anterior muscles using our protocol (22), modified to that originally described by aihara and miyazaki (23), and mice were studied 7 days later. mice were anesthetized with intraperitoneal administration of pentobarbital sodium (90 mg / kg of body wt). one leg was left unstimulated (basal / sham control) and the other leg was subjected to electrical stimulation using a grass s88 pulse generator for 15 min of contractions (train rate 1/s ; train duration 500 ms ; pulse rate 100 hz ; duration 0.1 ms at 27 v), as we have previously described (5). to study insulin - activated pas signaling, we injected mice with either a bolus of glucose that resulted in a physiological increase in plasma insulin concentrations (8) or an insulin bolus. for the glucose treatment, mice were anesthetized and administered a 20% glucose bolus (1.0 g of glucose / kg of body wt). for the insulin treatment, mice were anesthetized and injected with insulin (10 mu / g). immediately after contraction, 15 min after glucose administration, or 6 min after insulin stimulation, frozen muscle tissue was homogenized with a polytron (brinkman instruments) in lysis buffer, as described previously (12)., lysates were subjected to immunoprecipitation to remove as160 before separation by sds - page. as160 was immunoprecipitated with a rabbit polyclonal antibody (07741 ; millipore, billerica, ma). bead - antibody - protein complexes were washed 1 with lysis buffer, 2 with lysis buffer + 500 mmol / l nacl, and 1 with lysis buffer. proteins were eluted from protein g beads by adding laemmli buffer and heated for 5 min at 95c. for immunoblotting, lysates (50 g protein) the protein bands were scanned by imagescanner (amersham biosciences) and quantitated by densitometry (fluorchem 2.0 ; alpha innotech, san leandro, ca). protein expression was assessed with antibodies to as160 (07741 ; millipore), glut4 and glut1 (ab1346 and cbl242, respectively ; chemicon international), ampk 2 (07363 ; upstate biotechnology, inc.), and akt1/2, hexokinase ii, and anti-tubulin (sc1619, sc6521, and sc5286, respectively ; santa cruz biotechnology). serum - purified anti - tbc1d1 antibody was generated by cell signaling technology by immunizing rabbits (12). phosphorylation - specific antibodies included akt thr (9275), akt ser (4058), ampk thr (2535), and pas (9611) from cell signaling technology. horseradish peroxidase conjugated anti - rabbit, anti - mouse, and anti - goat antibodies (amersham biosciences) were used to bind and detect all primary antibodies. briefly, mice were fasted overnight and anesthetized with intraperitoneal administration of pentobarbital sodium (90 mg / kg of body wt). to examine contraction - stimulated glucose transport, muscle was contracted, as described above. to study insulin - stimulated glucose transport, mice were administered a saline or 20% glucose bolus (1.0 g of glucose / kg of body wt), along with [h]-2-deoxyglucose through the retro - orbital sinus. our previous studies have shown that this dosage stimulates a physiologic insulin response (75 u / ml) without inducing significant hypoglycemia (8). baseline blood samples were collected from the tail vein prior to retro - orbital injection. the blood samples were collected from the tail vein at 5, 10, 15, 25, 35, and 45 min after injection to determine blood glucose and [h]-2-deoxyglucose specific activity. subsequently, the animals were killed, and tibialis anterior muscles were removed and frozen in liquid nitrogen. muscles were subsequently lysed and glucose transport ([h]-2-deoxyglucose - p) was determined via a precipitation protocol adapted from ferr. when anova revealed significant differences, tukey post hoc test for multiple comparisons was performed. female icr mice 89 weeks old were purchased from taconic (hudson, ny) or charles river laboratories (wilmington, ma). all of the mice were housed with a 12-h light/12-h dark cycle and fed standard laboratory chow and water ad libitum. the mice were fasted overnight (9:00 p.m. to 8:00 a.m.) prior to study. protocols for animal use were reviewed and approved by the institutional animal care and use committee of the joslin diabetes center and were in accordance with national institutes of health guidelines. briefly, total rna was isolated from mouse tibialis anterior muscle using the rneasy fibrous tissue mini kit (74704 ; qiagen). rna was reverse transcribed to cdna using the accuscript high fidelity 1st strand cdna synthesis kit (200820 ; stratagene). the long form of tbc1d1 (ak122445) was amplified by pcr with the phusion hot start high fidelity polymerase (new england biolabs) using gene - specific primers : forward : 5-ggaaaatagtagcacgttttcctg-3 ; reverse : 5-ctctgcctctgacttccatctctcttg-3. the pcr product was gel purified with the qiaquick gel extraction kit (28706 ; qiagen) and ligated to the blunt ii topo cloning vector (k280020 ; invitrogen). the ligation product was transformed into top10 chemically competent escherichia coli (c404003 ; invitrogen). single clones were cultured and plasmid dna was extracted with the qiaprep spin miniprep kit (27106 ; qiagen) and sequenced in both the forward and reverse directions (brigham and women 's sequencing center, harvard medical school). a hemagglutinin tag was added to the nh2-terminus and xhoi sites to both termini of tbc1d1 by pcr cloning (phusion) using the following composite primers : forward : 5-tttctcgaggccaccatgtacccgtacgacgtgcccgactacgcg-3 ; reverse : 5-aaactcgagctctgcctctgacttccatctctcttg-3. the pcr product was gel purified, ligated to the blunt ii topo cloning vector, and transformed into top10 e. coli. the hemagglutinin - tbc1d1 open reading frame was cut from the blunt ii topo vector with xhoi and ligated into the xhoi site of the pcaggs mammalian expression vector. true clones were identified by ampicillin selection of transformed e. coli (top10) and sequencing purified plasmid. this pcaggs expression vector drives a target gene under the cytomegalovirus immediate - early enhancer chicken -actin hybrid (cag) promoter and has been demonstrated to have high activity in skeletal muscle (8). five tbc1d1 mutant constructs were generated : 1) tbc1d1 mutated to ala at four phosphorylation motifs (ser, thr, thr, and ser), rendering these sites incapable of being phosphorylated (4p) ; 2) tbc1d1 mutated to trp at arg (r125w) ; 3) tbc1d1 mutated to lys at arg within its rab - gap domain to eliminate tbc1d1 gap activity (r / k) ; 4) tbc1d1 double mutant containing both the 4p and r / k mutations ; and 5) tbc1d1 double mutant containing both the r125w and r / k mutations. tbc1d1 cdna constructs were sequenced to confirm accuracy, using the high - throughput dna sequencing service at brigham and women 's hospital. plasmid dna was amplified in e. coli top10 cells (invitrogen), extracted using an endotoxin - free plasmid mega kit (qiagen), and suspended in saline at 4 g/l. tbc1d1 plasmids (100 g) were injected into mouse tibialis anterior muscles using our protocol (22), modified to that originally described by aihara and miyazaki (23), and mice were studied 7 days later. mice were anesthetized with intraperitoneal administration of pentobarbital sodium (90 mg / kg of body wt). one leg was left unstimulated (basal / sham control) and the other leg was subjected to electrical stimulation using a grass s88 pulse generator for 15 min of contractions (train rate 1/s ; train duration 500 ms ; pulse rate 100 hz ; duration 0.1 ms at 27 v), as we have previously described (5). to study insulin - activated pas signaling, we injected mice with either a bolus of glucose that resulted in a physiological increase in plasma insulin concentrations (8) or an insulin bolus. for the glucose treatment, mice were anesthetized and administered a 20% glucose bolus (1.0 g of glucose / kg of body wt). for the insulin treatment, mice were anesthetized and injected with insulin (10 mu / g). immediately after contraction, 15 min after glucose administration, or 6 min after insulin stimulation, tibialis anterior muscles were dissected and frozen in liquid nitrogen. frozen muscle tissue was homogenized with a polytron (brinkman instruments) in lysis buffer, as described previously (12)., lysates were subjected to immunoprecipitation to remove as160 before separation by sds - page. as160 was immunoprecipitated with a rabbit polyclonal antibody (07741 ; millipore, billerica, ma). bead - antibody - protein complexes were washed 1 with lysis buffer, 2 with lysis buffer + 500 mmol / l nacl, and 1 with lysis buffer. proteins were eluted from protein g beads by adding laemmli buffer and heated for 5 min at 95c. for immunoblotting, lysates (50 g protein) the protein bands were scanned by imagescanner (amersham biosciences) and quantitated by densitometry (fluorchem 2.0 ; alpha innotech, san leandro, ca). protein expression was assessed with antibodies to as160 (07741 ; millipore), glut4 and glut1 (ab1346 and cbl242, respectively ; chemicon international), ampk 2 (07363 ; upstate biotechnology, inc.), and akt1/2, hexokinase ii, and anti-tubulin (sc1619, sc6521, and sc5286, respectively ; santa cruz biotechnology). serum - purified anti - tbc1d1 antibody was generated by cell signaling technology by immunizing rabbits (12). phosphorylation - specific antibodies included akt thr (9275), akt ser (4058), ampk thr (2535), and pas (9611) from cell signaling technology. horseradish peroxidase conjugated anti - rabbit, anti - mouse, and anti - goat antibodies (amersham biosciences) were used to bind and detect all primary antibodies. briefly, mice were fasted overnight and anesthetized with intraperitoneal administration of pentobarbital sodium (90 mg / kg of body wt). to examine contraction - stimulated glucose transport, muscle was contracted, as described above. to study insulin - stimulated glucose transport, mice were administered a saline or 20% glucose bolus (1.0 g of glucose / kg of body wt), along with [h]-2-deoxyglucose through the retro - orbital sinus. our previous studies have shown that this dosage stimulates a physiologic insulin response (75 u / ml) without inducing significant hypoglycemia (8). baseline blood samples were collected from the tail vein prior to retro - orbital injection. the blood samples were collected from the tail vein at 5, 10, 15, 25, 35, and 45 min after injection to determine blood glucose and [h]-2-deoxyglucose specific activity. subsequently, the animals were killed, and tibialis anterior muscles were removed and frozen in liquid nitrogen. muscles were subsequently lysed and glucose transport ([h]-2-deoxyglucose - p) was determined via a precipitation protocol adapted from ferr. means were compared by one - way or two - way anova. when anova revealed significant differences, tukey post hoc test for multiple comparisons was performed. to study tbc1d1 function in skeletal muscle, in vivo injection and electroporation were used to transfect mouse tibialis anterior muscles with recombinant wild - type tbc1d1 and five tbc1d1 mutants. the tbc1d1 mutants were 1) tbc1d1 mutated to trp at arg (r125w) ; 2) tbc1d1 mutated to ala at four phosphorylation motifs (ser, thr, thr, and ser) ; 3) tbc1d1 mutated to lys at arg within its rab - gap domain (r / k) ; 4) tbc1d1 double mutant containing both the r125w and r / k mutations ; and 5) tbc1d1 double mutant containing both the 4p and r / k mutations. we selected the 4p mutant sites because these sites are highly conserved and are predicted to be phosphorylated by ampk and/or akt (i.e., ser and thr by ampk, thr by akt, and ser by both ampk and akt) (12). in vivo dna injection and electroporation resulted in an approximate sevenfold increase in expression of tbc1d1 in mouse tibialis anterior muscle compared with endogenous tbc1d1 (fig. the magnitude of overexpression was comparable across all constructs, as each tbc1d1 variant was subcloned into the same pcaggs vehicle. tbc1d1 expression was significantly increased in mouse tibialis anterior muscles in response to in vivo cdna injection and electroporation. empty pcaggs vector (ev) and tbc1d1 cdna constructs (wild - type [wt ] and 4p, r125w, r / k, 4p / rk, and r125w / rk mutants) were injected into the tibialis anterior muscles of anesthetized mice, followed by in vivo electroporation. the animals were allowed to recover, and protein expression was assessed 1 week after injection. muscle proteins were separated by sds - page and immunoblotted with an anti - tbc1d1 antibody. the data are expressed as the means se ; n = 812/group. # p < 0.05 (vs. empty vector controls). we first determined the effects of overexpressing wild - type and mutant tbc1d1 proteins on the phosphorylation state of tbc1d1, using the pas antibody. because the pas antibody detects both as160 and tbc1d1, as160 was depleted from lysates by immunoprecipitation. after immunoprecipitation, as160 was not detected in the muscle lysates and there was no effect of the as160 immunoprecipitation on the tbc1d1 protein, as detected by immunoblotting (supplementary fig. 1, available in an online appendix at http://diabetes.diabetesjournals.org/content/early/2010/03/10/db09-1266/suppl/dc1). the as160-depleted lysates were used to study tbc1d1 pas signaling. as expected, basal phosphorylation was significantly increased in muscles overexpressing wild - type, r / k, r125w, and r125w / rk tbc1d1 mutants compared with empty vector controls (fig. pas phosphorylation in response to the three stimuli was greater in muscles overexpressing wild - type, r / k, r125w, and r125w / rk tbc1d1 (fig. 2a and b ; supplementary fig. 2a). pas phosphorylation in the 4p and 4p / rk tbc1d1-expressing muscle was not increased in the basal state compared with empty vector controls. furthermore, glucose, contraction, and insulin did not significantly increase pas phosphorylation in the 4p overexpressing muscles these data demonstrate that the recombinant tbc1d1 is functionally modified in vivo, and that the integrity of the tbc1d1 point mutations (4p and 4p / rk) was preserved after gene expression. the r125w and r / k mutations did not interfere with the ability of insulin and contraction to increase pas phosphorylation in the skeletal muscle. phosphorylation of tbc1d1 in transfected muscles. to determine whether expression of tbc1d1 altered tbc1d1 pas phosphorylation, ev and tbc1d1 cdna constructs were injected into tibialis anterior muscles followed by in vivo electroporation, and mice were studied 1 week later. a : mice were anesthetized and injected intravenously with saline () or glucose (+ ; 1.0 g of glucose / kg of body wt), and tibialis anterior muscles were obtained 15 min later. b : mice were anesthetized, one leg was sham treated () and the other leg was contracted in situ (+) for 15 min, and tibialis anterior muscles were dissected. muscle lysates were immunoprecipitated to deplete as160 and supernatants were separated by sds - page and immunoblotted with an anti - pas antibody. white bars represent basal treatment, and black bars represent glucose / insulin (a) or contraction (b) treatment groups. p < 0.05 (vs. basal) ; # p < 0.05 (vs. empty vector for respective treatment). although there is minimal expression of tbc1d1 in adipocytes (12,16), a previous study has shown that overexpression of wild - type tbc1d1 dramatically reduces insulin - stimulated glut4 translocation in 3t3l1 adipocytes (6). in contrast to these findings, we found that overexpression of wild - type tbc1d1 did not decrease basal or submaximal insulin - stimulated glucose transport in skeletal muscle (fig. overexpression of tbc1d1 altered insulin - stimulated glucose transport in mouse skeletal muscles. to determine whether expression of wild - type tbc1d1 and tbc1d1 mutants altered insulin - stimulated glucose transport, ev or tbc1d1 cdna constructs were injected into tibialis anterior muscles followed by in vivo electroporation. one week later, mice were anesthetized and administered a saline or 20% glucose bolus (1.0 g of glucose / kg of body wt) through the retro - orbital sinus to stimulate a physiologic insulin response. b : the effects of the rab - gap (r / k) mutants. white bars represent the basal treatment, and black bars represent the glucose / insulin treatment group. data are means se, n = 816 mice / group. p < 0.05 (vs. basal) ; # p < 0.05 (vs. empty vector for respective treatment). insulin and contraction increase tbc1d1 and as160 phosphorylation as detected in aggregate by the pas antibody (5,12), and abolishing as160 pas phosphorylation impairs insulin- and contraction - induced glucose transport (8). because of the structural similarity of tbc1d1 to as160, we hypothesized that tbc1d1 phosphorylation sites detected by the pas antibody would also be important for glucose transport. to address this question, we overexpressed the tbc1d1 4p construct that results in the loss of insulin- and contraction - stimulated pas phosphorylation (fig. 2) and measured basal and submaximal insulin - stimulated glucose transport in vivo. surprisingly, expression of the tbc1d1 4p mutant did not alter insulin - stimulated glucose transport (fig. genetic linkage analyses have shown a tbc1d1 r125w missense variant is linked to severe obesity in humans (18,19). because skeletal muscle is the tissue with the highest level of expression of tbc1d1, we tested the hypothesis that the r125w mutation would alter glucose transport in this tissue. we found that overexpression of the tbc1d1 r125w mutant in skeletal muscle significantly impaired insulin - stimulated glucose transport (fig. whether disruption of the rab - gap domain reverses the decrease in glucose transport associated with the r125w mutation, we expressed tbc1d1 containing both the r125w and rab - gap mutations in the muscles. figure 3b shows that glucose / insulin - stimulated glucose transport was normalized when the tbc1d1 r125w mutant was coexpressed with the mutated rab - gap domain. interestingly, expression of tbc1d1 with a disrupted rab - gap domain (r / k) significantly increased basal glucose transport without altering insulin - stimulated glucose transport (fig. the contraction protocol used was maximal, and therefore the increase in glucose transport with contraction was greater than with glucose - induced physiological insulin (figs. 3 and 4). similar to the effects of insulin, expression of wild - type tbc1d1 did not alter contraction - stimulated glucose transport (fig. however, in contrast to what was observed with glucose / insulin, muscles overexpressing the tbc1d1 4p mutant exhibited a significant decrease in contraction - stimulated glucose transport (fig. 4a). furthermore, although the expression of the tbc1d1 r125w mutation decreased insulin - induced glucose transport, it did not alter contraction - induced glucose transport (fig. overexpression of tbc1d1 altered contraction - stimulated glucose transport in mouse skeletal muscles. to determine whether expression of wild - type tbc1d1 and tbc1d1 mutants altered contraction - stimulated glucose transport, ev or tbc1d1 cdna constructs one week later, mice were anesthetized and contraction was performed by stimulation of the peroneal nerve. one leg was sham treated but unstimulated, and the other leg was contracted for 15 min. b : the effects of the rab - gap (r / k) mutants. white bars represent the basal treatment, and black bars represent the contraction treatment group. p < 0.05 (vs. basal) ; # p < 0.05 (vs. empty vector for respective treatment). tbc1d1 devoid of rab - gap activity significantly increased contraction - stimulated glucose transport by 60% (fig., disruption of the rab - gap domain of tbc1d1 resulted in reversal of the decrease in contraction - stimulated glucose transport that occurred with expression of the 4p mutant (fig. these results suggest that tbc1d1 plays an important role in contraction - stimulated glucose transport. for the contraction experiments, all treatment groups had a higher basal rate of glucose transport compared with the basal glucose transport in the insulin experiment. this is likely the result of contraction of one leg altering glucose transport of the control sedentary leg due to changes in blood flow or concentration of blood hormones. therefore, the higher basal rates of glucose transport in the contraction experiments may mask the more prominent effects of the rab - gap (r / k) mutant that are observed with the insulin experiments. akt plays a key role in mediating insulin - stimulated glucose transport, whereas the signaling mechanisms regulating contraction - induced glucose transport likely involve ampk and ampk - related kinases. to determine whether overexpression of the tbc1d1 constructs alter insulin- and contraction - stimulated akt and ampk signaling, akt - thr, akt - ser, and ampk thr phosphorylation were measured in muscles expressing the tbc1d1 constructs. 3) akt thr and akt ser phosphorylation were not altered in muscles expressing wild - type and mutant tbc1d1. in addition, contraction - induced ampk thr phosphorylation (supplementary fig these results indicate that tbc1d1 mutations alter glucose transport without interfering with akt and ampk signaling. as160 mutated at four pas phosphorylation sites significantly decreases insulin- and contraction - stimulated glucose transport in skeletal muscle (8). therefore, we determined whether overexpression of wild - type or mutant tbc1d1 would alter as160 protein expression and/or insulin- and contraction - stimulated as160 pas phosphorylation. as160 was immunoprecipitated from tibialis anterior muscle lysates expressing wild - type and mutant tbc1d1 and immunoblotted with the pas antibody. overexpression of tbc1d1 had no effect on as160 protein expression (supplementary fig. 6). glut proteins and glycolytic enzymes can be critical regulators of glucose metabolism in skeletal muscle. therefore, we determined whether tbc1d1 overexpression alters the protein expression of glut1, glut4, or hexokinase ii in the transfected muscles. as shown in supplementary fig. 6, there was no difference in expression among muscles injected with empty vector or any of the tbc1d1 constructs. therefore, tbc1d1 overexpression does not alter expression of these key regulatory proteins of glucose metabolism in skeletal muscle. to study tbc1d1 function in skeletal muscle, in vivo injection and electroporation were used to transfect mouse tibialis anterior muscles with recombinant wild - type tbc1d1 and five tbc1d1 mutants. the tbc1d1 mutants were 1) tbc1d1 mutated to trp at arg (r125w) ; 2) tbc1d1 mutated to ala at four phosphorylation motifs (ser, thr, thr, and ser) ; 3) tbc1d1 mutated to lys at arg within its rab - gap domain (r / k) ; 4) tbc1d1 double mutant containing both the r125w and r / k mutations ; and 5) tbc1d1 double mutant containing both the 4p and r / k mutations. we selected the 4p mutant sites because these sites are highly conserved and are predicted to be phosphorylated by ampk and/or akt (i.e., ser and thr by ampk, thr by akt, and ser by both ampk and akt) (12). in vivo dna injection and electroporation resulted in an approximate sevenfold increase in expression of tbc1d1 in mouse tibialis anterior muscle compared with endogenous tbc1d1 (fig. the magnitude of overexpression was comparable across all constructs, as each tbc1d1 variant was subcloned into the same pcaggs vehicle. tbc1d1 expression was significantly increased in mouse tibialis anterior muscles in response to in vivo cdna injection and electroporation. empty pcaggs vector (ev) and tbc1d1 cdna constructs (wild - type [wt ] and 4p, r125w, r / k, 4p / rk, and r125w / rk mutants) were injected into the tibialis anterior muscles of anesthetized mice, followed by in vivo electroporation. the animals were allowed to recover, and protein expression was assessed 1 week after injection. muscle proteins were separated by sds - page and immunoblotted with an anti - tbc1d1 antibody. we first determined the effects of overexpressing wild - type and mutant tbc1d1 proteins on the phosphorylation state of tbc1d1, using the pas antibody. because the pas antibody detects both as160 and tbc1d1, as160 was depleted from lysates by immunoprecipitation. after immunoprecipitation, as160 was not detected in the muscle lysates and there was no effect of the as160 immunoprecipitation on the tbc1d1 protein, as detected by immunoblotting (supplementary fig. 1, available in an online appendix at http://diabetes.diabetesjournals.org/content/early/2010/03/10/db09-1266/suppl/dc1). the as160-depleted lysates were used to study tbc1d1 pas signaling. as expected, basal phosphorylation was significantly increased in muscles overexpressing wild - type, r / k, r125w, and r125w / rk tbc1d1 mutants compared with empty vector controls (fig. pas phosphorylation in response to the three stimuli was greater in muscles overexpressing wild - type, r / k, r125w, and r125w / rk tbc1d1 (fig. 2a and b ; supplementary fig pas phosphorylation in the 4p and 4p / rk tbc1d1-expressing muscle was not increased in the basal state compared with empty vector controls. furthermore, glucose, contraction, and insulin did not significantly increase pas phosphorylation in the 4p overexpressing muscles (fig. these data demonstrate that the recombinant tbc1d1 is functionally modified in vivo, and that the integrity of the tbc1d1 point mutations (4p and 4p / rk) was preserved after gene expression. the r125w and r / k mutations did not interfere with the ability of insulin and contraction to increase pas phosphorylation in the skeletal muscle. phosphorylation of tbc1d1 in transfected muscles. to determine whether expression of tbc1d1 altered tbc1d1 pas phosphorylation, ev and tbc1d1 cdna constructs were injected into tibialis anterior muscles followed by in vivo electroporation, and mice were studied 1 week later. a : mice were anesthetized and injected intravenously with saline () or glucose (+ ; 1.0 g of glucose / kg of body wt), and tibialis anterior muscles were obtained 15 min later. b : mice were anesthetized, one leg was sham treated () and the other leg was contracted in situ (+) for 15 min, and tibialis anterior muscles were dissected. muscle lysates were immunoprecipitated to deplete as160 and supernatants were separated by sds - page and immunoblotted with an anti - pas antibody. white bars represent basal treatment, and black bars represent glucose / insulin (a) or contraction (b) treatment groups. p < 0.05 (vs. basal) ; # p < 0.05 (vs. empty vector for respective treatment). although there is minimal expression of tbc1d1 in adipocytes (12,16), a previous study has shown that overexpression of wild - type tbc1d1 dramatically reduces insulin - stimulated glut4 translocation in 3t3l1 adipocytes (6). in contrast to these findings, we found that overexpression of wild - type tbc1d1 did not decrease basal or submaximal insulin - stimulated glucose transport in skeletal muscle (fig. overexpression of tbc1d1 altered insulin - stimulated glucose transport in mouse skeletal muscles. to determine whether expression of wild - type tbc1d1 and tbc1d1 mutants altered insulin - stimulated glucose transport, ev or tbc1d1 cdna constructs were injected into tibialis anterior muscles followed by in vivo electroporation. one week later, mice were anesthetized and administered a saline or 20% glucose bolus (1.0 g of glucose / kg of body wt) through the retro - orbital sinus to stimulate a physiologic insulin response. insulin - stimulated glucose transport was measured in tibialis anterior muscles using [h]2-deoxyglucose. a : the effects of expressing the 4p and r125w mutants. b : the effects of the rab - gap (r / k) mutants. white bars represent the basal treatment, and black bars represent the glucose / insulin treatment group. data are means se, n = 816 mice / group. p < 0.05 (vs. basal) ; # p < 0.05 (vs. empty vector for respective treatment). insulin and contraction increase tbc1d1 and as160 phosphorylation as detected in aggregate by the pas antibody (5,12), and abolishing as160 pas phosphorylation impairs insulin- and contraction - induced glucose transport (8). because of the structural similarity of tbc1d1 to as160, we hypothesized that tbc1d1 phosphorylation sites detected by the pas antibody would also be important for glucose transport. to address this question, we overexpressed the tbc1d1 4p construct that results in the loss of insulin- and contraction - stimulated pas phosphorylation (fig. 2) and measured basal and submaximal insulin - stimulated glucose transport in vivo. surprisingly, expression of the tbc1d1 4p mutant did not alter insulin - stimulated glucose transport (fig. genetic linkage analyses have shown a tbc1d1 r125w missense variant is linked to severe obesity in humans (18,19). because skeletal muscle is the tissue with the highest level of expression of tbc1d1, we tested the hypothesis that the r125w mutation would alter glucose transport in this tissue. we found that overexpression of the tbc1d1 r125w mutant in skeletal muscle significantly impaired insulin - stimulated glucose transport (fig. 3a). to explore whether disruption of the rab - gap domain reverses the decrease in glucose transport associated with the r125w mutation, we expressed tbc1d1 containing both the r125w and rab - gap mutations in the muscles. figure 3b shows that glucose / insulin - stimulated glucose transport was normalized when the tbc1d1 r125w mutant was coexpressed with the mutated rab - gap domain. interestingly, expression of tbc1d1 with a disrupted rab - gap domain (r / k) significantly increased basal glucose transport without altering insulin - stimulated glucose transport (fig. the contraction protocol used was maximal, and therefore the increase in glucose transport with contraction was greater than with glucose - induced physiological insulin (figs. 3 and 4). similar to the effects of insulin, expression of wild - type tbc1d1 did not alter contraction - stimulated glucose transport (fig. 4a). however, in contrast to what was observed with glucose / insulin, muscles overexpressing the tbc1d1 4p mutant exhibited a significant decrease in contraction - stimulated glucose transport (fig. 4a). furthermore, although the expression of the tbc1d1 r125w mutation decreased insulin - induced glucose transport, it did not alter contraction - induced glucose transport (fig. overexpression of tbc1d1 altered contraction - stimulated glucose transport in mouse skeletal muscles. to determine whether expression of wild - type tbc1d1 and tbc1d1 mutants altered contraction - stimulated glucose transport, ev or tbc1d1 cdna constructs one week later, mice were anesthetized and contraction was performed by stimulation of the peroneal nerve. one leg was sham treated but unstimulated, and the other leg was contracted for 15 min. b : the effects of the rab - gap (r / k) mutants. white bars represent the basal treatment, and black bars represent the contraction treatment group. p < 0.05 (vs. basal) ; # p < 0.05 (vs. empty vector for respective treatment). tbc1d1 devoid of rab - gap activity significantly increased contraction - stimulated glucose transport by 60% (fig., disruption of the rab - gap domain of tbc1d1 resulted in reversal of the decrease in contraction - stimulated glucose transport that occurred with expression of the 4p mutant (fig. these results suggest that tbc1d1 plays an important role in contraction - stimulated glucose transport. for the contraction experiments, all treatment groups had a higher basal rate of glucose transport compared with the basal glucose transport in the insulin experiment. this is likely the result of contraction of one leg altering glucose transport of the control sedentary leg due to changes in blood flow or concentration of blood hormones. therefore, the higher basal rates of glucose transport in the contraction experiments may mask the more prominent effects of the rab - gap (r / k) mutant that are observed with the insulin experiments. akt plays a key role in mediating insulin - stimulated glucose transport, whereas the signaling mechanisms regulating contraction - induced glucose transport likely involve ampk and ampk - related kinases. to determine whether overexpression of the tbc1d1 constructs alter insulin- and contraction - stimulated akt and ampk signaling, akt - thr, akt - ser, and ampk thr phosphorylation were measured in muscles expressing the tbc1d1 constructs. 3) akt thr and akt ser phosphorylation were not altered in muscles expressing wild - type and mutant tbc1d1. in addition, contraction - induced ampk thr phosphorylation (supplementary fig. 4a) and akt thr and akt ser phosphorylation (supplementary fig. these results indicate that tbc1d1 mutations alter glucose transport without interfering with akt and ampk signaling. as160 mutated at four pas phosphorylation sites significantly decreases insulin- and contraction - stimulated glucose transport in skeletal muscle (8). therefore, we determined whether overexpression of wild - type or mutant tbc1d1 would alter as160 protein expression and/or insulin- and contraction - stimulated as160 pas phosphorylation. as160 was immunoprecipitated from tibialis anterior muscle lysates expressing wild - type and mutant tbc1d1 and immunoblotted with the pas antibody. glut proteins and glycolytic enzymes can be critical regulators of glucose metabolism in skeletal muscle. therefore, we determined whether tbc1d1 overexpression alters the protein expression of glut1, glut4, or hexokinase ii in the transfected muscles. as shown in supplementary fig. 6, there was no difference in expression among muscles injected with empty vector or any of the tbc1d1 constructs. therefore, tbc1d1 overexpression does not alter expression of these key regulatory proteins of glucose metabolism in skeletal muscle. under normal physiological conditions, skeletal muscle glucose transport is the rate - limiting step in glucose utilization, and impairment in insulin - stimulated glucose transport in this tissue is a major factor in the development of type 2 diabetes. despite the importance of skeletal muscle in metabolic health, tbc1d1 is a rab - gap protein and a paralog of as160, the latter protein having recently been recognized as a critical regulator of glucose transport in both adipocytes and skeletal muscle (8,13,15,25). although tbc1d1 is known to have the highest levels of expression in skeletal muscle and is phosphorylated in response to multiple factors that stimulate glucose transport in skeletal muscle (12), whether tbc1d1 functions in the regulation of glucose transport in adult skeletal muscle was not clear. a recent study using a mouse model with a tbc1d1 truncation mutation (sjl) showed that depletion of tbc1d1 in skeletal muscle increased fatty acid oxidation but decreased glucose utilization (17). because muscle from the sjl mouse has decreased glut4 expression, it is not known whether alterations in tbc1d1 function contributed to the inhibition of glucose uptake observed in the extensor digitorum longus muscle (17). in the current study, there was no change in glut4 expression, and therefore, tbc1d1 was established as a regulator of both insulin- and contraction - stimulated glucose transport in skeletal muscle. numerous phosphorylation sites have been identified on tbc1d1 (6,12,22), but the potential role of these sites in the regulation of glucose transport in skeletal muscle is not known. to determine whether tbc1d1 phosphorylation regulates glucose transport, we mutated ser, thr, thr, and ser to ala to inhibit their phosphorylation (4p). we chose these sites because they 1) are highly conserved phosphorylation sites on tbc1d1 ; 2) are not subject to splice variation ; and 3) represent predicted consensus sequences for both akt and ampk. of these sites, thr, a predicted akt motif, is phosphorylated in response to insulin, and corresponds to thr in as160 (12). tbc1d1 ser is a partial match for both the akt and ampk recognition motifs (12). given the importance of the akt sites on as160 in the regulation of glucose transport (8,13), we were surprised to find that expression of this tbc1d1 4p mutant had no effect on insulin - stimulated glucose transport. thus, our findings suggest that either tbc1d1 phosphorylation is not required for insulin - stimulated glucose transport due to the existence of another rab - gap protein or there are other akt sites on tbc1d1 that are critical to mediate insulin - induced glucose transport. in contrast, expression of the tbc1d1 4p mutant significantly decreased contraction - stimulated glucose transport and this decrease was completely reversed by concomitant disruption of tbc1d1 rab - gap activity. it is possible that ser and/or thr mediate this inhibition because these sites are predicted to be phosphorylated by ampk, and muscle contraction is a potent stimulus for ampk activation. taken together, our data suggest that phosphorylation of the putative ampk sites ser and/or thr, but not thr and ser, may be critical in regulating tbc1d1 rab - gap activity and glucose transport. these findings also raise the possibility that tbc1d1 phosphorylation on ser and/or thr function predominantly in the regulation of contraction effects on glucose transport, whereas the phosphorylation of as160 may be key in regulating insulin - stimulated glucose transport. a tbc1d1 r125w missense variant causes familial obesity predisposition in humans (18,19) ; however, the mechanism by which this mutation leads to obesity is not known. our finding that expression of the r125w mutation in skeletal muscle impairs insulin - stimulated glucose transport and our previous work showing that tbc1d1 is abundant only in skeletal muscle (12) suggest that metabolic dysfunction in skeletal muscle caused by alterations in tbc1d1 function could be a primary phenotypic manifestation of the tbc1d1 r125w missense variant. impairment of glucose transport in muscle could lead to increased fat accumulation in adipose tissue, and subsequent obesity. although the precise molecular mechanism by which the r125w mutation in tbc1d1 impairs insulin - stimulated glucose transport is not known, it is interesting to note that this mutation does not result in defects in upstream insulin signaling, as akt phosphorylation was normal. in contrast to insulin, expression of the tbc1d1 r125w mutant had no effect on contraction - stimulated glucose transport in mouse skeletal muscle, and this difference may provide important clues as to the mechanism by which the r125w mutation causes defects in glucose transport. this difference in r125 function for insulin- and contraction - stimulated glucose transport is likely due to distinct proximal signals for insulin and contraction. insulin - stimulated glucose transport involves insulin binding to its receptor and subsequent activation of insulin receptor substrate, pi 3-kinase, and akt (26,27). in contrast, contraction - stimulated glucose transport can not be inhibited by the pi 3-kinase inhibitor wortmannin, and although the exact mechanism is still not fully understood, it may be mediated through ampk or other ampk - related kinases (1,28,29). therefore, insulin and contraction may phosphorylate tbc1d1 on distinct sites, which may lead to different effects on its rab - gap activity or result in different affinities of tbc1d1 for other proteins. the r125 site is located in a tyrosine - binding domain in tbc1d1. given that this domain mediates binding of as160 with other proteins (25,3032), the r125w mutation may alter the affinity of tbc1d1 for proteins involved in insulin but not contraction signaling, resulting in impaired insulin - stimulated glucose transport. the mechanism by which the r125w mutation impairs glucose transport will be an important topic for future investigation. our finding that the impairment of insulin - stimulated glucose transport could be reversed by simultaneous disruption of rab - gap activity in tbc1d1 suggests a possible therapeutic approach to treat patients with this condition in the future. our results show that overexpression of wild - type tbc1d1 had no effect on both insulin- and contraction - stimulated glucose transport, in contrast to previous studies using adipocytes (6,16). the discrepancy is likely because there are very different expression levels of endogenous tbc1d1 in skeletal muscle and adipocytes. we find little to no expression of tbc1d1 in adipose tissue (12), and it has been reported that adipocytes have 20 times more as160 compared with tbc1d1 (16). thus, overexpression of a large amount of tbc1d1 in adipocytes may interfere with as160 phosphorylation, suppressing glut4 translocation, resulting in inhibition of glucose transport. if tbc1d1 functions as a brake to restrain glut4 translocation and adipocytes lack this protein, overexpressing this foreign protein may overwhelm the system, preventing glut4 translocation. in contrast, in mouse skeletal muscle, expression of endogenous tbc1d1 is so abundant that it is sufficient to inhibit glut4 translocation. therefore, overexpression of wild - type tbc1d1 in skeletal muscle did not inhibit glucose transport as it did in adipocytes. in addition, in 3t3-l1 adipocytes, mutation of r125w had no further effect on insulin - stimulated glucose transport compared with wild - type tbc1d1 (33). it is likely that in adipocytes, overexpression of wild - type tbc1d1 inhibits glucose transport enough to mask the effect of the r125w mutation on glucose transport. indeed, in our studies in skeletal muscle, expressing the tbc1d1 r125w mutant significantly decreased insulin - stimulated glucose transport, under conditions where there was no effect of wild - type tbc1d1. thus, the studies using adipocytes should be interpreted with caution and not extrapolated to skeletal muscle because tbc1d1 is minimally expressed in adipocytes and because the tbc1d1 splice variant expressed in the adipocyte study is not the variant expressed in skeletal muscle (6). the current work suggests that in addition to as160, tbc1d1 functions as a rab - gap protein regulating glucose transport in mouse skeletal muscle. in comparing tbc1d1 with as160, mutation of four akt phosphorylation sites on as160 inhibited both insulin- and contraction - stimulated glucose transport, and both of these effects were reversed by disruption of the rab - gap domain (8). in contrast, in the current study, mutations of four phosphorylation sites on tbc1d1, which consisted of both predicted akt and ampk sites, impaired only contraction - stimulated glucose transport. one explanation for this finding is that tbc1d1 phosphorylation may not mediate insulin - stimulated glucose transport in mouse skeletal muscle. the second possibility is that the mutation of the two putative akt sites (thr and ser) is insufficient to inhibit rab - gap activity of this protein, and that there must be one or more additional putative akt sites in tbc1d1 that also regulate its rab - gap activity after insulin stimulation. in future studies, it will be important to investigate the regulation and function of additional phosphorylation sites on tbc1d1. in addition, because different muscles have different relative levels of expression of as160 and tbc1d1, these two rab - gap domains may contribute differently in terms of glucose transport in various muscle types. in summary, we used in vivo gene injection and electroporation to overexpress wild - type and mutant tbc1d1 in mouse tibialis anterior muscles and evaluated the effect of tbc1d1 on glucose transport. our studies show that the r125w mutation in tbc1d1 impairs insulin - stimulated glucose transport and has no effect on contraction - stimulated glucose transport, which may be a major mechanism for the obesity associated with this mutation. in contrast, mutation of four conserved phosphorylation sites compromised only contraction - stimulated glucose transport. furthermore, we find that the rab - gap domain of tbc1d1 is critical in the regulation of glucose transport in muscle. tbc1d1 mediates stimuli - specific upstream signals, leading to regulation of glucose transport. taken together with the reported role of this protein in fatty acid metabolism, | objectivetbc1d1 is a member of the tbc1 rab - gtpase family of proteins and is highly expressed in skeletal muscle. insulin and contraction increase tbc1d1 phosphorylation on phospho - akt substrate motifs (pass), but the function of tbc1d1 in muscle is not known. genetic linkage analyses show a tbc1d1 r125w missense variant confers risk for severe obesity in humans. the objective of this study was to determine whether tbc1d1 regulates glucose transport in skeletal muscle.research design and methodsin vivo gene injection and electroporation were used to overexpress wild - type and several mutant tbc1d1 proteins in mouse tibialis anterior muscles, and glucose transport was measured in vivo.resultsexpression of the obesity - associated r125w mutant significantly decreased insulin - stimulated glucose transport in the absence of changes in tbc1d1 pas phosphorylation. simultaneous expression of an inactive rab - gtpase (gap) domain of tbc1d1 in the r125w mutant reversed this decrease in glucose transport caused by the r125w mutant. surprisingly, expression of tbc1d1 mutated to ala on four conserved akt and/or amp - activated protein kinase predicted phosphorylation sites (4p) had no effect on insulin - stimulated glucose transport. in contrast, expression of the tbc1d1 4p mutant decreased contraction - stimulated glucose transport, an effect prevented by concomitant disruption of tbc1d1 rab - gap activity. there was no effect of the r125w mutation on contraction - stimulated glucose transport.conclusionstbc1d1 regulates both insulin- and contraction - stimulated glucose transport, and this occurs via distinct mechanisms. the r125w mutation of tbc1d1 impairs skeletal muscle glucose transport, which could be a mechanism for the obesity associated with this mutation. |
the subclinical case was detected in 2011 during a contact investigation of a 40-year - old man suspected of having influenza a(h5n1) virus infection. the chickens roamed close to the sleeping area of the household members. the index case - patient, his daughter, and his daughter - in - law were involved in slaughtering and preparing the chickens. the index case - patient had fever, cough, dyspnea, and diarrhea that progressed over 2 days, leading to hospital admission. despite intensive care and treatment with oseltamivir and antibiotics, the disease progressed, and he died 2 days later. a throat swab taken from the index case - patient on day 3 of illness was tested by reverse transcription pcr, and results were positive for influenza a(h5n1) virus. hemagglutination inhibition (hi) and microneutralization (mn) tests for h5n1-specific antibodies were negative in samples taken during the acute phase of illness (technical appendix). on day 5 of illness of the index case - patient, a contact investigation was initiated. throat swab specimens were collected from 4 household members and 1 close contact of the index case - patient : his spouse (age 47 years), daughter (age 18 years), daughter - in - law (age 25 years), and grandson (age 1 year) and an unrelated man (age 43 years). infection control measures were initiated, and all household members were given oseltamivir (75 mg / d) for 1 week and instructed to seek immediate health care if fever or respiratory symptoms developed. results of hi testing of serum samples collected during the acute illness phase of the index case - patient were negative. the sample from the index case - patient s daughter, collected 6 days after the woman had slaughtered a chicken, was positive for influenza a / h5 by real - time rt - pcr, and virus was recovered on day 10 of inoculation on mdck cells (technical appendix). the woman had no signs or symptoms at the time the throat swab was collected, nor did she report any symptoms to health authorities during the subsequent week. chickens were also tested, and 4 chickens in the commune tested positive for influenza a(h5n1) virus by rt - pcr of throat and cloacal swab specimens. repeat throat swab specimens collected from the 4 household contacts 6 days after the initial collection yielded negative test results for influenza / h5. serologic testing showed seroconversion only in the woman with subclinical infection ; her hi titer increased from 80 are indicative of infection but must be confirmed by a second serologic test because of the possibility of cross - reactivity (1). the interpretation of results from a single serum sample is limited by the specificity or sensitivity of serologic tests, and viral shedding times may mean that infected cases may be missed. estimating the incidence of asymptomatic influenza a(h5n1) virus infection in humans exposed to sick poultry or human case - patients requires further careful study using early collection of swab samples and paired acute and convalescent serum samples. detailed methods of isolation and sequencing for influenza a(h5n1) virus from subclinical human case, vietnam. | laboratory - confirmed cases of subclinical infection with avian influenza a(h5n1) virus in humans are rare, and the true number of these cases is unknown. we describe the identification of a laboratory - confirmed subclinical case in a woman during an influenza a(h5n1) contact investigation in northern vietnam. |
a growing body of evidence suggests that routine mental health care varies strongly between different regions and providers, and that in many occasions, it does not correspond to the standards that the medical profession itself puts forward [12, 25, 29 ]. besides existing gaps between clinical practice and guideline recommendations, improving quality of care presents itself as an avenue to restrain the growth of medical expenditures by reducing costly complications and unnecessary procedures. these economic forces increase the desire for information evaluating the health benefits of investments in mental health care. in other words, better organisation and management of medical care would allow countries to spend their health budget more prudently. to improve care for their citizens and to realise these potential efficiency gains, policymakers are looking for methods to measure and benchmark the performance of their health care systems as a precondition for evidence - based health policy reforms. five mental disorders are among the ten leading causes of disability : depression, bipolar disorder, schizophrenia, obsessive - compulsive disorder, and alcohol abuse. for example, about 56% of national health service inpatient costs in england have been estimated to be attributable to schizophrenia. efforts to reduce these enormous costs and the burden for patients and relatives are urgently needed. with the development of treatment guidelines, there is growing hope that the quality of care will improve by diminishing inadequate care and increasing evidence - based practices. in mental health care, guidelines are intended for use by all physicians investigating, diagnosing and treating patients with mental illness, especially those with severe mental illness and a supposed unfavourable natural disease course. from the beginning, these practice guidelines have been used as standards against which routine care has been compared. thus, they also served as tools to detect practice variations across settings and across geographical areas, and to evaluate over- and underuse of services and interventions. the reasons for guideline non - adherence and for the gaps in quality detected by a variety of studies are complex and include areas that are only partly or completely out of the control of physicians and other healthcare providers. however, the methods for mental health quality assessment and improvement have been refined in the last years supported by increasing degrees of computerisation. although there seem to exist some structural measures of health care that have been shown to influence patient outcomes with sufficient reliability and validity, a paradigm shift has begun from developing and implementing measures of structural and process quality towards outcome quality. however, the challenging problem how to assess treatment quality now needs to be addressed in order to decide what type of measure will be best used for which specific purpose. to document the disease course, treatment effects and provider performance physician organisations, healthcare agencies, governments and other payers, consumers and researchers have created and implemented process measures that are typically rate - based and indicate the percentage of persons among the eligible population receiving adequate care. efforts to improve the quality of medical care must be measured with simple and reliable criteria. our article summarises the efforts and problems to develop quality indicators assessing the treatment of severe mental illness, and critically discusses whether increased measure performance and guideline implementation is likely to lead to better treatment quality and disease outcome. psychiatric interventions take place in complex bio - psycho - social interactions and settings that are not very well understood. various efforts have been undertaken to find valid parameters of the quality of care including the structural attributes of the settings in which care occurs, the processes of care, and the outcomes of care. before assessing quality, one has to decide how quality of care should be defined and this depends on whether one assesses only provider performance or also the contributions of patients and of the health care system. quality assessment may differ according to how broadly health and responsibility for health are defined ; whether the maximum effective or average care is sought ; and whether individual or social preferences define the optimum. today, psychiatric hospitals are, for example, often required to report their performance on standardised core measures, and to conduct both internal measurement - based as well as external quality improvement activities. public and private health care providers, consumers and accreditors may use the results several ways to encourage hospitals to improve their quality. in many countries, hospitals are provided with feedback systems comparing their performance with peer organisations, disclosing results publicly in an effort to influence purchaser or consumer decisions, or linking financial incentives to improved performance. their criteria, however, may vary according to the domains of care regarded as important, the available data sources, and the basis of comparison for the determination of quality. to increase comparability, the members of the oecd mental health care panel, consisting of international experts in mental health care, suggested a quality indicator set covering the most relevant domains of mental health care (primary care) by selecting four key aspects (http://www.oecd.org/els/health/technicalpapers) : treatment, continuity of care, coordination of care, and patient outcomes. [20, 21 ], the panel decided that the indicators should meet the following screening criteria : the indicator measures the technical quality provided, not interpersonal or consumer perspectives ; the indicator is focused on quality of care, not on cost or health care utilisation ; the indicator is built on a single item, not on a multi - item scale ; and the indicator is likely to be useful in quality assessment at the health care system level, rather than the provider level. in addition, hermann. argued that an indicator should likely be constructed from administrative data using uniform coding systems (e.g., icd or dsm codes), rather than requiring dedicated data collection or non - standardised data elements. the importance of a selected indicator was assumed to be made up of three different dimensions : the impact on health addressing the areas in which there is a clear gap between the actual and potential levels of health, the policy importance, and the susceptibility to be influenced by the health care system. for the latter, the question has to be addressed whether changes in the indicator will give information about the likely success or failure of policy changes. the scientific soundness of the indicators can be broken down into two dimensions : face validity and content validity. the face validity indicates if the measure is meaningful in a logical and clinical sense. the face validity of an indicator should be based on its basic clinical rationale, and on its past usage in national or other quality reporting activities. the content validity of an indicator addresses if the measure captures meaningful aspects of the quality of care. in addition, the feasibility of an indicator reflects the question of data availability and the burden of reporting. this last dimension should in particular address the issue whether the value of the information contained in an indicator outweighs the costs of data collection and reporting. for mental healthcare, some of the measure attributes conflict with each other. indicators with the best measurement properties such as utilisation data may not represent the breadth and diversity of the mental health care system in terms of processes, modalities, settings and interventions. for example, whereas psychopharmacological interventions have the broadest evidence base, other areas based on less rigorous studies may be similarly relevant for the long - term course of psychiatric disorders. specifications of quality indicators often require the use of approximations, and some measures are a proxy for a broader concept. for example, using hospital re - admission rates as a proxy for the quality of discharge planning assumes that hospital admissions are an unintended outcome. this builds on the ethics of a mental health care system that offers the least restrictive care which is effective. however, there may be research studies showing significant relations between re - admission rates and other measures of quality [1, 45 ]. targeted short - term hospital readmissions may even be an indicator of good quality avoiding long - term hospitalisations. thus, readmissions as quality indicators need to be evaluated in a more - differentiated manner. one of the indicators for provider performance selected by the mental health panel for primary care is the timely ambulatory follow - up after mental health hospitalisation, because the continuity of care was seen as an important aspect of quality in mental health. this indicator can be measured by the number of persons hospitalised due to a primary mental health diagnosis with an ambulatory mental health encounter or with a mental health practitioner within (a) 7 days, or (b) 30 days of hospital discharge. the importance of the indicator is outlined by the fact that most patients with a psychiatric disorder treated in an inpatient setting require follow - up ambulatory care to promote further recovery and prevent relapse. scheduling outpatient appointments proximally to discharge is generally recommended to address side effects that can result from inpatient medication changes, and to support compliance with the treatment plan. data indicate that there is wide variability in the duration between hospital discharge and the first ambulatory follow - up visit, some of which is related to patient factors (e.g., severity of illness) and some to system factors (e.g., availability of outpatient appointments). shorter gaps between discharge and aftercare may contribute to greater continuity of care and a decreased risk of relapse, although research evidence on this question is mixed. more continuity of care may be achieved by improvements in discharge planning interventions that have been shown to be effective in reducing rehospitalisation and in improving adherence to aftercare, making this indicator potentially useful in assessing the quality of an integrated care delivery system. a further quality indicator is the hospital readmission rate for psychiatric patients measured by the quota of the total number of readmissions to psychiatric inpatient care that occurred within (a) 7 days or (b) 30 days, divided by the total number of discharges from psychiatric inpatient care during a 12-month reporting period. hospital readmission rates are widely used as proxies for relapse or complications following an inpatient stay for psychiatric and substance use disorders. since they indicate premature discharge or lack of coordination with outpatient care, high readmission rates have led some inpatient facilities to examine factors associated with readmissions, including patient characteristics, length of stay, discharge planning, and links with outpatient care. given the high cost of institutional care, reducing readmission rates can have a substantial effect on mental health spending. however, the relation between readmission rates and other quality criteria are far from consistent. one study showed no association between readmission rates and clinical measures of treatment quality suggesting that it is not the success of the hospital intervention per se which influences the likelihood of readmission. this measure is therefore not a useful measure for one institution but rather for a whole mental health care delivery system, and indicates the quality of post - discharge outpatient treatment as well as the quality of inpatient treatment and information management between mental healthcare sectors. a further process measure proposed for provider performance measurement is the rate of persons with a mental illness receiving continuous medication treatment in the maintenance phase for disorders like major depression. in this case, the indicator may be specified as the number of persons treated with anti - depressant medication for a period of at least 180 days divided by the number of persons who are diagnosed with a new episode of depression. depressive disorders can impair personal, social and family functioning, decrease work productivity, and increase the risk of suicide. the world health organisation has estimated that by the year 2020, major depression will be the second leading disorder in terms of the global burden of disease. studies have consistently demonstrated that compared with their non - depressed counterparts, individuals with depression experienced impaired physical and role functioning, more workdays lost, and decreased productivity. many studies show heavy utilisation of health services, with hospitalisations accounting for a high proportion of costs. a range of antidepressant medications have been shown to be effective in ameliorating symptoms, and in improving quality of life and social functioning. however, adherence remains a problem when the choice for a medication trial has been taken with patients who discontinue their antidepressant early having a high risk to experience relapse or recurrence. randomised clinical trials have provided evidence that antidepressants need to be continued for 49 months after initiation to minimise the likelihood of relapse. the health system has considerable influence on this indicator of medication adherence with clinicians playing an important role in influencing patient attitudes to treatment by providing education, addressing concerns, and evaluating and treating side effects [6, 36 ]. it assesses the effectiveness of clinical management in achieving medication adherence as the basis of the effectiveness of an established dosage regimen by determining the percentage of adults who complete a period of continuation phase treatment adequate for defining a recovery according to the us agency for health care research and quality (ahrq) criteria. adherence indicators can be constructed from pharmacy data, which may be easily useful to identify patients who need assistance with medication adherence. at the same time, there is no consistent evidence that adherence to antidepressant medication dosages and other guideline recommendations are sufficient to improve patient outcomes. in one study, the authors found no differences in mean endpoint depression scores between a depression guideline intervention group and the control group, so that depression scores were only marginally better in the intervention group. other studies, however, showed considerable advantages of guideline - based intervention focusing on structured medication treatment and adherence compared to a group with treatment as usual in self - reported and physician - assessed depression scores after 12 months. a possible outcome indicator on the population level in primary care could be the mortality of persons with severe psychiatric disorders specified as the standardised mortality rate for persons with particular psychiatric disorders. individuals with schizophrenia and other severe mental illnesses have higher age- and sex - adjusted mortality rates than members of the general population. studies in some countries have found medical conditions and co - morbidities to be under - detected and under - treated in individuals with psychiatric disorders. such relative mortality rates, which are frequently used in cancer epidemiology studies, are well - accepted and plausible measures to indicate and evaluate the excess mortality in subgroups with certain diseases. they may also provide an estimate of the impact on longevity of these diseases. as there is no a priori biological reason why patients with mental health disorders should die prematurely, a large survival difference between different regions and different mental health care systems could point to shortfalls in the overall quality of medical care, not just mental health care, for this especially vulnerable group of patients. aggregate - level measures such as mortality or suicide rates are reliant on external and aggregate sources of data for interpreting results. inferences can only be made for a whole population and not for the individual patient. when there is no intrinsic standard, a process or outcome measure may be used to identify problems or outliers in the performance of a mental health care system, although such a measure can not determine the appropriateness of single interventions. for example, when hospitals are to be compared, statistical case - mix adjustment is needed to remove the influence of patient characteristics on the results in order to avoid unfair comparisons. in other areas, quality measurement is limited by the availability of data. to assess patient outcome, documentation systems for primary care as well as documentation systems for secondary mental health care may be used. this assessment should include instruments to record the first consultation / admission, weekly consultations, and discharge. for specialised care, these could assess sociodemographic data, diagnosis, disease - specific history, treatment course, and outcome. for major depressive disorder, such outcome assessments have been suggested to include the clinical global impressions (cgi) and the global assessment of functioning scale (gaf), the rating of depression scores by the self - rating patient health questionnaire (brief phq - d), and the beck depression inventory (bdi) as well as expert ratings using, i.e., the hamilton depression scale (ham - d). a therapist who is responsible for delivery of a clinical intervention, and thus has a stake in the outcome of the intervention, is not in a position to objectively assess the outcome. incentives will probably affect rating behaviour most strongly when the individual provider s job performance is directly assessed. thus, a rationale approach for quality measurement may combine the assessment of hard outcome parameters such as mortality, job integration, and long - term disease course including relapse and remission rates combined with an evaluation of process measures and treatment adherence. data suggest that the assessment of treatment adherence might have a more positive effect on clinical practice than routine outcome measurements. a cautionary note appears also necessary for payment for performance schemes in psychiatry using indicator systems. research indicates that clinicians are influenced by the implications of a new quality and outcomes framework indicator when recording diagnoses, disease severity, or treatment processes. thus, pay - for - performance or prospective payment schemes based on quality indicator performance may hold their promise to improve outcome quality only when such measures are valid and feasible, adopt a longitudinal perspective on quality management, and are successful at ensuring that all providers who are responsible for a particular patient s care are held accountable for the quality of care they provide. quality management measures are necessary to analyse weak points in routine care, detect opportunities for improvement of care, and check the implementation of guidelines. because some studies show that adherence to evidence - based guidelines in psychiatry can improve outcomes, whereas others have failed to do so, we further discuss this point in the following section. throughout the 1990s, educational initiatives for implementing guidelines were begun, and evidence - based guidelines have been formulated in many countries. several countries have initiated national clinical guideline programmes [16, 18, 36 ]. clinical guidelines aim to improve quality of care by advocating best - practice models and reducing treatment variation. some authors have even claimed that the term evidence - based guideline should only be used if a positive impact of guideline implementation on patient outcomes following guideline implementation has been shown [26, 42 ]. however, there is little evidence that guideline dissemination alone affects the behaviour of mental health clinicians or general practitioners. guideline implementation programmes using complicated and multifaceted procedures or participatory approaches appear to have an impact on professional behaviour. in a systematic review of psychiatric guideline implementation studies, the observed effects on provider performance or patient outcome after implementation were moderate and temporary in most cases. the studies with positive outcomes used complex multifaceted interventions or specific psychological methods to implement guidelines. interventions associated with better provider performance were multifaceted interventions with ongoing expert consultation, ongoing supervision, or interventions using marketing techniques and psychological theories to overcome guideline implementation obstacles. only one study using an intensive and costly intervention strategy showed a consistent positive and substantial improvement in the self- and physician - rated psychopathology of depression. there is, however, some evidence from observational studies that guideline adherence is associated with better outcomes. in a german multisite hospital study, treatment processes and patient outcomes patient structure and treatment processes showed a great variability between hospitals with mental state, chronicity of the disease, and other patient factors being the strongest predictors of clinical outcome. benchmarking hospitals, a poorer average clinical outcome was associated with lower guideline conformity in a variety of treatment domains, although it is not clear whether better guideline adherence in the hospitals with the best results was a causal factor for their enhanced performance. the paucity of studies showing positive effects of guideline implementation on patient outcome in mental health care should not discourage quality improvement initiatives based on guideline recommendations. it should be noted that programmes, which are directed only towards increasing guideline adherence, are too simplistic. measurement - based quality improvement needs organisational changes and may only be successful when positive incentives exist to continually improve treatment quality. they can be used to address suboptimal clinical outcomes, reduce the variability of care, and close the gaps between evidence - based guidelines and routine care. however, most indicators in mental health care are not empirically validated themselves, but are rather based on recommendations for interventions that have been evaluated in efficacy studies. due to a variety of national and other professional efforts, there is an extensive set of indicators available in mental health care that can be adapted for multiple purposes [19, 21 ]. among the basic setting cornerstones for quality indicators are inadequate variations in routine mental health care and a high degree of variability in guideline conformance rates. as an example, in severe mental disorders such as schizophrenia, there has been a trend towards polypharmacy in routine care not supported by evidence - based guidelines [2, 17 ]. reducing polypharmacy may reduce complications and side effects of antipsychotic treatment and therefore improve patient outcome. antipsychotic polypharmacy was suggested as an indicator of guideline adherence in the audits of the national institute of clinical excellence, specified as the number of individuals receiving only one antipsychotic at a time. based on reviews and meta - analyses, the research evidence to date is consistent with the goal of avoiding antipsychotic polypharmacy in patients who lack guideline - recommended indications for its use. two of the measures implemented by the joint commission on accreditation of healthcare organisations as a core measure set for hospital - based inpatient psychiatric services address antipsychotic polypharmacy. the second measure determines whether clinically appropriate justification has been documented supporting the use of more than one antipsychotic drug. this seems to be a pragmatically based measure of provider performance related to patient outcome. other quality indicators have to consider the regional mental healthcare system and need case - mix adjustment to avoid unfair comparisons. in general, quality indicators have to be meaningful, feasible and actionable, and address different dimensions of the mental health care system. studies show that there may be a differential effect of structured guideline implementation on the quality of provider performance and patient outcome, while the underlying causes for these differential effects are neither obvious nor easily explainable. for example, it is not clear why the texas medication algorithm project (tmap) intervention resulted in sustained improvement of patient outcomes in the depression study, but not in the schizophrenia study and not in the mania study. based on the results of guideline implementation studies, three implementation components may be necessary to improve patient outcomes by guideline implementation and other measurement - based quality improvement efforts : (1) ongoing support or feedback with an option to use expert consultation, (2) the use of specific psychological models to overcome obstacles to guideline implementation, or (3) social marketing techniques. rather than primarily relying on information, education and promotion of better quality of care, multifaceted guideline interventions should probably be specifically tailored to raising clinicians willingness to change, encouraging behaviour change through motivational techniques, reducing barriers through system reconfiguration, ensuring continued change, and establishing behavioural reinforcers. it should be noted that measures of rate - based processes and outcomes represent a subset of a broader range of approaches to quality assessment in mental health care. | in many occasions, routine mental health care does not correspond to the standards that the medical profession itself puts forward. hope exists to improve the outcome of severe mental illness by improving the quality of mental health care and by implementing evidence - based consensus guidelines. adherence to guideline recommendations should reduce costly complications and unnecessary procedures. to measure the quality of mental health care and disease outcome reliably and validly, quality indicators have to be available. these indicators of process and outcome quality should be easily measurable with routine data, should have a strong evidence base, and should be able to describe quality aspects across all sectors over the whole disease course. measurement - based quality improvement will not be successful when it results in overwhelming documentation reducing the time for clinicians for active treatment interventions. to overcome difficulties in the implementation guidelines and to reduce guideline non - adherence, guideline implementation and quality assurance should be embedded in a complex programme consisting of multifaceted interventions using specific psychological methods for implementation, consultation by experts, and reimbursement of documentation efforts. there are a number of challenges to select appropriate quality indicators in order to allow a fair comparison across different approaches of care. carefully used, the use of quality indicators and improved guideline adherence can address suboptimal clinical outcomes, reduce practice variations, and narrow the gap between optimal and routine care. |
renal cell carcinoma (rcc) is the most common malignancy arising in the kidney. in the united states alone, each year 39,000 people are diagnosed with rcc and 13,000 people die from the disease []. the classic triad of virchow (flank pain, hematuria and a palpable abdominal mass) is only seen in approximately 9% of newly diagnosed patients []. this considerably complicates the diagnosis, since the disease can present with a broad array of (paraneoplastic) symptoms []. as a consequence, 30% of patients will present with metastatic disease, whereas of the other 70% treated by nephrectomy, 3040% will eventually relapse []. the 5-year survival rate for small (less than 7 cm) tumors limited to the kidney (pt1 tumor) is more than 90% [], but prognosis for metastatic disease is bleak, with a median survival of only 10 months []. therefore, therapeutic strategies focus on immunotherapy, neoangiogenesis inhibitors and other targeted approaches. in this review, another approach using antibodies developed for targeting rcc is discussed and particularly their application in the diagnosis and therapy of rcc. since the first description of ehrlich to specifically guide cytotoxic therapy to cancer tissue [], much has been debated on the feasibility of this approach. development of the hybridoma technique [] allowed isolation of large quantities of antibodies with predefined specificity. with the identification of the tumor - associated target antigens, real progress has been made on developing treatment and/or diagnostic strategies using mabs. to date, no tumor specific antigen, i.e. an antigen expressed on all tumor cells which is not expressed by normal cells in the body, has been identified. tumor - associated antigens (taa) have been identified for a series of human tumor types []. these are either differentiation antigens, (transiently) expressed during organogenesis, or aberrantly expressed antigens, (transiently) expressed elsewhere in non - related normal tissue(s). expression of antigen on the primary tumor or metastases is generally heterogeneous. for tumor targeting with mabs this is a suboptimal feature, since not all cells can be targeted by the mab. heterogeneous expression between different tumor sites, varying degrees of expression in tumor cells of the same tumor and temporal modulation of taa - expression are considered major limitations of effective targeting of tumors with mabs. in addition to intratumoral heterogeneity of antigen expression, other parameters have been defined that may be equally important in hampering tumor targeting with mabs. these are : size of the tumor mass, the antigen density, the fate of antigen / antibody complex, presence of circulating antigen, mab format, mab dose, route of administration and mab circulating half - life []. mab targeting is complicated by large tumor blood vessels as well as impaired blood flow in the tumor by elevated interstitial fluid pressure (ifp) []. high vascular density is not equivalent to high perfusion rates in the tumor, which are required for optimal mab delivery. rcc has always been considered a highly vascularized tumor by morphologic standards. however, in comparison with normal kidney tissue rcc is poorly perfused [], thereby impeding adequate mab delivery to the tumor cells. these limitations of delivering the mab to tumor tissue have to be overcome in order to develop a suitable mab - based treatment strategy. several mechanisms to eradicate tumor cells by mabs are available : either via effector cells or complement dependent cytotoxicity or through conjugation of the mab to toxins, drugs or radionuclides. since antigen expression within tumors is heterogeneous, antigen - negative tumor cells may evade tumor cell lysis by effector cell- or complement - mediated cytotoxicity, which may eventually lead to tumor recurrence. the same applies to mabs conjugated to toxins or drugs, since internalization of a mab conjugated to a toxin or drug is required to mediate cell - killing []. radiolabeling of antibodies was developed in 1950, when eisen observed that proteins could be labeled with i without altering their immunological specificity []. besides i, other radionuclides (y, lu, re, re and cu) have since been investigated to induce tumor cell death (see table 1). the advantage of radiolabeled antibodies is that the mab does not have to bind to every tumor cell to induce cytotoxicity, since the radionuclides emit -particles, which can be effective for up to 50 or more cell diameters. this so - called crossfire effect can thus overcome heterogeneity of antigen expression, as the radiation destroys the antigen - negative cells as well. a disadvantage of this technique is the sensitivity of normal organs to radiation, particularly the bone marrow. the dose limiting toxicity of delivering high - dose radioimmunotherapy (rit), i.e. a radionuclide conjugated to a tumor - associated mab, is therefore generally hematological. table 1radionuclides used in radioimmunotherapy of clear cell renal cell carcinomaradionuclidehalf - life-average (kev) (kev)maximum range -particles in tissue (mm)advantagesdisadvantagesi8.0 days1923623.0easy labeling ; inexpensivehigh radiation burden to personnel / relatives ; hospital admittance requiredre90.7 h3621375.1out - patient treatment possible ; ideal gamma for imaginglaborious labelingy64 h935none12high - energy beta - emission ; prolonged tumor retention ; out - patient treatment possibleno imaging possiblelu6.7 days1492082.5prolonged tumor retention radionuclides used in radioimmunotherapy of clear cell renal cell carcinoma using the previously mentioned hybridoma technique a wide array of mabs against taas has been produced, e.g. mabs against carcino - embryonic antigen (cea) (mainly expressed in colorectal and medullary thyroid carcinomas), muc-1 (mainly ovarian and breast cancer), tag72 (mainly ovarian and colorectal cancer), cd-20 (non - hodgkin 's lymphoma (nhl)) and g250-antigen (rcc). in various clinical trials safety and efficacy of these newly developed mabs labeled with various radionuclides have been investigated []. the effector cell, complement and apoptosis inducing cytotoxicity of the mab give high intrinsic anti - tumor activity as well. extensive research has resulted in the first registered treatment with radiolabeled mabs directed against the surface antigen cd-20 expressed on b - cell nhl (y - labeled anti - cd20 mab ibritumomab tiuxetan (zevalin, biogen idec, boston ma, usa and schering, berlin, germany) and i - labeled anti - cd20 mab tositumomab (bexxar, gsk, philadelphia, pa, usa). in patients with solid tumors, therapeutic strategies with radiolabeled mabs however, as mentioned previously, tumor - related factors also play an important role. the most common types of solid malignancies targeted in clinical trials with rit have been epithelial cancers, e.g. colorectal cancer, ovarian cancer, medullary thyroid cancer, breast cancer, prostate cancer and rcc. results of these trials did not result in registration of radiolabeled mab preparations for regular treatment of these cancer types. however, patients entered in these trials often had bulky metastatic disease and had been heavily pretreated with chemotherapy and/or radiotherapy in most cases. partial responses and stabilization of previously progressive disease have been seen in few patients in most of these trials []. peptides have been used for radionuclide targeting of tumors to overcome the difficulties in tumor targeting with mabs mentioned above. these peptides have a high affinity for specific receptors that are expressed on the tumor cell. tumor targeting peptides have advantages over mabs, as they diffuse rapidly in target tissue and clear rapidly from the blood and from the non - target tissues. peptides are usually non - immunogenic and generally have a low toxicity profile. peptide receptor radionuclide imaging (prri) and therapy (prrt) are now under investigation. to date, the somatostatin (sst) analog in - labeled - octreotide (octreoscan, mallinckrodt tyco healthcare, petten, the netherlands) is the most successful radiopeptide for tumor imaging and has been the first to be approved for scintigraphic localization of primary and metastatic neuro - endocrine tumors expressing sst2 and sst5 receptor subtypes []. expression of these receptors was found in 72% of rcc samples analyzed, irrespective of histopathological subtype or grading of the tumor []. the use of [in]octreotide has been evaluated in patients with metastatic rcc. in this study, 68 rcc metastases in 9 patients, confirmed by diagnostic ct and/or x - ray images were evaluated. forty (59%) of the 68 known sites were visualized []. besides octreotide, a new series of peptides is now being evaluated for targeting of solid tumors. cholecystokinin (cck) analogues, vasoactive intestinal peptide (vip), neuropeptide y (npy), bombesin, glucagon - like peptide-1 (glp-1) and rgd peptides have shown promising preclinical tumor - receptor targeting (for review see reubi []). the gastrointestinal peptide gastrin acts as a neurotransmitter in the brain and as a regulator of various functions in the gastrointestinal tract []. these receptors are highly expressed in medullary thyroid carcinoma (mtc), enabling gastrin to visualize metastatic mtc with very high sensitivity []. rcc however, does not express these receptors and gastrin is therefore not suitable for rcc imaging []. vip, a member of the group of secretin - like peptides, is an important neurotransmitter in the gut. its actions are mediated by specific g protein - coupled receptors that can be internalized upon ligand binding []. although expression of the vip receptors has been found on nephroblastomas [], expression on rcc has not been determined. npy is a neurotransmitter that is predominantly found in the central nervous system, where it functions as a stimulator of feeding behavior and inhibition of anxiety []. more recently, expression of npy receptors has been found on rcc and nephroblastomas, suggesting a potential role for rcc targeting using radiolabeled npy []. bombesin has a high and specific affinity for the gastrin releasing peptide receptor (grp - r) and this receptor stimulates proliferation of tumor growth in various tumor types []. rcc has been found to have a high expression of grp - r []. targeting of rcc was done using a bombesin analogue labeled with lu. in this study, in vitro autoradiography showed specific uptake of the radioligand in five of the six rcc samples evaluated []. recently, glp-1 receptor expression in solid human tumors has been evaluated extensively and systematically. this study found no glp-1 receptor expression in 20 rcc tissue samples analyzed, excluding glp-1 receptor as a target for in vivo rcc imaging or therapy []. rgd peptides contain the amino acid sequence arg - gly - asp that has high and specific affinity for the v3 integrin []. this integrin is mainly expressed on proliferating endothelial cells, whereas it is not expressed on quiescent endothelial cells []. in growing tumors a continuous formation of new blood vessels is required. the expression of v3 has been found to increase with higher rcc tumor grades. of the rcc metastases examined, 2 of 14 showed high expression of v3, 8 of 14 showed weak expression and 4 of 14 did not express the v3 integrin []. to date, rgd peptides have not been evaluated for rcc imaging. in rcc, several mabs have been defined that are reactive with rcc - associated antigens []. cross - reactivity with non - kidney tissue was seen in some of these mabs, whereas others were only expressed in kidney / rcc. one of these mabs, which showed relative high tumor - to - blood ratios in mice with rcc xenografts is mab a6h []. this mab recognizes an antigen common to rcc, some lung and colon carcinomas, the proximal renal tubules but no other normal tissues in vivo []. in a clinical study, the imaging and rit potential of this mab was examined []. this low sensitivity was attributed to soluble antigen binding by the mab and the expression of antigen in normal tissue, thereby not allowing the mab to bind to tumor tissue. this clinical finding of antigen expression in normal tissue was not in line with the previous findings. after modification of the dosing regimen, the detection rate of metastatic lesions increased, but the number of detected lesions remained unsatisfactory. as a result, the use of mab a6h for diagnosis and treatment of rcc was discontinued. g250, a mab against a rcc - associated antigen has been investigated extensively, because the antigen which this mab recognizes showed remarkable tissue distribution and expression. the mab g250 was obtained after fusion of spleen cells from a mouse immunized with fresh rcc homogenates. the antigen that mab g250 targets has been designated in the literature as mn, ca ix and g250. of the 47 primary rcc specimens initially analyzed, 42 (89%) showed homogeneous g250-antigen expression, whereas four tumors showed heterogeneous expression and one tumor was g250-antigen - negative. of the eight metastases examined, g250-antigen expression was homogeneous in five (62%), heterogeneous in two, while one did not express the g250-antigen []. expression in normal tissues has been evaluated extensively and has been shown to be restricted to the (upper) gastrointestinal mucosa (stomach, ileum, proximal and middle colon) and gastrointestinal related structures (intra- and extrahepatic biliary system, pancreas) []. later studies showed an almost ubiquitous expression (> 90%) of g250-antigen in clear cell rcc (ccrcc), being the most prominent form of rcc (80% of cases). g250-antigen expression in the different histological subtypes of rcc was determined by rt - pcr and immunohistochemistry. all the clear cell tumors displayed g250-antigen mrna, but expression of g250-antigen by oncocytomas, chromophobe or papillary rcc was low or absent []. an early or a first event in the clear cell rcc tumorigenic pathway is mutations leading to loss of von hippel lindau protein (pvhl) in 5075% of sporadic rcc. in normoxic conditions pvhl is responsible for degradation of hypoxia inducible factor-1 (hif-1), which thereby can not bind to hif-1 to form hif-1. in hypoxic conditions, however, degradation does not occur and hif-1 can cause the transcription of a number of hypoxia - inducible genes. these include vascular endothelial growth factor (vegf), transforming growth factor- (tgf), erythropoietin, g250-antigen and others. clearly, expression of these proteins is advantageous for tumor growth. with the loss of functional pvhl in rcc this mutational loss of pvhl thus explains why g250-antigen is almost invariably upregulated in ccrcc. g250-antigen is also expressed in various other tumor types (e.g. cervix, lung) under hypoxic conditions. various animal and ex vivo experiments have shown the potential of the g250 mab as a targeting modality of rcc []. since g250-antigen is high and homogeneously expressed in rcc tissue, is restricted to a few normal tissues and other tumors, and a low mab dose is needed to obtain antigen saturation, mab g250 seemed a suitable candidate for further investigation in clinical studies. imaging and biodistribution were studied in 16 patients receiving 370 mbq i - labeled mg250 at escalating protein dose levels 1 week prior to nephrectomy. after 34 days clear delineation of tumors was seen in 12 patients, imaged with a gamma camera. ten of these tumors proved to be g250-positive, whereas the other two showed less than 5% g250-antigen expression. after nephrectomy, tumor samples were shown to have high and focal uptake of g250, up to 0.21 % injected dose / gram (% id / g). tumor targeting was not the result of blood pooling, as the blood volume marker tc - labeled human serum albumin showed significantly lower tumor uptake than [i]mg250 that had been administered earlier. therefore, this was indicative of true antibody targeting of the tumor by mg250 []. as good targeting of ccrcc by mg250 was seen in this study, a phase i / ii radioimmunotherapy (rit) dose escalation study was performed by divgi. patients in this study were treated with one high - activity - dose injection of [i]mg250. after reaching the maximum tolerated dose (mtd), another 15 patients were enrolled and treated at the mtd to monitor any possible therapeutic effects. in the phase i dose - escalation study, mtd was defined at 3330 mbq / m, due to hematological toxicity. transient hepatic toxicity occurred at dose levels of 1665 mbq / m and higher, but was not dose limiting. fourteen patients had grade 3 hepatic toxicity, that did not last for more than 2 weeks. a total of 33 patients was treated, 18 in the dose - escalating part of the study and another 15 patients at the mtd (3330 mbq / m), to evaluate therapeutic efficacy. of these 33 patients, 17 stabilized for 3 months, after which patients received other treatments, preventing further follow - up. three patients showed regression of some of their lesions, but no partial or complete responses were noted []. the formation of human anti mouse antibodies (hama) in all patients receiving mg250 prohibited retreatment. formation of immune complexes with rapid clearance of the radiolabeled mab to liver and spleen would have occurred in the case of multiple administrations, thereby limiting targeting of the mab to the tumor []. this, in combination with the high potential of g250 as a targeting agent in the treatment of metastasized rcc, led to the development of a chimeric form of g250 (cg250) []. this mab is composed of murine antigen - binding variable domains, that recognize the taa and human constant domains of heavy and light chains derived from the human igg1 isotype []. the rationale behind this construction was the decrease in immunogenicity of the antibody, potentially allowing multiple administrations. unlabeled g250 antibody facilitates antibody - dependent cellular cytotoxicity (adcc) of g250-antigen expressing cells, which leads to induction of lysis of these cells []. this finding led to a study where 36 patients with metastatic ccrcc received 50 mg cg250 weekly for 12 weeks. development of human anti chimeric antibody (haca) was low and not clinically significant. before treatment, 80% of patients were progressive. after treatment, 11 patients had stable disease and during follow - up one complete and one partial response were seen. the median survival of 15 months suggested that g250 may be able to immunomodulate the natural course of metastatic rcc []. based on these results, an adjuvant phase iii trial has been initiated in high - risk ccrcc patients who are nephrectomized and have no known metastases, using this treatment regimen. since the 1990s, high - dose bolus interleukin-2 (il-2) has been established as a first - line therapy for metastatic rcc. il-2 is a t - cell growth factor that is thought to play a critical role in t - cell dependent immune responses. high - dose bolus il-2 as therapy in metastatic rcc has had varying success, with responses in up to 15% of patients []. it was hypothesized that the immunological specificity of lymphokine - activated killer cells of patients receiving il-2 therapy may be enhanced through the co - administration of cg250 []. vice versa, co - administration of il-2 can enhance the therapeutic efficacy of g250 []. in a phase ii trial 35 patients with progressive ccrcc received weekly i.v. mean survival was 24 months in this trial, compared to 16.3 months median survival with high - dose il-2 therapy [], which also has toxic side effects. the authors considered it unlikely that the increased survival was due to the low - dose il-2, using a six - fold decrease of normal il-2 dose used to induce clinical efficacy. after cg250 became available, the pharmacokinetics, biodistribution, imaging characteristics and dosimetry of this new radiolabeled targeting vehicle was studied in a protein dose escalation study identical to murine g250. sixteen presurgical rcc patients received increasing doses of cg250 between 2 and 50 mg labeled with i, given i.v. a week before they underwent nephrectomy. highest tumor uptake was observed in the patients that received 5 and 10 mg [i]mg250, with focal tumor uptake as high as 0.52% id / g. at higher protein doses this suggested that antigen - saturation could have occurred at protein doses exceeding 10 mg. excellent images of g250-antigen positive tumors were obtained, with visualization of tumor lesions and metastases, seen earlier on ct or x - ray. dosimetric analysis showed a high radiation - absorbed dose to primary tumors as well as metastases (up to 1.9 cgy / mbq to primary tumor). up to 20 weeks post - injection, human anti chimeric antibody (haca) responses were seen in two patients, but titers were considered low and clinically irrelevant []. reducing the immunogenic properties of the antibody opened the possibility of multiple treatments. the mtd of [i]cg250 in metastatic rcc was determined in a phase i radioactivity dose escalation trial in patients with progressive metastatic rcc at study entry. twelve patients received 5 mg of cg250 labeled with 185 mbq i (scout dose). when accumulation of antibody was seen in any tumor site, patients received escalating radioactivity - doses of [i]cg250. in contrast to the trials performed with murine g250, no hepatic toxicity was seen. this was believed to be the result of saturation of the hepatic compartment by the diagnostic scout dose of [i]cg250. besides mild nausea without vomiting and transient fatigue (both grade 1 ctc), no other non - hematological side effects occurred. the mtd was observed to be 2220 mbq / m, with hematological toxicity as the dose - limiting factor. of the 8 patients receiving treatment, 1 showed stable disease and 1 had a partial response []. in subsequent studies two strategies were tested to optimize targeting of metastatic ccrcc with rit. fractionation of the dose was done in a phase i study by divgi., patients received 1110 mbq of [i]cg250 and whole - body activity was measured after 23 days. this was continued until a whole - body absorbed dose of 0.50 gy was reached. patients without disease progression were retreated after recovery from hematological toxicity. in subsequent cohorts, haca development was measured in two patients, altering pharmacokinetics and excluding them from further treatment. dose - limiting toxicity was again hematopoietic, with the mtd at 0.75 gy as whole - body absorbed dose. this trial therefore provided no evidence of a potential benefit of fractionation of rit doses in treating ccrcc. (2220 mbq / m), combined with the properties of cg250 allowing multiple administrations, led to a study where two sequential high doses of [i]cg250 treatment were given. three months later, rapid clearance of the mab by haca development was excluded by imaging of a scout dose of 185 mbq [i]cg250. when tumor targeting was seen again, the second high - dose injection [i]cg250 was given. mtd of the second rit proved to be again due to hematological toxicity and was set at 1665 mbq / m, being 75% of the mtd of the first infusion). subsequently, 15 patients were treated at this dose level to evaluate tumor response. in total, 29 patients entered the study, 11 were excluded due to grade 4 hematological toxicity after the first rit (n=3), palliative treatment (n=2), rapid progressive disease (n=2) or haca development (n=4). of the 18 patients evaluated (3 not receiving the second rit at mtd), 5 patients had stabilization of their disease, lasting 312 months. there proved to be an inverse correlation between the size of metastases and radiation absorbed dose. therapeutic radiation doses (more than 50 gy) [] were only guided to the lesions smaller than 5 g. the authors concluded that rit in rcc patients could best be given in the setting of small volume disease or as adjuvant therapy []. first, the targeting capabilities of [in]cg250 have been compared to those of [i]cg250. in nude mice - human tumor models, superior targeting of in over i had been shown []. as part of the cg250 antibody - antigen complex is internalized, intracellular [i]cg250 is metabolized and rapidly excreted by the tumor cell. metallic radionuclides, such as in, y and lu, are trapped in the lysosomes and residualize after internalization of the mab - antigen complex by the target cells []. to investigate whether this phenomenon may also occur in humans, five patients with metastatic rcc were i.v. injected with 185 mbq of [in]dtpa - cg250 on day 0 and 185 mbq of [i]cg250 on day 4. gamma images were made directly and on day 4 after both injections and compared (fig. [in]dtpa - cg250 images revealed more lesions than [i]cg250 (47 vs. 30) and quantitative analysis showed higher accumulation of [in]dtpa - cg250 in 20 of 25 lesions measured in terms of % id / g []. figure 1(a) [in]cg 250 immunoscintigram of a patient with metastatic ccrcc, acquired 6 days after injection of 185 mbq of [in]cg250. green arrows mark a lesion not seen on the fluorodeoxyglucose (fdg)-positron emission tomography (pet)-computed tomography(ct) images shown in (b). red arrows indicate the injection standard. the anterior image is shown in the left panel, (b) pet - ct scan of the same patient acquired after injection of 250 mbq of [f]fdg. (a) [in]cg 250 immunoscintigram of a patient with metastatic ccrcc, acquired 6 days after injection of 185 mbq of [in]cg250. green arrows mark a lesion not seen on the fluorodeoxyglucose (fdg)-positron emission tomography (pet)-computed tomography(ct) images shown in (b). red arrows indicate the injection standard. the anterior image is shown in the left panel, (b) pet - ct scan of the same patient acquired after injection of 250 mbq of [f]fdg. the therapeutic properties of cg250 labeled with four radionuclides have been tested in nude mice with human rcc xenografts. the four radionuclides under investigation were y and lu (both residualizing), and i and re (both non - residualizing). after determining the mtd for each radionuclide conjugated to cg250 in mice, an rit experiment was done comparing tumor growth and survival after treatment with each radiolabeled cg250 preparation. tumor growth was delayed most effectively by lu, followed by y and re and least by i (185, 125, 90 and 25 days, respectively). the best median survival was observed for lu (300 days), with the control group having a median survival of less than 150 days. these radionuclides should be considered better candidates for rit with cg250 than i []. based on these preclinical and clinical data (table 2), an ongoing phase i / ii [lu]dota - cg250 dose escalation rit study in progressive, metastatic rcc patients was designed. patients in whom cg250 targeting of rcc metastases is observed are treated with up to three cycles of [lu]dota - cg250 to determine mtd. so far, minor responses have been noted at the lower dose levels and dose escalation is ongoing. table 2phase i / ii radioimmunotherapy trials in clear cell renal cell carcinomareferenceradiophar - maceuticaltarget antigenmab typeno. of patientsresponsesspecial featuresdivgi.[][i]g250g250murine g2503317 sdsteffens.[][i]g250g250chimeric g250121 pr ; 1 sddivgi.[][i]cg250g250chimeric g250157 sdfractionated ritbrouwers.[][i]cg250g250chimeric g250275 sdtwo high - dose treatment phase i / ii radioimmunotherapy trials in clear cell renal cell carcinoma future strategies to improve clinical efficacy of cg250 could be : (1) tumor pretargeting ; (2) high dose rit with bone marrow support or transplant or (3) use of high linear energy transfer (let) particles emitting radionuclides. in pretarging rit, administration of the mab is separated from the injection of the radionuclide. this allows the unlabeled mab to bind to the tumor as well as to clear from circulation and normal organs. the radionuclide is administered in a second injection as a rapidly clearing agent with high affinity to the previously administered mab. affinity of the radionuclide binding to the mab may be achieved through, e.g. an avidin - biotin complex or with bispecific mabs (bsmabs). biotinylated mab and radionuclides can be coupled through an extremely avid interaction with avidin (for review see boerman. a bsmab (cg250 x dtin-1) was produced and pretargeting experiments in nude mice with rcc xenografts targeted with in - labeled bivalent peptide showed excellent tumor targeting []. to date, however, this approach has not been tested clinically for cg250. autologous marrow transplantation or peripheral blood stem cell reinfusion has been investigated as a means to overcome bone marrow toxicity in rit, thereby allowing administration of activity doses more than twice as high as without any bone marrow support []. the use of let (alpha) particle emitting radionuclides has the advantage of high cytotoxic potency, combined with a low range and thus not reaching most normal tissue surrounding the tumor. however, most alpha - emitters have a half - life of less than 1 h, which is hardly compatible with mabs targeting tumors. nevertheless, to date clinical impact of rit for treatment of ccrcc has been minimal. it remains to be established whether the use of more powerful radionuclides can lead to alteration of the clinical course of metastatic ccrcc. second, g250 may be more valuable in an adjuvant setting and/or as a diagnostic means. finally, with the advent of new treatment possibilities for rcc such as angiogenesis inhibitors, combination treatment with g250 rit and these new substances may play a role in more effective management of ccrcc. | abstractrenal cell carcinoma (rcc) is a radio- and chemotherapy resistant tumor, which has a very high morbidity and mortality when metastasized. the current treatment options demonstrate limited efficacy and severe side - effects. therefore, there is a need for new therapeutic strategies for rcc. as for other malignancies, monoclonal antibodies (mabs) targeting tumor - associated antigens have been developed for rcc. one of these, mab g250, targets the mn / caix / g250 antigen, which is ubiquitously expressed in clear cell rcc (ccrcc). ccrcc is the most common form of rcc with a prevalence of 80%. expression of g250 in normal tissue is restricted to the gastrointestinal mucosa and related structures, thereby making it a suitable candidate for targeting ccrcc. in several clinical studies the efficient accumulation of mab g250 in ccrcc has been demonstrated, resulting in high contrast images. g250-imaging could prove to be a valuable tool in diagnosing metastases in patients with a g250-antigen positive primary tumor and/or in the differential diagnosis of suspect kidney lesions. furthermore, the therapeutic efficacy of radiolabeled g250 has been investigated in a series of studies. thus far, most efforts have been devoted to g250 labeled with high doses of 131i. other radionuclides which may enhance the therapeutic index of this radiolabeled mab are currently under investigation. in our institution, an activity dose escalation study is currently ongoing to investigate the therapeutic potential of 177lu - labeled g250 in metastatic ccrcc patients. in this review, the current status of the diagnostic and therapeutic properties of radiolabeled antibodies in rcc is described. |
in today 's life, despite the recent advancements, notable risks yet threaten human beings, like the risk of burns which accounts for a high mortality in developed countries and leaves several people disabled. burn is an event that injures a huge number of victims each year and imposes irreplaceable physical, psychological, mental, economic, and social consequences, and even death. they are affected by disability for a longer period, compared to those with cancer and cardiovascular diseases. generally, 19,500 eases of burn events occur annually in the world, of which over 95% happen in less - developed countries. burns are the sixth cause of mortality in iran, as about 50,000 people suffer from burns in a year of whom 2600 develop acute complications and need critical care. therefore, burn injuries are among the most important hazardous events for health system in iran. there are five basic and important key points in the management of hazardous events, including epidemiological studies, prevention, injury biomechanism, treatment, and rehabilitation. measurement of distribution and frequency of the events and diseases is among the most important parts of an event or disease epidemiology as through interpretation of epidemiological data, the etiological factors of a hazard or disease and the individuals exposed to them are detected. in this way, the health status of a society is determined and preventive strategies are developed. consequently, the needed programs are planned to achieve the final goal of public health promotion. in recent years, numerous efforts have been made to prevent burns, such as conducting epidemiological studies, by which necessary interventions are purposefully administered in the treatment of burns. precise epidemiological information is needed for provision and equipment of burn centers, as well as for proper planning to prevent burns and reduce their complications and mortality. through adequate necessary information, the financial and human resources that are required it includes measurement of distribution and frequency of various factors related to burns, such as etiological causes of burns, and detection of the resulting mortality. as nurses play a major role in this part, the second part of the present study investigates the level of administration of nursing professional tasks in relation to burn patients to detect the existing defects in patients care, as well as nurses educational needs. as imam mosa kazem burn hospital is the only center in isfahan province and many other neighboring provinces, the data of the present study can be an appropriate source for detection of burn problems. due to the relatively high mortality associated with burns, our obtained results can play a role in prevention of such a problem and its complications, as well as in the establishment of burn care centers. the first part is a descriptive retrospective study that was conducted on all hospitalized patients in pediatric, men, and women burn wards, and icu1 and icu2 burn wards of imam mosa kazem burn hospital from april 2009 to april 2010. there were 2355 hospitalized patients. after reviewing their medical files regarding their etiology and related ward, those referring to the reconstructive surgery ward or those with no etiology of burns were excluded. finally, 836 patients files with burns as the chief complaint were reviewed by census sampling. associated variables of the study were extracted from patients files through a checklist whose content was modified and confirmed by experts of burns and epidemiology in isfahan university of medical sciences. all patients files contained similar forms and notes for measurement of patients burns through rule of nines. finally, the data were analyzed by spss and the results were extracted in the form of chart and diagrams. the second part of the study used a questionnaire, which, based on a checklist of nurses professional tasks in burn wards, introduces 27 duties of nurses concerning primary assessment, hypovolemic shock, inhalatory burns, sepsis, pain, psychological problems, nutrition, rehabilitation, team work, and information records. it has been prepared by the nursing association and approved by the ministry of health, and is the only existing reference for burns standard care in iran. the questionnaires were distributed among 50 hospital nurses to score each item of care from 1 to 10. then, the care was categorized into 10 general domains and the mean of each domain score was calculated, and the level of administration was investigated for each item of care. about 59.69% of the patients were male and 40.31% were female, and 50.40% were married and 49.60% were single. the highest percentage of patients were in the age groups 20 - 30 years (20 - 26%), 0 - 10 years (22.90%), and 10 - 20 years (17.50%). children and workers had the highest hospitalization rates (16.93% and 16.32%, respectively). the patients were referred from 16 provinces in iran, with the highest number of patients from the provinces of isfahan, charmahal bakhtiari, lorestan, khozestan, kermanshah, and ilam. about 28.98% of the patients were from rural areas and the rest of them were from urban areas. about 88 - 99% of the patients were iranians and 0.12% were non - iranians. the highest percentage of medical insurance programs covered by the patients was for rural area insurance (28.98%) and social security (28.86%), and 15.96% of the patients had no medical insurance coverage. the highest number of burns occurred in winter (29.11%), summer (26.57%), spring (23.31%), and fall (21.01%), and in the months of july (10.87%), february (10.39%), january (9.42%), and march (9.30%). the lowest number of burns occurred in november (6.04%), april (6.52%), and september (7%). numerous factors caused burns, including inflammable materials (24.06%), gas explosion (23.94%), and scalds (21.02%). concerning the etiology of burns, 59.33% of the burns occurred at home due to neglect, which had the highest frequency. this was followed by occupational burns (23.85%) and self - inflicted burns (9.09%). the upper limbs, lower limbs, trunk, face, and genital area were the most burned body parts. about 73.92% of the patients were discharged after a recovery period, 21.77% expired, 4.19% left the hospital without a discharge order, and 0.12% were transferred to other medical centers for further medical interventions. the highest mortality occurred among the individuals with burn percentages of 80 - 90%, 60 - 70%, and 40 - 50%, respectively. the major cause of morality in women and men burn wards and icu1 and icu2 was septicemia, and in the pediatric burn ward, it was hypovolemic shock. in women and men burn wards, inhalation burns were the second cause of mortality after septicemia, while in icu1 and icu2, hypovolemic shock was the cause of mortality after septicemia [figure 1 ]. causes of death in various wards in the second part of the study, the level of nursing professional task administration was investigated in the burn wards and its results have been presented in table 1. based on the results presented in the table, the lowest percentage of professional physical care was administered for sepsis. mean scores of various aspects of burn care, maximum possible score of each aspect, and percentage of implementation (based on nurses answers) most of the burns in the present study had occurred in subjects less than 30 years of age, which is consistent with the report of koushyar., in which 60% of the burns were reported in individuals less than 30 years of age, and sotoodehnejad., in which over 90% of the burns had occurred in people over 40 years of age. in a study conducted in the eastern mediterranean region in 1997 - 2007, one - third of the burns had occurred in people less than 5 years of age. this reveals that most of the burns had occurred in children, adolescents, and young adults, which is consistent with the present study. the present study revealed that burns are more prevalent in female homemakers, children, and workers, which agrees with the findings of samimi., who reported most of the burns in children, and pishnamazi, who reported burns to be more prevalent in female homemakers and workers. it can be concluded that children, female homemakers, and workers in the iranian society are at a higher risk of burns. the present study showed that burns occur commonly in people from urban areas than from rural areas, which is consistent with a study conducted in sari that reported the incidence of burns to be more in urban areas. meanwhile, our obtained results are not in line with the studies conducted in yasouj and zimbabwe which reported the burns to be more prevalent in rustic people. after all, it seems that the incidence of burns in rural and urban areas is different in different societies, which needs further studies, but may be due to the difference between rural and urban areas. in the present study, the incidence of burns was found to be more in summer and winter as well as in the months of july and february, compared to other seasons and months of the year. the prevalence of burns was also found to be less in fall, which is not in line with another study conducted in iran that reported the highest prevalence of burns in fall. therefore, it can be concluded that acute burns, which result in hospitalization, have almost the same prevalence in all months and seasons of the year and are not associated with a specific month or season. concerning the etiology of the burns, the most reported causes were flammable materials (kerosene, gasoline, gas oil, thinner, and alcohol), gas explosion, scalds, and flame, which is consistent with the results of two iranian studies reporting the most prevalent causes of burns in men as oil and its products, in children as scalds, and in women as flame. a literature review of south asian countries has reported the use of kerosene as the most prevalent cause of burns in these countries, which was also reported in the present study as one among the main factors. with regard to the findings of the present study and other studies, it can be concluded that although the causes of burns are known and several, the people are not cautious and ignore the safety instructions and standards in the use of flammable and explosive materials. unintentional burns at home and work, and self - inflicted burns were the most prevalent causes of burns in the present study. other studies in iran reported almost similar results, which include a study conducted in kerman that reported 85% of burn cases occurred at home of which 4% were self - inflicted. another study in mashhad reported at - home and self - inflicted burns to be the most prevalent causes of burns (57%). in the present study, the mean percentage of the burned area was found to be 31% and the mean percentage of the burns that resulted in patients expiry was found to be 51.3%, and patients mortality was 21%. in a study conducted in tehran, iran, the mean percentage of burns resulting in death was reported as 52.3%. in isfahan, the mean percentage of burns was reported as 33.9% and the mean percentage of burns leading to death was reported to be 69.5%. in studies conducted in france and turkey, the mean percentages of burns were reported to be 30% and 31.2%, respectively, and the mortalities were reported as 39% and 33.5%, respectively. these findings showed that mean burn percentages are almost the same in the studied countries, but the mortalities are different, which is possibly due to the difference in care, treatment, and following the standards. high mortality rate of burns can be due to resistance of bacteria, septicemia, and inappropriate routine care. the most prevalent causes of mortality were septicemia and hypovolemic shock in the present study. studies conducted in urmia and rasht also reported septicemia as the most prevalent cause of mortality due to burns. the causes that ranked second were burn severity and inhalation burns in these two cities, respectively. in the second part of the present study, the nurses themselves reported that they do their tasks concerned with septicemia more carelessly than the other tasks. therefore, by following sterile techniques while conducting procedures, detecting the bacteria that are resistant to antibiotics and disinfecting the environment, isolating the patients, and following standards in patients transfer and care, the mortality due to septicemia and even early complications of burns can be prevented to a large extent. the results of the second part of the study showed that in the viewpoints of nurses, the interventions associated with prevention of hypovolemic shock formed the second major care that was not administered properly in the care of acute burns. one of these important interventions in the burn patients is fluid therapy during their transfer, which, if conducted appropriately, can lower mortality and complications in these patients. reports have suggested that the process of transfer of burn patients should be performed by trained staff with appropriate equipments through an efficient coordination between the cent ers in such a way that the center admits the patient in less than 4 h after burn to prevent complications like hypovolemia, hypoxia, and hypothermia. as hypovolemic shock was the most common cause of death in pediatric age group in the present study, it can be concluded that children are more vulnerable to dehydration. so, the treatment team should administer precise fluid therapy, and in the first few days after burn, give more intensive care to these patients. patients transfer from distant areas to the hospital in the first few hours after burn and losing the golden time for fluid therapy can be the other causes of hypovolemic shock. based on the findings of the present study, with an emphasis on prevention, public education and increasing public knowledge about burns can be achieved through iranian national tv and radio. education and training ministry, with the cooperation of fire brigade organization can conduct firefighting and other related workshops. in addition, the ministry of oil and gas and the national standards organizations can reduce the burn events by improving the quality and supervising the safety of the products under their coverage. burn patients are hospitalized for a long time which leads to their disappointment and high financial burden. they also need reconstructive and plastic surgeries, rehabilitation, and physiotherapy due to the deformity of their limbs and face, which are all costly for both the patients and the hospital. therefore, the government, authorities, and the charities stakeholders should help these patients with their treatment costs, like in the case of an organization helping patients with chronic disease s. as already mentioned, one of the determining factors in prognosis of burns is infection. therefore, the committees of periodic infection control assessment in the hospital are suggested to inspect the bacteria that are resistant to routine antibiotics. with regard to the results of the second part of the study, it is suggested that the burn ward nurses be taught continuous educational courses on the prevention of septicemia and psychiatric nursing care given to the burn patients. we hope the present study can help the ministry of health, and treatment and burn clinics and hospitals. | background : many people suffer from burn injuries every year, and burns make the patients undergo surgeries and years of rehabilitation. burns lead to more years of disability, compared to cancer or heart diseases. epidemiologic studies are needed to reveal the span, impact, and related factors of burns to help take appropriate efforts to reduce its mortality and morbidity.materials and methods : this study was conducted in two phases. the first phase was a descriptive retrospective study conducted on 836 burn patients who were admitted to the main special burn hospital of isfahan, iran. data were collected from archived patients files using a checklist approved by the faculties of epidemiology and nursing. in the second phase, a survey was done based on the professional task checklist of burn ward nurses to assess the fulfillment of each task by the nurses.results:burns were found to occur more among those in the age groups of 20 - 30 (26.2%) and 0 - 10 years (22.9%). the most common causes of burns were flammables and gas explosions due to imprudence at home and workplaces, or self - infliction. mortality rate was 21.7% due to sepsis, shock, and inhalation injuries, respectively. nurses gave 19.78 out of 50 points (39.56%) to their performance in the prevention of sepsis.conclusions:based on the findings of this study, it can be concluded that there is still an increasing need for safety education and using environmental safety measures, as well as developing high - quality methods to transport burn patients and administer care to decrease the mortality and morbidity associated with burns. |
commonly it presents in males with mental retardation, facial abnormalities, cardiovascular involvement, and skeletal deformities. an anesthesiologist faces many unique challenges due to multi - system involvement and the problem is compounded by limited literature on this topic. we share our experience of successful management of a child with coffin lowry syndrome for ocular surgery. most of the associations of this syndrome may not have complete penetrance and may be clinically absent. however, in the case presented, all systemic ailments were present simultaneously which added to the complexity of anesthetic management. a 25-kg, 14-year - old boy, a known case of coffin lowry syndrome, presented with high myopia and retinal detachment associated vision deterioration. he was born at term without any history of birth asphyxia. since the age of 3 months, he had been suffering from recurrent chest infections requiring frequent hospital admissions and occasional mechanical ventilation. severe mitral regurgitation was also diagnosed, and by 3 years, he developed dilated cardiomyopathy (dcm) [figure 1 ] with heart failure. patient was on oral furosemide and digoxin therapy for last 10 years but had no sign of failure at presentation. the thyroxine had been tapered off 1 year back, and patient was clinically and biochemically euthyroid since then. at the age of 4 months, he developed recurrent myoclonic seizures for which combination of multiple anti - epileptics was administered, but they were stopped after he remained asymptomatic for a 5-year period. he complained of severe reflux gastritis and vomiting a year back for which daily ranitidine and metoclopramide were prescribed. it was not possible to communicate with him directly due to his mental status ; however, he seemed to reply appropriately to his father. since coffin lowry syndrome is an x - linked dominant trait, we tried to explore for similar family history, but nobody from the maternal or paternal lineage was found to possess any clinical manifestations. chest x - ray showing dilated cardiomyopathy on examination, an abnormal facies was evident with thick everted lips, increased intercanthal distance, and frontal bossing. scoliosis and pectus carinatum deformity were present. pitting pedal edema till ankle was present in both the feet. the air entry was normal in both lung fields and there were no added sounds. the airway was modified mallampati class iii, and there was a large tongue, high - arched palate, receding mandible, and buckteeth. echocardiography revealed mild mitral regurgitation (mr) with thickened mitral leaflets and severe pulmonary artery hypertension (pah) with mean pressure of 80 mmhg, which could not alone be attributed to the degree of mr seen on 2d echocardiography. a cobb 's angle of 42 without any cord involvement was seen on magnetic resonance image spine. written informed paternal consent was obtained and the child was allowed water till 2 h prior to surgery. in view of history of heart failure, a conservative approach toward preoperative fluids was adopted and no fluids were administered during the fasting period. patient was advised to continue ranitidine, metoclopramide, furosemide, and digoxin. after ensuring normal electrolyte values in the morning, oral midazolam syrup 12 mg was given half an hour prior to shifting to operating room. patient was shifted to the operating room, along with his father to keep him calm. after connecting standard monitoring, while maintaining his spontaneous ventilation, intravenous (iv) access was obtained and left radial artery was cannulated with a 22-g cannula. fentanyl 50 mcg and atracurium 10 mg were administered iv after which the child was hand ventilated ensuring low inspiratory pressure. no positive end expiratory pressure (peep)was used. confirming adequate depth of anesthesia, a 2.5 size proseal laryngeal mask airway (lma) was inserted and its placement confirmed. duration 70 min), he received 100 ml of ringer lactate and another 20 mcg of fentanyl. at the end of surgery, neuromuscular blockade was reversed, and once the patient was fully awake, the lma was removed. coffin lowry syndrome is a rare disease which presents clinically as hypotonia and lax joints at birth. as the child grows, facial and airway abnormalities and other systemic manifestations begin to appear. preoperative assessment of such mentally retarded children is difficult, thus parental presence and involvement is of utmost importance for assessment of growth history, natural history of the disease, and its current status. anomolies seen in coffin lowry syndrome typical facies and short fleshy fingers cardiac failure with dilated cardiomyopathy is rare in the pediatric age group. mitral valve disease with associated cardiomyopathy is known in this syndrome, making echocardiography evaluation mandatory. hypokalemia can precipitate digoxin - associated toxicity, and therefore preoperative electrolyte imbalance must be corrected. digoxin slows heart rate, increases inotropy, and may increase the regurgitant fraction in mr. this increase in regurgitant fraction can potentially cause heart failure. in case of bradycardia and hypotension, atropine or glycopyrrolate is preferred over drugs like ephedrine or mephentramine which cause peripheral vasoconstriction and increase regurgitation. severe pah in this patient may be attributable to a combination of scoliosis and moderate mr. nitrous oxide and peep were avoided as both are known to increase pulmonary vascular resistance. pre - induction arterial cannulation is difficult in children, and hence we preffered cannulating left radial artery after induction. this patient had high - arched palate and receding mandible, both of which are predictors of difficult airway. we preferred inhalational induction of anesthesia and muscle relaxant was used only after confirming mask ventilation. even though a flexible lma is preferred for ophthalmological surgeries, due to history of severe reflux, a proseal lma was used, keeping a fiber - optic bronchoscope standby. chances of failed lma insertion are less likely as most of the documented anomalies in this syndrome are limited to facial anatomy with no documented laryngeal anatomical abnormalities. good analgesia prevents any sympathetic stimulation and rise in peripheral resistance and myocardial stress ; hence, fentanyl and ketorolac were used. fluid maintenance in such patients with heart failure, dcm, and mr is challenging. we avoided liberal fluid administration as it can precipitate an increase in mr and cardiac decompensation. maintaining oral fluids should be started as soon as possible, in the postoperative period, as fluid management becomes easier and safer. our patient had history of seizures, but was asymptomatic and was not on anti - epileptic therapy at the time of presentation. coffin lowry syndrome patients can be on multiple anti - epileptics (enzyme inducers / inhibitors), which can alter anesthetic requirements. our patient had history of hypothyroidism, but was biochemically euthyroid at the time of presentation. literature does not document any association of hypothyroidism with the syndrome and it is difficult to comment whether these patients should be subjected to thyroid function tests routinely. postoperative management in icu ensures a safe postoperative course as these patients have multiple system involvement. utmost caution is needed while sedating these children in the icu as hypercarbia can worsen pah. to conclude, careful preoperative cardiovascular and neurological assessment, proper airway management, knowledge of different systemic involvement and drugs used, and low threshold for invasive monitoring ensure successful perioperative outcome of children with coffin lowry syndrome. | coffin lowry syndrome is a rare disease involving multiple organ systems. from the anesthesiologists point of view it involves mental retardation, seizures, difficult airway, cardiac abnormalities (pediatric dilated cardiomyopathy) and skeletal deformities. we share our experience of management of a child with coffin lowry syndrome and also discuss the problems faced during perioperative period. |
parkinson s disease (pd) is a common neurodegenerative disorder characterized by bradykinesia, rigidity, resting tremor, and postural instability.1,2 with progression of the disease, the response to levodopa decreases and various problems that are less dopa responsive (or dopa resistant) develop, such as cognitive dysfunction and speech and swallowing problems.3,4 studies have documented a very high prevalence of oropharyngeal dysphagia in patients with pd,5 which predisposes to aspiration pneumonia. pneumonia in turn is a major reason for hospitalization of patients with pd and it is the leading cause of mortality in patients with pd (in one prospective study accounting for 64% of deaths).6,7 pneumonia is a very common infectious disease and is one of the ten leading causes of death in the world.8 several risk factors for pneumonia in the general population, including chronic pulmonary disease, chronic heart failure, diabetes, chronic liver disease, chronic kidney disease, cochlear implants, cerebrospinal fluid shunts, splenic dysfunction, and hiv / aids, have been recognized.912 in pd, aspiration pneumonia is thought to be a multifactorial event, and aspiration alone is insufficient to cause pneumonia. other important factors include alterations in the bacterial flora of the oropharynx, as well as impaired pulmonary clearance and host resistance. to our knowledge, however, there have been no population - based studies exploring the risk factors for pneumonia in patients with pd. thus, we conducted a cohort study of patients with pd to identify the risk factors associated with pneumonia using a nationwide longitudinal population - based database. data for this study were derived from the 20002010 national health insurance research database (nhird), developed and managed by the taiwan national health insurance program for research purposes. the taiwan national health insurance program, since its introduction in 1995, has provided approximately 99% of taiwan residents with comprehensive and universal health care.13 we used one of the subsets of the nhird, composed of 1 million randomly selected subjects (constituting nearly 5% of the total taiwan population) drawn in 2000. the nhird includes data on patients demographic characteristics, diagnoses, and prescription claims (medication types, prescription dates, dosage, and duration supplied). the study was approved by the institutional review board at kaohsiung medical university hospital, and informed consent was waived by the institutional review board because the data obtained from the nhird have been de - identified. the pd cohort comprised patients who were newly diagnosed (between january 1, 2000 and december 31, 2009) based on the international classification of disease, ninth revision, clinical modification (icd-9-cm) diagnostic criteria (icd-9-cm code 332). patients were diagnosed by neurologists and received antiparkinsonian medication(s) (levodopa and decarboxylase inhibitor, entacapone, bromocriptine, pergolide, cabergoline, ropinirole, pramipexole, amantadine, or selegiline) with at least three consecutive outpatient clinic visits, which were characterized as regular follow up.14 exclusion criteria were as follows : age 80 years old table s2), which is comparable to the twofold increased risk of pneumonia in the general population.25,26 chronic kidney disease was associated with an increased risk of pneumonia among patients with pd in our study, similar to that in the general population, and this risk was particularly seen in older male patients.11,20 although chronic pulmonary disease is recognized as an important risk factor for pneumonia in the general population,20 for reasons that are unclear, this did not emerge as a risk factor in our study. of interest, we found that patients with pd who had received treatment for dental caries suffered less from pneumonia (especially in males aged < 70 years old) (table s3). poor oral health, including dental caries and periodontal diseases, is commonly observed in patients with pd, even in the early stages of the disease.2729 the high prevalence of impaired swallowing, periodontal diseases, and caries may lead to a greater risk of aspiration pneumonia.3033 maintenance of good oral hygiene and control of oral biofilm formation in the elderly reduce the number of potential respiratory pathogens in the oral secretions, which in turn reduces the risk of pneumonia.34 our findings suggest that patients with pd who received treatment for dental caries may have better oral health and a reduced risk of pneumonia than those who did not. although we can not determine based on the available data whether there were differences in dysphagia between the two groups, we believe our results highlight the potential importance of good oral health in reducing morbidity and mortality in patients with pd. in brief, patients with pd had similar risk factors for pneumonia hospitalization when compared to general population. our study found that chronic heart failure, chronic kidney disease, and dental caries were more significant risk factors for pneumonia hospitalization among patients with pd. the main strength of our study is that it provides information from a nationwide population - based cohort with a large sample size, and the results may provide a good representation of ethnic chinese patients with pd. to increase the accuracy of the diagnosis of pd, the study population was obtained by linking an ambulatory care expenditures database (neurologists and icd-9-cm code) and a prescription claims database (medical treatment for pd). moreover, covariates, including common underlying diseases (especially dental illness), were taken into consideration we did not have the opportunity to review all the medical charts of patients from the de - identified national institutes of health database. second, although we analyzed national health care records from a database of 1 million randomly selected subjects, there were still relatively few pd cases to allow us to make a more precise estimation of total pd populations in taiwan. third, information on other risk factors contributing to pneumonia, such as the severity of comorbidities, lifestyle factors, such as smoking and alcohol consumption, and biochemistry data were unavailable for retrieval from the database. other lifestyle - related pneumonia risk factors, including contact with children and nutritional status, were not included in the study. finally, it was difficult to distinguish between aspiration pneumonia and infectious pneumonia from the details available in the database. identification of risk factors for hospitalization with pneumonia among patients with pd in taiwan has highlighted chronic heart failure, chronic kidney disease, and oral hygiene as being associated with an increased risk of pneumonia. in particular, older female patients with pd with chronic heart failure and older male patients with pd with chronic kidney disease had a significantly higher risk of pneumonia. in contrast, male patients with pd had a diminished risk of pneumonia if dental caries were treated previously. early recognition and prompt management of comorbid physical diseases / risk factors in patients with pd may help to reduce the risk of hospitalization with pneumonia, and thus, the burden of the disease. icd-9-cm codes and atc classification system codes used in this study abbreviations : icd-9-cm, international classification of disease, ninth revision, clinical modification ; atc, anatomical therapeutic chemical. adjusted hazard ratio for pneumonia in the study population with pd stratified by age group and sex notes : adjusted age group, sex, geographic region of taiwan, urban level, monthly income, severity of physical condition, pd with dementia, and comorbidities, including chronic pulmonary disease, dental caries, chronic heart failure, and chronic kidney disease. abbreviations : pd, parkinson s disease ; hr, hazard ratio ; ci, confidence interval. adjusted hazard ratio for pneumonia in the study population with pd stratified by follow - up duration and sex notes : adjusted age group, sex, geographic region of taiwan, urban level, monthly income, severity of physical condition, pd with dementia, and comorbidities, including chronic pulmonary disease, dental caries, chronic heart failure and chronic kidney disease. abbreviation : pd, parkinson s disease. | objectivepneumonia is the leading cause of death in patients with parkinson s disease (pd). however, few studies have been performed to explore the risk factors for pneumonia development in patients with pd.methodswe conducted a nationwide population - based cohort study of patients with pd to identify the risk factors for these patients developing pneumonia. participants with newly diagnosed pd between 2000 and 2009 were enrolled from the 20002010 national health insurance research database in taiwan. we compared patients with pd with an incidence of hospitalization with pneumonia vs those without, and cox proportional hazard models were used to estimate the risk of pneumonia.resultsof the 2,001 enrolled patients (mean follow - up duration 5.8 years, range : 2.714.7 years), 381 (19.0%) had an incidence of hospitalization with pneumonia during the study period. multivariate cox proportional hazards analysis identified older age group (80 years of age, hazard ratio [hr ] = 3.15 [95% confidence interval 2.324.28 ]), male sex (hr = 1.59 [1.291.96 ]), certain geographic regions (northern, hr = 1.36 [1.041.78 ], southern and eastern, hr = 1.40 [1.051.88 ]), rural areas (hr = 1.34 [1.051.72 ]), chronic heart failure (hr = 1.53 [1.022.29 ]), and chronic kidney disease (hr = 1.39 [1.031.90 ]) as risk factors for hospitalization with pneumonia in patients with pd. however, treatment for dental caries was a protective factor (hr = 0.80 [0.640.99]).conclusionthe results of this study highlight risk factors that are associated with hospitalization with pneumonia, and, for the first time, suggest a link between treated dental caries and a diminished risk of hospitalization with pneumonia in patients with pd. |
the significance of chronic fibroproliferative diseases (fds) including pulmonary fibrosis, chronic kidney disease (ckd), inflammatory bowel diseases (ibd), and cardiovascular or liver fibrosis is rapidly increasing and they have become a major public health problem. according to current estimates nearly 45% of all deaths are attributed to fds ; thus, they are the leading cause of morbidity and mortality in developed countries [2, 3 ]. different fds share common features such as chronic inflammation which shows a correlation with the progression of fibrosis. in the injured organs chemotactic stimuli trigger the rapid recruitment of immune cells including macrophages and neutrophils. these infiltrating immune cells then produce numerous proinflammatory cytokines and growth factors, which trigger the activation of myofibroblasts (mfs), the main effector cells of tissue remodeling. under physiological conditions remodeling however, in the case of chronic fds the sensitive balance between the synthesis and degradation of extracellular matrix (ecm) components is disturbed, and the continuously activated mfs produce an excessive amount of ecm resulting in the replacement of parenchymal tissue by connective tissues. this chronic pathogenic remodeling process leads finally to the destruction of normal organ architecture and consequent decline of its function [5, 6 ]. despite the unmet medical need since inflammation plays an unequivocal role in the development of fibrosis, new therapeutic strategies targeting the inflammatory pathways may offer promising opportunities. thus, the aim of the present review is to summarize the main events of organ fibrosis with special focus on tissue remodeling - related inflammatory mediators, highlighting the potential pathomechanical role of the members of interleukin-10 (il-10) cytokine family. chronic inflammation, as a common hallmark of fds, is initially represented by the recruitment of neutrophils and macrophages ; however, almost all immune cell types including type 1 t helper (th1), th2, th17, regulatory t (treg) and b lymphocytes, and eosinophil and basophil granulocytes are involved in the process. these immune cells and also the injured inherent cells of the affected organ, such as endothelial and epithelial cells, release a wide range of inflammatory cytokines and growth factors [7, 8 ] including il-13 or transforming growth factor- (tgf-), which contribute either to the maintenance of chronic inflammation or to the proliferation and enhanced ecm production of mfs. mfs, as the main effector cells of organ fibrosis, are -smooth muscle actin (-sma) positive, spindle, or stellate - shaped cells lacking the epithelial or endothelial markers, such as cytokeratins or cluster of differentiation (cd) 31 [10, 11 ]. although the origin of mfs is controversial, they may arise by the phenoconversion of different cell types including fibroblasts, pericytes, stellate, smooth muscle, epithelial, endothelial, and stem cells or circulating progenitors [1820 ] (figure 1). after their activation mfs proliferate and produce an excessive amount of different ecm components including fibrillar collagens including collagens i, iii and glycoproteins such as fibronectin, fibrillin, elastin, and proteoglycans and nonfibrillar collagens including collagen iv, a main component of the basal membranes. however, the relative contribution of the different infiltrating and inherent cell types in the injured organ to the formation of mfs is still not clear. chronic inflammation leads to the release of a wide range of inflammatory mediators, which can contribute either to the stimulation (profibrotic) or to the inhibition (antifibrotic) of fibrosis (table 1). in the present section we discuss the biological role of the most well - studied mediators in the complex process of organ fibrosis. it is widely accepted that tgf-1 is the central element of the core pathway of organ fibrosis in most if not in all organs, including the airways, kidney, gastrointestinal tract, heart, and liver. tgf- is mainly derived from macrophages and fibroblasts ; however, other immune and nonimmune cells including dendritic cells, treg, cd8 t, or epithelial cells can also produce it. binding of tgf- to its receptor complex leads to the phosphorylation of the downstream signaling mediators small mothers against decapentaplegic homolog (smad)2/3 forming a complex with smad4 that translocates from the cytoplasm into the nucleus and induces the expression of its target genes. however, tgf- can also promote some noncanonical signaling pathways including the activation of extracellular signal - regulated kinase (erk)/cjun / p38 mitogen activated protein kinases. in response to the activation of these tgf--mediated signaling pathways mfs differentiate from their precursors and express -sma. other growth factors including platelet - derived growth factor (pdgf), connective tissue growth factor (ctgf), insulin - like growth factor (igf), fibroblast growth factor (fgf), and epidermal growth factor (egf) also influence the complex molecular interplay leading to the differentiation and increased ecm production of mfs [3537 ]. these growth factors have been also implicated in the pathomechanism of a number of fibrotic diseases including lung [38, 39 ], kidney [40, 41 ], intestinal [42, 43 ], heart, and liver fibrosis. th2-derived cytokines (il-4, il-5, il-6, il-13, and il-21) have distinct role in the regulation of organ fibrosis. one of the most studied th2 interleukins is il-13, which exerts a strong profibrotic effect in different fds. in animal models pulmonary overexpression of il-13 induced subepithelial airway fibrosis ; its inhibition with anti - il-13 antibody significantly reduced ecm deposition after bleomycin - induced lung fibrosis. previously it has been suggested that the biological role of il-13 is partially due to the profibrotic effects of il-4, as they share the common il-4r/signal transducer and activator of transcription 6 (stat6) signaling pathways. however, recently it has been demonstrated that il-13 still explicates its fibrosis - inducing effect when the canonic il-4r/stat6-mediated signaling pathway is blocked. indeed, il-13 has been demonstrated to activate an additional signaling mechanism through its own receptor (il-13r2) leading to organ fibrosis. other th2 cytokines including il-5 and il-21 can enhance the profibrotic effect of il-13 by increasing its production and/or the expression of its receptor [99, 100 ]. however, il-21 can promote tissue fibrosis also by inducing the differentiation of naive t cells to th17 cells. th17 cells produce a variety of different cytokines : among them il-17 is the most well - studied one. il-17 was shown to contribute to the development of fibrosis in different organs including the lung, kidney, intestine [103, 104 ], heart, and liver. elevated level of il-17 was found in human intestinal strictures and mfs expressed its receptor il-17rc during fibrosis associated with crohn 's disease (cd). indeed, il-17 induces the collagen production of subepithelial mfs and the expression of matrix metalloproteinase-3 (mmp-3), mmp-12, and tissue inhibitor of mmps (timp)-1 in the colon, which have significant effects on the ecm remodeling and tissue architecture [103, 104 ]. pharmacologic inhibition of the il-17-induced erk1/2 or p38 signaling pathways attenuated the collagen expression of mouse hepatic stellate cells [106, 107 ]. the profibrotic effect of il-17 was suggested in relation to skin fibrotic lesions as well. il-17 gene knockout (ko) mice had diminished bleomycin - induced skin fibrosis and il-17 deficiency attenuated skin thickness in a mouse model of scleroderma. in addition to the above described growth factors and cytokines, recently members of the il-10 family as a new group of fibrosis - related inflammatory mediators came into focus. cytokines of the il-10 family exert host defense mechanism ; they are essential for the maintenance of epithelial layer integrity and also facilitate tissue healing. il-10 cytokine family consists of nine related molecules : il-10, il-19, il-20, il-22, il-24, il-26, il-28a, il-28b, and il-29. these cytokines can be classified into three subfamilies with different biological functions : (1) il-10 subfamily represented by il-10 itself ; (2) il-20 subfamily (il-19, il-20, il-22, il-24, and il-26) which play a role in host defense mechanisms against bacteria ; (3) type iii interferons (ifns) : il-28a, il-28b, and il-29, which induce antiviral responses. initially, il-10 was described as a th2 cytokine but later it has been revealed that many other immune cells including th1, th17, treg, cd8 t, and b lymphocytes, macrophages, dendritic, natural killer, and mast cells also express il-10 [109, 110 ]. binding of il-10 dimers to their tetramer receptor consisting of two il-10r and two il-10r chains activates tyrosine kinase 2 and janus tyrosine kinase 1 (jak1), which phosphorylate il-10r. then stat3 binds to il-10r and gets phosphorylated by jak1. finally phosphorylated stat3 translocates into the nucleus and binds to the stat - binding elements in the promoters of various il-10 target genes. one of these il-10 responsive genes is the suppressor of cytokine signaling 3 (socs3), whose induction was correlated with decreased expression of tnf- and il-1. moreover, il-10 can affect the expression of other downstream effectors including mmp-9, inducible nitric oxide synthase, and ifn- [109, 111 ]. il-10 also inhibits the activation of antigen presenting cells through reducing the expression of major histocompatibility complex class ii. il-10 has a general suppressive effect ; it inhibits both the innate and adaptive immune responses, thus preventing increased exacerbations. thereby il-10 plays a significant role in the prevention of tissue damage which is a common element of chronic fds. indeed, wound repair results in scar formation in il-10 ko mice and on the contrary overexpression of il-10 modulates inflammatory responses at a wound site of adults more closely resembling the profile characteristic for the embryo. these observations suggest that by reducing the inflammatory response il-10 may inhibit the proliferation and collagen synthesis of the mfs as well. based on their overlapping target - cell profile and biological function, il-19, il-20, il-22, il-24, and il-26 were classified into the il-20 subfamily. cellular sources of the members of il-20 subfamily are immune cells including monocytes, lymphocytes, natural killer cells, and macrophages and also epithelial cells and fibroblasts. members of the il-20 subfamily show significant similarities in the structure of their receptor heterodimers. while il-19 binds to il-20ra / il-20rb, il-20 and il-24 bind either to il-20ra / il-20rb or to il-22ra1/il-20rb heterodimers. expression of the different receptor heterodimers can vary between tissues, which may explain the organ - specific effects of the members of this cytokine subfamily. il-10rb is ubiquitously expressed in the haematopoietic system, il-20ra and il-20rb are primarily distributed in the skin, lung, testis, ovary, and placenta, and il-22ra1 was shown to be present in the kidneys, intestine, liver, pancreas, and skin. similar to il-10, binding of the members of il-20 subfamily to their receptors activates the jak1/stat1 and stat3 signaling pathways. il-22 and il-24 both can act also through the akt, erk, jnk, and p38 signaling pathways [92, 116 ]. finally, the third subgroup of il-10 family is the subfamily of type iii ifns (il-28a, il-28b, and il-29) which signal through the ifn- receptor (ifnr). ifnr is a heterodimer consisting of an il-28r and an il-10r subunit and is present exclusively on the surface of epithelial cells. ligand binding to ifnr induces the activation of jak / stat signaling and antiviral activity on epithelial surfaces. unlike the other members of the il-10 family type iii ifns have no known effect on organ fibrosis. interstitial lung disease (ild) is a heterogenic group of disorders with different etiology. ild can be linked to a certain environmental exposure, including cigarette smoking, chemotherapy or radiation therapy, infection, or autoimmune diseases ; however, it can also appear without any known cause. in this case however, others such as ipf are primarily fibrotic and are associated with excessive deposition of ecm resulting in the disruption of the original architecture of the lung and loss of its volume. in general, patients with a known etiology of ild respond well to the targeted therapy especially when inflammation dominates ; however, they are difficult to treat when fibrosis comes into view. indeed treatment opportunities for ipf are limited ; lung transplantation is the only therapeutic option available in severe cases. level of il-10 was significantly increased in the lung and bronchoalveolar lavage (bal) of silica inhaled mice compared to controls. moreover, il-10 ko mice had more increased lung inflammation after intratracheal instillation of silica than wild type animals. moreover, genetic delivery of il-10 significantly attenuates the tgf- production in the lung of mice with bleomycin - induced pulmonary fibrosis. in humans greater percentage of peripheral cd4 t lymphocytes produced il-10 and higher serum levels of il-10 were found in patients with ipf than normal subjects. moreover, the extent of il-10 production correlated with forced vital capacity of the patients. similarly, comparing the protein concentration of il-10 in the bronchoalveolar lavage (bal) of patients with different types of ilds the highest level of il-10 was demonstrated in patients with ipf compared with sarcoidosis or hypersensitivity pneumonitis [5052 ]. similar to il-10 the protective role of il-22 is suggested in relation to fibrotic lung disorders. indeed, it has been demonstrated that recombinant il-22 treatment inhibits the phenoconversion of alveolar epithelial cells into mfs, thus reducing the number of ecm producing cells in a bleomycin - induced mouse model of lung fibrosis. administration of an anti - il-22 neutralizing antibody has also been shown to enhance pulmonary inflammation and ecm deposition in the same bleomycin - induced model of lung fibrosis. similar results were found in a mouse model of hypersensitive pneumonitis induced by repeated exposure to bacillus subtilis leading to lung fibrosis. namely, inhibition of il-22 resulted in enhanced collagen deposition in the lung, whereas treatment with recombinant il-22 inhibited lung fibrosis. these beneficial antifibrotic effects of il-22 suggest its potential as a novel therapeutic target in the treatment of pulmonary fibrosis. to the best of our knowledge there are no studies investigating the role of il-19, il-24, il-26, il-28, and il-29 in pulmonary fibrosis. the prevalence of ckds is estimated to be 816% worldwide and their number is rapidly increasing. the most common etiologies of ckds and renal fibrosis are diabetes mellitus (dm) and hypertension in the adult population [122, 123 ] and obstructive nephropathy in childhood. however, ckds irrespectively of their etiology always have an inflammatory component, which shows a strong correlation with the progression of fibrosis and the decline of renal function [125127 ]. recently, the connection between il-10 and renal fibrosis has been suggested. jin. demonstrated that after the onset of unilateral ureteral obstruction (uuo) more severe inflammation and fibrosis develop in the kidney of mice lacking il-10 than in wild type controls. following uuo they found increased infiltration of inflammatory cells and upregulation of inflammatory chemokines and cytokines including monocyte chemoattractant protein-1, rantes, tumor necrosis factor- (tnf-), il-6, il-8, or macrophage colony - stimulating factor in the kidney of il-10 knockout (ko) mice. in line with increased inflammation in the mice lacking il-10 they found a more pronounced collagen i deposition and increased expression of fibronectin, -sma, fibroblast - specific protein-1, vimentin, and mmp-2 supporting the development of renal fibrosis. in accordance with the observation of jin., rodell. demonstrated that local immunotherapy with il-10 in hyaluronic acid hydrogels reduces macrophage infiltration, the number of apoptotic cells, and the size of the fibrotic area as well, confirming the potential use of il-10 containing hydrogels in the local treatment of ckd [54, 55 ]. the participation of other members of the il-10 family like il-19 or il-20 in the pathomechanism of renal fibrosis is less unequivocal. elevated urinary level of il-19 and il-20 was observed in patients with ckd. in vitro treatment of human renal proximal tubular epithelial cells with nephrotoxic agents, including adefovir, dipivoxil, cisplatin, or ifosfamide, was shown to induce the expression of il-19. similarly, hgcl2 treatment of hk-2 human proximal tubular epithelial cell line resulted in increased presence of il-20 and its receptors. moreover, administration of either il-19 or il-20 induced the apoptosis of renal tubular epithelial cells in vitro. a recent study demonstrated that following renal ischaemia - reperfusion (i / r) injury the serum level of il-22 and also the expression of its receptor, il-22r1, in the renal proximal tubular epithelial cells are increased. treatment of the animals with recombinant il-22 or the overexpression of il-22 decreased the i / r - induced tubulointerstitial injury in the cortex and outer medulla and also the serum urea and creatinine levels compared to saline - treated control animals. indeed overexpression of il-22 upregulated the renal expression of b - cell lymphoma-2 (bcl-2) and downregulated that of bcl-2-associated death promoter in mice subjected to i / r injury. however, hypoxia is a known inducer of organ fibrosis ; to the best of our knowledge, there is no data in the literature directly supporting the role of il-22 in the pathomechanism of renal fibrosis. the involvement of other members of the il-10 cytokine family like il-24, il-26, il-28, and il-29 in renal fibrosis is completely unknown. intestinal fibrosis is a serious complication of ibd in both adults and children [128131 ] and more than 60% of patients with ibd require one or more operations over their lifetime, commonly because of stricture formation [128, 132 ]. however anti - inflammatory therapies reduce the symptoms of ibd, the recently available treatments of intestinal fibrosis are insufficient, and new therapeutic approaches are needed. similar to other chronic diseases experimental and clinical studies suggest the involvement of the members of il-10 family in intestinal fibrosis. the first study reporting elevated level of il-10 in the serum of patients with both active cd and ulcerative colitis (uc) was published in 1995. also increased expression of il-10 was found in the mucosa of patients with uc in remission. normal levels of il-10 in patients with ibd [57, 58 ] and lower expression of its receptors il-10r1 and il-10r2 in patients with remission were also revealed. moreover, loss of function mutations in the gene of il-10 or its receptor causes early onset of ibd with refractory colitis and perianal disease. in line with these findings decreased production of il-10 was observed in whole blood cell cultures of patients with severe phenotypes, compared with nonpenetrating, nonstricturing cd patients. similarly, dcs isolated from patients suffering from penetrating cd produced less il-10 in response to lipopolysaccharide (lps) stimulation compared to patients without complications. these observations suggest that defects in the production of the anti - inflammatory il-10 may represent a mechanism mediating the more severe manifestations of cd. despite the apparent discrepancy in the literature regarding the expression of il-10 in patients with ibd, the treatment with il-10 or il-10-inducing agents could be of particular benefit, because il-10 itself can suppress proinflammatory responses with a consequential limitation of tissue damage and may exert antifibrotic effects as well. recently clinical trials are in progress investigating the effect of the supplementation of il-10 in ibd (see more details later in the therapy section of this review) [60, 61 ]. indeed, il-19 ko mice were more susceptible to dss - induced experimental colitis than the wild type animals. the lack of il-19 in the il-19 ko mice correlated with the accumulation of macrophages. macrophages derived from il-19 ko mice produced significantly higher level of inflammatory cytokines including il-6, tnf-, and il-12 following lps stimulation compared to macrophages of wild type animals. in humans decreased production of il-19 was observed in the monocytes and peripheral blood mononuclear cells (pbmcs) of patients with active cd compared to those from healthy controls. moreover, administration of recombinant il-19 significantly decreased the production of tnf- in lps - treated monocytes and pbmcs of healthy controls but not in those of the patients with active cd. previous studies demonstrated that production of il-20 can be induced by lps, tnf-, and other proinflammatory cytokines [76, 115 ]. the number of epithelial and inflammatory cells expressing il-20 and il-20rb was increased in the mucosa of patients with active uc, but level of il-20 was decreased in the colonic mucosa of patients with uc in remission compared with patients with active uc and controls. elevated serum and mucosal level of il-22 was demonstrated in patients with active cd that correlated with disease severity [81, 131, 134, 135 ]. il-22 is a direct downstream effector cytokine of il-23, whose receptor was identified by the genome wide association study as an ibd - related gene. elevated level of il-23 was found in patients with active ibd and blocking of il-23 was effective in both prevention and treatment of active colitis, suggesting the potential of the il-23-il-22 pathway as a target of further therapeutic interventions. contrary to these findings in human ibd, studies on mouse models of colitis suggested the protective role of il-22 in the intestine. inhibition of il-22 by neutralizing antibodies in wild type mice or the lack of il-22 in ko mice with dextran - sodium - sulphate- (dss-) induced colitis resulted in an increased inflammation and epithelial damage of the colon leading to more severe weight loss of the animals [82, 83 ]. moreover, ril-22 treatment of colonic epithelial cells isolated from mice with dss - induced colitis induced activation of stat3 signaling pathway that regulates gut homeostasis and was shown to promote wound healing in an il-22-dependent manner. the expression of il-24 was shown to be significantly elevated in the mucosa of patients with active cd and uc compared to that of inactive ibd or controls, but the number of il-24-producing peripheral b, cd4 t, cd8 t cells and monocytes was increased only in patients with active cd but not in uc patients or controls. andoh. investigating the effect of il-24 treatment on the behaviour of ht-29 colonic epithelial cells found that il-24 activates the jak1/stat3 and also the socs3 signaling pathway and leads to increased expression of membrane - bound mucin-1, mucin-3, and mucin-4 supporting its suppressive effects on mucosal inflammation in ibd. level of il-26 was also elevated in the serum and inflamed region of the colonic mucosa of patients with cd and it was expressed by infiltrating immune cells mainly by th1 and th17 but not epithelial cells. il-26 can promote the expression of proinflammatory cytokines through the activation of stat1/3, erk1/2, jnk, and akt signaling pathways suggesting its proinflammatory role in ibd. to the best of our knowledge there are no data about the direct role of il-10 family in the pathomechanism of intestinal fibrosis ; however, the above - mentioned data suggest their relevance in aberrant intestinal tissue remodeling. the involvement of il-28a and il28-b and il-29 in ibd is still completely unknown. cardiac fibrosis is a common feature of different pathological conditions including myocardial infarction, pressure overload, hypertrophic cardiomyopathy, viral infections, toxic insults, or metabolic disturbances [63, 140, 141 ]. recently an in vivo experiment using il-10 ko and wild type mice suggested that lack of il-10 results in adverse tissue remodeling and more severe myocardial fibrosis in an isoproterenol - induced pressure overload - derived heart failure model. on the other hand, administration of recombinant il-10 improved cardiac remodeling and inhibited scar tissue formation and reduced the mortality of mice [62, 63 ]. the further in vivo studies confirmed the role of il-10 in tissue scaring using other animal models as well. in ischemia - induced mouse model of heart fibrosis impaired mobilization of bone marrow - derived endothelial progenitor cells, which are crucial in neovascularization and tissue repair, was observed in the heart of il-10 ko mice compared to wild type controls. moreover, il-10 treatment of the mice enhanced the survival of the endothelial progenitor cells leading to better myocardial recovery. similarly, il-10 treatment of the mice suffering from autoimmune myocarditis resulted in a significant decrease of myocardial inflammation and fibrosis. furthermore, the administration of il-10 prevented the relapse of the left ventricular function and increased the ejection fraction. the development of chronic cardiomyopathy in the experimental trypanosoma cruzi - infected dog model of chagas disease was strongly correlated with the production of il-10. indeed low level of il-10 and simultaneously high expression of ifn- and tnf- were observed in the acute cardiac infection phase, which correlated with the severity of heart inflammation and fibrosis in the chronic phase. however, our knowledge is limited, and elevated level of il-22 was demonstrated in the heart of mice with dilated cardiomyopathy and cardiac fibrosis. treatment of mice with il-22-specific antibody decreased the survival rate of the animals and exacerbated myocardial fibrosis suggesting the cardioprotective role of il-22 through the inhibition of myocardial fibrosis. the role of other members of the il-10 family including il-19, il-20, il-24, il-26, il-28, and il-29 in cardiac fibrosis is completely unknown. liver fibrosis is one of the major causes of morbidity and mortality worldwide with around 1.5 million deaths per year ; however, the exact pathomechanism is just partially understood. the main causes of liver fibrosis include fatty liver, alcohol abuse, biliary track disease, chronic viral infection, autoimmune disease, and toxicant exposure. the most studied members of the il-10 family related to liver fibrosis are il-10, il-20, and il-22. studies investigated the involvement of these cytokines mainly in alcoholic hepatitis, nonalcoholic and infection associated liver fibrosis. in the liver il-10 can be produced by a variety of cell types including hepatocytes, kupffer cells, sinusoidal endothelial cells, hepatic stellate cells, and lymphocytes and also its receptor is expressed by progenitor and hepatic stellate cells, the predominant cell types involved in liver fibrogenesis [66, 85 ]. higher hepatic tnf- levels and more severe liver fibrosis can be observed in the carbon tetrachloride (ccl4) treated il-10 ko mice than in the wild type animals [67, 68 ]. on the contrary, il-10 gene therapy reduced the expression of profibrotic genes, including tgf- and tnf-, and reversed the thioacetamide - induced hepatic fibrosis in mice. recent studies demonstrated that il-10 plays a protective role in alcoholic liver disease as well. similar to il-10, elevated level of il-20 was found in hepatocytes and hepatic stellate cells of patients suffering from liver fibrosis. however, contrary to the effect of il-10, recombinant il-20 treatment of mice enhanced the expression of the profibrotic cytokines including tgf- and tnf- and promoted the collagen synthesis of the liver. treatment with neutralizing antibody against il-20 or il20ra diminishes the ccl4-induced liver fibrosis in mice. a variety of studies reveal the relevance of il-22 in liver fibrosis demonstrating its protective role. in a chronic - binge ethanol feeding mouse model of alcohol induced liver injury the recombinant il-22 treatment of the animals ameliorated liver injury and alcoholic fatty liver through the activation of stat3 signaling pathway [85, 86 ]. in the same murine model of hepatic fibrosis administration of il-22 upregulated the expression of several antiapoptotic and antioxidant genes contributing to the attenuation of the oxidative stress. long - term administration of recombinant il-22 to mice with a high fat diet induced hepatic steatosis and diminished the tnf- level of the liver. overexpression of il-22 or recombinant il-22 treatment decreased the expression of alpha - smooth muscle actin (sma) in cultured hepatic stellate cells and also in the fibrotic liver of the mice with ccl4-induced liver fibrosis. in addition, il-22 promoted the senescence of hepatic stellate cells through the socs3 bound p53 and p21 signaling pathways, thereby ameliorating liver fibrosis. on the contrary, inhibition of il-22 with a neutralizing antibody reduced the activation of stat3 and led to the worsening of liver injury in a t cell - mediated hepatitis induced by concanavalin a. in humans, elevated level of il-22 was found in the serum and liver tissue of human patients with hcv - induced and alcoholic liver fibrosis. based on the results of wu. profibrotic effects of il-22 were proposed in humans in contrast with its antifibrotic role in mice suggesting that il-22 may have diverse functions in different species and disease states. the role of other members of the il-10 family including il-19, il-24, il-26, il-28, and il-29 needs to be elucidated in liver fibrosis. chronic fds affect hundreds of millions of people and are the leading cause of morbidity and mortality in the western world. despite the urgent medical need however, due to the efforts during the last few years there was a remarkable achievement in the treatment of organ fibrosis. the drugs which are currently under development target the key participants of the fibrosis pathway including tgf-, pdgf, igf, ctgf, angiotensin ii, or endothelin-1 (figure 2). among them, pirfenidone, targeting the tgf- pathway, was recently approved by the u.s. food and drug administration (fda) for the treatment of ipf. in phase iii clinical trial pirfenidone successfully reduced the progression of ipf and was associated with fewer deaths. however, tgf- and other key factors of organ fibrosis play also a crucial role in other significant biological processes, like embryogenesis, regulation of immune responses, or cancer development. therefore, cautions must be taken in case of organ fibrosis, which is often related to chronic diseases when the antifibrotic treatment needs to be maintained for years. therefore, besides the inhibition of the above - mentioned determinative pathways it seems to be preferable to alter new, more fibrosis - specific or endogenously antifibrotic pathways leading to fewer serious side effects and allowing life - long treatment of the patients. recently, the members of the il-10 family came into focus as possible new target molecules, which may alter the progression of organ fibrosis (table 2). different therapeutic strategies were developed to influence the effects of il-10, il-20, il-22, or il-20ra. among them, investigations aiming at the alteration of the il-10 mediated pathways are in the most advanced stage. indeed, after the successful preclinical experiments, clinical studies using human recombinant il-10 (rhuil-10) are already in progress for the treatment of ibd. a double - blind clinical trial enrolling patients with cd after intestinal resection demonstrated that administration of rhuil-10 for 12 consecutive weeks was safe and well tolerated. another double - blind placebo controlled trial with 95 active cd patients who received rhuil-10 (sc. ; 1, 5, 10, or 20 g / kg / day) for 29 days showed that 5 g / kg / day rhuil-10 treatment can induce clinical remission and endoscopic improvement in 23.5% of the patients compared to placebo - treated group where no remission was detectable. at the 20-week follow - up period the relapses requiring further therapeutic intervention were decreased by 15% in cd patients who were treated with 5 g / kg rhuil-10 compared to controls treated with placebo only. however, interestingly the higher than 5 g / kg / day doses of rhuil-10 treatment were less effective. cd patients treated with high doses (10 or 20 g / kg / day) of rhuil-10 had increased production of ifn- in whole blood cells and elevated serum neopterin levels, which may be responsible for the effectiveness of higher rhuil-10 doses in cd. moreover, high doses of rhuil-10 caused headache, fever, and anaemia. schreiber. examined 329 patients with therapy refractory cd and observed a clinical improvement in 46% of patients treated with 8 g / kg / day rhuil-10 compared with the 27% of placebo - treated control patients ; however, they did not find any significant differences in the clinical remission between rhuil-10 (1, 4, 8, and 20 g / kg) and placebo - treated groups. suggested that il-10 can be rather effective in the prevention of ibd ; however, there are several individual differences between the exact etiologies of the disease. moreover, il-10 may exert also an immunostimulatory effect, which may compensate its immunosuppressive qualities. local treatment with il-10-secreting lactococcus lactis (l. lactis) prevented the development of colitis in il-10 ko mice and reduced inflammation in the dss - induced mouse model of colitis without systemic side effects. in a human phase i trial ten cd patients were treated with a hil-10 sequence containing l. lactis twice a day for one week. the treatment was safe and reduced the disease activity without any side effects observed in the case of high systemic doses. similar to l. lactis treatment, replication - deficient adenoviral vectors directly delivered to gastrointestinal epithelial cells were also effective in murines through the improvement of local il-10 release [156, 157 ]. the above - mentioned data suggest that systemic administration of rhuil-10 may be a safe and well - tolerated treatment contributing to the clinical improvement of cd and the local il-10 therapy may have even more potential because of having fewer side effects. however, the direct effect of rhuil-10 on intestinal fibrosis that often appears in ibd had not been studied in humans. to the best of our knowledge, recently there are no human studies investigating the effect of recombinant il-10 and other family members on lung fibrosis. in rats, inhaled il-10 was shown to attenuate lps - induced pulmonary and systemic inflammation through the reduction of proinflammatory mediators including tnf-, il-1, il-6, and ifn- in the bal and plasma. moreover, after bilateral femoral fracture that induces systemic inflammation and impairs the lung function, inhalative administration of 50 g / kg / day recombinant mouse il-10 decreased the pulmonary infiltration of neutrophils and reduced the expression of the adhesion molecule icam-1 but had no significant effects on the systemic inflammatory response. in a double - blind, placebo - controlled study the effect of rhuil-10 was investigated in human patients with renal transplantation who received okt3, a monoclonal murine antibody targeting the epsilon chain of the cd3-t cell receptor complex that efficiently reverses graft rejection, as induction therapy. wissing. found that pretreatment with doses of up to 1 g / kg rhuil-10 was safe and significantly reduced the okt3-induced release of tnf-. moreover, short - term treatment of nephritic rats with intravenous (iv) rhuil-10 was effective in the inhibition of matrix deposition and reduced the protein level of -sma in antithymocyte 1 induced glomerulosclerosis but had no beneficial effects on proteinuria. in a mouse model of myocardial infarction subcutan (sc) administration of recombinant il-10 suppressed the expression of proinflammatory cytokines in the myocardium, reduced infarct size, attenuated infarct wall thinning, improved left ventricular functions, reduced the activity of mmp-9, and diminished fibrosis. in a randomized, double - blind trial twenty - four patients with chronic hepatitis c were sc. treated with either 4 or 8 g / kg rhuil-10 per day for 90 days. the therapy was safe and well tolerated, and administration of rhuil-10 normalized the serum levels of alanine aminotransferase (alt), improved liver histology, and reduced liver fibrosis. treatment of thirty patients with chronic hepatitis c - induced advanced fibrosis, who had failed antiviral therapy, resulted in a significant improvement of their serum alt, decreased hepatic inflammation, and fibrosis. however, long - term administration of rhuil-10 altered the cytokine profile of pbmcs promoting a th2 dominance and decreased the number of hcv - specific cd4 and cd8 t cells resulting in enhanced hcv viral burden due to the alterations in the host 's immunologic viral surveillance. moreover, in preclinical experiments the effect of treatment with recombinant il-22, anti - il-20, or anti - il-20ra monoclonal antibody was demonstrated to inhibit tgf- production or the excessive accumulation of ecm components in mouse models of liver fibrosis induced by chemical agents (ccl4) or mechanical bile duct ligation. however, results about the possible use of rhuil-10 as an antifibrotic drug are promising and further preclinical and clinical studies are needed to elucidate the precise role of the il-10 family in fibrosis and to estimate their potential therapeutic effectiveness. | importance of chronic fibroproliferative diseases (fds) including pulmonary fibrosis, chronic kidney diseases, inflammatory bowel disease, and cardiovascular or liver fibrosis is rapidly increasing and they have become a major public health problem. according to some estimates about 45% of all deaths are attributed to fds in the developed world. independently of their etiology the common hallmark of fds is chronic inflammation. infiltrating immune cells, endothelial, epithelial, and other resident cells of the injured organ release an orchestra of inflammatory mediators, which stimulate the proliferation and excessive extracellular matrix (ecm) production of myofibroblasts, the effector cells of organ fibrosis. abnormal amount of ecm disturbs the original organ architecture leading to the decline of function. although our knowledge is rapidly expanding, we still have neither a diagnostic tool to detect nor a drug to specifically target fibrosis. therefore, there is an urgent need for the more comprehensive understanding of the pathomechanism of fibrosis and development of novel diagnostic and therapeutic strategies. in the present review we provide an overview of the common key mediators of organ fibrosis highlighting the role of interleukin-10 (il-10) cytokine family members (il-10, il-19, il-20, il-22, il-24, and il-26), which recently came into focus as tissue remodeling - related inflammatory cytokines. |
after the operation, he presented with acute urinary retention requiring urinary catheterization, but pulled out the urinary catheter twice because of agitation due to alcoholic stop. for each procedure, a silicon foley catheter sizing 18 french was placed by the nurses without reported difficulty. on postoperative day 5 (the day following the third urinary catheterization), he presented with acute abdominal pain. on physical examination, there was abdominal guarding with hypotension. his temperature was normal, urine output in the foley was 200 ml and the blood parameters showed an acute inflammatory response (17000 leukocytes / mm, c - reactive protein : 17.5 mg / l), with normal creatinine and elevated urea (10 the laparotomy revealed a peritoneal perforation of the urinary bladder, with concomitant urinary peritonitis. the bladder had been perforated by the urinary catheter and there was neither diverticulosis nor tumor (figure 1). the urinary catheter was left in place for 15 days, and the patient resumed normal urination after its removal. the pathological examination confirmed the absence of tumor and the patient is well after 6 months of follow - up. figure 1intra - peritoneal rupture of the top of the bladder due to the contact with the urinary catheter. intra - peritoneal rupture of the top of the bladder due to the contact with the urinary catheter. intraperitoneal perforation of the urinary bladder may occur in particular situations such as trauma, iatrogenicity (cystoscopy, prostatectomy), cancer, radiotherapy, infections or inflammatory lesions (cystitis, schistosomiasis, tuberculosis) and diverticulosis. a few cases of idiopathic rupture that could have been related to the consumption of toxic substances (alcohol, drugs) have been reported. few cases of rupture after urinary catheterization without other predisposing factors have been described in the literature. the diagnosis is challenging because the symptoms are not specific (pain, hematuria, oliguria), as well as blood parameters (inflammatory syndrome, renal insufficiency), leading to misdiagnosis and inappropriate or late treatment. the medical history of this patient with several successive urinary catheters may suggest this diagnosis (even if no traumatic difficulty was reported during the catheterization). retrograde urethro - cystography or an abdominal computed comography scan with bladder opacification can establish the diagnosis and localize the site of rupture. the main factor for the therapeutic management the classical treatment for intraperitoneal rupture of the bladder is surgical repair and urinary rest (with a foley catheter left in place for one week at least). laparoscopy is possible in this setting and this approach would have been chosen if the perforation was diagnosed in our case. in selective cases (particularly in the absence of peritoneal signs), idiopathic intra - peritoneal rupture of the bladder is difficult to diagnose due to the non - specific clinical picture. despite it surgical treatment consists of bladder repair and a search for conditions that increase the risk of rupture (tumor, diverticulosis, infection). | intraperitoneal rupture of the bladder is a rare cause of peritonitis. intraperitoneal rupture of the bladder was diagnosed during an emergency laparotomy for suspected mesenteric ischemia. the patient had undergone iterative urinary catheterization after a vascular bypass. the perforation was excised and sutured and the patient was catheterized for urinary rest for 15 days. urinary catheterization is a possible cause of intraperitoneal rupture of the bladder. |
exposure to blood borne pathogens can lead to serious infections among health care workers (hcws). at least 26 different pathogens have been known to be involved in occupational infectious transmission. according to the world health organization (who) and the center for disease control (cdc), more than 85 million hcws have been reported to be continuously exposed to injury with contaminated sharp medical devices, worldwide. in spite of wide preventive programs and using of modern medical equipments, hcws are at high risk of exposure to blood borne pathogens like hepatitis c virus (hcv). hcv is a major public health problem that can cause severe medical outcomes such as acute hepatitis, chronic liver diseases and hepatocellular carcinoma. among all of health care workers who annually exposed to sharp contaminated devices, the number of those getting infected by hepatitis c virus (hcv) was found 926000 in the world. the annual incidence of occupational exposure to patients blood and body fluids has been reported 3% for hcv. the hcv transmission risk among hcws with needle stick injuries has been found 3 - 10%. the risk of hcv transmission with high levels of virus load in the source patient increases by more than tenfold. therefore infectious occupational diseases can be transmitted from patients to hcws and vice versa and also hcw 's families and community. according with standard and worldwide precautions, cdc and occupational health and administration (ohsa) suggest every patient should be considered as a possible carrier infection and to reduce the transmission of infectious agents by direct contacts, appropriate personal protective equipments must be performed. the present study was undertaken to evaluate the frequency and risk factors of infection with hcv, among laboratory personnel as a risk group for hepatitis c equation in isfahan iran. this descriptive cross sectional study was conducted from july to september 2010 among medical laboratory technologists (laboratory workers responsible for analyzing body fluids), medical laboratory technicians (laboratory workers responsible for sampling, preparing specimens) and laboratory staff (laboratory workers responsible for cleaning surface and equipment) who were potential for high risk exposures. the sample size includes 203 subjects and sampling was voluntary - basis using simple random sampling method. the laboratory health care workers were employed in the hospitals and medical laboratory centers in isfahan city. questionnaire was filled out by the subjects to collect personal and occupational injuries within the last 12 months including splashes to mucous, membranes hepatitis history, general health, and other contributing factors. five ml of venous blood was obtained from each participant and separated serum was sent to the laboratory of the infectious diseases and tropical medicine research center in a cold chain. hcv anti bodies were measured by means of enzyme linked immunosorbant assay (diapro kits, diagnostic bio probes s.r.j, italy) according to the manufacturer recommendations. statistical analysis was done by spss statistical software version 15 (inc, chicago, il). two hundred and three participants including 123 (62.1%) technologists, 40 (19%) lab technicians and 40 (19%) lab cleaning staff were studied. the age range of the study individuals was 20 - 69 years, and the mean and standard deviation were 35.8 and 9.54 respectively. of the 203 participants who studied, 75.1% had hand abrasions, 66.8% skin wounds and 29.8% had a history of eye splashing. in nobody of the participants the hcv - ab was detected (0%). according to the hcv prevalence among hcws, no risk factors could be evaluated. hepatitis c virus is a common cause of occupational blood- borne disease that transmitted from patients to health care workers and frequently leads to a chronic asymptomatic carrier condition for a long time before the development of symptomatic liver disease. therefore health care workers with hcv infection may be unaware of their disease or carrier condition and infect other persons like as their families or patients. in our study none of the medical laboratory heath care workers over the study period showed the presence of antibodies to hepatitis c virus and our finding was similar to a study that performed among health care workers of sudan. studied the general population of iran and estimated low hcv infection prevalence rate in iran (0.16% ; 95% confidence interval [ci ] : 0%-0.59%) that shows our result can be expected. however cdc reported that 1.4% of hospital workers are infected with hcv, the roots of hcv or hbv transmission to hcws have not been completely recognized. several studies in european countries have reported that hepatitis (hbv / hcv) transmission among hcws was significantly related to exposure of the patients open tissues to the blood of the workers. there are several reports of hcv transmission through percutaneous exposure contaminated needles, splashing fluids to mucus membrane and other sharp instruments in hcws.[1416 ] in our study 75.1% of participants had a history of abrasion on hand, 66.8% skin hurt and 29.8% had a history of eye splashing, that none of them leads to hcv infection. according to the investigations among hcws, surgeons, nurses, laboratory technicians, accidental emergency and cleaning staff have been recognized as having an increased risk of occupational infection with hcv. some explanations for infection transmission which is observed in hcws are less skilled hcws, high load of patients, insufficient protective devices, unexpected movement in patients and performing usual protocols by the less expertise employees. although there was nobody infected with hcv in our study, regular training and education teaching about risk of occupational contacts with body fluids and pre and post exposure management and planned vaccination is necessary in laboratory health care workers. laboratory health care workers and patients can both be comforted that laboratory personnel who are infected with hcv is extremely low frequency and the risk that a patient may become infected from laboratory worker is certainly low. however, performing universal precautions and reporting needle stick injuries should be performed. finally better devices and examinations should be supplied for detection of hcv - rna in negative hcv antibodies in high risk groups. | objectives : clinical laboratory health care workers can become infected through their occupation with blood - borne pathogens by percutanous injuries and mucocutaneous blood contacts such as cuts, needle sticks, splashes to mucous membranes or other body injuries. the purpose of this study was to determine the seroprevalence of, hepatitis c virus (hcv), and some of the risk factors in medical laboratory health care workers.methods:through a descriptive cross sectional study, 203 participants employed in the clinical laboratories of the city of isfahan, composed of medical laboratory technologists, technicians and cleaning staff were studied. participant data were obtained through a self - reporting questionnaire and the level of anti - hcv antibody was measured by enzyme linked immunosorbent assay (elisa). chi - square test was used to determine risk factors associated with infection.results:the mean age of the individuals (n = 203) was 35.8 9.54 years. there were 115 women (56.7%) and 88 men (43.3%). all of the subjects were negative for hcv ab.conclusions:hepatitis c infection is infrequent in laboratory health care workers in isfahan province. |
the dynamics of the disease has continued to change across geographical zones. in the past few decades, the etiology in developing countries like nigeria was mainly inflammatory and most commonly from gonococcal urethritis. the management has also undergone changes, passing from urethral dilatation to minimally invasive procedures like optical urethrotomy and then to open urethroplasty which is the preferred procedure with the highest success rate. the oldest and simplest form is urethral dilatation, which can be performed with a number of different devices and is generally considered a palliative maneuver. direct vision internal urethrotomy (dviu) was introduced by sachse, to treat urethral stricture using cold - knife incision. in general, open urethral reconstruction is the most successful management option for urethral strictures, but it requires surgical expertise, adequate operating room facilities and has a longer recovery period. choice of treatment is often affected by the experience of the surgeon, availability of treatment options, and cost. in nigeria, this can be explained by the documented higher success rate of these procedures, rather high failure rate of urethral dilatation and internal urethrotomy, and also by lack of experience with dviu in most of our centers. however, the number and types of open urethroplasties performed nationwide are yet to be fully ascertained so far. the aim of this study was therefore to determine the national practice patterns in the management of male urethral stricture disease by the open urethroplasty technique among reconstructive urologists in nigeria. a nationwide survey of practicing reconstructive urologists was performed during the 19 annual general meeting and scientific conference of the national association of urological surgeons of nigeria, held at ibadan, southwest nigeria in 2013 and by electronically mailed questionnaire. the questions were categorized into three groups : respondents demographics, nature of strictures, and method of diagnosis, stent practice patterns, and follow - up protocol. results were analyzed using microsoft exel 2007 microsoft corporation, washington and results expressed in simple proportions and percentages. a total of 55 of 82 questionnaires were completed and returned, giving a response rate of about 67.1%. in most of the respondents (urologists), 24 (43.6%) the practice pattern showed that 45.5% were in the employ of the federal teaching hospitals, 49.1% were employed in the state teaching / general hospitals, and 5.5% were in private practice. in most of the respondents, 23 (41.8%) had between 5 and 9 years of experience as a reconstructive urologist. respondents categorized by age, years of experience and number of urethroplasties performed in last year the estimated numbers of urethroplasties performed per year are shown in table 1. traumatic strictures accounted for the most common cause of strictures treated by respondents in 44 of 55 respondents (80.0%). in most of the respondents, 35 (63.7%) preferred to treat strictures after 36 months of diagnosis, 5 out of 55 respondents (9.2%) treated strictures between 0 and 2 months, 7 (12.7%) operated after 79 months after diagnosis, and 8 (14.5%) operated after 1012 months of diagnosis. thirty - seven of 55 (67.3%) respondents preferred the combination of retrograde urethrography (rug) and voiding cystourethrography (vcug) for the diagnosis of urethral stricture, while 18 of 55 (32.7%) used only rug. twenty - seven of 55 respondents (49.1%) preferred size 16 f silastic catheter while 28 of 55 respondents (50.9%) used size 18 f silastic catheter. thirty - one of 55 respondents (56.4%) preferred a 4 weeks stay, and 21 of 55 respondents (38.2%) left the stents for 3 weeks. three of 55 respondents (5.5%) left the stents for up to 5 weeks. the only investigative tool before removal of the stent was a peri - catheter urethrogram performed by 52 of 55 respondents (94.5%). of 55 respondents, 23 (41.8%) followed up their patients for a year while 20 (36.4%) expect to follow - up their patient for life. the most commonly used tool by the respondents to follow - up their patients after urethroplasty was uroflowmeter (36.6%) and international prostate symptom score (30.9%). the most common complication after open urethral repair was recurrence of the stricture as reported by 36.4% of the responders followed by fistula formation in 23.6% of the responders [table 2 ]. in some developed countries, the common practice in the treatment of anterior urethral stricture is to proceed to reconstructive surgery, only after a failed minimally invasive procedure. however, in nigeria, and in some developing countries, apart from urethral dilatation, open urethral reconstruction remains the most common mode of treatment for anterior urethral stricture. most of the urologists in this nigerian survey were within the age of 4049 years (43.6%) and almost half of the responders (49.9%) were under the employ of state - owned hospitals. compared with similar surveys of urologists in italy and america, the nigerian urologists were a decade younger. the majority (41.8%) had 59 years of experience as a reconstructive surgeon. for the purposes of this review, years of experience imply number of years postqualification as a urologist and does not signify experience with the practice of reconstructive urology or any additional training in the field. similarly, half of the responders (50%) perform between one and nine urethroplasties annually. overall, this relatively small numbers performed annually could hinder adequate skills acquisition and also raises the ethical issues of allowing such surgeons to perform such surgeries on patients without the requisite experience. it can be argued that for better results, such surgeries should be performed in specialized centers staffed with surgeons with necessary skills and experiences while not compromising the need to train younger urologists. an understanding of the underlying etiology of a stricture plays a significant role in making a choice for repair. whereas inflammatory strictures once accounted for the majority of urethral strictures in our environment, traumatic strictures from fall astride injuries, road traffic crash, and penetrating injuries to the urethra these strictures in most cases are expected to be single, short segment strictures, and easily amenable to single stage repairs either by excision and primary anastomosis or with graft repair. urethrography remains the gold standard for the diagnosis of urethral stricture although methods of evaluation vary from published data. sixty - seven percent of our responders preferred the combination of rug and vcug for the diagnosis of stricture, none of our responders used urethroscopy or sonourethrography for the diagnosis of stricture. in contrast to the italian and dutch national surveys, 16% of italian urologists and 72% of dutch urologist reported that urethrography as their diagnostic tool of choice. the reasons adduced for the low use of radiography by the italian urologist was that they considered urethrography invasive and not well accepted by italian patients. as a combined modality, rug and vcug offer a means for the diagnosis of urethral stricture especially if the stricture is complete, with a sensitivity of between 75% and 100%. it also provides additional information on concurrent pathology in the urethra such as fistula, false passage, and significant ductal reflux. both size 16 fr and 18 fr silastic catheters were used by the responders in almost equal proportions ; however, the duration of stenting varied significantly among nigerian urologists. the majority (56%) of our responders retained the stents for 4 weeks before removal. urethral healing is expected to occur after 21 days ; however, stenting can be extended for up to 28 days if there was leakage of contrast at the site of reconstruction after 21 days as demonstrated on a peri - catheter urethrogram. patients should ideally be follow - up for life because of the risk of restricturing. forty - two percentage of our responders would expect to follow - up their patients for only 1-year while 36% expected to follow - up their patients for life. this lack of uniformity in follow - up protocol means that it would be difficult to exactly define the success rate of the procedure as well as re - stricture rate for any particular procedure. the most commonly used tool in the literature for the follow - up of patients after urethroplasty is uroflowmetry. this low usage may be due to nonavailability of this method of assessment in our environment. anatomic assessment of the repair site either with urethrography or direct visualization provides the most accurate information with regards to success and the presence of recurrent stricture. the most common complication after open urethroplasty from this survey was recurrence of the stricture. thirty - six percentage of our responders experience recurrence as the most common complication after various procedures. it has been recommended that in order to improve outcome, surgeons should regularly audit their practice and make necessary adjustments. similarly, to optimize the management of urethral stricture disease, familiarity with indications and state - of - the - art performance of the procedures seem to be of paramount importance. firstly, the study is based on a questionnaire with a response rate of 67%. the sample size (55) surveyed is quite small compared to the total number of urologists in the country. the questionnaire also failed to reveal the skills that the responders acquired during training or any additional training in reconstructive urology. the implication of this is that the responders with similar skills and experience in reconstructive urology can not be compared. secondly, it would be difficult to ascertain the mean length of the strictures treated by our responded as well as the type of repair which was most commonly practiced. however, it is expected that this study will act as a framework for further study on national practice patterns among urologists in nigeria. there may also be a need for standardization of the practice of urethroplasty and the setting up of specialized centers with skilled and well - experienced staff. a combination of rug and vcug is the preferred method for the diagnosis of anterior urethral stricture disease. there are no standardized protocols for follow - up of patients after urethroplasty. to improve outcome, familiarity with state - of - the - art performance of the procedures seem to be of paramount importance. we recommend further studies to garner more information on the numbers and type of procedures performed nationwide which will serve as a baseline for determining practice patterns and planning strategies in the management of urethral stricture disease. (a)12 f (b)14 f (c)16 f (d)18 f (e)20 f f (f)22 f 9.how long do you leave the urethral catheter / stent in place after urethroplasty ? (a)1-week (b)2 weeks (c)3 weeks (d)4 weeks (e)5 weeks 10.do you routinely require any investigation before removal of catheter / stent (a)yes (b)no 11.if the answer to question 10 is yes, what procedure do you commonly employ before / during removal of stent ? (a)pericatheter urethrogram (b)pullout urethrogram (c)rug (d)mcug what is your best choice of catheter / stent size following urethroplasty ? (a)12 f (b)14 f (c)16 f (d)18 f (e)20 f f (f)22 f how long do you leave the urethral catheter / stent in place after urethroplasty ? (a)1-week (b)2 weeks (c)3 weeks (d)4 weeks (e)5 weeks do you routinely require any investigation before removal of catheter / stent if the answer to question 10 is yes, what procedure do you commonly employ before / during removal of stent ? (a)pericatheter urethrogram (b)pullout urethrogram (c)rug (d)mcug pericatheter urethrogram 12.what is the length of time you expect to follow - up your patient after urethroplasty?13.how frequently do you follow - up patients after urethroplasty?14.what investigative tool do you employ during follow - up of patients ? (a)uroflowmetry (b)rug (c)ipss (d)none (e)others 15.in your practice, what are the common complications after urethroplasty ? what is the length of time you expect to follow - up your patient after urethroplasty ? how frequently do you follow - up patients after urethroplasty ? what investigative tool do you employ during follow - up of patients ? (a)uroflowmetry (b)rug (c)ipss (d)none (e)others in your practice, what are the common complications after urethroplasty ? | objectives : to determine the national practice patterns in the management of male urethral stricture disease by the open urethroplasty technique.materials and methods : a questionnaire - based national survey of nigerian urologists was performed during the 19th annual general meeting and scientific conference of the national association of urological surgeons of nigeria, held at ibadan, southwest nigeria in 2013.results:a total of 55 respondents (67.1%) completed the questionnaire. about 43.6% were between the ages of 40 and 49 years. almost 41.8% had between 5 and 9 years of experience as a reconstructive urologist, and 50.9% performed 19 urethroplasties / year. a total of 80 responders reported trauma as the most common etiology for their strictures. about 63.7% preferred to treat strictures after 36 months of diagnosis and 67.3% of respondents preferred the combination of retrograde urethrography and voiding cystourethrography for the diagnosis of urethral stricture. stenting of the urethra was done after urethroplasty using size 16 fr of 18 fr silastic catheter ; however, the duration of stenting varied among urologists. about 41.8% followed up their patients for a year, and uroflowmetry was used by 36.6% of the responders to follow - up their patients. stricture recurrence was the most common reported complication by 36.4% of the respondents.conclusions:in nigeria, most urethral stricture diseases are treated by open urethroplasties. very few of these surgeries are performed annually by young urologists. there is no uniformity in the method of diagnosis, stenting, and follow - up after treatment. |
placenta percreta (with bladder invasion) is a rare obstetric condition with the risk of massive intraoperative hemorrhage. in these cases, the combination of a transverse uterine fundal incision and retrograde cesarean hysterectomy could be useful to minimize maternal hemorrhage and avoid severe bladder injury. placenta previa is an obstetric complication in which the placenta is inserted into the lower uterine segment. it can be complicated with abnormal placentation, the most serious of which is placenta percreta. placenta percreta is the rarest and most complicated variant of placenta accreta in which the placenta abnormally penetrates through the myometrium ; it is associated with heavy obstetrical hemorrhage and bladder injury [24 ]. severe hemorrhage can occur at the time of cesarean hysterectomy in cases with placenta percreta blood loss from 7000 ml to as high as 47,000 ml has been reported and this increases the difficulty of the operative technique. performing cesarean delivery and total abdominal hysterectomy in a patient with placenta percreta can be difficult and potentially life threatening. although several techniques to decrease the incidence of maternal morbidity and complications have been reported [710 ], the results have not been very satisfactory. we report here a case of placenta percreta where we attempted a novel surgical approach. we used a combination of a transverse uterine fundal incision and subsequent retrograde cesarean hysterectomy, with amputation of the anterior uterine wall and placenta, thereby successfully managing placenta percreta. a 32-year - old woman (gravida 5, para 3) at 32 weeks of gestation was diagnosed with placenta previa and was referred to our hospital from a private facility. ultrasonographic findings revealed multiple lacunae, and magnetic resonance imaging revealed the loss of uterine myometrium between the placenta and bladder wall (fig.1a and b). red arrows indicate the loss of uterine myometrium between the placenta and the bladder wall. based on these findings, we suspected placenta increta or percreta. at 33 weeks, she experienced a sudden onset of vaginal bleeding. blood loss was estimated about 100 ml and she was admitted to our hospital. she was received intravenous fluids and intramuscular betamethasone to stimulate fetal lung maturation. the day after admission, she experienced vaginal bleeding again and total blood loss was estimated to be over 300 ml. we decided to perform an emergency cesarean delivery using a transverse uterine fundal incision to deliver the fetus. laparotomy revealed large blood vessels and the placenta was observed through the anterior uterine wall (fig.2a). based on these findings, placenta percreta was suspected and separation of the bladder from the uterus was considered extremely difficult. we performed a transverse uterine fundal incision to avoid an incision into the placenta. after elevating the uterus outside of the abdominal wall, an ultrasound - guided transverse incision was made into the uterine fundus to avoid rupturing or producing a bulge on the fetal membrane (fig.2b). (a) large blood vessels and portions of the placenta were observed through the anterior uterine wall. (b) a transverse incision was made on the uterine fundus with minimal blood loss from the incision site. the placenta was not spontaneously delivered because of abnormal uterine adherence, vaginal bleeding continued. we initially attempted to perform hysterectomy using the bladder - filling technique ; but because of adherence between the anterior uterine wall and bladder, we could not strip the bladder from the uterus. because of our attempt to separate the bladder, bleeding suddenly increased from the placental site and the total blood loss reached over 3500 ml. a retrograde hysterectomy and placental amputation were successfully performed (as shown in fig.3a). second, the uterine artery and parametrial vessels were clamped and ligated while palpating the ureters, that contained an ureteral stent placed preoperatively. finally, the anterior uterine wall and placenta were amputated and resected to the uterine body. the placenta invaded the entire myometrium to the uterine serosa, confirming a diagnosis of placenta percreta. (c) a hematoxylin and eosin - stained section of the placenta (magnification 40). the upper edge of the section indicates the bladder side and the lower edge of the section indicates the uterine side. this finding suggests that the diagnosis is placenta percreta. following hysterectomy, bleeding continued and the remaining placental tissue was resected. during the resection the border between the bladder and placenta was visualized to separate the organs, and after complete resection of the placental tissue, we repaired the bladder. the total blood loss was approximately 7800 ml, including an estimated blood loss of 4000 ml from the placental site prior to hysterectomy ; 2000 ml during hysterectomy ; 1000 ml from the bladder wall after hysterectomy ; and 800 ml from other sources. the patient was transfused with 3360 ml of red blood cells ; 1800 ml of flash frozen plasma ; and 400 ml of platelets. postoperative blood pressure stabilized to 120/70 mmhg, and pulse rate was 90 beats / min. a complete blood count indicated a hematocrit level of 29.6% and a hemoglobin level of 10.6 g / dl. because of the substantial hemorrhage, considerable blood transfusions were required ; and she was subsequently placed in the intensive care unit. a hematoxylin and eosin - stained section of the placenta (fig.3c) revealed the placenta invaded the serosa of bladder. as shown in figure3b, the lower uterine segment was resected from the uterine body and the residual tissues were resected step by step from the bladder. we considered the placental tissue to be completely resected, and this was confirmed by a negative qualitative human chorionic gonadotropin test result obtained after discharge. the incidence of placenta accreta increases with the increase in the number of previous cesarean deliveries, and the number of cases of placenta percreta most likely increases as the number of cases of placenta accreta increases. although it is difficult to diagnose placenta accreta antenatally, an accurate diagnosis is one of the most important objectives for the successful management of maternal hemorrhage ; and this can be achieved using magnetic resonance imaging and ultrasonography [1518 ]. a transverse uterine fundal incision for the management of placenta accreta can effectively avoid an accidental incision into the placenta and consequently decrease the risk of heavy fetal and maternal hemorrhage. however, in these reports, the total number of cases involving placenta percreta was small, and few other reports have used this technique for placenta percreta. nevertheless, we considered it a very useful technique for this condition. in our patient, however, we could avoid an incision into the placenta, giving us time to strip the bladder from the uterus. if we had used a conventional method such as a vertical uterine incision, severe hemorrhage because of iatrogenic partial separation of the placenta may have occurred. these situations can easily lead to severe bladder injury or total cystectomy in the surgeon 's blind haste to control the massive bleeding. urologic complications occur in approximately 72% (39 of 54 cases) of cesarean hysterectomies performed during placenta percreta cases. urologic complications include bladder lacerations 26% (24 of 54 cases), urinary fistulas 13%, gross hematuria 9%, ureteral transection 6%, and a small capacity bladder 4%. another study reported that partial cystectomy (3 of 29 cases) or cystotomy (3 of 29 cases) are required for the treatment of placenta accreta and placenta percreta cases. therefore, the avoidance of severe bladder injury is of the utmost importance in cesarean hysterectomy for the management of placenta percreta. however, cesarean hysterectomy is extremely difficult, particularly when massive bleeding occurs, because the urgency of stopping the hemorrhage leaves little time to strip the bladder from the uterus. some reports have suggested using supracervical cesarean hysterectomy for placenta percreta to avoid severe bladder injury. however, supracervical hysterectomy may cause severe hemorrhage due to the large volume of placenta (approximately one - third of the total placenta) being amputated without ligation of the uterine artery and the parametrial vessels. in contrast, our method (fig.3a) likely reduced intraoperative hemorrhage because only approximately 5% of the placenta was retained, and the placenta was amputated after ligation of the uterine artery and parametrial vessels. in fact, in our case, we first opened the posterior vaginal wall and immediately clamped the parametrial vessels and uterine artery. compared with supracervical hysterectomy, retrograde hysterectomy may pose a higher risk of ureteral injury because of the uncertainty regarding the location of the ureters. as a result of preoperative placement of the ureteral stent in our case, we could palpate the ureters to avoid ureteral injury when opening the vagina and clamping and ligating the uterine artery and parametrial vessels. we, therefore, recommend that, in case of potential life - threatening hemorrhage during retrograde hysterectomy, a ureteral stent should be placed preoperatively to minimize the risk of ureteral injury. numerous studies have reported various methods for the postpartum management of placenta percreta, including conservative management, uterine artery embolization, balloon occlusion, uterine compression sutures, and cesarean hysterectomy [2,3,2226 ]. our patient experienced both preoperative and intraoperative vaginal bleeding and conservative management was not possible. while bleeding may be reduced with the use of the balloon occlusion technique, it was not used in our case because its safety has not yet been established. in summary, we successfully managed a case of placenta percreta with bladder invasion complicated by life - threatening hemorrhage using a transverse uterine fundal incision and retrograde hysterectomy without cystectomy. we found the following four strategies to be particularly useful ; (1) the placement of a transverse uterine fundal incision to minimize bleeding from the uterine incision ; (2) use of the bladder - filling technique to avoid bladder injury by allowing visual discrimination between the anterior uterine wall and the bladder ; (3) placement of an ureteral stent to facilitate palpation of the ureters, thus minimizing ureteral injury risk ; and (4) performing a retrograde hysterectomy, even if life - threatening hemorrhage occurs during hysterectomy. finally, retrograde hysterectomy and placental amputation should be performed to avoid severe bladder injury and to dramatically reduce hemorrhage. | key clinical messageplacenta percreta (with bladder invasion) is a rare obstetric condition with the risk of massive intraoperative hemorrhage. in these cases, the combination of a transverse uterine fundal incision and retrograde cesarean hysterectomy could be useful to minimize maternal hemorrhage and avoid severe bladder injury. |
it is known that 6070% of recurrent colorectal cancer involves the liver and that the liver is the only involved organ in 35% of cases. colorectal liver metastases used to be thought of as systemic disease that involves many organs and systems until recently when local liver therapy in the form of liver resection has been reported to results in 5-year survival of 2739% and even longer survival and cure [1, 2 ]. patients with liver metastases that are resectable but left untreated have average survival of 612 months and rarely longer than 2 years. over the last 20 years liver resection for colorectal metastases has seen many refinements ; the improvement in anaesthesia and postoperative care have reduced the morbidity and mortality with subsequent more aggressive surgical approach. strategies that have widened the indications for liver resection includes portal vein embolisation, staged liver resection, neoadjuvant chemotherapy, ablative procedures, and locoregional chemotherapy [47 ]. many studies have developed and validated scoring systems to predict prognosis and recurrence of colorectal metastases based on clinical and pathological data of large number of hepatic resections for colorectal metastases. these scores are based on variety of factors that include stage of the primary disease, time interval between diagnosis of the primary lesion and occurrence of liver metastases, level of cea immediately prior to liver resection, size and number of resected liver lesions, surgical resection margin, blood transfusion, and bilateral distribution of liver disease [1, 810 ]. various factors were found to have influence on disease recurrence and overall survival ; resection margins and lymph nodes involvement are common predictors of recurrence. other controversial factors are number and size of lesions, blood transfusion, and disease - free interval. this systematic review is conducted to evaluate the risk factors influence of overall long - term survival following hepatic resection for colorectal metastases. electronic search was performed and relevant reports were identified using electronic databases (medline 19502010 and embase 19802010), the search was restricted to human adults and english language literature. the search terms used were colorectal neoplasm, overall survival, and disease - free survival. all the references used in the published original and review studies were searched to identify more studies. to be included, studies had to meet the following criteria. population : patients with liver metastases from colorectal cancer who had liver resection as a curative treatment. exposure : surgical liver resection for metastases whether anatomic resection or segmental nonanatomic resection regardless of whether they had or did not have adjuvant chemotherapy. that included studies that evaluated patients ' survival following re - resection following primary liver resection. duplicate publications were excluded and wherever publications that evaluated the same population group were encountered, the report with the most relevant and comprehensive data was selected. two independent reviewers using standard data collection form extracted all relevant data from the full text articles. inconsistencies were resolved by discussion to reach a reasonable consensus. whenever missing data were encountered, the authors were contacted to request the data required to be included in the meta - analysis. methodological quality of the studies was evaluated independently by two reviewers using the newcastle - ottawa scale. a quality score was calculated on the basis of the following components : selection of the study groups (04 points), quality of the adjustment for confounding variables (02), and outcome of interest in the study population (03 points). odds ratio (or) of overall survival was used as the primary effect estimate in this meta - analysis. from the eligible studies that met the inclusion criteria, estimates of the or and its associated 95% confidence interval (ci) were calculated using the review manager software (version 5 for windows, copenhagen, denmark ; the nordic cochrane centre, the cochrane collaboration, 2008). data that could not be extracted directly were reconstructed indirectly by two reviewers when required. prespecified factors that was thought to affect the overall survival after liver resection for colorectal metastases were analysed. those included resection margin, tumour size, number of metastases, bilateral versus unilateral disease, t stage of the primary, lymph nodes positive primary versus lymph nodes negative, disease - free interval, cea level, and blood transfusion. heterogeneity between the included studies was appraised using the q measure for statistical significance and the i measure for the amount of heterogeneity, with p 25% showing important heterogeneity. a random effect model based on dersimonian - laird estimator was used wherever there was significant heterogeneity, and fixed effect model based on mantel - haenszel estimator was used when there was no significant heterogeneity. we conducted begg 's test and the harbord modified test to identify publication bias for small study effect, with p >.5 being statistically significant. the results of this systematic review were reported using the preferred reporting items for systematic reviews and meta - analyses (prisma) guidelines. the initial search revealed 164 titles, abstracts for those articles (figure 1) were reviewed, 28 articles were considered to be potentially useful for inclusion, and their full text was retrieved and reviewed. seventeen of these 28 articles were subsequently excluded from the meta - analysis as they did not meet the inclusion criteria. eleven original reports (all retrospective cohort studies) had enough data to investigate the role of different variables on overall long - term survival after liver resection for colorectal metastases. two studies were excluded due to the fact that they were a duplication of the same study population [19, 20 ]. one further study was excluded because it had no data that can be used in the meta - analysis. there was no statistical evidence of publication bias among the included studies based on the funnel plot used in review manager. the eleven studies reviewed 3442 patients who had liver resection for colorectal metastases, the eight included studies had a total number of 2387 patients, all studies reported five- and ten - year survival [2229 ]. these studies had variably reported the impact of different factors on overall survival ; these factors included resection margins, size of the largest liver lesion, number of liver lesions, distribution of lesions, cea levels prior to liver resection, lymph node status of the primary, satellite configuration of liver metastases, type of resection, extrahepatic disease, and whether the liver metastases were presented in a synchronous or metachronous to the diagnosis of the primary colorectal cancer. in the study of tomlinson. they reported that 34% of patients who are disease - free at 5 years after hepatic resection did experience recurrence. who reported 50- and 10-year disease - free survival of 26 and 23%, 28.2 and 25.4%, and 33.6 and 25.4%, respectively [22, 25, 26 ]. from these data since recurrence after 10-year of disease - free survival is rare ; ten - year survival could be considered as definitive cure. factors that influence disease - free survival and may predict that has been reported only in the study by minagawa., hence it was not suitable to conduct meta - analysis on this category of outcome. four studies reported adequate data to determine the relationship between positive and negative resection margin and long - term survival. the average overall survival for positive margin was 29%, which is significantly better compared with 20% of negative resection margin (p =.03) with odds ratio of 0.41 (95% ci 0.180.9). there was moderate degree of heterogeneity i 52% ; however, this heterogeneity was eliminated when prespecified sensitivity analysis was performed by elimination of the study by tomlinson. that had a wide 95% ci and that has little overlap with other studies. the result of the meta - analysis after this elimination showed or of 0.55 (0.95% ci 0.360.84) with p =.005, which ensures that the results of the meta - analysis are robust (figure 2). four studies reported the influence of wider negative margin of more than 1 cm compared with negative margin of 010 mm [2326 ]. wider resection margin had no beneficial effect on overall survival. pooled analysis for the likelihood of survival is shown in figure 3 (or = 1.11 ; 95% ci : 0.592.08 ; p =.75). the pooled estimate was robust : omission of individual study at a time did not change the statistical results (data not shown). six studies reported the effect of tumour size on overall survival [2226, 28 ] ; meta - analysis of the studies that reported analysis data on patients liver metastases more than 5 cm compared with patients who had liver lesions of 5 cm or less, showed no prognostic relationship between size of the resected tumour and overall patients ' survival. figure 4 shows the results of pooled estimate for survival in relation to tumour size (or = 0.73, 95% ci : 0.461.16 ; p =.18). there was moderate heterogeneity among the included studies i = 75%, the results were robust, omission of the study by giuliante four studies reported data on the effect of time interval between the diagnoses of the primary colorectal cancer and the occurrence of liver metastases on the overall patients ' survival. pooled estimate of the survival time after liver resection for colorectal metastases showed no significant prognostic relationship (or = 1.22 ; 95% ci : 0.751.99 ; p =.42) figure 5. there was moderate degree of heterogeneity (i = 68%). the analysis results were robust, exclusion of the study by scheele. removed the heterogeneity and the results and resulted in or = 1.03 ; 95% ci : 0.641.65, data not shown. six studies reported data to determine the relationship between lymph nodes metastasis status of the primary colorectal cancer and survival after liver resection for colorectal metastases [22, 23, 25, 26, 28, 29 ]. figure 6 shows the forest plot with pooled estimates of the odds ratio of survival in patients who had node positive primary disease compared with those who had node negative disease. the overall ten - year survival for node negative disease was 32% compared with 22% for nodes positive primary disease (or = 0.46 ; 95% ci : 0.260.79 ; p =.006), the pooled analysis showed significant heterogeneity i = 81% ; omission of the study by tomlinson. reduced the heterogeneity to insignificant level with results of odds ratio of 0.38 ; 95% ci : 0.236 ; p <.0001, which ensures the robustness of the pooled estimates of the effect. three studies reported data for determination of the relationship between types of liver resection (segmental or anatomic) [22, 25, 29 ]. figure 7 shows the forest plot of the pooled estimates of long - term survival for patients who had segmental resection compared with patients who had anatomic resection (right hepatectomy, left hepatectomy, or trisectionectomy). the type of resection did not have any significant impact on overall survival (or = 2.60 ; 95% ci : 0.887.63 ; p =.08), there was significant heterogeneity among the included studies. four studies provided data for determining the relationship of cea levels, prior to resection of colorectal liver metastases, and the overall patients ' survival [22, 23, 25, 26 ]. figure 8 shows the forest plot with the pooled estimate for likelihood of ten - year survival for patients with cea levels more than 50 ng / ml and those with cea levels less than 50 ng / ml. overall pooled survival for those with cea level less than 50 ng / ml is 37% compared with 19% for those with cea greater than 50 ng / ml. the results show statistically significant better survival in those with low cea levels (or = 2.27 ; 95% ci : 1.035.02 ; p =.04). there is significant heterogeneity (i = 72%) ; omission of the study by scheele., which looks like an outlier has removed the heterogeneity, and still the results were significant (or = 1.71 ; 95% ci : 1.152.55 ; p =.009 ; data not shown), which confirms the robust results of the analysis. five studies provided data to determine the effect of bilateral distribution of resected colorectal metastases on the ten - year survival [22, 25, 26, 28, 29 ]. ten - year survival was 36% compared with 18% in patients who had bilateral disease resected. figure 9 shows the forest plot of the pooled estimate for ten - year survival, the presence of bilateral disease in the liver leads to significantly reduced long - term survival (or = 1.64 ; 95% ci : 1.192.27 ; p =.003. seven studies reported data to determine the effect of the number of the resected liver lesions on long - term survival [2226, 28, 29 ] ; these studies compared survival in patients with four or less lesions versus patients who had more than four lesions and found that ten - year survival for patients with four or less lesion was 38% compared with 20% of those who had more than four lesions (or = 1.75 ; 95% ci : 0.873.51 ; p =.11) ; there was a significant heterogeneity i = 73% ; omission of individual studies made no statistical difference ; however, omission of three studies, scheele., minagawa., and tomlinson. [25, 26, 28 ] removes the heterogeneity and results in significantly better survival in patients who had four or less lesions (or = 2.26 ; 95% ci : 1.363.75 ; p =.002), data not shown. this made it not possible to make any valid conclusion about the effect of lesion 's number on overall survival (figure 10). six studies provided data to determine the relationship of timing of liver metastases ; whether it was found at the time of diagnosis of the primary colorectal cancer or afterward [22, 23, 2528 ]. figure 11 shows the forest plot with pooled estimate of likelihood of survival for patients who had liver metastases diagnosed synchronous with the primary colorectal cancer compared with those who developed liver metastases afterward (metachronous ; or = 0.77 ; 95% ci : 0.591.01 ; p =.06). the timing of liver metastases has no significant effect on long - term survival. four studies provided data for determining the relationship of blood transfusion, after liver resection, and long - term survival [2224, 29 ]. figure 12 shows the forest plot with the pooled estimate for long - term survival. patients who received two or less units had significantly better survival than patients who had more than two units (or = 3.69 ; ci : 1.797.60 ; p =.0004). omission of the study of giuliante. removed the heterogeneity, and the pooled estimate remained valid (or = 2.51 ; ci : 1.633.85 ; p < three studies provided data that compared the difference in survival between patients who had a single lesion resected and those who had more than a single lesion [22, 25, 29 ]. figure 13 shows the forest plot of the pooled estimates of survival for patients with single lesion compared to patients who had multiple lesions. patients with a single lesions had significantly better prognosis than those with multiple lesions (or = 1.56 ; 95% ci : 1.082.25 ; p =.02). three studies provided data that compared survival of patients who had satellite lesions along with larger lesion or lesions compared with those who had no satellite lesions [23, 24, 26 ]. figure 14 shows the forest plot of the pooled estimate of survival, the estimate shows significantly better survival for patients who had no satellite lesions compared with those who had satellite lesions (or = 0.37 ; ci : 0.180.77 ; p =.008), there was significant heterogeneity among the included studies. this systematic review and meta - analysis showed that factors that affect long - term survival following hepatic resection, for colorectal cancer metastases, include clear resection margins, advanced primary colorectal cancer with nodal metastases, cea levels, distribution of liver lesions, timing of diagnosis of liver metastases (synchronous or metachronous), quantity of blood transfusion, single lesion compared with multiple lesions, and presence or absence of satellite nodules close to the main lesion. patients who had clear resection margins had significantly better long - term survival than those with positive resection margins. patients who had early stage colorectal cancer with no lymph nodes metastases had better survival than those with lymph nodes metastases. it also showed that metachronous presentation of liver metastases is a good prognostic factor compared with synchronous presentation. a single liver lesion particularly in the absence of satellite nodules has better outcome than multiple lesions with or without satellite nodules ; however, when comparison was made between patients with more than four nodules and those with four or less lesions, there was no difference in overall long - term survival. cea levels less than 50 ng / ml, unilateral liver disease, and two units or less of perioperative blood transfusion were found to be favourable prognostic factors. there was no single prognostic factor of sufficient power to predict long - term survival and cure. other factors that were analysed in this meta - analysis and found not to have significant influence on long - term survival included a the width of resection margin, it was found that if the resection margin is clear there is no survival benefit from a wider resection margin more than 1 cm. whether the resected largest lesion was more or less than 5 cm did not have effect on survival. the interval between the diagnosis of the primary and liver recurrence less or more than 12 months did not seem to affect long term survival. whether the lesion removed in anatomic resection technique or segmental resection, number of resected lesions, and synchronous versus metachronous metastases, all had no effect on long - term survival. during the past two decades, liver resection for colorectal liver metastases has been increasing the standard of care whenever the disease is limited to the liver and is technically possible by leaving adequate liver remnants. there is overwhelming evidence to support the survival benefit with reports of actuarial 5-year survival of 2540% compared with patients who are treated only with chemotherapy who rarely survive up to five years [30, 31 ]. with technical advances and improved perioperative the mortality of liver resection is less than 5%, wei. this improvement makes liver resection a standard treatment for colorectal metastases [12, 20, 26, 28 ]. tomlinson. had investigated risk factors for 10-year survival and redesigned the original score devised by the same investigators. the original score had five components with one point for each component that includes tumour number more than four, size more than 5 cm, cea level more than 200 ng / ml, and disease - free interval less than 12 months and positive resection margin with one point for each. after analysing 10-year survival, patients fell in two groups ; the low risk group (02 points) who had 10-year survival of 21% and high - risk group (35 points) with 10-year survival of 10% (p no patients with positive resection margin had survived for 10 years, which seems to be the only factor that ruled out any possibility of cure. this raises the question of the benefit of neoadjuvant chemotherapy particularly now there are more effective agents that may increase the rate of complete resection, the effect of that approach on survival is not known. it was not possible in this review to analyse the effect of adjuvant chemotherapy on survival after hepatic resection for colorectal metastases. however, most of the study patients had been treated in the era when the standard chemotherapy was 5-flourouracil, which has limited effect, compared with the modern chemotherapeutic agents like irinotecan, oxaliplatin, and bevacizumab. this makes the evaluation of the pure curative benefit from surgical resection relatively difficult to assess. the improvement of outcome in liver resection has been attributed to a combination of factors such as aggressive surgical treatment, improved chemotherapy, and improvement of preoperative imaging and patient selection [3234 ]. repeat hepatectomy for recurrent hepatic disease following initial liver resection is being increasingly used, also staged liver resection and resection of isolated extrahepatic disease is being more and more utilised with encouraging results that lead to improvement in survival [4, 3437 ]. despite all the advances and improvements, it remains not possible to discriminate with reasonable certainty which subset of patients is likely to be cured or live more than 5 years. repeat liver resection gives overall survival results of up to 52% in one series and is comparable to the results of initial liver resection. improvement of preoperative staging by liberal use of helical ct scans and mri and the introduction of pet scan and pet ct have allowed for better patients selection by early detection of extrahepatic and bilobar disease [33, 3840 ]. many authors have reported using radiofrequency ablation in conjunction with surgery for nonresectable liver metastases either intraoperative or postoperative with variable results [21, 32 ], the impact of this technique on overall long - term survival remains questionable. the emergence of new chemotherapeutic agents such as oxaliplatin, irinotecan, and bevacizumab has increased the treatment option available for clinicians to deal with metastatic colorectal disease. these new agents have been used in conjunction with liver resection either as neoadjuvant or adjuvant manner in many studies but the effect of this approach on survival has not been test in a randomised controlled trial [5, 41, 42 ]. chemotherapy is being commonly used as an adjuvant agents following liver resection particularly in patients who had no previous chemotherapy, the survival benefit of this approach remains to be proved. another potential use is to downstage potentially resectable liver metastases, in this situation patients with advanced disease are offered resection if they show good response to chemotherapy, this strategy has been used with limited success [5, 34 ]. in conclusion this review defines 10-year survival and cure to be between 12% and 28% ; we described the factors that affect survival in this meta - analysis. there is no single factor that was of sufficient power to rule out cure with the possible exception of positive resection margin. this leads to the fact that patients ' selection for resection with disease limited to the liver or liver disease with resectable extrahepatic metastases remains a matter of trial and error particularly for patients who have marginal suitability for resection. this review also indicates that we need newer prognostic factors perhaps based on tumour biology that may discriminate between curable and noncurable metastatic colorectal cancer. an important limitation of this study that reflects the quality of the available data is the fact that raw data was not available to all the studies ' patients and the presence of heterogeneity among the included studies and the fact that those studies are generally retrospective reviews. also patients who had 10-year survival are likely to have been treated prior to the era of pet scan routine use and likely have been treated with old and less effective chemotherapeutic agents. | background. liver resection in metastatic colorectal cancer is proved to result in five - year survival of 2540%. several factors have been investigated to look for prognostic factors stratifications such as resection margins, node involvement in the primary disease, and interval between the primary disease and liver metastases. methods. we searched medline and embase for studies that reported ten - year survival. metaanalysis was performed to analyse the effect of recognised prognostic factors on cure rate for colorectal metastases. the meta - analysis was performed according to ottawa - newcastle method of analysis for nonrandomised trials and according to the guidelines of the prisma. results. eleven studies were included in the analysis, which showed a ten - year survival rate of 1236%. factors that have favourable impact are clear resection margin, low level of cea, single metastatic deposit, and node negative disease. the only factor that excluded patients from cure is the positive status of the resection margin. conclusion. predicted ten - year survival after liver resection for colorectal metastases varies from 12 to 36%. only positive resection margins resulted in no 10-year survivors. no patient can be excluded from consideration for liver resection so long the result is negative margins. |
the well - known diabetes control and complications trials (dcct) and united kingdom prospective diabetic studies (ukpds) have established glycosylated hemoglobin (hba1c) as a standard measure of average glucose control because of its strong correlation with the development of diabetes - related complications. however, in both basic and clinical studies in recent years, glycemic variability has become increasingly recognized as a risk factor for chronic diabetes complications. evidence is accumulating that high levels of glycemic variability have deleterious effects beyond those of sustained chronic hyperglycemia in terms of oxidative stress, diabetes complications, and cardiovascular outcomes [16 ], although some contradictory reports also exist. low levels of glycemic variability are therefore recommended as a component of overall glycemic control [811 ]. currently, efforts to reduce glycemic variability are largely frustrated by the lack of reference values. in our previous multi - center studies, we established normal reference ranges for continuous blood glucose parameters (the 24-h mean blood glucose and the percentage of time that subjects blood glucose levels were 7.8 mmol / l and 3.9 mmol / l within 24 h) through analysis of continuous glucose monitoring (cgm) data obtained from healthy chinese adults. the current paper reports on further analyses on these data, aiming to generate reference ranges for glycemic variability in normal chinese adults, which may facilitate clinical adoption. a total of 445 (out of 588 screened subjects) healthy subjects without related metabolic disorders were enrolled from 10 academic hospitals in china between october 2007 and july 2008. the inclusion / exclusion criteria and study methods have been described in detail in previous publication. the inclusion criteria were the following : 1) clinically stable condition with no previous medical history of diabetes, hypertension, dyslipidemia, coronary artery diseases or cerebral stroke ; 2) fasting plasma glucose 1.5-fold elevation of alanine aminotransferase, aspartate aminotransferase or direct bilirubin, or serum creatinine > 115 mol / l). as described in a previous publication, the cgms sensor was inserted into all subjects by the same specialist nurse at day 0 around 8:009:00 am in hospital. the first cgms calibration by finger - stick bg was performed after 1 hour of initialization. if no abnormal cgms situation was observed, the subjects was dismissed and they continued with cgm at home for 3 consecutive days. subjects were instructed to input at least 4 calibration readings per day. at day 3, around 8:009:00 am, subjects came to the hospital and had the cgms removed. all subjects completed a 3-day period of glucose monitoring using a continuous glucose monitoring system (cgms) (medtronic inc, northridge, ca), and 434 subjects had valid cgm data (figure 1). the parameters of glycemic variability included the mean amplitude of glycemic excursions (mage) and the standard deviation (sd) of blood glucose readings. mage was obtained by measuring the arithmetic mean of the differences between consecutive peaks and nadirs, with measurement in the peak - to - nadir direction ; only excursions > 1 sd were considered. for sd determination, the standard deviations of a total of 288 values collected during a 24-hour cgm period for each study subject were calculated. for 434 subjects, final values of both mage and sd data are expressed as the mean sd, except for skewed variables, which are presented as the median (interquartile range). the kolmogorv - smirnov test was used to determine the normality of the derived parameter s distribution within the population. a subgroup of 20 subjects was used to evaluate the cgms reproducibility. to balance the sex and age of these 20 subjects, a stratified sample scheme was adopted : subjects were divided into 10 ages by sex strata (i.e., 2029, 3039, 4049, 5059 and 6069 ; male and female). within each stratum as reported in a previous publication, a total of 434 healthy subjects (male 213, female 221 ; age 4314, 2069 years old ; bmi 21.81.7 kg / m, 18.524.9 kg / m) completed the study. mmol / l and 0.75 (0.42) mmol / l [median (interquartile range) ], respectively. spearman correlation analysis indicated a significant positive correlation between mage and sd (r=0.90, p0.05, table 1). the coefficients of skewness for these parameters were 1.18 and 0.79, respectively (figure 3). the 95 percentile values of 3.86 mmol / l for mage and 1.40 mmol / l for sd represent the upper limit of normal (table 1). two men and 2 women were randomly selected from each of the 5 age groups (2029, 3039, 4049, 5059 and 6069) for the evaluation of reproducibility. a total of 20 subjects with a mean age of 4316 (range, 2268) years and body mass index (bmi) of 22.21.8 kg / m underwent a second 3-day cgm evaluation 812 weeks after the initial monitoring. the values obtained for the 2 measurements were : 1.70 (0.82) mmol / l vs. 1.78 (0.87) mmol / l (first vs. second monitoring) for mage, and 0.75 (0.32) mmol / l vs. 0.82 (0.37) mmol / l [median (interquartile range) ] (first vs. second monitoring) for sd. no significant difference was observed in mage or sd between the first and second monitoring periods (both p>0.05). calculated from the 434 healthy subjects, mage and sd were 1.73 (1.08) mmol / l and 0.75 (0.42) spearman correlation analysis indicated a significant positive correlation between mage and sd (r=0.90, p0.05, table 1). the coefficients of skewness for these parameters were 1.18 and 0.79, respectively (figure 3). the 95 percentile values of 3.86 mmol / l for mage and 1.40 mmol / l for sd represent the upper limit of normal (table 1). two men and 2 women were randomly selected from each of the 5 age groups (2029, 3039, 4049, 5059 and 6069) for the evaluation of reproducibility. a total of 20 subjects with a mean age of 4316 (range, 2268) years and body mass index (bmi) of 22.21.8 kg / m underwent a second 3-day cgm evaluation 812 weeks after the initial monitoring. the values obtained for the 2 measurements were : 1.70 (0.82) mmol / l vs. 1.78 (0.87) mmol / l (first vs. second monitoring) for mage, and 0.75 (0.32) mmol / l vs. 0.82 (0.37) mmol / l [median (interquartile range) ] (first vs. second monitoring) for sd. no significant difference was observed in mage or sd between the first and second monitoring periods (both p>0.05). many different summary statistics have been proposed for accurate assessment of glycemic variability, including sd, coefficient of variation (% cv), interquartile range (iqr), percentage of glucose values within specified ranges, mage, m - value, mean of the daily differences (modd), the average daily risk range (adrr), and continuous overlapping net glycemic action over an n - hour period (conga - n). notwithstanding their apparent diversity, all of these parameters are derived from statistical interconversion and manipulation of sequential blood glucose levels. accordingly, various assessment parameters for glycemic variability, with different intrinsic characteristics and scopes of applicability, should be selected in specific clinical settings. in addition to sd, which is one of the most frequently used measurements of glycemic variability in statistics, mage obtained from cgm is currently generally regarded as the gold standard metric of glycemic variability. many ongoing studies use mage to investigate the relationship between glycemic variability in patients with diabetes and oxidative stress, chronic complications and pancreatic islet functions, and to evaluate the impact of treatment regimen on glycemic variability [24,21,22 ]. therefore, both sd and mage were analyzed in the current study. as seen in the current results previous studies showed that insulin secretion decreased with aging in people with normal glucose tolerance (ngt). moreover, glycemic variability (measured by mage) was found to be associated with postprandial beta - cell function in a study by kohnert.. it could therefore be assumed that the deterioration of beta - cell function with aging contributes to the association of age with glycemic variability observed in the current study. since the conduct of prospective follow - up studies on glycemic variability remains rather challenging, cgms data analyses in healthy subjects provide a feasible approach to establish normal reference values for these measurements. the population mean value of mage in this study was 1.96 mmol / l, comparable to the previous findings of monnier.. they reported that at the mean oxidative stress level (represented as 24-hour 8-isoprostane f2 excretion rate in urine) of 275 pg / mg creatinine, the corresponding glycemic variability (represented as mage) was 2.2 mmol / l (40 mg / dl) in healthy subjects. since distributions for both mage and sd values departed from normality, the 95th percentiles of the 2 parameters were used for defining normality in our study, and were set as the upper limits of the normal reference values (mage < 3.9 mmol / l and sd < 1.4 mmol / l, respectively). analyses of the relationship between these parameters and demographic characteristics revealed that mage and sd were independent of sex, but increased with age. we also recommend a unified cut - off point for the normal reference values of the 2 parameters for glycemic variability, similar to the normal glucose tolerance cutoff points recommended by ada or who, without regard for age or sex. further investigations are warranted to verify the normal reference values of the glycemic variability parameters established in this study. overall, the current concept of bg management calls for reduction of glycemic variability in patients with diabetes. this requires close monitoring of blood glucose levels with cgm, as well as judicious pharmacotherapy to minimize the risks of both postprandial hyperglycemia and hypoglycemia. the reference values of mage and sd parameters established in this study provide the preliminary targets for normalization of glycemic variability. its clinical application depends on validation in other populations, such as those with different glycemic metabolism. moreover, the impact of bmi on the mage and sd reference values needs further investigation. | summarybackgroundglycemic variability is increasingly recognized as an important issue in diabetes management. however, the lack of normative values may limit its applicability in the clinical setting.the objective of this study was to establish preliminary normal reference ranges for glycemic variability by analyzing continuous glucose monitoring (cgm) data obtained from healthy chinese adults.material/methodsthree-day cgm data were obtained from 434 healthy adults at 10 academic hospitals throughout china. glycemic variability was calculated as the 24-hour mean amplitude of glycemic excursions (mage) and standard deviations (sd) of blood glucose readings.results434 healthy subjects (male 213, female 221 ; age 4314, 2069 years old ; bmi 21.81.7 kg / m2, 18.524.9 kg / m2) completed the study. mage and sd values for the 434 healthy subjects were 1.73 (1.08) mmol / l and 0.75 (0.42) mmol / l [median (interquartile range) ], respectively. in both men and women, mage and sd tended to increase with age. neither mage nor sd showed a significant difference between men and women. values for both parameters were non - normally distributed within the population. the 95th percentiles of mage and sd were 3.86 and 1.40 mmol / l, respectively. these values were adopted as the upper limits of normal.conclusionsmage < 3.9 mmol / l and sd < 1.4 mmol / l are recommended as the normal reference ranges for glycemic variability in chinese adults. the values established in this study may facilitate the adoption of glycemic variability as a metric of overall glycemic control in diabetes. |
the purpose of the study was to evaluate the efficacy of a topical form of a carbonic anhydrase inhibitor (dorzolamide) on the foveal function and thickness in the eye of a patient with enhanced s - cone syndrome (escs) associated with macular cysts. twenty - eight - year - old polish man with escs and macular cysts appearance in the right eye was treated 3 times daily with 2.0 % dorzolamide drops for the period time equal to 6 months. monthly controls included : best corrected distance visual acuity (bcdva - logmar), foveal thickness (optical coherence tomography, oct) and foveal function (multi - focal electroretinography, mferg). before treatment, bcdva in the right eye was equal to 0.26 logmar, improved to 0.1 logmar during the first 3 months and remained stable for the next 3 months. after 6 months, foveal thickness decreased (from 482 to 224 m) and foveal function improved (the amplitude of p1-wave density increased from 34.8 to 107.3 nv / deg) and was between the ranges of normal values. the results of our short - term study suggest potential efficacy of topical dorzolamide treatment in escs patients with macular cysts. enhanced s - cone syndrome (escs) first described in 1990 by marmor. is a rare, slowly progressive autosomal recessive inherited retinal degenerative disorder. in most cases of escs, nr2e3 gene mutation is responsible for the disease development and leads to abnormal cones and rods differentiation. characteristic fundus changes usually progress from subtle pigmentary changes with white dots in the early stage to a more severe nummular pigmentary deposition around the vascular arcades accompanied by yellowish dots later on. cystic macular changes occur frequently, in about 5075 % of cases [3, 4 ]. enhanced s - cone syndrome is diagnosed on the basis of characteristic clinical fundus changes and pathognomonic features of the full - field electroretinography (erg). there were performed according to the international society for clinical electrophysiology of vision (iscev) standards such as the absence of rod response, morphologically similar waveforms of scotopic and photopic responses to the standard stimulus flashes (3.0 erg) and flicker reduction [4, 5 ]. the s - cone erg recording can be performed to confirm the pathophysiologic origin of the disease (the s - cone hyperactivity) and is characterized by abnormally large and delayed waveforms (relative to those with standard stimulation). retinal pigment epithelium (rpe) function is abnormal in escs retina, resulting in macular cysts development. dorzolamide (topical form of carbonic anhydrase inhibitor) may improve rpe metabolism by activating the pumping function and consequently, it reduces intraretinal cysts and rpe remains adherent to the retina [7, 8 ]. only 3 case report studies [911 ] described efficacy of topical (2.0 % dorzolamide) and/or oral (acetazolamide) carbonic anhydrase inhibitor in the treatment of macular cysts associated with escs. in these 3 patients, increase in visual acuity associated with normalization of foveal microanatomy (illustrated by oct) was observed. treatment time in these studies varied from 4 [9, 10 ] up to 27 months. during the follow - up, visual acuity, these promising initial results were the reason to implement dorzolamide therapy in a newly diagnosed escs patient presented in this report (table 1). a twenty - eight - year - old white male was sent to the ophthalmology clinic with the suspicion of escs and had following complaints : night vision problems since the childhood and decreased visual acuity since 2 years in the left eye and most recently in the right eye. initially, the patient s bcdva was equal to 0.26 in the right eye and 0.2 in the left eye on a logmar scale (early treatment diabetic retinopathy study chart). color vision (farnsworth panel-15 d) was normal in both eyes. clinical features of macular cysts were noticed in both eyes (fig. 1). macular cysts appearance was confirmed by the oct test (zeiss stratus oct, humphrey instruments model 3000, carl zeiss inc, dublin, ca, 2007). the cystoid spaces appeared as hyporeflective spaces, interspersed with high - signal septae which represented bridging retinal elements in both eyes. loss of the normal foveal depression was observed in the right eye (fig. 2). in the left eye, foveal contour became even convex and dome shaped, as previously described in the literature. fluorescein angiogram showed absence of any detectable leakage of the dye in the macular area in either eye.fig. 1fundus photographs, the right and left eye of the 28-year - old man with escsfig. 2optical coherence tomography (oct) images (above) and mferg recordings (below) of the right eye of the 28-year - old man with escs before and after a 6-month treatment with topical dorzolamide in comparison with the age - matched healthy control fundus photographs, the right and left eye of the 28-year - old man with escs optical coherence tomography (oct) images (above) and mferg recordings (below) of the right eye of the 28-year - old man with escs before and after a 6-month treatment with topical dorzolamide in comparison with the age - matched healthy control the erg (iscev) was recorded, using burian it showed the absence of rod response, reduced and delayed waveforms in response to white flash (0 db), both in scotopic and photopic conditions, reduced and delayed flicker recording (fig. 3). in the s - cone erg recordings, blue stimulus (455 nm, 5 ms, 80 cd / m, 16 cd s / m) was used in the orange background (590 nm, 560 cd / m). the recordings presented hyper - normal and delayed a- as well as b waves. in mferg (iscev) (retiscan system, roland consult, germany, 2009), delayed and reduced p1-wave amplitudes were observed in all six analyzed concentric rings. nr2e3 gene sequencing was not performed, because it is not necessary for escs diagnosis.fig. 3full - field erg of the escs patient full - field erg of the escs patient the patient was treated with topical form of 2.0 % dorzolamide 3 times daily for 6 months. left eye treatment was discontinued after 1 month because of inefficacy and cystoid macular changes progression. the probable reason of the left eye treatment failure was prolonged macular cysts appearance, which caused profound and irreversible rpe and cone photoreceptors dysfunction. examinations that were performed after 1st, 2nd, 3rd, 4th and 6th months of the treatment consisted of : bcdva, mferg and oct tests (5th month examination failed due to the absence of the patient). bcdva increased gradually during the treatment from 0.26 to 0.22 logmar at the 1st, to 0.14 logmar at the 2nd and to 0.1 logmar at the 3rd follow - up examination (fig. bcdva in the right eye remained stable during the next 3 months of treatment. except the 3rd follow - up examination, central retinal function, presented by p1 wave response density in mferg one of the possible explanations of this was unsystematic use of the drops in the 3rd month, which was confirmed by the patient. p1 wave implicit time during first 2 months of treatment shortened, but then it returned to the value before the treatment (fig. 4right eye foveal thickness (ft) and visual acuity (va) (above) ; implicit time (ms) and p1-wave response density (nv / deg) of mferg results (below) of escs patient, treated with dorzolamide during 6 consecutive visitstable 1.va logmar etdrsft (m)p1 density (nv / deg)implicit time (ms)m sdrangemedrangem sdrangenormal values0.0212 20192232134.5106.8148.239.2 2.13543.3 m mean, sd standard deviation, med median right eye foveal thickness (ft) and visual acuity (va) (above) ; implicit time (ms) and p1-wave response density (nv / deg) of mferg results (below) of escs patient, treated with dorzolamide during 6 consecutive visits m mean, sd standard deviation, med median the improvement of bcdva in the right eye corresponded with foveal thickness reduction from 482 m initially to 359 m at the 1st and to 226 m at the 2nd follow - up visit (fig. 2). at this point the main reason behind the decreased visual acuity in escs patients is cystoid changes in macular region. macular cysts early diagnosis may be of great value for escs patients, when at least partial rpe function is preserved. it is possible that by using topical dorzolamide, which provides an increased rpe pumping function and in consequence intraretinal fluid absorption, restoration of normal foveal morphology can be obtained and even in some cases, visual acuity may improve [911 ]. an extracellular membrane - bound carbonic anhydrase (ca xiv) was shown to be present in brain and retina (astrocytes, muller cells and rpe). carbonic anhydrase xiv is the target of carbonic anhydrase inhibitors that enhance subretinal fluid absorption in macular edema. carbonic anhydrase inhibitors usefulness was proven in the treatment of macular cystoid changes associated with diseases such as retinitis pigmentosa [1518 ], choroideremia and x - linked retinoschisis. to our best knowledge, this is the third case report study describing topical dorzolamide efficacy in macular cysts management in the escs patient, with the longest follow - up period equal to 6 months. dorzolamide treatment was applied, because we assumed that cystoid macular spaces were filled with endogenous interstitial retinal fluid undetectable to fluorescein angiography testing. obtained results are in agreement with previously published data [911 ], which presented the visual acuity improvement and foveal structure normalization after dorzolamide treatment. for the 1st time, mferg was used to evaluate foveal function during the topical dorzolamide treatment of macular cysts in escs. mferg is a well - known technique reflecting functional mapping of the retina including foveal region, especially bipolars and cone photoreceptors. to date, to estimate the macular cysts treatment efficacy of escs patients visual acuity, retinal thickness measurements (oct), contrast sensitivity and microperimetry were used [911 ]. the results of visual acuity measurements are subjective and allow only to estimate retinal function of about 1 central angle degree. hence, visual acuity examination is not precise enough to estimate retinal function from retinal regions covering macular cysts, which usually surpasses 1 angle degree. results of mfergs provided in this case useful information that visual acuity and foveal thickness improvement corresponded with partial increase in foveal function. assessment of foveal region function with mferg was previously described in patients with epiretinal membrane, retinitis pigmentosa, diabetic macular edema, uveitic macular edema and others [2224 ]. the results of our study suggest that the use of topical form of carbonic anhydrase inhibitor should be consider for a treatment of macular cysts associated with escs. | purposethe purpose of the study was to evaluate the efficacy of a topical form of a carbonic anhydrase inhibitor (dorzolamide) on the foveal function and thickness in the eye of a patient with enhanced s - cone syndrome (escs) associated with macular cysts.methodstwenty-eight-year-old polish man with escs and macular cysts appearance in the right eye was treated 3 times daily with 2.0 % dorzolamide drops for the period time equal to 6 months. monthly controls included : best corrected distance visual acuity (bcdva - logmar), foveal thickness (optical coherence tomography, oct) and foveal function (multi - focal electroretinography, mferg).results before treatment, bcdva in the right eye was equal to 0.26 logmar, improved to 0.1 logmar during the first 3 months and remained stable for the next 3 months. after 6 months, foveal thickness decreased (from 482 to 224 m) and foveal function improved (the amplitude of p1-wave density increased from 34.8 to 107.3 nv / deg2) and was between the ranges of normal values. implicit time of p1-wave remained prolonged.conclusions the results of our short - term study suggest potential efficacy of topical dorzolamide treatment in escs patients with macular cysts. |
the gradual wear of the occlusal surfaces of teeth is a normal process during the lifetime of a patient. however, excessive occlusal wear can result in pulpal pathology, occlusal disharmony, impaired function, and esthetic disfigurement.1 tooth wear can be classified as attrition, abrasion, and erosion depending on its cause. a differential diagnosis is not always possible because, in many situations, there exists a combination of these processes.2 therefore, it is important to identify the factors that contribute to excessive wear and to evaluate alteration of the vdo caused by the worn dentition.3 in many cases, the vertical dimension of occlusion (vdo) is maintained by tooth eruption and alveolar bone growth. as teeth are worn, the alveolar bone undergoes an adaptive process and compensates for the loss of tooth structure to maintain the vdo. therefore, vdo should be conservative and should not be changed without careful approach.4,5 especially, increasing the vdo in bruxers puts a severe overload on the teeth and often results in the destruction of the restorations or teeth themselves.4 however, the rehabilitation of the severely worn dentition is challenging when the space for restoration is not sufficient. in 1975, dahl.6 reported the use of a removable cobalt - chromium anterior occlusal device to an 18-year - old patient with advanced localized attrition to generate interocclusal space for subsequent restoration. the tooth movement involving a combination of orthodontic anterior teeth intrusion and eruption of the posterior teeth occurred. and long - term observations of this treatment were reported that the vertical relations were practically stable.7,8 nowadays this technique is replaced by using the adhesive resin9,10 or an overlay splint5, 11,12 instead of a cobalt - chromium device. management of worn dentition using fixed or removable prostheses is complex and among the most difficult cases to restore. assessment of the vertical dimension is important for the management, and careful comprehensive treatment plan is required for each individual case. articulated study casts and diagnostic wax - up can provide important information which is helpful for the evaluation of treatment options. tolerance of changes to vertical dimension of occlusion is usually confirmed with the clinical evaluation of the patient having a diagnostic splint or provisional prosthesis.13 this clinical report describes the treatment of a patient who was clinically monitored to evaluate the adaptation to the removable occlusal overlay splint during a 1 month trial period and the provisional restorations for 3 months.14,15 a 77-year - old woman was referred for the treatment of her severely worn dentition. her chief complaint was that she could not eat anything because her teeth were worn too much. intraoral examination revealed a generalized loss of dental substance that was greater in the maxillary left incisors and the mandibular right incisors. maxillary left canine and mandibular right canine were worn to the gingival level, and had got root canal treatments (fig. the anterior teeth had sharp enamel edges, dentinal craters, and attritional wear due to the loss of posterior support. the mandibular posterior teeth were missing, but she said that she did not use her old removable partial denture (rpd) as it was not comfortable. the mandibular rpd lost its retention and support because the regions that functioned as the rest and retentive undercut of abutment teeth had been fractured. the facial type of patient was square and her lip seemed to be under strong tension. the patient did not have temporomandibular disorder history and soreness of the mastication muscles, but the discrepancy between centric occlusion (co) and maximum intercuspal position (mip) was found when she was guided to cr with bimanual technique. the right mandibular condyle was flatter than the left one, but any specific disorder was not found (fig. 2). to determine whether vdo had been altered, the following aspects were investigated:1,5,11 loss of posterior support : mandibular posterior teeth were missing, and the patient did not use the mandibular partial denture. posterior collapse resulted in excessive wear and fracture of anterior teeth.history of wear : physiologic wear can be compensated by tooth eruption in general, but the accelerated wear may exceed the rate of eruption in addition, maxillary posterior base metal prostheses might accelerate the wear of mandibular acrylic resin teeth and unbalance of wear rate.phonetic evaluation : if the distance between the incisal edge of the mandibular incisors and lingual surface of the maxillary incisors is about 1 mm, it makes normal /s/ sound. the patient 's increased space altered /s/ sound to //.interocclusal rest space : the patient 's interocclusal rest space that was measured between nose tip and chin tip was 5 - 6 mm that was greater than the normal value, 2 - 4 mm.facial appearance : wrinkles and drooping commissures around mouth were observed. loss of posterior support : mandibular posterior teeth were missing, and the patient did not use the mandibular partial denture. history of wear : physiologic wear can be compensated by tooth eruption in general, but the accelerated wear may exceed the rate of eruption in addition, maxillary posterior base metal prostheses might accelerate the wear of mandibular acrylic resin teeth and unbalance of wear rate. phonetic evaluation : if the distance between the incisal edge of the mandibular incisors and lingual surface of the maxillary incisors is about 1 mm, it makes normal /s/ sound. the patient 's increased space altered /s/ sound to //. interocclusal rest space : the patient 's interocclusal rest space that was measured between nose tip and chin tip was 5 - 6 mm that was greater than the normal value, 2 - 4 mm. facial appearance : wrinkles and drooping commissures around mouth were observed. the possible causes of patient 's worn dentition that might include posterior interferences, parafunction, eating habit, and dental ignorance were explained to the patient. and the options of treatment plan were restoring mandibular edentulous posterior region with implants or removable partial denture, full mouth rehabilitation with metal ceramic restoration with or without crown lengthening procedure. the patient was scared of implant surgery, so the option of implant installation was excluded. also the patient did not want to get multiple crown lengthening procedures and endodontic treatments to restore the worn teeth in the insufficient space. as there was clinical evaluation of reduced vdo, full mouth rehabilitation with increasing vdo was planned. only severely worn teeth which were maxillary left canine and mandibular right canine were undergone crown lengthening procedures to obtain a sufficient clinical crown length and ferrule effect.16 the patient 's casts were mounted on a semi - adjustable articulator (hanau modular articulator ; whip mix corp., louisville, usa) using a face - bow record and an interocclusal record that was made with the aid of a lucia jig and polyvinylsiloxane occlusal registration material (exabite ii ; gc corp., the new vdo was set by 5 mm increase in the incisal guidance pin of the articulator. because the patient 's interocclusal rest space was 2 - 3 mm larger on the premolar area than normal distance, the actual increase were determined 3 mm in the anterior teeth and 1 - 2 mm in the posterior teeth. the splint was designed to offer bilateral contacts of all posterior teeth in centric relation and guides of the anterior teeth in excursive movement (fig. the adaptation of patient to the increased vdo was evaluated during 1-month trial period. no muscle tenderness and temporomandibular discomfort the method of increasing vdo with the splint was used to determine desirable vdo of the fixed interim prostheses. after taking cr record using lucia jig and wax - rim, diagnostic wax - up 4). autopolymerizing acrylic resin (alike ; gc america, alsip, usa) provisional crowns were fabricated using a vacuum formed matrix (drufolen h ; dreve dentamid gmbh, unna, germany) that was produced from the diagnostic wax - up, and mandibular provisional rpd was made to fit provisional crowns (fig. the provisional fixed restorations were cemented with temporary cement (freegenol temporary pack ; gc corp., tokyo, japan), and the patient 's adaptation was monitored. for three months, interim restorations were adjusted, and used as a guide for the definitive oral rehabilitation. during this period, the patient 's condition and functions, such as muscle tenderness, discomfort of tmj, mastication, range of the mandibular movements, swallowing, and speech, were evaluated. improvement in mastication, speech, and facial esthetics confirmed the patient 's tolerance to the new mandibular position with the restored vdo. the anterior guidance and posterior disclusion on excursive movement were established. adjusted occlusion was transferred to customized anterior guide table, which was made with acrylic resin (pattern resin ; gc corp, tokyo, japan)17(fig. 6). final preparation was performed, and definitive impressions were made with polyvinylsiloxane impression material (extrude ; kerr corp., romulus, germany). bite registration was taken using provisional crown and occlusal registration material (stonebite ; dreve dentamid gmbh, unna, germany) by half and half (fig. porcelain fused to metal restorations were made using customized anterior guide table and cemented with resin modified glass ionomer cement (fujicem ; gc america, alsip, usa). because the patient 's anterior guidance table was used in the production of definitie restoration, the amount of occlusal adjustment on the lingual surface of maxillary anterior teeth was minimal. the impression on posterior alveolar ridge was taken once more with the individual tray which is attached to the rpd framework, and the altered cast was made. after the adaptation of rpd framework and the trial of wax denture were done, the definitive mandibular rpd was fabricated and delivered with minor occlusal adjustment (fig. the anterior teeth protected the posterior teeth from excursive force and wear, and posterior teeth supported the bite force. in 1984, turner1 classified the treatment of a severely worn dentition by the amount of the loss of vdo and available space to restore. his classification and conventional treatment, which includes raising vdo with multiple crown - lengthening procedures, have been widely used up to present. however, the etiology of tooth wear is multifactorial, and clinical controlled trials of restorative and prosthodontic approaches are limited in quantity and quality. in addition, lack of evidence regarding the long - term outcomes of treatment methods and materials cause difficulty in clinical decision - making.18 because of these unclear guidelines, adhesive strategy, that is more conservative and reversible, is increasing.9,10,18 nonetheless, the composite resin restoration could not be used for the patient in this case. the remaining tooth structures were too small to have sufficient retention of composite resin, and the surveyed crowns to support rpd were necessary. therefore, the conventional treatment modality that includes a trial overlay splint, provisional restoration, careful monitoring, and definitive prosthesis, was chosen. in previous literature the trial period of overlay prostheses which are reversible and conservative is between 3 weeks and 5 months, and that of intensive fixed provisional prostheses is 2 - 6 months.1,5,9,11,19 in this case, the patient was carefully monitored for 1 month to evaluate the adaptation to the removable occlusal overlay splints.15 also the patient 's adaptation to the provisional restoration was monitored for 3 months.14 the trial period is relatively shorter than the other case report, but discomfort, wear, and muscle fatigue were not observed during that period. the increase of vdo was determined not by standardized esthetic golden proportion of anterior teeth but by patient 's physiologic factor like interocclusal rest space and speech. if the increase of vdo was decided arbitrarily without close evaluation, multiple complications would happen and longer treatment period might be needed. depending on the patient 's situation and adaptation ability, the interim period can be modified, and the careful evaluation and monitoring may shorten the overall treatment duration. the rehabilitation using restoration of anterior crowns and rpd providing posterior support is affordable and common for many patients who require the treatment of teeth wear because of reasons of economics and tradition.18 however, the restored anterior teeth can be easily exposed to excessive occlusal loads if the patient does not wear the rpd or resorption of residual ridge proceeds. because the compliance of patients in wearing free - end saddle dentures has been shown to be poor,20 the education on wearing rpd is necessary. in this clinical report, raising vertical dimension of occlusion using removable occlusal overlay splint and following fixed provisional based on accurate diagnosis showed successful full mouth rehabilitation for severely worn down dentition. | the severe wear of anterior teeth facilitates the loss of anterior guidance, which protects the posterior teeth from wear during excursive movement. the collapse of posterior teeth also results in the loss of normal occlusal plane and the reduction of the vertical dimension. this case report describes 77-year - old female, who had the loss of anterior guidance, the severe wear of dentition, and the reduction of the vertical dimension. occlusal overlay splint was used after the decision of increasing vertical dimension by anatomical landmark, facial and physiologic measurement. once the compatibility of the new vertical dimension had been confirmed, interim fixed restoration and the permanent reconstruction was initiated. this case reports that a satisfactory clinical result was achieved by restoring the vertical dimension with an improvement in esthetics and function. |
bone surrounding teeth, at times, may undergo alterations with respect to morphology either due to anatomical variations or during periodontal diseases resulting in osseous defects such as fenestrations and dehiscences. mucosal fenestration is a rare clinical pathologic entity where in the affected root portion of the tooth is exposed in the oral cavity owing to combined perforation of the overlying bone and mucosa. it is less prevalent that bony fenestration and is commonly seen to affect maxillary and mandibular incisors. mucosal fenestration is usually devoid of symptoms and not easily recognized by patients unless associated with problems like calculus deposits, hypersensitivity, pulpal involvement and esthetics. palato gingival or radicular groove is another developmental aberration known to maximally affect the palatal surfaces of maxillary lateral incisors but is often missed during a routine clinical examination. it is a malformation which presents as a groove / channel that begins in the central fossa, crosses the cingulum of the tooth and extends apically to or beyond the cemento enamel junction at varying distances and directions along the root surface. its unfavorable shape and location serves as a perfect bacterial harbor predisposing to pocket formation, concomitant bone loss and pulpal involvement thereby encouraging development of endodontic - periodontal lesions. depending on severity, the groove may render a direct communication between the periodontium and pulp cavity promoting effortless bacterial invasion. endodontic pathology progressing to involve periodontal tissues or vice versa is not very frequent, but when present can lead to complex situations, necessitating stringent interdisciplinary measures for correction. the following report describes the management of a rarely encountered maxillary lateral incisor that presented with failed previous endodontic treatment, and was affected by mucosal fenestration defect at the root apex coexistent with deep periodontal pocket coursing along a palato gingival groove on its palatal aspect. the tooth was severely compromised owing to long standing periodontal inflammation resulting in widespread periodontal destruction. this case report highlights the importance of establishing an accurate diagnosis and proper institution of multidisciplinary comprehensive treatment protocol for salvaging teeth with complex endodontic - periodontal problems. a 34-year - old female patient reported to the department of periodontics with exposed root in relation to maxillary right lateral incisor. the patient 's general medical condition (american society of anesthesiologists 1) was good. apical portion of the root was seen perforating the buccal cortical plate and overlying mucosa with the fenestration defect measuring approximately 4 mm 3 mm [figure 1a ]. the patient was unable to recollect the period of its occurrence but confirmed the presence of exposed root since 1-year. the patient had also not sought any dental treatment for the past 1-year despite being aware of the existing defect. plaque and calculus were seen deposited on the exposed root but no pus discharge was evident in the area [figure 1a ]. palatal examination revealed presence of a palatal groove possibly extending onto the root and an isolated periodontal pocket of > 5 mm [figure 1b and c ]. the tooth presented with grade i mobility. (a) preoperative defect in maxillary right lateral incisor showing exposed root tip covered with plaque and calculus. (b) palato gingival radicular groove seen extending beyond the cemento enamel junction in relation to maxillary right lateral incisor. (c) periodontal pocket depth of greater than 5 mm present adjacent to the groove. (d) root tip seen after removal of plaque and calculus radiographic examination of the concerned tooth revealed faulty root canal treatment in right maxillary lateral incisor and a lateral radiolucency on the mid mesial aspect of its root [figure 2a ]. a thin parapulpal radiolucent line was observed on the radiograph which confirmed the presence of a palato gingival groove. it was decided to repeat endodontic treatment in the affected tooth, treat the palato gingival groove and perform periodontal surgical correction of the fenestration defect by raising a full thickness flap. (a) preoperative radiograph showing incomplete obturation, para pulpal line adjacent to the root canal and radiolucency on mid mesial aspect of the root. (b) postobturation radiograph showing mineral trioxide aggregate in the entire canal the exposed root tip of right maxillary lateral incisor was cleared of the deposited plaque and calculus [figure 1d ]. previous gutta percha filling was removed from the canal with the help of a hand file and working length established. the canal was thoroughly cleaned and shaped using step back technique with 1580 stainless steel k files (dentsply maillefer, ballaigues, switzerland) under copious irrigation with 2.5% sodium hypochlorite. the canal was medicated with calcium hydroxide paste and the access cavity sealed for 1-week with a temporary restoration (cavit, premier dental co., philadelphia, pa, usa). on subsequent visit, the paste was flushed out of the canal using thorough irrigation with sterile water and dried. need for odontoplasty with respect to palato gingival groove was anticipated hence mineral trioxide aggregate (mta) (proroot mta ; dentsply tulsa dental, tulsa, ok) was used to fill the entire canal. wet mta was carried into the canal using amalgam carrier, advanced apically to the working length with a k file, 1 size smaller than the maf, and condensed properly using finger pluggers. incremental placement and compaction of mta was continued from apical to coronal area using subsequently larger hand files and pluggers until the level of cemento enamel junction. a moist cotton pellet was placed on mta and the cavity sealed with intermediate restorative material. retrograde filling was also done at the apex by direct access using mta. on third visit after 1-week paul, mn, usa) was placed over exposed mta and the access cavity permanently restored with bonded resin restoration [figure 2b ]. on fourth visit, palatally, a full thickness flap was raised using sulcular incision and the pocket curetted with gracey curettes number 1, 2 and 5, 6 (hu - friedy manufacturing co., chicago, usa). the palatogingival groove was saucerized and blended with the root surface using a fine diamond. the coronal extension of the groove was chemically conditioned using 10% polyacrylic acid and restored with type i glass ionomer cement (gic) (fuji 1, gc corporation, tokyo, japan). on raising the flap, it was observed that the entire labial surface of the root was denuded of bone. the mesial, distal and intercrestal bone were intact, but a bone defect was seen circumscribing the root apex [figure 3a ]. postpreparation of the recipient site with 17% ethylenediaminetetraacetic acid (pulpdent co., watertown, ma, usa) ; porous biphasic bone graft material containing hydroxyapatite and tricalcium phosphate (ossifi, equinox) to aid in bone regeneration was packed into the defect and over the denuded root surface [figure 3b ]. a bio resorbable collagen barrier membrane (healiguide, advanced biotech products (p) ltd., india) was placed over the bone graft and the flap repositioned and sutured to the adjacent tissue using interrupted sutures with 3 - 0 black silk [figure 3c and d ]. (a) osseous defect curetted around the root apex of 12 after resection of root. (d) flap sutured after approximation periodontal pack was placed over surgical site for protection and the patient prescribed a course of antibiotics and antiinflammatory medications. he was advised to rinse with 0.2% chlorhexidine twice in a day for 4 weeks and discharged after giving proper postsurgical instructions. complete closure of mucosal defect could be appreciated at follow - up appointment of 1-month [figure 4a and b ]. at 6 months, radiographic examination revealed a sound periapex and resolution of lateral radiolucency [figure 4c ]. all probing depths around the tooth continued to exist within normal limits and the tooth was no longer mobile. postoperative clinical photographs showing satisfactory healing (a) facial view (b) palatal view. (c) 6 months postoperative radiograph showing good peri apex and resolution of lateral radiolucency the exposed root tip of right maxillary lateral incisor was cleared of the deposited plaque and calculus [figure 1d ]. previous gutta percha filling was removed from the canal with the help of a hand file and working length established. the canal was thoroughly cleaned and shaped using step back technique with 1580 stainless steel k files (dentsply maillefer, ballaigues, switzerland) under copious irrigation with 2.5% sodium hypochlorite. the canal was medicated with calcium hydroxide paste and the access cavity sealed for 1-week with a temporary restoration (cavit, premier dental co., philadelphia, pa, usa). on subsequent visit, the paste was flushed out of the canal using thorough irrigation with sterile water and dried. need for odontoplasty with respect to palato gingival groove was anticipated hence mineral trioxide aggregate (mta) (proroot mta ; dentsply tulsa dental, tulsa, ok) was used to fill the entire canal. wet mta was carried into the canal using amalgam carrier, advanced apically to the working length with a k file, 1 size smaller than the maf, and condensed properly using finger pluggers. incremental placement and compaction of mta was continued from apical to coronal area using subsequently larger hand files and pluggers until the level of cemento enamel junction. a moist cotton pellet was placed on mta and the cavity sealed with intermediate restorative material. retrograde filling was also done at the apex by direct access using mta. on third visit after 1-week, the cotton pellet was removed. paul, mn, usa) was placed over exposed mta and the access cavity permanently restored with bonded resin restoration [figure 2b ]. on fourth visit, palatally, a full thickness flap was raised using sulcular incision and the pocket curetted with gracey curettes number 1, 2 and 5, 6 (hu - friedy manufacturing co., chicago, usa). the palatogingival groove was saucerized and blended with the root surface using a fine diamond. the coronal extension of the groove was chemically conditioned using 10% polyacrylic acid and restored with type i glass ionomer cement (gic) (fuji 1, gc corporation, tokyo, japan). on raising the flap, it was observed that the entire labial surface of the root was denuded of bone. the mesial, distal and intercrestal bone were intact, but a bone defect was seen circumscribing the root apex [figure 3a ]. the root tip was minimally resected and smoothened with a high speed diamond. the area around root tip postpreparation of the recipient site with 17% ethylenediaminetetraacetic acid (pulpdent co., watertown, ma, usa) ; porous biphasic bone graft material containing hydroxyapatite and tricalcium phosphate (ossifi, equinox) to aid in bone regeneration was packed into the defect and over the denuded root surface [figure 3b ]. a bio resorbable collagen barrier membrane (healiguide, advanced biotech products (p) ltd., india) was placed over the bone graft and the flap repositioned and sutured to the adjacent tissue using interrupted sutures with 3 - 0 black silk [figure 3c and d ]. (a) osseous defect curetted around the root apex of 12 after resection of root. (b) bone graft placed in and around the defect. (d) flap sutured after approximation periodontal pack was placed over surgical site for protection and the patient prescribed a course of antibiotics and antiinflammatory medications. he was advised to rinse with 0.2% chlorhexidine twice in a day for 4 weeks and discharged after giving proper postsurgical instructions. complete closure of mucosal defect could be appreciated at follow - up appointment of 1-month [figure 4a and b ]. at 6 months, radiographic examination revealed a sound periapex and resolution of lateral radiolucency [figure 4c ]. all probing depths around the tooth continued to exist within normal limits and the tooth was no longer mobile. postoperative clinical photographs showing satisfactory healing (a) facial view (b) palatal view. (c) 6 months postoperative radiograph showing good peri apex and resolution of lateral radiolucency fenestrations and dehiscences are not very frequently observed defects of the alveolar bone, but when present can create great difficulties for both the clinician and the patient. alveolar fenestrations usually exist undetected in the oral cavity until accompanied by the loss of overlying mucosa. combined loss of bone and soft tissue results in denuded and clinically exposed root surfaces. their existence can jeopardize several common dental procedures like endodontic treatment, periodontal or peri radicular surgeries as well as implant placement. recognition of these pathologies prior to any procedure, is therefore, important to prevent any intra or postoperative complications and improve overall prognosis. several etiologies speculated in the development of alveolar fenestrations include (1) anatomic factors like extreme labial prominences of root tips, tooth malpositions, thin or nonexistent buccal cortical plates and (2) pathologic factors like trauma, heavy occlusal forces, orthodontic movement or chronic peri apical inflammation. once alveolar deficiency has occurred, loss of overlying soft tissue may be seen in areas of thin keratinized gingiva on exposure to mechanical injury or extension of underlying pathology. contributing factors in the development of fenestration / dehiscence in this case seemed to be multifactorial most likely being periodontal inflammation associated with palato gingival groove, chronic peri radicular pathology, previous endodontic treatment, thin cortical plate and labial prominence of the root. exposure of the root was followed by plaque and calculus deposition, which further prevented mucosal approximation. to generate an environment that would promote periapical healing and repair of hard and soft tissue defects with respect to fenestration, it was critical to first treat the tooth endodontically, followed by root resection and peri apical curettage. endodontic treatment aimed at eliminating all bacteria and toxins from the root canal system prior to surgical correction. the entire canal was then obturated using mta to thoroughly seal all canal walls and any channels existing as a communication between the canal and radicular groove. this unique sealing property, combined with high alkaline ph of 12.5 after curing provides a suitable mechanism for bacterial neutralization and inhibition within the canal system. also, diffusion of calcium ions from mta through the dentinal tubules inhibits bacterial colonization and survival at the root surface, which when coupled with osteogenic activity is known to promote new cementum deposition and regenerate periodontal ligament. root resection was done to aid containment of root within its alveolar housing and at the same time improve accessibility and visibility around the apex for curettage. bone grafting with guided tissue regeneration (gtr) on the facial aspect of the root was performed with the aim of regenerating the lost attachment apparatus and provide root coverage. use of resorbable collagen membrane in gtr membrane therapy has proved to be highly successful when treating endodontic - periodontal lesions in communication and has shown good results relating to bone growth and connective tissue attachment. porous hydroxyapatite employed as bone graft in this case, has earlier in the literature, known to serve as a suitable bone graft material owing to its excellent bone conductive properties. when combined with high porosity, it permits migration of osteogenic cells from existing bone surfaces into adjacent bone thereby providing useful potential in treatment of periodontal defects. palatogingival groove, one of the several morphological variations, serves as an easy pathway for bacterial penetration leading to periodontal inflammation, epithelial attachment breakdown and progression into periodontal pocket. palatogingival groove is often poorly recognized owing to its entrance concealed under the gingival tissue or plaque. a thorough examination to uncover such defects is highly recommended before devising any treatment plan. on careful exploration, a palate gingival groove may be felt as a notch which may or may not be associated with an isolated pocket. further, its presence can be ascertained on radiographs, commonly seen as dark lines extending along the length of the root, parallel to or superimposed over the root canal termed as parapulpal lines. prognosis and treatment of palato gingival grooves is greatly dictated by the depth, direction and extent of grooves. closed debridement coupled with odontoplasty is usually successful for shallow defects that do not extend down for considerable distances along the length of the root whereas open flap debridement is recommended for more deeper and tortuous grooves. simple saucerization has shown to be helpful in eliminating shallow grooves with great success. here, in this case, the groove was classed as moderate hence simple root planning and curettage of the pocket with radiculoplasty in the cervical part of the groove followed by restoration with gic was able to provide an effective seal at the radicular entrance. true combined endodontic - periodontal pathology mostly necessitates institution of restorative, endodontic and periodontal therapies in combination to achieve the best treatment outcomes. collective treatment rendered in this case which included endodontic therapy, apical root coverage and coronal closure of the groove prevented bacterial colonization and penetration in both retrograde and orthograde directions thereby providing an environment favorable for healing and repair. attachment of periodontal ligament with repair of mucosal fenestration and periapical healing could be appreciated both clinically and radiographically. combined endodontic - periodontal problems offer great challenges to a clinician with respect to diagnosis and treatment. these complex lesions usually have a hopeless prognosis, especially when periodontal pathology is chronic in nature and associated with extensive loss of attachment. in the present case report, thorough knowledge and identification of disease etiology followed by institution of comprehensive interdisciplinary treatment protocol were able to render satisfactory results and restore healthy state in a tooth affected with coexistent palatogingival groove and mucosal fenestration defects. | mucosal fenestrations, wherein the tooth root apices are clinically discernible in the oral cavity subsequent to loss of overlying alveolar bone and mucosa, are rare pathologic entities. palato gingival grooves- anatomic aberrations are also infrequent occurrences that notoriously predispose to periodontal pathologies of varying extent. both conditions independently are known to popularly affect maxillary lateral incisors. coexistent fenestration defect and palato gingival groove in the same tooth is extremely rare and undoubtedly is a perfect combination to precipitate severe endodontic - periodontal consequences. in this report, a 34-year - old patient presented to the dental department with complaint of esthetics in relation to exposed root of right maxillary lateral incisor. on closer inspection, a palato gingival groove in addition to fenestration defect was evident on the root surface along with a periodontal pocket of > 5 mm. an interdisciplinary treatment was instituted which included endodontic treatment followed by root end resection, osseous bone graft placement and guided tissue regeneration procedures for repair of mucosal fenestration defect. debridement of the palatal pocket, with saucerization of the groove and restoration with glass ionomer cement were simultaneously employed to correct the palatal defect. |
the superior shoulder suspensory complex (sssc) is an extremely important structure in terms of shoulder biomechanics, and is composed of a ring of bony and soft tissues at the superior aspect of the shoulder. it comprises the glenoid process, the coracoid process, the coracoclavicular ligament, the distal end of the clavicle, the acromioclavicular joint, the coracoacromial ligament, and the acromial process. single traumatic disruptions of the sssc, such as type i distal clavicle fractures, are common, but double disruptions are known to be rare.[13 ] double disruption can occur in combination with bone and soft tissue injuries, and leads to construct instability. therefore, they usually require operative treatment. on the other hand, it is difficult to conceive of triple sssc disruption during a single traumatic event. in fact, only two non - english case reports were found in a comprehensive literature search. here, we present the case of a patient with combined fractures of the coracoid and acromion with acromioclavicular separation after a severe in - car traffic accident. a 27-year - old man with no previous shoulder problems experienced direct trauma during an in - car traffic accident. although no definite neurovascular abnormalities of the right upper extremity were found, he had lower extremity paraplegia below the t8 level. in addition, he had dyspnea because of bilateral hemothorax and pleural effusion caused by direct traumatic lung injury. initial radiographs of the right shoulder showed comminuted fractures in the right scapular superior border, acromion, and coracoid process, and a humerus shaft fracture with acromioclavicular joint separation [figure 1 ]. fracture of the acromion and acromioclavicular joint separation had substantial displacement and fracture of the coracoid is regarded as type i according to ogawa 's classification. these fractures were also confirmed by computed tomography (ct) scan with 3d reconstruction [figure 2 ]. radiographs demonstrating comminuted fractures in the right scapular superior border, acromion, and coracoid process, and a humerus shaft fracture with acromioclavicular ligament separation preoperative 3d ct images : anterior view (a) and medial view (b) on the 7 day post - injury, surgery for the shoulder was undertaken on the triple disruption injury of the sssc and fracture of the humeral shaft. under general anesthesia, with the patient in beach chair position, a saber cut approach was adopted from the posterior border of the acromion to the coracoid. prior to the fixation of sssc injuries, intramedullary nailing (synthesis, paoli, pa, usa) of the humeral fracture was performed. the entry point of the nail was established through the detachment of the anterior deltoid and fracture site of the acromion. after humeral shaft fixation, the coracoid fracture was reduced and fixed with one cancellous screw [figure 3 ] (6.5 mm 50 mm, 16 mm half thread ; synthes australia, north ryde, australia). the acromial fracture was then reduced and fixed with tension band wiring [figure 4 ] (tension band pin system, acumed, hillsboro, or, usa), and finally, the acromioclavicular joint was fixed with three transarticular steinmann pins [figure 5 ]. after meticulous reattachment of the deltoid, the wound was closed in layers [figure 6 ]. radiograph showing fixation of the coracoid process with a cancellous screw (black arrow), the clavicle (arrowhead), and the acromion (white arrow) the acromial fracture was fixed using the tension band wiring technique the acromioclavicular joint was fixed using three transarticular s - pins immediate postoperative radiograph postoperatively, the patient was immobilized with an abduction brace for 6 weeks. during this period, shoulder joint motion, including pendulum exercise, was not allowed. however, shrugging of the affected shoulder, and active hand and wrist motion were encouraged immediately after surgery. at 6 weeks post - injury,, transarticular s - pins were removed under fluoroscopic guidance and local anesthesia at an outpatient clinic. he was followed for 5 years, and had a good functional outcome at the last follow - up [figures 7 and 8 ]. fractures of the scapula are relatively rare injuries and account for 35% of all fractures involving the shoulder girdle and 1% of all fractures. direct trauma to the lateral or posterosuperior aspect of the forequarter is the most common mechanism of injury. fractures of the coracoid process account for 25% of all scapular fractures, and the mechanisms involved determine fracture sites. according to ogawa. type i fractures, which are proximal to the coracoclavicular ligament, are usually associated with other injuries, such as acromioclavicular separation (the most frequent) scapular fractures, deltoid lacerations, clavicle fractures, and acromion fractures. type i fractures are more common but unstable, and thus require open reduction and internal fixation. however, avulsion fractures and type ii fractures which are distal to the coracoclavicular ligament could be caused by contractions of the coracobrachialis, the short head of the biceps, and the pectoralis minor. it would appear that type i coracoid fracture commonly occurs with concomitant acromioclavicular separation by a similar mechanism as acromioclavicular coracoclavicular separation. in this case, the coracoclavicular ligament is usually saved. previous studies mentioned that double disruption of the sssc is a rare injury around the shoulder. however, if one considers the structures involved in common injuries at the sssc, for example, the acromioclavicular and coracoclavicular ligaments for acromioclavicular coracoclavicular separation, and the clavicle and coracoclavicular ligament for type ii distal clavicle fractures, double disruption of the sssc should not be regarded as a rare injury. in ring structure concept, like the pelvis, it is more reasonable to think if the ring is broken in one area and the fragments displaced, then there must be a fracture or dislocation in another portion of the ring. however, triple disruption of the sssc, which is breakage of the ring in three different locations, is a very rare injury indeed. a comprehensive literature search showed this type of injury has only been reported twice in the non - english literature, and both of these cases had a displaced fracture of the coracoid process associated with fracture of the acromion and a minimally displaced fracture of the distal clavicle. on the other hand, our patient had a displaced fracture of the coracoid process associated with fracture of the acromion, and the separation of the acromioclavicular joint. in the two reported cases, the etiologies were a traffic accident in one like our case and a fall from height in the other. we consider that these triple disruptions could not have been caused by a single impact to the shoulder and that rather they were caused by multiple impacts during one severe traumatic event. our case had a fracture of humeral shaft on the ipsilateral humerus and vertebral fractures. although associated injuries were not mentioned in one report, the other did mention multiple associated rib fractures. in structures with the ring concept, complete disruptions at more than two other locations cause instability, triple disruption of the structure was a definite indication for surgery. in conclusion, triple disruption of the sssc is an extremely rare injury caused by high - energy trauma and it is often associated with other injuries. | the superior shoulder suspensory complex (sssc) is an extremely important structure composed of a ring of bone and soft tissues at the superior aspect of the shoulder. double disruption leads to instability of the construct and usually requires operative treatment. triple disruption of the sssc is extremely rare and is encountered in high - energy trauma cases often in association with other injuries. the authors experienced a case of triple disruption involving the acromion, coracoid process, and acromioclavicular separation. this type of sssc disruption is unlikely to have been caused by a single impact and is rather caused by multiple impacts during one traumatic event. |
citius, altius, forties which is latin for faster, higher, and stronger. with this basis, we too have adopted the same in our quest for a decalcifying agent. lesions affecting hard tissues need intricate, technique - sensitive methodology for interpretation and diagnosis. the pulpal soft tissue can only be assessed in decalcified sections which otherwise in ground section is not possible as it is lost. the aim of decalcification is to remove calcium salts from mineralized tissue using chemical solutions like acids and chelating agents, while preserving the organic portions. so, an ideal decalcifying agent should be fast;be good and;do good. though some agents remove the calcium ions completely and rapidly so, we present here the comparative evaluation of different decalcifying agents with respect to rate of decalcification, the effect of decalcifying agents on the dental tissue, and its influence on the staining characteristics. the quest for at most near ideal decalcifying agent begins. the aims and objectives of the study were : to evaluate the fastest decalcifying agent;to evaluate the decalcifying agents based on its effect on the organic content and integrity of the soft tissue;to evaluate the staining characteristics of the teeth decalcified in the different decalcifying agents;to compare and contrast between the decalcifying agents. to evaluate the fastest decalcifying agent ; to evaluate the decalcifying agents based on its effect on the organic content and integrity of the soft tissue ; to evaluate the staining characteristics of the teeth decalcified in the different decalcifying agents ; to compare and contrast between the decalcifying agents. freshly extracted, noncarious, not attrited, 24 natural teeth were obtained from patients aged 4045 years.[35 ] the teeth, fixed in 10% formalin, included incisors, canines, premolars, and molars, and were used to analyze the six different decalcifying agents namely 5% nitric acid, perenyi 's fluid, formalin - nitric acid solution, neutral ethylene diamine tetra acetic acid (edta) decalcifying solution, 10% formic acid, and 5% trichloroacetic acid. each decalcifying agent was used to decalcify an incisor, a canine, a premolar, and a molar each. decalcifying agents were subjected to repeated agitation and replaced by freshly prepared agents as indicated by the chemical method of end point of decalcification for 5% nitric acid, perenyi 's fluid, formalin nitric acid solution, and 5% trichloroacetic acid. for both neutral edta and 10% formic acid, strong liquor ammonia is added drop by drop to 5 cm of decalcifying agent until it turns alkaline to litmus.if solution went cloudy, it indicated presence of calcium and the solution was replaced with fresh solutionif solution does not turn cloudy, 5 cm of saturated ammonium oxalate is added to the solution.if solution remains clear for 30 min, it was concluded that end point of decalcification was reached and the procedure is completed. strong liquor ammonia is added drop by drop to 5 cm of decalcifying agent until it turns alkaline to litmus. if solution went cloudy, it indicated presence of calcium and the solution was replaced with fresh solution if solution does not turn cloudy, 5 cm of saturated ammonium oxalate is added to the solution. if solution remains clear for 30 min, it was concluded that end point of decalcification was reached and the procedure is completed. end point of neutral edta was assessed radiographically wherein the opacity suggested incomplete decalcification [figure 1 ]. the physical method of probing the teeth subjected to neutral edta with a needle was also suggestive of incomplete decalcification. radiograph of a molar decalcified using neutral edta at the end of 91 days, before processing the speed of decalcification was graded from 15 [1-slowest and 5-fastest].[35 ] all the teeth were washed under running tap water for 10 min (neutral edta decalcified teeth was washed for 2 h) and continued with routine processing, paraffin wax infiltration, and embedding ; sectioning and staining with hematoxylin and eosin. the stained sections were observed under the microscope and were graded from 14 [1-poor, 2-fair, 3-good, and 4-excellent ] based on the following criteria : ease of sectioning;hard - tissue staining;soft - tissue staining both cytoplasmic and nuclear staining;soft - tissue attachment;soft - tissue shrinkage and;pulpal organization. hard - tissue staining ; soft - tissue staining both cytoplasmic and nuclear staining ; soft - tissue attachment ; soft - tissue shrinkage and ; parameter 1 : speed of decalcification : 5% nitric acid decalcified teeth the fastest and neutral edta was the slowest [graph 1].parameter 2 : soft - tissue integrity : with respect to soft - tissue integrity, features like soft - tissue attachment, soft - tissue shrinkage, and pulpal organization, teeth decalcified with neutral edta exhibited excellent results, and the ones decalcified with 5% nitric acid scored the least [graph 2, figures 2 and 3].parameter 3 : staining quality : neutral edta and 5% trichloroacetic acid decalcified teeth stained the best, and 5% nitric acid and perenyi 's fluid stained the worst [graph 3, figures 4 and 5].overall, neutral edta scored over the other agents and perenyi 's fluid scored the least [table 1 ]. parameter 1 : speed of decalcification : 5% nitric acid decalcified teeth the fastest and neutral edta was the slowest [graph 1 ]. parameter 2 : soft - tissue integrity : with respect to soft - tissue integrity, features like soft - tissue attachment, soft - tissue shrinkage, and pulpal organization, teeth decalcified with neutral edta exhibited excellent results, and the ones decalcified with 5% nitric acid scored the least [graph 2, figures 2 and 3 ]. parameter 3 : staining quality : neutral edta and 5% trichloroacetic acid decalcified teeth stained the best, and 5% nitric acid and perenyi 's fluid stained the worst [graph 3, figures 4 and 5 ]. overall, neutral edta scored over the other agents and perenyi 's fluid scored the least [table 1 ]. time in days taken for decalcification soft - tissue integrity of the various decalcifying agents tooth decalcified using neutral edta decalcifying solution under high power 5% nitric acid decalcified tooth in low power staining characteristics of the different decalcifying agents high - power view of a tooth decalcified in 5% trichloroacetic acid tooth decalcified using perenyi 's fluid under low power cumulative scores of the decalcifying agents based on the various parameters the process of decalcification is done to study the structure of tooth, pulp calcifications, and also to evaluate the biological response of dental pulp to restorative materials. for many years, scientists have tried to introduce new decalcifying substances or tried to modify known decalcification agents, in order to meet the criteria of a good decalcifying agent which ensures complete removal of calcium;causes minimal damage to cells and tissues;causes non impairment to subsequent staining and;decalcifies at reasonable speed. ensures complete removal of calcium ; causes minimal damage to cells and tissues ; causes non impairment to subsequent staining and ; decalcifies at reasonable speed. most authors have compared two to four decalcifying agents, sometimes varying the methods used and by employing a lot of permutations and combinations of the methods and agents, mainly to decalcify bone.[2810 ] in the present study, we attempted to compare the efficacy of six decalcifying agents, its rate of decalcification, its effect on organic and inorganic components of teeth, and staining characteristics. the speed factor of the decalcifying agents was the highest with 5% nitric acid and lowest by neutral edta decalcifying solution, which was in accordance with literature. also noted, based on chalky white appearance of enamel, was the initial rate of decalcification 4 days after the start of the procedure. contrary to the overall increased time taken by neutral edta, it was noted that teeth decalcified in it had lost its enamel faster than 5% nitric acid. when sectioning it was noted that there was crumbling of tissue decalcified in 5% nitric acid and perenyi 's fluid which also contains nitric acid. also, tissue was indistinct when observed under microscope as also noted by zappa. with respect to 7% nitric acid. teeth decalcified with neutral edta responded the best to microtome knife, hence deceiving the physical and radiological methods of testing end point of decalcification with respect to neutral edta. in terms of efficacy of agents with respect to soft - tissue integrity and hard- and soft - tissue staining, excellent results were actually obtained with the slowest decalcifying agent, i.e., neutral edta. soft - tissue attachment and soft - tissue shrinkage, as reported by zappa., suggest that formic acid and nitric acid produce worst results in contrast to the results obtained from our study, wherein formic acid gave good results as it showed minimal soft - tissue shrinkage and minimal loss of tissue [figure 6 ]. the pulp organization with its extra - cellular matrix and histological zones were clearly distinct and excellent in teeth decalcified with neutral edta and 5% trichloroacetic acid. 10% formic acid decalcified tooth in low power the overall superior results obtained with neutral edta may be attributed to the mechanism of capturing metallic ions like calcium which binds to the chelating agent. this means that the calcium ions from the external layer of the apatite crystals will be removed. when all calcium ions from the outer layer of apatite crystals are removed, they will be replaced by ions from the deeper layers. in this way, the crystal size decreases gradually, producing an excellent preservation of tissue components.[46 ] the quality of decalcified sections and rate of decalcification depends on factors like fixation concentration of decalcifying agent used, temperature, pressure, agitation, electric current, microwave radiation, tissue suspension, and size and type of tissue.[469 ] in a study by waerhaug, bone and teeth were decalcified rapidly under vacuum. the time taken for decalcification was reduced to one - tenth. changes in temperature at which decalcification occurs also varies the time taken for complete decalcification. vongsavan., in his study on cat and rat teeth, reported a faster process of decalcification in microwave oven at 37 2c than in room temperature or conventional oven. another study by pitol. showed that microwave - aided decalcification showed to be more effective than the traditional methods in aspects like reduction of time period for decalcification, good morphology of bone tissue, and an increase of calcium release using microwaving. warshawsky and moore studied the effects of decalcifying agents, namely nitric acid, formic acid, and edta, on number and stainabilty of gram - positive bacteria. the number of studies using edta was greater than the other decalcifying agents, especially when the need was for academic interests and research.[1619 ] to conclude, harder the structure, hardest to decalcify. we propose, in cases of urgent requirement, use of acids may be employed although with poor tissue integrity. for preservation and presentation, when time is not a factor, use of neutral edta may be advocated for its excellent soft - tissue integrity and quality of staining. an agent that seemed to balance both of the factors of time and tissue integrity was formalin - nitric acid solution [figure 7 ]. low - power view of a tooth decalcified in formic acid - nitric acid solution regardless of the solution used, methods of decalcification share their characteristic of being accelerated when additional factors are employed. the present study was done with respect to different decalcifying agents only and none of the factors were employed, thus standardizing the procedure. future studies in which the factors can be varied and decalcifying agent can be kept constant, might bring us to the near ideal decalcifying agent. | context : in routine histopathology, decalcification of bone and teeth is often an essential and important step during tissue processing. various decalcifying agents have been used in the past. the rate of decalcification and the effect of decalcifying agents on the tissue and its staining characteristics are two important parameters which influence the selection of decalcifying solutions. though some agents remove the calcium ions completely and rapidly, they adversely affect the staining characteristics and may also damage the organic components. there have been very few studies which have systematically evaluated the efficacy of these agents in decalcifying dental hard tissues.aims:the present study was done to evaluate the rate of decalcification of six different decalcifying agents and also their effect on staining characteristics on dental hard tissues.materials and methods : six decalcifying agents namely, neutral ethylene diamine tetra acetic acid (edta) decalcifying solution, 5% nitric acid, perenyi 's fluid, formalin nitric acid, 5% trichloracetic acid, and 10% formic acid were used to decalcify 24 natural teeth (four in each solution). the endpoint of decalcification was evaluated by radiographic and chemical methods. the decalcified teeth were then routinely processed, sectioned, and stained with hematoxylin and eosin stains.results:neutral edta was the most considerate to the soft and hard tissues and 5% nitric acid was the least considerate to the tooth structure.conclusions:neutral edta, though being the slowest decalcifying agent among the six agents used in the study, gave excellent results for soft - tissue integrity, and best quality of both soft - tissue and hard - tissue stainings. |
breast cancer metastasis to the liver the liver is a common site of breast cancer metastasis, along with bone and lung. liver metastasis in breast cancer patients is an independent prognostic factor for very poor outcomes, as median survival of breast cancer patients with liver metastasis ranges from 4.8 to 15 months. in contrast, breast cancer patients with lung or bone metastasis have median survival rates of 9 to 27.4 months and 16.3 to 56 months, respectively. metastasis is a multistep process, referred to as the metastatic cascade, which begins with tumor cell dissemination in the primary tumor and ends with patient mortality due to the seeding and outgrowth of circulating tumor cells within a distant organ. rodent models of metastasis have revealed that the metastatic cascade is remarkably inefficient, with only 0.02 - 10% of circulating tumor cells establishing overt metastasis. one major bottleneck of metastatic inefficiency is dictated by the unique tissue microenvironments at secondary sites, called metastatic niches, highlighting the importance of understanding site - specific metastasis. the metastatic niche is unique to the site of recurrence, and is, in part, characterized by deposition of distinct extracellular matrix proteins, infiltration of various immune cell populations, and altered tissue homeostasis including dysregulated production of numerous cytokines, chemokines, and growth factors. thus, an understanding of the tissue specific metastatic niche precedes an understanding of how to target metastatic disease. further, improved models of liver metastasis will be essential to identifying novel targets and effective treatments for breast cancer patients with liver metastases. established models to study breast cancer metastasis to the liver currently available models to study breast cancer metastasis to the liver include human cancer cell xenografts in immune compromised mice. these models typically use well - studied human breast cancer cell lines such as mcf-7 and mda - mb-231 and nude, rag1, or scid immune compromised murine hosts. xenograft models provide the advantage of involving human derived cancer cell lines, however, given the recent appreciation for immune cells in metastasis and in therapeutic resistance, the study of metastasis in a fully immune competent host is paramount. models to study breast cancer metastasis to the liver in immune competent hosts include orthotopic injection of syngeneic tumor cells (e.g., 4t1 and d2a1 cell lines) into the mammary fat pad, with or without surgical resection of the primary tumor, and subsequent assessment of metastasis. of note, the rate of liver metastasis from orthotopic transplant models is very low or non - existent compared to other metastatic sites such as lung, or occurs after lung metastasis is established, complicating the study of liver - specific metastasis. tumor explants from spontaneous genetically engineered breast cancer models can be re - injected into the mammary fat pads of nave hosts as syngeneic tumor cells. for example, it was recently reported that spontaneous tumors from k14ecadp53 mice, which model invasive lobular breast carcinoma, develop tumors when orthotopically injected into wildtype hosts. following surgical resection of these tumors once they reach 15 mm, 18% of the mice progressed to liver metastasis. a third approach to model liver metastasis utilizes spontaneous metastasis in genetically engineered mice. to date, reports of spontaneous murine models of breast cancer metastasis that readily spread to the liver are uncommon. exceptions include the h19-igf2, the p53mmtv - cre wap - cre, and the k14ecadp53 genetically engineered mouse models, where liver metastasis develops in a low percentage of mice. thus, while genetically engineered mouse models facilitate the study of all stages of the metastatic cascade, providing powerful and clinically relevant models, they are limited due to low rates of liver metastasis. several metastasis models bypass the initial steps of the metastatic cascade including dissemination of tumor cells from the primary tumor and intravasation. these models permit investigation into the later steps of the metastatic cascade, from extravasation to establishment of tumors at secondary sites. the intracardiac injection model delivers tumor cells into the left ventricle, which distributes tumor cells into the circulatory system via the aorta. intracardiac injection requires ultrasound guided imaging of the injection site or other imaging modalities such as bioluminescence of luciferase tagged cells to confirm successful injection. tumor cell injection via the left ventricle may result in bone, brain, lung, and/or liver metastasis, amongst other organs. because of multi - organ metastases, these mice frequently need to be euthanized prior to development of overt liver metastasis, negating the ability to fully investigate metastatic growth within the liver. an alternative approach that significantly minimizes the development of multi - site metastasis is the intrasplenic injection model. intrasplenic injection delivers tumor cells via the splenic vein that joins with the superior mesenteric vein to become the portal vein. animals can be monitored for outgrowth of metastatic lesions in the liver because formation of metastases at other sites is rare, and as a result, the animal 's overall health is maintained. however, it is important to note that the intrasplenic model requires splenectomy to avoid splenic tumors, a procedure that impacts immune function. for example, myocardial ischemia reperfusion injury is characterized by infiltration of ly6c monocyte subsets that originate from the spleen and are responsible for phagocytic and proteolytic activity during the wound healing following ischemia. with splenectomy further, splenectomy has been shown to reduce primary tumor growth and lung metastases in a non - small cell lung cancer model, specifically through a reduction in the number of circulating and intra - tumor ccr2cd11bly6c monocytic myeloid cells. additionally, splenectomy following intrasplenic injection of colon cancer cells resulted in reduced levels of anti - tumor natural killer cells in mesenteric lymph nodes and elevated liver metastasis. in sum, these findings suggest that splenectomy compromises the immune system 's role with subsequent consequences for metastatic cell fate. portal vein injection model of liver metastasis to investigate breast cancer metastasis to the liver in a fully immune competent host, under conditions where mice are not compromised due to multi - organ metastases, a portal vein injection model was developed. intraportal injection models have been used previously to study liver metastasis of colorectal and melanoma cell lines ; here we describe application of the intraportal injection to model syngeneic mammary tumor cell metastasis. this model can be used to study the later stages of the metastatic cascade including breast cancer cell extravasation and seeding, tumor cell fate decisions regarding death / proliferation / dormancy, and outgrowth into overt lesions. in this model, syngeneic mammary tumor cell lines are injected via the portal vein of immune competent balb / c female mice, a method that delivers tumor cells firstly and directly to the liver without removal of the spleen. to develop this model, the use of four mammary tumor cell lines that range in their metastatic capability from low to high were employed : d2.or, d2a1, and 4t1, and have employed d2a1 tagged with green fluorescent protein (d2a1-gfp) to investigate early time - points after tumor cell injection. 4t1 is a highly metastatic cell line derived from the 410.4 tumor that spontaneously arose in an mmtv balb / c female mouse and metastasizes to lung, liver, brain, and bone from mammary fat pad primary tumors. d2a1 tumor cells were also originally derived from a spontaneous mammary tumor arising in a balb / c host after transplant of d2 hyperplastic alveolar nodule cells, and are confirmed to be metastatic from the primary tumor to the lung. d2.or tumor cells are a non - metastatic sister line to the d2a1 line and, although they escape the primary tumor and arrive at secondary sites, they rarely establish distant metastases. additionally, it is important to avoid use of commonly employed pain management drugs including non - steroidal anti - inflammatory drugs (nsaids) during or following the surgical procedure. nsaids have anti - tumor activity in certain breast cancers, and some classes of nsaids increase the risk of hepatotoxicity, potentially compromising the study of liver metastasis and the liver metastatic niche. further, studies suggest that nsaids directly influence the tissue microenvironment, reducing pro - metastatic extracellular matrix proteins tenascin - c and fibrillar collagen. alternatively, the use of an opioid derivative, buprenorphine, was used because of its efficacy in rodent pain management and due to the lack of evidence that opioids have anti - tumor activity. this portal vein injection model was optimized for smaller injection volumes of 5 - 10 l to avoid unnecessary damage to the liver. the model was also optimized to include needles with smaller diameter (32 gauge) and use of hemostatic gauze immediately following injection to minimize blood loss during the procedure. in contrast to these optimized injection parameters, cell numbers should be determined on an individual basis, based on the tumorigenic potential of the cell line. however, starting at 10,000 cells / injection for long - term studies is recommended. for shorter endpoints (e.g., 24 hr post - injection) considerably more tumor cells (e.g., 1 x 10- 1 x 10) may be used if warranted. in summary, the portal vein injection model detailed here represents a useful tool for the study of breast cancer metastasis to the liver and circumvents a number of the limitations of other liver metastasis models. this model facilitates study of tumor cell extravasation, seeding, early fate decisions of survival, proliferation, and dormancy, and metastatic outgrowth in immune competent murine hosts. all animal procedures in this article were reviewed and approved by the oregon health & science university institutional animal care and use committee. prepare the scissors, forceps, and hemostat by autoclaving at 124 c for 30 min, 1 - 2 days prior to the planned surgeries. ensure access to autoclaved or sterile bedding, cages, and food for post - surgical recovery. wipe down all surfaces of the surgical area with 10% bleach, including the heating pad, light source, anesthesia tubing and nose cone, and any other part of the surgical suite that will be in close proximity to the surgical procedure while it is being performed.in the aseptic surgical area, place the cleaned heating pad with sterile drape, light source, anesthesia tubing and nosecone, insulin syringes, 1 ml syringes, bupivacaine, artificial tears, sterile saline, 2 x 2 " sterile gauze sponges, 4 x 4 " sterile gauze, hemostatic gauze cut into 0.5 - 1 cm pieces, scissors, forceps, hemostat, 4 - 0 vicryl sutures with taper needle, and 50 ml 2% chlorhexidine gluconate in an autoclaved container.ensure that there is room in this space for prepared tumor cells stored on ice.on the bench adjacent to the surgical area, prepare the recovery area with a second heating pad and clean cages with sterile bedding. note : this area can also house items such as a bead sterilizer. wipe down all surfaces of the surgical area with 10% bleach, including the heating pad, light source, anesthesia tubing and nose cone, and any other part of the surgical suite that will be in close proximity to the surgical procedure while it is being performed. in the aseptic surgical area, place the cleaned heating pad with sterile drape, light source, anesthesia tubing and nosecone, insulin syringes, 1 ml syringes, bupivacaine, artificial tears, sterile saline, 2 x 2 " sterile gauze sponges, 4 x 4 " sterile gauze, hemostatic gauze cut into 0.5 - 1 cm pieces, scissors, forceps, hemostat, 4 - 0 vicryl sutures with taper needle, and 50 ml 2% chlorhexidine gluconate in an autoclaved container. ensure that there is room in this space for prepared tumor cells stored on ice. on the bench adjacent to the surgical area, prepare the recovery area with a second heating pad and clean cages with sterile bedding. one - hour prior to the planned injections, treat balb / c female mice aged 8 - 15 weeks with 100 l of 0.015 mg / ml buprenorphine, subcutaneously, for pain management. note : this injection protocol may be applied to any strain of female or male mouse at any age, using the appropriate cell lines for changes to the strain. prepare the tumor cells for injection based on protocols for the cell line or tumor explant of choice. test all tumor cell lines prior to administration for the presence of murine pathogens to reduce the risk of introducing such pathogens into the animal colony. for syngeneic balb / c tumor cell lines including d2a1, d2.or, and 4t1 tumor cells, thaw cells into a 10 cm tissue culture plate 3 days prior to injection such that the following day cells are at ~ 90 - 100% confluency.1 day following tumor cell thaw wash cells once with 1x phosphate buffered saline (pbs) and trypsinize the confluent tumor cells using 2 ml of 0.05% trypsin at 37 c for 5 min. add 8 ml of complete media (dmem high glucose, 10% fetal bovine serum, 2 mm l - glutamine, and 1x penicillin / streptomycin) and passage 1:10 into a fresh 10 cm dish with 10 ml of complete media.on the day of the injections, wash cells once with 1x pbs and trypsinize as described above.resuspend trypsinized cells in 8 ml of complete media, spin for 5 min at 1,500 x g, remove the media and resuspend in 5 ml 1x pbs.count cells on a hemocytometer using trypan blue exclusion for viability assessment. resuspend cells for injection in 1x pbs at a pre - determined concentration and volume. note : 5 - 10 l is recommended as smaller injection volumes prevent unnecessary damage to the liver.keep cells on ice for the duration of the injections. following completion of injections, return a sample of cells to the laboratory and place in culture in complete media for 1 day to ensure viability. for syngeneic balb / c tumor cell lines including d2a1, d2.or, and 4t1 tumor cells, thaw cells into a 10 cm tissue culture plate 3 days prior to injection such that the following day cells are at ~ 90 - 100% confluency. 1 day following tumor cell thaw wash cells once with 1x phosphate buffered saline (pbs) and trypsinize the confluent tumor cells using 2 ml of 0.05% trypsin at 37 c for 5 min. add 8 ml of complete media (dmem high glucose, 10% fetal bovine serum, 2 mm l - glutamine, and 1x penicillin / streptomycin) and passage 1:10 into a fresh 10 cm dish with 10 ml of complete media. on the day of the injections, wash cells resuspend trypsinized cells in 8 ml of complete media, spin for 5 min at 1,500 x g, remove the media and resuspend in 5 ml 1x pbs. resuspend cells for injection in 1x pbs at a pre - determined concentration and volume. note : 5 - 10 l is recommended as smaller injection volumes prevent unnecessary damage to the liver. keep cells on ice for the duration of the injections. following completion of injections, return a sample of cells to the laboratory and place in culture in complete media for 1 day to ensure viability. place the mouse under anesthesia with 2 - 2.5% isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro - ethane) delivered in oxygen. ensure complete anesthetization by assessing for a reaction to a toe pinch, and then maintain anesthesia at 2 - 2.5% isoflurane. note : it is important to monitor the animals breathing rate and adjust the isoflurane flow - rate accordingly throughout the procedure. place a small amount of artificial tears or vet ointment over each eye to avoid excessive drying of the eyes during the surgical procedure. remove hair on the ventral left side of the rodent from the second rib space down to the 4 inguinal mammary gland nipple by wiping the area with chemical depilatory. allow the depilatory to sit for 1 - 2 min and then remove completely with gauze and h2o. this step can be done 1 - 2 days in advance to save time if numerous surgeries are planned. take one 2 x 2 " sterile gauze sponge (soaked in 2% chlorhexidine) and wipe down the mouse at the site of hair removal. sterilize the entire surrounding area, including the tail, to minimize bacterial contamination of instruments. wipe the site of hair removal and surrounding area down with an alcohol prep pad. repeat 2% chlorhexidine and alcohol steps once more and finish with a final chlorhexidine wipe down for a total of three 2% chlorhexidine and two alcohol prep pad washes. do the final chlorhexidine wipe such that the chemical is not dripping around the surgical site to avoid getting chlorhexidine on internal organs. note : application of large amounts of chlorhexidine and alcohol to the skin and surrounding fur may result in a significant drop in body temperature. using sterile gloves and a sterilized scalpel with sterile blade, make a single 1-inch incision into the skin between the median and sagittal planes on the left side of the mouse, starting just below the ribs and ending just above the plane of the fourth inguinal mammary gland teat. using autoclaved or bead sterilized scissors and forceps, make a similar 1-inch incision into the peritoneum. avoid cutting into the mammary fat pad and ensure not to cut the intestines, liver, or diaphragm. place a 4 x 4 " gauze pad soaked in sterile saline on the left side of the mouse, where the incision was made, such that internal organs can be placed on the gauze and not come into contact with the surrounding skin or surgical area. prepare tumor cells by pipetting up and down several times as tumor cells will settle during preparation of the mouse. push on the syringe until tumor cells are at the tip of the needle and the plunger is at the appropriate volume for injection ; avoid injection of air bubbles. wipe the outside of the needle with a sterile alcohol pad to remove any external tumor cells. hold the median side of the incision, including skin and peritoneal lining, aside with the forceps and use a sterile cotton swab to carefully pull the large and small intestines out, placing them on the sterile gauze soaked in sterile saline. cover the internal organs in the saline soaked gauze to maintain internal moisture and sterility. have an assistant, also wearing sterile gloves, hold the intestines wrapped in the saline soaked gauze gently out of the way with a sterile cotton tipped swab to fully reveal the portal vein. additionally, it may be necessary to use the autoclaved hemostat or forceps to hold tissue aside on the median side of the incision. insert the needle loaded with tumor cells ~ 3 - 5 mm into the portal vein ~ 10 mm below the liver at an angle 10% weight loss / gain, scruffiness, loss of attention to surroundings, abdominal edema / ascites, pale eyes and ears, or a hunched position. note : daily health checks are particularly important when developing the model for use with a new cell line or cell concentration until the timeline of metastasis is well understood. at study end - point, euthanize mice by co2 inhalation followed by cervical dislocation. note : alternative methods of euthanasia approved by the investigators ' oversight committee may be used, including perfusion with 1x pbs while under anesthesia. perfusion of the liver is performed by cannulating the portal vein, snipping the inferior vena cava, and pushing 1x pbs through the liver vasculature at a rate of 4 ml / min for 1 - 2 min to remove circulating blood and leukocytes. note : depending on the endpoint for the study, the cell line, and the concentration used, overt liver metastasis may or may not be apparent at necropsy. extract the liver with scissors and forceps by first removing the gallbladder, then cut through the inferior vena cava superior to the liver. cut through the vena cava, portal vein, and hepatic artery inferior to the liver. formalin fix liver in 10% neutral buffered formalin for 48 hr while shaking at room temperature. process tissues through a series of alcohols in increasing concentration and xylene ; paraffin embed the fixed tissue. note : alternatively, the liver may be snap - frozen by placing tissue in a cryomold with optimum cutting temperature (oct) formula and freezing on dry ice pellets submerged in 95% ethanol. using a microtome, cut into the paraffin embedded tissue block such that a match - head size of tissue is revealed.cut five 4-micron serial sections, this constitutes the first level for analysis.cut through 250 microns of tissue, throw these sections in the waste.at 250 microns cut a second level of five 4-micron serial sections.repeat as necessary to section through the entire liver. hematoxylin and eosin stain the first section of every level to assess for metastasis. note : for snap - frozen tissue use a cryostat to cut sections. using a microtome, cut into the paraffin embedded tissue block such that a match - head size of tissue is revealed. cut through 250 microns of tissue, throw these sections in the waste. at 250 microns hematoxylin and eosin stain the first section of every level to assess for metastasis. note : for snap - frozen tissue use a cryostat to cut sections. note : detection of micrometastatic disease will require tumor cell specific staining. remove mice from study if there is tumor growth at the incision site, as this indicates tumor cell leakage into the peritoneal cavity following injection. prepare the scissors, forceps, and hemostat by autoclaving at 124 c for 30 min, 1 - 2 days prior to the planned surgeries. ensure access to autoclaved or sterile bedding, cages, and food for post - surgical recovery. wipe down all surfaces of the surgical area with 10% bleach, including the heating pad, light source, anesthesia tubing and nose cone, and any other part of the surgical suite that will be in close proximity to the surgical procedure while it is being performed.in the aseptic surgical area, place the cleaned heating pad with sterile drape, light source, anesthesia tubing and nosecone, insulin syringes, 1 ml syringes, bupivacaine, artificial tears, sterile saline, 2 x 2 " sterile gauze sponges, 4 x 4 " sterile gauze, hemostatic gauze cut into 0.5 - 1 cm pieces, scissors, forceps, hemostat, 4 - 0 vicryl sutures with taper needle, and 50 ml 2% chlorhexidine gluconate in an autoclaved container.ensure that there is room in this space for prepared tumor cells stored on ice.on the bench adjacent to the surgical area, prepare the recovery area with a second heating pad and clean cages with sterile bedding. wipe down all surfaces of the surgical area with 10% bleach, including the heating pad, light source, anesthesia tubing and nose cone, and any other part of the surgical suite that will be in close proximity to the surgical procedure while it is being performed. in the aseptic surgical area, place the cleaned heating pad with sterile drape, light source, anesthesia tubing and nosecone, insulin syringes, 1 ml syringes, bupivacaine, artificial tears, sterile saline, 2 x 2 " sterile gauze sponges, 4 x 4 " sterile gauze, hemostatic gauze cut into 0.5 - 1 cm pieces, scissors, forceps, hemostat, 4 - 0 vicryl sutures with taper needle, and 50 ml 2% chlorhexidine gluconate in an autoclaved container. ensure that there is room in this space for prepared tumor cells stored on ice. on the bench adjacent to the surgical area, prepare the recovery area with a second heating pad and clean cages with sterile bedding. one - hour prior to the planned injections, treat balb / c female mice aged 8 - 15 weeks with 100 l of 0.015 mg / ml buprenorphine, subcutaneously, for pain management. note : this injection protocol may be applied to any strain of female or male mouse at any age, using the appropriate cell lines for changes to the strain. prepare the tumor cells for injection based on protocols for the cell line or tumor explant of choice. test all tumor cell lines prior to administration for the presence of murine pathogens to reduce the risk of introducing such pathogens into the animal colony. for syngeneic balb / c tumor cell lines including d2a1, d2.or, and 4t1 tumor cells, thaw cells into a 10 cm tissue culture plate 3 days prior to injection such that the following day cells are at ~ 90 - 100% confluency.1 day following tumor cell thaw wash cells once with 1x phosphate buffered saline (pbs) and trypsinize the confluent tumor cells using 2 ml of 0.05% trypsin at 37 c for 5 min. add 8 ml of complete media (dmem high glucose, 10% fetal bovine serum, 2 mm l - glutamine, and 1x penicillin / streptomycin) and passage 1:10 into a fresh 10 cm dish with 10 ml of complete media.on the day of the injections, wash cells once with 1x pbs and trypsinize as described above.resuspend trypsinized cells in 8 ml of complete media, spin for 5 min at 1,500 x g, remove the media and resuspend in 5 ml 1x pbs.count cells on a hemocytometer using trypan blue exclusion for viability assessment. resuspend cells for injection in 1x pbs at a pre - determined concentration and volume. note : 5 - 10 l is recommended as smaller injection volumes prevent unnecessary damage to the liver.keep cells on ice for the duration of the injections. following completion of injections, return a sample of cells to the laboratory and place in culture in complete media for 1 day to ensure viability. for syngeneic balb / c tumor cell lines including d2a1, d2.or, and 4t1 tumor cells, thaw cells into a 10 cm tissue culture plate 3 days prior to injection such that the following day cells are at ~ 90 - 100% confluency. 1 day following tumor cell thaw wash cells once with 1x phosphate buffered saline (pbs) and trypsinize the confluent tumor cells using 2 ml of 0.05% trypsin at 37 c for 5 min. add 8 ml of complete media (dmem high glucose, 10% fetal bovine serum, 2 mm l - glutamine, and 1x penicillin / streptomycin) and passage 1:10 into a fresh 10 cm dish with 10 ml of complete media. on the day of the injections, wash cells resuspend trypsinized cells in 8 ml of complete media, spin for 5 min at 1,500 x g, remove the media and resuspend in 5 ml 1x pbs. resuspend cells for injection in 1x pbs at a pre - determined concentration and volume. note : 5 - 10 l is recommended as smaller injection volumes prevent unnecessary damage to the liver. keep cells on ice for the duration of the injections. following completion of injections, return a sample of cells to the laboratory and place in culture in complete media for 1 day to ensure viability. place the mouse under anesthesia with 2 - 2.5% isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro - ethane) delivered in oxygen. ensure complete anesthetization by assessing for a reaction to a toe pinch, and then maintain anesthesia at 2 - 2.5% isoflurane. note : it is important to monitor the animals breathing rate and adjust the isoflurane flow - rate accordingly throughout the procedure. place a small amount of artificial tears or vet ointment over each eye to avoid excessive drying of the eyes during the surgical procedure. remove hair on the ventral left side of the rodent from the second rib space down to the 4 inguinal mammary gland nipple by wiping the area with chemical depilatory. allow the depilatory to sit for 1 - 2 min and then remove completely with gauze and h2o. this step can be done 1 - 2 days in advance to save time if numerous surgeries are planned. take one 2 x 2 " sterile gauze sponge (soaked in 2% chlorhexidine) and wipe down the mouse at the site of hair removal. sterilize the entire surrounding area, including the tail, to minimize bacterial contamination of instruments. wipe the site of hair removal and surrounding area down with an alcohol prep pad. repeat 2% chlorhexidine and alcohol steps once more and finish with a final chlorhexidine wipe down for a total of three 2% chlorhexidine and two alcohol prep pad washes. do the final chlorhexidine wipe such that the chemical is not dripping around the surgical site to avoid getting chlorhexidine on internal organs. note : application of large amounts of chlorhexidine and alcohol to the skin and surrounding fur may result in a significant drop in body temperature. using sterile gloves and a sterilized scalpel with sterile blade, make a single 1-inch incision into the skin between the median and sagittal planes on the left side of the mouse, starting just below the ribs and ending just above the plane of the fourth inguinal mammary gland teat. using autoclaved or bead sterilized scissors and forceps, make a similar 1-inch incision into the peritoneum. avoid cutting into the mammary fat pad and ensure not to cut the intestines, liver, or diaphragm. place a 4 x 4 " gauze pad soaked in sterile saline on the left side of the mouse, where the incision was made, such that internal organs can be placed on the gauze and not come into contact with the surrounding skin or surgical area. prepare tumor cells by pipetting up and down several times as tumor cells will settle during preparation of the mouse. push on the syringe until tumor cells are at the tip of the needle and the plunger is at the appropriate volume for injection ; avoid injection of air bubbles. wipe the outside of the needle with a sterile alcohol pad to remove any external tumor cells. hold the median side of the incision, including skin and peritoneal lining, aside with the forceps and use a sterile cotton swab to carefully pull the large and small intestines out, placing them on the sterile gauze soaked in sterile saline. cover the internal organs in the saline soaked gauze to maintain internal moisture and sterility. have an assistant, also wearing sterile gloves, hold the intestines wrapped in the saline soaked gauze gently out of the way with a sterile cotton tipped swab to fully reveal the portal vein. additionally, it may be necessary to use the autoclaved hemostat or forceps to hold tissue aside on the median side of the incision. insert the needle loaded with tumor cells ~ 3 - 5 mm into the portal vein ~ 10 mm below the liver at an angle 10% weight loss / gain, scruffiness, loss of attention to surroundings, abdominal edema / ascites, pale eyes and ears, or a hunched position. note : daily health checks are particularly important when developing the model for use with a new cell line or cell concentration until the timeline of metastasis is well understood. at study end - point, note : alternative methods of euthanasia approved by the investigators ' oversight committee may be used, including perfusion with 1x pbs while under anesthesia. perfusion of the liver is performed by cannulating the portal vein, snipping the inferior vena cava, and pushing 1x pbs through the liver vasculature at a rate of 4 ml / min for 1 - 2 min to remove circulating blood and leukocytes. note : depending on the endpoint for the study, the cell line, and the concentration used, overt liver metastasis may or may not be apparent at necropsy. extract the liver with scissors and forceps by first removing the gallbladder, then cut through the inferior vena cava superior to the liver. cut through the vena cava, portal vein, and hepatic artery inferior to the liver. formalin fix liver in 10% neutral buffered formalin for 48 hr while shaking at room temperature. process tissues through a series of alcohols in increasing concentration and xylene ; paraffin embed the fixed tissue. note : alternatively, the liver may be snap - frozen by placing tissue in a cryomold with optimum cutting temperature (oct) formula and freezing on dry ice pellets submerged in 95% ethanol. using a microtome, cut into the paraffin embedded tissue block such that a match - head size of tissue is revealed.cut five 4-micron serial sections, this constitutes the first level for analysis.cut through 250 microns of tissue, throw these sections in the waste.at 250 microns cut a second level of five 4-micron serial sections.repeat as necessary to section through the entire liver. note : detection of micrometastatic disease will require tumor cell specific staining. using a microtome, cut into the paraffin embedded tissue block such that a match - head size of tissue is revealed. cut through 250 microns of tissue, throw these sections in the waste. at 250 microns hematoxylin and eosin stain the first section of every level to assess for metastasis. note : for snap - frozen tissue use a cryostat to cut sections. note : detection of micrometastatic disease will require tumor cell specific staining. remove mice from study if there is tumor growth at the incision site, as this indicates tumor cell leakage into the peritoneal cavity following injection. the portal vein injection model, in which tumor cells are delivered directly to the liver via a surgical procedure, allows for tumor cell injection into the portal vein. under antiseptic conditions, in an anesthetized mouse, a ~1-inch surgical incision is made on the left side of the mouse between the median and sagittal planes, starting just above the plane of the fourth inguinal mammary gland teat and ending just below the ribs. the large and small intestines are gently pulled through the incision to provide visualization of the portal vein (figure 1a). accurate anatomical identification of the portal vein and successful intra - portal injection can be confirmed by practicing the injection protocol with india ink or a similar dye. correct injection via the portal vein will result in the ink being delivered immediately and specifically to the liver, and will not result in india ink spread to the lung (figure 1b). further, using d2a1 mouse mammary tumor cells tagged with gfp, dispersal of tumor cells throughout the liver is apparent at ninety minutes post - injection, confirming portal vein injection delivery to the liver (figure 1c). at higher magnification it becomes apparent that at 90 min post - injection, tumor cells are found within sinusoids, as well as within the liver parenchyma in close proximity to portal triads, where the portal vein blood enters the liver (figure 1c). these data suggest that active tumor cell extravasation is occurring at 90 min post - tumor cell injection. taken together, these data confirm that the portal vein injection model delivers the injection volume directly to the liver, with ink or tumor cells dispersed throughout the liver and no appreciable transport of injection volume to the lung. to assess robustness of the portal vein injection model, three separate syngeneic mouse mammary tumor cell lines were tested in adult female balb / c mice. these mammary tumor lines were selected based on their characterized behavior in mammary fat pad models and include the highly aggressive and metastatic 4t1 cell line, the less aggressive metastatic d2a1 line, and the low / non - metastatic d2.or line. 2,000 and 10,000 cells per injection were tested with no notable differences in the time to development of overt metastasis with these low cell concentrations. for these studies surrogate markers of metastasis were used such as lack of grooming, pallor, and weight loss to justify necropsy, upon which the presence or absence of liver metastases were confirmed by visual assessment of the liver and other organs to confirm intra - portal delivery. these data confirm previous reports that the 4t1 and d2a1 cell lines represent more aggressive mammary tumor lines, as shorter metastasis free survival rates are observed compared to mice injected with the less aggressive d2.or line (figure 2a). mice injected with 4t1 or d2a1 tumor cells developed overt liver metastasis by ~ 30 - 40 days post - injection, and some developed metastasis as early as 18 days post - injection (figure 2a), whereas only one mouse injected with d2.or cells had developed overt liver metastasis by study end, which was 60 - 65 days post - tumor cell injection. metastases were subsequently confirmed by sectioning through the liver in 250 m levels and analyzing hematoxylin and eosin (h&e) stained sections (figure 2b - d). in addition to detection of overt metastatic lesions in the mouse liver, the portal vein model can also be utilized to study earlier events in the metastatic cascade including detection of single cells / cell clusters following extravasation, and formation of micro - metastatic lesions. multiplex immunofluorescence - staining was used to detect 4t1, d2a1, and d2.or mammary tumor cells in liver when they are present as single cells or micro - metastatic lesions, as h&e is not sufficient to confirm the presence of small lesions. figure 3a shows a representative balb / c mouse liver with a putative micrometastatic foci of d2a1 tumor cells that are positive for the epithelial keratin ck18, negative for the pan - immune marker cd45, and negative for the hepatocyte marker heppar-1. hepatocytes also stain positive for ck18, necessitating use of heppar-1 in this staining panel. one important note is that bile duct epithelium and liver progenitor cells stain positive for ck18, requiring careful discrimination between tumor cells and bile ducts, particularly when assessing periportal regions (figure 3a). an alternative to multiplex immunofluorescence to identify single disseminated cells and micrometastatic foci is to utilize syngeneic mammary tumor lines tagged with enhanced green fluorescent protein (egfp) and/or luciferase and perform ihc for the tag (figure 1c). due to the immunogenicity of egfp, luciferase, and other proteins, it is essential to use mouse models that are tolerized to these proteins, such as the novel immune competent " glowing head " mouse that expresses egfp and luciferase in the anterior pituitary gland. for identification of macrometastatic lesions of untagged syngeneic lines such as the d2a1 tumor line, the ck18/heppar-1/cd45 multiplex immunofluorescence is ideal (figure 3b). figure 1:portal vein injection delivers tumor cells directly to the liver. a) portal vein injection ; the incision is made between the median and left sagittal planes, from above the plane of the fourth inguinal mammary gland teat and ending just below the rib cage. b) balb / c liver with pbs injection (left). following india ink injection via the portal vein, the liver (middle) but not the lung (right) takes up ink. c) representative thin section images of d2a1 mouse mammary tumor cells tagged with gfp in a balb / c mouse liver at 90 min post - injection of 1 x 10 tumor cells via the portal vein. livers were formalin fixed, paraffin embedded, sectioned, and stained with anti - gfp antibody for tumor cell detection. top panel shows dark brown stained tumor cells (arrows) dispersed throughout the liver, asterisk denotes non - specific hepatocyte staining around central veins ; scale bar = 300 m. lower panels show representative images of tumor cells at 90 min post - injection still closely associated with vasculature ; scale bar = 50 m. figure 2:outgrowth of mouse mammary tumor cell lines in the liver following portal vein injection. a) kaplan - meier curve showing metastasis - free survival rates in mice injected with 2,000 - 10,000 4t1, d2a1, or d2.or mouse mammary tumor cells. mice were injected with tumor cells and monitored for signs of metastasis including lack of grooming, pale eyes, and weight changes. metastasis was confirmed at time of necropsy and by h&e on histological sections of the livers. representative h&e images of b) 4t1 lesions at 21 days post - injection of 10,000 cells, c) d2a1 lesions at 26 days post - injection of 10,000 tumor cells, and d) d2.or lesions at 59 days post - injection of 10,000 cells ; scale bars = 75 m. no evidence of metastasis in other organs or at the surgical incision site was apparent in any mouse on these studies. figure 3 : detection of single cells and metastatic lesions in the mouse liver using multiplex immunofluorescence. a) representative multiplex immunofluorescence of d2a1 tumor cells in a balb / c mouse liver at 90 min post - injection using the portal vein injection model. staining was done using a multiplex kit. from left to right shows dapi ; cd45 to mark leukocytes ; heppar-1 to mark hepatocytes ; and ck18 to mark tumor cells, hepatocytes, and bile duct epithelium. merged image shows a putative cluster of ck18heppar-1cd45 d2a1 tumor cells closely associated with a portal triad. arrow = d2a1 tumor cells, asterisk = bile duct epithelium ; scale bar = 25 m. b) a representative overt d2a1 metastatic lesion using the same staining panel as in a. tumor cells are ck18 whereas adjacent hepatocytes are ck18heppar-1 ; scale bar = 25 m. images were captured on a microscope with 20 x 0.8, 40 x 1.3, and 60 x 1.4 objectives and ccd camera, using the microscope software. the balb / c mouse portal vein injection model permits the study of mammary cancer lesions in the liver in the absence of confounding multi - organ metastasis and in a fully immune competent host. our protocol is an advancement of previously published surgical procedures that permit access to the portal vein for injection of tumor cells directly into the liver. one advancement we have made is to significantly reduce the number of injected tumor cells from 1 x 10 cells / injection down to 10,000 tumor cells / injection. we have also expanded the model for the study of breast cancer metastasis to the liver. using this protocol, two mammary cancer cell lines with known metastatic potential develop liver metastases with shorter latency than a more quiescent mammary tumor cell line. further, at early time points, tumor cells are distributed throughout the liver parenchyma as single or small groups of single cells after tumor cell injection. the model is poised to address questions of metastatic efficiency including tumor cell extravasation, cell survival, dormancy, and proliferation all phenotypes that contribute to the development of micro - metastatic and overt metastatic disease in the liver. it is important to consider numerous aspects of the portal vein injection protocol prior to initiating studies. carefully deciding on cell lines, cell concentration, total cell number, and end - points of interest based on smaller exploratory studies is highly recommended. further, the use of immune competent hosts and syngeneic cell lines is of utmost importance for understanding host - tumor cell interactions. the newly developed " glowing head " mouse that expresses egfp and luciferase from the anterior pituitary gland is an important tool for eliminating host responses to exogenous egfp and luciferase, proteins often used to tag mammary tumor lines. use of the " glowing head " mouse and syngeneic tagged tumor cells will facilitate easy identification of single disseminated cells and micrometastatic foci by ihc without the concern of inflammatory responses to egfp or luciferase. similarly, choosing pain management strategies carefully to ensure minimal anti- or pro - tumor impact from the drug treatment regimen is strongly recommended. critical steps in this protocol include maintaining sterile conditions throughout surgeries to ensure that infection does not occur, as this will confound any results. it is also important that the needle is properly placed in the portal vein to ensure that tumor cells are delivered to the liver. tumor growth at the skin incision site is the best indicator that improper needle placement occurred. finally, it is critical that blood loss from the portal vein is adequately controlled and ceases entirely prior to suturing the animal. the use of hemostatic gauze greatly diminishes the risk of uncontrolled blood loss from the portal vein following injection. in our hands, procedural related mortality due to blood loss from the portal vein following injection it is important to note that the portal vein injection model does not replicate the full metastatic cascade, but is limited to the study of tumor cell extravasation, tumor cell - niche interactions following extravasation and tumor growth. models that accurately replicate the full metastatic cascade to the liver, such as occurs in patients, are urgently needed. an additional limitation of the portal vein injection model is that it is confounded by the impact of surgery on the host, with wound healing known to impact disease progression. the portal vein injection model represents an improvement on other injection models to study liver metastasis, including intracardiac and intrasplenic models. specifically, the portal vein injection model allows for the study of a larger range of disease progression than the intracardiac model, which is often limited by concomitant metastases in other tissues. further, the portal vein model is not complicated by removal of the spleen, as is done in the intrasplenic model. the portal vein injection model may prove a useful tool for the study of liver metastasis in general. liver metastasis is the most frequent site of metastasis in adenocarcinomas overall, with particularly high rates in pancreatic cancers (85% of metastases are to the liver), colon and rectal adenocarcinomas (> 70%), as well as stomach and esophageal (> 30%). although spontaneous and orthotopic primary tumor models of pancreatic and colon adenocarcinomas more readily metastasize to the liver, the portal vein injection model may prove useful to understanding the metastatic process of these cancers as controlled delivery of tumor cells permits biochemical, molecular and histological assessments at specified times after tumor cell arrival. in summary, the portal vein injection model represents an important improvement on available liver metastasis models of breast cancer, and may also be applicable to the liver metastasis field in general. | breast cancer is the leading cause of cancer - related mortality in women worldwide. liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary - site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. to address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. this model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. the optimized portal vein protocol employs small injection volumes of 5 - 10 l, 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. the portal vein injection approach in balb / c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested ; high - metastatic 4t1 cells, moderate - metastatic d2a1 cells, and low - metastatic d2.or cells. concentrations of 10,000 cells / injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4t1 and d2a1 lines, and > 55 days for the less aggressive d2.or line. this model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas. |
penicillium marneffei can cause a life - threatening systemic fungal infection in human immunodeficiency virus (hiv)-infected patients, and is prevalent in southeast asia, including thailand, northeastern india, southern china, hong kong, vietnam, and taiwan. in 1956, the first p. marneffei isolation was reported in the bamboo rat 's liver (1) and the first human infection was in a 61-yr - old american missionary who lived in southeast asia and suffered from hodgkin 's lymphoma in 1973 (2). in 1988, the first disseminated p. marneffei infection case was reported in a hiv - infected patient (3). p. marneffei might cause systemic infection in an immunocompetent host, but most of the reported cases of disseminated infection were related to hiv - infected patients. in endemic areas, the p. marneffei infection is regarded as an acquired immune deficiency syndrome (aids) marker, or an " aids defining illness, " following tuberculosis and cryptococcosis in frequency (4). however, as overseas travel increases, sporadic cases of p. marneffei infection have been reported among americans, europeans, australians, japanese, and africans who resided in or visited the endemic areas (5). here we report on the first case of p. marneffei in a korean hiv - infected patient who had been living in the endemic area. a 39-yr - old male patient visited the emergency room on may 24, 2010 with fever, cough, and multiple papules that had been on his face for two weeks. he was told that he had been infected with hiv and miliary tuberculosis was suspected after a chest computed tomography (ct) scan at a hospital in thailand. the patient denied any history of homosexual contact. upon arrival at the emergency room, his mental status was alert and the diffuse vesiculopapular lesions were easily observed on face. his vital signs were : blood pressure of 151/90 mmhg, a body temperature of 37.1, a heart rate of 116 beats per minute, and a respiration rate of 27 breaths per minute. his oxygen saturation was 90% and arterial blood gas analysis revealed ph 7.46, paco2 34 mmhg, pao2 55 mmhg, and hco3 24 mm on room air. his breathing sounds were coarse with crackles in the bilateral lung field. upon laboratory examination, his complete blood cell counts were : white blood cell count 4,100/l (neutrophil 92.0%, lymphocytes 4.8%), hemoglobin 11.4 g / dl, and platelet 134,000/l, while erythrocyte sedimentation rate and c - reactive protein were 114 mm / hr and 90 mg / l, respectively. peripheral cd4 + t lymphocyte count and hiv - rna viral load were 7 cells/l and 457,392 copies / ml. a chest x - ray revealed diffuse reticulonodular opacities in the entire lung field. chest ct scans revealed bilaterally distributed micronodules and consolidation with a ground - glass opacity pattern on a dependent portion of his lungs (fig. suspecting tuberculosis, anti - tuberculosis medications of isoniazid, rifampin, ethambutol, and pyrazinamide were started. in addition, the results of the trans - bronchoscopic lung biopsy were initially reported as pneumocystis jirovecii and trimethoprim - sulfamethoxazole was added to his regimen. one week after admission, vesiculopapular facial lesions exhibited central hemorrhagic changes (fig. 1b) and spread to the neck, trunk, and upper extremities. deoxycholate amphotericin - b (0.7 mg / kg) was started and antiretroviral therapy (art) with abacavir, lamivudine, and efavirenz was also implemented for the treatment of hiv infection. considering the typical skin lesions and fungal organisms in the biopsy, a disseminated p. marneffei infection was suspected. fungal culture of skin tissue obtained by biopsy revealed colonies of green - gray belts with red pigmentation in sabouraud dextrose agar plate (fig. 2a) and mycelia was seen in the microscopic examination after staining with lactophenol cotton blue by touching clear scotch tape to the colony (fig. to identify the fungal species, we performed sequencing of the internal transcribed spacer (its) and 26s rrna gene regions. the its region (including the 5.8s rrna gene) and the 26s rrna gene d1/d2 domains were amplified with the primer pairs of pits - f / pits - r and nl1/nl4, respectively (6). sequence similarity searches were performed using basic local alignment search tool (blast), which revealed a complete (100%) match with p. marneffei. after 10 days of amphotericin - b administration, the patient exhibited clinical and radiological improvement. the amphotericin - b was changed to oral itraconazole at 400 mg / day after 14 days of the amphotericin - b treatment. after eight weeks, the dose of itraconazole was modified to 200 mg / day and was maintained for an additional six months. we reported the first case of p. marneffei in a korean, hiv - infected patient who had lived in a p. marneffei endemic area. primary human infections of p. marneffei have been known to be acquired by inhalation of spores from soil which are then disseminated to the skin, the reticuloendothelial system and the gut by hematogenous spread and other organ systems. penicilliosis in hiv - infected patients may be misdiagnosed as mycobacterium tuberculosis because multiple nodules and mediastinal lymphadenopathy are radiologic findings of penicilliosis (7, 8). fungal cultures or direct staining of infected tissues is required to differentiate penicilliosis from tuberculosis. in addition, diffuse alveolar shadows have been observed in about 10% of patients infected with p. marneffei (4, 9), which may reveal intense parenchymal opacification like acute respiratory distress syndrome. therefore, penicilliosis may also be misdiagnosed as pneumocystis jirovecii pneumonia (pcp) (10). sputum examination may also be helpful to differentiate pcp from a p. marneffei infection (11, 12). in our case, an initial chest ct scan showed bilaterally distributed micronodules and consolidation with a diffuse ground - glass opacity pattern and a trans - bronchoscopic lung biopsy result was initially misinterpreted as pneumocystis jirovecii. therefore, we suspected a pulmonary tuberculosis and pcp co - infection. clinical improvement, however, was not obvious despite anti - tuberculosis medication and trimethoprim - sulfamethoxazole administration. this led us to suspect the p. marneffei infection, considering typical skin lesions revealed fungal organisms in the biopsy and an aggravated pulmonary infiltration. the proportion of patients with skin lesion has been reported between 28% and 71% in disseminated penicilliosis (4, 9). skin lesions present as multiple flesh - colored, dome - shaped papules in the early stage, similar to molluscum contagiosum (13). however, simple papules observed on the face, trunk, neck and extremities frequently ulcerate over time and change with central necrotic umbilication (5, 14). molluscum contagiosum resolves by itself in six months to two years in hiv - infected patients in accordance with immune function improvement after art. but skin lesions of p. marneffei are not expected to improve without active antifungal treatment. p. marneffei mainly affects people with impaired cellular immunity (i.e. hiv - infected patients) (15) and the severity and clinical manifestations depend on the patient 's immunity (16). in patients with normal immunity, p. marneffei mostly causes mild and localized infections, but it can cause severe disseminated infections with generalized lymphadenopathy and persistent fever in immunocompromised hiv - infected patients (5). it is known that the prognosis of penicilliosis can be improved if immunity could be recovered during the antifungal treatment in immunocompromised patients (9, 15). there have been two case reports of p. marneffei infection in the republic of korea. one case was a continuous ambulatory peritoneal dialysis - associated peritonitis which revealed improvement after catheter removal and antifungal therapy (17). the second case was a disseminated p. marneffei infection which occurred after a visit to china to receive a liver transplant, and that area of china is a p. marneffei endemic area (18). lymphocytopenia, which is considered a marker of impaired cellular immunity, was observed in both the liver transplant recipient (lymphocyte count 410 cells/l) and our hiv - infected patient (lymphocyte count 197 cells/l, cd4 + t lymphocyte count 7 cells/l). while both patients also exhibited a disseminated p. marneffei infection, the liver transplant recipient died due to refractory viral and bacterial infections, despite intensive treatment. in previous reports, the mortality rate of patients with p. marneffei infection was reported as 13.5% (7/52) in hiv - infected patients and 60% (6/10) in non - hiv infected patients (9, 15). in our case, the patient exhibited a good clinical outcome although he initially presented severe disseminated penicilliosis. we suppose that recovery depends upon the immune recovery related with art and is similar to findings of previous studies which revealed a better outcome in hiv - infected penicilliosis patients compared to the non - hiv - infected patients. the authors experienced the first case of p. marneffei in a korean hiv - infected patient. we suppose that it is important to consider p. marneffei when immunocompromised patients with histories of visits to areas of endemicity reveal respiratory disease, generalized lymphadenopathy, and central umbilicated vesicular skin lesions. | penicillium marneffei may cause life - threatening systemic fungal infection in immune - compromised patients and it is endemic in southeast asia. a 39-yr - old hiv - infected male, living in laos, presented with fever, cough, and facial vesiculopapular lesions, which had been apparent for two weeks. ct scans showed bilateral micronodules on both lungs ; pneumocystis jirovecii was identified by bronchoscopic biopsy. despite trimethoprim - sulfamethoxazole and anti - tuberculosis medications, the lung lesions progressed and the facial lesions revealed central umbilications. biopsy of the skin lesions confirmed disseminated penicilliosis, with the culture showing p. marneffei hyphae and spores. the p. marneffei was identified by rrna pcr. a review of the bronchoscopic biopsy indicated penicilliosis. the patient completely recovered after being prescribed amphotericin - b and receiving antiretroviral therapy. this is the first case of penicilliosis in a korean hiv - infected patient. it is necessary to consider p. marneffei when immunocompromised patients, with a history of visits to endemic areas, reveal respiratory disease. |
inflammatory bowel disease (ibd) is a chronic gastrointestinal inflammatory disorder characterized by alternating relapses and remissions. the two most common types of ibd are crohn 's disease (cd) and ulcerative colitis (uc), which are characterized by exacerbated uncontrolled intestinal inflammation that contributes to worsening of the life quality of the patients and require prolonged medical and/or surgical interventions. the inflammation associated with cd can discontinuously affect all the gastrointestinal tract, from the mouth to the anus, but it is more often localized to the distal small bowel and/or colon. samples of inflamed bowel obtained from patients with active cd show transmural inflammation with an important accumulation of acute and chronic inflammatory cells within the mucosa, submucosa, and muscularis propia. on the other hand, uc is characterized by a nontransmural inflammation, just localized within the rectum and the large bowel. typically, the inflammation is restricted to the mucosa and submucosa, with cryptitis and crypt abscesses, although the inflammatory cell composition is similar to cd. the clinical presentation in these intestinal conditions mostly depends on disease location and is characterized by diarrhoea, abdominal pain, fever, bowel obstruction, passage of blood, and/or mucus [1, 2 ]. unfortunately, the aetiology of ibd is not fully understood, although there is a general agreement that ibd is the result of a complex combination of four main factors : multiple genetic variations, alterations in the composition of the intestinal microbiota, changes in the surrounding environment, and overreactivity of the intestinal mucosal immune response. thus, genetically susceptible patients build up an exaggerated and uncontrolled immune response in the gastrointestinal tract towards an altered intestinal microbiota that turns into a chronic intestinal inflammation. similarly to other inflammatory conditions, a broad spectrum of inflammatory mediators is responsible of the pathophysiology of ibd, including cytokines, chemokines, leukotrienes, and prostaglandins, together with reactive oxygen and nitrogen species. their synthesis and release are severely altered, which participate in the different phases of the inflammatory process that take place in the gut. considering all the above, ibd treatment pursues two clear goals : firstly, to promote the symptom remission during the acute flare, and secondly, to maintain the remission and control the chronic inflammation to prevent or hold up the reactivation of the intestinal inflammatory process. it is evident that suppression of the exaggerated immune response is crucial for the management of ibd patients. actually, this is the major aim of the pharmacological therapy, which includes aminosalicylates (sulfasalazine or mesalamine), immunosuppressants (glucocorticoids, azathioprine, methotrexate, and cyclosporine a), and biologicals (infliximab or adalimumab). nevertheless, despite the efficacy shown by these drugs, the important rate of side effects may even limit their necessary long - term use. therefore, the development of new therapies that combine efficacy and safety in human ibd therapy is needed. in this regard, the use of alternative therapies has emerged as a common approach in gastrointestinal diseases ; actually, a study described that almost half of ibd patients have ever taken or currently use complementary remedies. different factors may contribute to this situation, including the lack of a complete response to standard therapy and the general feeling about a better safety profile of traditional remedies, in combination with the appreciation of an improved control of their disease [1012 ]. there are many different types of alternative and/or complementary therapies, although the botanical drugs are very relevant for the treatment of the intestinal inflammation. this can be mainly related to their safety, since they have been taken from ancient times, in addition to their reputed efficacy, most probably due to the presence of different active components that can concurrently target several pathways or mediators of the inflammatory response. however, most of these uses have an empirical basis, and in consequence, it is necessary to properly evaluate these botanical drugs to consider them as an adequate strategy to treat ibd. the aim of the present review is to provide scientific arguments that would support the use of medicinal plants as alternative and/or complementary therapy in human ibd. for this purpose, we have focused our attention on those botanical drugs evaluated in human ibd by clinical trials, most of them based on preclinical studies performed in experimental models of colitis. in addition, the mechanisms that may be involved in their intestinal anti - inflammatory effects will be analysed, as well as the main components that can account for the reputed beneficial effects, with a special consideration to polyphenols, including flavonoids, phenylpropanoids, and stilbenes. in fact, these compounds have been well characterized by their antioxidant properties that may prevent the damage caused by reactive oxygen and nitrogen species, which have been proposed to be key for the pathogenesis of these intestinal diseases. the physical barrier of the intestinal epithelium is complemented by a well - evolved mucosal innate immune system, which is poised to defend against pathogenic incursions, and limits inflammatory responses to maintain a state of hyporesponsiveness to commensal bacteria. the epithelial - cell layer is comprised of absorptive and secretory cells, goblet cells, and paneth cells. goblet cells contribute to the formation of the protective mucus layer. under physiological conditions, lamina propria hosts a large number of different immune cells, including macrophages (m), dendritic cells (dcs), mast cells, neutrophils, eosinophils, natural killer (nk), nkt cells, and t and b cells. all of these cells coexist in perfect equilibrium that confers tolerance and protection at the same time (figure 1). the presence of either pathogenic bacteria or the disruption of the epithelial - cell barrier may result in inflammation and dysregulation of mucosal homeostasis : cells of the innate immunity, such as macrophages and dendritic cells, are specialized in identifying microorganism 's molecular patterns by using the pattern recognition receptors (prr), such as toll - like receptors (tlr). moreover, newly recruited monocyte - derived macrophages (and other innate host defense cells) generate cytokines and chemokines to recruit monocytes and other leukocyte populations to contain the inflammation. when innate immunity is no longer able to counteract the pathogen aggression, the adaptive immune response is triggered : dcs migrate to the mesenteric lymph nodes, where they present the antigen to naive t cells and, depending on the factors released by dcs, induce the t cell differentiation. t cells are key players of adaptive immune response that cooperate with other cells and molecules from innate immune system to generate an effective response in order to eliminate the invading pathogens. upon contact with apcs, naive cd4 + cells have the potential to differentiate into different t helper (th) subtypes ; this process is controlled by the effector cytokines produced by apcs : in presence of il-12 into th1 and in presence of il-4 into th2, with il-10 and tgf-, they induce regulatory t cells (itreg) and with il-6, il-1, and tgf-, they induce th17 cells [3235 ]. each th subtype exerts specific functions : th1 cells are essential to eliminate intracellular pathogens, th2 cells mediate allergic reaction and they confer protection against parasites ; and th17 cells contribute to removing extracellular bacteria and fungi [36, 37 ]. then, the interaction between t and b leads to the production of antibodies upon the contact with t cell or dc [38, 39 ]. although their main function is the antibody production, b cells can also act as antigen presenting cell and, moreover, they are able to produce cytokines and are involved in maintaining mucosal immune homeostasis. furthermore, activated leukocytes, such as neutrophils, which are infiltrated in the inflamed mucosa not only generate different proinflammatory cytokines, but also induce oxidative reactions, which markedly alter the redox equilibrium within the gut mucosa, and maintain inflammation by inducing redox - sensitive signalling pathways and transcription factors. moreover, several inflammatory molecules generate further oxidation products, leading to a self - sustaining and autoamplifying vicious circle, which eventually impairs the gut barrier. in consequence, and as a result of this complex immune cell activity, ibd, similarly to other inflammatory conditions, is characterized by the involvement of a broad spectrum of inflammatory mediators, including cytokines, chemokines, leukotrienes, and prostaglandins, which actively participate in all the phases of the inflammatory process : initiation, progression, and resolution, when it occurs. during the active phases of ibd, some chemokines are consistently increased : il-8 and its receptor, monocyte chemoattractant protein- (mcp-) 1 and mcp-3, and macrophage inflammatory proteins (mip). chemokines mediate the recruitment of leucocyte effector populations to the sites of immune reaction and tissue injury since they tightly control leukocyte adhesion and migration across the endothelium, being also able to trigger multiple inflammatory actions including leukocyte activation, granule exocytosis, production of metalloproteinases for matrix degradation, and upregulation of the oxidative burst. similarly, the upregulated expression of different adhesion molecules in ibd, such as the intercellular adhesion molecule- (icam-) 1, the lymphocyte function - associated antigen- (lfa-) 1, the macrophage 1 antigen (mac-1), the vascular cell adhesion molecule- (vcam-) 1, the very late antigen- (vla-) 4, and p- and e - selectins, collaborates in the recruitment of granulocytes and lymphocytes through blood vessels. the fact that these mediators control t - cell differentiation and regulation has made them to be considered as central points of potential intervention to control the inflammatory response. il-12, il-18, and il-23 have a crucial function in th1 differentiation and chronic activation, whereas other cytokines, such as tnf-, il-1, and il-6, augment the inflammatory response by recruiting other cells and enhancing the production of inflammatory mediators. moreover, il-23 is induced by pattern recognition receptors (prrs), whose sustained activation drives chronic intestinal inflammation. although it was initially linked to the preferential expression of th17 responses, it can promote a wide range of pathological responses in the intestine, mediated either by t cells or by excessive innate immune activation. il-23-mediated enhancement of th1 and th17 responses is consistent with the increased levels of ifn-, il-17, and il-22 observed in the chronically inflamed intestine [46, 47 ]. when considering the il-17 cytokine family, a group of cytokines that includes at least six members, il-17a, il-17b, il-17c, il-17d, il-17e (or il-25), and il-17f, acts both in vitro and in vivo as potent proinflammatory cytokines. il-17 can induce the expression of proinflammatory cytokines (like il-6 and tnf-), chemokines (including keratinocyte chemoattractant (kc), mcp-1, and mip-2), and matrix metalloproteases, which mediate tissue infiltration and tissue destruction, thus playing a key role in human ibd. finally, it has been reported that an altered production of anti - inflammatory cytokines, including il-10 and tgf-, can account to the pathogenesis of ibd, because they are considered as key regulators of immunological homeostasis and inflammatory responses in the gut [51, 52 ]. the growing interest about the potential role that medicinal plant extracts may play in intestinal inflammatory conditions has promoted the development of different clinical studies, thus trying to evaluate their potential efficacy and safety. it is important to note that nowadays most botanical drugs go through a similar rigorous testing as pharmaceutical medicines, in an attempt to avoid inconsistent conclusions. unfortunately, different factors associated with the design, execution, and interpretation of these clinical trials still make it difficult to easily get clear conclusions with the different strategies to be evaluated against these pathologies. among these factors, the clinical heterogeneity of both intestinal conditions, uc and particularly cd, can be highlighted, as well as the selection of appropriate therapeutic end points to evaluate the efficacy. in spite of all these concerns, there are positive examples of successful human - controlled trials within the literature of botanical drugs. although the preclinical studies reporting the beneficial effects of plant extracts on experimental models of colitis are numerous, only a few plant extracts have been used in different clinical assays, which will be described in the present review (table 1). the main botanical drugs were aloe vera, andrographis paniculata, artemisia absinthium, boswellia serrata, cannabis sativa, and curcuma longa. aloe vera (xanthorrhoeaceae) is a tropical plant used in traditional medicine all over the world, mainly for its gel, which is the leaf pulp mucilaginous, aqueous extract. the aloe vera juice has been reported to exert anti - inflammatory activity ; therefore, it has been empirically used for the treatment of uc patients (table 1), being considered as the most popular botanical drug. this was confirmed when the gel was tested in the dextran sulphate sodium (dss) model of experimental colitis in rats, since it produced an amelioration of the colonic tissue injury induced by dss, being related to a downregulation of the inflammatory mediators, including cytokines, and attenuation of the immune cell recruitment. among the different components of the gel, which comprise acetylated mannans, polymannans, anthraquinone c - glycosides, anthrones, anthraquinones (emodin), and lectins, with reputed biological activities, in the same study it was proposed that the chromone aloesin seemed to be essential for the control of the intestinal inflammatory process. it was reported that aloesin, mainly, but also aloin and aloe - emodin (figure 2) were able to reduce myeloperoxidase (mpo) activity, an enzyme involved in neutrophil activity, an effect that can account in inhibiting the progression of ibd. moreover, aloesin is a strong inhibitor of leukotriene b4 (ltb4) that can activate and recruit the inflammatory cells in the injured tissue. also, these compounds significantly decreased the expression of proinflammatory cytokines, like tnf- and il-1, in the colonic segment in a dose - dependent manner, being aloesin again the most effective. it is known that this chromone derivative blocks the activation of the nf-b pathway, thus inhibiting the expression of related proinflammatory genes, including tnf-. based on the effect observed in retina ganglion cells, the anti - inflammatory effect of aloe - emodin and aloin metabolite, in dss rat colitis, maybe is due to map kinase pathways phosphorylation inhibition. andrographis paniculata (acanthaceae) can be mainly found in india and sri lanka, as well as in south and south - eastern asia, where its extracts are used as anti - inflammatory remedies. hmpl-004 is a proprietary extract from this plant that has been evaluated for its intestinal anti - inflammatory effects in human trials [15, 16 ] (table 1). the phytochemical analyses of the extracts from andrographis paniculata reveal that the main known components are diterpene lactones, principally andrographolide (figure 3) and its derivatives, which have been reported to exert anti - inflammatory properties through inhibition of the transcription factor nf-b [59, 60 ]. particularly, andrographolide reacts with reduced cysteine 62 of p50 subunit forming a covalent adduct blocking the bond of nf-b oligonucleotide to nuclear proteins. nf-b activation promoted the increased expression and synthesis of different proinflammatory mediators involved in the inflammatory response associated with ibd, including chemokines, cytokines, and adhesion molecules, in the different cell types that participate in the altered immune response in these intestinal conditions. for instance, when stimulated - endothelial cells were treated with andrographolide, the reduction of adhesion molecule e - selectin was observed preventing the e - selectin - mediated leukocyte migration. andrographolide is also able to suppress the expression of inducible nitric oxide synthase (inos) as observed in raw 264.7 cells and, as a consequence, there is a reduction of no production. this inhibition of no is due to the blockage of the synthesis and also to the reduction of the stability via a posttranscriptional mechanism. andrographolide prevents the reactive oxygen species (ros) production by neutrophils through the modulation of a protein kinase c- (pkc-) pathway. this confers to andrographolide the capacity to downregulate leukocyte integrin mac-1 (m2 cd11bcd18) that it has been reported to be upregulated by ros. besides, andrographolide exerts immunomodulatory properties, most likely affecting the innate immune cells, including macrophages and dendritic cells, but also t cells, by downregulating the production of proinflammatory cytokines [6466 ]. it has been observed that andrographolide reduced significantly, in a dose - dependent manner, the ifn- production in concanavalin a - stimulated murine t cell in vitro, whereas its effects on il-2 inhibition were partial. moreover, it can reduce the erk1/2 phosphorylation that is associated with a reduction of ifn production. in another study, the ability of andrographolide to interfere with the dcs maturation and with their capacity to present antigen to t cells has been showed. when the dcs were treated with the compound and then were mixed with lymphocytes for allogeneic stimulation, il-2 release and proliferation were reduced. the extract of a. paniculata also contains andrograpanin (figure 3), a hydrolysate of neoandrographolide (another bicyclic diterpenoid lactone), which also showed anti - inflammatory activity. it was able to reduce the mrna expression of several genes, including tnf-, il-16, il-12p35, and il-12p40, in a dose - dependent manner. in particular, the reduction of il-12p35 and il-12p40 proteins was lower than their mrna levels, suggesting that andrograpanin applies the major changes at a posttranscriptional level for these two genes. all these studies could justify the inhibitory effects that the extracts of andrographis paniculata may exert on multiple immune cells (dc, macrophages, and t cells) that are implicated in disease development and progression in uc and cd. supporting this, michelsen. reported the intestinal anti - inflammatory effects of the extract hmpl-004 in a t cell driven experimental model of chronic colitis, by inhibiting the proliferation and/or differentiation of nave t cells, as well as the th1/th17 responses that are activated in intestinal inflammation, being these effects associated with reduced expression of the different proinflammatory cytokines, including tnf, il-1, ifn, and il-22. artemisia absinthium (compositae), commonly known as wormwood, is widely distributed all over the world and it is described in different pharmacopoeias, with leaves and stems being used for medicinal purposes. this botanical drug is usually standardised based on its content in the dimeric guaianolides absinthins, being considered as a high - quality wormwood when it contains at least 0.2% of absinthin. two different clinical trials have reported the beneficial effect of this botanical drug in cd [17, 18 ] (table 1). tnf- is considered to play a key role in the pathogenesis of cd, which supports the high efficacy obtained with the biologicals acting as tnf- inhibitors, like infliximab and adalimumab, for severe cases of cd. the results obtained in these clinical trials showed that wormwood administration promoted the clinical improvement of the symptoms in all the patients, whereas no amelioration in the disease was observed in the placebo group. the beneficial effect induced by wormwood was associated with a significant decrease in tnf- serum levels in comparison with those obtained in the placebo group. the suppression of tnf-, as well as of others proinflammatory cytokines like il-1 or il-6, by wormwood extracts has been reported in vitro as well. among the components of artemisia absinthium, the flavonoid 5,6,3,5-tetramethoxy 7,4-hydroxyflavone (p7f) (figure 4) has been isolated that has been shown to exert anti - inflammatory effects. in fact, p7f was able to inhibit the expression of cox-2 and inos in lps - stimulated raw 264.7 cells. as a consequence of this inhibition, a reduced production of pge2 and no in the same cells has been observed. moreover, p7f decreased the activation of nf-b, induced by lps, probably through its antioxidant properties. another compound also isolated from this plant, 20,40-dihydroxy-60-methoxychalcone, known as cardamonin (figure 4), has shown to dose - dependently inhibit no release and inos expression in lps - stimulated raw 264.7, as well as nf-b activation. all these results would support the use of artemisia absinthium, at least as complementary therapy, in ibd. the oleo - gum resin from boswellia serrata (burseraceae), or indian frankincense, is a traditional ayurvedic remedy used to treat inflammatory diseases, including uc. as claimed by a survey performed in germany, approximately 36% of ibd patients have been administered with boswellia serrata extracts to treat their intestinal condition, reporting positive therapeutic effects, and they have been assayed in different clinical trials [1921 ] (table 1). among the different chemical compounds of this resin, triterpenes are the most abundant (3060, depending on its origin), with boswellic acids being the major constituents (figure 5), which are thought to largely contribute to the pharmacological activities such as anti - inflammatory and antiarthritic effects ascribed to this crude drug. in vivo experiments performed in the acetic acid model of rat colitis have proposed that the antioxidant properties of the extracts from boswellia serrata may also account for their intestinal anti - inflammatory effect [74, 75 ]. it has been shown that boswellia serrata extract reduced the lipid peroxidation, while it increased the levels of superoxide dismutase (sod), thus ameliorating the oxidative stress associated with intestinal inflammation. thus, in vitro experiments have shown that these compounds decreased the leukotriene formation by blocking the 5-lipoxygenase pathway which can account in the beneficial effect showed by this botanical drug since leukotrienes have been clearly involved in the pathogenesis of ibd. similarly, the anti - inflammatory effects of boswellic acids seem to involve the inhibition of different cellular pathways including those related to the transcription factor nf-b activation, which has been described to induce the expression and/or to activate the function of many proinflammatory cytokines, like tnf, il-1, and il-6, that are crucial for the development and the maintenance of intestinal inflammation [77, 78 ]. surprisingly, contradictory results have been reported when the effectiveness of boswellia extracts was assayed in dss- or trinitrobenzene sulfonic acid- (tnbs-) induced experimental models, since no efficacy was demonstrated in ameliorating colitis in these models. furthermore, in the same study, the ability of different boswellic acids to enhance the basal and the il-1-stimulated nf-b activity in intestinal epithelial cells was demonstrated, as well as the potential hepatotoxic effect of boswellia, claiming for an special attention with the use of this botanical drug in ibd and other inflammatory disorders. on the contrary, a semisynthetic form of acetyl-11-keto--boswellic acid lessened the disease activity index in dss colitis, and it managed to diminish the recruitment of leucocytes and platelets, maybe due to its ability to prevent p - selectin upregulation, thus protecting colonic mucosa from dss insult. the beneficial effects showed by this derivative were similar to those obtained with the standard corticosteroid dexamethasone. cannabis sativa (cannabaceae) has been long employed for the treatment of different diseases, especially for chronic pain and different neurological conditions [81, 82 ]. moreover, this botanical drug has treated different gastrointestinal conditions including anorexia, emesis, abdominal pain, diarrhea, and diabetic gastroparesis. it has been reported to contain over 60 different cannabinoid compounds, which are responsible for the biological activities reported for cannabis sativa. in addition, experimental evidence suggests that the endogenous cannabinoid system is involved in most of the major immune events, including those located in the gastrointestinal tract [85, 86 ]. for this reason, it was proposed that the activation of this system by cannabinoids might have a therapeutic role in human ibd. however, and although its use is common in ibd patients, there are few controlled studies that evaluate the exact role of cannabis in ibd [22, 23, 88 ] (table 1). the mechanisms involved in the intestinal anti - inflammatory effects of cannabis can be related to the capacity of cannabinoids to downregulate the production and release of different proinflammatory mediators including tnf, il-1, and nitric oxide, thus restoring the altered immune response that occurs in ibd. most probably, these effects would be related to cannabinoid receptors type 1 (cb1) activation that mediates essential protective signals and counteracts proinflammatory pathways, since it has been reported that the severity of two different experimental models of colitis, induced by the intrarectal infusion of 2,4-dinitrobenzene sulfonic acid (dnbs) or by oral administration of dss, are higher in cb1-deficient mice (cb1(/)) than in wild - type. lack of cb1 receptors rendered mice more sensitive to inflammatory insults, indicating a protective role of the cb1 receptors during inflammation induction. consistently, the administration of a specific cb1 antagonist to these wild - type mice before colitis induction resulted in a similar degree of intestinal damage to that seen in cb1(/) mice, whereas the administration of a cannabinoid receptor agonist protected from dnbs - induced colitis in mice. supporting these observations, both -tetrahydrocannabinol (thc) and cannabidiol (cbd) (figure 6), two of the main components of cannabis, were able to exert beneficial effects in the tnbs model of acute colitis in rats, with a similar efficacy to that shown by sulphasalazine, which was used as a positive control. particularly, treatment with thc and combined treatment with cbd was able to reduce macroscopic damage score. both alone and in combination, thc and cbd reduced the mpo activity similarly to treatment with sulphasalazine. unfortunately, the main concern with the use of cannabis in ibd can be derived from the indiscriminate binding of the cannabinoids to the receptors in the central nervous system, which may result in serious side effects including dizziness, dry mouth, nausea, fatigue, somnolence, euphoria, vomiting, disorientation, drowsiness, confusion, loss of balance, and even hallucination. it is evident that the manipulation of the endocannabinoid system can be helpful for the management of human ibd ; however, additional research needs to be carried out to consider cannabis (and cannabinoids) as a suitable medicine in these gastrointestinal conditions. this may be the case of cannabidiol, which possesses anti - inflammatory and immunomodulatory properties, as demonstrated in experimental models of rodent colitis, although it lacks of any cognitive or psychoactive effects. it is an indian spice obtained from the rhizomes of the plant, which has been long used in ayurvedic medicine for the handling of different inflammatory diseases. curcumin (figure 7) is the main active component of turmeric that also provides its vibrant yellow colour. numerous pharmacological activities have been reported for curcumin, including antioxidant, antimicrobial, anticancer, and anti - inflammatory properties. regarding the latter, curcumin has been suggested to exert positive effects in ibd, although just a few clinical studies have shown the power of curcumin to prevent and/or ameliorate this condition. clinical studies showed that the concurrent administration of curcumin and standard drugs improved their efficacy [24, 25 ] (table 1) and seemed to be well tolerated after a prolonged administration, which can be considered as a secure medication for maintaining remission and preventing relapse. the preclinical studies performed with curcumin have shown its efficacy in different experimental models of colitis, either chemically induced or in knockout mice, and following different protocols of administration, that is, preventative or curative [97102 ]. besides, all these studies have rendered important information about the possible mechanisms implicated in these beneficial effects. among these, the antioxidant properties of curcumin, derived from its ability to scavenge free radicals, may play a prominent role, given the oxidative stress that characterizes these intestinal conditions. in addition, curcumin can also modify multiple signaling pathways, especially the kinases mapk and erk, thus affecting the expression of different proteins implicated in the intestinal inflammatory cascade, like mpo, cox-2, inos, or lox. additionally, a modulation of the altered immune response has been proposed to occur after curcumin administration in experimental colitis, by attenuating the production of proinflammatory cytokines, like tnf, il-1, il-12, or ifn, but increasing of the expression of anti - inflammatory cytokines [98, 101 ]. other mechanisms involved in the beneficial effects of curcumin can be related to an inhibitory effect of nf-b activity. in fact, it has been reported that elevated levels of nf-b in ibd proportionately amplified the production of inflammatory cytokines and resulted in mucosal damage, which in turn can upregulate the production of this transcription factor, promoting a recurring feedback loop of inflammation. in consequence, the ability of curcumin to modulate nf-b activation may prevent the inflammatory response, thus preventing colonic mucosa damage. in conclusion, the results reviewed show that botanical drugs might prompt clinical remission and a clinical response in ibd patients. in particular, botanical drugs significantly induce clinical remission in cd patients and clinical response in uc patients ; however, there was not a significant induction of clinical remission in uc patients and an obvious clinical response in cd patients. the results of subanalyses taking into account the plant type demonstrate that only artemisia absinthium and boswellia serrata were able to induce clinical remission, while aloe vera induced a clinical response. however, none of the plants confirmed endoscopic or histological efficacy. on the other hand so, although some of the botanical drugs may have clinical efficacy in patients with ibd, there is too limited evidence to make any strong conclusions. however, botanical drugs could still be safer than synthetic drugs, despite the fact that they are not completely devoid of risk. the potential additional benefits of botanical drugs could be that patients accept them, besides their efficacy, acceptable safety, and comparatively low cost. patients all over the world seem to be prone to use botanical drugs, and their efficacy has been now evaluated in multitude of clinical trials in the management of uc. however, the evidences are still partial, intricate, and puzzling and unquestionably related to both benefits and side effects. first of all, there should be a deeper knowledge of their composition, the active compounds that are responsible of their properties, and their side effects or toxicity. it is also important to control the harvest of the right plants, their quality, and the later processing to ensure the stability of the active components. therefore, there is a need for a regulation that establishes the quality standards of the botanical drugs that are sold. secondly, further controlled clinical trials, with larger number of patients, are required to evaluate the potential efficacy and toxicity of botanical drugs in the treatment of uc. in this regard, it is also important to let the doctors know any evidence so they can prescribe these botanical drugs with the maximal guarantee. | crohn 's disease and ulcerative colitis are the two most common categories of inflammatory bowel disease (ibd), which are characterized by chronic inflammation of the intestine that comprises the patients ' life quality and requires sustained pharmacological and surgical treatments. since their aetiology is not completely understood, nonfully efficient drugs have been developed and those that show effectiveness are not devoid of quite important adverse effects that impair their long - term use. therefore, many patients try with some botanical drugs, which are safe and efficient after many years of use. however, it is necessary to properly evaluate these therapies to consider a new strategy for human ibd. in this report we have reviewed the main botanical drugs that have been assessed in clinical trials in human ibd and the mechanisms and the active compounds proposed for their beneficial effects. |
orthodontics is a specialty that relies heavily on patient s cooperation for a successful end result.1 personal perception of the need for orthodontic treatment may be influenced by variety of social, economical, and cultural factors.2 during the last 25 years, the kingdom of saudi arabia has had a marked increase in population and a massive development in various aspects of life.3 consequently an increase in the demand for health services including oral health became evident.3 as growing public interest in oral health increased the demand for orthodontic treatment also became more noticeable in dental practices.4 demand for orthodontic therapy may be influenced by the patient s perceived need for treatment and the anticipated improvement in self - image.5,6 lew has stated that the practitioners should focus their attention beyond orthodontic mechanotherapy to the most subjective aspects of patient s discomfort, and attitude toward treatment.7 several studies have concentrated on clarifying the role of malocclusion on an individual s perception and satisfaction with dental or facial appearance.8 - 10 enhancing appearance and improving psychological status have been identified as an important motivating factors behind the decision to initiate orthodontic treatment.5,11 - 15 traditional methods of estimating orthodontic treatment need are mainly based on normative need assessed by professionals using occlusal or cephalometric measurements. this shortcoming is serious because there are considerable difference between professional and patient s perceptions of dental appearance and need for the treatment.16,17 while the orthodontist prioritizes the function and occlusion in consultation, the patient might perceive other factors to be equally important to initiate the treatment.18 as the ultimate goal of a health service is to meet the public needs, professional measurements can be supplemented band related to individual s self - perception of occlusion and need for treatment.19 the objective of the study is to determine whether there is any correlation between demand and need for orthodontic treatment among patients in sattam bin abdulaziz university (sau) dental college clinic. this study also provides a baseline data on the demand and need for orthodontic treatment among a saudi population, which is important for planning public orthodontic dental services in the kingdom. for the first time in the history of prince sau dental college specialty clinic, a cross - sectional survey was carried out to determine whether there is any correlation between demand and need for orthodontic treatment at the university dental college orthodontic specialty clinic. in total 107 subjects were given to the questionnaire of orthodontic demand and only 100 (94%) eventually responded out of which 75 (75%) were males and the rest 25 (25%) were females. the research instrument was a self - administered questionnaire for the demand part and index of orthodontic treatment need (iotn) to assess the orthodontic treatment need. this survey was completed during regular orthodontic specialty clinical sessions, with an average time of 10 min. the questionnaire was carefully designed to maximize the response rate and minimize the missing data, the attitudes to malocclusion, and orthodontic treatment demand were determined from the response to fixed choice questionnaire with six open questions to know the orthodontic treatment need. the questions on demand were selected from a previous similar study, and adapted to the prince sau dental college context. the iotn is one of the most widely used occlusal indices worldwide.20 data were analyzed using the statistical software program, predictive analytics software statistics version 22.0 (spss inc., chicago, il, usa). samples included in the study were saudi nationals with angle s class i, class ii, and class iii malocclusions, with the age limit of 10 - 30. the subjects excluded were non saudi arabian nationals, and subjects below the age of 10 years, and above the age of 30 years, subjects with vertical and horizontal periodontal bone loss, mentally retarded, and behavior disorder that reduced their ability for self - determination, as well as those who disagree to participate or whose legal representatives did not authorize the participation in the study. ethical clearance was obtained from the college ethical clearance committee 2 weeks prior to the beginning of the study. tables 1 - 3 and graphs 1 - 4 shows the distribution of response obtained to the first questionnaire (the questionnaire on demand) on the attitude of subjects to malocclusion between males and females. as shown in table 1 and graph 1, 21.3% of males and 48% of females expressed the satisfaction with the arrangement of their teeth mean while 64% of males and 52% of females expressed dissatisfaction with the arrangement of their teeth and 14.6% of males and none of the females expressed that they are not sure regarding the arrangement of their teeth. as shown in table 2 and graph 2, 82.66% of males and 84% of females expressed that their teeth needed to be straightened while 4% of males and females expressed that their teeth needed not be straightened, and 13.33% of males and 12% of females expressed that they are not sure whether to straighten their teeth. as shown in table 3 and graph 3, 86.66% of males and 92% of females expressed that well aligned teeth are important for overall facial appearance while 2.66% of males and 4% females expressed that well aligned teeth are not important for overall facial appearance, while 8% of males and 4% of females expressed that they have no opinion regarding this matter. as shown in graph 4, 74.66% of males and 84% females expressed that they came to the clinic at their own wish, while 22.66% of males and 16% of females expressed that they had come to the clinic at the opinion of their parents and 2.66% of males and none of the females expressed that they had come to the clinic at the opinion of their relatives and friends. using spearman s rank correlation coefficient (tables 4 - 6) the following results are obtained regarding the correlation between demand and need for orthodontic treatment among patients in sau dental college clinic. demand for orthodontic treatment : do you consider well aligned teeth important for an overall facial appearance ? correlations using spearman s rho for demand and need among study subjects. a significant correlation (0.482) was observed for female patients with orthodontic demand (2) and treatment need (1) at 0.05 level of significancea significant correlation (0.268) was observed in male patients with orthodontic demand (2) and treatment need (1) at 0.05 level of significancea significant correlation (0.325) was observed in male patients with orthodontic demand (4) and treatment need (5) at 0.05 level of significancea significant correlation (0.326) was observed for study subjects (both males and females) with orthodontic demand (2) and treatment need (1) at 0.05 level of significancea significant correlation (0.240) was observed for study subjects (both males and females) with orthodontic demand (2) and treatment need (2) at 0.05 level of significancea significant correlation (0.292) was observed for study subjects (both males and females) with orthodontic demand (4) and treatment need (5) at 0.05 level of significance a significant correlation (0.482) was observed for female patients with orthodontic demand (2) and treatment need (1) at 0.05 level of significance a significant correlation (0.268) was observed in male patients with orthodontic demand (2) and treatment need (1) at 0.05 level of significance a significant correlation (0.325) was observed in male patients with orthodontic demand (4) and treatment need (5) at 0.05 level of significance a significant correlation (0.326) was observed for study subjects (both males and females) with orthodontic demand (2) and treatment need (1) at 0.05 level of significance a significant correlation (0.240) was observed for study subjects (both males and females) with orthodontic demand (2) and treatment need (2) at 0.05 level of significance a significant correlation (0.292) was observed for study subjects (both males and females) with orthodontic demand (4) and treatment need (5) at 0.05 level of significance our results clearly show that patients with high orthodontic demand are at a high priority for orthodontic treatment. there was a gender difference in the subjective assessment of dental appearance in this sample of saudi arabian patients with 48% females, and 21.3% of males expressed satisfaction with the arrangement of their teeth. this frequency is not consistent with satisfaction of 61.9% and 63% reported in polish and latvian children by grzywacz,21 and liepa.22 respectively. 52% of the female subjects and 64% of the male subjects felt their teeth arrangement was unsatisfactory while none of the female subjects and 14.6% of the male subjects were undecided, but graber and lucker,23 reported that small percentage of american children who considered their teeth to be unattractive. a statistical significant correlation was determined for female patients with orthodontic demand (2) as they were in significant requirement of treatment needs (1) (less demand shows less need), and a significant correlation was determined for male patients with orthodontic demand (2) as they were in significant requirement of treatment needs (1) (less demand shows less need), and significant correlation was determined for male patients with orthodontic demand (4) as they were in significant requirement of treatment needs (5) (more demand shows more need), a significant correlation was determined for patients with orthodontic demand (2) as they were in significant requirement of treatment needs (1). patients with demand need 2 require minor or no orthodontic treatment, and a significant correlation (0.292) was determined for the patients with orthodontic demand (4) as they were in significant requirement of treatment needs (5). salonen.,24 had reported that the awareness of malocclusion was higher among younger than older subjects and among those who had severe malocclusion. however, studies have shown that dissatisfaction with dental appearance was generally related to the severity of the occlusal irregularities. 92% of female subjects and 82.66% of male subjects expressed desire to straighten their teeth whereas 4% of female and male subjects were not interested in orthodontic treatment and 12% of female subjects and 13.33% of the male subjects were uncertain. this study supported the observation of gravely,25 who reported that girls were more aware of malocclusion than boys and were prepared to accept treatment. shaw26 and pietil and pietil,27 also reported that dissatisfaction with dental appearance was more common among females than males. holmes,28 in his study of english children also reported that a greater proportion of females perceived themselves as having less attractive dentitions and greater treatment need despite any objective evidence to support this view. 92% of female subjects and 82.66% of male subjects considered well - aligned teeth to be important for overall facial appearance which suggested the high awareness of dental esthetics among females than among males. further studies are awaited with wider sample size in the future to validate the results. | background : the objective of the study is to determine whether there is any correlation between demand and need for orthodontic treatment among patients in sattam bin abdulaziz university (sau) dental college clinic. this study also provides a baseline data on the demand and need for orthodontic treatment among a saudi population, which is important for planning public orthodontic dental services in the kingdom.materials and methods : an epidemiological descriptive survey was conducted using two sets of questionnaire in the orthodontic clinic of prince sau, al - kharj among saudi subjects with angle s class i, class ii, and class iii malocclusions, between the ages of 10 and 30 years for a period of 6 months with purposive sampling method.results:using spearman s rank correlation coefficient a significant correlation (0.482) was observed in male and female patients respectively with orthodontic demand (2) and treatment need (1) at 0.05 level of significance. a significant correlation (0.326) was observed for the study subjects (both males and females) with orthodontic demand (4) and treatment need (1) at 0.05 level of significance. a significant correlation (0.325) was observed in male patients with orthodontic demand (4) and treatment need (5) at 0.05 level of significance.conclusions:patients with the higher orthodontic demand required high treatment needs and vice versa. |
this cancer is widespread in males with 1/5 of men being affected throughout life. every year however, only 20% of them will lead to demise of the patient with an approximately 9-year reduction in expected survival. thus, prostate cancer remains the second cause of oncologic death in males in europe and in the usa. because of patient - to - patient heterogeneity in the clinical behavior of this disease, prognostic markers that may help tailor therapeutic strategies to individual clinical situations are continuously re - assessed. this frequent reassessment leads to modifications of clinical criteria utilized for the selection of therapeutic strategies which, in turn, make difficult the comparison and interpretation of clinical results among different therapeutic strategies. in particular, there is a well known discrepancy between post - surgical progression after radical prostatectomy and pathological staging. often local or systemic progression is observed in patients theoretically at low risk such those staged as pt2n0. thus, current pathological staging can not comprehensively evaluate neoplasm extent, aggressiveness and risk of progression. clinical recurrence after surgical excision could be ascribed to microscopic nodal metastases and/or micro - invasion at the margins of resection missed by pathological staging. to complement pathological staging, new diagnostic and prognostic markers (shbg, p53, bcl2 human callicrein-2, psma, and so forth) are under evaluation. reverse tanscriptase polymerase chain reaction (rt - pcr) for determination of prostate - specific antigen (psa) can detect a single prostate cancer cell in a tissue sample. the high sensitivity of this technique may increase the accuracy of staging in low tumor burden cases and, consequently, be a more appropriate molecular marker for the determination of the effectiveness of a neoadjuvant therapy such as maximum androgenic blockage (mab) before radical prostatectomy. in fact, this therapeutic approach is believed to induce a down staging of the disease. however, it has been questioned whether pre - operative hormonal blockade may introduce a bias in pathological staging due to the iatrogenically induced tissue changes. in this study, we assessed the incidence of micro - metastases at surgical margins and pelvic lymph nodes in patients submitted to radical prostatectomy after neoadjuvant mab or to radical prostatectomy alone. we compared traditional histopathological staging to molecular detection of psa using taqman - based quantitative real - time pcr (qrt - pcr) never used before for this purpose. this is a report about an open randomized monocentric pilot study with parallel arms, approved by the ethics committee of our institution. thirty - three < 80 year - old patients were accrued from july 2000 to june 2002. all were clinically staged as having a previously untreated, histologically confirmed t1-t2 prostate cancer. patients were randomly assigned to neoadjuvant hormonal therapy plus radical prostatectomy (arm a) or radical prostatectomy alone (arm b). clinical and pathological stages were determined according to the 1997 american joint committee - international union against cancer tnm system. pre - treatment evaluation included clinical history, digital rectal examination, serum psa determination using hybritech technique, routine blood tests, trans - rectal ultrasound, confirmation of out - side diagnosis, abdominal computerized tomography and radioisotope bone scan. patients in both arms underwent pelvic lymphadenectomy and radical retro - pubic prostatectomy 55 to 70 days from randomization. neoadjuvant therapy consisted of : nilutamide 150 mg daily until surgery and subcutaneous administration of buserelin depot 9,9 mg on the day of randomization. all patients underwent radical retro - pubic bladder neck sparing prostatectomy with or without nerve sparing. bilateral pelvic (iliac and obturator) node dissection was consistently performed and one right and one left node were taken proximal to the iliac bifurcation, and preserved in liquid nitrogen for subsequent qrt - pcr analysis. in addition, the right and left lateral margins of resection were submitted for pathological evaluation after dissection of a small wedge for cryostatic preservation (at least 0,5 cm of lenght). in all cases surgeons and pathologists have checked the integrity of prostatic capsule in order to be sure that these strips had been removed outside the capsule (non containig prostatic tissue). in addition, the expression of the tumor associated antigen psa was quantified by means of qrt - pcr in surgical margins and lymph nodes. total rna was then isolated using rneasy mini kits (qiagen, santa clarita, ca) following manufacturer 's guidelines. one g of total rna was reverse transcribed into cdna using 1 l of oligo - dt primers (1 g/l, gibcobrl), 5 l first strand buffer (gibcobrl), 2 l 0.1 m dtt, 2 l magnesium sulfate (gibcobrl), 1 l of 10 mm dntp and 1 l superscript - ii (ss - ii, gibcobrl). the reaction mixture was heated to 65c for 10 minutes before adding ss - ii, then synthesis was continued with 1 hour at 42c. the expression of psa and of a housekeeping gene (i.e., -actin) was assessed by qrt - pcr as performed by the abi prism 7700 sequence detection system (perkin elmer, foster city, ca). probes and primers (see table 1) were designed to span intron - exon junctions in order to avoid genomic dna amplification. probes and primers used to generate a standard curve for each gene, 1 l of cdna obtained from normal prostate was added to the following reagents : 1 l taqman primers (10 m, perkin elmer), 5 l 10 pcr buffer (perkin elmer), 11 ml mgcl2 (25 mm), 1 ml dnpt (10 mm) and 0.5 l amplitaq gold (perkin elmer) in a final volume of 50 l. the thermal cycler conditions for this pcr reaction were as follows : after 10 minutes at 95c, 15 seconds at 95c followed by 1 minute at 60c were repeated 40 times. the equation describing the standard curves was used to extrapolate the absolute copy number of psa and -actin. quantitative real time pcr of sample cdna and standard amplified cdna was conducted in a 25 l final volume mixture containing primers, probe and 1 taqman master mix (perkin elmer) at optimized concentrations (probe : 200 nm, primers : 400 nm). the taqman abi prism 7700 sequence detection system allows running 96 samples at a time. thermal cycler parameters included 2 minutes at 50c, 10 minutes at 95c and 40 cycles involving denaturation at 95c for 15 seconds and annealing / extension at 60c for 1 minute. to ensure that probes and primers in use did not amplify genomic dna, we extracted genomic dna from a normal prostate biopsy and tested it with the taqman primers. given the small size of the amplicons (< 100 bp), a fluorimager 595 (molecolar dynamics, sunnydale, ca) scanning system was used to detect the fluorescence of the vistra green fluorescent intercalator, which was adopted instead of ethidium bromide to visualize the bands of the agarose gel after electrophoresis. an argon laser for excitation (488 nm) and an emission band - pass filter 530 df30 were applied. for qrt - pcr purposes, probes were labeled with a fluorescent reporter dye at the 5'-end (6-carboxyfluorescein, 6-fam ; emission max = 518 nm) and a quencher dye at the 3'-end (6-carboxytetramethylrhodamine, tamra ; emission max = 582 nm). probe and primers were designed to obtain amplicons less then 100 bp in length in order to enhance pcr efficiency. furthermore, they were designed to span an intro - exon junction in order to avoid genomic dna amplification. since both the amount of total rna added to each reverse transcription reaction tube (based on waive length absorbance) and its quality (i.e. degradation) are not reliable parameters as measures of starting material, transcripts of a housekeeping gene were quantified as endogenous control. beta - actin (-actin) is one of the most used aspecific housekeeping genes, but according to the experimental conditions, other internal references can be adopted (e.g. leukocyte markers). for each experimental sample the value of both the target and the housekeeping gene are extrapolated from the respective standard curve. the target value is then divided by the endogenous reference value to obtain a normalized target value independent from the starting material amount. patients were seen for follow up 50 days after surgery and every 3 months thereafter during the first year, every 6 months during the subsequent four years and then annually. at each visit serum psa statistical analysis was performed on 29 valuable patients randomized and included in the study. for each patients (arm a or b) qrt - pcr results from lateral surgical margins and pelvic nodes were analyzed. statistical evaluation and data elaboration was made using sas 's software (statistical analysis system). continuity adjust chi - squared test was used to assess differences in qrt - pcr results between arm a and b. p values of = 0.05 were considered statistically significant. this is a report about an open randomized monocentric pilot study with parallel arms, approved by the ethics committee of our institution. thirty - three < 80 year - old patients were accrued from july 2000 to june 2002. all were clinically staged as having a previously untreated, histologically confirmed t1-t2 prostate cancer. patients were randomly assigned to neoadjuvant hormonal therapy plus radical prostatectomy (arm a) or radical prostatectomy alone (arm b). clinical and pathological stages were determined according to the 1997 american joint committee - international union against cancer tnm system. pre - treatment evaluation included clinical history, digital rectal examination, serum psa determination using hybritech technique, routine blood tests, trans - rectal ultrasound, confirmation of out - side diagnosis, abdominal computerized tomography and radioisotope bone scan. patients in both arms underwent pelvic lymphadenectomy and radical retro - pubic prostatectomy 55 to 70 days from randomization. neoadjuvant therapy consisted of : nilutamide 150 mg daily until surgery and subcutaneous administration of buserelin depot 9,9 mg on the day of randomization. all patients underwent radical retro - pubic bladder neck sparing prostatectomy with or without nerve sparing. bilateral pelvic (iliac and obturator) node dissection was consistently performed and one right and one left node were taken proximal to the iliac bifurcation, and preserved in liquid nitrogen for subsequent qrt - pcr analysis. in addition, the right and left lateral margins of resection were submitted for pathological evaluation after dissection of a small wedge for cryostatic preservation (at least 0,5 cm of lenght). in all cases surgeons and pathologists have checked the integrity of prostatic capsule in order to be sure that these strips had been removed outside the capsule (non containig prostatic tissue). in addition, the expression of the tumor associated antigen psa was quantified by means of qrt - pcr in surgical margins and lymph nodes. total rna was then isolated using rneasy mini kits (qiagen, santa clarita, ca) following manufacturer 's guidelines. one g of total rna was reverse transcribed into cdna using 1 l of oligo - dt primers (1 g/l, gibcobrl), 5 l first strand buffer (gibcobrl), 2 l 0.1 m dtt, 2 l magnesium sulfate (gibcobrl), 1 l of 10 mm dntp and 1 l superscript - ii (ss - ii, gibcobrl). the reaction mixture was heated to 65c for 10 minutes before adding ss - ii, then synthesis was continued with 1 hour at 42c. the expression of psa and of a housekeeping gene (i.e., -actin) was assessed by qrt - pcr as performed by the abi prism 7700 sequence detection system (perkin elmer, foster city, ca). probes and primers (see table 1) were designed to span intron - exon junctions in order to avoid genomic dna amplification. probes and primers used to generate a standard curve for each gene, 1 l of cdna obtained from normal prostate was added to the following reagents : 1 l taqman primers (10 m, perkin elmer), 5 l 10 pcr buffer (perkin elmer), 11 ml mgcl2 (25 mm), 1 ml dnpt (10 mm) and 0.5 l amplitaq gold (perkin elmer) in a final volume of 50 l. the thermal cycler conditions for this pcr reaction were as follows : after 10 minutes at 95c, 15 seconds at 95c followed by 1 minute at 60c were repeated 40 times. the equation describing the standard curves was used to extrapolate the absolute copy number of psa and -actin. quantitative real time pcr of sample cdna and standard amplified cdna was conducted in a 25 l final volume mixture containing primers, probe and 1 taqman master mix (perkin elmer) at optimized concentrations (probe : 200 nm, primers : 400 nm). the taqman abi prism 7700 sequence detection system allows running 96 samples at a time. thermal cycler parameters included 2 minutes at 50c, 10 minutes at 95c and 40 cycles involving denaturation at 95c for 15 seconds and annealing / extension at 60c for 1 minute. to ensure that probes and primers in use did not amplify genomic dna, we extracted genomic dna from a normal prostate biopsy and tested it with the taqman primers. given the small size of the amplicons (< 100 bp), a fluorimager 595 (molecolar dynamics, sunnydale, ca) scanning system was used to detect the fluorescence of the vistra green fluorescent intercalator, which was adopted instead of ethidium bromide to visualize the bands of the agarose gel after electrophoresis. an argon laser for excitation (488 nm) and an emission band - pass filter 530 df30 were applied. for qrt - pcr purposes, probes were labeled with a fluorescent reporter dye at the 5'-end (6-carboxyfluorescein, 6-fam ; emission max = 518 nm) and a quencher dye at the 3'-end (6-carboxytetramethylrhodamine, tamra ; emission max = 582 nm). probe and primers were designed to obtain amplicons less then 100 bp in length in order to enhance pcr efficiency. furthermore, they were designed to span an intro - exon junction in order to avoid genomic dna amplification. since both the amount of total rna added to each reverse transcription reaction tube (based on waive length absorbance) and its quality (i.e. degradation) are not reliable parameters as measures of starting material, transcripts of a housekeeping gene were quantified as endogenous control. beta - actin (-actin) is one of the most used aspecific housekeeping genes, but according to the experimental conditions, other internal references can be adopted (e.g. leukocyte markers). for each experimental sample the value of both the target and the housekeeping gene are extrapolated from the respective standard curve. the target value is then divided by the endogenous reference value to obtain a normalized target value independent from the starting material amount. patients were seen for follow up 50 days after surgery and every 3 months thereafter during the first year, every 6 months during the subsequent four years and then annually. at each visit serum psa statistical analysis was performed on 29 valuable patients randomized and included in the study. for each patients (arm a or b) qrt - pcr results from lateral surgical margins and pelvic nodes were analyzed. statistical evaluation and data elaboration continuity adjust chi - squared test was used to assess differences in qrt - pcr results between arm a and b. p values of = 0.05 were considered statistically significant. one dropped out for noncompliance, one for pneumonia, one for allergy to nilutamide and one because the sample was inadequate to perform qrt - pcr analysis. of 29 patients, 15 (51,7%) underwent neo - adjuvant mab treatment (arm a) and 14 (48,3%) received prostatectomy alone (arm b). five of the 29 patients (17,2%) experienced biochemical recurrence after a mean follow - up of 19 months, all of these patients belonged to arm b. in the neo - adjuvant arm a 5/15 (33,3%) patients were staged as pt2a - g2. one of 15 patients (6,6%) was staged as pt2a - g3 and resulted positive at qrt - pcr. four of the 15 patients (26,8%) were pt2b - g2 : 2 resulted negative and 2 positive at qrt - pcr. five of the 15 (33,3%) patients were pt3b - g2 and 4 resulted negative and 1 positive to qrt - pcr (see table 2). the ratio of psa mrna expressing patients according to qrt - pcr in arm a was of 26,6% (4/15). in arm b, 4/14 (28,6%) patients were pt2a - g2 of this 3 negative and one positive at qrt - pcr analysis ; 6/14 (42,8%) patients were pt2b - g2, one negative and 5 positive to qrt - pcr ; 4/14 (28,6%) patients were pt3b - g2, one negative and 3 positive for psa expression in periprostatic tissues (see table 3) pathological and qrt - pcr data of arm b patients. overall 64,3% (9/14) of the patients had evidence of psa mrna expression in arm b, while 26.6% (4/15) resulted molecularly positive in arm a (fisher exact test = 0.04). pathological and molecular finding of the patients who had biochemical recurrence are reported in table 4. analysis was performed on patients from arm a or b whether they had or had not cumulative evidence of psa mrna expression by qrt - pcr in lymph nodes and/or lateral surgical margins. nine of 14 (64,29%) patients expressed psa in arm b and four of 15 (26,67%) in arm a (see table 5). continuity adjust chi - square (a chi - square modified for not continuous variable) was calculated and resulted in 0.0965, this value trend to statistical significance. this results suggests a difference between arm a and b, although not statistically significant (see table 6). in table 7 value founded show the risk to be positive if not treated with neoadjuvant therapy respect to treated arm ; the risk to have positive surgical margins or nodes to qrt - pcr is 2,41 higher in not treated patients (arm b). in the neo - adjuvant arm a 5/15 (33,3%) patients were staged as pt2a - g2. one of 15 patients (6,6%) was staged as pt2a - g3 and resulted positive at qrt - pcr. four of the 15 patients (26,8%) were pt2b - g2 : 2 resulted negative and 2 positive at qrt - pcr. five of the 15 (33,3%) patients were pt3b - g2 and 4 resulted negative and 1 positive to qrt - pcr (see table 2). the ratio of psa mrna expressing patients according to qrt - pcr in arm a was of 26,6% (4/15). in arm b, 4/14 (28,6%) patients were pt2a - g2 of this 3 negative and one positive at qrt - pcr analysis ; 6/14 (42,8%) patients were pt2b - g2, one negative and 5 positive to qrt - pcr ; 4/14 (28,6%) patients were pt3b - g2, one negative and 3 positive for psa expression in periprostatic tissues (see table 3) pathological and qrt - pcr data of arm b patients. overall 64,3% (9/14) of the patients had evidence of psa mrna expression in arm b, while 26.6% (4/15) resulted molecularly positive in arm a (fisher exact test = 0.04). pathological and molecular finding of the patients who had biochemical recurrence are reported in table 4. analysis was performed on patients from arm a or b whether they had or had not cumulative evidence of psa mrna expression by qrt - pcr in lymph nodes and/or lateral surgical margins. nine of 14 (64,29%) patients expressed psa in arm b and four of 15 (26,67%) in arm a (see table 5). continuity adjust chi - square (a chi - square modified for not continuous variable) was calculated and resulted in 0.0965, this value trend to statistical significance. this results suggests a difference between arm a and b, although not statistically significant (see table 6). in table 7 value founded show the risk to be positive if not treated with neoadjuvant therapy respect to treated arm ; the risk to have positive surgical margins or nodes to qrt - pcr is 2,41 higher in not treated patients (arm b). radical prostatectomy is widely performed in early prostate cancer and it may lead to cure as long as the cancer is confined to the prostate and all neoplastic cells are removed. clinical staging is inaccurate and a significative number of patients with prostate cancer defined as ct1 - 2 are found to have positive surgical margins. this in turn is associated with increased chances of cancer recurrence and progression within 5 years from the surgical resection of the primary tumor. neoadjuvant hormonal therapy may reduce the rate of margin positiveness, therefore, decreasing the chance of observing a local recurrence after radical prostatectomy. however, it has been suggested that after neoadjuvant hormonal therapy residual foci of atrophic glands can be difficult to identify with hematoxylin and eosin staining, increasing the possibility that pt may be under staged [13 - 15 ]. clinical randomized studies adopting neoadjuvant therapy before radical prostatectomy have demonstrated a reduction of the risk of leaving positive surgical margins in pt1 - 2 patients at the time of the surgical procedure and this reduced risk is proportional to the length of treatment. the primary end point of these studies was to assess whether neoadjuvant therapy can increase overall survival. indeed, there is no published evidence that patients undergoing mab before radical prostatectomy have a clear benefit. at the same time the use of pcr methods for determination of psa gene expression in surgical periprostatic samples has suggested new staging strategies based on higher sensitivity. it is now possible to obtain information about presence of prostatic tissue in extra glandular structures with more sensitivity compared with conventional histology. traditional methods to study protein or gene expression in tumor biopsies include mainly immunohistochemistry and polymerase chain reaction (pcr). these techniques are not quantitative and allow only a semi quantitative evaluation based on the subjective judgment of the observer, which makes the comparison between samples inaccurate (unreliable). flow cytometry is highly reliable to determine the levels of expression of a given antigen. however, it requires tissue desegregation procedures, which can damage cell surface antigens, and can not be performed in case of hypo cellular specimens. to measure of the amount of gene expression these techniques are cumbersome, time consuming and require multiple manipulations of the samples, increasing the risk of carrying over contamination. quantitative real - time pcr (qrtpcr) allows a highly sensitive measurement of the transcriptional levels of the gene of interest in a few hours with minimal handling of the samples. in addition, qrt - pcr is stringently sequence dependent because of the labelled probe. therefore, it yields indirect sequence confirmation that the amplified product is the gene of interest. finally, the utilization of intro - exon junction spanning probes avoids mis - calculation of gene level expression due to genomic dna contamination. this technique has been mainly used for viral load measurement, but recently it has been employed for eukaryotic studies and in particular analysis of human mrna expression in various clinical and therapeutic conditions. in this study, we adopted qrt - pcr for the sensitive identification of psa mrna level in areas where it should not be present such as positive surgical margins and/or draining lymph nodes. in our experience, neoadjuvant therapy for 3 months before surgical intervention appears to affect disease staging. in fact, according to qrt - pcr results, the rate of lymph nodes and/or surgical margins in which psa mrna could be detected was significantly lower (26.6%) in arm a (mab plus surgery) as compared to that found in arm b (surgery alone) (64,3%). all the 5 patients that experienced biochemical evidence of recurrence (17,2%) belonged to arm b. although, psa mrna was detected by qrt - pcr in 60% of patients who experienced biochemical recurrence six of the remaining 8 patients in arm b had also detectable psa mrna. this finding suggests that identification of psa mrna expression at the surgical margin or draining lymph - nodes is more a marker of the ability of the neoadjuvant therapy to sterilize the patient before surgical intervention than an independent predictor of recurrence. in fact, the most significant difference in psa mrna expression was noted between the two randomization arms where in the neo - adjuvant protocol only 4 of 15 patients were positive while 9 of 14 were positive in the surgical treatment alone arm (fisher test p2 value = 0.02). thus, qrt - pcr appears to yield useful information about the effectiveness of a given therapy and it may be more sensitive, compared to conventional histology, in identifying presence of viable prostate carcinoma cells in surgical resection margins and draining lymph nodes. finally, this study seems to confirm that neoadjuvant therapy with nilutamide 150 mg + buserelin depot for 3 months before surgical intervention induces clinically relevant down staging, at least in the short term. if these results were confirmed in a larger series of patients, we believe that gene expression of psa in surgical periprostatic samples might be taken into consideration as a novel and reliable indicator of minimal residual disease after neoadjuvant therapies. in this case, the molecular detection of cancer cells might represent an indication to adjuvant treatments in order to sterilize microscopic foci of tumor possibly responsible for local recurrence. | backgroundwe assessed the incidence of micro - metastases at surgical margins (sm) and pelvic lymph nodes (ln) in patients submitted to radical retropubic prostatectomy (rp) after neoadjuvant therapy (nt) or to rp alone. we compared traditional staging to molecular detection of psa using taqman - based quantitative real - time pcr (qrt - pcr) never used before for this purpose.methods29 patients were assigned to nt plus rp (arm a) or rp alone (arm b). pelvic ln were dissected for qrt - pcr analysis, together with right and left lateral sm.results64,3% patients of arm b and 26.6% of arm a had evidence of psa mrna expression in ln and/or sm. 17,2% patients, all of arm b, had biochemical recurrence.conclusionsqrt-pcr may be more sensitive, compared to conventional histology, in identifying presence of viable prostate carcinoma cells in sm and ln. gene expression of psa in surgical periprostatic samples might be considered as a novel and reliable indicator of minimal residual disease after nt. |
systemic sclerosis (ssc) is an autoimmune disease that involves the connective tissue of skin and internal organs with a remarkable heterogeneity in the disease course and affected organs, resulting in high morbidity and mortality. the disease is characterized by vascular dysfunction and injury and by overproduction and accumulation of collagen and other extracellular matrix proteins, resulting in the thickening of the skin and fibrosis of the affected organs [1, 2 ]. the pathogenetic mechanisms involve three interactive components represented by severe and diffuse endothelial cell damage, immune system dysfunction, and fibroblasts activation. endothelin-1 (et-1) has been described to play a role in fibrosis, angiogenesis, and inflammation, all major features of ssc [3, 4 ]. indeed et-1 level is elevated in the serum and tissues of ssc patients, especially in diffuse ssc patients, and serum levels have been shown to correlate with the extent of vascular damage and cutaneous fibrosis [36 ]. et-1 is the major isoform of three endothelin isoforms and is a soluble mediator that exerts a potent vasoconstrictor effect. et-1 has been firstly described in endothelial cells and in vascular smooth muscle cells, where hypoxia, cold exposure, low shear stress, angiotensin ii, cytokines, and growth factors may facilitate its production [7, 8 ]. many cells can produce et-1 including fibroblasts and myofibroblasts, mast cells, monocytes / macrophages, polymorphonuclear leukocytes, and dendritic cells [914 ]. transforming growth factor- (tgf-) and et-1 itself, with an autocrine loop, are able to induce et-1 production in fibroblasts and myofibroblasts [9, 15 ]. there are at least three et-1 receptors : eta, etb, and etc [8, 16, 17 ] ; however, the function of etc is poorly known ; eta and etb are expressed on the majority of cells that actively contribute to ssc pathogenesis, such as fibroblasts, myofibroblasts, vascular smooth muscle cells, and platelets, while endothelial cells selectively express etb [68, 16 ]. upon binding receptors on vascular smooth muscle cells, et-1 is able to induce vasoconstriction, cell growth, and proliferation, leading to lumen narrowing at arterial and arteriolar level [1820 ]. moreover, et-1 facilitates fibroblasts transdifferentiation into myofibroblasts and induces the production of both collagen and et-1 probably through an autocrine mechanism [9, 15, 2126 ]. there is increasing evidence that et-1 may play a pivotal role in inflammation in several human diseases including chronic renal disease, asthma [2730 ], and sepsis (reviewed in) ; however, the mechanisms by which et-1 induces the activation of the innate and adaptive immune systems have not been fully elucidated so far. saleh and pollock suggested that et-1 can directly activate neutrophils and can induce the production of chemoattractant factors, such as monocyte chemoattractant factor-1 (mcp-1), and the synthesis of cell adhesion molecules, such as soluble intercellular adhesion molecule-1 (icam-1). moreover et-1 seems to be associated with the activation of transcription factors such as nf-b and the production of proinflammatory cytokines including tumor necrosis factor - alpha (tnf-), interleukin- (il-) 1, and il-6. little is known on et-1 receptors expression on immune cells with the exception of a few data on cells of the innate immune system, such as dendritic cells and monocytes [1014, 32 ]. the expression of et-1 receptors on adaptive immune effectors cells (t and b lymphocytes) has not been investigated so far and therefore little is known on the possible role of et-1 as a mediator of inflammatory responses. in the last decade, orally active et-1 receptor antagonists (eras) were developed and approved for clinical use. two orally active eras are currently approved, the dual receptor antagonist, bosentan, and the selective eta receptor antagonist, ambrisentan [3335 ]. both eras are used in the treatment of pulmonary arterial hypertension (pah) whereas only bosentan has been shown to be effective in the prevention of new scleroderma - related digital ulcers (dus). in addition to the effects on vasculature and fibrosis [3638 ], it has been recently reported that et-1 blockade using bosentan may also have some anti - inflammatory effects (, reviewed in). in particular, bosentan seems to be able to suppress the et-1-induced production of tnf- and other proinflammatory mediators by monocytes in vitro. in vivo, bosentan significantly reduces il-6, icam-1, and pro - brain natriuretic peptide (pro - bnp) serum levels in patients with pah and leads to the normalization of soluble adhesion molecules in ssc - associated pah [40, 41 ]. inflammation is deeply involved both in the early phase of ssc pathogenesis and in the progression of vascular damage and fibrosis. therefore, we aimed at investigating the role of et-1 as possible mediator of inflammatory damage in ssc. since immune effectors cells, such as t and b lymphocytes, monocytes, and neutrophils, are important players of inflammation in ssc, we aimed at clarifying the possible role played by et-1 receptors in immune cells activation. in this paper, we studied the presence of et-1 receptors on t and b lymphocytes, monocytes, and neutrophils by facs analysis. we also analysed the effects of et-1 receptors engagement in order to verify the proinflammatory activity of et-1 and the potential anti - inflammatory effects of eras. we studied a cohort of 41 patients (5 males and 36 females, mean age : 57 14 years) affected by ssc, attending the unit of autoimmunity diseases at the university hospital of verona, italy. ssc diagnosis was performed in accordance with the american college of rheumatology / european league against rheumatism classification criteria for systemic sclerosis [42, 43 ]. patients were classified according to the following clinical features : limited (lssc) or diffuse (dssc) cutaneous form of ssc (32 patients with lssc and 9 with dssc) and presence or absence of ischemic digital ulcers, pah, and interstitial lung disease (ild). blood samples (20 ml) were collected in heparinized falcon tubes (becton dickinson, nj, usa) from both patients and control subjects. a written informed consent was obtained from all the participants to the study and the study was approved by the local ethical committee. all clinical investigations have been conducted according to the principles expressed in the helsinki declaration. blood samples obtained from patients and controls were diluted with 20 ml of phosphate buffered saline (pbs) solution. mononuclear cells isolated by density gradient centrifugation using lymphoprep ficoll - isopaque (axis - shield, oslo, norway) were washed twice with pbs and suspended in tubes containing 1 million cells for flow - cytometry (facs) analysis. analysis of monocytes and lymphocytes was carried out in different tubes ; cells used for monocytes staining were preincubated with mouse serum (dako, glostrup, denmark) for 10 minutes at room temperature. each sample was incubated for 1 hour at 4c with eitherrabbit polyclonal anti - eta (acris antibodies gmbh, herford, germany) or sheep polyclonal anti - etb (lifespan biosciences, seattle, wa, usa) antibodies. phycoerythrin- (pe-) conjugated goat anti - rabbit igg monoclonal (0.25 mg / ml) was used as a secondary antibody for eta (r&d systems, minneapolis, mn, usa) and pe - conjugated donkey anti - sheep igg monoclonal (0.2 mg / ml) was used as a secondary antibody for etb (r&d systems) and incubated for 30 minutes at 4c. samples were also stained for 20 minutes at room temperature in a dark room with allophycocyanin- (apc-) conjugated anti - cd3 or anti - cd14 or anti - cd19 antibodies (bd biosciences, san jose, ca, usa). the sensitivity of fluorescence detectors was set and monitored using calibrite beads (becton dickinson) according to the manufacturer 's recommendations ; 20.000 cd3 +, cd14 +, or cd19 cells per sample were, respectively, acquired in live gating. flowjo 8.8.2 software (tree star, ashland, or) was used to analyze the data. expression of eta or etb was calculated as the difference between mean fluorescence intensity (mfi) of cells stained with primary plus secondary antibodies and mfi of their negative control (cells stained with secondary antibodies) : mfi. in order to assess receptors expression on activated cd4 + and cd8 + t cells, pbmc isolated from 4 patients and 4 controls were stimulated for 24 hours with anti - cd3/cd28 antibodies coated microbeads - dynabeads human t - activator (dynal, oslo, norway), according to the manufacturer 's recommendations. cells were cultured in rpmi-1640-glutamax - i, supplemented with 10% fetal calf serum (fcs), 100 u / ml penicillin, and 100 microg / ml streptomycin (all purchased from life technologies, carlsbad, ca). in order to identify cd4 + and cd8 + t lymphocytes, we incubated cells with a mixture of the following antibodies : percp - conjugated anti - cd3, apc - h7-conjugated anti - cd4, and apc - conjugated anti - cd8 antibodies. activated cells were detected by incubating cells with fitc - conjugated anti - cd25 antibodies ; all reagents were purchased from bd biosciences. cells were previously stained with anti - eta and anti - etb primary and secondary antibodies as previously described and samples were acquired on a facscanto cytometer flowjo 8.8.2 software was used to analyse data. the variation in receptors surface exposure was expressed as the difference between activated cells mfi and unstimulated cells mfi (mfi). mononuclear cells from healthy donors buffy coats were isolated by density gradient centrifugation using lymphoprep ficoll - isopaque. cd4 + t cells were obtained through negative selection using cd4 + t cell isolation kit ii (miltenyi biotec) and midimacs starting kit, including macs ld column and macs separator (miltenyi biotec), following manufacturer 's instructions. total rna was extracted from cd4 + t cells using trizol reagent (gibco brl, billings, mt, usa) following the manufacturer 's protocol. first - strand cdna was carried out using the super script iii system (invitrogen, carlsbad, ca, usa), with random hexamers, according to the manufacturer 's recommendations. fibroblasts cdna was used as positive control for the detection of eta- and etb - coding mrna. cd4 + t cells and fibroblasts cdna were amplified with eta and etb specific primers : eta forward 5-atgcacaactattgcccaca-3, eta reverse 5-ggacaggatccagatggaga-3 ; etb forward 5-gcacatcgtcattgacatcc-3, etb reverse 5-cagagggcaaagacaaggac-3 (sigma - aldrich, saint louis, mo, usa). eta forward 5-atgcacaactattgcccaca-3, eta reverse 5-ggacaggatccagatggaga-3 ; etb forward 5-gcacatcgtcattgacatcc-3, etb reverse 5-cagagggcaaagacaaggac-3 (sigma - aldrich, saint louis, mo, usa). vimentin was used as pcr reaction - control. amplification was performed using the amplitaq gold pcr mastermix system (applied biosystems, foster city, ca, usa). cdna was amplified using the primers specific for eta and etb receptors and for vimentin using the geneamp pcr system 9700 thermal cycler (applied biosystems) and the amplification reaction was carried out as follows : 10 minutes at 95c followed by 40 cycles of denaturation (45 seconds at 94c), annealing (30 seconds at 53c for eta and 55c for etb and for vimentin), and extension (1 minute at 72c and 7 minutes at 72c to stop reaction). amplicons (length : 447 bp for eta, 558 bp for etb, and 266 bp for vimentin) were run on agarose gel (1.5%) and revealed using versadoc video documentation system (bio rad, hercules, ca, usa). in order to study the cytokine production in response to eta and etb stimulation by et-1 in cd4 + t cells, we seeded cd4 + cells in microplates : 1 million cd4 + t cells per well were seeded in 24-well plates and different conditions were carried out in duplicate. cells were incubated (a) without et-1 and receptors antagonists (control sample) ; (b) with et-1 alone ; (c) with eta antagonist (bq123) and et-1 ; (d) with etb antagonist (bq788) and et-1 ; (e) with bq123 plus bq788 and et-1. cells were incubated with bq123 and bq788 at the concentration of 10 m for 45 minutes and with et-1 at concentration of 10 m for 24 hours. we then measured interferon- (ifn-), il-4, and il-17 concentrations in cell culture supernatants by enzyme - linked immunosorbent assay (elisa) (quantikine human ifn- immunoassay, quantikine human il-4 immunoassay, and quantikine human il-17 immunoassay, resp. sunrise absorbance reader for microplates (tecan, salzburg, austria) was used to determine optical density for each sample. we isolated neutrophils from healthy donors buffy coat in order to study surface expression of eta and etb by flow - cytometry and the transcripts for eta and etb by rt - pcr. highly purified granulocytes (neutrophils > 96.5%) were isolated and prepared under endotoxin - free conditions using lymphoprep ficoll - isopaque. neutrophils were further enriched by positively removing all contaminating cells with mab against cd3, cd56, cd19, cd36, cd49d, and gly - a using a custom - made easy - sep kit (stemcell technologies, vancouver, bc, canada) to reach more than 99,7% purity. one million neutrophils were suspended in tubes for facs analysis. staining for eta- and etb neutrophils were seeded in microplates and incubated with or without 100 ng / ml ultrapure e. coli lipopolysaccharide (lps) (invivogen, san diego, ca) and with or without et-1. therefore we used 4 different conditions : (a) neutrophils without any stimulus as negative control, (b) neutrophils incubated with et-1, (c) neutrophils incubated with lps alone, (d) and neutrophils incubated with both et-1 and lps. cells were cultured in rpmi-1640-glutamax - i, supplemented with 10% fetal calf serum (fcs), 100 u / ml penicillin, and 100 microg / ml streptomycin. interleukin-8, tnf-, vascular endothelial growth factor (vegf), ifn-, il-17, and matrix metallopeptidase 9 (mmp-9) released in the supernatants of cultured neutrophils were assessed by elisa at two different time points (3 and 10 hours). correlations between et - a and et - b cell surface expression and clinical features were assessed with nonparametric test and multivariate analysis. t and b lymphocytes as well as monocytes and neutrophils express eta and etb on their surface, using facs analysis ; the data were obtained as a difference of mean fluorescence intensity between samples incubated with primary and secondary antibodies and their negative controls incubated with secondary antibody alone (figure 1). in addition some of the data were validated by reverse transcription - pcr in cd4 + t cells and neutrophils (figure 2). in both patients and controls, t lymphocytes and monocytes showed a higher surface expression of eta (patients : mfi = 100.61 45.21 and 212.24 64.27, resp. ; when compared to etb (patients : 46.85 29.78 and 91.14 27.44, resp. ; controls : mfi = 49.23 29.16 and 98.74 54.66, resp.) (p < 0.001) (table 1). these data indicate that surface et-1 receptors distribution on t cells and monocytes of ssc patients is similar to the one observed in healthy donors. patients affected by dssc showed a lower etb surface expression on t cells when compared to patients affected by lssc (28.6 17.9 versus 51.9 31.1) (p < 0.01) ; a similar pattern of surface expression was observed on monocytes (74.4 29.6 versus 97.2 24.5) (p < 0.05). no significant difference in eta expression by t lymphocytes and monocytes was observed in the diffuse or limited form of disease (94.8 48.2 versus 99.1 42.1 and 251.2 116.3 versus 199.3 34.5, resp.). eta and etb surface expression were not modified by bosentan treatment, both on t cells (eta : 97.9 52 versus 102.6 44.3 ; etb : 47.9 17.7 versus 47.6 32.3) and on monocytes (eta : 240.7 130.6 versus 205.1 34.9 ; etb : 89.6 22.9 versus 93.1 27.7), suggesting that bosentan therapy does not induce an increased et-1 receptors expression. eta and etb surface expression on t cells and monocytes did not correlate with the presence or absence of dus (t cells : 121.4 69 versus 98.8 41.6 and 40.8 20.1 versus 48.6 31, resp. ; monocytes : eta : 221 4.3 versus 211.3 69.6 ; etb : 80.4 25.3 versus 93.6 27.5) (table 2). patients with pah had a lower etb surface expression on monocytes when compared to patients without pah, although the difference was not statistically significant (77.2 23.4 versus 96.9 27.3) ; this difference was significant when considering patients with the limited subset of the disease (77.6 17.6 versus 102.3 24.4 ; p < 0.05) (table 2). furthermore, eta expression was lower on t cells of lssc patients with ild when compared to t cells of patients without ild (77.8 34.2 versus 111.6 43.9 ; p < 0.05) (table 2). eta and etb expression on b lymphocytes were similar in patients and healthy donors (table 1). in ssc patients eta surface expression was higher than etb and was not influenced by the treatment with bosentan (eta : 281.33 43.47 and etb : 161.33 43.97 ; p < 0.05 versus eta : 270.00 28.16 and etb : 171.33 35.47 ; p < 0.05). eta and etb surface expression were similar in the diffuse or limited form of disease and were not influenced by the presence of pah, ild, and dus. neutrophils presented the same pattern of expression of et-1 receptors in ssc patients and control subjects (table 1). as already shown on the entire t cell population, both cd4 + and cd8 + t cell subsets isolated from ssc patients and control healthy donors express eta and etb on their surface. a higher eta expression on resting cd4 + and cd8 + t cells was also confirmed. upon activation, we found a decreased expression of eta and an increased expression of etb (figure 3). we tested the levels of inf-, il-4, and il-17 in the supernatants of cd4 + t cells obtained from ssc patients and controls. cells were either incubated without any stimulus or incubated with et-1 in the presence or absence of et-1 receptors blockade. after 24 hours of incubation with et-1, inf- concentration was 9.5 times higher than in the supernatants of cells cultured without et-1 (7.6 pg / ml versus 0.8 pg / ml ; p < 0.05). following selective eta or etb blockade before et-1 stimulation remarkably, the simultaneous dual et-1 receptors blockade, which mimics in vitro the effects of bosentan treatment, caused a marked reduction of inf- concentrations in the cells culture supernatants. interleukin-4 levels did not change in the supernatant of cells exposed to et-1 for 24 hours. in the presence of selective inhibition of eta, il-4 levels increased in a more significant manner than in the presence of etb blockade (711.42 102.2 pg / ml and 694.47 99.8 also, the double receptors blockade induced the production of il-4 (682 100.6 pg / ml versus 60.8 80.2 pg / ml ; p = 0.02). remarkably, we observed a slight increase in il-17 level following the incubation with et-1. however, this effect was not modified by partial or complete et-1 receptor blockade (table 2). levels of mmp-9, il-8, tnf-, vegf, ifn-, and il-17 were evaluated in the medium of neutrophils isolated from 4 healthy donors incubated with et-1 or lps or with et-1 and lps for 1, 3, or 10 hours (table 3). a 1-hour incubation with et-1 induced a marked increase of mmp-9 (165.1 ng / ml versus 46.4 ng / ml ; p < 0.05), whereas, at the same time point, we did not observe significant changes in the production of the other soluble molecules analysed, as shown in table 4. after 3 hours of incubation with both et-1 and lps, neutrophils released a higher amount of il-8 when compared to the concentration detected following the incubation with et-1 alone or lps alone (67.7 versus 7.7 versus 59.2 pg / ml, resp.), whereas the levels of the other soluble mediators remained unchanged. the concentration of tnf- released in the supernatants following a 10-hour incubation with et-1 was higher than the levels detected in the medium of cells stimulated with et-1 and lps or lps alone (23.4 pg / ml versus 2.1 pg / ml versus undetectable level ; p < 0.05). at the same time point, incubation with et-1 alone induced a higher production of il-17 compared to the stimulation with both et-1 and lps or lps alone (3.5 pg / ml versus undetectable level ; p < 0.05). finally, the simultaneous stimulation with et-1 and lps induced a higher secretion of il-8 and mmp-9 in the supernatant (134.6 pg / ml and 220.7 ng / ml, resp.) when compared to the concentration reached with et-1 (16.6 pg / ml and 63.7 ng / ml, resp.) or lps alone (93.8 pg / ml and 205.3 ng / ml, resp.). taken together, these data indicate that et-1 is able to induce neutrophils to release proinflammatory mediators. in the present study, we aimed firstly at analysing the cellular surface distribution of et-1 receptors in the different immune cell subsets and secondly at dissecting the mechanisms by which the et-1 signalling network may participate in the inflammatory responses in ssc. et-1 is a potent vasoconstrictor which plays a fundamental role in key pathogenetic aspects of ssc such as vascular damage and fibrosis and treatment with eras exerts beneficial effects on vasculopathy [36, 37 ]. more recently, amelioration of inflammatory parameters during era treatment has been reported (reviewed in), thus implying an important role for et-1 in inflammation, another important aspect of ssc pathology. indeed inflammation plays a pivotal role in early ssc ; however, it may influence also different phases of ssc. the presence of et-1 receptors on dendritic cells and polymorphonuclear cells has been already reported [68, 16 ], often with conflicting results, whereas very little is known on eta and etb expression on t and b lymphocytes. moreover, a proinflammatory role for etb expressed by monocytes / macrophages has been hypothesized on the basis of increased production of inflammatory mediators (tnf-, prostaglandin e2, and il-1) upon et-1 stimulation [11, 44 ]. we show here that all the immune cells studied (b and t lymphocytes, monocytes, and neutrophils) express et-1 receptors both in normal subjects and in ssc patients with a difference in the relative expression of either eta or etb in the different cell types analysed. in particular, b lymphocytes and neutrophils show the same pattern of expression in healthy controls and in ssc patients, without any significant difference related to the clinical features of the disease. since et-1 serum levels are higher in dssc than lssc patients and they correlate with the extent of vascular damage and cutaneous fibrosis, we may hypothesize that at least part of et-1 profibrotic effects is preferentially mediated by the engagement of eta [3, 16 ]. interestingly, we noticed that, in lssc patients, a lower etb expression on monocytes correlates with the presence of pah and a lower eta expression on t cells correlates with ild. we can therefore hypothesize that a different pattern of receptor expression on immune cells is associated with a different functional activity that may contribute to the development of pah or ild. a recent multicenter, placebo - controlled trial investigating new drug therapies for idiopathic pulmonary fibrosis compared the effects of ambrisentan, a selective eta antagonist, to placebo on disease progression. the study showed that the treatment was associated with an accelerated decline in pulmonary function tests, increased hospitalizations, and higher mortality. since a selective eta inhibition, such as the one obtained with ambrisentan, seems to accelerate pulmonary disease, we may suggest that an imbalanced expression of et-1 receptors with a diminished expression of eta on immune cells may predispose ssc patients to develop ild possibly through an increased etb - mediated stimulation on t cells. the results of this study are in accordance with our findings of a lower eta expression on t cells of ssc patients with ild. however, the precise role played by eta stimulation and inhibition in the progression of pulmonary fibrosis remains unclear. we next evaluated whether the presence of an inflammatory microenvironment could influence the relative expression and/or distribution of et-1 receptors and, to this aim, we stimulated t cells with anti - cd3/cd28 antibody - coated microbeads. stimulation resulted in reduced expression of eta and increased expression of etb on cd4 + and cd8 + t cells, thus suggesting that these cells, once activated, modulate receptor surface expression by overexpressing etb and downregulating eta. these results support the hypothesis that etb signalling plays a major role in inflammation and, as a consequence, that dual et-1 receptors blockade may represent a more suitable therapeutic strategy in ssc. we have then investigated the functional effects of et-1 stimulation on cd4 + t cells and found that et-1 is able to induce a proinflammatory response since the engagement of both eta and etb induced an ifn- secretion 9.5 times higher than the one observed in basal condition. ifn- production was markedly reduced following dual receptor blockade, a situation which resembles the effect of bosentan treatment. these findings suggest the need of eta - etb receptors cooperation to obtain an inflammatory response in cd4 + lymphocytes. these results are in agreement with the observation that simultaneous blockade of both eta and etb in scleroderma fibroblasts is necessary in order to suppress collagen production. in addition, et-1 stimulation of cd4 + t cells leads to a mild increase of il-17 concentration in cell supernatants, suggesting again an important role for et-1 in inducing the production of proinflammatory cytokines. finally, the receptors blocking induces the production of the anti - inflammatory cytokine il-4. finally, it is interesting to note that neutrophils activated with lps are able to increase the production of proinflammatory molecules after stimulation with et-1, thus giving further support to the proinflammatory effects of et-1 also on cells of innate immunity. all together, these data indicate that et-1 behaves also as a proinflammatory molecule through a synergistic action on eta and etb. therefore, a dual receptor blockade strategy is likely to better control inflammation and fibrosis than a selective receptor blockade. in conclusion, our results, besides generating useful insight in the understanding of et-1 effects on immune cells in healthy donors and in ssc patients, provide a rationale for the use of dual receptor antagonist in the early stages of ssc, when inflammation is prominent. | endothelin-1 (et-1) plays a pivotal role in vasoconstriction, fibrosis, and inflammation, the key features of systemic sclerosis (ssc). et-1 receptors (eta and etb) are expressed on endothelial cells, smooth muscle cells, and fibroblasts, but their presence on immune cells has not been deeply investigated so far. endothelin receptors antagonists such as bosentan have beneficial effects on vasoconstriction and fibrosis, but less is known about their potential anti - inflammatory effects. we studied the expression of et-1 receptors on immune cells (t and b lymphocytes, monocytes, and neutrophils) and the link between et-1 and inflammation in patients with ssc. we show here that et-1 exerts a proinflammatory effect in cd4 + t cells, since it induces an increased ifn- production ; preincubation with antagonists of both receptors reduces ifn- production. moreover, following et-1 stimulation, neutrophils produce proinflammatory mediators, thus amplifying the effects of activated cd4 + t cells. our data indicate that et-1 system is involved in the pathogenesis of inflammation and fibrosis typical of ssc, through the activation of t lymphocytes and neutrophils and the consequent release of proinflammatory and profibrotic cytokines. these findings suggest that dual et-1 receptors antagonist therapy, besides its effect on vasculopathy, has a profound impact on the immune system favouring antiinflammatory and antifibrogenic effects. |
the patient was placed in the supine position with the knee in a dangling position at around 70 of flexion. the un - operated left leg was supported with circumferential operative leg holders with sufficient padding at the fibular head to prevent common peroneal nerve palsy caused by the pressure4). first, the patellar apex, patellar tendon and lateral femoral condyle were identified by palpation. the lateral triangle, bounded by the lateral border of the patellar tendon, the medial circumference of the lateral femoral condyle, and distally by the superior rim of the anterior horn of the lateral meniscus, was palpated. once the optimum site for the anterolateral arthroscopic portals are selected, a longitudinal skin incision approximately 4 - 5 mm long was made with a scalpel (no. subsequently, a vertical incision about 3 - 4 mm was made at the lateral capsule. the sheath was inserted with a careful rotatory movement, avoiding injury to the cartilage and synovial covering of the anterior cruciate ligament. when the sheath penetrated the fibrous capsule and tissue resistance declined, the knee joint was extended and the sheath was carefully advanced into the medial portion of the suprapatellar pouch. the " needle technique " was used to make an anteromedial instrumental portal. by constantly visualizing the needle, the relationship between the skin opening and capsular opening was assessed by monitoring the changes in the relative direction of the scope and the instruments. in an extended knee position (20), the capsular opening migrated proximally and the direction from the skin to in a more flexed knee position, the capsular opening migrated distally and the direction from the skin to the capsule moved caudally (fig. the capsular opening migrated slightly laterally and medially, respectively. the commonly used position in knee arthroscopy is valgus extended knee for the examination of the medial meniscus and varus flexed knee (figure of " 4 " position) for the lateral meniscus. at 30 of knee flexion with valgus stress, the direction from the skin to the capsule changed cephalad with a slight lateral tilt. at 90 of knee flexion with varus stress, the direction from the skin to the capsule changed to a caudal with a slight medial tilt. to confirm the mismatch between the skin and the capsule according to various knee positions, capsular suture was carried out using roll wire (no. 2, zimmer, warsaw, in, usa). the skin was temporarily sutured with staples (skin stapler, dimensions 6.93.9 mm, johnson & johnson company, cincinnati, oh, usa). the relative positions were confirmed by intraoperative real - time c - arm images in various knee position, as described (figs. 2, 3). by knowing the direction of portals according to the knee positions, it was possible to access the portals with instruments without changing to the original dangling position or further violating the capsule. even for experienced surgeons, changing the arthroscopic instruments through the knee portals can be a cumbersome process. this is due to the mismatch of the initial relationships between the skin and soft tissue orifices according to the knee position. in these situations, changing the knee position into the original (index) position or further spreading the portal opening using a mosquito may lead to prolonged operation time and further violation of the capsule. by understanding the relative skin - capsular mismatch in arthroscopic portals according to knee positions, instrument insertion can be facilitated with the adjustment of the direction of instrument insertion or skin traction. we believe that knowing the direction of portals according to knee position is useful during arthroscopic procedures. the small size of the capsular incision coupled with the elimination of frequent capsular violation limits fluid extravasation, which can complicate a straightforward procedure and lead to increased postoperative swelling. in this series, it was accessible to all portals without any difficulty. various authors have reported new techniques and approaches for easy access of the arthroscopic instruments into the different regions of the knee compartments with their own limitations. kim and kim6) reported different locations of portals to visualize the different regions of the compartments and concluded that the locations should be flexible as per the need of the surgeon rather than fixed. ahn and ha7) showed that a posterior trans - septal portal further improves the visualization of the posterior compartments. although different orientation of portals are available for better visualization of specific intra - articular regions, most of arthroscopic surgeries can be managed by using anterolateral, anteromedial and posteromedial portals. this note describes the relationships between skin and soft tissue opening according to knee positions. based on our experience of handling more than two thousand arthroscopy surgeries, we have established that correct understanding of the skin - capsular mismatch minimizes the soft tissue injury and indirectly reduces the operative time. the limitation of this study is that the degree of skin - capsular mismatch could not be shown quantitatively. further studies with a large number of patients and an appropriate measuring method could additionally enhance the quality of the paper. | damage to soft tissues, chondral surfaces, and the menisci may result from imprise or overly aggressive establishment of portals in arthroscopic knee surgeries. in this note, we address the relationship between the skin and the capsule at portal sites according to knee positions. understanding the skin - capsular mismatch may facilitate arthroscopic procedures and indirectly reduce the operation time. |
a luminal narrowing in the arterial vessel wall is induced by injury that is leading to stenosis and development of intimal hyperplasia. the balloon coronary angioplasty has represented a revolutionary treatment that led to the birth of interventional cardiology. this new special surgery process with the exclusion surgical intervention produces ischemia reperfusion, inflammation and final to restenosis with an occlusion of arterial lumen. proven indications for endarterectomy as recoil are moderate - to - high grade stenosis, rapidly progressive stenosis with lesions revealing greater than 70 percent in diameter of stenosis in carotid diseases. clinical results reveal primary success rates, less complication, and restenosis rates comparable with those of surgical endarterectomy. the coronary stents, which were first developed in the mid-1980s, have ultimately replaced the old balloon angioplasty as the preferred method of performing percutaneous transluminal coronary angiography (ptca) intervention. after the observed improvements (in angiographic and clinical outcomes) 171 interventions on 151 patients were performed as a single surgeon 's experiences and long - term follow - up was completed in 109 patients. the combined survival rate was 85%, recurrent stenosis - free 88% at 5 years, respectively. reduction of the incidence of clinical events was only possible if a low perioperative complication rate was accomplished. nowadays involve a coronary stent, and interventional cardiologists are faced with a wide choice of coronary stents to implant, such as bare metal stents and drug - eluting stents. coronary stenting only became a widely accepted technique. in spite of some relevant strong points such as greater than 90% success rate and the repeatability of the procedure, several major drawbacks still persist, including restenosis within the treated vessel [58 ]. the restenosis phenomenon is currently the object of intensive research in different areas of biomedical field and therapy. restenosis means the reoccurrence of stenosis, a narrowing of a blood vessel, leading to restricted blood flow. restenosis usually sets to an artery or other large blood vessel that has become narrowed. this phenomenon is grown up after balloon intervention with an enhanced inflammatory processes and the blood vessel subsequently becomes renarrowed. this term is common in vascular surgery, cardiac surgery, and angioplasty, all branches of medicine that frequently treat narrowing of blood vessels. restenosis can be defined as a reduction in the circumference of the lumen of 50% or more and had a high incidence rate (2550%) in patients who had undergone balloon angioplasty, with the majority of patients needing further angioplasty within 6 months. damage to the blood vessel wall by angioplasty triggers physiological response that occurs immediately after tissue trauma, is thrombosis. a blood clot forms at the site of damage and further hinders blood flow with an inflammatory response. the second stage tends to occur 36 months after surgery and is the result of proliferation of cells in the intima, a smooth muscle wall in the vessel. the artery can react to the stent, perceive it as a foreign body, and respond by mounting an immune system response (see physiology below) which leads to further narrowing near to or inside the stent. drug - eluted stents substantially reduce the occurrence of restenosis but the clinical studies indicate a slight incidence rate of recurrence (5%). obviously restenosis remains a problem. restenosis is mainly due to neointima formation, which is caused primarily by the effects of vsmc proliferation and migration. cell proliferation after stenting occurs both early, as part of the acute injury response, and late, located around stent struts. whereas some neointima formation is necessary for vessel healing after stenting, excessive neointima formation narrows the lumen. the function of endothelium plays a central role in destroyed balance and later the maintaining of vascular health by virtue of the endothelial cells. development of restenosis has demonstrated high proliferation activity of vsmc from the tunica media to the intima which is the occurrence of internal elastic lamina rupture [16, 17 ]. to summarize, the adverse cardiovascular events are associated to endothelial dysfunction and activated proliferation of vsmc. atherosclerosis is coexistence with occlusion of arterial wall and the etiology of the disease distinct of restenosis. it is known as vascular disease in which an artery wall thickens as a result of the accumulation of cholesterol. it is a chronic inflammatory response, caused largely by the accumulation of macrophages and lymphocytes and promoted by high level of oxidized low - density lipoproteins. it is commonly referred to as a hardening of the arteries and caused by the formation of multiple plaques within the arteries. most commonly, the soft plaques suddenly create ruptures causing the formation of a thrombus and incidentally leading to death of the tissues (infarction). the most important risk factors of mih and restenosis are ischemia / reperfusion injury, shear stress, inflammation, diabetes, oxidative stress, hypertension, modulation of cytokine, and c - reactive protein [crp ] level, together with other environmental stimuli such as smoking. those factors which appear to have impact on the evaluation of restenosis will be briefly mentioned in this review. i / r injury plays a significant role in the pathophysiology of restenosis by inducing endothelial dysfunction. the arterial blood vessel is vulnerable to i / r injury which depends on the ischemic time, hemodynamic status of patients, and the reperfusion itself. i / r injury of endothelial cells (ecs) apparently provide an initial trigger and subsequently causes enhanced proliferation of vsmcs. thus, diminution of i / r injury would be a great benefit not only by inhibiting direct cellular injury but also indirectly through the mentioned factors that influence several processes, for example, the immune response. the injury can block the artery or result in poor perfusion of reperfused tissue of blood vessel. time - dependent increased level of il-6 and tnf - alpha has detected in ptca patients. vascular homeostasis on arterial walls is highly dependent on the blood flow and shear stress. endothelial cells act as sensors of shear stress and regulate its level by adapting the arterial dimensions to blood flow. shear stresses control and modify function of cells in arterial wall. in the presence of several risk factors, low shear stress contributes to endothelial dysfunction, whereas normal - to - high shear stress results in protecting the function of blood vessel. high shear stress upregulates the expression of endothelial genes and proteins which are protected against dysfunction, and by low shear stress an opposite effect is produced. disturbed flow has been proven to result in postsurgical neointimal hyperplasia, in - stent stenosis, and aortic valve calcification. the immune system plays important roles in restenosis. endothelial injury and activation elicits the release of proinflammatory cytokines, chemokines, and expression of adhesion molecules, which fosters immune cell recruitment and transmigration of immune cells across the ec barrier and into the intima. the current review will not delve into details regarding the role of immunity in development of restenosis [2527 ]. thromboembolism, governed by several risk factors (hypertension, diabetes, obesity, smoking, etc.) the phenomena of thromboembolism overlap with the phenomena of atherothrombosis and/or restenosis. elevated c - reactive protein (crp) level associated with inflammation may promote the restenosis process. exogenous crp induces the expression of adhesion molecules and decreases endothelial nos (enos) production. furthermore, crp upregulates smc angiotensin - i receptors and thereby increases the level of the reactive oxygen species as well as proliferation. in addition, monocytes / macrophages exposed to crp increase the release of tissue factor, which potentially stimulates cell migration and adhesion to endothelial cells extending inflammatory process and finally results in restenosis. epidemiological evidence shows that high level of serum homocysteine is associated with an increased risk of cardiovascular diseases. hyperhomocysteinemia affects primarily the endothelium, which in turn results in reduced endothelial no production, decreased arterial response to vasodilators, and increased expression of procoagulant factors. hypertension, smoking, and diabetes mellitus are risk factors associated to the development of restenosis. hypertension causes endothelial injury by promoting the formation of intimal hyperplasia. in spite of our increasing knowledge on the nature and effect of numerous risk factors, partially covered above, further studies are clearly needed to establish important features of their mode of action as well as their interactions in the pathogenesis of myointimal hyperplasia and restenosis. we suggest that i / r injury and hypoxia are one of the initiative risk factors of restenosis, acting by disturbing the normal functioning of the endothelial monolayer. as a result, endothelial cells release enhanced levels of inflammatory cytokines and growth factors, leading to phenotype switch of the vascular smooth muscle cells, followed by their increased migration and/or proliferation. the endothelium regulates many important functions in the cardiovascular system including vascular tone, coagulation, and inflammatory responses. in addition, the endothelium limits local thrombosis by producing tissue plasminogen activator, maintaining a negatively charged surface, and secreting heparans, no, and thrombomodulin. no, a paracrine factor produced by the endothelial no synthase enzyme (enos) is well known as vasodilator. in addition, its effect is crucial for several other processes as well, involving the prevention of platelet aggregation / adhesion and reduction of the expression of several proinflammatory genes. pharmacological inhibition or genetic deficiency of enos hampers endothelium - dependent vasodilatation, impairs tissue blood flow, and raises the blood pressure. no plays a key role in maintaining vascular homeostasis, by exerting a wide variety of effects on the endothelial and smooth muscle cells of the vessel wall. it stimulates endothelial cell proliferation, mainly by potentiating the mitogenic effect of vascular endothelial growth factor. on the contrary, in the case of vmscs, no inhibits their proliferation and migration. as a consequence, enhanced no production in the vessel wall slows down or even reverses restenosis [37, 38 ]. beside no, mitogens (e.g., pdgf) produced by ecs are also important regulators of vsmcs. selective inhibition of pdgf production by low - density lipoprotein has potentially profound implications for the clinical control of mih. in summary, endothelial dysfunction (mechanical injury to the endothelium) is an important early feature of vascular injury with serious impact on the pathogenesis of restenosis. induced proliferation of vsmcs contributes to the pathobiology of restenosis and linked also to other cellular processes such as inflammation, apoptosis, and matrix alterations. in fact, beside inflammation enhanced proliferation of vsmcs is one of the key processes of restenosis and atherosclerosis. enhanced proliferation of vsmcs is most probably the consequence of phenotype switch of these cells. phenotype switch is a complex cellular mechanism providing the possibility of adult smooth muscle cells to fluctuate reversibly between a contractile and an immature (also called proliferative) phenotype. in response to various inner and environmental signals, these two phenotypes exert different activities : contractile smooth muscle cells stiffen, shorten and relax, whereas the immature ones have a tendency to proliferate, to migrate, and to synthesize extracellular matrix components. it is to be noted, that within both phenotypes functional diversity occurs, that is, distinct populations might coexist which respond differently to given stimuli. molecular mechanisms underlying the functional plasticity of the contractile phenotype are centered on (a) remodeling of the actomyosin filaments constituting the contractile apparatus, and (b) variation of the expression of proteins regulating cell responses to environmental cues. numerous proteins belong to this latter group, involving receptors, signaling effectors, ion channels, and so forth, preferentially sequestered in caveolae, flask - shaped invaginations of the plasma membrane. caveolae components are differently organized in the distinct myocyte phenotypes ; they are more abundant in contractile vascular myocytes. in contractile vsmcs they regulate processes leading to functional plasticity, and they are suggested to be involved the integration of events leading to phenotype switch. at least two transmembrane adhesion receptors, the integrins and the dystrophin - glycoprotein complex (dgc) are present in caveolae, which constitute a link between the extracellular matrix (ecm) components, (e.g., laminins) and the intracellular actin filament system, being therefore key players in the communication of the cells with their environment. noticeably, different types of integrins and dgc components are expressed in vsmcs of different phenotypes (for a review, cf.). in the course of restenosis, one of the first steps of the lesion development is the focal accumulation of vsmcs within the intima. the local inflammatory milieu can induce expression of collagenase and inhibit expression of proteolytic inhibitors. the vsmc population in intimal lesions has been proposed to arise from medial vsmcs, adventitial cells, preexisting intimal clones, and precursor cells derived from the vessel wall itself or from circulating vascular progenitors. principal factors among these are platelet - derived growth factor (pdgf), somatomedin - c, epithelial growth factor, and insulin. lipoproteins may be also important substrates for vsmc proliferation, while heparin directly inhibits vsmc protein synthesis [51, 52 ]. the degree of vsmc proliferation appears to be dependent mainly on the degree of initial injury and partially, but not in all cases on loss the confluence of overlying endothelium. heparin and other ec products appear to inhibit vsmc proliferation independently of platelet - vsmc interaction. platelets may play a role in the early response to arterial injury and development of mih, but their long - term role appears to be minor [52, 53 ]. hypertension results in a marked proliferation of vsmc enhancing the proliferative response to injury. structural organization and signaling of vsmcs and thereby phenotype switch are influenced by externally applied mechanical force as well. the predominant mechanical force acting this way drug therapy directed at the components of the signaling pathways influenced by stretch may prevent myointimal hyperplasia and thereby restenosis. recently, we have discovered that hypoxia ; one of the cardinal risk factors of restenosis influences both the endothelial monolayer and the vsmcs. in the last decades surgery techniques have been routinely performed [2, 56 ]. the restenosis is one of the most common and dangerous postoperative complication of these interventions. pathology of coronary diseases in general comprises several distinct features involving, among others, degenerative and cell proliferation / differentiation processes. cell proliferation and differentiation disturb the cellular morphogenesis leading to the formation of a multilayered compartment internally to the elastic membrane of the arterial wall, composed of cells expressing alpha - smooth - muscle actin. myointimal hyperplasia, or neointima formation, and it is the leading pathological mechanism leading to the narrowing of the arterial lumen. it is destructive event of the postoperative complications after angioplasty, bypass operations, or stenting as well [60, 61 ]. several attempts have been made to interfere with the development of postoperative intimal hyperplasia, of which application of drug - eluting stents (des) seems to be the most promising approach [57, 62 ]. our vascular surgeon team has been performing endovascular interventions since decades. in a comprehensive retrospective study [3, 61 ], it was shown that the 5-year patient survival rate after carotid endarterectomy (cea) was 85%, and the restenosis - free rate among the survivors was 88%. physiological and pathophysiological factors leading to restenosis were studied. changes in the levels of two acute phase proteins, plasma fibrinogen, and serum c - reactive protein (s - crp) were determined in 117 patients with severe carotid stenosis after eversion endarterectomy. during the postoperative period sharp, these findings indicate that the removal of atherosclerotic plaques from the carotid arteries markedly decreases the production of the two acute phase proteins. the role of mannose - binding lectin (mbl2) genotype in restenosis after eversion endarterectomy in patients with severe carotid atherosclerosis has been also studied. mbl is thought to influence the pathophysiology of the cardiovascular system by decreasing the risk of advanced atherosclerosis and by contributing to enhanced ischemia reperfusion injury. our data show that carotid duplex scan (cds) values in patients homozygous for the normal (a) mbl2 genotype were significantly higher 14 months postsurgery as compared to the values measured 6 weeks after surgery. on the other hand, the frequency of mbl2 variant genotype was significantly higher in patient 's not experiencing restenosis as compared with patients with restenosis. these results indicate that reoccurrence of stenosis after carotid endarterectomy is influenced by genetic factors and imply that mbl2 contributes to the pathophysiology of this condition. in our previous study, we analyzed the relationship of c3 complement component with early restenosis, detected by cds, after eversion endarterectomy by analysing three noncomplement acute - phase reactants (aprs) (c - reactive protein, haptoglobin, and alpha2-hs glycoprotein / fetuin - a) as control. in mbl2 a / a allele carriers c3 levels increased during the follow - up period and correlated with the occurrence of restenosis at 14 months postsurgery. even after adjusting for mbl2 genotype, age and gender, patients with high c3 levels had nearly five - fold higher odds for the development of significant restenosis (> 50% reduction in diameter) by contrast, no such associations were detected between early restenosis and the noncomplement aprs. c3 is thus associated with the development of an early restenosis after eversion endarterectomy, which is related to an intact mbl lectin pathway. these results suggest that c3 levels probably have clinical importance and indicate that the regulation of c3 differs from noncomplement aprs. we investigated whether early postoperative changes in serum vascular endothelial growth factor (vegf) and platelet - derived growth factor (pdgf) concentrations are in correlation with the mbl2 genotype in the context of the development of restenosis. the data indicated that pronounced significant increases in both vegf and pdgf - predicted restenosis exclusively in patients who were homozygous for the normal mbl2 genotype. in this group, the adjusted odds ratio of restenosis at 14 months was 27.7 (2.4 to 317.2) in patients with high early vegf and pdgf increases, while in patients with low early growth factor increases the odds ratio was 9.2 (1.4 to 58.7). these findings indicate that in patients disposed to restenosis by mbl2 genotype pathologic processes leading to enhanced production of vegf and pdgf during the very early postoperative period exert more harmful effect as compared to patients with the protective mbl2 genotype. considering the impact of mbl2 genotype on the occurrence of early restenosis after cea, the role of c1-inhibitor (c1-inh), known as an inhibitor of the lectin pathway, has been also studied. the aim of the study was to determine whether the c1-inh levels have predictive value for restenosis after cea. patients with > 50% restenosis had significantly lower c1-inh levels at 6 weeks and at 4 days postsurgery. c1-inh levels at 6 weeks correlated inversely with the cds values at 14 months (r = 0.3415), but only in mbl2 a / a homozygotes (r = 0.5044). patients with mbl2 a / a and low c1-inh levels at 6 weeks postsurgery had 13.9 times higher risk developing an early restenosis. these data indicate that mbl2 genotyping together with measuring the c1-inh level at 6 weeks postsurgery should be useful for identifying patients with high risk for early carotid restenosis. pharmacological treatments currently in use include anticoagulants, antiplatelet agents, immunosuppressants, and antiproliferation agents. unfortunately, none of these approaches has been conclusively shown to prevent coronary restenosis after balloon angioplasty or graft restenosis after peripheral arterial bypass. consequently, the treatment of vascular stenosis remains today predominantly within the domain of the surgery. however, numerous successful animal experiments and promising proposals, as well as initial clinical results have been published lately. here we review some remarkable contributions to this field. as unfolded earlier, hypoxia is one of the risk factors which initiate the pathological mechanism leading to restenosis. the obvious target cells of restenosis therapy are the permanent proliferating vsmcs and the endothelial cells with injured function. the possibility of influencing the process of the phenotype switch of vsmcs to proliferative form during pathological, ischemic conditions would open up new vistas in the prevention and/or therapy of restenosis. techniques addressing directly the proliferating vsmcs or injured endothelial cells have demonstrated significant degrees of clinical success. a recent review has summarizied the present status of cellular and animal studies aimed at preventing restenosis by using protein kinase c (pkc) inhibitors. these agents are expected to exert therapeutically beneficial effects on restenosis causing factors of various kinds, including vsmcs. angiogenesis, that is, the growth of new blood vessels from existing host vessels, is recognized more and more as an important factor in the growth and progression of restenosis. the phenomenon occurs when the intima thickens beyond the diffusion limits of oxygen and nutrients, as a response to tissue hypoxia and consists of growth and extension of adventitial blood vessels into the intima. under hypoxic conditions the hypoxia - inducible factor alpha blood vessels are destabilized, and this leads ultimately to vessel regression. in the presence of vascular endothelial growth factor (vegf), other molecules and growth factors (vegf and fgf) have also been shown to induce neovascularization and thereby increase nutrient perfusion. vegf has therapeutic potential in cardiovascular gene therapy, the enhancement of arterioprotective endothelial functions, prevention postangioplasty restenosis. whereas the production of no in endothelial cells is induced by vegf, the proliferation of vascular smooth muscle cell is inhibited. inhibition of neointimal hyperplasia may also be achieved by gene transfer of endothelial no synthase (enos), pgi synthase, or cell - cycle regulators cyclin - dependent kinase inhibitors, p53, and transcription factors such as nuclear factor kappab. it has been suggested that bone marrow - derived endothelial progenitor cells (epcs) or circulating endothelial progenitor cells play a role in neovascularization [73, 74 ]. nevertheless, it is not yet clear whether these progenitor cells should be considered as therapeutic targets in vascular diseases. iroxanadine (5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridil)-4h-1,2,4-oxadiazine) is a drug candidate with a marked cytoprotection by heat shock protein (hsp) coinduction. it has been originally synthesized by biorex - hungary (brx-235), and the worldwide rights have been recently sold by cytrx / california / usa to orphazyme aps / novo a / s, copenhagen, denmark. in a recent work we have shown that brx-235 stimulates the migration of ecs via p38 stress - activated - protein - kinase (sapk) and enhanced expression of hsps and heat - shock transcription factors. after treatment by brx-235, the vigorous proliferation of mih cells decreased while the proliferation of deregulated ec cells increased. this finding harmonizes with a clinical report demostrating compromised ec proliferation, and hsp72 expression was upregulated in vsmcs. neointima formation due to enhanced vmsc proliferation is suggested to be associated with reduced cyclic guanosine monophosphate signaling. we have undertaken to study the effect of the inhibition of cyclic guanosine monophosphate degradation on neointima formation in a rat model of endarterectomy, by using the selective phosphodiesterase-5 inhibitor vardenafil as a pharmacological inhibitor. the results were evaluated by conventional microscopy with hematoxylin and eosin staining and by immunohistochemical analysis to confirm neointima formation and the local cyclic guanosine monophosphate content. immunohistochemical analysis demonstrated intensive staining for transforming growth factor beta1 (tgf beta1) and alpha - smooth muscle actin in the control neointima. vardenafil significantly reduced the stenosis grade (24.64% versus 54.12% in the control group) and expression of tgf beta1, as well as alpha - smooth muscle actin in the neointima. on the basis of these results it was suggested to consider the treatment with vardenafil as a new possibility to prevent neointimal hyperplasia after endarterectomy. stent insertion, one of the most common surgical techniques applied in vascular diseases, is too often followed by early, as well as late restenosis. to attenuate this complication, instead of conventional stents, drug - eluting stents (dess) stents generally attenuate endothelial recovery, altering thereby the natural biology of the vessel wall and increasing the associated risk of stent thrombosis. dess, similarly to brachytherapy, target proliferating vsmcs at the site of injury and have been successful in reducing the formation of neointimal lesion [8082 ]. dess releasing various antiproliferative drugs represent also a useful strategy for the prevention of restenosis. vegf-2 gene - eluting stents accelerate reendothelization, thereby offering an alternative strategy for the prevention of restenosis. this type of treatment, when applied in hypercholesterolemic rabbits, resulted in transgene expression in the vessel wall and a 2.4-fold increase in no production by the ecs, along with improved functional recovery of stented segments. follow - up studies, however, show the possible onset of late stent thrombosis. delayed arterial healing most probably associated with poor endothelialization of stent struts seems to be the underlying mechanism of this complication. as recent results indicate, balloon angioplasty and stent implantation might still go hand in hand with severe postoperative complications. several drugs have been suggests to use in des, for example, antitumor agents (paclitaxel, doxorubicine, rapamycin, erythromycin, etc.), cholesterin lowering drugs (statins), immunosuppressants (cyclosporin), anti - inflammatory agents (rapamycin), antibiotics (amphotericin b), and natural products (shikonin). finally, we briefly summarize the achievements of gene therapy, as an emerging approach to counteract the pathological processes leading to restenosis. none of the gene therapy agents have been approved for clinical use up to now, but several promising animal experiments and preclinical studies have been recently published. the great potential advantage of the gene therapy resides in its potential to ensure specific, controlled expression of selected proteins within the targeted cell types. the panoply of gene therapy possibilities, among others the potential of promoting a functional epithelium, inhibiting smc proliferation and therapeutic angiogenesis has been summarized in a review. adenoviruses (ads) and adeno - associated viruses (aavs) are currently the most efficient vectors for delivering therapeutic genes into the vascular system [86, 87 ]. gene therapy is often associated with the drug - eluting stent (des) technique ; that is, the therapeutic gene is delivered by the stent [85, 87 ]. along this on the basis of successful animal application of inos gene transfer, a single - blind, multicenter clinical study has been recently performed by inos lipoplex gene therapy. the results suggest that the technique applied provide a safe therapeutic principle for the prevention of coronary restenosis. as a conclusion, we predict that significant clinical benefits are to be expected when using complex therapeutically approaches, which stimulate physical and functional recovery of the endothelial monolayer and in the same time inhibit the proliferation of vascular smooth muscle cells. | the vascular disease involves imbalanced function of the blood vessels. risk factors playing a role in development of impaired vessel functions will be briefly discussed. in ischemia / reperfusion (i / r), ischemic hypoxia is one of the cardinal risk factors of restenosis. various insults are shown to initiate the phenotype switch of vsmcs. the pathological process, leading to activated inflammatory process, complement activation, and release of growth factors, initiate the proliferation of vsmcs in the media and cause luminal narrowing and impaired vascular function. the review summarizes the alteration process and demonstrates some of the clinical genetic background showing the role of complement and the genotypes of mannose - binding lectin (mbl2). those could be useful markers of carotid restenosis after stent implantation. gene therapy and therapeutic angiogenesis is proposed for therapy in restenosis. we suggest a drug candidate (iroxanadine), which ensures a noninvasive treatment by reverse regulation of the highly proliferating vsmcs and the disturbed function of ecs. |
the purpose of this study was to explore differences between spirituality (including spiritual experiences and spiritual well - being), depressive symptoms, and quality of life among elders with and without heart failure. patients were recruited from the heart failure out - patient department of the main hospital and from a family practice clinic from the regional clinics. institutional review board approval was obtained from the patient care facility and from the university. a convenience sample of heart failure (hf) patients and non - hf patients who attended either the heart failure clinic, for the hf group, or a family practice clinic for the non - hf group was obtained. the inclusion criteria for this study were that all the patients were ; (a) 65years or older, (b) able to speak and read english, in addition, the hf patients were nyha classification ii to iv. the exclusion criteria for the study were (a) patients who were cognitively impaired ; and (b) no diagnosis of hf among the non - hf group. the physicians and nurse practitioner identified potential subjects and invited eligible patients to participate in the study. the researchers explained the study to all subjects and informed consent was obtained prior to completion of the questionnaires. following data collection, each subject was given $ 5 as a token of appreciation. spirituality was measured using two instruments, the daily spiritual experiences scale (dses) which measures spiritual experiences, and the spirituality index of well - being (siwb) which measures spiritual well - being. these two spirituality instruments were chosen as they measured a broad range of spiritual dimensions, and have strong psychometric properties. depressive symptoms were measured by the center for epidemiologic studies depression scale (ces - d). the sf-12 was selected rather than the longer sf-36 as time for completion was a consideration. the dses consists of 16 items in a six - point likert - type format. each item asks the respondent to choose how often they experience the spirituality concept referred to in the statement. content validity was supported by the use of qualitative interviews and focus groups, as well as a review of current spirituality scales. the authors reported that each of the 16 items significantly loaded on one of two factors within the tool. in the two studies, the internal consistency reliability was.94 and.95. with an average completion time of less than two minutes for the entire scale, it is very practical for both clinical and research administration (underwood and teresi 2002). the spirituality index of well - being (siwb) is a 12 item scale to measure the effect of spirituality on subjective well - being (daaleman and frey 2004). items 1 to 6 address self - efficacy while items 7 to 12 address life scheme. a multi - site cross - sectional survey with 523 adult outpatients at primary care clinics in the midwest each of the 6 item subscales showed good reliability, =.86 for self - efficacy and =.89 for life scheme. test retest reliability for the total scale was.79 and.77 and.86 for each of the subscales respectively. factor analysis showed that the two factor model accounted for 56% of the total variance ; self - efficacy, factor 1, accounted for 27.2% of the variance while life scheme, factor 2, accounted for the remaining 28.6% (daaleman and frey 2004). it was designed to measure four dimensions of depressive symptoms : depressed affect, positive affect, somatic and retarded activity, and interpersonal (radloff 1977). the ces - d consists of 20 items that measure depressed mood, feelings of worthlessness, loss of appetite, poor concentration, sleep disturbance, and feelings of helplessness. the ces - d is a 4-point rating scale with a time frame for reporting symptoms in the past week. the possible range of total scores varies between 0 and 60, with a higher score indicating more depressive symptoms. a score of 16 or higher is designated as a standard cutoff point to determine the presence of significant depressive symptoms. the cronbach s alpha reliability coefficient for the 20 items of the ces - d was.85 for the general population and test - retest reliability values ranged from.51 to.67 (radloff 1977). the ces - d has been reported to have good internal consistency (cronbach 's alpha) with a range of.87 to.92 in the elderly (davidson 1994 ; williamson and schulz 1992). sf-12 health survey (qualitymetric 2006) is a valid and reliable self - administered, 12-item scale developed from the sf-36 that measures the perceived health - related quality of life for eight concepts (physical functioning, role - physical, bodily pain, general health, vitality, social functioning, role - emotional, and mental health) of two major domains, that of physical and mental health. sf-12 has been used for measuring health status and monitoring health outcomes in both general and specific populations. the test - retest reliability was reported as.64 to.89 (ware 1996), and validity was supported with a strong correlation (r = 0.94) with sf-36 (hurst 1998). spirituality was measured using two instruments, the daily spiritual experiences scale (dses) which measures spiritual experiences, and the spirituality index of well - being (siwb) which measures spiritual well - being. these two spirituality instruments were chosen as they measured a broad range of spiritual dimensions, and have strong psychometric properties. depressive symptoms were measured by the center for epidemiologic studies depression scale (ces - d). the sf-12 was selected rather than the longer sf-36 as time for completion was a consideration. the dses consists of 16 items in a six - point likert - type format. each item asks the respondent to choose how often they experience the spirituality concept referred to in the statement. content validity was supported by the use of qualitative interviews and focus groups, as well as a review of current spirituality scales. the authors reported that each of the 16 items significantly loaded on one of two factors within the tool. in the two studies, the internal consistency reliability was.94 and.95. with an average completion time of less than two minutes for the entire scale, it is very practical for both clinical and research administration (underwood and teresi 2002). the spirituality index of well - being (siwb) is a 12 item scale to measure the effect of spirituality on subjective well - being (daaleman and frey 2004). items 1 to 6 address self - efficacy while items 7 to 12 address life scheme. a multi - site cross - sectional survey with 523 adult outpatients at primary care clinics in the midwest was conducted to determine the scale psychometrics. each of the 6 item subscales showed good reliability, =.86 for self - efficacy and =.89 for life scheme. test retest reliability for the total scale was.79 and.77 and.86 for each of the subscales respectively. factor analysis showed that the two factor model accounted for 56% of the total variance ; self - efficacy, factor 1, accounted for 27.2% of the variance while life scheme, factor 2, accounted for the remaining 28.6% (daaleman and frey 2004). it was designed to measure four dimensions of depressive symptoms : depressed affect, positive affect, somatic and retarded activity, and interpersonal (radloff 1977). the ces - d consists of 20 items that measure depressed mood, feelings of worthlessness, loss of appetite, poor concentration, sleep disturbance, and feelings of helplessness. the ces - d is a 4-point rating scale with a time frame for reporting symptoms in the past week. the possible range of total scores varies between 0 and 60, with a higher score indicating more depressive symptoms. a score of 16 or higher is designated as a standard cutoff point to determine the presence of significant depressive symptoms. the cronbach s alpha reliability coefficient for the 20 items of the ces - d was.85 for the general population and test - retest reliability values ranged from.51 to.67 (radloff 1977). the ces - d has been reported to have good internal consistency (cronbach 's alpha) with a range of.87 to.92 in the elderly (davidson 1994 ; williamson and schulz 1992). sf-12 health survey (qualitymetric 2006) is a valid and reliable self - administered, 12-item scale developed from the sf-36 that measures the perceived health - related quality of life for eight concepts (physical functioning, role - physical, bodily pain, general health, vitality, social functioning, role - emotional, and mental health) of two major domains, that of physical and mental health. sf-12 has been used for measuring health status and monitoring health outcomes in both general and specific populations. the test - retest reliability was reported as.64 to.89 (ware 1996), and validity was supported with a strong correlation (r = 0.94) with sf-36 (hurst 1998). the sample consisted of 84 subjects, 44 with hf and 40 in the non - hf group. in the total sample and in both groups there was a significant difference in gender between the two groups ; in the hf group, there were significantly more men (n=30, 68.2%) (x = 21.84, p =.000). in the total sample, all of the black / non hispanic subjects were in the hf group (n = 13, 29.5%) ; x = 14.15, p = 0.003). fifty percent (n = 22) of the subjects in the hf group were classified as nyha class ii ; 31.8% were class iii ; and 18.3% were class iv. these results are included in table 2. means, standard deviations (sd), and ranges were computed for the dependent variables of spiritual experiences, spiritual well - being, quality of life (physical and mental), and depressive symptoms in the total sample, the hf group, and non - hf group. the hf group reported more daily spiritual experiences and more spiritual well - being than the non - hf group. there were significant differences between the hf and non - hf groups on gender and ethnicity. after controlling for gender and ethnicity, spiritual well - being and physical component of quality of life were significantly different between the hf groups and the non - hf group. the hf group had significantly more spiritual well - being than the non - hf group (f = 5.48, p = 0.02). while controlling for gender and ethnicity, the physical component of quality of life in the hf group was significantly lower than that of the non - hf group (f = 13.14, p = 0.001)., there were no significant differences in daily spiritual experiences, mental component of quality of life, and depressive symptoms between the hf and non - hf groups. the overall percentage of males in the sample was 44%. however, 68.2% of the hf group was male compared to 17.5% in the non - hf group. westlake and dracup (2001) had 68.9% males from two heart failure clinics in their study. hardin (2003) had 70% males in their study of 29 heart failure patients. also heart disease is more common or at least diagnosed more frequently in men rather than women. almost all (92.5%) of the non - hf group were white / non - hispanic. this can be accounted for as the family practice clinic was situated in a predominately white neighborhood. in the heart failure group, the present study has a larger percentage of black subjects compared to any of the previous studies of hf patients. in the study conducted by westlake and dracup, beery (2002) had a predominately caucasian sample as 90% were european american and 10% african american. westlake (2002) had a sample with caucasian (84%), 15% hispanic, and 2% african american when they investigated quality of life in hf patients. in this study, in the hf group, however, as the total hf group included only 44 patients, inter - group comparisons were not made on any of the main variables. westlake and dracup (2001) had two - thirds of their sample in classes iii and iv heart failure. in the westlake s (2002) study, the majority of patients being evaluated for cardiac transplantation were either nyha class ii or iii. spiritual well - being was higher in the hf group than the non - hf group, even after controlling for gender and ethnicity. previously researchers have identified spiritual dimensions among heart failure patients at different stages of their illness. this present study is the first to compare spirituality among hf patients and non - hf patients. this finding of higher spiritual well - being may be related to the illness experience. it is of note that there were no differences in daily spiritual experiences once gender and ethnicity were controlled. on the sf-12, this is not an unexpected finding as these patients are likely to have more physical symptoms than the non - hf group. however, one limitation of this study is that co - morbidities or medications were not measured in either group. there were no differences between the two groups for the mental composite scores once gender and ethnicity were controlled. a previous study found that the mental composite score was significantly related to spiritual, religious, and existential well - being while the physical composite score was not related (beery 2002). in addition, there were no differences in depressive symptoms after controlling for gender and ethnicity. in previous research, patients with hf had high rates of depression and depressive symptoms (havranek 1999 ; skotzko 2000). in summary, this study extends previous research on spirituality and well being among elders. controlling for the effects of gender and ethnicity in this sample, differences between elders with hf and those without hf were found on spiritual well - being and physical quality of life. the current study has the largest percentage of blacks with heart failure, thus adding to the literature. future research is needed to further clarify dimensions of spirituality, including spiritual well - being and spiritual experiences and their relationship to quality of life in older persons. also, it is recommended that future studies include persons from different cultural and ethnic backgrounds. for comparison purposes, future research should address heart failure patients who are hospitalized as well as those who are outpatients. | heart failure is a chronic debilitating disease that affects all aspects of a person s life, including physical, mental and spiritual dimensions. the associations among these dimensions, and the relationship to overall health status, have not been clearly identified. the purpose of this quantitative, descriptive study was to explore differences between spirituality, depressive symptoms, and quality of life among elders with and without heart failure. a total of 44 elders with heart failure and 40 non - heart failure elders completed several questionnaires including : the daily spiritual experiences scale (dses), spirituality index of well - being (siwb), center for epidemiologic studies depression scale (ces - d), and sf-12 health survey. there were significant differences in the groups on gender and ethnicity ; thus these variables were controlled in the analyses related to the dependent variables. after controlling for gender and ethnicity, there were significant differences in the physical component of quality of life and spiritual well - being. the heart failure patients had significantly lower physical quality of life but more spiritual well - being than the non - heart failure patients. there were no significant differences in daily spiritual experiences, mental component of quality of life, and depressive symptoms between the two groups. |
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