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limb traumas are among the most painful and stressful events , that anybody may experience during his or her life . so long as pain control is concerned , morphine remains the gold standard for analgesia . it has been the standard analgesic for many years , and the effects of newer analgesics are often expressed in comparison with the effect of morphine . the search for good analgesics , however , should not only focus on analgesia . as far as analgesia is concerned , morphine is an excellent drug ; but morphine lacks quality of an ideal analgesia due to its side effects . these side effects include sedation , respiratory suppression , nausea and vomiting and pruritus , which may be avoided by substituting other drugs or combinations with the same effects and lower or no adverse effects such as ketamine with or without midazolam . first synthesized in 1963 during the search for the ideal anesthetic , ket - amine was so named because it is a derivative of an amine . and compared with the r ( - ) isomer , the s ( + ) isomer has a fourfold greater affinity for the nmda receptor , twice the analgesic potency , and fewer psychomimetic effects . ketamine is useful as a general anesthetic for trauma patients , because it preserves sympathetic reflexes that help support blood pressure in patients who have lost blood . because it does not interfere with respiratory drive , it is also widely used in resource - poor set - tings where intubation and intraoperative mechanical ventilation are unfeasible . the analgesic action of low , subanesthetic doses of ketamine predominantly de - rives from its activity - dependent , noncompetitive blockade of the glutaminergic nmda - receptor channel complex , through binding at phen - cyclidine ( pcp)-binding sites in the ion channel . experimental and clinical evidence indicates that ketamine reduces opioid - induced tolerance and hyperalgesia ; however , the mechanisms involved are only partially understood . low - dose ketamine decreased morphine - resistant pain , reduced dosage requirements in opioid - tolerant patients , decreased hyperalgesia after remifentanil infusion , and reduced hyperalgesia and allodynia along surgical incisions . traditionally , they have been considered to lack analgesic action ; however , benzodiazepines reduce the minimum alveolar anesthetic concentration of inhaled anesthetics . this is of interest that the neurophysiological mechanisms of the effects of benzodiazepines ( i.e. midazolam ) on nociception have been studied in animals , but the conclusions often are conflicting . according to these studies midazolam may suppress pain pathways of the spinal cord by its specific action and that effect is not due to the action of the drug on supraspinal systems . in this study we had two main objectives : ( 1 ) to compare the efficacy of two analgesic regimens regarding pain control in trauma patients ( closed fractures : defined as fractures with intact skin and soft tissue above the fracture site ) ; ( 2 ) to compare the incidence of adverse events , categorized as respiratory , nausea , and vomiting , and agitation in conjunction with hemodynamic alterations between groups receiving low dose iv ketamine plus midazolam versus iv morphine . we performed a prospective , randomized , single - blind , non - inferiority clinical trial in the emergency department ( ed ) of a university medical center from december 2011 until february 2013 ( clinicaltrials.gov identifier : nct01807429 ) . the main reason for which we considered this study as a single blind was the possibility of a physician bias resulting from ketamine - induced nystagmus . the study was approved by the hospital ethics committee , and written informed consent was obtained from each patient . the sample size was determined about 236 patients ; considering type 1 error rate as 0.05 , statistical power as 80% and common variance as 1.8 for detecting = 1 as the fixed non - inferiority margin ( pain score in terms of visual analogue score ( vas ) ) . two hundred and thirty - six patients of both genders sustaining closed fracture of the extremities ( i.e. , long bones ) who were between the ages of 18 and 60 years , and in good health or with only mild systemic diseases ( american society of anesthesiologists grade 1 or 2 ) were recruited for this study as a convenience sample because the availability of such a population in the ed . patients were eligible for inclusion if they presented a trauma with a severe acute pain defined as a vas score of at least 60/100 ( or 6 ) ; were aged between 18 and 60 years ; and were without acute respiratory , hemodynamic , or neurologic compromise ( respiratory distress signs , systolic blood pressure < 90 mmhg , glasgow coma score < 15 ) . the patients with a psychiatric history ; chronic respiratory , and acute pulmonary infection ; renal , or hepatic failure ; known ketamine sensitivity ; known opioid allergies ; treatment of chronic pain or treatment with opioids ; incapacity to understand the vas ; the use of drugs that affect the central nervous system ; chemical substance abuse ; chronic pain ; pregnancy ; morbid obesity ; increased intracranial pressure ; cardiovascular , hepatic , renal or ocular pathology ; thyroid disease , and pregnancy were not included in the study . patients , who had already received an opioid analgesic ( either by self - administration or by another attending physician or emergency medicine service ( ems ) ) , were also not included in our study . a table of random numbers determined the randomization sequence , using a restricted randomization scheme to ensure roughly equal numbers in each group . the threshold for administration of analgesics in this study was severe acute pain , defined as a vas of at least 6 . eligible patients were randomly allocated to receive either morphine ( 0.05 - 0.1 mg / kg ) followed by additional doses of 3 mg every 5 - 10 minutes or ketamine - midazolam ( 300 - 500 mcg / kg ketamine and 0.03 mg / kg midazolam ) . the latter regimen was also repeated as needed , until pain relief was obtained as defined by a vas score not exceeding 30/100 . the consort flow diagram the patients were assessed at the time of arrival at the ed and every 10 minutes for a total interval of 30 minutes , and the following variables were carefully monitored : visual analogue pain score ( vaps ) , pulse oxymetry ( spo2 ) , respiratory rate ( rr ) , blood pressure ( sbp , dbp ) , heart rate ( hr ) , nausea and vomiting , screaming , cursing , nightmares , and unpleasant hallucinations . pain intensity was verified using a 10 cm ( 100 mm ) vas , anchored by no pain at one end and by worst possible pain at the opposite end . the vas , after teaching the patient as to determining their pain score visually or numerically , was used to evaluate pain , with the patient attributing a value that corresponded to the level of his or her pain . the threshold for efficient analgesia the statistically significant analgesia , however , was held to be as a vas score of 30/100 or lower . respiratory adverse events were defined as oxygen desaturation ( spo2 < 90% ) , apnea ( a minimum 20 seconds transient cessation of breathing ) , or laryngospasm . thirty minutes after the first injection , patients , physicians , and nurses were queried about their level of satisfaction , from 1 ( least satisfied ) to 5 ( most satisfied ) , with a likert scale . the responses were categorized as satisfied ( likert scale score of 4 or 5 ) and not satisfied ( score of 1 , 2 , or 3 ) . primary outcome measure was the percentage of patients with pain relief ( with a vas score of 30/100 or lower ) 30 minutes ( t30 ) after the first injection ( t0 ) . secondary outcomes included pain score comparisons every 10 minutes within the first 30 minutes and comparison of adverse events as mentioned above . thirty minutes after initial analgesic dose , if the patient still did not reach a visual analogue score of 30 mm or less , he or she was excluded from the study . in this situation the patient was considered as having adequate pain relief and was assigned a vas score of 0 . when pain was initially too severe to obtain a vas ( patient refusal ) , it was scored 100 . quantitative variables were expressed as mean sd and qualitative variables as number ( percentage ) . two independent samples t - test was used as the main statistical method for comparing the main outcomes . for all main outcomes the percentage changes from baseline values were calculated and compared between two groups . within group comparisons chi - square test also was used for comparing the qualitative variables between two studied groups . we performed a prospective , randomized , single - blind , non - inferiority clinical trial in the emergency department ( ed ) of a university medical center from december 2011 until february 2013 ( clinicaltrials.gov identifier : nct01807429 ) . the main reason for which we considered this study as a single blind was the possibility of a physician bias resulting from ketamine - induced nystagmus . the study was approved by the hospital ethics committee , and written informed consent was obtained from each patient . the sample size was determined about 236 patients ; considering type 1 error rate as 0.05 , statistical power as 80% and common variance as 1.8 for detecting = 1 as the fixed non - inferiority margin ( pain score in terms of visual analogue score ( vas ) ) . two hundred and thirty - six patients of both genders sustaining closed fracture of the extremities ( i.e. , long bones ) who were between the ages of 18 and 60 years , and in good health or with only mild systemic diseases ( american society of anesthesiologists grade 1 or 2 ) were recruited for this study as a convenience sample because the availability of such a population in the ed . patients were eligible for inclusion if they presented a trauma with a severe acute pain defined as a vas score of at least 60/100 ( or 6 ) ; were aged between 18 and 60 years ; and were without acute respiratory , hemodynamic , or neurologic compromise ( respiratory distress signs , systolic blood pressure < 90 mmhg , glasgow coma score < 15 ) . the patients with a psychiatric history ; chronic respiratory , and acute pulmonary infection ; renal , or hepatic failure ; known ketamine sensitivity ; known opioid allergies ; treatment of chronic pain or treatment with opioids ; incapacity to understand the vas ; the use of drugs that affect the central nervous system ; chemical substance abuse ; chronic pain ; pregnancy ; morbid obesity ; increased intracranial pressure ; cardiovascular , hepatic , renal or ocular pathology ; thyroid disease , and pregnancy were not included in the study . patients , who had already received an opioid analgesic ( either by self - administration or by another attending physician or emergency medicine service ( ems ) ) , were also not included in our study . a table of random numbers determined the randomization sequence , using a restricted randomization scheme to ensure roughly equal numbers in each group . the threshold for administration of analgesics in this study was severe acute pain , defined as a vas of at least 6 . eligible patients were randomly allocated to receive either morphine ( 0.05 - 0.1 mg / kg ) followed by additional doses of 3 mg every 5 - 10 minutes or ketamine - midazolam ( 300 - 500 mcg / kg ketamine and 0.03 mg / kg midazolam ) . the latter regimen was also repeated as needed , until pain relief was obtained as defined by a vas score not exceeding 30/100 . the consort flow diagram the patients were assessed at the time of arrival at the ed and every 10 minutes for a total interval of 30 minutes , and the following variables were carefully monitored : visual analogue pain score ( vaps ) , pulse oxymetry ( spo2 ) , respiratory rate ( rr ) , blood pressure ( sbp , dbp ) , heart rate ( hr ) , nausea and vomiting , screaming , cursing , nightmares , and unpleasant hallucinations . pain intensity was verified using a 10 cm ( 100 mm ) vas , anchored by no pain at one end and by worst possible pain at the opposite end . the vas , after teaching the patient as to determining their pain score visually or numerically , was used to evaluate pain , with the patient attributing a value that corresponded to the level of his or her pain . the threshold for efficient analgesia the statistically significant analgesia , however , was held to be as a vas score of 30/100 or lower . respiratory adverse events were defined as oxygen desaturation ( spo2 < 90% ) , apnea ( a minimum 20 seconds transient cessation of breathing ) , or laryngospasm . thirty minutes after the first injection , patients , physicians , and nurses were queried about their level of satisfaction , from 1 ( least satisfied ) to 5 ( most satisfied ) , with a likert scale . the responses were categorized as satisfied ( likert scale score of 4 or 5 ) and not satisfied ( score of 1 , 2 , or 3 ) . primary outcome measure was the percentage of patients with pain relief ( with a vas score of 30/100 or lower ) 30 minutes ( t30 ) after the first injection ( t0 ) . secondary outcomes included pain score comparisons every 10 minutes within the first 30 minutes and comparison of adverse events as mentioned above . thirty minutes after initial analgesic dose , if the patient still did not reach a visual analogue score of 30 mm or less , he or she was excluded from the study . in this situation the patient was considered as having adequate pain relief and was assigned a vas score of 0 . when pain was initially too severe to obtain a vas ( patient refusal ) , it was scored 100 . we performed a prospective , randomized , single - blind , non - inferiority clinical trial in the emergency department ( ed ) of a university medical center from december 2011 until february 2013 ( clinicaltrials.gov identifier : nct01807429 ) . the main reason for which we considered this study as a single blind was the possibility of a physician bias resulting from ketamine - induced nystagmus . the study was approved by the hospital ethics committee , and written informed consent was obtained from each patient . the sample size was determined about 236 patients ; considering type 1 error rate as 0.05 , statistical power as 80% and common variance as 1.8 for detecting = 1 as the fixed non - inferiority margin ( pain score in terms of visual analogue score ( vas ) ) . two hundred and thirty - six patients of both genders sustaining closed fracture of the extremities ( i.e. , long bones ) who were between the ages of 18 and 60 years , and in good health or with only mild systemic diseases ( american society of anesthesiologists grade 1 or 2 ) were recruited for this study as a convenience sample because the availability of such a population in the ed . patients were eligible for inclusion if they presented a trauma with a severe acute pain defined as a vas score of at least 60/100 ( or 6 ) ; were aged between 18 and 60 years ; and were without acute respiratory , hemodynamic , or neurologic compromise ( respiratory distress signs , systolic blood pressure < 90 mmhg , glasgow coma score < 15 ) . the patients with a psychiatric history ; chronic respiratory , and acute pulmonary infection ; renal , or hepatic failure ; known ketamine sensitivity ; known opioid allergies ; treatment of chronic pain or treatment with opioids ; incapacity to understand the vas ; the use of drugs that affect the central nervous system ; chemical substance abuse ; chronic pain ; pregnancy ; morbid obesity ; increased intracranial pressure ; cardiovascular , hepatic , renal or ocular pathology ; thyroid disease , and pregnancy were not included in the study . patients , who had already received an opioid analgesic ( either by self - administration or by another attending physician or emergency medicine service ( ems ) ) , were also not included in our study . a table of random numbers determined the randomization sequence , using a restricted randomization scheme to ensure roughly equal numbers in each group . the threshold for administration of analgesics in this study was severe acute pain , defined as a vas of at least 6 . eligible patients were randomly allocated to receive either morphine ( 0.05 - 0.1 mg / kg ) followed by additional doses of 3 mg every 5 - 10 minutes or ketamine - midazolam ( 300 - 500 mcg / kg ketamine and 0.03 mg / kg midazolam ) . the latter regimen was also repeated as needed , until pain relief was obtained as defined by a vas score not exceeding 30/100 . the consort flow diagram the patients were assessed at the time of arrival at the ed and every 10 minutes for a total interval of 30 minutes , and the following variables were carefully monitored : visual analogue pain score ( vaps ) , pulse oxymetry ( spo2 ) , respiratory rate ( rr ) , blood pressure ( sbp , dbp ) , heart rate ( hr ) , nausea and vomiting , screaming , cursing , nightmares , and unpleasant hallucinations . pain intensity was verified using a 10 cm ( 100 mm ) vas , anchored by no pain at one end and by worst possible pain at the opposite end . the vas , after teaching the patient as to determining their pain score visually or numerically , was used to evaluate pain , with the patient attributing a value that corresponded to the level of his or her pain . the threshold for efficient analgesia the statistically significant analgesia , however , was held to be as a vas score of 30/100 or lower . respiratory adverse events were defined as oxygen desaturation ( spo2 < 90% ) , apnea ( a minimum 20 seconds transient cessation of breathing ) , or laryngospasm . thirty minutes after the first injection , patients , physicians , and nurses were queried about their level of satisfaction , from 1 ( least satisfied ) to 5 ( most satisfied ) , with a likert scale . the responses were categorized as satisfied ( likert scale score of 4 or 5 ) and not satisfied ( score of 1 , 2 , or 3 ) . primary outcome measure was the percentage of patients with pain relief ( with a vas score of 30/100 or lower ) 30 minutes ( t30 ) after the first injection ( t0 ) . secondary outcomes included pain score comparisons every 10 minutes within the first 30 minutes and comparison of adverse events as mentioned above . thirty minutes after initial analgesic dose , if the patient still did not reach a visual analogue score of 30 mm or less , he or she was excluded from the study . in this situation the patient was considered as having adequate pain relief and was assigned a vas score of 0 . when pain was initially too severe to obtain a vas ( patient refusal ) , it was scored 100 . quantitative variables were expressed as mean sd and qualitative variables as number ( percentage ) . two independent samples t - test was used as the main statistical method for comparing the main outcomes . for all main outcomes the percentage changes from baseline values were calculated and compared between two groups . within group comparisons chi - square test also was used for comparing the qualitative variables between two studied groups . two hundred and thirty - six patients were selected , among whom were 207 males ( 87.3% ) , and 29 females ( 12.2% ) . the average age was 32.6 12.8 with extremes of 60 years and 18 years . the patients were divided into two groups : g1 : 116 patients receiving ketamine - midazolam ( 33.8 14.1 ) and g2 : 120 patients receiving morphine alone ( 31.5 11.4 ) . the median for body weight among g1 patients was 69 kg , and among g2 patients it was 70 kg [ table 1 ] . demographic characteristics of two studied groups the vas score at t30 was significantly decreased compared with vas score at t0 , in both groups ( p < 0.0001 ) . no statistically significant difference , however , was observed between two groups ( p < 0.16 ) . the mean value of the vaps between two groups showed no meaningful difference ; in other words the efficacy of both regimens was the same in terms of pain control . the mean value of dbp revealed no significant difference in both groups ; whereas regarding the mean value of rr and spo2 a meaningful difference was encountered between two groups . furthermore , the mean value of the sbp and hr depicted a meaningful difference ; this means that sbp and hr mean values increased 30 minutes after taking ketamine - midazolam but with morphine these values were decreased [ table 2 ] . nausea , vomiting , agitation , or hallucination was not noted in both groups . with reference to satisfaction from pain control process , no significant difference was seen between two groups [ table 3 ] . comparing the mean of analgesic in two groups in terms of main studied outcome variables satisfaction rates among physicians , nurses , and patients the most significant finding achieved in this study is that there is no meaningful difference between low - dose ketamine - midazolam and morphine with respect to pain control in patients sustaining closed limb fracture(s ) . we found that hypoventilation and increased heart rate / blood pressure were more prominent in group morphine and group ketamine plus midazolam , respectively . the changes noted in cardiopulmonary system , however , were congruent with what we already knew according to the previous evidence - based researches . the emergency atmosphere and ed are somehow stressful per se ; so ketamine - midazolam regimen could be a better choice in terms of pain control in limb trauma patients , while it imposes a minimal risk on cardiopulmonary systems ( like respiratory depression and apnea ) . it also makes itself a reasonable choice in opium - addicted patients , in group ketamine - midazolam inflicted with drug tolerance because of its morphine - sparing effects . regarding the effects of ketamine and morphine on pain on the one hand and their interaction , on the other , so many studies have been yet accomplished . in 2003 , fj et al published an article as to comparing the quality of intravenous patient controlled analgesia ( pcia ) of low - dose morphine plus ketamine with morphine in a group of patients scheduled for elective abdominal hysterectomy . contrary to our research , this study compared morphine consumption and morphine sparing effect of ketamine . they concluded that morphine plus ketamine pcia , in doses used in this study ( 7 mg / kg morphine plus 14 mg / kg ketamine as a bolus ) provided analgesia inferior to that of morphine pcia , but may improve the respiratory side effect profile of morphine . one reason for this conclusion , of course , could be the ultra - low dose of ketamine . laeben et al reported a study about low - dose ketamine for analgesia in the emergency department ( ed ) . however , there was concern about the patient group in this study which may interfere with their conclusion when applied to the general population . compared to our study , the majority of the patients in this research had the record of chronic pain medication use or illicit drug abuse . tolerance or dependence of opioids or opioid - like agents was frequently seen in these patients , which make them become a drug seeker in the ed , and thus , analgesia or so - called improvement of pain for them is more complicated . in our study , however , anybody with history of chronic drug abuse was excluded . galinski assessed management of severe acute pain in emergency settings in an article titled as the aim of the study was to compare in emergency settings two analgesic regimens , morphine with ketamine ( k group ) or morphine with placebo ( p group ) , for severe acute pain in trauma patients . at t30 , the main problem with this study , nonetheless , seems to be possible ketamine - induced nystagmus , and a resultant bias pursued , which we tried to resolve it , as mentioned earlier . similarly , babak garaei and coworkers in iran did a randomized , placebo - controlled clinical trial , in opioid abusers based on their daily opioid consumption . lithotripsy was performed under moderate sedation with intermittent bolus doses of remifentanil ( 0.2 g / kg ) to alleviate pain . the incidences of bradypnea , apnea , nausea , vomiting , and hemodynamic changes were not statistically different between the ketamine and placebo groups . they concluded that preemptive low - dose ketamine ( 0.1 mg / kg ) as a bolus has opioid - sparing effects in opioid abusers undergoing moderate sedation . what has not been studied in the above mentioned researches , however , is the comparison of morphine versus ketamine regarding ed pain control . they all used placebo in their studies but this intervention did not necessarily create an unethical issue in these studies , because all the patients received analgesic irrespective of taking ketamine . regarding midazolam , our goals were to decrease pain as toshinobu sumida et al followed in their study , beside its already known effects on anxiety and ketamine induced agitation , known as emergence phenomenon. of course , probable analgesic effects of midazolam are yet to be investigated by more precise , double - blind studies in the future . another issue that makes our sample more different from its predecessors is that it is a noninferiority trial . the term noninferiority trial is commonly used to refer to a randomized clinical trial in which a new test treatment is compared with a standard active treatment rather than a placebo or untreated control group . one starting principle is that no patient is denied a known effective treatment by entering a clinical trial . in our study we noted no adverse pulmonary effects like hypoventilation as caused by morphine , in group ketamine - midazolam . the effect of ketamine on respiratory rate is in accordance with the findings of presson et al . alfentanil induced a decrease in respiratory rate , without affecting tidal volume and respiratory drive . firstly , ketamine caused subjective side effects in all subjects ( e.g. , strange feeling , body feels tight , arms and legs strange , body feels heavy , etc ) that might have caused general arousal , thereby stimulating respiration indirectly . secondly , being an nmda receptor antagonist , ketamine may antagonize the effect of opioids on ventilation as the effect of opioids on the control of breathing may be through inhibition of glutaminergic transmission . the ketamine dose was , therefore , high enough to have an analgesic effect , but lower than a dose that causes hallucinations . finally , we recommend more extensive , double - blinded studies with lower therapeutic range of ketamine , in the future which may clarify more exactly the real effect and safety of ketamine plus midazolam compared with morphine . we studied only adults between the ages of 18 and 60 years , and thus our data can not apply to elderly patients who are perhaps more likely to experience the sympathomimetic effects of ketamine . furthermore , according to the available data and medical textbooks , the incidence of the closed fractures of the extremities tends to be more at the extremes of ages ( e.g. , femoral head , intertrochanteric or distal radius fractures in the elderly and suprachondylar humeral fractures in children , both of them not involved in the study ) . closed limb fracture because of its low - energy nature ( vas of less than 6 ) , most of the times does not need to be managed with analgesics ; and if a patient who has been probably a drug seeker would apply for analgesic or declared that his pain score was above 6 . we could not , unfortunately , trust his or her real intent , despite receiving analgesics and being assigned for this study . another limitation was the ketamine induced nystagmus that made us perform this study as a single - blind trial , in fear for the probable physician bias while monitoring the patients . we studied only adults between the ages of 18 and 60 years , and thus our data can not apply to elderly patients who are perhaps more likely to experience the sympathomimetic effects of ketamine . furthermore , according to the available data and medical textbooks , the incidence of the closed fractures of the extremities tends to be more at the extremes of ages ( e.g. , femoral head , intertrochanteric or distal radius fractures in the elderly and suprachondylar humeral fractures in children , both of them not involved in the study ) . closed limb fracture because of its low - energy nature ( vas of less than 6 ) , most of the times does not need to be managed with analgesics ; and if a patient who has been probably a drug seeker would apply for analgesic or declared that his pain score was above 6 . we could not , unfortunately , trust his or her real intent , despite receiving analgesics and being assigned for this study . another limitation was the ketamine induced nystagmus that made us perform this study as a single - blind trial , in fear for the probable physician bias while monitoring the patients . we concluded that low - dose iv ketamine - midazolam has the same analgesic effects as iv morphine concerning pain control in adult patients sustaining closed limb fracture(s ) , with less pulmonary adverse events . whether midazolam has significant analgesic effects , however mehdi nasr isfahani , awat feizi : substantial contributions to the conception or design of the work ; or the acquisition , analysis , or interpretation of data for the work . mehdi nasr isfahani : drafting the work or revising it critically for important intellectual content . omid ahmadi , mehdi nasr isfahani : final approval of the version to be published . omid ahmadi , mehdi nasr isfahani : agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved .

background : we assessed the effects of low - dose iv ketamine - midazolam versus morphine on pain control in patients with closed limb fracture(s ) ; and also compared the incidence of adverse events ( cardio - pulmonary ) between two groups.materials and methods : this prospective , single - blind , non - inferiority trial randomized consecutive emergency department ( ed ) patients aged 18 - 60 years to two groups : receiving 300 - 500 mcg / kg ketamine plus 0.03 mg / kg midazolam , or 0.05 - 0.1 mg / kg morphine . visual analogue score ( vas ) and adverse events were verified during an interval of 30 minutes.results:two hundred and thirty six patients were selected , among whom 207 were males ( 87.3% ) . the average age was 29 2 , ( range , 18 - 60 years ) . the vas score at t30 ( i.e. , 30 minutes after initial analgesic dose ) was significantly decreased compared with vas score at t0 , in both groups . no statistically significant difference , however , was observed between the two groups ( 6.1 1.1 versus 6.2 1.0 ; p = 0.16 ) . with regard to systolic blood pressure and respiratory rate , however , a meaningful difference was noted between the two groups ( 1.5 6.4 versus 2.1 6.6 ; p = 0.000 for sbp , and 0.2 1.1 versus 1.1 6.1 ; p = 0.048 for rr).conclusion : low - dose intravenous ketamine plus midazolam has the same analgesic effects as morphine on pain control in trauma patients with closed limb fracture(s ) , in addition to less respiratory adverse events .

INTRODUCTION MATERIALS AND METHODS Study procedures and outcome measure Study design and Patients Study analysis RESULTS DISCUSSION AND CONCLUSIONS Limitations CONCLUSIONS AUTHOR'S CONTRIBUTIONS

a pregnant 23-year - old female with a six - year history of type 1 diabetes presented with a complaint of increased blur in both eyes for the previous two months . best corrected visual acuity was 20/40 ou . slit - lamp examination was entirely unremarkable in both eyes . dilated funduscopic examination was significant for flame - shaped , dot , and blot hemorrhages in the posterior segment , with associated macular edema and exudates bilaterally ( figures 1a and 1b ) . optical coherence tomography ( oct ) revealed a foveal thickness of 578 5 microns and 667 8 microns in the right and left eye , respectively ( figures 2a and 2b ) . all oct scans were performed with the stratus optical coherence tomograph ( zeiss - humphrey inc , dublin , ca ) . examination findings were consistent with bilateral , nonproliferative diabetic retinopathy and clinically significant macular edema . after careful deliberation with the patient and her obstetrician , a decision was made to treat the bilateral macular edema with intra - vitreal triamcinolone acetonide injection . the patient received 0.05 ml of triamcinolone acetonide 40 mg / ml in the left eye initially and in the right eye one week later . best - corrected visual acuity was 20/20 and 20/25 in the right and left eye , respectively . repeat oct revealed a foveal thickness of 159 5 microns and 202 6 microns in the right and left eye , respectively ( figures 3a and 3b ) . slit - lamp examination and goldman applanation tonometry did not reveal any intraocular hypertension or significant lenticular changes at this visit or at any point after triamcinolone injection . the patient delivered a full - term , healthy baby boy weeks prior to this examination . progression of diabetic retinopathy during pregnancy has been described previously by several authors.1,2 visual impairment in these cases can result from both proliferative ( eg , vitreous hemorrhage , retinal detachment ) and nonproliferative etiologies ( eg , retinal hemorrhage , papillopathy , macular edema).1 although macular edema may regress in some cases after delivery , in other cases edema can persist and can be associated with severe and persistent visual dysfunction.1 data from large , randomized clinical trials have established the benefits of argon laser photocoagulation for clinically significant macular edema.3 focal laser photocoagulation of actively leaking blood vessels or grid laser for areas of diffuse permeability can decrease clinically significant edema . however , laser photocoagulation in close proximity to the fovea increases the risk of inducing iatrogenic central scotoma as a result of thermal injury to the tissues , or subsequent glial proliferation . there are many reports of off - label use of intravitreal triamcinolone in cases of persistent and refractory diabetic macular edema.4 improved visual acuity and decreased foveal thickness have been documented by serial oct after a single injection . however , these effects do not appear to persist beyond 34 months without repeated administration.5 a medline search using keywords pregnancy revealed no case reports or case series documenting the treatment of clinically significant macular edema with intravitreal corticosteroids in a pregnant patient . side effects of systemic corticosteroid administration are well known . major ophthalmic complications of intravitreal corticosteroid injection include , but are not limited to , cataract formation and increased intraocular pressure . although corticosteroid equivalents can be measured in the aqueous three months after a single intravitreal injection , it is not known how much corticosteroid is released into the systemic circulation after a single intravitreal injection.6 thus , it is hard to estimate the systemic effects of intravitreal corticosteroid therapy . to date , there are no reports of teratogenic outcomes with systemic corticosteroid use in pregnant human females . however , teratogenic effects have been observed in many species receiving equivalent systemic human doses.7 many authors have noted maxillofacial deformity and , in particular , cleft palate in mice which have received corticosteroids early in gestation . the area of greatest thickening was within the fovea of both the right and left eyes , with little edema observed in the extrafoveal area . laser photocoagulation to this area carried a significant risk of inducing a permanent central scotoma . given the patient s late stage of pregnancy , we felt that intravitreal steroid posed little risk to either mother or fetus . we were able to achieve resolution of macular edema and improved visual acuity with a single intravitreal dose to each eye . in conclusion , we propose that intravitreal triamcinolone injection may be a viable treatment modality for management of clinically significant macular edema in pregnant patients . the safety profile with administration of this medication is enhanced if the steroid is administered after the first trimester . we suggest a multidisciplinary approach and consultation with an obstetrician whenever using corticosteroid therapy in pregnant patients .

we present a case of diabetic macular edema in a pregnant patient treated with a single intravitreal injection of triamcinolone acetonomide . initial presentation and serial examinations after treatment included visual acuity , slit - lamp examination , indirect ophthalmoscopy , and optical coherence tomography . resolution of visual acuity and macular edema were present six weeks after injection and persisted throughout the duration of the pregnancy without further intervention . no adverse outcomes for either mother or fetus were noted . to our knowledge , this is the first report of intravitreal triamcinolone administration in this patient population to be published in the medical literature .

Case report Discussion

it is the most frequent cause of acute liver failure when acetaminophen overdose is included and is therefore medically relevant , exhibiting a high morbidity and mortality . it is also of economic importance and represents the second most frequent cause for drug withdrawal from the pharmaceutical market . many agents , for example , anti - infectives , neurologic , cholesterol - lowering or pain - modifying substances , can act as hepatotoxins ( table 1 ) . they can either damage the liver directly in a dose - dependent and therefore predictable fashion , or idiosyncratically by immunologic mechanisms or metabolic activation independent of the dose . the latter is unpredictable , but unfortunately the more common event , if serious liver injury caused by acetaminophen / paracetamol ( apap ) overdose is not accounted for . symptoms of affected patients are non - specific and include nausea , discomfort in the right upper abdominal quadrant , dark urine , or fatigue . alt , alanine aminotransferase ; alp , alkaline phosphatase ; r , ratio ; uln , upper limit normal . to substantiate the relationship between an administered drug and liver injury , other causes of acute hepatocellular injury need to be excluded . these comprise viral hepatitis ( hepatitis a virus [ hav ] , hepatitis b virus [ hbv ] , hepatitis c virus [ hcv ] , hepatitis e virus [ hev ] , epstein - barr virus [ ebv ] , cytomegalovirus [ cmv ] ) , ischemic liver injury , autoimmune hepatitis , budd - chiari syndrome , wilson 's disease and , in cases of cholestatic liver injury , other causes of extrahepatic or intrahepatic cholestasis , such as choledocholithiasis , malignancy , primary biliary cirrhosis , or sclerosing cholangitis . after exclusion of these differential diagnoses , physicians have to consider dili in patients with an unexplained increase in liver enzymes . currently dili is categorized into hepatocellular ( r - value [ alt / uln alp / uln ] [ alanine aminotransferase / upper limit of normal alkaline phosphatase / upper limit of normal ] 5 ) , cholestatic ( r - value 2 ) , or mixed - type injury ( r - value 2 - 5 ) by the respective enzyme pattern in blood ( table 1 ) . the temporal relation to the administration of a potential hepatotoxin and the exclusion of other differential causes are the cornerstones for establishing a diagnosis of dili . for causality assessment , different tools such as . however , new diagnostic biomarkers for liver injury are currently under investigation in various mechanistic and pre - clinical models and also in large clinical trials . several consortia are dedicated to evaluating new biomarkers in the diagnosis and management of dili . although the objectives of the various endeavours differ , the translation of preclinical mechanism - based findings into clinical liver safety assessment offers a unique opportunity to move the field forward . the us drug - induced liver injury network ( dilin ) recruits patients with idiosyncratic dili in both retrospective and prospective study registries . the international drug - induced liver injury consortium ( idilic ) is a multinational scientific group coordinated by the university of newcastle and university of nottingham . predicting dili by genetic risk factors and identifying potential hepatotoxins before they lead to severe liver injury are the primary goals of investigation . for this purpose , dna isolated from patients with dili is analysed , for example , in genome - wide association studies ( gwas ) . genetic associations have been shown for flucloxacillin - induced dili and the human leukocyte antigen ( hla)-b*5701 allele . with this predisposition , an 80-fold increase in the risk of developing dili pharmacogenetic testing of hla - b*5701 is mandatory in patients scheduled to receive abacavir to prevent hypersensitivity . donaldson and co - workers identified hla class ii genotypes in association with amoxicillin / clavulanate associated dili . for lumiracoxib , a cyclooxygenase-2 ( cox-2 ) selective inhibitor , hla - dr and hla - dq genotypes were found to be predictive for dili . lapatinib , a tyrosine kinase inhibitor , has been associated with dili in carriers of the hla allele dqa*02:01 . an association between dili caused by ximelagatran , a direct thrombin inhibitor , and hla - drb1 * 0701 and hla - dqa1 * 02 was reported . in the case of alt elevations caused by ximelagatran , colony - stimulating factor 1 receptor ( csf1r ) , the receptor for the respective cytokine , was increased significantly in plasma , leading to a new quest for early identification tools . the safe - t consortium ( safer and faster evidence - based translation ) is focused on the identification of new biomarkers for dili . funded by the innovative medicines initiative ( imi ) the consortium aims to identify new tools to predict , detect and monitor drug - induced organ toxicity in a more specific , sensitive and predictive manner . academic centres , pharmaceutical companies and small- to medium - sized enterprises collaborate to promote a personalized medicine approach in the prediction and management of dili . toxicities to other organs of interest are analysed in drug - induced kidney injury ( diki ) and drug - induced vascular injury ( divi ) work packages . another imi project , called mip - dili ( mechanism - based integrated systems for the prediction of drug - induced liver injury ) , focuses on the development of preclinical test systems that integrate multiple preclinical data types to improve prediction of dili in man . the predictive safety testing consortium ( pstc ) was created by the critical path institute and aims to qualify new biomarkers for the detection and monitoring of drug - induced toxicity in preclinical and clinical studies . in this respect , pstc is the preclinical counterpart of the safe - t consortium , since both aim to qualify biomarkers that allow the separation of patients with a self - limiting course of dili from those that progress to severe dili and ultimately liver failure . this prognostic assessment is currently performed by use of hy 's law that predicts a 10% risk of fatality in cases in which a 3-fold elevation of alt above the upper limit of normal ( uln ) is accompanied by a 2-fold elevation of bilirubin . food and drug administration ( fda ) introduced the edish program ( evaluation of drug - induced serious hepatotoxicity ) in 2004 , which has since been implemented to assess dili in clinical trials . new biomarkers should improve patient safety and also reduce drug attrition due to toxicity . further investigated and proposed markers for dili include liver injury markers such as micrornas ( mirna ) , mechanistic biomarkers such as hmgb-1and cytokeratin-18 fragments , and metabolites from urine and serum . circulating serum mirnas such as mir-122 and mir-192 are liver - specific and are proposed as potentially sensitive and specific markers of liver injury . with a short half - life hmgb-1 is a molecular pattern protein marking necrosis : in the acetylated form it indicates immune activation . ck18 and mirna-122 were evaluated as superior biomarkers compared to alt in terms of sensitivity and specificity in dili . ck18 together with hmgb-1 point towards the mechanism of hepatocellular death in apap overdose , notably the degree of necrosis vs. apoptosis . the performance of combinations of biomarkers is likely to surpass that of a single biomarker alone , depending on the context of its use . thus , the balance between apoptosis and necrosis may be indicative of the subsequent course the patient will follow , given that the liver is unique in its ability to activate defence mechanisms against toxic injury . idiosyncratic dili is usually detected in a late stage of drug development often after drug approval in phase iv studies or pharmacovigilance surveillance . an understanding of the pathophysiologic processes in this type of hepatotoxicity is of special interest to both pharmaceutical companies and regulatory authorities . in cases of apap hepatotoxicity , levels of mitochondrial and nuclear dna could differentiate patients who developed acute liver failure , but subsequently recovered , from those who died . these mechanistic markers could provide further insight into the pathophysiological mechanisms of dili , allowing early detection and outcome prediction . in vitro generated hepatocytes derived from skin biopsies of patients with an idiosyncratic reaction could be used to study the unique features of the affected individuals ' hepatocytes , as well as the genotype - phenotype correlation in dili . these new techniques and models hold great promise to improve drug safety with back - translation from bedside to bench . until now , the management of dili has usually consisted of initial withdrawal of the suspected drug . targeted therapy with antidotes , such as n - acetylcysteine in paracetamol ( acetaminophen ) overdose , is possible in defined cases . re - challenge with the causative agent can lead to an augmented adverse effect , especially in immunologically mediated dili , and is therefore not recommended . preventive or prophylactic measures against dili include patients ' education and alerting health care professionals towards liver enzyme monitoring under therapy with a potentially hepatotoxic drug . pharmacogenetic tests , including next - generation sequencing , proteomic or metabolomic approaches for preselecting susceptible patient populations , and tailoring drug therapy to individual patients , have not yet been approved for routine clinical practice . to what extent these new markers will change clinical practice for the prevention of dili remains to be seen . ongoing efforts aim to overcome the gap between bench and bedside towards the early detection of dili and the identification of patients at risk . important results from major consortia such as dilin , safe - t , mip - dili , pstc and idilic can be expected in 2015 .

drug - induced liver injury ( dili ) is a potentially fatal adverse event with significant medical and economic impact . many drugs , especially anti - infective , neurologic or pain - modifying substances , act as hepatotoxins . with cardiovascular toxicity , liver toxicity is one of the two leading causes for drug withdrawal from the market . the liver can be affected directly , in a predictable and dose - dependent manner , or idiosyncratically , independent of the dose and therefore unpredictable.currently dili is a diagnosis of exclusion that physicians have to bear in mind in patients with an unexplained increase of liver enzymes . the type of injury is categorized into hepatocellular , cholestatic , or mixed by the respective enzyme pattern of injury . symptoms of affected patients can mimic any other liver disease.therefore , new diagnostic and prognostic biomarkers for early liver injury are currently being evaluated in multi - centre clinical trials that are conducted by international consortia and other initiatives . pharmacogenetic testing , next - generation sequencing , proteomics , metabolomics and mechanistic markers can help to preselect susceptible patient populations and tailor drug therapy to individual patients . proposed dili indicators that are under investigation include micrornas , cytokeratin-18 ( ck18 ) , high mobility group box protein 1 ( hmgb-1 ) , and several other biomarkers . these developments can change clinical practice , and improve patients ' safety and management . however , they have not been translated into clinical practice or approved for routine use yet . management of dili usually consists of initial withdrawal of the suspected drug and if applicable administration of specific antidotes , such as n - acetylcysteine . however , the overall management of dili could change in the near future with the advent of novel diagnostic and prognostic dili markers .

Introduction Diagnosing DILI: future biomarkers Management and outlook Disclosures

need can be defined as whatever is required for health or comfort ; though there are various ways to look at it as normative , felt , expressed , fundamental , etc . a person with psychiatric disorder needs all things that anybody needs to lead a good life . a person 's need at a given point of time is likely to be influenced by different factors including type of illness , stage of illness , response to treatment , age , gender , socioeconomic status , support system , and place of stay . some studies , however , show that needs of persons with severe mental illness are not related to age , gender and education . whereas persons recovered from these illnesses may express many needs ; persons with severe negative symptoms may express minimal needs or no needs . numerous scales and schedules are available to explore rehabilitation needs of persons with psychiatric illness . these include needs for care assessment , camberwell assessment of needs ( can ) , and perceived rehabilitation needs questionnaires for people with schizophrenia . the most commonly used schedule in indian studies for assessing rehabilitation needs are rehabilitation needs assessment scale ( rnas ) and can . rnas schedule collects purely qualitative information from patients ( on 6 items ) and caregivers ( on 2 items ) , but does not require observer rating . can is a family of questionnaires to assess needs of persons with mental health problems . it has clinical practice versions , research version , and short appraisal schedule version ( cansas ) for persons with severe mental illness . the indian studies have assessed rehabilitation needs in out - patient or in - patient set - up or both . studies have assessed rehabilitation needs from caregivers of persons with chronic mental illnesses in a community sample and in inpatients . the patients in these studies were suffering from schizophrenia , chronic psychosis , severe mental illness comprising bipolar disorder , schizophrenia , and psychosis not otherwise specified ( nos ) . the main needs expressed in these studies were need for employment ( 65% ) and help for family ( 55% ) ; accommodation for in - patients and help for family for outpatients ; social skills ( 80% ) , employment ( 65% ) , providing help to families ( 60% ) ; help for family and modification in families attitude toward them ; attitude modification , employment and help for family and help for family , employment , vocational training and psychosocial modification . a retrospective case records based study on needs of chronic psychiatric patients admitted in a tertiary hospital , reported the need for the development of community outreach programs , especially in areas where no psychiatric services are available . ( 2010 ) reported that patients expressed the need for welfare benefits , information about condition , money , help for psychotic symptoms and psychological distress . in the same study , caregivers expressed the need for help in welfare benefits , psychological distress , money , company , and intimate relationships . caregivers in community sample expressed needs in areas of help for the family , employment and occupation , psychosocial modification and skills training . caregivers of in - patients with schizophrenia patients reported felt need in managing the behavior and social - vocational problems of patients ; health issues of caregivers ; education about schizophrenia ; rehabilitation ; and managing sexual and marital problems of patients . in a study of 101 patients with schizophrenia , 42 had high unmet needs . this was associated with poverty , lower education , and persistent psychopathology . in an indian psychiatric society multicentric study on assessment of health care needs of persons with severe mental illness , patients reported needs in money , welfare benefits , transport , information about the illness and treatment , relief of psychological distress , company , household skills , and intimate relationships . psychiatric rehabilitation services ( prs ) at the national institute of mental health and neurosciences ( nimhans ) , bengaluru runs a day - care center for in - patients and outpatients . at any given point of time , the center has a range of vocational sections including candle making , bakery , bamboo , weaving , plastic molding , tailoring , horticulture , carpentry , leather , printing , crafts , recreation , domestic skills , and computer section . nine female patients admitted in a closed ward for many years ( 2 - 27 years ) due to difficulty in discharging them attend various sections of their choice during the day . the current study was planned to assess rehabilitation needs of this special population to help us understand and cater to their needs . to assess the rehabilitation needs of chronic female inpatients attending day - care in prs , nimhans , bengaluru , karnataka , india . to assess the rehabilitation needs of chronic female inpatients attending day - care in prs , nimhans , bengaluru , karnataka , india . due to unique needs of this special population , an interview schedule was developed by expert consensus [ box 1 ] . focused group discussion was done among the authors of the paper , after going through available scales / schedules . the schedule included needs like accommodation , safety , diet , grooming , privacy , illness management , family , relationships , sexual , recreation , education , occupation , vocational training , financial , emotional , spiritual and any other needs . in addition to whatever facilities you are availing here , do you need anything else ? before applying the interview schedule . rehabilitation needs of chronic inpatients as the patients resided in the hospital , their interactions were limited to nursing staff , hospital attenders , vocational instructors in day - care and treating team . the perspectives of nursing staff in patient 's ward , vocational section instructor of the day - care section attended by the patient and treating psychiatrist ( senior resident in psychiatry ) were also independently sought . nine female inpatients hospitalized for more than 1-year and attending day - care in prs were included in this study . sociodemographic details were collected using proforma designed for the study . the diagnosis made by the treating unit was noted . due to unique needs of this special population , an interview schedule was developed by expert consensus [ box 1 ] . focused group discussion was done among the authors of the paper , after going through available scales / schedules . the schedule included needs like accommodation , safety , diet , grooming , privacy , illness management , family , relationships , sexual , recreation , education , occupation , vocational training , financial , emotional , spiritual and any other needs . in addition to whatever facilities you are availing here , do you need anything else ? before applying the interview schedule . rehabilitation needs of chronic inpatients as the patients resided in the hospital , their interactions were limited to nursing staff , hospital attenders , vocational instructors in day - care and treating team . the perspectives of nursing staff in patient 's ward , vocational section instructor of the day - care section attended by the patient and treating psychiatrist ( senior resident in psychiatry ) were also independently sought . nine female inpatients hospitalized for more than 1-year and attending day - care in prs were included in this study . sociodemographic details were collected using proforma designed for the study . the diagnosis made by the treating unit was noted . four patients were diagnosed with schizophrenia , four patients with bipolar affective disorder and one patient was diagnosed with mental retardation with epilepsy with psychosis nos . sociodemographic details detailed interview about rehabilitation needs was not possible for the patient with mental retardation with epilepsy with psychosis nos due to her symptomatic status . hence , rehabilitation needs have been mentioned for eight patients from patient 's perspective [ table 2 ] and nine patients from perspective of nursing staff , treating team and vocational instructors [ tables 35 ] . rehabilitation needs : patient 's perspective ( n = 8) rehabilitation needs : nursing staff 's perspective for nine patients rehabilitation needs : treating team 's perspective for nine patients rehabilitation needs : vocational instructor 's perspective for nine patients the patients were staying in the closed ward as either they did not have any close family members ( 5 ) or their family was not willing to take them home currently ( 4 ) . they were attending the following vocational sections : bamboo ( 3 ) , computer ( 2 ) , tailoring ( 1 ) , mat weaving ( 1 ) , leather ( 1 ) , and craft section ( 1 ) . to the question in addition to whatever facilities you are availing here do you need anything else ? 8 out of 9 reported that they did not want anything else . most of the patients and all hospital staff interviewed felt that the hospital provided the patients safe , clean accommodation , and enough privacy . in terms of food , both patients and hospital staff felt that breakfast was provided early . the treating doctors felt that more nutritious and less oily food should be provided to these patients ; though one of the doctors felt that the food given here was okay as the patient had never complained . patients expressed unmet needs in grooming such as talcum powder , soap , paste , shampoo , ornaments , etc . which they required when they went out of the ward for day - care . patients expressed unmet needs in recreation like listening to music , watching tv , and reading books . everyone interviewed expressed the need for increasing monthly incentives , which can enable patients to have food of their choice occasionally and buy items for grooming . treating psychiatrists and vocational section instructors wished that the patients should get more incentives but did not specify any amount . one of the doctors was not aware about how much incentives patient was getting and could not comment . the nursing staff felt that only two patients needed to stay in closed ward and rest of them could be placed outside or sent out to work . some interesting suggestions by the treating doctors were about having facilities of listening to music and beauty - parlor facilities for the patients . the most commonly expressed rehabilitation needs were more incentive for attending day - care , change in food timing , more variety in food , avenues for recreation and more involvement by family . five out of nine patients were symptomatic , but were referred to day - care as the treating team felt that they were fit to engage in activities . the patient who could not be interviewed in detail was highly productive in the section she was attending . probably they were not asked about the needs previously or they might not have felt the need to express themselves or they did not think that these needs might be fulfilled . this highlights the fact that patients may not spontaneously come out with their needs and have to be specifically asked about it . both patient and staff felt that patients had enough privacy , even though they stayed in general wards ( 15 - 20 patients in one ward ) and used common toilets . the possible reason is that patients may have got used to living in the same environment . the hospital staff felt that seven of them could be sent for work , but only four among those seven patients expressed interest in working . others were 40 or more than 40 years of age ( 5 of them in 40s and 2 in 60s ) . the nursing staff expressed concern regarding the change in societal attitude towards patients , but only three patients expressed the need for change . patients again might have got used to the environment in the hospital , and their interaction with the society had reduced significantly . a similar view was not expressed by either treating team or vocational instructors as they might be with the patient for limited time , but nursing staff were observing these patients more closely . vocational instructors expressed need that patients should get more incentives , have more variety in food and they should be engaged in work . the patients had material needs like variety and taste of food , some grooming items , and an increase in incentives . it is possible that patients had got used to living in the current situation with limited needs and were unaware about increasing costs of things in the outside world . the hospital authorities were sensitized about this , and the incentive has recently been increased to rs . most of the patients had expressed passive recreation needs ( listening to music , watching tv ) ; they did not express much active recreation ( going out , playing games etc . ) . this suggests that they might have got used to spend time just sitting in the ward in their free time . we had included sexual needs in the schedule , but we did not probe it specifically as it was a sensitive issue . many of the expressed needs could be met if the patients were placed back in the community . some of them could not adjust in nongovernmental organizations when they were placed outside and came back to the hospital again . in future , careful admissions should be planned to avoid families abandoning patients in the ward . for prolonged admissions where family members can not stay with the patient , families should make regular visits to meet the patients and should take them out for a short duration . in those with poor family support , attempts to reintegrate patients back to the community should be pursued at the earliest . else , patients may become comfortable in the hospital , which may make attempts in community reintegration difficult . as evidenced in the present study , long - term hospitalization can reduce aspirations for independent living of patients . for chronic patients who are hospitalized due to poor family support , the hospital atmosphere such patients are in a state of handicaptivity who are left with little options other than accepting the security of a hospital due to lack of better available alternatives . national human rights commission , india ( 2012 ) recommends that authorities may find some jobs for fully recovered patients on nominal remunerations within the hospital to rehabilitate them . positive social role models , avenues to take up a career / studies , fulfillment of social milestones ( like getting married , being financially independent , becoming parents ) and support in their pursuits is necessary to help them in their road to recovery . it is important to develop services like supported housing , supported education and supported employment to cater to their complex needs . such supported approaches will be helpful in social reintegration , reduce stigma , improve quality life , and reduce disability and dependency on the hospital set - up . small sample size , interviews were not audio - taped , emotional needs were not asked from patients once the nursing staff expressed about them . small sample size , interviews were not audio - taped , emotional needs were not asked from patients once the nursing staff expressed about them .

background : female patients hospitalized for a long duration in psychiatric hospitals are a special population with unique needs.aims and objectives : to assess rehabilitation needs of chronic female inpatients attending psychiatric rehabilitation services in a tertiary care psychiatric hospital.materials and methods : rehabilitation needs of nine chronic female inpatients were assessed with an interview schedule developed by expert consensus . the needs were elicited from the patients . perspectives of nursing staff , vocational instructors , and treating psychiatrists were also sought.results and conclusion : most patients expressed the need for more incentives for working in day - care , variety in food and grooming items . the nursing staff felt many patients could be placed outside , and the family members should come more frequently to meet them . vocational instructors felt that patients need more incentives , variety in food and work . treating psychiatrists said that major barriers in discharging and placing them were nonavailability or poor involvement of family members . services like supported housing , supported education and supported employment are necessary to cater to their complex needs .

INTRODUCTION Aim MATERIALS AND METHODS Interview schedule Sample RESULTS DISCUSSION Limitations CONCLUSION Financial support and sponsorship Conflicts of interest

guidelines would be the syntheses of the best available evidences , in addition to professional judgment and patient preferences , support decision - making by clinicians , managers and policy makers about the organization , delivery and improvement of health care [ 1 - 8 ] . according to the world health organization s ( who ) definition , appropriately developed guidelines , based on the best available evidences , should assist providers and recipients of health care and other shareholders to make informed decisions . recommendations [ in guidelines ] might relate to clinical interventions , public health activities , or government policies [ 9 - 11 ] . the first clinical guideline have produced by the uk s national institute for clinical excellence was on schizophrenia , in australia date to the late 1970s , when the state health authority began endorsing guideline booklets [ 12 , 13 ] . most of the guidelines have established in high - income countries but progress in developing such national programs in low and middle income countries has still been lagging behind . they have sought for improving the quality of care provided to the patients , reducing variability and containing the health care costs [ 15 , 16 ] . most organizations were under pressure to produce more guidelines in a shorter time with increasingly limited resources . achieving those goals was difficult , and despite the development of clinical guidelines in many countries , solving the problems of cost and quality as well as variation in care has remained a challenge to health systems . the who has recognized the need to use more rigorous processes to ensure that health care recommendations have informed by the best available research evidence . systematic approach to considering the use and/or modification of guideline(s ) produced in one cultural and organizational setting for application in a different context . customizing evidence - informed guideline recommendations for local application has demanded both methodological expertise and an intimate knowledge of the intended clinical practice environment . dedicated guideline development bodies might have greater capacity to synthesize evidence but often had limited access to detailed contextual information . however , the cultural and organizational differences between and within countries could lead to legitimate variation in recommendations , even when the evidence base would be the same . the means that guidelines produced in one setting might not necessarily be appropriate for another , without careful consideration and/or contextualization . guidelines have produced and used in the complex environment of a health care system with its cultural , ethical , economical , legal and other aspects ; these aspects have need to be taken into consideration in each country . guidance has needed to be transparent , systematic , and adapted to the local contexts . the first national programs of guideline development have started in the usa in the 1980s . national programs have also sought with the expectation that it will be easier and more fruitful to implement national guidelines than local or society developed clinical guidelines . transparent , cost - conscious and patient - centered guidelines based on the best available evidence could help establishing these quality and practice measures . thus , use of guidelines might be optimized by improving their format and content . research has shown that guideline format and content influence perceptions about and use of guidelines specifically , these intrinsic guideline qualities have shown to promote greater understanding of how users could apply the recommendations , stimulating confidence in users , ability to practice the recommended behavior , leading to greater intent to use guidelines and actual use [ 13 , 25 - 27 ] . in iran , the authorities already have thought of producing and localizing the knowledge products such as clinical evidence - based practice guidelines . in addition to legislation , a number of clinical guidelines have also produced and localized . legislator would be responsible for policymaking , planning and supervising on social , economic , cultural and politic , register and presentation of policies , strategies and activities related to training of employees for medical groups , research , health , treatment and medical services , welfare organization social security organization have defined as ministry of health and medical education ( mohme ) responsibility and the islamic consultative assembly in article number 36 ( section b ) in fifth 5 years development plan , ratified in january 5 , 2011 , policy making , planning and supervision of health sector is the responsibility of mohme and consider it as national priority of islamic republic of iran . mohme has recognized as policy maker and supreme supervisor of national health based on the article number 38 ( section h ) , which all of the governmental and nongovernmental sectors in health areas should follow and obey its rules and regulations . despite the development of guidelines in many countries , the cultural and organizational differences between countries , the problems of cost and quality and limited resources guidelines that have produced in one setting might not necessarily would be appropriate for another , our national works have conducted within the context of international and national guidelines in developed countries , which has aimed to harmonize guideline development methods in order to reduce duplication of efforts and to ensure efficient use of resources and produced national health guideline in islamic republic of iran . for improving the quality of care provided to the patients , reducing variability and containing the health care costs , mohme should provide and distribute the guidelines base on its accountability to legal responsibility . the development of guideline recommendations in a typical professional society setting has occurred at committee meetings , under the direction of a chairperson . guideline developers have divided in to 2 groups ( include core and main group ) . the principal aims of the core group were as below : coordinate and provide technical support on guidelines development , organize training on guideline production for main group , provide administrative support for the work of the guidelines review committee . collaborate with other organizations and international networks that provided methodological expertise in relation to guideline development , adaptation and implementation . in addition , for each guideline a technical team which contains of author of guideline and co - workers have designated , who have competency in scientific and literary editing considering cost effectiveness as well as efficacy and equity . the team has included medical specialists in their respective fields , statistician , public health practitioner and epidemiologist as methodologist . in order to adjust and conform , the authors of the guidelines , systematically searched databases of twenty - two international sites which have specifically supported by governments as shown in " table1"with the related keywords to the special subject(s ) . then they have selected at least five sources of them that were more relevant for each subject . then key questions have identified for each guideline , and main table prepared for it including blank space for the answers of questions by each resources . in front of each question , there was a space for entering of iranian experts final opinion for each question , there were at least five answers . gathering and summarizing the replies base on general consensus ( iranian experts ) have considered for the answer of the each question ( making group decision and reaching consensus ) . sometimes , some of the guidelines have sent to scientific institution or experts of other universities to have more idea in this regard ( respected peers - those who have not been members of the guideline panel but were experts in the same field - should review guidelines for scientific validity . these outside reviewers should be acknowledged at the end of the guideline document ) . at the end of this process , the authors have organized and finalized local guideline in a unified frame work . the figure presents an overview of the steps . at the end of this process , national health guidelines have written by team and all of guidelines had an identical framework : name of guideline : in persian and english health professional target groups : in governmental and non - governmental sectors ( private public , cooperative , ngo ) centers , specially family doctors introduction and preface : should have the important points of epidemiology , cost , mortality , incidence , prevalence and burden of disease(s ) in iran and the world . preventive interventions : focusing on preventing risk factors of diseases . for each group of health provider the duties and responsibilities diagnostic interventions : have pots of history , physical examination , least priorities of laboratory - test and imaging . proper care interventions : should have appropriate cost effectiveness , following , referring of patients and determine the responsibilities of rehabilitation . implementation : responsibilities of mohme , universities of medical sciences and health services and other organization have defined for executing the guidelines . in purpose of final editing , the guideline has returned to national main team for more investigating . guidelines have also reviewed by the guidelines review committee for content and format . each guideline group has submitted its final draft to the guidelines committee for approval . the main authors group has finalized the text and draft has forwarded to the idsa governing council for editing and final approval then for registration by a special i d number . in less than 5 months , more than 500 authors in whole country have prepared for educating co- workers about guidelines development and approximately 150 guidelines have provided in three volumes of the book published and distributed . at the beginning of each book the law and main national health policies that support the guidelines have notified . according to this supreme documents executing of all the strategies which have approved by mohme in the field of health and treatment areas , including prevention , diagnosis , test requests , medical treatment , surgical interventions , follow - up , record keeping , patient information and health promotion stratification of medical services and each guideline had a specific 4 components national i d number , containing official automation code , year of publication , and unique guideline code and version code . guideline should be changed base on new evidences every 3 - 5 years ; the version code all will be updated . the guideline leader could determine whether the scope of change warranted a full - scale revision of the guideline . the importance of guidelines as decision - making tools , for promoting evidence - informed practice , and serving as one of the foundations for improving health care efforts , has been shown scientifically . health systems guidance has shown the potential to improve decision - making and enable more efficient use of resources with consequent improvements in the health of populations . however , such guidance needed rigorous and transparent processes of production and evidence - based approaches to ensure its dissemination and uptake . using clinical practice guidelines , especially when they have prepared by adoption method , of course it needed extensive planning and coordination in different parts of the system fortunately promising approaches have been taken in our country in recent years . the primary target groups have been the health professionals and the main responsibility for developing guidelines should rest with them and their organizations . other interested parties - patients , funders , and policy makers - could be involved whenever appropriate . paying attention these objective , guidelines should be based firmly on scientific evidences , interpreted through professional experience and complemented by expert opinion whenever was necessary . in the developing countries such as iran adapting health guidelines in short and medium- term could be a proper way to obtain necessary and enough capacity to design national original ones . also , several guidelines in each different topic would be available to be modified by our country , but , repetition alone was useless except in epidemic and special diseases . the revision of original guidelines as an alternative could be beneficial . besides , using the modified guidelines could base on social economy . international evidences have shown that using national guidelines could increase the quality of care in the health systems and avoid many mistakes and medical errors in developing countries . according to this fact that national guidelines have minded very essential means for policy making in health care , mohme should provide and distribute the guidelines based on its accountability to legal responsibility . therefore , for all the governmental and nongovernmental sectors , institutions and organizations execution of them would be mandatory . in addition , we could consider our modifying and adapting health guidelines experiences as an innovation for developing countries .

backgroundguidelines have produced and used in complex environment of health care system with its ethical , economical , legal and other aspects ; that should be taken into account in any country . modifying the format and content of guidelines might facilitate their usage and lead to improved quality of care and cost containment . we have produced this tool for explained above purpose.methodsa coordinating national team has settled at the office of minster of health and medical education , supported by a guideline review committee . an innovative and appropriate approach for adapting national health guidelines has consisted of eight steps , have defined for preparing the draft of each guideline a technical team which , including main author , her / his co - workers have nominated . the authors of each topic have systematically searched databases of the proposed twenty - two international sites , and then have selected at least five sources of them that were more relevant . the final recommendations have proposed by agreement of technical team and guideline review committee.resultsin less than 5 months , more than 500 authors in whole country have selected to prepare guidelines and , approximately 150 guidelines have provided in three volumes of the published and distributed book . each guideline had a national i d number , constant forever ; all topics should be reviewed every 3 - 5 years.conclusionnational health guideline(s ) would be essential means for policy making in health system and increased the cost containment and quality of care . ministry of health and medical education should provide and distribute the guidelines based on its accountability to legal responsibility .

Introduction Materials and Methods Results Discussion Conclusion

orthognathic surgery can affect the oropharyngeal airway through skeletal displacement and changes in the positions of the hyoid bone and tongue1 . the main concern regarding pharyngeal dimensional changes caused by orthognathic surgery is patient sleep quality1 . up to 25% of adults suffer from obstructive sleep apnea ( osa ) ( apnea - hypopnea index [ ahi ] 5/h ) , and 10% of patients have moderate to severe osa ( ahi 15/hr)23 . maxillomandibular advancement surgery has been shown to safely and effectively treat osa4 , but the efficacy of other kinds of orthognathic surgery on the upper airway is not well understood . two - dimensional ( 2d ) radiograph - based evaluations of the upper airway have revealed that mandibular setback surgery narrows the pharyngeal airway , whereas bimaxillary surgery has a milder or non - narrowing effect156 . one study reported a significant decrease in the pharyngeal airway six months after bimaxillary surgery7 . others used computed tomography ( ct ) to evaluate the effect of bimaxillary surgery on class iii patients and found no significant decrease in the pharyngeal airway89 . cone - beam ct ( cbct ) is an accurate and reliable tool for upper - airway evaluation10 . in this study , we aimed to assess changes in the upper airway space following bimaxillary orthognathic surgery to correct skeletal class iii malocclusion and to determine whether changes in 2d measurements correlate with three - dimensional ( 3d ) measurements on cbct images . this was a prospective cohort study conducted at mashhad university of medical sciences ( mashhad , iran ) from april to june 2012 . all study activities were performed in accordance with the ethical guidelines set by the ethical committee of mashhad university of medical sciences , which conform to the declaration of helsinki . the aims and procedures of the study we included healthy subjects with no previous medical problems , anteroposterior discrepancy > 10 mm , and a need for bimaxillary orthognathic surgery to correct a class iii malocclusion . patients were excluded if they had any previous craniofacial syndrome or dentofacial trauma or if they did not require maxillary impaction as part of their surgical plan . twelve patients participated in the study ( five males and seven females ) with an age range of 16 - 35 years ( mean age , 20.4 years ) and normal body mass index ( bmi ) of 18 - 25 kg / m . two patients were excluded during the experiment because one did not complete follow - up , and the medical documents of the other were lost ( one male and one female ) . all patients underwent bimaxillary orthognathic surgery ( maxillary advancement by lefort i osteotomy and mandibular setback by bilateral sagittal split ramus osteotomy [ bssro ] ) and rigid fixation with titanium miniplates and screws . the mean amounts of maxillary advancement and mandibular setback were 4.4 mm and 6.55 mm , respectively . maxillomandibular fixation was applied for a two - week period for all patients and was followed by elastic therapy to establish optimal occlusion . two upper airway cbct scans were performed for all patients one week before and two to four months after surgery . all cbcts were performed by the same technician at the same clinic , using the same machine and the same settings ( 80 kvp with 6 ma and 0.32 mm slice thickness , planmeca promax 3d s ; planmeca oy , helsinki , finland ) . to provide a reproducible cbct image before and after surgery all cbct segmentations and measurements were performed with the same workstation ( hp xw9400 ; hewlett - packard , palo alto , ca , usa ) and same software ( planmeca romexis 2.8.1.r ; planmeca oy ) . the anterior boundaries of the volume space of interest were the vomer bone , soft palate , base of the tongue , and anterior wall of the pharynx . the posterior , lateral , superior , and inferior boundaries were the posterior wall of the pharynx , the lateral walls of the pharynx , the roof of the nasopharynx , and the plane crossing the vallecula perpendicular to the posterior wall of the pharynx , respectively . 1 , table 1 ) using the mid - sagittal plane view , a mid - sagittal slice was selected and used as a 2d measurement reference ; the landmarks , which are described in fig . 2 and table 2 , were used to perform 2d measurements as previously described by jakobsone et al.9 the wilcoxon one - sample test was used to compare the mean measurements of the airway pre- and post - surgery . spearman 's rank correlation coefficient was used to test associations between cbct volumetric and linear measurements . this was a prospective cohort study conducted at mashhad university of medical sciences ( mashhad , iran ) from april to june 2012 . all study activities were performed in accordance with the ethical guidelines set by the ethical committee of mashhad university of medical sciences , which conform to the declaration of helsinki . the aims and procedures of the study we included healthy subjects with no previous medical problems , anteroposterior discrepancy > 10 mm , and a need for bimaxillary orthognathic surgery to correct a class iii malocclusion . patients were excluded if they had any previous craniofacial syndrome or dentofacial trauma or if they did not require maxillary impaction as part of their surgical plan . twelve patients participated in the study ( five males and seven females ) with an age range of 16 - 35 years ( mean age , 20.4 years ) and normal body mass index ( bmi ) of 18 - 25 kg / m . two patients were excluded during the experiment because one did not complete follow - up , and the medical documents of the other were lost ( one male and one female ) . all patients underwent bimaxillary orthognathic surgery ( maxillary advancement by lefort i osteotomy and mandibular setback by bilateral sagittal split ramus osteotomy [ bssro ] ) and rigid fixation with titanium miniplates and screws . the mean amounts of maxillary advancement and mandibular setback were 4.4 mm and 6.55 mm , respectively . maxillomandibular fixation was applied for a two - week period for all patients and was followed by elastic therapy to establish optimal occlusion . two upper airway cbct scans were performed for all patients one week before and two to four months after surgery . all cbcts were performed by the same technician at the same clinic , using the same machine and the same settings ( 80 kvp with 6 ma and 0.32 mm slice thickness , planmeca promax 3d s ; planmeca oy , helsinki , finland ) . to provide a reproducible cbct image before and after surgery all cbct segmentations and measurements were performed with the same workstation ( hp xw9400 ; hewlett - packard , palo alto , ca , usa ) and same software ( planmeca romexis 2.8.1.r ; planmeca oy ) . the anterior boundaries of the volume space of interest were the vomer bone , soft palate , base of the tongue , and anterior wall of the pharynx . the posterior , lateral , superior , and inferior boundaries were the posterior wall of the pharynx , the lateral walls of the pharynx , the roof of the nasopharynx , and the plane crossing the vallecula perpendicular to the posterior wall of the pharynx , respectively . 1 , table 1 ) using the mid - sagittal plane view , a mid - sagittal slice was selected and used as a 2d measurement reference ; the landmarks , which are described in fig . 2 and table 2 , were used to perform 2d measurements as previously described by jakobsone et al.9 the wilcoxon one - sample test was used to compare the mean measurements of the airway pre- and post - surgery . spearman 's rank correlation coefficient was used to test associations between cbct volumetric and linear measurements . the 2d measurements showed no significant decrease in airway space , whereas there was a significant increase in distance between posterior nasal spine ( pns ) and the posterior wall of the pharynx ( p=0.004 ) . the volumetric measurements of the upper airway space increased in total volume , but the change was not statistically significant . hypopharyngeal and oropharyngeal areas decreased , and nasopharyngeal area increased , but none of these changes were significant . we found that 2d and 3d measurements were significantly correlated with changes in u - mpw ( distance between the uvula [ u ] and the middle pharyngeal wall [ mpw ] , which represents the oropharyngeal airway space ) and oropharyngeal volume ( opv ) ( r=0.83 , p=0.004 ) , as well as with changes in p - upw and degree of maxillary advancement ( r=0.72 , p=0.02 ) . the 2d and 3d changes in the upper airway space that were estimated from cbct scans are shown in tables 3 and 4 . our results indicated that there were no significant decreases in the upper airway , but a significant increase in pns - upw ( distance between the pns and the upw , which represents the nasopharyngeal airway space ) in 2d measurements was noted . although total and oropharyngeal volumes increased , and nasopharyngeal and hypopharyngeal volumes decreased , these changes were not significant . we found correlations between changes in u - mpw and opv ( r=0.83 , p=0.004 ) as well as between changes in p - upw and amount of maxillary advancement ( r=0.72 , p=0.02 ) . one of the advantages of ct scanning is that it offers better delineation between soft tissue and air based on different hounsfield unit ( hu ) densities9 , allowing more accurate measurement of the upper airway . herein , we used the categorizations and boundaries of upper airway levels described by jakobsone et al.9 and lowe et al.11 . our upper airway volume calculations were performed automatically using software that calculated airway volume in a segmented manner based on hu density differences . several authors have demonstrated the feasibility of automatic segmentation and dimension estimation from cbct images for upper airway volume measurement12 . other researchers have used ct , cbct , and cephalometric imaging techniques to assess correlations between 2d and 3d measures and volumetric measurements , but they did not find any statistically significant results913 . the cephalograms were taken in an upright position , and no special measures were performed to obtain standardized radiographs in relation to respiration phase9 . in all cases , the acquisition time for the ct scans was too long for patients to hold their breath . the cephalograms were performed with the patient in an upright position , while ct scans were performed in a supine position9 . several studies found that changes in posterior airway space are associated with changes in body position among osa patients1415 and healthy individuals1516 . furthermore muto et al.17 showed that a 10-degree increase in craniocervical inclination enlarged the posterior airway space by approximately 4 mm . therefore , any increase in craniocervical inclination following mandibular setback17 should be considered during cephalometric imaging with a natural head position . our study was not affected by these limitations because all 2d and 3d measurements were conducted on cbct scans that were performed by the same radiologist , using the same machine , with the patients in the same position . however , the main limitation of our study was the small number of patients . although the only procedure we performed on the mandible for this study was bssro , another common procedure in other studies is intraoral vertical ramus osteotomy ( ivro ) , which has been evaluated for its effect on the upper airway . kawamata et al.18 did not find significant differences between the effects of bssro and ivro on posterior airway space . the only significant change in the 2d measurements was the distance between the pns and the upper pharyngeal wall , which was consistent with previous studies919 . the volumetric measurements showed increases in total and oropharyngeal volumes and decreases in nasopharyngeal and hypopharyngeal volumes , but none of these changes were significant . these findings are in contrast with those of kim et al.20 , who found a significant decrease in total pharyngeal airway volume after bimaxillary surgery in class iii patients . this discrepancy can be attributed to the surgery procedure , in which they performed posterior maxillary impaction and rotated the occlusal plane . however , our results supported the findings of jakobsone et al.9 because of the similar boundaries and areas used to measure the volumes . we found a correlation between change in pns - upw and the amount of maxillary advancement in 2d measurements . the only significant correlation between 2d and volumetric measurements was between changes in u - mpw and oropharyngeal volume . kim et al.20 found correlation between hyoid bone position and airway volume and between changes in palatal plane angle and decrease in total airway space . postoperative pharyngeal airway space ( pas ) values should be compared with osa development values in order to assess osa risk , particularly among patients with osa syndrome9 . a cephalometric - based study conducted by kollias and krogstad21 reported that the normal range of pas values was 14.84.4 mm for males and 12.13.7 mm for females . pas values < 5 mm were reported to correlate with a high apnea index , independent of bmi22 . only one patient , whose preoperative pas minimum was 7.37 mm , had a pas < 5 mm ( 4.58 mm ) three months after surgery , which might be clinically important . there are few studies on the evaluation of polysomnographic value changes after orthognathic surgery for treatment of class iii patients , and the findings from these studies are controversial232425 . gokce et al.23 reported an improvement in sleep quality among class iii patients after bimaxillary orthognathic surgery for treatment . in contrast , hasebe et al.24 reported two cases of mild osa after mandibular setback surgery , and foltn et al.25 concluded that bimaxillary surgery for correction of class iii malocclusion increased upper airway resistance . upper airway changes are not statistically significant but are nevertheless clinically important in some patients because of their associated risk with osa .

objectivesany change in maxilla and mandible position can alter the upper airway , and any decrease in the upper airway can cause sleep disorders . thus , it is necessary to assess airway changes after repositioning of the maxilla and mandible during orthognathic surgery . the purpose of this study was to evaluate linear and volumetric changes in the upper airway after bimaxillary surgery to correct class iii malocclusion via cone - beam computed tomography ( cbct ) and to identify correlations between linear and volumetric changes.materials and methodsthis was a prospective cohort study . cbcts from 10 class iii patients were evaluated before surgery and three months after . the wilcoxon one - sample test was used to evaluate the differences in measurements before and after surgery . spearman 's rank correlation coefficient was used to test the correlation between linear and volumetric changes.resultsthe results show that the nasopharyngeal space increased significantly , and that this increase correlated with degree of maxillary advancement . no significant changes were found in volumes before and after surgery . a correlation was found between linear and volumetric oropharyngeal changes.conclusionbimaxillary surgical correction of class iii malocclusion did not cause statistically significant changes in the posterior airway space .

I. Introduction II. Materials and Methods 1. Participants 2. Inclusion criteria 3. Exclusion criteria 4. Imaging procedure 5. 2D and 3D 6. Data analysis III. Results IV. Discussion V. Conclusion

palatal tremor is a rare movement disorder characterized by rhythmic contraction of the soft palate . palatal tremor is most often symptomatic , secondary to brainstem or cerebellar disease and , in rarer cases , is categorized as essential in the absence of documented brain lesions.1 some authors have documented hypertrophic degeneration of the inferior olives in secondary palatal tremor , but its precise role in causing palatal tremor has not yet been clarified and this finding is currently controversial.2 essential palatal tremor generally affects children of both genders , whereas secondary palatal tremor is most commonly observed in adult males . essential palatal tremor is bilateral and usually disappears during sleep , whereas secondary palatal tremor is more frequently unilateral and persists even during sleep . patients with essential palatal tremor usually have an ear click , which is absent in the symptomatic form . although essential palatal tremor has a benign course and usually disappears spontaneously , it is very annoying for the patient , so several treatments have been administered , including anticonvulsants and anxiolytics , with varying therapeutic effects . at present no specific treatment has been established , although successful treatment by botulinum has recently been reported.3 there have also been reports in the literature of cases of palatal tremor which have been described as psychogenic because they were associated with psychological or psychiatric disorders . in this form , known as psychogenic palatal tremor , the abnormal movement is intermittent , increases during attention , decreases during distraction , and is under voluntary control.4 we describe the case of a 12-year - old boy with palatal tremor presenting clinical features of symptomatic essential and psychogenic palatal tremor , thus suggesting a neuropsychopathological continuum between the different forms of palatal tremor . the case was a 12-year - old boy , the only child of unrelated parents . there was a positive family history of psychiatric disorders , in that the father has a personality with borderline and narcissistic traits , poor impulse control , and multiple motor tics , and the mother had a major , recurrent depressive disorder , and histrionic and paranoid personality traits . moreover , the emotional climate in this family was characterized by a high level of expressed emotion with over involvement and criticism , and with a very conflictual relationship between the parents.5 the patient was born by elective cesarean delivery at the eighth month of gestation after a pregnancy characterized by gestational diabetes treated with insulin . he showed mild motor developmental delay and inadequate performance at primary school , and difficulty in relating to peers . he came to our attention about one year earlier due to the emergence of behavioral disturbances characterized by oppositional defiant behavior and aggression , episodes of psychomotor agitation with dysphoria , and aggression towards others . about two years before these events , when the patient was 10 years old , his parents had started to hear a clicking sound coming from the boy , but no research was carried out until our observations . we performed clinical and instrumental assessments , including physical , neurological , and otolaryngological examinations , laboratory tests , audiological and neuroimaging studies , and a psychodiagnostic assessment , including the iq rating scale ( wechsler intelligence scale for children iii ) and interviews administered to the patient and his parents using the child behavior checklist and schedule for affective disorders and schizophrenia for school - age children present and lifetime version . the neurological examination showed only hypotonia of the facial muscles causing drooling , but no other clinical signs , except for the presence of rhythmical bilateral palatal movements , mainly on the right side , associated with a clicking sound that could easily be heard when listening close to the child s ear . the patient seemed to be very annoyed and concerned about this ear click , which was present with the mouth both open and closed . nevertheless , it was under voluntary control , and the patient was able to voluntarily induce it or stop it for a few minutes . moreover , the ear click was less audible or had a changed frequency during the performance of complex motor and cognitive tasks . otolaryngological examination with fibroscopy detected a bilateral palatal tremor at a frequency of 80 cycles per minute . laboratory tests , including a complete blood count , antinuclear antibody studies , thyroid tests , plasma copper , ceruloplasmin , plasma amino acids , serum lactate and pyruvate , ammonium , and transaminases , were normal . audiometry and electroencephalography were also normal , as were brain magnetic resonance imaging , angiographic magnetic resonance imaging , and proton magnetic resonance spectroscopy , focusing particularly on study of the brainstem . during spontaneous observation , the patient showed a reduced adaptive and relational capacity , with his relationships being characterized by oppositional behavior that sometimes culminated in episodes of aggression and psychomotor agitation . his moods and affectivity were extremely unstable , featuring sudden swings and a poor control of emotions and impulses , in particular at moments of frustration and deferral of pleasure . psychosomatic and anxiety symptoms were also detected . at the wisc - iii test , he obtained an iq of 65 ( verbal iq = 81 , performance iq = 56 ) , expressing mild mental retardation . during the previous year , several therapies had been attempted , including anticonvulsants and antipsychotic drugs , with variable results . topiramate and valproic acid treatment showed no effect on either the behavioral disturbances or the palatal tremor , and carbamazepine therapy showed a transient effect that disappeared for about two months but then recurred . due to an increased frequency of episodes of agitation , chlorpromazine therapy was started , then carbolithium was added , resulting in an improvement of the behavioral disturbances . treatment with piracetam , the effectiveness of which in essential palatal tremor was recently reported by campistol - plana et al,3 was ineffective . it was not possible to administer treatment with botulinum toxin due to the patient s limited cooperation . after six months of treatment with carbolithium , chlorpromazine , and family psychotherapy , the behavioral disturbances were further improved and the ear click was audible only on the right side and had started to fluctuate , disappearing spontaneously for days , reappearing spontaneously in stressful situations . over time , the patient had developed a greater ability to inhibit the palatal tremor voluntarily and to induce it on request or by thinking about it , sometimes to attract attention , thus demonstrating voluntary control . palatal tremor is an abnormal movement of the soft palate and was previously known as palatal myoclonus but more recently has been renamed palatal tremor because this corresponds better with the electrophysiological characteristics of the rhythmic pattern . the heterogeneous nature of palatal tremor covers a wide spectrum of clinical pictures , including symptomatic essential and psychogenic forms . secondary palatal tremor can be a consequence of vascular , infectious , degenerative , traumatic , or neoplastic lesions of the brainstem or cerebellum . this form is often associated with neurological deficits and , once present , persists throughout life and does not disappear during sleep or even in coma . most symptomatic cases occur in adulthood , while a few cases have been reported in childhood in the course of krabbe s disease,6 a cerebellar tumor,7 and encephalitis.8 in secondary palatal tremor , abnormal hypertrophy of the inferior olives has been detected , but its precise role in causing palatal tremor is controversial and not yet clearly demonstrated . recently shaikh et al9,10 have shown , through a mathematical model , that oculopalatal tremor oscillations originate in the hypertrophic inferior olive and are amplified by learning in the cerebellum . in essential palatal tremor , neurological investigations and brain neuroimaging are normal , while an ear click is present , perceived as objective tinnitus , that does not occur in the symptomatic form . deuschl et al11 demonstrated that , in secondary palatal tremor , the movement of the soft palate is caused by contraction of the levator veli palatini and in essential palatal tremor , by contraction of the tensor veli palatini which induces secondary closure of the eustachian tubes , causing a clicking sound that is audible by the examiner . essential palatal tremor is more frequently bilateral , occurs mainly in children , and usually disappears when the patient s mouth is open and during sleep , although in some cases it persists even during sleep . the pathophysiology of essential palatal tremor remains unclear ; it is generally benign , persists for months or years , and in children it tends to fluctuate and disappear spontaneously . essential palatal tremor has been observed in monozygotic male twins , in one of whom symptoms developed at the age of 14 years and in the other at 37 years , suggesting a genetic etiology of the disorder.12 fernandez - alvarez13 included essential palatal tremor in the class of transient movement disorders , a heterogeneous group of primary pediatric movement disorders with a limited duration of expression over time , none or mild disability , and a spontaneously fading disorder . in fact , the abnormal movement can be voluntarily induced or inhibited , and can also be discontinued when the patient is concentrating on motor and cognitive tasks , performing movements , reading , thinking , or doing calculations . some authors have suggested that voluntary control of essential palatal tremor could be due to a certain degree of cortical control that is otherwise absent in the symptomatic forms , while other authors have suggested that it could be due to a psychogenic origin of the disorder . this form is often associated with psychopathology , responds to placebo and improves with psychotherapy . furthermore , the movements have a variable frequency , and unlike the other forms of palatal tremor , increase during attention and decrease when the patient is distracted . nevertheless , psychiatric symptoms , in particular anxiety disorders , have also been reported in the literature for some cases of essential palatal tremor . after a review of the literature in relation to different forms of voluntary control , samuel et al14 suggested that rather , than being a uniform pathogenic entity , essential palatal tremor may be a heterogeneous disorder including different forms ; in particular , a form of conversion disorder , a tic disorder , or a series of completely involuntary movements , as described in most patients with essential palatal tremor . moreover , the same authors suggested a correlation between secondary palatal tremor and essential palatal tremor , in view of the fact that functional magnetic resonance imaging studies have shown an activation of the inferior olives during palatal movements in the essential form.15 these studies suggest an involvement of the same structures in essential palatal tremor and secondary palatal tremor . in addition , in some cases of essential palatal tremor , an association with minor ailments has been reported , including respiratory infections , otitis , tonsillitis , fever , minor head trauma , and headache . the clinical characteristics of our patient seem to support this hypothesis of a continuum between the different forms of palatal tremor ( table 1 ) . he has a palatal tremor with an ear click , audible in both ears , which is usually present in the essential form but is rare in the symptomatic form . the palatal tremor persisted during sleep , which is common in the symptomatic form but rare in the essential form . furthermore , neuroimaging studies did not document lesions in the brainstem or cerebellum , which are typically present in the symptomatic form . in addition , in our patient , pre - perinatal risk factors were present , namely a family history of psychiatric disorders , motor developmental delay , mental retardation , hypotonia of the facial muscles , and behavioral disturbances . even if voluntary modulation of palatal tremor has been reported in literature in some cases of essential palatal tremor , our patient has developed possible voluntary control , suggesting the acquisition of special motor skills rather than an inhibition of involuntary movements , and this seems suggestive of a psychogenic etiology.16 limited data are available about psychogenic movement disorders in children , but clinical findings suggesting a psychogenic cause similar to that in adults , including inconsistent character of the movements , exacerbation with stress and while paying attention , and disappearance when distracted , as well as a response to placebo and improvement with psychotherapy . the underlying psychiatric diagnosis was a conversion disorder in most of the cases described in literature.17 in our patient , the disorder also shared some features of psychogenic movement disorders , such as a discontinuous ear click , association with psychopathological disorders , an increase during periods of attention and disappearance at moments of distraction , as well as voluntary control of the movement . in conclusion , the clinical characteristics of our patient suggest that there may be a neuropsychopathological continuum between different forms of palatal tremor , but this hypothesis requires further investigation , especially functional neuroimaging and genetic studies .

palatal tremor is a rare movement disorder characterized by rhythmic contractions of the soft palate . it is most often symptomatic , secondary to brainstem or cerebellar disease and , in rarer cases , is categorized as essential in the absence of documented brain lesions . there have also been reports in the literature of cases of palatal tremor described as psychogenic because they were associated with psychological or psychiatric disorders . we describe the case of a 12-year - old boy with palatal tremor presenting clinical features of symptomatic essential and psychogenic palatal tremor , thus suggesting a neuropsychopathological continuum between the different forms of disease .

Introduction Case report Discussion

clinical clues apparent on examination can expand or narrow the differential diagnosis substantially , even when the patient is seen first for management of a life - threatening condition in the intensive care unit ( icu ) . we present a female patient with presumed chronic inflammatory demyelinating polyneuropathy ( cidp ) in whom recognition of hypertrichosis , clubbing and papilledema suggested poems ( polyradiculoneuropathy , organomegaly , endocrinopathy , m - protein and skin abnormalities ) syndrome . a 35-year - old female patient was seen by the icu consult services for management of impending respiratory failure . she had presented with cough , sputum , worsening limb weakness and orthopnea for one - week . weakness was symmetrical in all four limbs and she was unable to move any of her limbs across the bed . she was unable to roll to one side or lift her head off the pillow . she had presented with insidious progressive weakness of all limbs 7 months prior to the current symptoms to another hospital . cerebrospinal examination was acellular and showed raised proteins ( 1.3 g / dl ) , without any oligoclonal bands . human immunodeficiency virus enzyme linked immunosorbent assay ( elisa ) , venereal disease research laboratory antibodies and antinuclear antibodies by elisa were negative . she was initiated on 2 g / kg intravenous immunoglobulins monthly , along with 0.5 mg / kg steroids , 1.5 mg / kg azathioprine and osteoporosis prevention therapy . weakness remained static till one - week prior to the time of her current presentation . she had no previous history of smoking , alcohol or other drug abuse or animal exposure . there was also no relevant family history . on examination , she was afebrile , normotensive , with a respiratory rate of 40 cycles / min and pulse rate of 106 beats / min . there was evidence of accessory muscle use , with flaring of alae nasi and paradoxical movement of the diaphragm on inspection . general examination also showed bilateral pitting pedal edema to the level of the knee , grade 2 pandigital clubbing , diffuse skin hyperpigmentation with mucosal sparing and hypertrichosis [ figure 1 ] . neurological examination confirmed normal mental status and muscle weakness : power in both lower limbs was graded 1/5 and in the upper limbs was 2/5 , according to the medical research council ( mrc ) scale . arterial blood gas showed respiratory academia due to acute respiratory acidosis ( ph 7.3 , pao280 mm hg on 4 l / min oxygen , paco250 mm hg , hco328 meq / l ) . computed tomography of the head was normal . bilateral lower limb venous doppler did not show any evidence of venous thrombosis and quantitative d - dimers ( immunoturbidometry , lister metropolis ) were negative . she was unable to complete a forced vital capacity maneuver or perform breath hold required for respiratory muscle testing . sniff measurements and ultrasonography of diaphragmatic muscles were not performed given the obvious paradoxical movements clinically . composite clinical photographs showing grade 3 clubbing ( left ) , with severe distal weakness and a correctible claw - hand deformity and hypertrichosis ( right ) summary of clinical investigations in the index patient she was initiated on parenteral piperacillin - tazobactam , azithromycin , enoxaparin , oxygen at 4 l / min ( approximate fio20.4 ) with bilevel noninvasive ventilation ( niv ) ( bipap 14/4 cmh2o , resmeds9 vpap auto ) . plasmapheresis with 2 l exchanges using 5% albumin replacement was started on alternate days for possible worsening of cidp and was continued for six sessions . the simultaneous features of clubbing , hypertrichosis and papilledema along with progression of weakness despite treatment prompted further evaluation . radiographs of the skull , pelvis , and spine [ figure 2 ] showed osteosclerotic lesions over the right femur and spine . ultrasound examination showed splenomegaly ; there was no evidence of hepatomegaly , enlarged nodes or ascites . g / l ) with serum albumin levels of 2.43 g / dl ( normal 4 - 6 g / dl ) . free lambda chain levels were elevated at 29.60 mg / l ( normal , 5.71 - 26.3 mg / dl ) with normal free kappa chain value of 11 mg / l ( normal 3.3 - 19.4 mg / l ) . serum 2-microglobulin was 3550 ng / ml ( normal 670 - 2143 ng / ml ) . bone marrow biopsy from the right iliac crest showed normocellular marrow , absence of lymphoid aggregates or plasma cell rimming and megakaryocyte hyperplasia . composite radiographs of the pelvis and hips ( left ) showing a sclerotic lesion over the right femur and thoraco - lumbar spine ( right ) , showing another lesion over the lumbar spinous processes ( black arrows ) poems syndrome is a rare paraneoplastic syndrome because of an underlying plasma cell disorder ( pcd ) . the pathogenesis of poems syndrome involves -chain producing monoclonal plasma cell clone from the sclerotic bony lesions . vascular endothelial growth factor ( vegf ) levels are high but may not be the sole pathogenic mechanism , as anti - vegf therapy has shown inconsistent results . the diagnosis of poems syndrome is made by the dispenzieri criteria [ table 2 ] . the differential diagnosis includes vasculitis , connective tissue disease , monoclonal gammopathy of undetermined significance , smoldering multiple myeloma ( mm ) , mm or solitary plasmacytoma . the composite clinical and laboratory findings [ table 1 ] differentiates poems syndrome from these other disorders . plasma vegf level of greater than 200 pg / ml has a specificity of 95% and sensitivity of 68% in support of diagnosis of poems syndrome . this entity can be distinguished from other pcds by the prominent neuropathy , minimal to absent renal disease , thrombocytosis and organomegaly . diagnostic criteria for the poems * syndrome treatment is directed at abolishing the underlying plasma cell clone . in patients without diffuse marrow involvement and/or > 3 skeletal lesions , radiation to the isolated sclerotic lesions half of nonresponders or progression occurs within 12 months of radiation . in those with disseminated bone marrow involvement , chemotherapy with melphalan - dexamethasone , lenalidomide , and autologous stem cell transplantation thalidomide and bortezomib have activity in poems syndrome but have potential to worsen the neuropathy . the index patient presented with findings of advanced peripheral and phrenic nerve neuropathy as a result of the delay of 18 months prior to diagnosis . she was managed with local radiotherapy to proximal right femur and lumbosacral area ( 40 gy , 2 gy / cycle , total 20 cycles ) , thromboembolism prophylaxis , limb and respiratory rehabilitation , percutaneous gastrostomy feeds and nocturnal bilevel niv . she improved objectively 6 weeks after completion of radiotherapy with upper limb power improving to 3/5 and lower limb power to 2/5 and absent paradoxical respiratory movements . she remains on follow - up at 6 months with clinical assessment monitoring the weakness , sensory loss and pulmonary function testing by spirometry .

we present a female patient who was referred for management of respiratory failure . she was being evaluated and managed as worsening chronic inflammatory demyelinating polyneuropathy with type 2 respiratory failure . initial examination showed hypertrichosis , clubbing and papilledema along with severe distal and proximal motor - predominant weakness with impending respiratory failure . she was managed with noninvasive ventilation ( niv ) and plasmapheresis awaiting diagnostic investigations . immunofixation showed an m band and free lambda chain levels were elevated . radiographs showed the classic osteosclerotic lesions of poems ( polyradiculoneuropathy , organomegaly , endocrinopathy , m - protein and skin abnormalities ) syndrome . six weeks after commencing radiotherapy to the osteosclerotic lesions , the patient responded favorably and remains off nocturnal niv support .

Introduction Case Report Discussion

however , there are several controversies in the operative indication , surgical approach and the best treatment method . the gold standard in the treatment of distal radius fracture remains uncertain.1 cast treatment with or without closed reduction has long been considered a viable option for treatment of distal radius fractures . however , the results are often unsatisfactory with restricted function and disabilities.23 several studies have reported good anatomical and functional results of distal radius fractures treated with external fixation but also described high incidence of pin tract infection , cosmetic deformity , and nerve injury.4 traditional open reduction and internal fixation often results in extensive soft tissue dissection and periosteal injury and may be associated with high rates of infection , delayed union and nonunion.45 fortunately , the technique of minimally invasive plate osteosynthesis ( mipo ) recently emerged and this technique can minimize soft tissue stripping and damage to the vascularity of the bone.6 it can lead to a reduction in complications caused by conventional treatment . mipo has been widely used in the treatment of the femoral and tibial fractures.78 more recently its use has also been expanded for the fixation of upper extremity fractures.9101112 in 2005 , imatani et al.13 first reported that five patients of distal radius metaphysis comminuted fracture were treated by mipo in which reduction and internal fixation was completed by two longitudinal incisions ( 3 cm ) on the palmar side of distal forearm . inspired by the report , we designed two kinds of incisions ( 2 cm ) according to the distance between the main fracture line and the wrist for further minimizing soft tissue related complications , such as tendon injury , nerve dysfunction , and vascular compromises.14 besides , mini - incision can offer better cosmetic results.1516 plate fixation of distal radius fractures usually involves two kinds of incisions : palmar and dorsal . the palmar approach has been demonstrated to be more superior because it can avoid the complications caused by the attrition of the extensor tendon in the dorsal approach.171819 in addition , the cortex on the palmar side of distal radius is wide and flat which benefits the exposure of fracture fragments and placement of the plate , especially for a die - punch type of fracture . importantly , the operative scar of the wrist is not very obvious , leading to an excellent appearance.20 a palmar locking plate has superior biomechanics compared with traditional dorsal locking plate.21 we reviewed our experience in treating distal radius fractures using the palmar locking plate and two kinds of incisions in the mipo technique , in order to assess the functional outcome and complications following this method . 22 patients with distal radial fractures ( 10 left , 12 right ) were treated by the modified mipo technique with a t - shaped palmar locking plate . an informed consent was taken from the patients and the approval of the ethics committee of hand surgery hospital was obtained . our indications for using mipo were type a2 , type a3 , type b1 , type b3 , type c1 and type c2 distal radial fractures . patients with type a1 , b2 or c3 of distal radius fracture were not included in this study . patient was placed on a conventional table in the supine position under brachial plexus block . once a satisfactory reduction was achieved , one or two 1.5 mm kirschner wires were obliquely inserted from the styloid process of radius to the proximal ulnar side to maintain the reduction , sometimes the kirschner wires were inserted from the opposite direction as well . it is necessary to add another k wire , parallel to the articular surfaces of the distal radius for a type c fracture . the two skin incisions of 2 cm each were made [ figure 1 ] . for patients with a distance between the main fracture surface and articular surface of distal radius within 1 cm , if this distance was more than 1 cm , distal skin incision was made on the radial side along the proximal wrist crease , parallel to flexor carpi radialis . after cutting the superficial and deep fascia on the radial side of flexor carpi radialis , the flexor carpi radialis and the radial artery were retracted to the radial or ulnar side respectively . then the flexor pollicis longus was retracted to the ulnar side to expose the pronator quadratus . for a few patients with unsatisfied closed reduction , the incision should be made on the fracture end to help reduction under direct vision . a suitable , oblique , t - shaped palmar locking plate was inserted through the incision extraperiosteally . after satisfactory fracture reduction and fixation of the plate , a 2 cm proximal skin incision was made on the radial side of the flexor carpi radialis . the superficial and deep fascia was cut to retract the radial artery and the flexor pollicis longus was dissected using forceps to expose the proximal end of the locking plate . intraoperative clinical photographs showing ( a ) one horizontal and one vertical incision were made at the palmar side . ( b ) the locking screws were inserted to fix the plate.(c ) intraoperative x - ray showed the reduction of the fracture on the lateral and anteroposterior view plaster splints were not applied to any patient postoperatively . we encouraged the patients to exercise their shoulders , elbows , metacarpophalangeal joints , interphalangeal joints 2 or 3 days after operation . patients were advised to perform extension and flexion movement of the affected wrist joints after 3 - 5 days post surgery . postoperative x - rays ( a ) anteroposterior ( b ) lateral views at 2 months followup demonstrating a healed distal radius fracture patient was placed on a conventional table in the supine position under brachial plexus block . once a satisfactory reduction was achieved , one or two 1.5 mm kirschner wires were obliquely inserted from the styloid process of radius to the proximal ulnar side to maintain the reduction , sometimes the kirschner wires were inserted from the opposite direction as well . it is necessary to add another k wire , parallel to the articular surfaces of the distal radius for a type c fracture . the two skin incisions of 2 cm each were made [ figure 1 ] . for patients with a distance between the main fracture surface and articular surface of distal radius within 1 cm if this distance was more than 1 cm , distal skin incision was made on the radial side along the proximal wrist crease , parallel to flexor carpi radialis . after cutting the superficial and deep fascia on the radial side of flexor carpi radialis , the flexor carpi radialis and the radial artery were retracted to the radial or ulnar side respectively . then the flexor pollicis longus was retracted to the ulnar side to expose the pronator quadratus . for a few patients with unsatisfied closed reduction , the incision should be made on the fracture end to help reduction under direct vision . a suitable , oblique , t - shaped palmar locking plate was inserted through the incision extraperiosteally . after satisfactory fracture reduction and fixation of the plate , a 2 cm proximal skin incision was made on the radial side of the flexor carpi radialis . the superficial and deep fascia was cut to retract the radial artery and the flexor pollicis longus was dissected using forceps to expose the proximal end of the locking plate . intraoperative clinical photographs showing ( a ) one horizontal and one vertical incision were made at the palmar side . ( b ) the locking screws were inserted to fix the plate.(c ) intraoperative x - ray showed the reduction of the fracture on the lateral and anteroposterior view plaster splints were not applied to any patient postoperatively . we encouraged the patients to exercise their shoulders , elbows , metacarpophalangeal joints , interphalangeal joints 2 or 3 days after operation . patients were advised to perform extension and flexion movement of the affected wrist joints after 3 - 5 days post surgery . postoperative x - rays ( a ) anteroposterior ( b ) lateral views at 2 months followup demonstrating a healed distal radius fracture the average age of the patients was 65 years ( range 37 - 80 years ) . the mean time from injury to surgery was 7 days ( range 0.5 hour-10 days ) . they were all closed fracture and were classified according to ao / asif system , including type a2 ( n=5 ) , type a3 ( n=3 ) , type b1 ( n=4 ) , type b3 ( n=7 ) , type c1 ( n=2 ) , and type c2 ( n=1 ) . all 22 patients were followed up for an average time of 12 months ( range , 10 - 18 months ) . according to wrist function evaluation criteria described by dienst et al.,22 13 patients obtained excellent results , six cases had good results and three patients had moderate results . none of the patients had poor results [ figure 4 ] . one patient had anesthesia in the thenar eminence and this symptom disappeared after 3 months . the extension and flexion of the wrist and the supination and pronation of the forearm were all good since the first mipo techniques were developed for subtrochanteric and distal femoral fractures , these methods were modified and adapted for use in other fracture types as well , including the distal tibia,2324 humeral shaft925 and the foot.26 in this study , we adopted a modified mipo technique that used a palmar locking plate through two small incisions to treat distal radial fractures . incision length was determined according to the distance between the main fracture line and the articular surface of distal radius . a prospective , randomized study analyzed 90 patients with displaced intraarticular distal radius fractures with an average of 4-year followup to evaluate the functional outcomes of three modes of treatment ( plaster immobilization , external fixation , and open reduction with internal fixation ) . good or excellent results were achieved in 43% , 80% , and 63% in each group , respectively.4 similarly , another prospective , randomized study reported that good or excellent outcomes of conventional closed reduction and immobilization with or without norian srs ( skeletal repair system ) cement in the management of intra or extraarticular distal radial fractures were 45%.27 it appears that the functional outcome was similar when compared our results with that using external fixation . pin tract infection was the major complication of external fixation of fractures and the rates of infection ranged from 0.5% to 30%.28 some bacteria , usually staphylococcus aureus and staphylococcus epidermidis , often colonized at the site of external fixation pins28 that resulted in these infection . in our study , there were no infections . conventional closed reduction using only simple plaster cast immobilization is insufficient to maintain the reduction and cherubino et al . observed a secondary loss of reduction during plaster cast immobilization in 35% of cases.2 a traditional open surgical approach may increase the risk of nonunion , delayed healing and infection due to extensive soft tissue stripping.30 in addition , the complication of nerve injuries are commonly reported after operative treatment of distal radius fractures . the incidence has been estimated from 0% to 17% , with the median nerve being the most commonly injured followed by the radial and ulnar nerves.31 however , in present study , no damage of radial artery and median nerve occurred in all 22 patients postoperatively since our longitudinal incisions were usually along the radial side of the flexor carpi radialis and our surgery was performed strictly in the safety window area to avoid accidental injury.32 besides , the radial artery and median nerve were not exposed during the operation in current study 27% cases developed complications in our study including anesthesia ( 4.5% ) , delayed healing ( 4.5% ) , pain ( 9% ) and limited extension and rotation ( 9% ) 27% complication rate associated with mipo used in our study for various types of fractures shows that this method is not suitable for all distal radius fractures . a specific treatment method and selection of individuals will certainly reduce the number of complications . the limitations of this study include the small sample size , the short followup time , and the absence of a control group . in conclusion , two small incisions to implant a palmar locking plate by mipo for distal radius fractures give good functional results . but further research also should be carried out to explore more suitable treatment for specific individuals .

background : fractures of distal radius are common injury in all age groups . cast treatment with or without close reduction is a viable option . however , the results are often unsatisfactory with restricted function . the open reduction and internal fixation often results in extensive soft tissue dissection and associated high rates of infect and delayed / nonunion . the distractor / external fixator have reported good functional and anatomical results but the incidence of pin traction infection nerve injury and cosmedic deformity are high . we introduced a modified operative technique for minimally invasive plate osteosynthesis ( mipo ) for distal radial fracture and evaluated the functional outcomes and complications.materials and methods:22 distal radial fractures ( 10 left , 12 right ) were treated using the mipo technique and two small incisions with a palmar locking plate from august 2009 to august 2010 . the wrist function was assessed according to dienst wrist rating system , and postoperative complications were recorded.results:according to dienst wrist rating system , 13 patients showed excellent results , 6 cases showed good results and 3 patients had moderate results . no patient had poor results . thus , the excellent and good rate was 86.4% . one patient had anesthesia in the thenar eminence and this symptom disappeared after 3 months . one patient had delayed healing in the proximal wrist crease . two patients had mild pain on the ulnar side of the wrist and two patients had limited wrist joint function.conclusion:the mipo technique by using two small palmar incisions is safe and effective for treatment of distal radial fractures .

I M Operative procedure R D

insulin plays a critical role in glucose homeostasis by regulating the balance between glucose production by the liver and glucose uptake by muscle and adipose tissues . in adipocytes and myocytes , insulin regulates glucose transport by controlling the translocation of the glucose transporter , ( glut)4 . ir has a rather loose definition , but generally refers to the fact that tissues do not respond sufficiently to physiological insulin concentrations . t2d patients usually have hyperinsulinemia , and poor insulin sensitivity is a common characteristic of obesity and hyperlipidaemia . insulin initiates its effects by binding to the extracellular -subunit of insulin receptor ( insr ) , resulting in autophosphorylation of the intracellular -subunit . once activated , the insr recruits and phosphorylates intracellular substrates , including insr substrate ( irs ) family proteins and shc . phosphorylated tyrosine residues on irs and shc then recruit downstream signaling molecules containing src homology 2 ( sh2 ) domains , such as the p85 subunit of phosphatidylinositol 3 kinase ( pi3k ) , which activates akt - mediated signaling , and growth factor receptor - binding protein 2 ( grb2 ) , which leads to the activation of mitogen - activated protein kinase ( mapk ) signaling pathway . in addition to over 20 tyrosine residues , irs proteins contain more than 50 potential serine / threonine phosphorylation sites . in general , irs serine / threonine phosphorylation inhibits insulin signaling by antagonizing tyrosine phosphorylation . increased serine / threonine phosphorylation of irs-1 accelerates its dissociation from the insr and downstream signaling molecules , induces mislocalization , and accelerates its degradation by the ubiquitin - proteasome pathway . multiple irs serine kinases are activated during ir , resulting in increased irs serine phosphorylation and impaired insulin signaling . increased irs serine phosphorylation in ir states , including obesity and t2d , akt ( akt1 , akt2 , and akt3 ) , also known as pkb ( pkb , pkb , and pkb ) , is a serine / threonine kinase activated by pi3k downstream of growth factors and various cellular stimuli . many molecules involved in akt signaling are the key therapeutic targets for the treatment of human diseases , including t2d and cancer . akt mediates the bulk of insulin 's action , including glycogen , lipid , and protein synthesis , cell survival , and the anti - inflammatory response . akt2 activation is closely correlated to glut4 translocation via insulin - activated pi3k signals in adipocytes , and t2d patients have reduced akt activation in adipocytes and skeletal muscle . akt2 knockout mice have impaired insulin action in liver and skeletal muscle and develop diabetes with hyperglycemia , hyperinsulinemia , glucose intolerance , and impaired muscle glucose uptake . in contrast to the decrease in pi3k - akt activity , the mapk pathway is relatively unaffected by ir . mapk pathway activation by insulin signaling is responsible for gene expression , cell growth , and mitogenesis . mapk acts as an irs serine kinase in certain conditions ; therefore , inappropriate activation of mapk may worsen ir by increasing serine phosphorylation of irs and interfering with insulin signaling . insulin , a peptide secreted by pancreatic beta cells , enters the central nervous system by crossing the blood brain barrier in a regulated and saturable fashion , although de novo synthesis of insulin in the brain is still under debate . insrs are widely expressed in the brain , including in the olfactory bulb , cerebral cortex , hippocampus , hypothalamus , and amygdala . insrs are more concentrated in neurons relative to glial cells and are especially high in post - synaptic densities . brain insulin signaling plays critical roles in the regulation of food intake , body weight , reproduction , and learning and memory . intranasal insulin administration improves working memory in both human and animal studies , and intrahippocampal delivery of insulin improves hippocampal - dependent spatial working memory . in addition , insr mrna and protein levels are increased in the hippocampus ca1 region in association with short - term memory formation after a spatial memory task , suggesting that neuronal insulin sensitivity could be enhanced during learning . disruption of insulin signaling , however , makes neurons more vulnerable to metabolic stress , thus accelerating neuronal dysfunction . defective insulin signaling is associated with decreased cognitive ability and the development of dementia , including ad . poor cognitive performance in diabetes and ad are associated with a decrease in insr expression and cerebrospinal fluid ( csf ) insulin levels . a recent study demonstrated decreases in the phosphorylation of similar insulin signaling molecules in both ad and t2d patient brains , and this decrease was more severe in the brains of the patients with both ad and t2d . decreased insulin signaling , including altered kinase activity and irs expression , in ad gets worse with disease progression , and increased basal irs-1 phosphorylation , a key signature of ir , is evident in the ad brain . interestingly , the brain regions with the highest densities of insr , such as the hippocampus and temporal lobe , are also the major targets of neurodegeneration in ad . therefore , impaired insulin signaling caused by ir can have a profound effect on cognitive decline and the development of ad . multiple studies report that patients with mets have an increased risk of developing ad compared to age- and gender - matched controls , and accumulating evidence suggests that ad is closely related to dysfunction of both insulin signaling and glucose metabolism in the brain , prompting some investigators to refer ad as type 3 diabetes , or an insulin resistant brain state . the incidence of both t2d and dementia rise in later life , increasing the prevalence of the comorbidity of these age - related diseases . indeed , t2d predicts cognitive decline in older adults and is related not only to vascular dementia ( vd ) , but also to ad . one of the pioneering epidemiological studies about the connection between t2d and dementia is the rotterdam study , which demonstrates that t2d almost doubles the risk of dementia ad ; interestingly , people who were insulin - treated ( therefore , with more severe diabetes ) at baseline were at the highest risk . another study on a japanese - hawaiian cohort further showed a 1.5- , 1.8- , and 2.3-fold increased risk of total dementia , ad , and vd , respectively , for people with diabetes . the religious order study demonstrated a 65% increased risk of ad among t2d patients . among the diabetes - related factors , higher levels of 2-hour post - load plasma glucose , fasting insulin , and homeostasis model assessment - estimated ir ( homa - ir ) were associated with increased risk for senile plaques after adjustment for age , sex , systolic blood pressure , total cholesterol , bmi , habitual smoking , regular exercise , and cerebrovascular disease . a detailed analysis of 14 high quality longitudinal studies from medline and embase searches further demonstrates that individuals with t2d have a greater than two - fold increased risk of developing ad compared to individuals without t2d , adjusted for age , sex , education , and vascular risk factors ( including a history of stroke , hypertension , and heart disease ) . the duration of diabetes is also a risk factor for increased cognitive decline , and this may be related to the length of exposure to high levels of insulin combined with the severity of disease . the converse is also true , as patients with ad are also more likely to develop diabetes . the mayo clinic ad patient registry reveals that 80% of ad patients have either t2d or an impaired fasting glucose level . animal studies also demonstrate the connection between diabetes and ad . we have reported that db / db mice , a model of t2d , exhibit age - dependent increases in tau phosphorylation and cleavage . inducing type 1 diabetes ( t1d ) and ir by streptozotocin ( stz ) treatment or by feeding with a high fat diet ( hfd ) in ad animal models exacerbates both amyloid and tau accumulation . high - fat and high carbohydrate diet feeding of ad rats significantly increased hyperphosphorylated tau and total tau mrna compared to rats with t2d or ad alone , and there was also a significant difference in spatial memory deficits between ad and ad+t2d groups . obesity is a pathologic state defined by an excessive accumulation and maintenance of adipose tissue . bmi is a simple index of weight - for - height that is commonly used to classify overweight and obese adults ( kg m ) . worldwide , obesity has nearly doubled between 19802008 , with ~35% and ~11% of adults currently overweight or obese , respectively ( http://www.who.int/mediacentre/factsheets/fs311/en/ ) . in the united states , the epidemic is even worse ; 35% of adults and 15% of children were obese in 2010 and 80% of obese individuals are insulin resistant . obesity has been associated with several processes related to the acceleration of aging , including the excessive production of free radicals , oxidation , and inflammation . visceral adipose tissue is a metabolically active endocrine organ , and dysfunction in this organ is responsible for increased plasma free fatty acids ( ffas ) . the inappropriate accumulation of lipids in muscle and liver due to abnormal fatty acid metabolism is one of the main features of ir . visceral fat is also infiltrated with inflammatory cells and secretes proinflammatory cytokines , such as interleukin 6 and tumor necrosis factor- , which are implicated in the development of ir . furthermore , these proinflammatory cytokines produced by adipocytes can cross the blood - brain barrier ( bbb ) and induce neuroinflammation and subsequent neurodegeneration . increased inflammation induces accelerated a deposition and/or decreased clearance and facilitates the polymerization of tau . midlife obesity ( measured by bmi ) consistently demonstrates a strong and independent association with late - onset dementia and ad . in a 30 year longitudinal study involving 6583 members of kaiser permanente of northern california , central obesity ( sagittal abdominal diameter ) alone doubled the risk of dementia after adjusting for age , sex , race , education , marital status , diabetes , hypertension , hyperlipidemia , stroke , heart disease , and medical utilization . studies examining later onset obesity , however , have generated mixed results . in the cardiovascular health study , which involved people 65 years or older , underweight individuals ( bmi<20 ) had an increased risk of dementia ( hazard ratio [ hr]=1.62 ) , whereas obese individuals ( bmi>30 ) exhibited a reduced risk ( hr=0.63 ) relative to normal bmi controls . another study found a decreased risk of dementia with higher bmi in subjects over 76 years old , and bmi and dementia further exhibited a u - shaped correlation in individuals younger than 76 years . rats fed with diets high in saturated or unsaturated fat for 3 months display impairments in learning and memory tasks , and mice fed with a hfd have impaired spatial working memory , as assessed by a t - maze . furthermore , brains of hfd - fed mice exhibit dysfunctional akt signaling and increased irs serine phosphorylation , a marker of ir , and mice fed with a high fat / high cholesterol diet display increased app c - terminal fragment accumulation and increased tau phosphorylation . these results suggest that obesity - induced peripheral ir alters central insulin signaling and leads to ad - like cognitive impairment . dyslipidemia is an important component of ir . because insulin plays a critical role in lipid metabolism by stimulating lipogenesis and suppressing lipolysis , perturbations in insulin signaling lead to accelerated lipolysis and increased production of ffas . approximately 30% of total body cholesterol is contained in the brain ; therefore , slight changes in lipid metabolism may have profound effects on cognitive function . cholesterol is the key component of the plasma membrane , and the processing of app into a occurs in the plasma membrane . the interaction between cholesterol and app in the plasma membrane is critical for a production and clearance . in tg2576 ad mice , a high - fat / high - cholesterol diet significantly increased the production of a , and cholesterol lowering drugs reduced this brain amyloid load by more than two - fold . lowering cholesterol levels in vitro also stimulates non - amyloidogenic processing of app , thereby reducing a production . a meta - analysis of 18 prospective studies ranging from 329 years revealed a consistent association between high midlife total cholesterol and an increased risk of ad and dementia . interestingly , no evidence was found to support an association between total cholesterol later in life and ad . cohorts of the seven countries study by a finnish group concluded that high serum total cholesterol is an independent risk factor for ad , while another finnish study further demonstrated that high midlife cholesterol levels are positively connected with an increased risk of ad later in life . further , a retrospective cohort study of members of the kaiser permanente medical care program of northern california showed that midlife cardiovascular risk factors including smoking , hypertension , high cholesterol , and diabetes are all positively connected with increased dementia in later life , with diabetes and high cholesterol being the strongest risk factors . despite these findings , however , the connection between cholesterol and dementia is still complex and inconclusive , as some studies show no correlation , and others even demonstrate a protective role for cholesterol . abnormal phosphorylation of tau has been implicated as a mechanism of ad pathophysiology since the mid-1980s . tau is commonly regulated by post - translational modifications , including phosphorylation , glycation , glycosylation , sumoylation , o - glcnacylation , and cleavage . in ad , tau is abnormally phosphorylated at its over 80 serine / threonine residues , which leads to the aggregation of tau filaments , appearing as nfts , in cell bodies and proximal dendrites . several kinases , including glycogen synthase kinase-3 beta ( gsk3 ) , cyclin - dependent kinase 5 , mapk , and microtubule affinity - regulating kinases , and phosphatases , such as protein phosphatase 2a , are responsible for tau phosphorylation . these kinases and phosphatases are the targets of insulin regulation . as in peripheral tissues , ir mostly affects pi3k - akt signaling in the brain , and chronic hyperinsulinemia prevents insulin - stimulated akt phosphorylation in cortical neuron cultures . increased basal akt phosphorylation is also present in the cortex of t2d db / db mice , and ex vivo insulin stimulation could not increase cortical akt phosphorylation as it did in non - diabetic control mouse ( db+ ) . gsk3 is one of the key signaling molecules downstream of akt and is a major tau kinase . therefore , the precise regulation of akt signaling is critical for both amyloid and tau neuropathology in ad . impaired glucose metabolism due to ir can affect tau pathology via the dysregulation of o - glcnacylation . similar to phosphorylation , o - glcnacylation is a dynamic post - translational modification involving the attachment of n - acetyl - d - glucosamine ( glcnac ) moieties to the hydroxyl group of serine and threonine residues . o - glcnacylation is affected by nutrients in circulation , especially glucose ; therefore , the accumulation of excess energy associated with obesity and ir can result in the dysregulation of o - glcnacylation . furthermore , in some cases , o - glcnacylation may occur at or near the residues that can also be phosphorylated . tau has at least 12 o - glcnacylation sites which are mostly inversely correlated with phosphorylation status . recent reports demonstrate that reduced brain glucose metabolism and o - glcnacylation leads to increased tau phosphorylation in both in vivo and in vitro models . thus , the failure of proper insulin signaling can promote the accumulation of neurofibrillary tau , disrupt neuronal cytoskeletal networks and axonal transport , and lead to a loss of synaptic connections and progressive neurodegeneration . a recent study examining 157 human brain autopsies demonstrated that irs-1 serine phosphorylation is increased in multiple sites in ad , and in other tauopathies such as pick 's disease , corticobasal degeneration and progressive supranuclear palsy . furthermore , increased irs-1 serine phosphorylation was frequently co - expressed with pathologic tau in neurons and dystrophic neurites . similar results were observed in transgenic ps19 tau mouse brains , where abnormally increased irs-1 serine phosphorylation co - localized with tangle - bearing neurons . conversely , inducing ir by feeding mice a hfd increased tau phosphorylation and impaired insulin signaling , and high fat - feeding of animal models of ad exacerbated the pathologies . after 23 wk on hfd , appswe / ps1de9 mice displayed severe hyperinsulinemia along with increased tau phosphorylation , a levels , and amyloid burden . hfd - feeding from an early age in thy - tau22 mice also potentiated spatial learning deficits and significantly increased tau phosphorylation , and direct disruption of insulin signaling by inducing t1d using stz or irs-2 knockout increased tau pathology . in addition , stz injections in pr5 tau transgenic mice greatly increased insoluble hyperphosphorylated tau and the later deposition of nfts , features not observed in control pr5 mice . together , these results suggest that ir accelerates the onset and increases the severity of ad , especially in situations with a predisposition to developing tau pathology . brain insulin signaling plays a critical role in the regulation of food intake , body weight , reproduction , and learning and memory , and defective insulin signaling is associated with decreased cognitive ability and the development of dementia and ad . ad is characterized by improper expression and processing of app and the accumulation of insoluble neurotoxic a into subsequent senile plaques . studies show that insulin signaling regulates multiple steps of the amyloid cascade and affects a aggregation in the brain . insulin increases the transcription of anti - amyloidogenic proteins , such as the insulin - degrading enzyme ( ide ) and -secretase , and stimulates a clearance . while gsk3 is the major tau kinase , gsk3 increases a production by stimulating -secretase activity . constitutively active akt inhibits app trafficking and a secretion through feedback inhibition of irs and pi3-k . app is modified by o - glcnac in a region that may affect its degradation , and a recent report demonstrated that o - glcnacylation of app encourages non - amyloidogenic -secretase processing , thus decreasing a secretion . conversely , a can affect insulin signaling by competing with or reducing the affinity of insulin binding to its own receptor or by regulating intracellular signaling . soluble a binds to the insr in hippocampal neurons to inhibit receptor autophosphorylation and subsequent activation of pi3k / akt , and a derived diffusible ligands ( addls ) induce the abnormal expression of insr and interrupt insulin signaling , thereby potentially contributing the development of central ir . in addition , a inhibits insulin signaling by c - jun n - terminal kinase ( jnk)-mediated increases in irs serine phosphorylation . intracellularly , a prevents the interaction of pyruvate dehydrogenase lipoamide kinase isozyme 1 ( pdk1 ) with akt to inhibit akt activation . therefore , a feed - forward interaction between impaired insulin signaling and increased a production exacerbates ad pathology in the presence of ir . in a recent cross - sectional study of the wisconsin registry for alzheimer 's prevention that included187 late middle - aged adults , higher ir levels were connected with increased amyloid deposition , as measured by pittsburgh compound b uptake . inducing ir in rats using fructose - containing water also enhanced a production by increasing the expression of -secretase , stimulating -secretase activity , and decreasing ide levels . in cultured primary cortical neurons , insulin reduced a buildup by enhancing the conversion of oligomers to monomers and also prevented a oligomer - induced synaptic toxicity at the level of both synapse composition and structure . in contrast , inhibiting insr activity by transfecting a kinase - dead mutant receptor or a tyrosine kinase inhibitor increased a oligomer aggregation ; similar results were obtained through either insr knock - down or pi3k pathway inhibition . thus , dysfunctional insulin signaling due to ir accelerates amyloid pathology , both in human and animal models . in summary , the resulting impaired insulin signaling , which mainly impacts the pi3k / akt pathway , then increases app processing / a levels and tau phosphorylation . finally , increased a further disrupts insulin signaling to exacerbate ad pathology and cognitive decline ( figure 1 ) . given the close correlation between brain ir and cognitive impairment , therapeutic approaches using anti - diabetic drugs to improve insulin levels or signaling have been tested to treat ad . in rats , intracerebroventricular ( icv ) administration of insulin improved spatial learning and memory and reversed stz - induced cognitive decline , and intranasal delivery of insulin reduced hfd - induced tau phosphorylation , whereas subcutaneous injection only had a minimal effect . although systemic insulin infusion demonstrated beneficial effects on memory , it is not a viable long - term option for ad patients due to adverse hypoglycemic effects , including cognitive deficits and neuronal death . intranasal delivery of insulin , however , is a safe and effective alternative to increase csf insulin concentrations without affecting systemic insulin and glucose levels , and studies have demonstrated the beneficial effects of intranasal insulin on memory function . intranasal insulin delivery for 8 wk improved delayed word recall test in cognitively normal young adults , and a single - dose intranasal insulin administration improved verbal memory in subjects with ad and mild cognitive impairment ( mci ) . in a subsequent study , chronic intranasal insulin for 21 days resulted in enhanced declarative memory and selective attention performance in early ad patients . intranasal insulin also increased plasma a40/a42 ratios , suggesting decreased amyloid pathology levels . likewise , daily intranasal insulin treatment for 4 months in 104 adults with early ad or amnestic mci also improved delayed memory and preserved caregiver - rated ability to carry out daily functions . these studies provide compelling evidence that intranasal insulin may be a promising novel therapeutic approach for early ad and mci patients ; however , it should also be noted that some studies suggest that intranasal insulin treatment is effective only in individuals with apoe - 4-negative genotypes . thiazolidinediones ( tzds ) , rosiglitazone , and pioglitazone are ligands for peroxisome proliferator - activated receptors ( ppars ) , a family of nuclear receptors that regulate the transcription of genes involved in lipid and glucose metabolism . tzds have been used as an anti - diabetic drug since the 1990s due to their beneficial effects on glucose homeostasis , which include increasing insulin sensitivity , reducing blood glucose levels and improving lipid metabolism . a number of animal and clinical studies further suggest that tzds improve cognitive function by enhancing insulin action . early treatment with ppar agonists effectively prevented icv - stz - induced neurodegeneration and its associated learning and memory deficits , and rosiglitazone similarly improved learning and memory and reduced ide mrna levels in tg2576 ad mice . chronic treatment with rosiglitazone also facilitated a clearance , reduced amyloid plaques , decreased tau phosphorylation , and improved cognitive function in ad mouse models , and pioglitazone prevented ir and a overproduction that are associated with fructose - drinking in rats . some studies , however , show inconsistent results depending on gender and/or genotype of the selected animal models . likewise , there are mixed results from human clinical trials with tzds . an early study demonstrated that mild ad or amnestic mci patients who received rosiglitazone for 6 months exhibited better delayed recall and selective attention , and rosiglitazone protected cognitive decline in older individuals with both t2d and mci . similar to intranasal insulin treatment , the effect of rosiglitazone was effective only on the individuals with apoe - 4-negative genotypes . a multicenter trial proved no effect of rosiglitazone on brain atrophy or cognitive function in ad patients , and two large phase iii clinical trials also demonstrated no evidence of clinically significant efficacy in cognition or global function , regardless of apoe - 4 genotype . two prospective randomized open - labelled studies demonstrated that pioglitazone induced cognitive and functional improvements and stabilization of the disease in the individuals with mild ad and mci with t2d . however , another study evaluating the safety of pioglitazone as the primary outcome exhibited no significant treatment effect on cognition after 6 months of treatment in non - diabetic ad patients . incretins , glucose - dependent insulinotropic peptide ( gip ) and glucagon - like peptide-1 ( glp-1 ) , are a group of gastrointestinal hormones secreted by intestinal epithelial cells in response to food intake that can affect whole body glucose utilization . glp-1 receptors are widely expressed throughout the body , including in the pancreas , intestines , heart , and lungs , and in both the central and the peripheral nervous system . the activation of glp-1 receptors leads to the facilitation of glucose utilization and antiapoptotic effects in various organs , and two long - lasting glp-1 analogues , exenatide ( byetta ) and liraglutide ( victoza ) , are approved for the treatment of t2d . more importantly , glp-1 can readily cross the bbb and enhance insulin signaling in the brain . therefore , glp-1 analogues are an attractive therapeutic approach to improve ir in ad and in t2d because they can activate pathways common to insulin signaling and facilitate brain synaptic plasticity , cognition , and cell survival . excendin-4 prevented glucose - induced tau hyperphosphorylation or a-mediated toxicity in cultured neurons , and subcutaneous injection of liraglutide for 30 days reduced icv - stz - induced tau hyperphosphorylation and significantly improved learning and memory in mice . two months of liraglutide injections in app / ps1 mice reduced plaque load , app and a oligomer levels , and overall inflammation and increased ide levels . furthermore , long - term potentiation was significantly enhanced and liraglutide also improved cognitive function , as measured by novel object recognition and morris water maze tests . liraglutide not only has a protective effect at an early stage of ad ( 7 mo ) , but it is also able to reverse ad - related changes in older ( 14 mo ) app / ps1 mice . moreover , liraglutide reduced tau phosphorylation and restored akt and gsk3 phosphorylation in a hfd - induced model of t2d in rats . other glp-1 analogues demonstrated similar beneficial effects on ad pathologies and cognitive function in ad mouse models . although the first small clinical trial of liraglutide in ad patients did not lead to any improvement in cognition or changes in a deposition , as measured by pittsburgh compound b pet , a recent clinical trial of exenatide in parkinson 's disease patients demonstrated clinically relevant improvements in motor and cognitive measures . currently two additional clinical trials are in progress with exenatide ( http://clinicaltrials.gov/ct2/show/nct01255163?term ) and liraglutide ( http://clinicaltrials.gov/ct2/show/nct01843075?term ) in mci and early ad patients , and completion is estimated in 2016 and 2017 , respectively . recent evidence supports the contention that ad may be a slow - progressing brain metabolic disease , and numerous studies demonstrate an intricate connection between mets and ad . individuals with mets features , such as t2d and obesity , have a higher risk of developing ad , while ad patients often develop hyperglycemia and ir . ir due to impaired insulin signaling is a common characteristic of both mets and ad , and likely represents the key link between the two diseases . insulin signaling regulates a and tau , and a has negative effects on insulin signaling ; therefore , dysfunctional insulin signaling can enhance a and tau pathology , and increased a production can further exacerbate ir . thus , several diabetes treatments that enhance insulin signaling are being tested for therapeutic potential in ad and dementia , and even though the results from the tzd clinical trials were disappointing , intranasal insulin and glp-1 analogues are still being actively pursued as a potential treatments for ad and have exhibited some promising results . intranasal insulin , however , is only effective in early ad and mci patients , and individuals with the apoe - 4 allele do not respond well . in addition , exenatide and liraglutide are still in the early stages of therapeutic development , and large clinical trials are currently in progress . effective ad and mci treatment demands the development of specific biomarkers to support the diagnosis of these conditions as early as possible . currently , total tau , hyperphosphorylated tau , and a40/a42 ratios in the csf exhibit over 80% specificity as biomarkers of ad . continued research to discover the precise mechanism of how ir contributes to the onset and progression of ad , is also imperative for the development of improved therapeutic interventions . given the current obesity epidemic among all ages and increased life expectancy , there is a critical need to understand the underlying causes of cognitive impairment due to ir , which may be the key link for the increased incidence of ad in individuals with metabolic disorders .

metabolic syndrome ( mets ) is a cluster of cardiovascular risk factors that includes obesity , diabetes , and dyslipidemia . accumulating evidence implies that mets contributes to the development and progression of alzheimer 's disease ( ad ) ; however , the factors connecting this association have not been determined . insulin resistance ( ir ) is at the core of mets and likely represent the key link between mets and ad . in the central nervous system , insulin plays key roles in learning and memory , and ad patients exhibit impaired insulin signaling that is similar to that observed in mets . as we face an alarming increase in obesity and t2d in all age groups , understanding the relationship between mets and ad is vital for the identification of potential therapeutic targets . recently , several diabetes therapies that enhance insulin signaling are being tested for a potential therapeutic benefit in ad and dementia . in this review , we will discuss mets as a risk factor for ad , focusing on ir and the recent progress and future directions of insulin - based therapies .

Metabolic syndrome and insulin signaling Insulin signaling in the brain MetS and AD IR and tau IR and A Insulin signaling as a therapeutic target of AD Conclusion

studies that only infuse oxytocin into participants and then make claims about human behavior are suspect . this approach does not identify what the brain itself is doing during social interactions , including neurochemical promotion and inhibition of oxytocin synthesis and dose - response relationships between oxytocin and behavior . the key question is whether the brain produces its own oxytocin during the behavior being studied ; if so , the causal relationship between oxytocin and a particular behavior can be demonstrated via an infusion study . but the reverse is not true : infusing oxytocin or any drug into the brain and observing a change in behavior does not mean that this is how the brain works it simply means that a drug has changed behavior , as many drugs do . my studies complete the causal circle by measuring what the brain does naturally and then intervening in this system pharmacologically to show that the behavior can be provoked . after years of experiments , i now consider oxytocin the neurologic substrate for the golden rule : if you treat me well , in most cases my brain will synthesize oxytocin and this will motivate me to treat you well in return . this is how social creatures such as humans maintain themselves as part of social groups : they play nice most of the time . ( why people do not play nice is a fascinating story we also have studied ; see zak , 2012 for evidence ) . but i m a skeptic at heart , so i always want to measure the behavioral effects of oxytocin rather than simply ask people s opinions about how they feel . the experience i had watching million dollar baby caused me to wonder if movies , in addition to direct personal interactions , would cause oxytocin release . to test this , my colleague jorge barraza edited a set of a short video clips that we obtained with permission from st . one version shows a father talking to the camera while his 2-year - old son , ben , who has terminal brain cancer , plays in the background . the story has a classic dramatic arc in which the father is struggling to connect to and enjoy his son , all the while knowing that the child has only a few months to live . the clip concludes with the father finding the strength to stay emotionally close to his son until he takes his last breath . we also developed a video of the same father and son spending a day at the zoo . this version does not mention cancer or death , but the boy is bald ( from his chemotherapy ) and is called miracle boy once during the clip . this video lacks the tension induced by the typical story form but includes the same characters . this version was used as a control story to see what the brain does when any video is being watched . in our first study of narratives , we took blood before and after participants watched one of the two versions of the video.11 we found that the narrative with the dramatic arc caused an increase in cortisol and oxytocin . tellingly , the change in oxytocin had a positive correlation with participants feeling of empathy for ben and his father . heightened empathy motivated participants to offer money to a stranger who was in the experiment . flat narrative of ben and his father at the zoo did not increase oxytocin or cortisol , and participants did not report empathy for the story s characters . these findings suggest that emotionally engaging narratives inspire post - narrative actions in this case , sending money to a stranger . , we did not know for sure that oxytocin was the reason participants cared about the people in the video , just that oxytocin and empathy were correlated . our previous study pointed to oxytocin as the biological instrument that puts people in thrall to a story . to assess the causal impact of oxytocin on narrative immersion , we ran a study using public service announcements ( psas ) in which participants received intranasal infusions of synthetic oxytocin or a placebo . this time around , we decided to test a larger set of video narratives . we wanted stories that most people would not have seen before and ones that could elicit a prosocial action at a cost ( such as a donation ) . we found a rich trove of public service announcements from the united kingdom that are well - produced and engaging . the experiment used sixteen psas that ran for thirty or sixty seconds on four topics : smoking , drinking to excess , speeding , and global warming . to incentivize people to pay attention to the videos , each of the participants was paid five dollars if they could correctly answer a factual question about the ad immediately after watching it . for example , was there a car in the video ? then , our software asked participants if they would like to donate some of the five dollars they had just earned to a charity associated with the cause shown in the psa . none of the psas solicited donations , they simply told stories about social issues . computer software presented all the videos and post - video questions and we used random participant identifiers so that one s donation behavior was kept private . forty people received either 40 iu of oxytocin or an equivalent amount of normal saline ( placebo ) . participants started watching the videos after an hour - long period during which the synthetic oxytocin diffuses from the sinuses into the brain . we found that those who received oxytocin donated , on average , 56 percent more money to charity compared with participants who received the placebo.12 this confirmed the causal role of oxytocin on post - narrative prosocial behavior . we discovered that participants who were given oxytocin showed substantially more concern for the characters in the psas . this increased concern motivated them to want to help by donating money to a charity that could alleviate the suffering these stories depicted . it is as if the brain is lazy and is using a monkey see , monkey do approach to assess appropriate social behaviors . ( indeed , the brain seeks to conserve energy by using default pathways a kind of laziness . ) the psas seemed to persuade viewers that ( for example ) nowadays the humans are very concerned about drinking too much , so as a human , i , too , should be concerned . such responses are what social creatures with social brains do . and yet , participants understand that the stories are fictional and are portrayed by professional actors . the money donated to charity can not help these actors out of their fictional binds . the money might help prevent the harm depicted in the psas from happening to an unknown other person , but this is a big if . nevertheless , oxytocin makes people want to help others in costly and tangible ways . in another experiment,12 we sought to replicate our earlier study by taking blood samples before a group of forty - two participants ( who were not in the oxytocin infusion study ) watched one of the uk psas . we measured the change in oxytocin and in a fast - acting arousal hormone with a long name that is abbreviated acth . when the psa elicited an increase in both acth and oxytocin , donations were 261 percent higher than when one or both of these biomarkers did not rise . the change in acth correlated with the amount of attention people paid to the story . this finding makes sense : if we do not attend to a story , it will not pull us into its narrative arc . attention is a scarce neural resource because it is metabolically costly to a brain that needs to conserve resources . if a story does not sustain our attention , then the brain will look for something else more interesting to do . we also found that the change in oxytocin was associated with concern for the characters in the story , replicating our earlier finding . if you pay attention to the story and become emotionally engaged with the story s characters , then it is as if you have been transported into the story s world . this is why your palms sweat when james bond dodges bullets . and why you stifle a sniffle when bambi s mother dies . narratives that cause us to pay attention and also involve us emotionally are the stories that move us to action . more generally , stories with a dramatic arc fit the requirements for high - impact narratives . this structure sustains attention by building suspense while at the same time providing a vehicle for character development . the climax of the story keeps us on the edge of our neural seats until the tension is relieved at the finish . theorists including aristotle ( poetics , 335 bce ) , gustav freytag ( die technik des dramas , 1863 ) , and joseph campbell ( the hero with a thousand faces , 1949 ) have contended that the rising and falling tension of dramatic performances facilitate the audience s emotional connection to the characters . now let s get down to brass tacks : why are there so many dreadful movies ? humans have known about the three - act structure and mythos , pathos , and ethos for 2,500 years . department of defense wanted to know why narratives are persuasive and supported our research and that of other labs as well . attention is easy to measure rapidly , via a quickened heartbeat or sweat coming from eccrine glands in the skin . nature provided a solution . while we were mostly interested in oxytocin in the brain , the stimulus - induced co - release of oxytocin in the brain and blood meant we could measure changing activity in regions with densities of oxytocin receptors . the vagus nerve ( the longest cranial nerve , which innervates the heart and gut ) is chock - full of oxytocin receptors . with a bit of algorithmic fiddling , scientists can measure the activity of the vagus using an electrocardiogram ( ecg ) . we confirmed that the change in oxytocin in blood correlates with changes in vagus nerve activity . we returned to the story of the dying child ben because it is a reliable way to stimulate oxytocin release . this time we measured cardiac activity using an ecg and sweat using an electrodermal sensor on the fingers . because we were developing a system that might be used in a war zone , we built in redundancies . attention was measured using both heart rate and skin conductance changes from sweat on the fingers ; emotional resonance was quantified using two measures of changes in the brain s relaxation response driven by the vagus nerve . the exciting part was that we could measure both effects up to one thousand times a second with off - the - shelf wireless technologies . but it is not so simple to isolate the effects of a story from everything else the brain is doing to keep you upright , breathing , and conscious . all neuroscience studies need to extract the neurologic signal produced by a stimulus during an experiment from the background noise of all other neural activity . to give you a sense of the scope of this problem , for every thirty people we test for an hour each , we collect a terabyte of peripheral neurologic data . most of this data is not relevant to understanding why people respond to stories , but the faint traces that are relevant must be extracted and processed with extraordinary care . once we did all this , the data told us several interesting things.13 first among them is that the brain does not work like the hypothetical story structure known as freytag s pyramid , in which strictly rising action leads to a climax , and then strictly falling action occurs as the story resolves . even for the one - hundred - second ben video , one s attention waxes and wanes . the brain is attending to the story and then doing a quick search of the rest of the environment , and then refocusing on the story as the tension rises . nevertheless , the peak attentional response occurs in the climax , when ben s father reveals that ben is dying . that s a bombshell to which people pay attention . after about thirty seconds , vagal activity begins to increase as viewers get to know and then begin to empathize with ben and his father . not only were we able we track what the brain is doing millisecond by millisecond during a story , we used the neurologic data to build a predictive model of donations to a childhood cancer charity our measure of story impact . the statistical model we built predicts whether a participant would donate money with 82 percent accuracy . that is , by measuring how your peripheral nervous system responds to a story , we can almost perfectly predict what you ll do before you do it . the participants who , for whatever reason , either lost interest in the video or did nt form an emotional connection to ben and his father almost never donated money to charity . the money will not save ben and it wo nt offer relief to his father . it seems that once we are attentive and emotionally engaged , our brains go into mimic mode and mirror the behaviors that the characters in the story are doing , or might do . as social creatures we are biased toward engaging with others , and effective stories truth be told , ben s story is as near to a perfect high - impact narrative as there is . we wondered if neurologic data could identify bad stories , too . and what about stories that may be distasteful but that are still desirable to watch ? i m glad i did , but i do nt have much desire to watch it again . our next study tested stories about hot - button issues to see how people reacted to potentially disagreeable topics . we used first - person narratives from storycorps , a nonprofit that collects and distributes personal stories . we choose six stories on racism , gun control , and the terrorist attacks of september 11 . for our narrative impact measure , we invited participants to donate some of their earnings to a charity associated with the topic of the story . just as in the ben story , we confirmed that stories that sustain attention and generate emotional resonance produce post - narrative donations even stories on difficult topics . to the brain , good stories are good stories , whether first - person or third - person , on topics happy or sad , as long as they get us to care about their characters . psycholinguists have shown that effective stories induce transportation into the narrative.14 transportation happens when one loses oneself in the flow of the story just like i did while watching million dollar baby . to understand the psychological effects of stories , we included surveys of narrative transportation and concern for story characters in the storycorps study . this shows why stories affect behavior after the story has ended : we have put ourselves into the narrative . even a week after the experiment , accurate story recall was predicted by a single measure : narrative transportation . you may be thinking that we have a money - centric approach to assessing when people are moved by a story . let s try a different approach : we ll have thousands of people rate stories instead . the stories we used were tv commercials . conveniently , this is just what usa today asks readers to do on super bowl sunday : vote for the commercials they like the best . about five thousand people voted for their favorite commercials in 2014 , and the style and content of these short narratives vary from the unusual to poignant to just plain silly . this gave us a chance to further refine our algorithms and test them against what people say they like . usa today does not simply provide a ranking of commercials ; it has its readers rate them on a one to ten scale . my group derived a quantification of narrative engagement using neurologic data so we , too , could rate story quality . we estimated the relative contribution of attention and emotional resonance on story impact from our corpus of studied stories . we call this measure a story s zest ( for zak engagement statistic ) . by estimating each super bowl ad s zest , we could compare the usa today readers ad likability with the zest measure of brain activity . three days after the 2014 super bowl , sixteen participants watched the top ten super bowl commercials in random order in my lab while we measured their peripheral neurologic activity . there was no correlation at all between what usa today readers said they liked and a commercial s zest . so we ran another study using usa today s top ten 2013 super bowl commercials and found exactly the same thing : zero correlation . these findings suggest that people are unable to articulate what they like and do not like . perhaps this should not surprise us . in a classic study , psychologist and economics nobel laureate daniel kahneman found that people s preferences for things they have not experienced are largely unformed.15 watching the super bowl commercials myself , i sensed why it is hard to articulate what one likes . the best super bowl commercial in 2014 , according to usa today readers , produced for budweiser beer . in the first ten seconds , one sees a puppy nuzzling the nose of a clydesdale horse . one immediately recognizes the clydesdale as the budweiser icon , and this tells viewers what they can expect from the ad . the suspense is gone , and our neurologic measures show that people s attention wanders starting fifteen seconds into the commercial . without attention , the hoped - for emotional resonance with the ad s characters ( and presumably the brand ) fails to occur . but ask people what they like and , gosh , they see puppies and horses and wide open country and , well , of course we love these images . the commercial is dull . in all our studies we ruled out effects that might influence zest , including movement , cars , buildings , attractive men and women , and many other factors . department of defense s funding of the emerging science of narrative jump - started the field.16 17 storytellers have always known that attention and emotion are important to develop during a narrative , but now we have ways to measure these responses directly rather than rely on incohate impressions such as entertaining or fascinating . yet , even with millennia of practice , creating a great story is difficult . the emerging science of narrative can guide the art , but it can not replace it . the narrator in million dollar baby describes the heroine , maggie s , desire to be a boxer as ... the magic of risking everything for a dream that nobody sees but you .

editor s note : the man behind the discovery of the behavioral effect of a neurochemical in the brain called oxytocin wondered if the molecule might motivate people to engage in cooperative behaviors . in a series of tests using videos , his lab discovered that compelling narratives cause oxytocin release and have the power to affect our attitudes , beliefs , and behaviors .

Provoking the Brain Narrative Immersion Attention-getting Do We Know a Good Story When We See One?

enamel demineralization is a significant problem and can cause major clinical complications of orthodontic treatment with fixed appliances . prevalence of white spot lesions after orthodontic treatment was reported to vary from one - third up to 96% in patients undergoing fixed appliance therapy . the placement of fixed orthodontic appliances creates a favorable environment for the accumulation of microorganisms , which causes enamel demineralization or exacerbates the effects of any pre - existing caries . the high prevalence of carious lesions might be due to the high cariogenic challenge existing in the plaque around orthodontic appliances . prevention of enamel decalcification and remineralization of enamel through orthodontic treatments is a critical issue . topical fluoride in various forms ( toothpaste , mouthrinse , gels , varnishes , and fluoride - releasing cements ) has been used extensively in the prevention of demineralization around orthodontic brackets . the use of topical fluorides in addition to fluoride toothpaste appears to reduce the incidence of decalcification in patients undergoing orthodontic treatment with fixed appliances . a systematic review examining 90 studies concluded that the optimum results were obtained when orthodontic patients with fixed appliances had used daily mouth rinsing with a 0.05% sodium fluoride mouthrinse . the effectiveness of these products and methods of prevention is directly related to the patients compliance . patients compliance and their cooperation in orthodontic treatment and oral hygiene are considered a problematic matter . orthodontists do not implement the available evidence in order to prevent enamel demineralization during fixed - appliance treatment . the application of fluoride varnish ( fv ) can be considered an efficient preventive method to enhance enamel resistance against the cariogenic challenges during orthodontic therapy . clinicians should consider applying fv on areas of enamel that exhibit demineralization or are at risk of demineralization in patients with poor oral hygiene . one topical application of fv with a high concentration can decrease enamel lesion depth adjacent to bonded brackets by about 40% for 3 months . to prevent decalcification , it has been recommended to use casein phosphopeptide - amorphous calcium phosphate ( cpp - acp ) , which may assist remineralization and can maintain high concentration gradients of calcium and phosphate ions and ion pairs into the subsurface lesion , thus leading to high rates of enamel remineralization . the presence of cpp - acp agent delays the biofilm formation and favors the nucleation and crystallization of calcium phosphates , possibly in apatitic form , in matured biofilms . the application of cpp - acp before bonding improves the shear bond strength ( sbs ) to demineralized enamel . the application of teeth mousse ( cpp - acp ) ( tm ) , naf , or tm / naf can significantly prevent enamel demineralization when composite resin is used for bonding . in an attempt to reduce the numbers of procedural steps and chair time when bonding orthodontic brackets to enamel , the self - etching primers ( seps ) were developed . several in vitro studies observed the efficacy of sep as an agent to prepare enamel for bonding orthodontic attachments . clinical failure rate of sep was evaluated and it was concluded that using sep in routine orthodontic clinical practices had brought about significant results . the results of in vivo , randomized cross - mouth clinical trial suggested that enamel pre - treatment with ideal 1 sep system ( gac orthodontic products ) results in unacceptably high bond failure rates when compared with conventional enamel acid etching and , as such , it can not be recommended for clinical use . however , the pre - treated enamel by sep transbond plus ( 3m / unitek ) has resulted in acceptable sbs values for clinical use . it was suggested that the sep should achieve adequate bond strengths when applied to dry enamel surfaces . bonding systems with seps may offer potential benefits compared with conventional acid etchings and primers because of the fewer irreversible changes to the enamel surface . an in vitro bond strength testing of materials used in orthodontic bonding will produce more reliable guidance for the clinical orthodontist . the aim of this study was to evaluate the effect of the application of either fluoride varnish ( fv ) or amorphous calcium phosphate ( acp ) as preventive method on shear bond strength ( sbs ) at the same time of their bonding in vitro using self - etching primer ( sep ) as an agent for enamel pre - treatment fv . the null hypothesis was that the application of either fv or acp at the same time of the bonding , using sep will have no effect on the sbs . the aleppo university scientific and ethics committee authorized the authors to proceed with the project using the materials and methods described in the manuscript . sixty sound human premolars freshly extracted for orthodontic purposes were collected and stored in a solution of 10% formaldehyde solution ( epenhuysen chemie b.v . , drachten the netherlands ) and then in distilled water until use . the criteria for tooth selection included an intact buccal enamel surface , not subjected to any pre - treatment by chemical agents such as phosphoric acid , hydrogen peroxide , no cracks due to the presence of the extraction forceps , and no caries . each tooth was cleaned and polished using pumice for 10 s. teeth were placed in acrylic boxes . a mounting jig was used to align the facial surface of each tooth in order to be as perpendicular with the bottom of the mold as possible . the specimens were randomly divided into five groups ( n=12 ) : group 1 : rubbed by sep transbond plus ( 3 m unitec ) for 5 s ( control group)group 2 : rubbed for 5 s by sep 5 min after the application of acp ( tm - gc)group 3 : rubbed for 10 s by sep 5 min after the application of acpgroup 4 : rubbed for 5 s by sep 5 min after the application of fv ( durashield 5% sodium fv , usa)group 5 : rubbed for 10 s by sep 5 min after the application of fv group 1 : rubbed by sep transbond plus ( 3 m unitec ) for 5 s ( control group ) group 2 : rubbed for 5 s by sep 5 min after the application of acp ( tm - gc ) group 3 : rubbed for 10 s by sep 5 min after the application of acp group 4 : rubbed for 5 s by sep 5 min after the application of fv ( durashield 5% sodium fv , usa ) group 5 : rubbed for 10 s by sep 5 min after the application of fv a piece of wax equal in size to the base of a bracket was placed at the center of the buccal surface of each tooth as a guide for the placement of orthodontic brackets . the wax was gently removed from the teeth , and then sep was applied to the teeth in each group according to the protocol above . stainless steel metal brackets ( mini sprint - brackets ; forestadent company , pforzheim , germany ) were used . the brackets were bonded to the teeth using a light - curing composite ( system rmo / mono - lok2 bonding ) and polymerized with a light - curing unit ( bluephase led ivoclar vivadent , irradiance 380515 nm ) for40 s. the area of the bracket base surface was 12.4 mm as given by the manufacturer . a zwick / roell z020 universal testing machine ( zwick gmbh and co , germany ) with a 500 n load cell was used to test sbs . the specimens mounted in their acrylic blocks were secured to the lower grip of the machine ( fixed head ) . to maintain a consistent debonding force , a custom - made blade was fixed in the upper grip ( movable head ) connected to the load cell . the blade was positioned in such a way that it touched the bracket and the force applied to the ligature groove between the bracket base and the wings . each tooth was oriented with the testing device as a guide so that its labial surface was parallel to the force during the shear strength test . the debonding forces of the brackets were recorded in n and then converted in mpa by taking into account the surface area of the bracket base . the surfaces of the teeth were then examined with a stereomicroscope ( meiji techno , japan , saitama , japan ) at 7 magnification to evaluate the mode of failure and enamel fracture . adhesive remnant index ( ari ) scores were recorded for each specimen to determine the mode of failure . the ari scale ranges from 0 to 3 and the scores were classified as : 0:no adhesive is remaining on the enamel surface,1:less than half of the adhesive is remaining on the enamel surface,2:more than the half of the adhesive is remaining on the enamel surface , and3:the entire adhesive is remaining on the enamel surface . no adhesive is remaining on the enamel surface , less than half of the adhesive is remaining on the enamel surface , more than the half of the adhesive is remaining on the enamel surface , and the entire adhesive is remaining on the enamel surface . a one - way analysis of variance ( anova ) and tukey 's post hoc test at a level of confidence ( p<0.05 ) was used to statistically analyze the sbs results . wallis test , and mann whitney u test was used to determine which group in fact differs . a piece of wax equal in size to the base of a bracket was placed at the center of the buccal surface of each tooth as a guide for the placement of orthodontic brackets . the wax was gently removed from the teeth , and then sep was applied to the teeth in each group according to the protocol above . stainless steel metal brackets ( mini sprint - brackets ; forestadent company , pforzheim , germany ) were used . the brackets were bonded to the teeth using a light - curing composite ( system rmo / mono - lok2 bonding ) and polymerized with a light - curing unit ( bluephase led ivoclar vivadent , irradiance 380515 nm ) for40 s. the area of the bracket base surface was 12.4 mm as given by the manufacturer . a zwick / roell z020 universal testing machine ( zwick gmbh and co , germany ) with a 500 n load cell was used to test sbs . the specimens mounted in their acrylic blocks were secured to the lower grip of the machine ( fixed head ) . to maintain a consistent debonding force , a custom - made blade was fixed in the upper grip ( movable head ) connected to the load cell . the blade was positioned in such a way that it touched the bracket and the force applied to the ligature groove between the bracket base and the wings . each tooth was oriented with the testing device as a guide so that its labial surface was parallel to the force during the shear strength test . the debonding forces of the brackets were recorded in n and then converted in mpa by taking into account the surface area of the bracket base . the surfaces of the teeth were then examined with a stereomicroscope ( meiji techno , japan , saitama , japan ) at 7 magnification to evaluate the mode of failure and enamel fracture . adhesive remnant index ( ari ) scores were recorded for each specimen to determine the mode of failure . the ari scale ranges from 0 to 3 and the scores were classified as : 0:no adhesive is remaining on the enamel surface,1:less than half of the adhesive is remaining on the enamel surface,2:more than the half of the adhesive is remaining on the enamel surface , and3:the entire adhesive is remaining on the enamel surface . no adhesive is remaining on the enamel surface , less than half of the adhesive is remaining on the enamel surface , more than the half of the adhesive is remaining on the enamel surface , and the entire adhesive is remaining on the enamel surface . a one - way analysis of variance ( anova ) and tukey 's post hoc test at a level of confidence ( p<0.05 ) was used to statistically analyze the sbs results . wallis test , and mann whitney u test was used to determine which group in fact differs . the mean shear bond ( mpa ) , standard deviation , and minimum / maximum values for each group are shown in table 1 . the mean sbs of g1 ( control group ) was 10.004.48 mpa , g2 was 5.714.37 mpa , g3 was 7.474.44 mpa , g4 was 4.42.39 mpa , and g5 was 3.980.83 [ figure 1 ] . when the means of sbs were compared using one - way anova , multiple comparison [ tukey 's honestly significant difference ( hsd ) test ] [ table 2 ] showed that the mean bond strength findings of groups 2 , 4 , and 5 were significantly lower than those of the control group . however , no significant difference was found to exist between g3 and the control group . shear bond strength findings ( mean , standard deviation , maximum , and minimum ) bar chart diagram showing the mean sbs values for all four groups tested ( 1 = g1 sep 5 s ; 2 = g2 sep 5 s and acp ; 3 = g3 sep 10 s and acp ; g4 = sep 5 s and fv ; g5 = sep 10 s and fv ) comparing shear bond strength findings between the groups by one - way anova ( p<0.05 ) and multiple comparison ( tukey 's hsd test ) the results of the ari testing are recorded in table 3 . wallis test showed that there was a significant difference in ari between the groups ( p<0.001 ) . whitney test showed that g1 was found to have a significantly higher ari score than all groups : g2 ( p=0.001 ) , g3 ( p=0.005 ) , g4 ( p<0.001 ) , and g5 ( p=0.001 ) . groups 2 and 3 , 2 and 4 , 2 and 5 , 3 and 4 , 3 and 5 , and 4 and 5 did not differ in ari scores ( p=0.229 , p=0.66 , p=1.00 , p=0.105 , p=0.229 , and p=0.66 , respectively ) . additional computation of pearson correlation coefficients ( 0.543 ) showed strong correlation between bond strength and ari scores within or across all groups ( p<0.001 ) . the hypothesis that there would be no difference in mean sbs between groups whether fv was used at the same time of the application of sep in different times ( 5 s and 10 s ) or an sep only was used was not accepted . this was partially accepted only when the calcium phosphate was used at the same time of the application of sep for 10 s. the results of kimura 's study suggested that there was no difference in bond strength of orthodontic brackets to enamel treated with fv or not when he applied the fv 10 days before the etching materials . the application of fv does not affect the bond strength of orthodontic brackets to enamel with conventional or sep systems . the application of a cpp - acp containing remineralizing paste ( tm ) for 60 min daily for 7 days before etching did not affect sbs to enamel for either the total etch ( single bond ) or sep adhesives . the findings suggest that the sbs of resin to enamel using self - etching priming adhesive may be decreased if the enamel is treated with cpp - acp ( tm ) . baysal and uysal evaluated the effect of cpp - acp application on the sbs of orthodontic brackets bonded to demineralized enamel surface and concluded that pretreatment of enamel with cpp - acp improves the sbs . this is not in agreement to the finding of this study where the application of ( tm ) did not affect sbs when brackets were bonded to normal enamel . taking into account that the minimum accepted sbs value for clinical using was 6 mpa , the results of this study show that the application of calcium phosphate ( tm ) as preventive material for enamel demineralization at the same time of the use of sep for 5 s or 10 s can be used clinically . in addition , the application of sep for 10 s has resulted in higher sbs when compared to its application for 5 s. several studies evaluated the role of fluoride in enamel decalcification prevention . the protective effects of f on the reduction of enamel decay have also been observed . it has been confirmed that f reduces the solubility of calcium hydroxyapatite , balances the rates of demineralization and remineralization , and has an antimicrobial feature as well . in order to prevent enamel demineralization , fv and calcium phosphate this application had limited the etching areas and eased the removal of the composite remains after bonding , where these remains should be accumulated above the applied preventive materials . this has resulted in protected and smooth enamel surfaces compared with conventional bonding methods and topical application preventive materials . the suggested technique of applying of fv or calcium phosphate , sep , and bonding should give us the following advantages : decreased dental plaque accumulation around orthodontic brackets , delayed biofilm formation , and antimicrobial and enamel remineralization roles of fluoride , calcium , and phosphate ions . the failure at the enamel adhesive interface decreases the probability of enamel damage by reducing the required mechanical removal of the residual adhesive after debonding . the results of the current study showed reduction in the ari values when the calcium phosphate was applied before the application of sep . this technique has a clinical application as a preventive method in minimizing the enamel damage after treatment . the reduced sbs values obtained in the current study , which are unacceptable values for clinical use , after application of fv can be explained by the contamination of bonding area by varnish . however , we can use fv immediately after bonding as a demineralization agent , an antibacterial agent , and to reduce in streptococcus mutans counts in the dental plaque . the application of acp ( tm ) around bonding area with sep for 10 s did not affect sbsthe application of fv around bonding area with sep for 5 and 10 s affected sbs . the application of acp ( tm ) around bonding area with sep for 10 s did not affect sbs the application of fv around bonding area with sep for 5 and 10 s affected sbs .

aim : to evaluate the effect of the application of either fluoride varnish ( fv ) or amorphous calcium phosphate ( acp ) as preventive method on shear bond strength ( sbs ) at the same time of their bonding in vitro using self - etching primer ( sep ) as an agent for enamel pre - treatment fv.materials and methods : sixty human bicuspids were randomly divided into five groups : g1 was rubbed by sep for 5 s , g2 for 5 s by sep and acp , g3 for 10 s by sep and acp , g4 for 5 s by sep and fv , and g5 for 10 s by sep and fv . stainless steel metal brackets were bonded . a zwick / roell z020 universal testing machine ( zwick gmbh and co , germany ) with a 500 n load cell was used to test sbs . sbs values were analyzed using one - way analysis of variance ( anova ) and tukey 's post hoc tests ( p0.05 ) . differences in adhesive remnant index ( ari ) values between groups were calculated.results:the mean sbs values were 10.004.48 mpa , 5.714.3 mpa , 7.474.44 mpa , 4.42.39 mpa , and 3.980.83 mpa for groups 15 , respectively . significant differences in sbs values between all groups were found . the mean sbs values of groups 2 , 4 , and 5 were significantly lower than that of the g1 . no significant difference was found between g3 and g1 . significant difference in ari between the groups was found ( p<0.001 ) and g1 had a significantly higher ari.conclusion:the results suggested that the application of acp at the same time of using sep for 10 s has no effect on sbs .

INTRODUCTION MATERIALS AND METHODS Application of FV or ACP (TM-GC) and SEP Bonding Procedure Debonding Strength Testing Statistical Analysis RESULTS DISCUSSION CONCLUSIONS

an 8-year - old girl was admitted to the pediatric department of the balaji hospital , chrompet , chennai with the complaints of with high grade fever of 4 days duration associated with nonbilious vomiting , watery stools , abdominal pain , and reduced appetite . a complete blood count showed a total white cell count of 3000 with a differential count of 78% neutrophils , 20% lymphocytes , 2% eosinophils . liver function tests showed a total serum bilirubin of 0.5 , direct of 0.2 and indirect of 0.3 . serum glutamic pyruvic transaminase - 49 , serum glutamic oxaloacetic transaminase - 117 , albumin - 3.7 and globulin - 2.5 . serum electrolytes revealed a sodium level of 122.1 , potassium of 3.54 and chloride 92.6 . ascites , hepatosplenomegaly , mesenteric lymphadenopathy and gall bladder thickening were obtained in abdominal ultrasound . a diagnosis of multi - drug resistant typhoid fever with thrombocytopenia , hyponatremia , mesenteric adenitis and ascites was made . thrombocytopenia is prevalent in 1015% of children with typhoid fever according to a study done by ur rab and beig of the department of pediatrics of j.n . ascites is an underreported finding in enteric fever according to sinha of shishuniketan kolkata , india as published in indian pediatrics . mild hyponatremia has been reported in typhoid fever as are multi - drug resistant cases . we are not aware of a combination of all these complications in a single patient , as revealed by a literature search using the internet .

a rare case of typhoid presenting with thrombocytopenia , hyponatremia , ascites mesenteric adenitis , and multi - drug resistance is being presented in this article . an 8-year - old girl was admitted with a history of fever , vomiting , abdominal pain and loose stools . clinical examination revealed fever and hepatosplenomegaly . investigations showed leucopenia , thrombocytopenia and hyponatremia . blood widal was positive , and blood culture grew salmonella typhi . ultrasound abdomen revealed ascites , hepatosplenomegaly , mesenteric lymphadenopathy and thickening of the gall bladder . she was treated with ciprofloxacin intravenously for 6 days and when the fever persisted injection ceftriaxone was added . ciprofloxacin was given intravenously for a total of 15 days and injection ceftriaxone was given for 12 days . even then , the fever persisted and hence oral azithromycin was added . fever subsided completely in 3 days with azithromycin and she became asymptomatic without fever , loose stools , abdominal pain or anything on follow - up after 3 months .

Case Report Investigations Diagnosis Treatment Discussion

polymorphous low - grade adenocarcinoma ( plga ) has been known as a comparatively less aggressive malignant tumor that predominantly occurs in the minor salivary glands . it has been reported to occur rarely in the palate and the prognosis of this lesion is far better than adenocarcinoma as the regional metastasis would be very minimal . a clear difference between the biologic behaviors of adenocarcinoma and plga has been reported in literature . a 63-year - old female reported at our institute with a swelling in relation to her upper left back region of her jaw for three months , gradual in onset and associated with pain . after clinical and radiological examination , the differential diagnosis were consolidated abscess or minor salivary gland tumor . the post - operative follow up showed no evidence of recurrence and the healing was satisfactory . the site of predilection of plga is more in favor of palate ( 49 - 77.8% ) followed by either upper lip or buccal mucosa ( 7.4 - 13.4% ) . there are sporadic reports of metastasis sometimes even transformation to a high grade adenocarcinoma , sometimes ending in mortality . cervical lymph node metastasis is rare with reported incidence of 5 - 15% and is more commonly seen in recurrent tumor than the initial diseases . extra palatal plgas present with significant papillary growth or arising from ventral surface of tongue frequently metastasize to cervical lymph nodes . distant metastasis is very rare with an incidence of 7.5% and the site involved is the lung which is attributed to the inadequate control of the disease . evans and batsakis first described plga as a distinct entity in 1984 as it is characterized as a tumor with low grade biologic behavior and diverse architectural pattern ( 1 ) . previously lobular carcinoma or terminal duct carcinoma of minor salivary glands were the terms used to describe the histological appearance of this tumor ( 2 , 3 ) . plga is an indolent tumor , exclusively arising in the minor salivary glands ( 4 , 5 ) . waldron et al . has reported that it is the third most common intra oral malignancy occurring in minor salivary glands with an incidence rate of 26.4% among intraoral malignancies ( 6 ) . though it is common in minor salivary glands , two cases have reported in parotid gland ( 7 ) and one in the sublingual gland ( 8) . as the tumor is asymptomatic , many times it is only diagnosed during routine diagnostic evaluation ( 9 ) . a 63-year - old patient had reported at our institute with the chief complaint of swelling in relation to her upper left back tooth region for 15 days which has associated with pain for 3 days . there was no history of any secondary changes and the pain associated with the swelling was of a severe , throbbing type in relation to the middle third of face , which was aggravated on chewing food , causing difficulty in food intake . her general examination has shown that review of her systems were normal except for bilateral palpable submandibular lymph nodes of size 1 2 cm which were soft , tender and mobile with no fixation to the underlying structures . intraorally , on inspection , a solitary diffuse swelling of size 3 2 cm was present in relation to the left posterior part of the hard palate ( figure 1 ) . the swelling has extended anteriorly up to the 1 molar and posteriorly to the soft palate . the surface of the lesion appeared smooth with necrotic slough seen in the buccal vestibule . on palpation , all the inspectory findings in relation to the site , size and extent of the lesion have confirmed . there was also a root stump in relation to the left second molar which was tender on percussion . the provisional diagnosis was given as a minor salivary gland tumor with 2 other differential diagnosis namely palatal abscess and consolidated abscess in relation to left second molar . routine hematologic investigations were normal and the radiologic investigations included a maxillary occlusal radiograph which had revealed an ill - defined radiolucency of size 1 2 cm present in relation to left third molar with ill - defined borders and an opg has revealed a root stump in relation to left third molar , there was no evidence of erosion or invasion of the underlying structures . the ct scan revealed involvement of the posterior aspect of the left hard palate , the alveolar process and buccal vestibule and multiple bilateral lymph nodes with possibility of pathology . an incisional biopsy of the lesion has done , and the histopathological features were indicative of uniform tumor cells exhibiting moderate eosinophilic to amphophilic cytoplasm , vesicular nuclei and occasional prominent nucleoli . cytologically bland tumor cells have shown various patterns like nest , ductular , tubular and papillary patterns ( figure 2 ) showing apoptotic bodies . a treatment plan of surgical excision of the primary lesion with immediate obturator placement was made . accordingly the patient was administered general anesthesia and through an intraoral approach , palatal and buccal mucoperiosteal flaps were elevated and the lesion has exposed . partial maxillectomy was done maintaining a tumor free margin of 2 cm and including the involved alveolar process . regular follow up was done postoperatively , initially every day to change the gauze packing and later review was done every month . the wound healing was satisfactory showing healthy granulation tissue at the periphery of the lesion ( figure 3 ) . since there were no signs of recurrence , after 6 months of follow up , the patient was given a definitive functional obturator . plga is a distinct entity due to its architectural diversity , cytological uniformity , and indolent clinical behavior ( 2 , 10 ) . its clinical behavior is characterized by slow rate of growth , absence of symptoms , less aggressiveness , minimal metastatic potential and good prognosis ( 11 ) . the lesion described in this patient was similar to the ones previously described in literature in terms of the location , indolent course , minimal symptoms , delayed detection and histopathological features . from the past literature it is seen that plga is more frequent in females than in males with the female to male ratio being 3:1 ( 12 ) . also adults from the age group of the 3 and 7 decades were more frequently affected with the peak prevalence being in the 5 and 6 decades . in accordance with the prevalence of plga in literature site of predilection is more in favor of the palate ( 49 - 77.8% ) followed by either the upper lip or buccal mucosa ( 7.4 - 13.4% ) and rarely could involve floor of mouth , lower lip , alveolar ridge and tongue ( 1 , 13 - 15 ) . literature has shown that plga could also arise in lung ( 16 ) maxilla ( 17 ) , parotid gland ( 18 ) and submandibular gland ( 19 ) . in our case the tumor had presented as an asymptomatic swelling with smooth margins and surface and was seen in the soft palate extending anteriorly to the hard palate . there were sporadic reports of metastasis sometimes even transformation to a high grade adenocarcinoma , sometimes ending in mortality as reported by evans et al . cervical lymph node metastasis was rare with reported incidence of 5 - 15% and was more commonly seen in recurrent tumor than the initial disease ( 17 ) . extra palatal plgas had presented with significant papillary growth or arising from the ventral surface of the tongue and frequently metastasize to cervical lymph nodes ( 18 ) . distant metastasis was very rare with an incidence of 7.5% and the site involved is the lung which has attributed to the inadequate control of the disease ( 19 ) . in our case neck and chest imaging many times a small incisional biopsy specimen was sufficient for diagnosis if the lesion was small or multiple incisional biopsy samples might be necessary for large tumors especially in case the differential diagnosis includes a salivary gland malignancy ( 1 , 20 ) . the tumor margins should also be examined to assess the presence of infiltration ( 1 , 16 , 17 ) . in our patient , postoperative histopathological analysis of the excised specimen had indicated a plga which had bone invasion . if positive or close surgical margins is there , post - operative radiotherapy is recommended but it has not shown to alter outcome in patients without neck node metastasis . the recurrence rate for plga is minimal following wide excision and if present radical excision is warranted ( 12 ) . in our case partial maxillectomy was followed by placement of an obturator . in general minor salivary gland tumors more case reports are needed to understand the site , age , sex , and prevalence and histological typing of this tumor which would help in the proper diagnosis and appropriate management of polymorphous low grade adenocarcinoma . wide local excision should be the ideal treatment of choice . if positive or close surgical margins is there , post - operative radiotherapy is recommended but it has not shown to alter outcome in patients without neck node metastasis . the recurrence rate for plga is minimal following wide excision and if present radical excision is warranted ( 12 ) . in our case more case reports are needed to understand the site , age , sex , and prevalence and histological typing of this tumor which would help in the proper diagnosis and appropriate management of polymorphous low grade adenocarcinoma .

introductionpolymorphous low - grade adenocarcinoma ( plga ) has been known as a comparatively less aggressive malignant tumor that predominantly occurs in the minor salivary glands . it has presented as a painless , slow growing tumor in the oral cavity . it has been reported to occur rarely in the palate and the prognosis of this lesion is far better than adenocarcinoma as the regional metastasis would be very minimal . a clear difference between the biologic behaviors of adenocarcinoma and plga has been reported in literature.case presentationa 63-year - old female reported at our institute with a swelling in relation to her upper left back region of her jaw for three months , gradual in onset and associated with pain . after clinical and radiological examination , the differential diagnosis were consolidated abscess or minor salivary gland tumor . an incisional biopsy was done and the lesion was diagnosed as plga . the lesion was treated by wide excision and reconstruction with immediate obturator . the post - operative follow up showed no evidence of recurrence and the healing was satisfactory.conclusionsthe site of predilection of plga is more in favor of palate ( 49 - 77.8% ) followed by either upper lip or buccal mucosa ( 7.4 - 13.4% ) . there are sporadic reports of metastasis sometimes even transformation to a high grade adenocarcinoma , sometimes ending in mortality . cervical lymph node metastasis is rare with reported incidence of 5 - 15% and is more commonly seen in recurrent tumor than the initial diseases . extra palatal plgas present with significant papillary growth or arising from ventral surface of tongue frequently metastasize to cervical lymph nodes . distant metastasis is very rare with an incidence of 7.5% and the site involved is the lung which is attributed to the inadequate control of the disease .

Introduction Case Presentation Conclusions 1. Introduction 2. Case Presentation 3. Discussion 3.1. Treatment 3.2. Conclusions

lymphatic filariasis is common in tropical countries and is endemic in india with most infections caused by wuchereria bancrofti . the microfilaria are not just confined to the lymphatic system , but also associated with other organs , subcutaneous tissues , and serous cavities like pleura and pericardium . demonstration of microfilariae ( mf ) in the peripheral blood smear or in the tissues is the mainstay of diagnosis . filariasis acutely presents with fever , adenolymphangitis , funiculitis , epididymitis , orchitis , or pericarditis . lymphedema , hydrocele , elephantiasis and chyluria , and pleural or pericardial effusion are features of chronic infection . our case was a rare as recurrent hemorrhagic pericardial effusion and tamponade , which was first treated with diethyl carbamazine and finally albendazole . she complained of cough and breathlessness on exertion and low grade fever since 3 weeks and generalized weakness since 1 week . on examination , she was severely dyspneic and had pulsus paradoxus . echocardiogram showed massive pericardial effusion with cardiac tamponade [ figure 1 ] . apical four - chamber view showing large pericardial effusion with right atrial collapse ( left ) and parasternal short axis view showing large pericardial effusion with right ventricular outflow tract ( rvot ) diastolic collapse ( right ) pericardiocentesis was done and 1,300 ml of hemorrhagic fluid was collected . results of complete blood picture were : total count - 6,800/mm , polymorphs - 67% , lymphocytes - 23% , eosinophils - 06% , monocytes - 04% , platelet count - 160,000/mm , hemoglobin - 11 g% , erythrocyte sedimentation rate ( esr ) - 40 1 h. , human immunodeficiency virus ( hiv)-nonreactive , and hepatitis b surface antigen ( hbsag)-negative . complete urine examination , blood sugar , urea , creatinine , and electrolytes ; all were in normal range . cytological analysis of fluid showed - 7,000 cells / mm , ( neutrophils - 35% , lymphocytes - 65% , plenty of red blood cells ( rbc ) ) , negative for malignant cells , sugar - 61 mg% , proteins - 6 g% , albumin - 2.5 g% , adenosine deaminase ( ada ) - 23 u / l , acid - fact bacilli ( afb ) - negative ; microscopy and routine cultures were negative for bacteria . while screening the smears , a few large , long , smooth - walled , refractile unstained worm - like structures were observed . the organisms were identified as mf of w. bancrofti in the smears stained by leishman 's stain [ figure 2 ] . cytology showed plenty of rbc , occasional pus cells , and microfilaria of w. bancrofti [ figure 3 ] . the patient improved rapidly by next day and effusion was grossly reduced by the 2 day . pericardial fluid cytology smear showing microfilaria of wuchereria bancrofti with occasional pus cells ( leishman stain , 1000 ) pericardial fluid cytology smear showing microfilaria of w. bancrofti with occasional pus cells ( leishman stain , 40 ) it is transmitted by the culex mosquito and is caused by two closely related nematodes , w. bancrofti and brugia malayi , which are responsible for 90 and 10% of the cases , respectively . microfilaria have been confined not only to the lymphatic system but also associated with other organs , subcutaneous tissues , and serous cavities like pleura and pericardium . pericardial effusion is a rare manifestation of filariasis , and recurrent effusion with tamponade is even more rare . our case was of massive pericardial effusion established as filarial in origin on demonstration of mf in pericardial aspirate . it might have lodged itself through blood stream subsequent to development of pericarditis . as mf are capable of penetrating the tissues of the vector , they can freely move in and out of small , delicate blood , and lymphatic capillaries and tissues of the vertebrate host . peripheral blood smears may be negative for mf in cases of secondary manifestations of filariasis . in this case patient developed effusion despite being treated with dec suggests that it may not be 100% effective in all cases as dec does not act on microfilaria residing in lymphatics , and they might have migrated to pericardial space causing recurrent effusion . she was finally cured with alternate combination of ivermectin and albendazole , which makes it even more interesting . although mf in unusual sites are considered incidental findings , on the basis of existing evidences we suggest that some of the cases of recurrent pericardial effusion , especially those from endemic areas , might be of filarial origin . it is therefore emphasized that a thorough examination of the aspirated fluid for mf is warranted in all patients with unexplained pericardial effusion .

filariasis presenting with pericardial effusion with tamponade is rare . we report a case of a 30-year - old female who was admitted with severe dyspnea and chest pain since 2 days . echocardiogram showed massive pericardial effusion with tamponade . pericardial fluid aspiration drained 1.2 l of hemorrhagic fluid . cytology examination revealed microfilaria of wuchereria bancrofti . she was treated with diethyl carbamazine and discharged . six weeks later , she presented again with massive pericardial effusion with cardiac tamponade . pericardiocentesis was done . cytology examination revealed microfilaria of w. bancrofti . this time she was treated with ivermectin and albendazole and cured . hemorrhagic effusion resolved completely . though relatively uncommon , tropical diseases must always be considered in the etiological diagnosis of recurrent pericardial effusion .

INTRODUCTION CASE REPORT DISCUSSION

meningioma is the most common type of benign brain tumor , whereas ectopic meningioma , although reported , is rare . the head and neck region is the most common ectopic site whereas the scalp , skin , orbit , paranasal sinuses , salivary glands , and intraosseous or intradiploic regions can also be affected [ 25 ] . in view of their rarity , ectopic meningiomas of the skull the main differential diagnoses are fibrous dysplasia and osteoid osteoma , the most common being benign primary tumors . we report herein the case of a 62-year - old female patient with a pure ectopic intraosseous meningioma without dural invasion . a 62-year - old female patient presented with a history of classical migraine for the last 30 years . six month before the diagnosis , her headaches had changed their characteristics to a continuous unilateral ( right side ) pain of increasing intensity . the patient reported no nausea or vomiting , which usually followed her typical migraines . also , the patient noted a growing lump on the right parietal side and was referred to our service by her primary care physician . on neurological examination , the patient was alert and oriented , complaining of moderate headache . a hard , slightly painful , elliptical prominence without clearly defined margins was detected on her right parietal bone , which measured approximately 7 8 cm . mri images showed an osteoblastic lesion in the right parietal bone diploe , with possible involvement of both cortical layers and without dural extension ( fig . the patient was scheduled for elective surgery in the following week . the surgical procedure consisted of a right parietal incision and craniotomy and craniotomy was performed with a clear 1-cm margin and skull convexity was reconstructed with a titanium mesh ( fig . ten months after surgery , the patient remains asymptomatic and shows no signs of recurrence ( fig . ectopic intraosseous meningiomas can also be described as intradiploic or calvarial and may appear as osteoblastic , osteolytic lesions [ 4,1014 ] or mixed lesions on plain x - rays and computed tomography scans . further investigation by mri can identify ectopic meningiomas , which do not exhibit the usual paramagnetic contrast enhancement . the clinical presentation of the present case is similar to that found in previously reported cases of skull tumors , usually headaches and an often painless , palpable mass on the scalp or skull . involvement of other ectopic sites such as the paranasal sinuses and orbit usually manifests as pain and proptosis , whereas pain and a palpable mass are common when the tumor affects more distant sites . histologic examination usually presents meningothelial meningiomas , but the microcystic or lipomatous variations are also reported . immunohistochemical staining is usually positive for progesterone receptor , epithelial membrane antigen as in our described case , and s100 . in 1960 , hoye et al . reviewed the latest case reports and proposed the classification of ectopic meningiomas into four types : ( 1 ) intracranial tumors with extracranial extension ; ( 2 ) meningiomas originating in cranial nerve sheaths ; ( 3 ) extracranial tumors without any connection to cranial nerve foramina ; ( 4 ) intracranial benign lesions with extracranial metastases . in 2000 , lang et al . described a similar , but simpler classification : type 1 , purely extracalvarial tumors ; type 2 , purely calvarial tumors , and type 3 , calvarial tumors with extracalvarial extension . each type is further divided according to location into skull base ( s ) or calvarial ( c ) lesions . our case is classified as type 3 in hoye s classification and type 2 in lang s classification , since the lesion was restricted to the skull ( inner and outer tables ) , showing no dural invasion on histopathological examination , supporting the hypothesis that the tumor originated in the diploe layer . some theories have been offered to explain how a meningioma can appear distant from the usual arachnoid cap cells ( meningocytes ) . one theory suggests that embryological remains of neuroectodermal tissue , which should develop into cap cells , can expand or erroneously migrate to other tissues . other theories that could account for some cases are the spreading of these remnant cells due to trauma , dural lesions , or cells entrapped in cranial sutures and cranial nerve sheaths . some authors proposed the occurrence of dedifferentiation of cells in extracranial tissues , which develop into neoplastic meningocytes . none of these theories could be confirmed in the present case , but a probable intradiploic origin is suggested . overall , most meningiomas , including ectopic tumors , are benign ; therefore , their complete surgical excision should be the rule . partial resection of residual lesions can be monitored radiologically , while adjuvant therapies such as radiation may provide an alternative for symptomatic and surgically difficult cases . written informed consent was obtained from the patient for publication of this case report and accompanying images . a copy of the written consent is available for review by the editor - in - chief of this journal . written informed consent was obtained from the patient for publication of this case report and accompanying images . rbv : drafted and designed the manuscript and participated in surgery ; pthf : drafted and coordinated the manuscript ; rll : participated in surgery and reviewed the literature for previous cases ; vzm and fol : analyzed the pathological and histological materials ; frr : coordinated surgery and grammatical review .

highlightsintraosseous meningiomas may be confused with fibrous dysplasia and/or osteoid osteoma.lesions appear either as osteoblastic or osteolytic lesions.usually presents as a painless palpable mass associated with headaches.embryological remnants of neuroectodermal tissue or cellular dedifferentiation seem to justify the lack of dural connections.complete surgical excision is usually curative .

Introduction Presentation of case Discussion Conclusions Conflict of interest Funding Ethical approval Consent Authors contributions

according to the taiwan department of health , the cumulative death rates from coronary artery disease , stroke , and diabetes are 152 cases per 100,000/yr , which nearly equals the death rate from malignancies . these ' modern diseases ' are a huge burden , not only to the patients themselves , but also to their families and society . this alarming trend is not unique to taiwan , and has been reported to occur in many other parts of the world . therefore , the prevention and early detection of coronary heart disease and diabetes has become a major public health issue . the well - known risk factors for these diseases include excess body weight , hypertension , hyperlipidemia , and hyperglycemia . indeed , the clustering of these risk factors was first noted in 1966 ( 1 ) . in 1988 , reaven introduced the term syndrome x , which consisted of hyperinsulinemia , hypertension , dyslipidemia , hyperglycemia , and resistance to insulin - mediated glucose uptake ( 2 ) . he suggested that insulin resistance ( ir ) plays an important role in the etiology and clinical course of patients with diabetes mellitus , hypertension , and coronary heart disease ( 3 ) . in 1998 , the world health organization ( who ) also recognized the importance of this clustering and further defined the clinical characteristics of the ' metabolic syndrome ' ( mets ) ( 4 ) . however , such criteria are not practical for routine clinical use because one of the major criteria , ir , requires measurement by a hyperinsulinemic euglycemic clamp . therefore , the national cholesterol education program adult treatment panel iii ( atp iii ) provided a simpler definition in 2001 , in the hope that it could be used even in general practice ( 5 ) . it had been shown that patients with mets , as defined by either who or atp iii criteria , have more severe ir as compared to the general population ( 6 , 7 ) . however , the cut - off values of these diagnostic criteria were originally determined arbitrarily , have never been stratified according to a weighted clinical effect , and may vary in different ethnic groups . moreover , it is unknown which of the five clinical characteristics amongst the atp iii criteria , if any , is related to more severe ir . in the current study , we measured ir directly by an insulin suppression test ( ist ) . subjects were placed into quartiles based on the level of each of the mets clinical characteristics . then , the steady state plasma glucose ( sspg ) level resulting from the ist was compared between the different quartiles representing one clinical characteristic as well as between all the different clinical characteristics . furthermore , factor analysis ( 8 , 9 ) , a multivariate statistical tool , could reduce a considerable number of inter - correlated variables to a smaller set that accounts for most of the variances between the data . by this mean , a set of dimensions ( sometimes called factors ) which are not easily observed in the original variable could be identified . therefore , it was also used to investigate which of mets factors are related to ir ( sspg levels).thus , we are able to determine the severity of ir between the quartiles representing each clinical characteristic , but also determine which clinical characteristic of mets was associated with the most severe ir when compared with other clinical characteristics within the chinese population . five hundred sixty four subjects , 250 males and 314 females aged 20 - 75 yr , were enrolled during routine health evaluations at the tri - service general hospital between 1995 and 1999 . subjects with a history of diabetes , hypertension , hyperlipidemia , or other significant medical or surgical diseases were excluded . subjects who were placed on medications which affect insulin sensitivity were also excluded from the study . the study was approved by the hospital ethics committee , and the nature , purpose , and potential risks of the study were explained to the patients before obtaining their consent to participate . since reaven suggested that the upper 25% in the general population are ir , we divided our subjects into four groups regarding each of five clinical characteristics of the mets and then evaluated the ir in each quartile ( 2 ) . the ability of insulin to dispose of a glucose load was estimated by modification of the ist , as described by shen et al . one catheter was used for the administration of a 180 min infusion of somatostatin ( 250 g / hr ) , insulin ( 25 mu / m / min ) , and glucose ( 240 mg / m / min ) . blood was collected every 30 min initially , and then at 10 min intervals from 150 - 180 min of the infusion , to determine the steady state plasma insulin ( sspi ) and sspg concentrations for each individual . the sspi concentrations were comparable in all individuals , thus the sspg concentrations provided the measure of efficacy of insulin in promoting disposal of the infused glucose load . the samples obtained at -5 and 0 min were analyzed for fasting plasma glucose ( fpg ) , fasting plasma insulin ( fpi ) , and lipid levels . plasma glucose levels were determined using the glucose oxidase method ( ysi 203 glucose analyzer ; scientific division , yellow spring instrument company , inc . , yellow spring , oh , u.s.a . ) . insulin levels were measured by a commercial solid phase radioimmunoassay kit ( 11 ; coat - a - count insulin kit ; diagnostic products corporation , los angeles , ca , u.s.a . ) . the intra- and inter - assay coefficients of variance for insulin were 3.3% and 2.5% , respectively . both triglyceride ( tg ) and total cholesterol ( tc ) levels were measured using the dry , multilayer analytical slide method in the fuji dri - chem 3000 analyzer ( fuji photo film corporation , minato - ku , tokyo , japan ) . serum high density lipoprotein cholesterol ( hdlc ) concentration was determined by an enzymatic , cholesterol assay method following dextran sulfate precipitation . analysis was performed using spss for windows , version 10.0 ( spss ; chicago , il , u.s.a . ) . data were tested for normal distribution using the kolmogorov - smirnov test and for homogeneity of variances with levene 's test . independent student 's t - test was used to evaluate gender - based physical and metabolic differences . the one - way anova with the bonferroni method as a post hoc test was also applied to compare differences between the clinical characteristics in each quartile . since age was considered as the confounding covariate , each variable of interest ( i.e. , body mass index [ bmi ] , tg , hdlc , fpg , and blood pressure ) was first adjusted for age by employing the analysis of covariance . the derived residuals ( adjusted variables ) were then again used for analyses after the unadjusted data were examined . all statistical tests were two - sided and p - values less than 0.05 were considered to indicate statistical significance . furthermore , exploratory factor analysis was conducted to examine the relationships among the variables which constituted mets . principle factor analysis was employed to transform the original variables into a new set of components which are independent of each other . then number of components to be retained was based on scree plot analysis ( factors above the break in the curve were retained ) and eigenvalue criteria ( 1.0 ) . such two modalities have been adopted to identify the minimum number of components ( 8 , 12 , 13 ) . five hundred sixty four subjects , 250 males and 314 females aged 20 - 75 yr , were enrolled during routine health evaluations at the tri - service general hospital between 1995 and 1999 . subjects with a history of diabetes , hypertension , hyperlipidemia , or other significant medical or surgical diseases were excluded . subjects who were placed on medications which affect insulin sensitivity were also excluded from the study . the study was approved by the hospital ethics committee , and the nature , purpose , and potential risks of the study were explained to the patients before obtaining their consent to participate . since reaven suggested that the upper 25% in the general population are ir , we divided our subjects into four groups regarding each of five clinical characteristics of the mets and then evaluated the ir in each quartile ( 2 ) . the ability of insulin to dispose of a glucose load was estimated by modification of the ist , as described by shen et al . one catheter was used for the administration of a 180 min infusion of somatostatin ( 250 g / hr ) , insulin ( 25 mu / m / min ) , and glucose ( 240 mg / m / min ) . blood was collected every 30 min initially , and then at 10 min intervals from 150 - 180 min of the infusion , to determine the steady state plasma insulin ( sspi ) and sspg concentrations for each individual . the sspi concentrations were comparable in all individuals , thus the sspg concentrations provided the measure of efficacy of insulin in promoting disposal of the infused glucose load . the samples obtained at -5 and 0 min were analyzed for fasting plasma glucose ( fpg ) , fasting plasma insulin ( fpi ) , and lipid levels . plasma glucose levels were determined using the glucose oxidase method ( ysi 203 glucose analyzer ; scientific division , yellow spring instrument company , inc . , yellow spring , oh , u.s.a . ) . insulin levels were measured by a commercial solid phase radioimmunoassay kit ( 11 ; coat - a - count insulin kit ; diagnostic products corporation , los angeles , ca , u.s.a . ) . the intra- and inter - assay coefficients of variance for insulin were 3.3% and 2.5% , respectively . both triglyceride ( tg ) and total cholesterol ( tc ) levels were measured using the dry , multilayer analytical slide method in the fuji dri - chem 3000 analyzer ( fuji photo film corporation , minato - ku , tokyo , japan ) . serum high density lipoprotein cholesterol ( hdlc ) concentration was determined by an enzymatic , cholesterol assay method following dextran sulfate precipitation . analysis was performed using spss for windows , version 10.0 ( spss ; chicago , il , u.s.a . ) . data were tested for normal distribution using the kolmogorov - smirnov test and for homogeneity of variances with levene 's test . independent student 's t - test was used to evaluate gender - based physical and metabolic differences . the one - way anova with the bonferroni method as a post hoc test was also applied to compare differences between the clinical characteristics in each quartile . since age was considered as the confounding covariate , each variable of interest ( i.e. , body mass index [ bmi ] , tg , hdlc , fpg , and blood pressure ) was first adjusted for age by employing the analysis of covariance . the derived residuals ( adjusted variables ) were then again used for analyses after the unadjusted data were examined . all statistical tests were two - sided and p - values less than 0.05 were considered to indicate statistical significance . furthermore , exploratory factor analysis was conducted to examine the relationships among the variables which constituted mets . principle factor analysis was employed to transform the original variables into a new set of components which are independent of each other . then number of components to be retained was based on scree plot analysis ( factors above the break in the curve were retained ) and eigenvalue criteria ( 1.0 ) . such two modalities have been adopted to identify the minimum number of components ( 8 , 12 , 13 ) . as expected , the male subjects in the current study had higher blood pressures and lower hdlc levels than the female subjects . the other mets clinical characteristics measured did not differ significantly between male and female subjects ( table 1 ) . we further divided the male and female subjects into quartiles according to the measured levels of each mets clinical characteristic , with subjects in the upper quartile having the highest levels and those in the lower quartile having the lowest levels . in the current study , we used two different methods to evaluate the importance of ir with respect to the mets . then , since there were six mean sspg values derived from each clinical characteristic ( i.e. , bmi , systolic blood pressure [ sbp ] , diastolic blood pressure [ dbp ] , fpg , tg , and hdlc ) , we further compared the sspg values derived from all clinical characteristics . the results of comparisons of the four quartiles for males and females are shown in fig . 1 , 2 , respectively . in males , the upper quartiles from the bmi and fpg clinical characteristics had the highest sspg levels as compared to the other three quartiles . the upper quartiles of the dbp and tg clinical characteristics were only greater than the lower two quartiles . in females , the sspg levels in the upper quartile of the bmi and tg clinical characteristics were significantly greater than the other lower three quartiles . while the sspg levels were highest in the sbp and fpg clinical characteristics , they were only higher than the third quartile ( fig . 2 ) . in order to evaluate which of the highest quartiles representing any of the mets clinical characteristics had the highest sspg levels , we further compared the upper quartiles of the bmi , sbp , dbp , fpg , hdlc , and tg clinical characteristics . 3 ) , except the sspg level was greater in the upper quartile of the bmi clinical characteristic than in the upper quartile of the hdlc clinical characteristic ( 12.370.47 vs. 9.520.52 finally , table 2 displays the results of factor analysis of core metabolic variables among the study subjects . 4 graphically depicts the same results with the percentage of variance explained by each factor . a two - factor solution , which was supported by the retention criteria described in the method , was obtained . these factors could be interpreted as 1 ) a " insulin resistance " factor , with a positive loading of bmi , fpg , tg , sspg and an inverse loading of hdlc , and 2 ) a " blood pressure " factor with a positive loading of bmi and both the sbp and dbp . the two - factor solution explained about 56% of the total variance in these subjects ( 28.4% factor 1 and 27.3% factor 2 ) . the data herein showed that , in general , the trend of higher sspg levels was associated with more mets clinical characteristics ( fig . 1 , 2 ) . however , only the bmi , fpg , and tg in both males and females , dpb in males , and sbp in females had significantly higher sspg levels in the upper quartile as compared to the lower quartiles . these results suggested that among the five clinical characteristics of the mets measured , bmi , fpg , and tg might be best related to ir and therefore , are the first clinical characteristics to be detected as abnormal . for example , cheal et al . found that among all the mets clinical characteristics , bmi and tg were best correlated with sspg ( 14 ) . by using factor analysis to evaluate the relationships between mets clinical characteristics and ir , meigs et al thus , he further suggested that ir should be the central focus in assessing the mets ( 8) . it has been long - recognized that ir is clearly associated with obesity , whether generalized or central ( 15 - 17 ) . the detrimental influence of central adiposity on ir is thought to be mediated by intra - abdominal fat deposition , in which hypertrophied adipocytes are resistant to anti - lipolytic action of insulin . as a consequence , elevated levels of free fatty acids may induce ir in the peripheral tissues and liver ( 18 ) . also , the preponderance of enlarged fat cells , as occur in abdominal obesity , may increase the risk of glucose intolerance and hypertriglyceridemia and thus account for the metabolic derangements ( 19 , 20 ) . it is interesting to note that the relationships between ir and some of the mets clinical characteristics were affected by gender . in our study , for example , the upper quartile of dbp in males had significantly higher sspg levels than the lower three quartiles . in contrast , instead of dbp , the sspg was higher in the upper quartile of sbp in females . these results suggested that the relationships between blood pressure and sspg were less consistent in our study . it is difficult to meaningfully compare this finding with other reports , since most of the other reports adjusted both sex and age when discussing the relationships between blood pressure and ir ( 17 , 21 , 22 ) . other than blood pressure , the relationships between ir and hdlc were also different between the genders . fig . 1 , 2 show that the levels of sspg did not differ between any quartiles representing the hdlc clinical characteristic . 3 , when comparing the upper quartiles of all the mets clinical characteristics , the upper quartile of the hdlc clinical characteristic had the lowest sspg level , but was only significant in males . it is well - recognized that ir is related to high tg and low hdlc ( 23 , 24 ) ; however , most of these reports showed that gender had no effect on the relationships involving ir . found that ir was more strongly related to hdlc in males than females , as evidenced by the intravenous glucose tolerance test ( 24 ) . this unique finding was further confirmed in another study conducted in subjects with morbid obesity . the correlations between ir and tg or hdlc were stronger in males than in females ( 25 ) . both hdlc and sbp were shown to be related to sspg levels in males , our data further imply that males might be more prone to develop mets when in middle age , as suggested by cook et al . at present , there are two possibilities to explain the metabolic differences which existed between males and females in our study . this in turn will further increase the risk of cardiovascular disease and diabetes ( 28 , 29 ) . second , male subjects tend to have more upper truncal adiposity ( i.e. , a higher waist - to - hip ratio [ whr ] ) than female subjects . in the current study , since we observed differences between each mets clinical characteristic , we did not adjust the effect of bmi on other risk factors , which is a departure from other studies ( 23 - 25 ) . it is generally agreed that there is a positive correlation between blood pressure and ir . using the euglycemic insulin clamp , this relationship has been repeatedly demonstrated in many different studies ( 17 , 21 , 22 ) . for instance , ferrannini et al . reported that male gender , age , and ir were independently associated with sbp , whereas dbp was related to age , ir , and fpi , but not to the bmi ( 17 ) . they also found that dbp had a higher correlation with ir than sbp ( r=0.34 and 0.18 , respectively ) . however , the same conclusion was not always reached in studies involving other ethnic groups ( 30 ) and the relationships did not exist when adjusted for body fat content ( 31 ) . the discrepancies of these results may be due to the fact that blood pressure per se is not clustered with ir . other possibilities such as the different ethnic groups or the methods used to measure adiposity might also play a role . in the ferrannini study , and as done herein , bmi was used to quantify adiposity . at the same time , toft et al . used whr and suggested that in subjects with the same bmi , changes in whr may still influence the results of variables associated with insulin sensitivity because the accumulation of visceral fat leads to altered insulin clearance ( 31 ) . in the peiris study , the clearance rates of insulin were similar between obese and non - obese subjects ( 32 ) ; however , it was inversely correlated with the whr in obese subjects . therefore , peiris et al . pointed out that whr is better associated with diminished insulin clearance than bmi ( 32 ) . it has been shown that the homeostasis model assessment of ir ( homa - ir ; 33 , 34 ) , which is calculated from fpg and fpi , is a useful surrogate for ir in healthy and diabetic subjects . it has proven to be highly correlated with the gold standard , the euglycemic hyperinsulinemic clamp ( 33 ) . at present , it is generally recognized that one of the main underlying pathophysiologies of glucose intolerance is ir . however , in subjects who already have ir , due to the compensatory increased secretion of the insulin , the fpg may persist within the normal range . that is why the study done by hollenbeck and reaven showed that there was no relationship found between fpg and ir ( 35 ) . on the contrary , surprisingly , in our study , the sspg seemed to be higher as the fpg increased . this finding suggested that even in the early stages before mets develops , fpg is already elevated in subjects with an elevated ir . the results of the factor analysis are similar to most of the other studies . in these studies , usually there are three to four factors being identified ; the " insulin resistant factor " , " obese factor " , " lipid factor " , and/or " blood pressure factor " ( 8 , 9 , 13 , 36 - 39 ) . to the present , one of the most important studies using factor analysis to explore the relationship between ir and other common risk factors for cardiovascular diseases was published by hanley et al . , they have used the intravenous glucose tolerance test to evaluate insulin sensitivity ( si ) . they have also identified two factors in the igt and ngt subjects : the " metabolic factor " ( comprising bmi , waist , si , tg , and hdlc ) ; and the " blood pressure factor " . the only differences are fpg was not loaded in factor 1 and bmi was not loaded in factor 2 . it could be noted that the clustering patterns in our study were not only similar to hanley 's study ( 40 ) but also to the most of other studies with different severities of impaired glucose metabolism and ethnic groups ( 13 , 36 , 38 , 39 ) . the clustering of the sspg , bmi , fpg and tg could further confirm that the ir is more correlated with lipids profile and bmi , but less with the blood pressure . it should be noted and emphasized that this was a hospital - based cohort study . however , the purpose of this study was to observe the relationships between ir and mets , thus , this drawback should not affect the conclusions drawn from the present study . our data suggested that adiposity , higher fpg , and hypertriglyceridemia are more strongly associated with ir in chinese subjects and the importance of these clinical characteristics should be reemphasized . when considering each of the mets clinical characteristics , subjects with a higher bmi may have the highest ir .

the impact the metabolic syndrome ( mets ) components on the severity of insulin resistance ( ir ) has not been reported . we enrolled 564 subjects with mets and they were divided into quartiles according to the level of each component ; and an insulin suppression test was performed to measure ir . in males , steady state plasma glucose ( sspg ) levels in the highest quartiles , corresponding to body mass index ( bmi ) and fasting plasma glucose ( fpg ) , were higher than the other three quartiles and the highest quartiles , corresponding to the diastolic blood pressure and triglycerides , were higher than in the lowest two quartiles . in females , sspg levels in the highest quartiles , corresponding to the bmi and triglycerides , were higher than in all other quartiles . no significant differences existed between genders , other than the mean sspg levels in males were greater in the highest quartile corresponding to bmi than that in the highest quartile corresponding to hdl - cholesterol levels . the factor analysis identified two underlying factors ( ir and blood pressure factors ) among the mets variables . the clustering of the sspg , bmi , triglyceride and hdl - cholesterol was noted . our data suggest that adiposity , higher fpg and triglyceride levels have stronger correlation with ir and subjects with the highest bmi have the highest ir .

INTRODUCTION MATERIALS AND METHODS Subjects Laboratory evaluation Statistical analysis RESULTS DISCUSSION

quality of life ( ql ) may be defined as an individual s perception of their well - being . health - related ql reflects the impact of a healthy state on a person s ability to lead a fulfilling life , and covers the individual s satisfaction in physical , functional , psychological , and social domains.1 studies2,3 have shown that ql worsens after brain damage ( vascular lesions , head injury , infections ) . changes in ql in the poststroke period do not seem to be age-4 or sex - dependent , but rather are due to the onset of depression5 and a low level of reacquisition in motor functions , above all in the upper limbs.6 ql improves within the family environment.7 aphasia is the loss of or radical change in voluntary speech as a consequence of damage to the left cerebral hemisphere , and generally causes significant changes in a patient s self - image and family , social , and work relationships.8 the impairment in language skills due to aphasia causes problems in verbal expression , auditory comprehension , reading , and writing . aphasia is a prominent cause of limitation on communication activities , such as using the phone or writing a letter . aphasia will have relatively little direct impact upon the performance of domestic activities of daily living , but it will particularly affect complex social activities , such as work and participating in community activities and leisure activities involving other people . studies have documented high levels of depression9 and social exclusion,10 and low levels of leisure and other social activities,11 social contacts,12 and ql13,14 among patients with aphasia ( pwa ) . significant correlations have been found between degree of aphasia and the social , emotional , mobility , and total scores of ql , measured with the nottingham health profile.15 in a population - based study , lam and wodchis16 found that aphasia exhibited the largest negative influence on ql , followed by cancer and alzheimer s disease . pwa reported significantly worse ql than nonaphasic patients , since aphasia influenced independence , social relationships , and access to their environment . le dorze and brassard17 have described the consequences of aphasia by analyzing the personal accounts of aphasic individuals , relatives , or friends . these consequences were classified into one of three categories : disabilities , handicaps , and coping behaviors . in the first group , the handicaps included changes in situations of communication , in interpersonal relationships , loss of autonomy , and restriction of activities . coping behaviors , however , were adopted by pwa and their relatives to adjust the undesired effects of the various disabilities and handicaps they experienced . although a number of stroke - specific ql scales have been developed , most exclude stroke survivors with aphasia , subjects most prone to social isolation and exclusion . this is due to the difficulties faced in proposing a questionnaire to subjects with severe comprehension and expression deficits . in regard to the studies on the impact of aphasia on patient s life , there are two paradigms . from a qualitative perspective , ethnographic methods , like participant and nonparticipant observations and analysis of artifacts such as diaries , can reflect the everyday experiences of pwa and their social inclusion , but they do not suggest how patients feel their ql to be . from a quantitative perspective , many authors18,19 have evaluated the ql of patients with severe aphasia , asking proxy respondents to report on their partners health - related quality of life ( hrql ) . also in this case , evaluating one s emotional distress , aphasia severity , communication and activity limitations , other medical problems , and social aspects all influenced ql . the extent of aphasia ( severity , language impairment , communication disability ) was associated with or predictive of lower hrql in seven of the eight reviewed studies . hilari et al20 evaluated the ql in patients with and without aphasia , testing a stroke - specific hrql scale ( the stroke and aphasia quality of life scale [ saqol-39 ] ) in a generic stroke sample that included patients both with and without aphasia . the saqol-39 generic stroke scale measures hrql after stroke in three domains : physical , psychosocial , and communication . this scale demonstrates good internal consistency , test retest reliability and validity , and adequate responsiveness to changes in a sample of patients with acute and chronic stroke . the information obtained from ql measures can be useful to identify patients problems , determine treatment priorities , manage interventions , and monitor disease periods . the first aim of this observational study was to conduct a preliminary evaluation of the psychometric properties ( validity , reliability ) of a ql questionnaire for aphasics ( qlqa ) , in which we focused particularly on difficulties in interpersonal relationships , on loss of independence , and on abilities in daily life as a result of language disorders . moreover , in the aphasic group , we studied how qlqa was sensitive to the severity and type of aphasia and to the time from onset . finally , we tried to identify the specific role of linguistic deficits on ql , minimizing the effects of motor impairment . the qlqa scores of pwa with hemiparesis were compared to those of a control group affected by a right - brain injury or a neurological peripheral disease and motor impairment , but with no linguistic or communicative problems . a total of 164 consecutive patients with neurological central disease admitted to our neurorehabilitation unit for cognitive and physical rehabilitation from 2011 to 2012 were examined . eighteen of them were excluded for bilateral or multiple lesions identified by neurodiagnostic scans ( computerized axial tomography or magnetic resonance imaging ) . the experimental group consisted of 146 pwa and right hemiparesis , all cared for by a caregiver . inclusion criteria were aphasia resulting from unilateral left - hemisphere stroke , unknown prestroke history of severe cognitive decline , or mental health problems . no patients presented with prestroke neuropsychological deficits , psychiatric disorders , history of alcohol or drug abuse , head injury , or tumoral lesions . patients were excluded if they did not speak italian premorbidly a control group consisted of 37 subjects with right - hemisphere cerebral damage , hemiparesis , and varying degrees of unilateral spatial neglect . written consent all the patients underwent an initial screening to evaluate the presence and type of linguistic disorders and the severity of motor impairment . all aphasic patients were evaluated by the aachener aphasia test ( aat)21 to analyze language deficits and to diagnose type of aphasia . for this study , the patients underwent the following series of tests , administered by a speech therapist : the subtest of spontaneous speech of the aat,21 structured in six parts ( communicative behavior , articulation and prosody , automatic language , semantics , phonology , syntax ) , with a score range from 0 to 5 the token test22 to assess verbal comprehension with 36 items raven s coloured progressive matrices,23,24 which provides a nonverbal measure of intellectual abilities ; the patient must logically complete a given visual spatial pattern , choosing from a set of six alternatives a scale of functional independence the functional independence measure25,26 ( fim ) divided into scores for motor fim ( 13 items ) and cognitive fim ( five items ) another scale of functional independence the functional assessment measure ( fam)2729 cognitive subscale a compound of fourteen items evaluating comprehension , expression , reading , writing , speech intelligibility , social interaction , emotional status , adjustment to limitation , employability , problem solving , memory , orientation , attention span , and safety judgment hrql questionnaires give outcome measures that evaluate the impact of health on a person s ability to lead a fulfilling life , and generally incorporate the individual s perceptions of physical , mental / emotional , family , and social functioning . although a number of stroke - specific ql scales have been developed , measures that ecologically identify both the effects of linguistic communicative disability and the quality of everyday life are still needed for pwa . they could be useful to bridge the gap between linguistic rehabilitation and the patient s real use of residual means of communication or ability to perform the daily life activities in which language is involved . in this way , they could link the individual rehabilitation training to outcomes of increased functional autonomy in social and communicative environments , according to the recent world health organization international classification of functioning , disability and health ( icf).30 one example of these measures is the functional outcome questionnaire for aphasia,31 used in the us . it consists of 32 items , and is focused on the efficacy of the aphasic s verbal and nonverbal communicative abilities . this scale assesses the ability to communicate basic needs and new information and the ability to make routine requests ; it also investigates the issues of communication and comprehension . the saqol-3932 scale , recently translated into italian and also into spanish,33,34 is a more complete ql assessment ( it includes measurement of general , physical , and psychosocial health and vitality ) , even if there are few linguistic items compared to the total number ( nine of 39 ) . our qlqa has the aim of addressing a lack of ql measures in the italian language , including , as with previous ones , measurement of several domains , such as physical , psychological , communication , and social participation . engell et al35 developed a pictorial procedure for rating ql to minimize the influence of aphasia . they transformed an existing ql inventory the modified german version of the sickness impact profile36 into a picture - based representation . the authors selected the aachen quality of life inventory ( alqi),37 a german - language adaptation of the sickness impact profile . the alqi items were transformed into a pictorial version to maximize aphasic patients understanding of the verbal statements , as well as to permit them to give nonverbal responses . this procedure allowed for self - rating of aphasic patients in parallel to a proxy rating by caregivers . there was in fact also a written version for the caregivers that corresponded item by item to the patients pictorial version . the limited use of these assessments has negative effects on the course of clinical welfare . in italy , stroke rehabilitation still focuses more on the improvement of physical autonomy rather than on a global increase in neuropsychological functions , which are often affected . rehabilitation therapy should aim not only to regain communicative linguistic abilities , but also to help in adapting to the disability and to encourage social integration and personal well - being , thus improving ql . our ql questionnaire was developed by means of the aforementioned conceptual considerations and analysis of literature studies and of the previous ql and functional questionnaires for aphasic patients , in order to point out the aphasic disease , handicap and coping behavior in everyday life . the questionnaire consists of 37 questions that are important for daily well - being , chosen from a larger repertoire by a mixed group of pwa and their caregivers and members of the italian aphasia association of our region ( puglia ) . we consequently based the qlqa more on the percentage of residual abilities performed by the individuals with aphasia rather than on their perceived ql , which was difficult to investigate , considering their communicative problems . express the disability , mainly as relationship problems and loss of autonomy caused by linguistic deficits ; it consists in a majority of questions regarding language - related activities and abilities consider verbal comprehension difficulties include items of residual functional disability due to motor deficits include items on psychological problems that occur both in the poststroke phase and later , partly as a result of brain damage but also the social and individual aftereffects of aphasia highlight relational and nonrelational residual problems due to language deficits . the questions , formulated as easily and succinctly as possible , assess the ability to perform basic functions and convey health problems , the psychological changes due to the disability and the ability to socialize , the ability of linguistic and contextual comprehension , and expression in routine daily activities . the items are scored on a 5-point scale , with 0= the individual is able to successfully perform the behavior 0% of the time , 1= the individual is able to successfully perform the behavior 25% of the time , 2= the individual is able to successfully perform the behavior 50% of the time , 3= the individual is able to successfully perform the behavior 75% of the time , and 4= the individual is able to successfully perform the behavior 100% of the time . the qlqa was administered by a speech therapist using verbal or pragmatic means ( gestures and drawings ) when the patient showed comprehension deficits , in the presence of the caregiver . when there was no agreement in the answers , we considered those of the caregiver more reliable , so excluding the influence of the patients reduced consciousness of their own limits or anxiety / depression state . demographic and clinical variables of aphasic sample and control subjects were evaluated with descriptive statistics ( means , standard deviations , etc),using student s t - test for analyzing the differences between two samples . due to the ordinal nature of the scale , differences in the qlqa scores and in other tests of both groups were analyzed using nonparametric tests ( mann whitney u ) . we used standard psychometric methods3840 to evaluate internal consistency , test retest reliability , and construct validity ( convergent and discriminant ) of qlqa in 146 aphasic patients . a principal - component analysis ( pca , a type of factor analysis ) was used to determine the number and the type of domains underlying the ql of aphasic patients and reduce the number of items in the qlqa to those best measuring it . in order to evaluate convergent and discriminant validity , a multiple correlation among the scores of the qlqa subtests ( communication , autonomy , and psychological condition ) , resulting from pca , and the fim and fam scores and the correct linguistic tests scores were calculated by spearman s correlation coefficient . internal consistency was calculated using cronbach s -test and test retest reliability using intraclass correlation coefficient ( icc ) . data analyses were carried out with spss 18.0 for windows ( ibm , armonk , ny , usa ) . a total of 164 consecutive patients with neurological central disease admitted to our neurorehabilitation unit for cognitive and physical rehabilitation from 2011 to 2012 were examined . eighteen of them were excluded for bilateral or multiple lesions identified by neurodiagnostic scans ( computerized axial tomography or magnetic resonance imaging ) . the experimental group consisted of 146 pwa and right hemiparesis , all cared for by a caregiver . inclusion criteria were aphasia resulting from unilateral left - hemisphere stroke , unknown prestroke history of severe cognitive decline , or mental health problems . no patients presented with prestroke neuropsychological deficits , psychiatric disorders , history of alcohol or drug abuse , head injury , or tumoral lesions . patients were excluded if they did not speak italian premorbidly a control group consisted of 37 subjects with right - hemisphere cerebral damage , hemiparesis , and varying degrees of unilateral spatial neglect . written consent all the patients underwent an initial screening to evaluate the presence and type of linguistic disorders and the severity of motor impairment . all aphasic patients were evaluated by the aachener aphasia test ( aat)21 to analyze language deficits and to diagnose type of aphasia . for this study , the patients underwent the following series of tests , administered by a speech therapist : the subtest of spontaneous speech of the aat,21 structured in six parts ( communicative behavior , articulation and prosody , automatic language , semantics , phonology , syntax ) , with a score range from 0 to 5 the token test22 to assess verbal comprehension with 36 items raven s coloured progressive matrices,23,24 which provides a nonverbal measure of intellectual abilities ; the patient must logically complete a given visual spatial pattern , choosing from a set of six alternatives a scale of functional independence the functional independence measure25,26 ( fim ) divided into scores for motor fim ( 13 items ) and cognitive fim ( five items ) another scale of functional independence the functional assessment measure ( fam)2729 cognitive subscale a compound of fourteen items evaluating comprehension , expression , reading , writing , speech intelligibility , social interaction , emotional status , adjustment to limitation , employability , problem solving , memory , orientation , attention span , and safety judgment hrql questionnaires give outcome measures that evaluate the impact of health on a person s ability to lead a fulfilling life , and generally incorporate the individual s perceptions of physical , mental / emotional , family , and social functioning . although a number of stroke - specific ql scales have been developed , measures that ecologically identify both the effects of linguistic communicative disability and the quality of everyday life are still needed for pwa . they could be useful to bridge the gap between linguistic rehabilitation and the patient s real use of residual means of communication or ability to perform the daily life activities in which language is involved . in this way , they could link the individual rehabilitation training to outcomes of increased functional autonomy in social and communicative environments , according to the recent world health organization international classification of functioning , disability and health ( icf).30 one example of these measures is the functional outcome questionnaire for aphasia,31 used in the us . it consists of 32 items , and is focused on the efficacy of the aphasic s verbal and nonverbal communicative abilities . this scale assesses the ability to communicate basic needs and new information and the ability to make routine requests ; it also investigates the issues of communication and comprehension . the saqol-3932 scale , recently translated into italian and also into spanish,33,34 is a more complete ql assessment ( it includes measurement of general , physical , and psychosocial health and vitality ) , even if there are few linguistic items compared to the total number ( nine of 39 ) . our qlqa has the aim of addressing a lack of ql measures in the italian language , including , as with previous ones , measurement of several domains , such as physical , psychological , communication , and social participation . engell et al35 developed a pictorial procedure for rating ql to minimize the influence of aphasia . they transformed an existing ql inventory the modified german version of the sickness impact profile36 into a picture - based representation . the authors selected the aachen quality of life inventory ( alqi),37 a german - language adaptation of the sickness impact profile . the alqi items were transformed into a pictorial version to maximize aphasic patients understanding of the verbal statements , as well as to permit them to give nonverbal responses . this procedure allowed for self - rating of aphasic patients in parallel to a proxy rating by caregivers . there was in fact also a written version for the caregivers that corresponded item by item to the patients pictorial version . the limited use of these assessments has negative effects on the course of clinical welfare . in italy , stroke rehabilitation still focuses more on the improvement of physical autonomy rather than on a global increase in neuropsychological functions , which are often affected . rehabilitation therapy should aim not only to regain communicative linguistic abilities , but also to help in adapting to the disability and to encourage social integration and personal well - being , thus improving ql . our ql questionnaire was developed by means of the aforementioned conceptual considerations and analysis of literature studies and of the previous ql and functional questionnaires for aphasic patients , in order to point out the aphasic disease , handicap and coping behavior in everyday life . the questionnaire consists of 37 questions that are important for daily well - being , chosen from a larger repertoire by a mixed group of pwa and their caregivers and members of the italian aphasia association of our region ( puglia ) . we consequently based the qlqa more on the percentage of residual abilities performed by the individuals with aphasia rather than on their perceived ql , which was difficult to investigate , considering their communicative problems . express the disability , mainly as relationship problems and loss of autonomy caused by linguistic deficits ; it consists in a majority of questions regarding language - related activities and abilities consider verbal comprehension difficulties include items of residual functional disability due to motor deficits include items on psychological problems that occur both in the poststroke phase and later , partly as a result of brain damage but also the social and individual aftereffects of aphasia highlight relational and nonrelational residual problems due to language deficits . the questions , formulated as easily and succinctly as possible , assess the ability to perform basic functions and convey health problems , the psychological changes due to the disability and the ability to socialize , the ability of linguistic and contextual comprehension , and expression in routine daily activities . the items are scored on a 5-point scale , with 0= the individual is able to successfully perform the behavior 0% of the time , 1= the individual is able to successfully perform the behavior 25% of the time , 2= the individual is able to successfully perform the behavior 50% of the time , 3= the individual is able to successfully perform the behavior 75% of the time , and 4= the individual is able to successfully perform the behavior 100% of the time . the qlqa was administered by a speech therapist using verbal or pragmatic means ( gestures and drawings ) when the patient showed comprehension deficits , in the presence of the caregiver . when there was no agreement in the answers , we considered those of the caregiver more reliable , so excluding the influence of the patients reduced consciousness of their own limits or anxiety / depression state . demographic and clinical variables of aphasic sample and control subjects were evaluated with descriptive statistics ( means , standard deviations , etc),using student s t - test for analyzing the differences between two samples . due to the ordinal nature of the scale , differences in the qlqa scores and in other tests of both groups were analyzed using nonparametric tests ( mann whitney u ) . we used standard psychometric methods3840 to evaluate internal consistency , test retest reliability , and construct validity ( convergent and discriminant ) of qlqa in 146 aphasic patients . a principal - component analysis ( pca , a type of factor analysis ) was used to determine the number and the type of domains underlying the ql of aphasic patients and reduce the number of items in the qlqa to those best measuring it . in order to evaluate convergent and discriminant validity , a multiple correlation among the scores of the qlqa subtests ( communication , autonomy , and psychological condition ) , resulting from pca , and the fim and fam scores and the correct linguistic tests scores were calculated by spearman s correlation coefficient . internal consistency was calculated using cronbach s -test and test retest reliability using intraclass correlation coefficient ( icc ) . data analyses were carried out with spss 18.0 for windows ( ibm , armonk , ny , usa ) . table 1 shows the demographic and clinical variables of the entire sample of aphasic patients . the 146 aphasic subjects ( 67 female and 79 male ) had a mean age of 68.4 years , a mean education of 6.96 years , and a mean poststroke period before assessment of 399.16 days . most patients ( 80.2% ) were married and had a caregiver , 17 aphasic subjects were widowed but had a child as a caregiver , while twelve patients were single . the language assessment by aat classified 102 patients as nonfluent pwa ( 61 with global aphasia , 39 with broca s aphasia , and two with transcortical motor aphasia ) , and 38 patients with a fluent form of aphasia ( 25 with wernicke s aphasia , twelve amnesic , one with a transcortical sensorial aphasia ) . olkin measure ( kmo ) verified the sampling adequacy for the analyses ( kmo = 0.924 ) , and all kmo values for individual items were > 0.6 , which is well above the acceptable limit of 0.5 . bartlett s test of sphericity =4,674.195 , p<0.001 indicated that correlations between items were sufficiently large for pca . the final model explained 59.5% of the variance and included three factors : the first factor , named communication , comprises 22 items evaluating the patient s ability to express and understand in real life and pragmatic situations ; the second factor , named psychological condition , includes six items evaluating the impact of language deficits on emotional status ; and the third factor , named autonomy , includes nine items assessing the independence of the subject in activities of daily life . we assessed the qlqa reliability , ie , the internal consistency and test retest reliability . to assess the extent to which qlqa items measure the format and homogeneity of the scales , we calculated the internal consistency using cronbach s -coefficients ( table 3 ) for the whole scale and for each of the three subscales . the internal consistency of the qlqa was high ( =0.96 , criteria for acceptability = cronbach s >0.70 ) . the qlqa subscales showed similarly high reliability , ranging from 0.79 for psychological condition to 0.97 for communication subscale and 0.89 for autonomy subscale . the test retest reliability , defined as the stability of an instrument over time , was evaluated by administering this test at a 3-day interval to a small sample of 14 ( 10% of complete sample ) aphasic patients with similar characteristics to those of the overall sample in terms of age , sex , marital status , and aphasia . retest reliability for both overall ( icc = 0.98 ) and subscale scores ( communication icc = 0.95 , autonomy icc = 0.85 , psychological condition icc = 0.65 ) . iccs < 0.40 were seen as indicating poor agreement , 0.400.75 fair - to - good agreement , and 0.761.00 excellent agreement.39 qlqa total , communication , and autonomy subscales had excellent reliability . the lower test retest reliability of the psychological condition subscale can be lined up with a physiological variation of the patient s mood state . the convergent validity was calculated by correlating the qlqa subscales and total scores with tests that assessed the same or similar constructs . high correlation coefficients were found between qlqa total , the communication and autonomy subscales , and fim ( respectively , r=0.69 , r=0.62 , r=0.67 ) and fam scores ( respectively , r=0.75 , r=0.73 , r=0.64 ) . all these scales in fact evaluated the patients autonomy both for motor and in cognitive aspects . high correlations were found also between qlqa total and communication subscale and aat scores ( respectively , r=0.58 , r=0.63 ) . a significant correlation was found with fam emotional status and social interaction subscales and qlqa psychological condition subscale . to evaluate the discriminant validity , we calculated the correlation coefficients between the qlqa total and subscale scores and the scores of tests assessing different functions . low correlations were found between the qlqa total and subscale scores and raven s coloured progressive matrices score . for the qlqa psychological condition subscale , we found low correlation coefficients , showing the specificity of this subscale . subjects with aphasia ( n=146 ) were divided into two groups based on the severity of language disorders , according to aat assessment . a total of 112 patients had severe aphasia , and 34 had mild language disorders . whitney test to compare the qlqa , linguistic tests , and fim scores of severe and mild patients . severe patients , apart from having a more severe linguistic deficit , were significantly more impaired in motor and cognitive autonomy , based on fim data ( p<0.001 ) . significant differences were found in communicative , autonomy , and total qlqa scores ( p<0.001 ) : eighty - four aphasic patients were assessed within 3 months from stroke , and 62 patients were evaluated in a chronic stage of disease ( after 3 months ) . the two groups were different for age ( p<0.01 ) and time from stroke ( p<0.001 ) , and were comparable for educational level ( table 6 ) . linguistic and motor conditions were significantly different between the groups . no difference was found in raven s test score . qlqa patients evaluated in the acute stage showed greater impairment in communication , autonomy , and total score . finally , we evaluated the specific role of linguistic deficits on ql in two groups of patients with and without aphasia in the acute stage of disease . since the time from stroke has an influence on ql of patients , we chose to compare the qlqa scores of 84 acute pwa with hemiplegia to those of a control group of 37 subjects with motor impairment and without aphasic problems , assessed within 3 months from stroke . the two groups were compared in the following series of tests : raven s coloured progressive matrices the control patients had a mean age of 64.79 years , a mean education of 7.69 years , and a mean time from stroke of 114.19 days . pwa and controls were similar for age , educational level , sex , and motor autonomy . significant differences were found in time from stroke ( p<0.001 ) , cognitive ( p<0.001 ) and total fim scores ( p<0.001 ) , and token test ( p<0.001 ) . statistically significant differences between pwa and control subjects were found regarding qlqa total and subscale scores ( communication , autonomy , and psychological condition ) . we assessed the qlqa reliability , ie , the internal consistency and test retest reliability . to assess the extent to which qlqa items measure the format and homogeneity of the scales , we calculated the internal consistency using cronbach s -coefficients ( table 3 ) for the whole scale and for each of the three subscales . the internal consistency of the qlqa was high ( =0.96 , criteria for acceptability = cronbach s >0.70 ) . the qlqa subscales showed similarly high reliability , ranging from 0.79 for psychological condition to 0.97 for communication subscale and 0.89 for autonomy subscale . the test retest reliability , defined as the stability of an instrument over time , was evaluated by administering this test at a 3-day interval to a small sample of 14 ( 10% of complete sample ) aphasic patients with similar characteristics to those of the overall sample in terms of age , sex , marital status , and aphasia . retest reliability for both overall ( icc = 0.98 ) and subscale scores ( communication icc = 0.95 , autonomy icc = 0.85 , psychological condition icc = 0.65 ) . iccs < 0.40 were seen as indicating poor agreement , 0.400.75 fair - to - good agreement , and 0.761.00 excellent agreement.39 qlqa total , communication , and autonomy subscales had excellent reliability . the lower test retest reliability of the psychological condition subscale can be lined up with a physiological variation of the patient s mood state . the convergent validity was calculated by correlating the qlqa subscales and total scores with tests that assessed the same or similar constructs . high correlation coefficients were found between qlqa total , the communication and autonomy subscales , and fim ( respectively , r=0.69 , r=0.62 , r=0.67 ) and fam scores ( respectively , r=0.75 , r=0.73 , r=0.64 ) . all these scales in fact evaluated the patients autonomy both for motor and in cognitive aspects . high correlations were found also between qlqa total and communication subscale and aat scores ( respectively , r=0.58 , r=0.63 ) . a significant correlation was found with fam emotional status and social interaction subscales and qlqa psychological condition subscale . to evaluate the discriminant validity , we calculated the correlation coefficients between the qlqa total and subscale scores and the scores of tests assessing different functions . low correlations were found between the qlqa total and subscale scores and raven s coloured progressive matrices score . for the qlqa psychological condition subscale , we found low correlation coefficients , showing the specificity of this subscale . the convergent validity was calculated by correlating the qlqa subscales and total scores with tests that assessed the same or similar constructs . high correlation coefficients were found between qlqa total , the communication and autonomy subscales , and fim ( respectively , r=0.69 , r=0.62 , r=0.67 ) and fam scores ( respectively , r=0.75 , r=0.73 , r=0.64 ) . all these scales in fact evaluated the patients autonomy both for motor and in cognitive aspects . high correlations were found also between qlqa total and communication subscale and aat scores ( respectively , r=0.58 , r=0.63 ) . a significant correlation was found with fam emotional status and social interaction subscales and qlqa psychological condition subscale . to evaluate the discriminant validity , we calculated the correlation coefficients between the qlqa total and subscale scores and the scores of tests assessing different functions . low correlations were found between the qlqa total and subscale scores and raven s coloured progressive matrices score . for the qlqa psychological condition subscale , we found low correlation coefficients , showing the specificity of this subscale . subjects with aphasia ( n=146 ) were divided into two groups based on the severity of language disorders , according to aat assessment . a total of 112 patients had severe aphasia , and 34 had mild language disorders . whitney test to compare the qlqa , linguistic tests , and fim scores of severe and mild patients . severe patients , apart from having a more severe linguistic deficit , were significantly more impaired in motor and cognitive autonomy , based on fim data ( p<0.001 ) . significant differences were found in communicative , autonomy , and total qlqa scores ( p<0.001 ) : mild pwa in fact had a better ql than severe patients . eighty - four aphasic patients were assessed within 3 months from stroke , and 62 patients were evaluated in a chronic stage of disease ( after 3 months ) . the two groups were different for age ( p<0.01 ) and time from stroke ( p<0.001 ) , and were comparable for educational level ( table 6 ) . qlqa patients evaluated in the acute stage showed greater impairment in communication , autonomy , and total score . finally , we evaluated the specific role of linguistic deficits on ql in two groups of patients with and without aphasia in the acute stage of disease . since the time from stroke has an influence on ql of patients , we chose to compare the qlqa scores of 84 acute pwa with hemiplegia to those of a control group of 37 subjects with motor impairment and without aphasic problems , assessed within 3 months from stroke . the two groups were compared in the following series of tests : raven s coloured progressive matrices the control patients had a mean age of 64.79 years , a mean education of 7.69 years , and a mean time from stroke of 114.19 days . pwa and controls were similar for age , educational level , sex , and motor autonomy . significant differences were found in time from stroke ( p<0.001 ) , cognitive ( p<0.001 ) and total fim scores ( p<0.001 ) , and token test ( p<0.001 ) . statistically significant differences between pwa and control subjects were found regarding qlqa total and subscale scores ( communication , autonomy , and psychological condition ) . the results of this preliminary psychometric examination suggest that the qlqa is a reliable and valid measure of ql in people with aphasia . the internal consistency was found to be high . the data of convergent and discriminant validity are not univocal : the correlation coefficients are not very high , and sometimes there were correlations also with different measures . this could be due to the ecological value of the qlqa : as in daily life , qlqa comprehension is influenced and improved by pragmatic aids , unlike token test performances , causing the absence of any relationship between pwa subjective estimation of their comprehension difficulties and the token test results.41 moreover , also other measures used for this study , such as fam , evaluate the patient s functional autonomy in multiple cognitive domains that do not leave out of consideration the use of language . this could explain , for example , the correlations that we found between qlqa total , subscales , and fam subscales . the qlqa differs from other ql questionnaires due to the greater importance given to the reduction of autonomy caused by aphasia , independently of motor deficits , as shown by the differences between pwa and controls . our results showed that the time from stroke and the severity of aphasia influence the patient s functional and communicative autonomy , but not the psychological condition . the mild and the chronic aphasic patients had a better quality of life than severe and acute ones , underlining the fact the passing of time helps patients with language disorders to adapt themselves to the new condition . in the literature , other studies,20,42 reported low - to - moderate improvements in ql after stroke . the physical and communication domains present greater improvement between acute stroke and 3 months , while psychosocial well - being takes longer to improve poststroke . regarding severity of language disease , engell et al35 found a correlation between the alqi ratings and the performance scores of the aat . total , physical , and psychosocial scores were significantly correlated with communicative and systematic failures in spontaneous language , but not with articulation disorders . moreover subjects with aphasia , independently of severity and time , tend towards social isolation and demand that other family members manage personal and family problems . our results agree with many studies in the literature that underline that the frequency of depression in aphasic patients is higher than in other stroke survivors.9,43 this test was given to pwa in the presence of a caregiver and with the aid of a speech therapist to overcome comprehension / expression deficits and avoid the problem of missing data . this could be a valid method when using these questionnaires in future , since severe pwa have until now been excluded from similar evaluations . other studies18 suggested that proxy respondents can provide reliable information on the hrql of pwa at the chronic stage of disease , even if self - report is more valid than any proxy report . however , using proxies may be a useful way to obtain information on the hrql of patients with severe aphasia . with qlqa , the differences among emotional , social , and communicative aspects of pwa and patients with motor deficits clearly emerge . this has an important effect on rehabilitation : on the one hand , it justifies the equal importance of language and motor rehabilitation ; on the other , it explains the reasons for a greater incidence of stress among pwa and their family members.44 hrql scales are essential in stroke assessment and outcome measurement . with patients subjective evaluation of their functioning and well - being , the speech - language pathologists and related professionals get a more holistic picture of how stroke and aphasia has affected patients lives , and they can make more informed decisions on what needs to be targeted in intervention . lastly , these findings suggest that people with stroke , and particularly those with aphasia , need long - term service provision that takes into account their affected mood , through community - based interventions , eg , participation in personally relevant meaningful activities . the use of qlqa in the assessment of people with aphasia focuses on the frequency of activities of life , depending on the correct use of language . it may point out gains in linguistic and communicative behaviors , due to rehabilitation training and/or personal ability of coping with disease limitation , and it shows daily personal aspects of discomfort , which are rarely evidenced by the patients , while leading them to isolation and passivity . timely interventions by speech - language pathologists and occupational therapists , psychological support to patients and caregivers , and changes in communicative environments are needed to promote socialization , according to the conceptual models of the icf . as aphasia causes communication impairment and altered relationships , for aphasic people the aim of rehabilitation should be to improve either the disability ( making language and communication more efficient ) or the possibilities of coping behaviors , intervening in the social sphere , and reinforcing self - esteem and the desire to return to personal autonomy . a limitation of this study lies in the comparison of the qlqa with measures of functional autonomy . it could be interesting also to compare the qlqa psychological condition subscale with scales of mood state , in order to increase the construct validity of our questionnaire . as is common with new measures , further research is needed to confirm its psychometric properties in independent samples . moreover , it is necessary to evaluate the test retest reliability in a larger sample of pwa . our next objective is to evaluate the ql of aphasic patients through a multicentric longitudinal study , in order to assess how ql changes with the evolution of language disease . qlqa is a valid measure of ql in pwa , contributing to a better distinction between severe and mild aphasia and also to the variations in ql depending on the time interval from stroke . improvement in the severity of language deficits also causes an improvement in ql , except for the psychological condition . this underlines the important role of aphasia in social isolation and emotional distress of patients . the regular use of ql scales in the assessment of pwa highlights their disability , handicap , and ability to cope . qlqa would be a useful tool in planning rehabilitation with a view to achieving greater functional autonomy in social and communicative environments .

backgroundquality of life ( ql ) can be defined as the individual s perception of their own well - being . aphasia is the most important potential consequence of stroke and has a profound effect on a patient s life , causing emotional distress , depression , and social isolation , due to loss of language functions.aimsto draw up a ql questionnaire for aphasics ( qlqa ) focusing particularly on difficulties in interpersonal relationships and on the loss of independence as a result of language disorders . we reported the results of a psychometric evaluation of this measure . moreover , we experimentally focused on the differences in qlqa between patients affected only by neurological motor impairment and hemiparetic patients with aphasia ( pwa ) in order to verify the specific role of aphasia on ql . we also explored if the qlqa is sensitive to the severity of aphasia and to the time elapsing from the stroke.methodsa total of 146 consecutive pwa and 37 control subjects were enrolled to evaluate the reliability ( internal consistency and test retest reliability ) and validity of the qlqa , using standard psychometric methods . patients were divided into acute ( within 3 months since stroke ) and chronic ( beyond 3 months ) groups , and into mild and severe according to the severity of aphasia . the experimental group of only acute pwa was compared to control subjects , with right hemispherical lesion and without aphasia in qlqa total and partial scores.resultsthe qlqa had good internal consistency and test retest reliability . acute and chronic pwa and mild and severe ones differed in qlqa total , communication , and autonomy subscales . no differences were found in psychological condition . between aphasic and control patients , significant differences were found in all qlqa subscales.conclusionthe qlqa is a valid measure of ql in pwa , contributing to a better distinction between severe and mild aphasia , and it is sensitive also to the variations in ql depending on the time interval from stroke .

Introduction Materials and methods Subjects Materials Quality of life questionnaire for aphasics Psychometric analysis Results Reliability Validity Convergent and discriminant validity Sensitivity to the severity of aphasia Sensitivity to time from stroke Differences between PWA and control subjects Discussion Conclusion

the patient was a 23-year - old woman , a known case of arnold - chiari malformation with peripheral neuropathy and muscular atrophy , who presented with headache , drowsiness , decreased vision , and severe gait dysfunction lasting for several years . brain magnetic resonance imaging ( mri ) confirmed a hypointense - signal mass in the left hemisphere of the cerebellum causing mass effects on the fourth ventricle , which shifted it , accompanied with dilation of third and lateral ventricles . hypertensive cerebrospinal fluid ( csf ) form of hydrocephaly was seen in the supratentorial region , but the fourth ventricle was normal . the possibility of aqueduct stenosis or obstruction was mentioned ( fig . 1 ) . cervical spine mri revealed a normal spinal column with herniation of the cerebellar tonsil ( fig . 2 ) . brain magnetic resonance imaging . a hypointense signal mass in the left hemisphere of the cerebellum causing mass effects on the fourth ventricle , which shifted it , accompanied with dilation of third and lateral ventricles . tonsillar herniation to the cervical spine is seen . with the impression of tonsillar herniation and cerebellar tumor in the sense of arnold - chiari syndrome , external ventricular drainage with external reservoir a cream - white tight mass was seen , which could not be extracted by suction and thus was totally resected . simultaneously , regarding the arnold - chiari malformation and tonsillar herniation , the patient underwent suboccipital decompression and microscopic laminectomy of c2 vertebrae with excision of foramen magnum and duraplasty . gross examination revealed multiple pieces of irregular cream tissue totally measuring 2.5 2.0 0.7 cm . microscopic assessment confirmed a granulomatous inflammation composed of aggregation of epithelioid histiocytes associated with giant cells and lymphocyte cuffing foci of caseating necrosis compatible with tuberculoma . postoperative spiral chest computed tomography ( ct ) scan showed a normal pulmonary parenchyma without evidence for pulmonary tuberculosis , thus , the patient had a primary extrapulmonary cerebellar tuberculoma ( fig . the patient has been followed to now ; the neurological symptoms were alleviated 6 months subsequent to the surgery . chiari malformations are rare congenital anomalies with an estimated prevalence of 0.1 to 0.5%5 ; however , the true frequency is unknown . in most cases , due to the small posterior fossa the main pathogenesis of this malformation remains the subject of debate and involves patients presenting with a wide spectrum of clinical symptoms.1 arnold - chiari syndrome is usually detected prenatally or at birth , as it is nearly always associated with lumbosacral or thoracic myelomeningocele.1 weakness , stridor , apneic spells , aspiration , and dysphagia are the common manifestations in infancy,6 followed by progressive hydrocephalus in childhood.1 it may be associated with other syndromes like syringomyelia and scoliosis.7 8 our case presented with headache , drowsiness , decreased vision , and severe gait dysfunction lasting for several years . arnold - chiari syndrome should be considered in any fetus or newborn with clinical evidence of a spinal myelomeningocele . neuroimaging plays the main role in confirming the diagnosis , and mri is the best imaging modality for evaluation.9 mri of the brain along with the entire spinal cord ( cervical ) is appropriate to demonstrate downward displacement of the inferior cerebellar vermis and medulla through the foramen magnum into the upper cervical canal . despite these typical findings , brain mri in our case showed an unusual mass in the left cerebellar hemisphere causing mass effect . the patient underwent the most common procedure for arnold - chiari malformations , which is posterior decompression via suboccipital craniotomy with duraplasty.10 11 simultaneously , regarding the cerebellar mass with the primary impression of cerebellar tumor , posterior fossa craniotomy and resection of the mass were performed . postoperative histopathologic evaluation showed that the mass was in fact a granulomatous tuberculoma . because no pulmonary involvement was found in either clinical manifestation or pulmonary ct scan , it was diagnosed as a primary extrapulmonary cerebellar tuberculoma mimicking a cerebellar tumor . although tuberculosis is considered primarily a pulmonary disease , it can affect any organ system . however , central nerves system ( cns ) involvement is so rare , it is associated with potentially devastating complications ; it affects both immunocompetent and immunologically incompetent populations . , only a few numbers of cases with cerebellar tuberculoma have been reported , and most of them are secondary to the pulmonary involvement12 and occurred in immunocompromised patients.13 14 primary cerebellar tuberculoma is rare in an immunologically incompetent patient like our case . to our knowledge , this is the first reported case of primary cerebellar tuberculoma mimicking a posterior fossa tumor in a patient with arnold - chiari malformation . depending upon the involved part of the cns , various spectrums of clinical manifestations intracranial tuberculoma may present as either a solitary or multiple lesions in the brain parenchyma . a ring - enhanced area on ct scan or mri is a characteristic appearance , but when there are no accompanied clinical manifestation or laboratory findings for tuberculosis , it would be so difficult to differentiate it from other cns tumors . despite being rare , cns involvement of tuberculosis always should be kept in mind in any patient with neurological complaints from regions with a high endemic rate of tuberculosis , either in those being immunocompetent or immunologically incompetent .

chiari malformations are a congenital heterogeneous group of disorders characterized by anatomic anomalies of the cerebellum , brain stem , and craniocervical junction associated with downward displacement of the cerebellum , alone or with lower medulla , into the cervical spine canal . the patient was a 23-year - old woman , a known case of arnold - chiari malformation with peripheral neuropathy and muscular atrophy , who presented with headache , drowsiness , decreased vision , and severe gait dysfunction lasting for several years . brain magnetic resonance imaging confirmed a hypointense signal mass in the left hemisphere of the cerebellum causing mass effects on the fourth ventricle , which shifted it , accompanied with dilation of third and lateral ventricles .

Case Report Discussion Conclusion

asthma is a chronic inflammatory airway disease characterized by marked eosinophilic inflammation , leading to progressive airway remodeling and impaired airflow . therefore , asthma treatment includes the use of anti - inflammatory drugs to reduce the progression of airway impairment at an early stage . the inhaled corticosteroid ( ics ) is a standard anti - inflammatory treatment for patients with asthma . moreover , a combination therapy with ics and long - acting agonists ( labas ) improves respiratory function and asthma symptoms and also reduces the frequency of usage of short - acting agonists in comparison with ics alone.1,2 the global initiative for asthma ( gina ) guidelines recommend that once asthma control has been achieved and maintained for at least 3 months , step - down therapy can be attempted under careful monitoring . for ics / laba combination therapies , the guidelines suggest to reduce the dose of ics while continuing laba because few studies suggest that low - dose ics with laba is more effective in reducing the frequency of exacerbations than fixed doses of ics without laba . on the other hand , the us food and drug administration recommends that laba be discontinued once asthma control has been achieved or not be used when asthma control is achieved with low - to - medium doses of ics for safety concerns.3,5 meta - analyses on safety of laba in asthma clinical trials suggest that laba monotherapy may increase the risk of death related to asthma.4 the present randomized prospective study compared the long - term outcomes of step - down therapy with ics / laba or ics alone for asthmatic patients who had achieved well - controlled asthma levels with ics ( fluticasone)/laba ( salmeterol ) combination therapy . between january 2011 and january 2013 , adult patients with mild - to - moderate persistent asthma were recruited at the showa university northern hospital , yokohama , kanagawa , japan . based on the guidelines , patients with intermittent , mild moderate , or moderate persistent asthma were included in the study . patients had achieved well - controlled asthma for at least 6 months using two puffs daily of salmeterol xinafoate ( 50 g)/fluticasone propionate ( fp ; 250 g ) combination ( sfc ) . exclusion criteria included the use of other laba drugs , symptoms of chronic obstructive pulmonary disease ( copd ) , and being a current smoker . we verified that the brinkman indexes of enrolled ex - smokers were < 100 for differentiating asthma and copd in ex - smokers . all of them underwent high - resolution computed tomography scanning , and attending doctors and radiologist confirmed that they had no attenuation areas . additionally , considering the reversibility of forced expiratory volume in 1 second ( fev1 ) following a bronchodilator administration of > 12% at the initial examination , asthma was diagnosed in ex - smokers . the study was approved by the showa university northern yokohama hospital ethics committee , and all patients gave written informed consent . , patients were randomized into two groups using the envelope method : patients who received step - down therapy with sfc 100 g ( two puffs daily ) comprised the sfc group and patients who received step - down therapy with fp 500 g daily comprised the fp group . the use of histamine receptor blocker and leukotriene receptor antagonists was allowed as long as the prescription was not changed during this study . the maintenance of > 80% compliance was verified during regular visits . over the 12-month follow - up period after initiation of the step - down therapy , patients who experienced exacerbation and required the use of short oral systemic corticosteroids or those who needed to modify the treatment due to poor asthma control were treated as dropouts and their data were excluded from analysis . patients were monitored every 2 months for changes in respiratory function , asthma control test ( act ) score , and the concentration of fractional exhaled nitric oxide ( feno ) before the initiation of the step - down therapeutic protocol ( ie , at baseline ) and over the following 12 months . monitored respiratory functions included percent forced expiratory volume in 1 second ( % fev1 ) , maximal expiratory flow rate at 50% of the vital capacity ( % fef50 ) , and maximal expiratory flow rate at 25% of the vital capacity ( % fef25 ) . in addition , the fev1 absolute decline rate was compared between the groups at 12 months . the act test was used to verify that the patient maintained well - controlled asthma levels , characterized by an act score 20 , according to the gina guidelines.6 feno was measured by a portable device , nobreath ( bedfont scientific ltd , maidstone , uk).7 data were collected before the initiation of the step - down therapeutic protocol ( ie , at baseline [ 0 weeks ] ) , as well as after 2 months , 4 months , 6 months , 8 months , 10 months , and 12 months . next , comparisons between the sfc group and the fp group at specific time points were performed using the mann , patients were randomly assigned ; however , there was a significant difference in baseline % fef25 values between the sfc and fp groups . therefore , we evaluated respiratory functions using relative changes over time and not absolute values . likewise between january 2011 and january 2013 , adult patients with mild - to - moderate persistent asthma were recruited at the showa university northern hospital , yokohama , kanagawa , japan . based on the guidelines , patients with intermittent , mild moderate , or moderate persistent asthma were included in the study . patients had achieved well - controlled asthma for at least 6 months using two puffs daily of salmeterol xinafoate ( 50 g)/fluticasone propionate ( fp ; 250 g ) combination ( sfc ) . exclusion criteria included the use of other laba drugs , symptoms of chronic obstructive pulmonary disease ( copd ) , and being a current smoker . we verified that the brinkman indexes of enrolled ex - smokers were < 100 for differentiating asthma and copd in ex - smokers . all of them underwent high - resolution computed tomography scanning , and attending doctors and radiologist confirmed that they had no attenuation areas . additionally , considering the reversibility of forced expiratory volume in 1 second ( fev1 ) following a bronchodilator administration of > 12% at the initial examination , asthma was diagnosed in ex - smokers . the study was approved by the showa university northern yokohama hospital ethics committee , and all patients gave written informed consent . during this prospective randomized study , patients were randomized into two groups using the envelope method : patients who received step - down therapy with sfc 100 g ( two puffs daily ) comprised the sfc group and patients who received step - down therapy with fp 500 g daily comprised the fp group . the use of histamine receptor blocker and leukotriene receptor antagonists was allowed as long as the prescription was not changed during this study . the maintenance of > 80% compliance was verified during regular visits . over the 12-month follow - up period after initiation of the step - down therapy , patients who experienced exacerbation and required the use of short oral systemic corticosteroids or those who needed to modify the treatment due to poor asthma control were treated as dropouts and their data were excluded from analysis . patients were monitored every 2 months for changes in respiratory function , asthma control test ( act ) score , and the concentration of fractional exhaled nitric oxide ( feno ) before the initiation of the step - down therapeutic protocol ( ie , at baseline ) and over the following 12 months . monitored respiratory functions included percent forced expiratory volume in 1 second ( % fev1 ) , maximal expiratory flow rate at 50% of the vital capacity ( % fef50 ) , and maximal expiratory flow rate at 25% of the vital capacity ( % fef25 ) . in addition , the fev1 absolute decline rate was compared between the groups at 12 months . the act test was used to verify that the patient maintained well - controlled asthma levels , characterized by an act score 20 , according to the gina guidelines.6 feno was measured by a portable device , nobreath ( bedfont scientific ltd , maidstone , uk).7 data were collected before the initiation of the step - down therapeutic protocol ( ie , at baseline [ 0 weeks ] ) , as well as after 2 months , 4 months , 6 months , 8 months , 10 months , and 12 months . next , comparisons between the sfc group and the fp group at specific time points were performed using the mann , patients were randomly assigned ; however , there was a significant difference in baseline % fef25 values between the sfc and fp groups . therefore , we evaluated respiratory functions using relative changes over time and not absolute values . forty asthmatic patients met the inclusion / exclusion criteria and were randomly assigned to the sfc group ( n=20 ) or the fp group ( n=20 ) . the two groups exhibited similar baseline characteristics , except for the elevated % fef25 of the sfc group ( p<0.001 ; table 1 ) . one - third of the patients in each group were ex - smokers with no signs or diagnosis of copd according to exclusion criteria . the average act scores were consistent with well - controlled asthma , and lung functions were compatible with mild - to - moderate disease severity . in the sfc group , there were three drop - out patients in the fp group . in all cases , the mean period until dropout was 8 months in the sfc group and 8.3 months in the fp group . there was no significant difference in the dropout rate of the sfc and fp groups ( log - rank sum test ; figure 1 ) . accordingly , the following analyses were performed on a total of 35 patients for the sfc group ( n=17 ) and the fp group ( n=18 ) . the two step - down therapeutic protocols did not affect act scores , which remained relatively stable over the 12-month follow - up period ( figure 2 ) . likewise , there was no significant change in feno levels before and after the initiation of the step - down therapeutic protocols between the two groups ( figure 3 ) . collectively , these data suggest that bronchial inflammation was equally controlled by low - dose ics in the sfc group and high - dose ics in the fp group . regarding lung function , % fev1 was not affected by the sfc step - down therapeutic protocol ( figure 4 ) . in contrast , % fev1 significantly decreased within the first 2 months after the initiation of the fp step - down therapeutic protocol and then remained stable during the remaining follow - up period . accordingly , there was a significant difference in % fev1 between the fp and sfc groups over the entire 2- to 12-month period ( p<0.02 ) . the fev1 absolute decline rate of the sfc group was 18.0 ml / year , whereas that of the fp group was 144 ml / year ( figure 5 ) . the fev1 decline rate of the sfc group was significantly slower than that of the fp group as is the case with the relative changes in % fev1 ( p<0.05 ) . the sfc step - down therapeutic protocol did not significantly affect % fef50 over the entire 12-month follow - up period ( figure 6 ) . in contrast , the fp step - down therapeutic protocol induced a progressive decrease in % fef50 values , which became significantly different from those of the sfc group during the 812-month period ( p<0.03 ) . on the other hand , the two step - down therapeutic protocols did not affect % fef25 values , except for significantly lower values in the fp group than those in the sfc group at the 12-month visit ( p<0.05 ; figure 7 ) . these data suggest that both % fef50 and % fef25 respond to laba discontinuation by a gradual decrease over time ; however , % fef50 represents a more sensitive marker of treatment response . this study is based on the current gina guidelines that recommend step - down therapy once asthma control has been achieved and maintained for 36 months . meta - analyses suggest that reducing the ics dose while maintaining laba is more likely to maintain asthma under control and to reduce the rate of severe exacerbation than discontinuing laba while maintaining the ics dose for several months.811 however , a few studies have reported the adverse effects of long - term laba inhalation , which may worsen airway inflammation and increase the risk of death related to exacerbation,4,12 which is in agreement with us food and drug administration s warnings . these studies suggest that the most appropriate step - down therapeutic protocol would be to reduce both ics and laba in tandem , instead of reducing or eliminating only one of them . accordingly , the present study compared the long - term outcomes of a balanced step - down therapeutic protocol ( from sfc 250 g to sfc 100 g , two puffs each day ) and an ics - only protocol ( fp 500 g daily ) . the patients were monitored for changes in act scores , feno levels , and respiratory function over a 12-month period . the first parameters remained stable during the 12-month follow - up , regardless of the step - down period . in contrast , the % fev1 of the fp group decreased significantly immediately after discontinuing laba . this result suggests that laba is effective in improving lung functions in mild - to - moderate persistent asthmatic patients , despite their conditions being well controlled for at least 6 months as reported in other studies.1,2 on the other hand , feno levels did not increase in the sfc group despite ics dose reduction . in addition , there were no significant differences between the two groups in act and dropout rate over the entire year . it appears that laba is also effective in terms of improving respiratory functions in patients with mildly or moderately persistent asthma , and low - dose ics may be enough for such patients to relieve their inflammations even if they continue laba for a long period . on the other hand , the formoterol and corticosteroids establishing therapy study reported that in patients with severe asthma ( not mild or moderate asthma as in this study ) , the exacerbation rate is increased significantly in those who were treated with low - dose ics and laba combination therapy compared with those who were treated with high - dose ics alone.2 it appeared that low - dose ics was not enough for severe asthma , and it became the cause of potential revival of bronchial inflammation . this suggests that it is important to consider how to step down the treatment while evaluating the inflammatory conditions of asthma . the impact of the step - down therapeutic protocol on the status of the peripheral bronchi was assessed by the measurements of forced expiratory flow rates . the standard 250 mg sfc treatment is known to improve % fef25 and % fef50 in asthmatic patients . the present study shows that stepping down from 250 mg to 100 mg sfc did not significantly affect % fef25 or % fef50 values of the asthmatic patients , suggesting that their asthma remained under control for at least 12 months . in contrast , the absence of laba in the fp step - down therapeutic protocol caused a gradual decrease in % fef50 , reaching 90% of baseline values after 12 months . these data suggest that the continuous use of low - dose laba with low - dose ics may prevent the progression of peripheral airway impairment . it has been reported that airway remodeling progresses only for airway contraction even if respiratory tract inflammation is controlled.1315 in the sfc group , airway inflammation was relieved to the same extent as in the fp group , and the continuous use of laba may prevent airway contraction . as a result , in conclusion , this study demonstrates that in patients with mild - to - moderate asthma , well controlled with 250 mg sfc , a balanced step - down therapeutic protocol with 100 mg sfc efficiently maintains the feno level , act score , and % fev1 , % fef25 , and % fef50 values stable for at least 12 months . in contrast , switching to high - dose ics alone led to a rapid increase in airflow impairment in the upper respiratory tract and a gradual deterioration of peripheral airflow over 12 months . these data suggest that step - down therapeutic protocols should include low - dose laba to prevent the progression of airflow impairment and improve the long - term prognosis of asthmatic patients under step - down therapy .

purposea combination therapy with inhaled corticosteroid ( ics ) and a long - acting agonist ( laba ) is the standard treatment for asthmatic patients , and step - down treatment is recommended once control has been achieved . however , little data exist that evaluate the long - term outcomes after step - down treatment.objectiveto compare the long - term outcomes of step - down therapy with ics / laba or ics alone for asthmatic patients who have achieved well - controlled asthma by the ics ( 250 g fluticasone)/laba ( 50 g salmeterol ) combination ( sfc , two puffs per day).patients and methodswe randomized 40 well - controlled patients with asthma receiving sfc ( 250 g ) to two groups ; one group of patients received step - down therapy with low - dose sfc ( 100 g , two puffs daily ) and another group of patients received step - down therapy with high - dose fluticasone propionate ( fp ) alone ( 500 g , daily ) . the two groups were monitored over 12 months for changes in asthma control test scores , respiratory function ( percent forced expiratory volume in 1 second , maximal expiratory flow rate at 50% of the vital capacity [ % fef50 ] , and maximal expiratory flow rate at 25% of the vital capacity [ % fef25 ] ) , and the concentration of fractional exhaled nitric oxide.resultsthere was no significant difference in the dropout rate between the sfc and fp groups . low - dose sfc maintained the stability of all parameters over 12 months , whereas the fp group exhibited a rapid 5% decrease in forced expiratory volume in 1 second within 2 months after discontinuation of salmeterol ; furthermore , after 10 months , there was a gradual decrease in % fef50 and % fef25.conclusionthis study suggests that a balanced step - down protocol , including both ics and laba , is essential in providing long - term stability to patients with mild - to - moderate well - controlled asthma .

Introduction Material and methods Study subjects Study design Outcome parameters Data analysis Results Discussion Conclusion

a 51-year - old man ( height 183 cm , weight 96 kg ) was admitted for radical nephrectomy with the diagnosis of renal cell carcinoma . he had undergone surgery for varicose veins under general anesthesia 3 months prior ; his tobacco use was 1 pack / day for 30 years and alcohol consumption was soju 1 bottle / day , 3 - 4 days / week . a preoperative chest x - ray revealed multiple hematogenous metastatic nodules and emphysema on both lung fields . he had no history of dyspnea and no wheezing sounds on his lung exam , however . preoperative laboratory results were the following : hemoglobin was 13.5 g / dl , hematocrit was 42.3% , ast / alt were 10/20 iu , and bun / creatinine were 12.8/0.8 mg / dl . his preoperative blood pressure was 120/80 mmhg and heart rate was 72 beats / minute . after arriving at the operating theater , ecg , noninvasive blood pressure measurement , pulse oximeter , and capnography were applied . initial vital signs in the operation room were blood pressure 110/73 mmhg , heart rate 65 beats / min , respiratory rate 18 breaths / min . for the induction of anesthesia , propofol 120 mg and rocuronium bromide 50 mg were injected intravenously , and 6 l / min of o2 and desflurane were inhaled . anesthesia was maintained with o2 and n2o ( fio2 50% ) , desflurane 5.0 - 7.0% , and an intravenous target controlled infusion of remifentanil with a target of 2.0 - 3.0 ng / ml . after arterial catheterization , the patient was placed in a lateral decubitus position with a kidney rest on the lower part of the iliac crest . the operation table was flexed by 30 degrees , and the patient 's buttocks were fastened with a hip strap , with careful attention paid to prevent the femur head from encountering pressure . after a trocar was inserted via a retroperitoneal approach , pneumoperitoneum was produced until the abdominal insufflation pressure was 11 mmhg , and 3 port laparoscopic radical nephrectomy was performed . there was no change in vital signs at initiation of the surgery , and vital signs during surgery were maintained , with blood pressure 90 - 100/55 - 65 mmhg , heart rate 60 - 75 beats / min , respiratory rate 12 - 15 breaths / min , peak inspiration pressure 18 cmh2o , end tidal co2 38 mmhg , and sao2 97% . two branches of the right renal artery and one branch of the right renal vein were ligated . four hours after initiation of surgery , the operating procedure was changed to open nephrectomy , because one branch of the right renal artery entered into the back of the kidney . after ligation of the right renal artery , the right kidney and the oncologic mass were resected , and the peritoneal wall was closed . during the operation , an arterial blood gas analysis was performed and hemoglobin , hematocrit , and electrolytes were checked . at one hour and 20 minutes after laparotomy , labs showed a hemoglobin level of 7.8 g / ml and hematocrit of 23.0% , so 420 ml of packed red blood cells were transfused . the mean operative time was 7 hours , and blood loss and urine output were 800 ml and 400 ml , respectively . the total amounts of fluids administered were 2,100 ml of crystalloid solution and 500 ml of colloid solution . after reversing muscle relaxation and making sure the patient had an adequate amount of spontaneous breathing , the endotracheal tube was removed . the patient was transported to the postanesthetic care unit . after awakening , he complained of back pain on the left side . examination showed slight redness on his flank that extended to the buttocks , but localized swelling and warmth were not observed . due to the postoperative low back pain , patient control analgesia ( pca ) was administered in doses of 1 ml . an arterial blood gas analysis was performed while the patient was spontaneously breathing with mask oxygenation at 8 l / min . the results of the blood gas analysis showed a ph of 7.40 , paco2 of 43.1 mmhg , pao2 of 98 mmhg , and hco3 of 22.6 his vital signs were stable , and the patient was transferred to the icu . on postoperative day 1 , laboratory tests revealed ast / alt of 497/175 iu , bun / creatinine of 27/3.5 mg / dl , creatinine phospokinase ( cpk ) of 21,817 iu / l , and lactate dehydrogenase ( ldh ) of 1,973 iu / l . the patient 's urine output decreased to 83 ml during 24 hours and did not respond to diuretics . on postoperative day 2 , the patient had persistent pain in the area of his left flank , and laboratory tests revealed ast / alt of 432/176 iu , bun / creatinine of 40.9/5.1 mg / dl , and his urine output was still low at 254 ml over 24 hours . renal ultrasonography results did not show any left kidney abnormality , such as pelvicouretral dilatation indicating hydronephrosis or obstruction . on postoperative day 3 , bun / creatinine increased to 51/6.8 mg / dl , and urine output was less than 10 ml . at that point , on postoperative day 4 , cpk was 7,091 iu / l , ldh was 2,161 iu / l , and serum myoglobin was more than 3,000 ng / ml . aldose was 43.2 u / l , and the patient had persistent pain of the left flank . on postoperative day 8 , bun / creatinine level increased to 50/10.8 mg / dl and then gradually decreased . on postoperative day 20 , bun / creatinine level decreased to 23/2.0 mg / dl and continued to drop , and urine output returned to normal . cpk and ldh levels also dropped to 127 iu / l and 572 iu / l on postoperative day 15 . he was discharged 41 days after surgery and has had no problems since that time . rhabdomyolysis can be defined as a reversible or irreversible injury due to degeneration of the cell membrane or an injury to the sarcolemma of skeletal muscle , resulting in leakage of its components into the blood or extracellular environment [ 1 - 4 ] . injury to the sarcolemma may be caused by hypoxia , reperfusion injury , or direct injury to the cell membrane , causing leakage of intracellular proteins into the extracellular environment . leaked substances include cpk , ldh , myoglobin , aldose , phosphate , and potassium , resulting in increased plasma concentrations of these substances . criteria for the diagnosis of rhabdomyolysis includes the absence of myocardial ischemia , five times more than normal cpk levels ( greater than 1,000 ) , or presence of myoglobinuria . rhabdomyolysis occurs more frequently in males , as in this case , and is prevalent in patients in their late 40s . postoperative rhabdomyolysis is known to be associated with ruptured abdominal aortic aneurysm surgery , revascularization of vascular occlusion , and urology surgery . risk factors include male gender , high body mass index , prolonged surgery , lateral decubitus position , and flexed lateral decubitus position . additional risk factors include kidney disease , extracellular dehydration , and hypoperfusion of the kidney . the most common sites include the muscles around the lumbar spine and gluteus , as in this case [ 7 - 9 ] . symptoms of rhabdomyolysis include pain , tenderness , edema , weakness of the affected muscles , and muscle pain related to passive stretch . myoglobinuria occurs when myoglobin levels in plasma exceed 0.5 - 1.5 mg / dl and is characterized by dark brown urine . in some cases of rhabdomyolysis , however , myoglobinuria intermittently occurs and thus myoglobin can not be detected in the urine . urine may not test positive for myoglobin because these proteins are metabolized to bilirubin , excreted quickly and appear only temporarily in the urine . in this case , we could not grossly detect myoglobinuria , mostly likely because we could not distinguish the characteristic dark brown color of myoglobinuria from postoperative hematuria . obtaining the patient 's serum myoglobin level could not have been useful , because serum myoglobin can be increased and normalized in a short period of time . the cpk level is known to be the most sensitive test for rhabdomyolysis , because it is increased in proportion to the amount of muscle damage . in this case , we measured cpk , ldh , ast , and alt on a daily basis and monitored the patient 's progress . complications of rhabdomyolysis include persistent muscle weakness , atrophy , and nerve dysfunction , with acute renal failure as the most serious complication . acute renal failure occurs in 15 - 46% of cases of rhabdomyolysis and death occurs in 3.4% of cases . the mechanism of acute renal failure in patients with rhabdomyolysis is not clearly known , but it is thought to be a complex mechanism that includes myoglobin - induced vasoconstriction , cytotoxicity of the heme molecule , and blockage of renal tubules with precipitated myoglobin that results in reverse pressure . dehydration and acidosis as a result of fluid loss are also a part of this phenomenon . the flexed lateral decubitus position , called the kidney position or nephrectomy position , is a position commonly used in urological surgery . studied the association between rhabdomyolysis and operative position and concluded that rhabdomyolysis is associated with the lateral position and long lasting surgery . glassman et al . suggested the possibility that placing a patient in a rotated supine position instead of the flexed lateral position would reduce the occurrence of rhabdomyolysis . it has also been suggested that intraperitoneal co2 infusion for laparoscopic surgery resulting in abdominal pressure of 12 - 15 mmhg can further add to low perfusion pressure among the obese , so it is possible that in our case laparoscopic surgery served as one of the factors causing low perfusion . permissive hypertension would provide improved perfusion of compressed tissue and prevent the development of rhabdomyolysis . in general , surgeons want to establish a blood pressure that is low enough to prevent blood from disturbing the operative field and avoid fluid overload , which could cause postoperative pulmonary edema . laparoscopic surgery and low blood pressure might be precipitating factors for rhabdomyolysis , however , because they perturb perfusion to muscles under pressure . in the anesthesiologist 's view , the disease to distinguish from postoperative rhabdomyolysis is malignant hyperthermia . in this case , increased co2 production , which is the earliest and most sensitive indicator of malignant hyperthermia , was not observed and the body temperature was normal . also , there was no rigidity of the masseter muscle and no tachycardia , arrhythmia , and tachypnea , signs that are known to occur within 5 minutes of induction of anesthesia in malignant hyperthermia . finally , the patient 's cpk level was higher than those of other hyperthermia cases reported in the country . treatment of rhabdomyolysis includes aggressive fluid resuscitation with central venous pressure monitoring , use of mannitol , and infusion of sodium bicarbonate to alkalinize the urine and stop the progression of renal failure . when renal failure occurs and it is indicated , hemodialysis is important to recover renal function . the criteria for hemodialysis in patients with rhabdomyolysis includes serum creatinine more than 1.5 mg / dl , base deficit less than -4 , serum cpk more than 5,000 iu / l , and the presence of myoglobinuria . sharp et al . reported that renal failure requiring dialysis occurs in 9.5% of patients with postoperative rhabdomyolysis and that mortality is 3 times higher in patients with rhabdomyolysis who need hemodialysis than in those who do not . also , it is known that muscle swelling can delay muscle ischemia and cause a second surge of cpk , so vigilance must not be relaxed after the initial treatment . if patients have surgery again at a later date , anesthesiologists should proceed with care , because rhabdomyolysis reoccurs in 11% of patients . in conclusion , it is important to note that anesthesiologists should pay attention to positioning during surgery , because prevention methods are critical to reduce the risk of rhabdomyolysis . that is , appropriate pads on body parts under pressure should be installed during surgery and lowering the kidney rest could be effective even before the end of the operation . more study is needed on the efficacy of devices that measure abdominal pressure ; studies are also needed to test how well commercial products like vacuum bean bags or gel cushioning pads , which are designed to distribute the pressure of large areas , can prevent excessive local pressure . in rhabdomyolysis , stable hemodynamic status should be maintained , because hypotension and acidosis may act as contributing factors during anesthesia . if possible , intentional hypertension should be tried in patients who are at risk for rhabdomyolysis . reduction of operating time by surgeons and interruption of surgery at regular intervals to normalize flexed areas might help prevent rhabdomyolysis . in patients with the possibility of postoperative rhabdomyolysis , it is important to observe the patient 's progress and have an early suspicion of rhabdomyolysis , especially when the patient 's pain is out of proportion to the clinical situation and skin symptoms and an abnormal urine color are found .

rhabdomyolysis is a rare but potentially lethal clinical syndrome that results from acute muscle fiber necrosis with leakage of muscle constituents into blood . this devastating disease could be due to muscle compression caused by urologic positioning for a lengthy nephrectomy . in this regard , laparoscopic renal surgery may be a risk for the development of rhabdomyolysis . this phenomenon of massive muscle necrosis can produce secondary acute renal failure . the risk factors have to be managed carefully during anesthetic management . here , we report a case of a patient with rhabdomyolysis that developed in the flexed lateral decubitus position during laparoscopic nephrectomy .

Case Report Discussion

in 1990 , the direct gene transfer of plasmid dna into mouse muscle in vivo without the need for a special delivery system was demonstrated . furthermore , intramuscular inoculation with plasmid dna encoding reporter genes induced protein expression within the muscle cells . this study provided evidence for the idea that naked dna could be delivered in vivo to direct protein expression . subsequently , a further study reported the gene expression a year or more after intramuscular injection of plasmid dna . since these initial studies , many more experiments have been carried out to evaluate different factors that determine the efficiency of gene transfer and immunogenicity of plasmid dna . furthermore , plasmid dna has been used to immunise against a variety of diseases ( known as dna vaccination ) . alternatively , plasmid dna has been used to treat genetic diseases and similar factors may affect the efficacy of this gene therapy . dna vaccines usually consist of plasmid vectors ( derived from bacteria ) that contain heterologous genes ( transgenes ) inserted under the control of a eukaryotic promoter , allowing protein expression in mammalian cells . an important consideration when optimising the efficacy of dna vaccines is the appropriate choice of plasmid vector . the basic requirements for the backbone of a plasmid dna vector are a eukaryotic promoter , a cloning site , a polyadenylation sequence , a selectable marker and a bacterial origin of replication . a strong promoter may be required for optimal expression in mammalian cells . for this , some promoters derived from viruses such as cytomegalovirus ( cmv ) or simian virus 40 ( sv40 ) have been used . a cloning site downstream of the promoter should be provided for insertion of heterologous genes , and inclusion of a polyadenylation ( polya ) sequence such as the bovine growth hormone ( bgh ) or sv40 polyadenylation sequence provides stabilisation of mrna transcripts . the most commonly used selectable markers are bacterial antibiotic resistance genes , such as the ampicillin resistance gene . however , since the ampicillin resistance gene is precluded for use in humans , a kanamycin resistance gene is often used . finally , the escherichia coli cole1 origin of replication , which is found in plasmids such as those in the puc series , is most often used in dna vaccines because it provides high plasmid copy numbers in bacteria enabling high yields of plasmid dna on purification . various reports have described the strength of promoter / enhancers or other transcriptional elements in dna vaccines ( see table 1 ) [ 5 - 20 ] . in general , virally - derived promoters have provided greater gene expression in vivo than other eukaryotic promoters . in particular , the cmv immediate early enhancer - promoter ( known as the cmv promoter ) has often been shown to direct the highest level of transgene expression in eukaryotic tissues when compared with other promoters . for example , in one study a plasmid expressing human immunodeficiency virus type 1 ( hiv-1 ) gag / env under the regulation of the cmv promoter / enhancer was compared to a comparable plasmid utilising the endogenous akv murine leukemia long terminal repeat . analysis of the immune responses in macaques injected with the plasmids showed that the cmv - containing plasmid elicited higher gag- and env - specific humoral and t - cell proliferative responses , reflecting the greater transcriptional activity of the cmv promoter . furthermore , it has been demonstrated that inclusion of the cmv intron a improved the level of expression of transgenes expressed by the cmv promoter or other promoter / enhancers . it is thought that the beneficial effect of introns on expression is primarily due to an enhanced rate of polyadenylation and/or nuclear transport associated with rna splicing . however , some widely used virally - derived promoters , such as the cmv promoter , may not be suitable for some gene therapy applications since treatment with interferon- or tumour necrosis factor- may inhibit transgene expression from dna vaccines containing these promoters . for example , the desmin promoter / enhancer , which controls expression of the muscle - specific cytoskeletal protein desmin , was used effectively to drive expression of the hepatitis b surface antigen priming both humoral and cellular immunity against the antigen . these responses were shown to be of a comparable magnitude to those in mice immunised with comparable dna vaccines containing the cmv promoter . other tissue - specific promoters that have been studied include the creatine kinase promoter , also specific to muscle cells , and the metallothionein and 1,24-vitamind(3)(oh)(2 ) dehydroxylase promoters , both of which are specific to keratinocytes . comparison of promoters used in dna expression studies in vitro and in vivo since the rate of transcriptional initiation is generally increased by the use of strong promoter / enhancers , the rate of transcriptional termination may become rate - limiting . in addition , the efficiency of primary rna transcript processing and polyadenylation is known to vary between the polyadenylation sequences of different genes . thus , the polyadenylation sequence used within a dna vaccine may also have significant effects on transgene expression . for example , it was demonstrated that the commonly used sv40 polyadenylation sequence was less efficient than the minimal rabbit -globin and bovine growth hormone polyadenylation sequences in mouse liver , although addition of a second sv40 enhancer downstream of the sv40 polyadenylation signal did increase expression to a level comparable to the other signals . therefore , it is possible that the strategy of inserting a second sv40 enhancer downstream of a sv40 polyadenylation sequence may be utilised in the construction of more efficient vectors . sequences flanking the aug initiator codon within mrna influence its recognition by eukaryotic ribosomes . as a result of studying the conditions required for optimal translational efficiency of expressed mammalian genes , it has been proposed that this defined translational inititiating sequence ( gcca / gccaugg ) should be included in vertebrate mrnas located around the initiator codon . it has also been suggested that efficient translation is obtained when the -3 position contains a purine base or , in the absence of a purine base , a guanine is positioned at + 4 . therefore , the expression level of these genes might be increased by the insertion of a kozak sequence . codon bias is observed in all species , and the use of selective codons in genes often correlates with gene expression efficiency . in general , taxonomically - close organisms , such as e. coli and salmonella enterica serovar typhimurium , for example , use similar codons for their protein synthesis whereas taxonomically - distant organisms , such as e. coli and saccharomyces cerevisiae , utilise very different codons . nagata et al . studied the effect of codon optimisation for mammalian cells of cytotoxic t - lymphocyte ( ctl ) epitopes derived from the intracellular bacterium , listeria monocytogenes , and the parasite plasmodium yoelii , and reported that the codon optimisation level of the genes correlated well with translational efficiency in mammalian cells . the greatest deviation from random codon usage in an organism occurs in the most highly expressed genes as a result of selection for codons that maximise translational efficiency . thus , differences between codon usage in a heterologous gene and the host organism may affect expression . to improve expression of human immunodeficiency virus type 1 gp120 from a dna vaccine vector , andr et al . generated a synthetic gp120 sequence in which most of the wild - type codons were replaced with codons from the resulting construct showed increased in vitro expression of gp120 compared to the wild - type sequence . in addition , significantly increased antibody titres and ctl reactivity were observed following administration of the vector containing the synthetic sequence . similarly , a dna vaccine vector encoding a synthetic epitope of listeriolysin o with mammalian codon usage showed higher translation efficiency than a vector containing the wild - type sequence in murine cells . furthermore , the first dna vaccine was capable of inducing specific cd8 t cells able to confer partial protection against challenge with l. monocytogenes where the second dna vaccine could not . a number of other studies have reported that increased immune responses may be obtained by dna vaccination with a transgene sequence with optimised codon usage . in this review the methodologies by which antigen expression has been optimised to date , i.e. optimisation of vector and transgene sequences , have been discussed . it is clear that transgene expression may be increased through the use of optimised promoters and polya sequences . however , in some circumstances it may be necessary to optimise dna vaccines to produce reduced transgene expression . for example , the weaker sv40 promoter has been used rather than the cmv promoter to drive expression of antigens that induce cell death upon overexpression . such tissue - specific expression systems may be able to produce stable expression by reducing the probability of inducing an immune response to the transgene . it may be possible to design vectors for gene therapeutic purposes that avoid inducing unwanted immune responses against the encoded antigen by using tissue - specific promoters . restricting the site of expression of genes should minimise the risks related to aberrant expression of a gene product . furthermore , it should be possible to develop expression systems where gene products are only expressed in the critical cell types for dna vaccination or gene therapy , for example , dendritic cells ( dcs ) . as a better understanding of the proteins whose expression is limited to dcs is obtained , novel expression systems will be generated . finally , through increased knowledge of the regulation of expression of antigens , it is now possible to produce multivalent systems whereby multiple antigens may be expressed from a single dna vaccine vector . it is clear that the optimisation of antigen expression is an important consideration in dna vaccine vector design . however , it is important to recognise that other aspects of vector design may influence the efficacy of the vaccine / gene therapy . a rational approach to improve the efficacy of dna vaccination or gene therapy would optimise the : ( i ) vector backbone dna sequence ; ( ii ) transgene sequence ; ( iii ) co - expression of stimulatory sequences ; ( iv ) delivery system used for the vector ; ( v ) targeting of the vector for appropriate immune stimulation . the backbone of a dna vaccine vector could be further modified to enhance immunogenicity via the manipulation of the dna to include certain sequences , so that the dna itself will have an adjuvantising effect . dna vaccine vectors contain many cpg motifs ( consisting of unmethylated cpg dinucleotides flanked by two 5 ' purines and two 3 ' pyrimidines ) that , overall , induce a th1-like pattern of cytokine production , and are thought to account for strong ctl responses frequently seen following dna vaccination . it is possible to augment responses to dna vaccine vectors by incorporating cpg motifs into the dna backbone of the plasmid . alternatively , immune responses may be modulated or enhanced by the co - expression of stimulatory molecules or cytokines or through the use of localisation or secretory signals [ 47 - 49 ] , or ligand fusions [ 50 - 54 ] to direct antigens to sites appropriate for immune modulation . finally , a variety of routes of administration of dna vaccines have been studied , including intramuscular , intradermal , subcutaneous , intravenous , intraperitoneal , oral , vaginal , intranasal and , more recently , non - invasive delivery to the skin ( reviewed by gurunathan et al . ) . the approaches outlined above will together allow for the rational and optimised design for dna vaccines and gene therapy vectors . the ability to improve antigen expression through the use of optimisation of regulatory elements , kozak sequences and codon usage is highlighted in this review , as part of this rational approach .

dna vaccination is a relatively recent development in vaccine methodology . it is now possible to undertake a rational step - by - step approach to dna vaccine design . strategies may include the incorporation of immunostimulatory sequences in the backbone of the plasmid , co - expression of stimulatory molecules , utilisation of localisation / secretory signals , and utilisation of the appropriate delivery system , for example . however , another important consideration is the utilisation of methods designed to optimise transgene expression . in this review we discuss the importance of regulatory elements , kozak sequences and codon optimisation in transgene expression .

Review Regulatory elements Kozak sequences Codon usage Conclusions Competing interests Authors' contributions

osteoarthritis ( oa ) is a chronic musculoskeletal disease characterized by loss of articular cartilage and changes in the subchondral bone . several risk factors ( e.g. , age , female gender , hypothyroidism , race , genetic susceptibility , and obesity ) are associated with oa1 . thyroid hormones might be of key importance in the maintenance of articular cartilage and play a role in the pathogenesis of oa3 . radiographic oa is characterized by osteophytes , sclerosis , and joint space narrowing , which is a result of cartilage erosion and subchondral sclerosis4 . ultrasonography is also used to evaluate the condition of the articular cartilage , and pre - oa evaluation of the knee by ultrasound may have prognostic value5 . the aim of the present study was to investigate the effects of hypothyroidism on femoral cartilage thickness by using ultrasound , which is a valid and reliable method in this regard and has previously been found to be useful in the early diagnosis of knee oa6 , 7 . forty patients diagnosed with hypothyroidism and 30 age- , gender- , body mass index ( bmi)- , smoking status- , and physical activity - matched healthy subjects were enrolled . subjects with a history of knee trauma or previous knee surgery ; additional ( other than hypothyroidism ) systemic and/or chronic diseases including diabetes mellitus and rheumatoid arthritis ; and any abnormal laboratory results regarding renal , hepatic , thyroid , or parathyroid function were excluded . all subjects were informed about the study procedure , and they provided consent to participate.this study was approved by the local ethics committee of recep tayyip erdogan university medical school . the thickness of the femoral articular cartilage was measured using a 7- to 12-mhz linear probe ( eizo nanao corporation , esaote , italia ) while the subjects comfortably sat on the examination table with their knees in maximum flexion , and the probe was placed in the axial plane on the outer edge6 , 8 . three mid - point measurements were taken from each knee , from the right lateral condyle ( rlc ) , right intercondylar area ( ria ) , right medial condyle ( rmc ) , left medial condyle ( lmc ) , left intercondylar area ( lia ) , and left lateral condyle ( llc ) ( fig . ( suprapatellaraxial view ) demonstrating femoral cartilage measurementsrlc : right lateral condyle ; ria : right intercondylar area ; rmc : right medialcondyle ; llc : left lateral condyle ; lia : left intercondylar area ; lmc : left medial condyle ) . the cartilage thickness was interpreted as the distance between the thin hyperechoic line at the synovial space / cartilage interface and the sharp hyperechoic line at the cartilage - bone interface7 . ultrasonogram ( suprapatellaraxial view ) demonstrating femoral cartilage measurements rlc : right lateral condyle ; ria : right intercondylar area ; rmc : right medialcondyle ; llc : left lateral condyle ; lia : left intercondylar area ; lmc : left medial condyle all statistical analyses were performed using spss version 16.0 . the normal distribution of continuous variables was tested using the kolmogorov - smirnov test . student s t - test was used to compare normally distributed data , and the mann - whitney u test was used to compare non - normally distributed data . eighty knees of the 40 patients with hypothyroidism ( 29 females and 11 males ) and 60 knees of the 30 healthy subjects ( 22 females and 8 males ) were examined in this study . the demographic characteristics of the participants are shown in table 1table 1.characteristics of study population ( mean standard deviation ) patients ( n=40)control ( n=30)age ( years)25.50 3.3424.46 3.15gender , n ( % ) female29 ( 72.5)22 ( 73.3)male11 ( 27.5)8 ( 26.7)bmi ( kg / m)26.38 1.3825.45 3.06disease duration ( months)30.3 15.4physical activity , n ( % ) physical activity34 ( 85.0)25 ( 83.3)no physical activity6 ( 15.0)5 ( 16.7)smoking status , n ( % ) smokers7 ( 17.5)6 ( 20.0)non - smokers33 ( 82.5)24 ( 80.0)*significant at p<0.05 . the mean age of the patients with hypothyroidism was 25.50 3.34 years , while that of the healthy subjects was 24.46 3.15 years ( p>0.05 ) . the mean bmi of the former was 26.38 1.38 kg / m , while that of the latter was 25.45 3.06 kg / m ( p>0.05 ) . the mean disease duration in the patients with hypothyroidism was 30.32 15.41 months . gender , smoking status , and physical activity were similar between the groups ( p>0.05 for all ) . * significant at p<0.05 table 2table 2.comparison of femoral cartilage thickness ( mm ) between patients with hypothyroidism and healthy controls ( mean standard deviation)patients ( n=40 ) control ( n=30)rlc ( mm)1.83 0.141.88 0.08ria ( mm)1.89 0.111.92 0.09rmc ( mm)1.79 0.111.83 0.08llc ( mm)1.86 0.121.91 0.08lia ( mm)1.88 0.141.93 0.10lmc ( mm)1.77 0.12 * 1.83 0.08rlc : right lateral condyle ; ria : right intercondylar area ; rmc : right medial condyle ; llc : left lateral condyle ; lia : left intercondylar area ; lmc : left medial condyle . patients with hypothyroidism had thinner femoral cartilage than the healthy controls at all measurement sites , but the differences were not statistically significant except in the case of the lmc ( p<0.05 ) . rlc : right lateral condyle ; ria : right intercondylar area ; rmc : right medial condyle ; llc : left lateral condyle ; lia : left intercondylar area ; lmc : left medial condyle . hypothyroidism is known to be associated with oa and inflammatory forms of arthritis and connective tissue diseases , which can cause arthritis9 . the clinical findings of hypothyroidism include epiphyseal disgenesis , aseptic necrosis , and viscous non - inflammatory arthropathy particularly affecting the knees , wrist joints , and hands10 . the thyroid hormones are essential for endochondral ossification and stimulating the expression of the gene that controls chondrocyte maturation and matrix synthesis3 . mature chondrocytes in the cartilage synthesize type ii collagen and proteoglycans , which begin to degradation in early oa . as this degradation progresses oa involves the entire joint , including the subchondral bone and articular cartilage , and the prevalence of periarticular soft tissue lesions is high14 , 15 . in addition to degradation and loss of the articular hyaline cartilage , progressive thickening , remodeling , and sclerosis of the subchondral bone ; formation of osteophytes ; and chronic inflammation of the synovial membrane are other features of oa17 . ultrasound is an inexpensive noninvasive method for imaging the musculoskeletal system that is readily accepted by patients . it can be provide information about the joint cartilage , synovitis , periarticular soft tissues , and bony cortical abnormalities in peripheral joints with oa . it seems to be a promising and accurate method for monitoring cartilage response to drug therapy in oa18 . cartilage defects may predict cartilage loss in asymptomatic knee oa and can indicate early oa , which is demonstrated by a loss in the sharpness of the margin19 . lee et al . reported a significant correlation between ultrasonographic measurements and histologic grading of oa femoral condylar cartilage6 . a previous study reported that low hemoglobin levels had a negative effect on femoral cartilage thickness as detected by ultrasonography in healthy subjects20 . ultrasound is also useful to monitor the effect of biologic therapy in rheumatoid arthritis and can be used to evaluate both inflammatory and destructive changes21 . tsai et al . suggested that ultrasound could be used to monitor changes in the cartilage of patients with rheumatoid arthritis22 , and tunc et al . revealed that the femoral cartilage is thinner on the hemiparetic side of stroke patients23 . the results of the present study show the negative effects of hypothyroidism on the thickness of the distal femoral cartilage . to the best of the authors knowledge , this is the first study exhibiting thinning of the distal femoral cartilage in correlation with hypothyroidism . the sample size was relatively small , and only the thickness and not the volume of the cartilage was measured , although it has been shown that differences in cartilage volume result primarily because of differences in joint surface areas ( epiphyseal bone size ) rather than cartilage thickness24 . overall , on the basis of the study findings , patients with hypothyroidism seem to have thinner femoral cartilage . thus , the presence and severity of femoral cartilage thinning may be useful for early diagnosis of knee oa . ultrasonography is a noninvasive , cost - effective method to measure the thickness of the articular cartilage , and screening of joint cartilage by ultrasonography may enable early diagnosis of knee oa in patients with hypothyroidism .

[ purpose ] the aim of this study was to investigate the effects of hypothyroidism on femoral cartilage thickness by using ultrasound , which has been found to be useful in the early diagnosis of knee osteoarthritis . [ subjects and methods ] forty patients diagnosed with hypothyroidism and 30 age- , gender- , smoking status , physical activity- , and body mass index - matched healthy subjects were enrolled . the thickness of the femoral articular cartilage was measured using a 7- to 12-mhz linear probe . three mid - point measurements were taken from each knee at the lateral condyle , intercondylar area , and medial condyle . [ results ] age , gender , body mass index , smoking status , and physical activity were similar between the groups , but patients with hypothyroidism had thinner femoral cartilage than the healthy controls at all measurement sites . nonetheless , the differences were not statistically significant ( except in the case of the left medial condyle ) . [ conclusion ] ultrasonographic measurement of femoral cartilage thickness may be useful in the early diagnosis of knee osteoarthritis in patients with hypothyroidism .

INTRODUCTION SUBJECTS AND METHODS RESULTS Discussion

maintaining the health of australia 's ageing population is a priority for governments and healthcare providers , not only to improve quality of life but also to minimise the burden of ill health on existing resources . ensuring that older people get the recommended amount of daily physical activity is a key way to improve and maintain health in this important group . just 40% of australian adults who aged 5574 years achieve the recommended amount of physical activity each week , and after 75 years , the proportion falls to 24% ; however identifying physical activities that are enjoyable and accessible for older people can be challenging . many older adults prefer to exercise alone or with others of their own age , and more than half of older australians undertake their physical activity in unstructured forms such as walking to local shops . barriers to exercise for older people can include cost , time , and difficulty , travelling to group programs . it is nonweight bearing and therefore has less impact on the joints than jogging or other running sports , and several longitudinal epidemiological studies have shown significant risk reduction for all - cause and cancer mortality , cardiovascular disease , colon and breast cancer , and obesity morbidity in middle - aged and elderly cyclists [ 79 ] . cycling may also contribute to improved quality of life for older people , by enhancing social networks and building empowerment , and can be incorporated easily into a daily routine . despite these benefits , cycling participation by older adults in australia is low . in contrast to many european cities where older people continue to cycle , in australia just 18% of those who aged over 50 cycled in the past year , compared with 50% of those aged 1029 and 39% of those aged 3049 years . a small number of published studies have investigated the barriers and facilitators of adult cycling in australia , but none have looked specifically at the important and growing population of older people . these studies found that poor confidence and cycling ability are concerns for inexperienced riders [ 14 , 15 ] . lack of cycle paths is often cited as a barrier to regular cycling , as is fear of cars and negative attitudes from motorists [ 14 , 16 , 17 ] . this study investigated the motivators , barriers and facilitators of cycling in older people who participated in a pilot cycling program . this qualitative study was part of a cycling promotion study among older people , looking at improvements in leg strength and balance as risk factors for falls . adults who aged 5075 years , who were willing to cycle for two or more hours per week over a 12-week period , were recruited through local advertising and word of mouth in a discrete geographic area of sydney ( canada bay ) , australia . canada bay is an innerwest suburb of sydney , located around 8 km west of the city . the area scores 1,076.5 on the seifa index of disadvantage , indicating that it is less disadvantaged that the national average , and the median / average age of the population is 39 years of age , 2 years above the australian average . the site was chosen for this study to build on existing relationships between one of the researchers ( cm ) and the community centre and bicycle user group in that area . we aimed to recruit around 20 participants for the pilot study because it was feasible to support this quantity with the resources available , and the researchers felt that around 20 participants would be sufficient for the purposes of this trial . semistructured interviews were conducted one - on - one before participants attended a cycling skills course and 12-weeks later . the study 's chief investigator conducted baseline interviews , and follow - up interviews were conducted by another author . the chief investigator was experienced in qualitative interviewing techniques , is personally within the age bracket for participation in the study , and is an experienced cyclist who rides to work daily . the second interviewer has less experience in qualitative interviewing techniques , is younger than the study participants , and cycles to work often . five themes were investigated ; motivators and barriers were the focus of the baseline interview , while the follow - up interview focused on benefits / outcomes , enablers , and cycling promotion . interviews were audio - recorded but not transcribed , and analysed by an individual researcher using thematic analysis . interviews were coded and then data linked by codes were collated into potential themes . in this way , the researcher generated a thematic map of the entire analysis . the person conducting the analysis was younger than the study participants , is female , and cycles to work occasionally . participants were supported to complete their 2 hr / week of cycling by providing cycling skills training , mentors , and a resource pack . the program commenced with a cycling skills course to develop participants ' cycling ability , safety knowledge , and confidence , as poor skills and confidence are known barriers to taking up cycling across all ages . the course was delivered by accredited trainers bikewise , and funded by the city of sydney council ( see http://www.bikewise.com.au/ ) . the course lasted 4.5 hours and covered basic bicycle control skills ( starting , braking , and turning ) , route planning , and on - road riding skills . participants were matched with mentors from their local area , who were experienced cyclists and could be contacted either directly or via a google group . the research team also made brief informal phone calls to participants at one and six weeks to maintain participant engagement and identify any additional support required . strong social support and encouragement have been shown to enable and motivate people to take up and sustain physical activity , and these interventions targeted these constructs . participants received a resource pack which included local cycling maps , information about local bicycle user groups , and where and when local group rides were held . these interventions targeted the potential barrier of participants not knowing where to ride and also encouraged participants and supported social connectedness . this study received ethical approval on september 7 , 2011 from the university of sydney research integrity human research ethics committee ( protocol no . : 09 - 2011/14065 ) . seventeen participants were recruited , ranging from 49 to 72 years of age ( mean 61 years ) . the majority ( twelve ) were female . most ( twelve ) had not ridden in the past year , but fifteen were undertaking some form of physical activity ; however the amount varied widely , ranging from a little bit of walking to one woman running 50 km per week . two participants withdrew from the program for personal reasons after the baseline interview , and researchers were unable to schedule a suitable time for a followup interview with four participants . the four participants who were unable to attend a follow - up interview were at the younger end of the age bracket ( more likely to be in their 50s ) and more likely to be working . as the sample was self - selected , all enrolled participants were highly motivated to start cycling . the main reason for wanting to cycle was for exercise , and the biggest perceived barrier was fear of cars and traffic . this study was advertised as a way to potentially improve balance and leg strength , and this should be considered a motivator for people to take part . all participants cited exercise as a major reason to take up cycling , as many did not like gyms and thought that cycling would be a more fun way to exercise , and would fit more easily into their lifestyle . if it fits into my lifestyle , then i 'll do it and love it ... but if i have to ... go to the gym ... then i 'm not very good at following through if it fits into my lifestyle , then i 'll do it and love it ... but if i have to ... go to the gym ... then i 'm not very good at following through ( 59yo female ) . some participants added that cycling is a form of exercise that they can continue to do now that they are older.we [ my wife and i ] used to run ... but i ca n't run anymore because i damaged one of my knees so i 'm past running , so that 's why we 're thinking well bikes will be good we [ my wife and i ] used to run ... but i ca n't run anymore because i damaged one of my knees so i 'm past running , so that 's why we 're thinking well bikes will be good all participants also mentioned enjoyment as a reason to cycle , but this was not as strong a motivator as exercise . cycling was perceived as a fun way to spend time with family or make new friends . participants expected to enjoy being outdoors and anticipated the feeling of freedom that cycling brings.i think it 's quite nice because i see my husband going off and it 's like he has a social group and ... they always seem to stop somewhere nice for coffee ... i think that would be quite nice to be in a similar sort of social group ( 67yo female ) . i think it 's quite nice because i see my husband going off and it 's like he has a social group and ... they always seem to stop somewhere nice for coffee ... i think that would be quite nice to be in a similar sort of social group ( 67yo female ) . joy , exercise , enjoyment , getting out , you know ? getting out and getting about ( 59yo female ) . around a third intended to use the bicycle for local utility trips . motivators included convenience , exercise , enjoyment , and the environment.i drive to work , i drive to dad , i drive back from dad , i drive to church , i drive ... even to the shops now ... i 'm just driving all the time and i 'm hating it ... i 'd love to ... just jump on my bike to go and get the milk ( 59yo female ) . i drive to work , i drive to dad , i drive back from dad , i drive to church , i drive ... even to the shops now ... i 'm just driving all the time and i 'm hating it ... i 'd love to ... just jump on my bike to go and get the milk ( 59yo female ) . several participants had plans to go on a cycling holiday with family members and were building their confidence and fitness.i said to my son ... next year , why do n't you meet me in copenhagen and we can cycle up the east coast ? i said to my son ... next year , why do n't you meet me in copenhagen and we can cycle up the east coast ? the primary barrier to cycling for all participants was fear of cars and riding on streets . most would only consider riding on cycle paths and very quiet roads , and many mentioned friends who had had a close call while cycling on roads.i've got a lot of friends ... and i ca n't find a single friend who thinks that it 's safe enough for them to be on the road i 've got a lot of friends ... and i ca n't find a single friend who thinks that it 's safe enough for them to be on the road many were not confident in their riding ability , mentioning they might be a bit wobbly on the bike to begin with because they had not ridden in years and had never ridden a bicycle with gears . many were relying on the cycling skills course to practice and to build confidence.i feel very nervous about riding a bike because i do n't feel i can have adequate control over it ... in terms of wobbling where there 's traffic i feel very nervous about riding a bike because i do n't feel i can have adequate control over it ... in terms of wobbling where there 's traffic interestingly , participants did not feel particularly concerned about falling off their bikes as older people . participants were certainly aware that they may fall , but they reported that their fear was no worse than when they were younger . even when asked about being frail or having brittle bones , this group were not particularly concerned with this aspect of cycling . of these , one had been unable to cycle due to an unrelated injury , and one found she lacked the skill to cycle so she gave up on her 2 hours / week target . all of the remaining nine participants had met their 2 hr per week target and many had done much more . most were cycling for recreation and fitness , and just over half had also used the bicycle to travel locally to work , the gym , or local shops and events . all who had met their target reported a very positive experience from cycling.you enjoy it , you 're getting to work , you 're missing the traffic , it makes you feel more healthy in your mind ... and you end up stronger ! you enjoy it , you 're getting to work , you 're missing the traffic , it makes you feel more healthy in your mind ... and you end up stronger ! overwhelmingly , taking up cycling was a liberating , fun experience , and many participants reported that it felt lovely to be outdoors , exercising in nature . many recalled cycling in childhood and recaptured a sense of joy and freedom.i loved the wind blowing through your hair , that was lovely ! ... that was the exhilarating part of it ( 71-year - old female ) . i loved the wind blowing through your hair , that was lovely ! ... that was the exhilarating part of it this was a distressing realisation for this participant and she expressed disappointment in herself . at baseline , this participant explained that she had fallen from her bike recently and had lost her riding confidence . she hoped that participating in this program would give her the resources and motivation that she needed to get back on the bicycle . many participants also enjoyed a benefit to their social life as a result of cycling . they had a new experience to talk about with friends and inspired others to take up cycling themselves . in addition , cycling plugged participants into a new community , allowing them to interact with people outside their usual social group . the best example of this was one local cycling group , where two participants rode on weekends with a father and his two daughters . participants loved interacting with the two young girls , as well as strengthening their own friendship . finally , most of the women expressed a strong sense of pride at their cycling achievements and felt empowered by overcoming their fear of cycling and improving their skills , especially as an older person . friends told them they were an inspiration , which heightened the positive association.some of my friends , they 're amazed that i 've gone out ( well , i am too ! ) ... she said ( 71yo female ) . some of my friends , they 're amazed that i 've gone out ( well , i am too ! ) ... she said participants were not asked about this feeling specifically ; it came up spontaneously in their interviews . feeling proud and/or empowerment was not mentioned by any of the male participants in their interviews . participants reported feeling more energetic , having a better mood , and being able to relax and sleep better after a bike ride . this improvement in mental health was the most commonly reported health - related benefit of cycling ; however they also reported improved cardiovascular fitness and feeling stronger , especially in the legs . one individual 's diabetic control had improved so much that he was using less insulin . the strongest determinants of confidence were bike - handling skills and knowing their intended route . the majority began cycling in parks , before moving to cycle paths , and eventually back - roads as confidence increased . while finding the confidence to cycle was a struggle for some , the cycling skills course was a hugely positive experience for all participants , and for many it was the main factor in getting them back on a bike.fantastic ! if it was n't for that , i would n't be cycling now participants were the happiest when they were grouped with other older riders or riders with a similar level of skill , as they did not feel they were holding the group back and did not feel embarrassed.i did not want to be the duffer in the class three participants attended the course a second time , and two more asked whether they were allowed to attend again , emphasising the importance of repetition and reinforcement for older riders . they valued the safety knowledge they gained through the course , such as road positioning and how to approach intersections , and practicing basic skills , which improved their confidence . many had recommended the course to friends and colleagues and could not speak about it more highly.it's probably the best course i 've ever done in my life ( 66yo male ) . ( 66yo male ) . due to low confidence , participants needed to feel secure about where they were riding . mentors played a big role by either providing cycle maps or showing the way . for example , one participant 's mentor rode with her to work one day , and after that she was able to ride to work a few times a week , always along that same route . local cycle maps were also highly valued , but one participant could not use the map given because she could not locate her house on it . participants were impressed with the well - maintained cycling facilities available in local parks , and some had not known such facilities existed until they saw them on the map.well , i was amazed [ when i saw the map ] at the extent of bike paths and cycle ways in sydney ( 71yo female ) . well , i was amazed [ when i saw the map ] at the extent of bike paths and cycle ways in sydney i 'm more concerned about traffic than anything , so if anyone in sydney wants to build more cycleways , i think that 's wonderful ! i 'm more concerned about traffic than anything , so if anyone in sydney wants to build more cycleways , i think that 's wonderful ! many participants felt that a media campaign about the benefits of cycling would encourage more older people to cycle , but that campaign would need to be backed - up with more cycle ways . other suggestions included advertising in seniors magazines and at seniors groups and word of mouth or presentations from other older cyclists . they felt the messages needed to be that cycling is achievable , fun , and beneficial for health and that it could be done safely . despite this , participants noted that many older people would never be responsive to this type of encouragement as cycling is perceived as too dangerous and some older people simply will not undertake physical activity . the strategies used to promote cycling in this study were simple and inexpensive , mainly linking motivated people to existing resources , but had a high success rate for increasing cycling participation . this work explored motivators , barriers , and outcomes to taking up or increasing cycling and highlighted successful strategies for promoting cycling in older people . key findings included that cycling promotion for this age group must focus on improving confidence and must consider the need for reinforcement and repetition . encouragement for bicycle user groups to reach out to older people , widespread availability of cycling maps , and advertising the multiple benefits of cycling are also helpful . continued improvement to cycle paths was strongly supported by participants . fear of cars and traffic is a strong barrier to cycling across all age groups so investment in infrastructure should also have benefits across the population . the participant who found she was unable to cycle adds weight to the theory that confidence is extremely important in cycling promotion . this participant was given additional time , resources , and encouragement ( she was lent a bike to use and strongly encouraged by her mentor ) ; however even with these additional resources she was not able to overcome her fear of falling off her bike again although she persisted in trying . none of the other participants mentioned previous bad experiences with cycling , indicating that a previous bad experience may well cause a significant loss of confidence which would need additional , tailored strategies to overcome . another interesting finding is that the sense of empowerment and pride was felt very strongly by female participants but not even mentioned by men . it is possible that men may be inherently more physically confident and assume that they will be able to ride without problems while for women this is a realisation rather than an assumption . it could also be that both sexes felt empowered and proud of cycling but that women were more likely to talk about it at interview , especially as the topic was not specifically discussed or explored . contrary to our own preconceived ideas and that of colleagues who we spoke to informally , the participants themselves did not express concerns about being frail and bicycle riding . there may be a feeling among the general population that older people need to be physically cautious that is not held by older people themselves , or worse that older people participating in physical activity are being reckless . if this view is held widely , it may partly explain why older people are not routinely targeted and encouraged in physical activity campaigns , despite the many benefits that physical activity provides in older age . our study showed that exercise is a strong initial motivator when encouraging older people to cycle , as are sociability and fun , but the best outcomes are empowerment and improved quality of life . participants preferred to be in a group with others like themselves and some needed the reinforcement and repetition of attending the cycling skills course a second time . following our program , the city of sydney has set up a cycling skills course specifically for older people , called rusty riders . having a mentor for support and encouragement and to ride with a participant along a new route was another effective method to support new riders , and for some new riders , group rides were a fun way to experience the social aspect of riding , gain confidence , and learn about where to ride while getting their physical activity . a public health campaign focusing on the health benefits of cycling and showing personal stories of other older cyclists might be effective and is worthy of further research . local cycle maps were highly valued and might be improved if someone ( perhaps a mentor ) could interpret the map and highlight a route for an older person to take . as with all qualitative work , our findings are specific to the older people in our study group and not necessarily representative of the broad older population in sydney . however , the broad themes identified in this group of older adults appear to be readily applicable to older adults generally . similarly as with all qualitative work , differences between interviewers may have influenced the way the interviews were conducted and the way participants related to the interviewers . interview data were analysed by one researcher , which may be seen as a weakness to the study ; however analysis of qualitative data involves interpretation of study findings , and this process is inherently subjective . it is well accepted in qualitative research that a definitive , objective view of social reality does not exist and different researchers may well interpret the same data differently . therefore validation by an additional researcher would not necessarily give a correct interpretation , simply another , equally valid one . the participants who were lost to followup were all contacted multiple times , but the researchers were unable to schedule a time which was convenient for them for their followup interview . on reflection , these participants were more likely to be younger and working , meaning they were probably busy and had less time to devote to the study . unfortunately , we are unable to say whether these participants had actually increased their cycling behaviour but an area for further research may be investigating the differences for cycling promotion in working , compared with nonworking older adults . other areas for further research could expand on our finding that empowerment , pride , and external feedback ( admiration from peers ) were very important positive outcomes for female participants but not mentioned by men . another relevant topic would be looking at the use of electric bikes ( ebikes ) as enablers ( or perhaps deterrents ) to cycling in this age group . this study suggests that cycling can have a positive influence on the quality of life of older adults , especially through a sense of empowerment and pride , broadening and invigoration of social networks , and simple pleasure . older people could successfully be encouraged to cycle relatively cheaply and easily by linking them with existing resources in an age - targeted way . while additional cycling infrastructure remains an important goal , in the meantime , programs such as the one used here are simple and low - cost to implement . cycling promotion should centre around improving older people 's cycling confidence through improving skills and practice and repetition , promoting existing cycle paths , increasing safe cycle paths , and promoting cycling to older adults for its health , social , and well - being benefits .

introduction . cycling can be an enjoyable way to meet physical activity recommendations and is suitable for older people ; however cycling participation by older australians is low . this qualitative study explored motivators , enablers , and barriers to cycling among older people through an age - targeted cycling promotion program . methods . seventeen adults who aged 5075 years participated in a 12-week cycling promotion program which included a cycling skills course , mentor , and resource pack . semistructured interviews at the beginning and end of the program explored motivators , enablers , and barriers to cycling . results . fitness and recreation were the primary motivators for cycling . the biggest barrier was fear of cars and traffic , and the cycling skills course was the most important enabler for improving participants ' confidence . reported outcomes from cycling included improved quality of life ( better mental health , social benefit , and empowerment ) and improved physical health . conclusions . a simple cycling program increased cycling participation among older people . this work confirms the importance of improving confidence in this age group through a skills course , mentors , and maps and highlights additional strategies for promoting cycling , such as ongoing improvement to infrastructure and advertising .

1. Introduction 2. Methods 3. Ethical Approval 4. Results 5. Discussion 6. Conclusions

briefly , diagnosis of type 1 diabetes was made according to criteria by the australasian pediatric endocrine group diabetes register and national guidelines . baseline assessment and retinal photography was performed at the initial visit during the recruitment period . of 1,159 participants who had retinal photographs taken at their baseline visits , we excluded those with ungradable photographs ( n = 215 , 18.6% ) , due to either poor quality of retinal photographs or having less than four big vessels that could be traced by the computer - assisted program , leaving 944 ( 81.4% ) participants included in analyses for this report . key diabetes - related characteristics assessed included pubertal stage , duration of diabetes , and levels of a1c , cholesterol , and blood pressure , which were collected at the baseline visits via interviews , clinical examinations , and laboratory investigations following standardized protocols ( 1 ) . bmi was calculated by dividing subject 's weight ( in kilograms ) with the square of height ( in meters ) . systolic ( sbp ) and diastolic ( dbp ) blood pressure were measured after resting for 5 min , sitting in an upright position using a standard sphygmomanometer with an appropriately sized cuff . a1c and total plasma cholesterol levels were measured following standardized laboratory procedures ( 1 ) . retinal photography was performed according to a standardized protocol , as detailed elsewhere ( 1 ) . briefly , stereoscopic retinal photographs in seven standard fields of the early treatment diabetic retinopathy study ( etdrs ) were taken on film from both eyes after pupil dilation , using a topcon fundus camera ( trc 50-vt ; tokyo optical , tokyo , japan ) ( 1 ) . retinopathy was assessed by an ophthalmologist and defined as present when any microaneurysm / retinal hemorrhage was found in either eye ( 1 ) . for measuring retinal microvascular geometric properties , right - eye retinal photographs of each patient were digitized and analyses were performed using a semiautomated computer - assisted image program ( singapore i vessel assessment or siva , singapore ) . retinal photographs that were centered on the optic disc were viewed on two 19-inch monitors with a resolution of 1,280 1,024 . for each retinal photograph , a trained grader , masked to participants ' identities , applied the program to measure retinal microvascular geometric parameters within a concentric zone between the optic disc margin and two optic disc diameters away from the optic disc margin . the grader allowed the software to detect the center of the optic disc and divided the region into three subzones ( a , b , and c ) surrounding the optic disc , each zone corresponding to 0.5 , 1.0 , and 2.0 optic disc diameters away from the optic disc margin , respectively . once the optic disc and the three concentric subzones were considered appropriately located , the grader executed the program to trace all vessels . however , the grader checked each graded image to see if all arterioles and venules were correctly identified , based on information of parent vessels , crossing between arterioles and venules and the color of the vessels . graders were trained and tested for his / her ability in identifying arterioles and venules by a senior grader . during the grading process of this project , 100 randomly selected images were used to test intra- and intergrader reliabilities in classification of arterioles and venules , with value 0.950.99 . images were considered poor quality if they were blurred or had an incomplete representation of zone c and as ungradable if there were less than four gradable large arterioles or venules . the software combined the individual measurement into summary indexes of tortuosity , branching angles , optimality deviation , and ldr for arterioles and venules separately , as described below : vessel tortuosity ( fig . 1 ) reflects the shape of the vessel and is expressed as a curvature tortuosity index ( 4 ) , calculated from the integral of the total squared curvature along the path of the vessel divided by the total arc length . increased tortuosity has been linked with hypertension ( 8) and diabetic retinopathy ( 9 ) , while decreased tortuosity has been associated with ischemic heart disease related death ( 10 ) , ageing , and hypertension ( c. y. cheung , e. lamoureux , l. xu , w. hsu , m. l. lee , q. p. lau , j. j. wang , p. mitchell , t. y. wong).branching angle represents ( in degrees ) the angle between two daughter vessels ( 5 ) and is thought to be related to blood flow efficiency , energy cost of bulk flow , and diffusion distance ( 11 ) . ( 5 ) proposed that the optimal value for the branching angle is 75 degrees ( fig . 1 ) , and increased angles have been related to decreased blood flow ( 12 ) , while decreased angles are associated with ageing and hypertension ( 11).optimality deviation is determined when the junctional exponent is calculated as d1 + d2 = d0 , where d0 , d1 , and d2 are diameters of the parent , larger , and smaller daughter vessels , respectively ( 11 ) . the greater the value of x , the larger the daughter arterioles are relative to the parent vessel . junctional exponent provides an index of caliber sizes of two daughter vessels relative to the parent vessel and is considered to represent an optimality state of microvascular networks ( 5 ) . it has been proposed that in an optimal state , the value of junctional exponent is three ( 5 ) , and optimality deviation represents the deviation from this value.ldr is calculated as the length from the midpoint of the first branch to the midpoint of the second branch divided by the diameter of the parent vessel at the first branch ( 6 ) . ldr is a measure of diameter changes that are independent of refractive magnification power of the eye ( 6 ) . 1 ) reflects the shape of the vessel and is expressed as a curvature tortuosity index ( 4 ) , calculated from the integral of the total squared curvature along the path of the vessel divided by the total arc length . normal vessels are generally straight and smooth ( 4 ) . increased tortuosity has been linked with hypertension ( 8) and diabetic retinopathy ( 9 ) , while decreased tortuosity has been associated with ischemic heart disease related death ( 10 ) , ageing , and hypertension ( c. y. cheung , e. lamoureux , l. xu , w. hsu , m. l. lee , q. p. lau , j. j. wang , p. mitchell , t. y. wong ) . branching angle represents ( in degrees ) the angle between two daughter vessels ( 5 ) and is thought to be related to blood flow efficiency , energy cost of bulk flow , and diffusion distance ( 11 ) . ( 5 ) proposed that the optimal value for the branching angle is 75 degrees ( fig . 1 ) , and increased angles have been related to decreased blood flow ( 12 ) , while decreased angles are associated with ageing and hypertension ( 11 ) . optimality deviation is determined when the junctional exponent is calculated as d1 + d2 = d0 , where d0 , d1 , and d2 are diameters of the parent , larger , and smaller daughter vessels , respectively ( 11 ) . the greater the value of x , the larger the daughter arterioles are relative to the parent vessel . junctional exponent provides an index of caliber sizes of two daughter vessels relative to the parent vessel and is considered to represent an optimality state of microvascular networks ( 5 ) . it has been proposed that in an optimal state , the value of junctional exponent is three ( 5 ) , and optimality deviation represents the deviation from this value . ldr is calculated as the length from the midpoint of the first branch to the midpoint of the second branch divided by the diameter of the parent vessel at the first branch ( 6 ) . ldr is a measure of diameter changes that are independent of refractive magnification power of the eye ( 6 ) . measures of retinal vessel tortuosity , branching angle , and optimality deviation . a comparison of optimal ( a ) and less optimal ( b ) arrangement of arteriolar geometry . a : tortuosity of 0.00 , branching angle of 72 , and optimality deviation of 0.16 . b : tortuosity of 47.2 10 , branching angle of 130 , and optimality deviation of 0.61 . a high - quality color representation of this image is available online . all of statistical procedures were performed using intercooled stata 10.1 for windows ( statacorp , college station , tx ) . retinal microvascular geometry ( tortuosity , branching angle , optimality deviation , and ldr ) of the right eye of each patient were used as dependent , continuous variables . we used ancova to compare mean retinal vessel geometric parameters by age - group ( 1214 and 1420 years ) , sex ( female and male ) , pubertal stage ( stage 1 to stage 5 ) , bmi levels ( 18.0 , 18.121.0 , 21.125.0 , and 25.1 kg / m ) , sbp ( 120 vs. 120 mmhg ) , cholesterol ( 3.7 , 3.84.3 , 4.44.8 , and 4.9 mmol / l ) , duration of diabetes ( 5.0 , 5.110.0 , and 10.1 years ) , and a1c ( 8.5 vs. > 8.5% ) ( 1 ) ( see supplementary tables in the online appendix , available at http://care.diabetesjournals.org/cgi/content/full/dc10-0055/dc1 ) . multiple linear regression models were subsequently used to examine the independent determinants of retinal microvascular geometric parameters as continuous variables . we constructed three models : model 1 included age and sex ; model 2 additionally included bmi , sbp , cholesterol , duration of diabetes , a1c ( all assessed as per sd change ) , presence of retinopathy , and retinal arteriolar caliber ( in models for arteriolar geometric parameters ) or venular caliber ( in models for venular geometric parameters ) ; and model 3 included all variables in model 2 after excluding subjects with diabetic retinopathy . finally , sensitivity analyses were performed by including and excluding subjects with poor - quality images . key diabetes - related characteristics assessed included pubertal stage , duration of diabetes , and levels of a1c , cholesterol , and blood pressure , which were collected at the baseline visits via interviews , clinical examinations , and laboratory investigations following standardized protocols ( 1 ) . bmi was calculated by dividing subject 's weight ( in kilograms ) with the square of height ( in meters ) . systolic ( sbp ) and diastolic ( dbp ) blood pressure were measured after resting for 5 min , sitting in an upright position using a standard sphygmomanometer with an appropriately sized cuff . a1c and total plasma cholesterol levels were measured following standardized laboratory procedures ( 1 ) . retinal photography was performed according to a standardized protocol , as detailed elsewhere ( 1 ) . briefly , stereoscopic retinal photographs in seven standard fields of the early treatment diabetic retinopathy study ( etdrs ) were taken on film from both eyes after pupil dilation , using a topcon fundus camera ( trc 50-vt ; tokyo optical , tokyo , japan ) ( 1 ) . retinopathy was assessed by an ophthalmologist and defined as present when any microaneurysm / retinal hemorrhage was found in either eye ( 1 ) . for measuring retinal microvascular geometric properties , right - eye retinal photographs of each patient were digitized and analyses were performed using a semiautomated computer - assisted image program ( singapore i vessel assessment or siva , singapore ) . retinal photographs that were centered on the optic disc were viewed on two 19-inch monitors with a resolution of 1,280 1,024 . for each retinal photograph , a trained grader , masked to participants ' identities , applied the program to measure retinal microvascular geometric parameters within a concentric zone between the optic disc margin and two optic disc diameters away from the optic disc margin . the grader allowed the software to detect the center of the optic disc and divided the region into three subzones ( a , b , and c ) surrounding the optic disc , each zone corresponding to 0.5 , 1.0 , and 2.0 optic disc diameters away from the optic disc margin , respectively . once the optic disc and the three concentric subzones were considered appropriately located , the grader executed the program to trace all vessels . however , the grader checked each graded image to see if all arterioles and venules were correctly identified , based on information of parent vessels , crossing between arterioles and venules and the color of the vessels . graders were trained and tested for his / her ability in identifying arterioles and venules by a senior grader . during the grading process of this project , 100 randomly selected images were used to test intra- and intergrader reliabilities in classification of arterioles and venules , with value 0.950.99 . images were considered poor quality if they were blurred or had an incomplete representation of zone c and as ungradable if there were less than four gradable large arterioles or venules . the software combined the individual measurement into summary indexes of tortuosity , branching angles , optimality deviation , and ldr for arterioles and venules separately , as described below : vessel tortuosity ( fig . 1 ) reflects the shape of the vessel and is expressed as a curvature tortuosity index ( 4 ) , calculated from the integral of the total squared curvature along the path of the vessel divided by the total arc length . normal vessels are generally straight and smooth ( 4 ) . increased tortuosity has been linked with hypertension ( 8) and diabetic retinopathy ( 9 ) , while decreased tortuosity has been associated with ischemic heart disease related death ( 10 ) , ageing , and hypertension ( c. y. cheung , e. lamoureux , l. xu , w. hsu , m. l. lee , q. p. lau , j. j. wang , p. mitchell , t. y. wong).branching angle represents ( in degrees ) the angle between two daughter vessels ( 5 ) and is thought to be related to blood flow efficiency , energy cost of bulk flow , and diffusion distance ( 11 ) . ( 5 ) proposed that the optimal value for the branching angle is 75 degrees ( fig . 1 ) , and increased angles have been related to decreased blood flow ( 12 ) , while decreased angles are associated with ageing and hypertension ( 11).optimality deviation is determined when the junctional exponent is calculated as d1 + d2 = d0 , where d0 , d1 , and d2 are diameters of the parent , larger , and smaller daughter vessels , respectively ( 11 ) . the greater the value of x , the larger the daughter arterioles are relative to the parent vessel . junctional exponent provides an index of caliber sizes of two daughter vessels relative to the parent vessel and is considered to represent an optimality state of microvascular networks ( 5 ) . it has been proposed that in an optimal state , the value of junctional exponent is three ( 5 ) , and optimality deviation represents the deviation from this value.ldr is calculated as the length from the midpoint of the first branch to the midpoint of the second branch divided by the diameter of the parent vessel at the first branch ( 6 ) . ldr is a measure of diameter changes that are independent of refractive magnification power of the eye ( 6 ) . 1 ) reflects the shape of the vessel and is expressed as a curvature tortuosity index ( 4 ) , calculated from the integral of the total squared curvature along the path of the vessel divided by the total arc length . normal vessels are generally straight and smooth ( 4 ) . increased tortuosity has been linked with hypertension ( 8) and diabetic retinopathy ( 9 ) , while decreased tortuosity has been associated with ischemic heart disease related death ( 10 ) , ageing , and hypertension ( c. y. cheung , e. lamoureux , l. xu , w. hsu , m. l. lee , q. p. lau , j. j. wang , p. mitchell , t. y. wong ) . branching angle represents ( in degrees ) the angle between two daughter vessels ( 5 ) and is thought to be related to blood flow efficiency , energy cost of bulk flow , and diffusion distance ( 11 ) . ( 5 ) proposed that the optimal value for the branching angle is 75 degrees ( fig . 1 ) , and increased angles have been related to decreased blood flow ( 12 ) , while decreased angles are associated with ageing and hypertension ( 11 ) . optimality deviation is determined when the junctional exponent is calculated as d1 + d2 = d0 , where d0 , d1 , and d2 are diameters of the parent , larger , and smaller daughter vessels , respectively ( 11 ) . the greater the value of x , the larger the daughter arterioles are relative to the parent vessel . junctional exponent provides an index of caliber sizes of two daughter vessels relative to the parent vessel and is considered to represent an optimality state of microvascular networks ( 5 ) . it has been proposed that in an optimal state , the value of junctional exponent is three ( 5 ) , and optimality deviation represents the deviation from this value . ldr is calculated as the length from the midpoint of the first branch to the midpoint of the second branch divided by the diameter of the parent vessel at the first branch ( 6 ) . ldr is a measure of diameter changes that are independent of refractive magnification power of the eye ( 6 ) . a comparison of optimal ( a ) and less optimal ( b ) arrangement of arteriolar geometry . a : tortuosity of 0.00 , branching angle of 72 , and optimality deviation of 0.16 . b : tortuosity of 47.2 10 , branching angle of 130 , and optimality deviation of 0.61 . a high - quality color representation of this image is available online . all of statistical procedures were performed using intercooled stata 10.1 for windows ( statacorp , college station , tx ) . retinal microvascular geometry ( tortuosity , branching angle , optimality deviation , and ldr ) of the right eye of each patient were used as dependent , continuous variables . we used ancova to compare mean retinal vessel geometric parameters by age - group ( 1214 and 1420 years ) , sex ( female and male ) , pubertal stage ( stage 1 to stage 5 ) , bmi levels ( 18.0 , 18.121.0 , 21.125.0 , and 25.1 kg / m ) , sbp ( 120 vs. 120 mmhg ) , cholesterol ( 3.7 , 3.84.3 , 4.44.8 , and 4.9 mmol / l ) , duration of diabetes ( 5.0 , 5.110.0 , and 10.1 years ) , and a1c ( 8.5 vs. > 8.5% ) ( 1 ) ( see supplementary tables in the online appendix , available at http://care.diabetesjournals.org/cgi/content/full/dc10-0055/dc1 ) . multiple linear regression models were subsequently used to examine the independent determinants of retinal microvascular geometric parameters as continuous variables . we constructed three models : model 1 included age and sex ; model 2 additionally included bmi , sbp , cholesterol , duration of diabetes , a1c ( all assessed as per sd change ) , presence of retinopathy , and retinal arteriolar caliber ( in models for arteriolar geometric parameters ) or venular caliber ( in models for venular geometric parameters ) ; and model 3 included all variables in model 2 after excluding subjects with diabetic retinopathy . finally , sensitivity analyses were performed by including and excluding subjects with poor - quality images . of 944 patients in this study , 170 ( 14.7% ) had retinopathy ( all classified as mild nonproliferative retinopathy ) . the baseline characteristics of participants with and without gradable right - eye retinal photographs are shown in table 1 and retinal microvascular measurements are shown in table 2 . baseline characteristics , according to the gradability of retinal photograph data are age and sex - adjusted means se or % . retinal microvascular parameters in 944 children and adolescents aged 1220 years with type 1 diabetes data are means sd or median ( interquartile range ) . table 3 shows that older age was associated with decreased arteriolar and venular tortuosity , and female patients on average had larger arteriolar branching angle than male patients . after adjusting for age , sex , sbp , cholesterol level , duration of diabetes , a1c level , retinopathy , and vessel caliber , the following associations were evident : 1 ) increasing diabetes duration was associated with increased arteriolar branching angle ( p 0.002 ) and increased optimality deviation of arterioles ( p = 0.014 ) , 2 ) high a1c ( > 8.5 vs. 8.5% , ) was associated with increased arteriolar tortuosity ( p = 0.018 ) , 3 ) increasing sbp was associated with decreasing arteriolar ldr ( p = 0.001 ) and nonsignificantly decreasing venular ldr ( 0.05 < p < 0.10 ) , and 4 ) increasing total cholesterol level was associated with increasing arteriolar ldr ( p = 0.014 ) and decreasing optimality deviation of venules ( p = 0.07 ) . the observed significant associations largely remained after excluding eyes with retinopathy ( n = 170 , model 3 ) ( table 3 ) . associations of baseline and diabetes - related factors with retinal microvascular geometry * data are mean difference ( 95% ci ) of retinal parameters per sd increase in duration ( 3.3 years ) or 8.5 vs. > 8.5% a1c level and per 1-sd increase in sbp ( 12 mmhg ) , cholesterol ( 0.9 mmol / l ) , and bmi ( 3.5 kg / m ) using linear regression models . p , p value of coefficient adjusted for age and sex ; p , p value of coefficient adjusted for age , sex , sbp , cholesterol , duration , a1c , retinopathy , and retinal vessel caliber ; p , p value of coefficient , adjusted for age , sex , sbp , cholesterol , duration , a1c , and retinal vessel caliber , excluding subjects with retinopathy . there were no significant interactions between age , sex , blood pressure , bmi , cholesterol , duration of diabetes , and a1c level . analyses after excluding subjects with relatively poor image quality ( n = 129 , 11.1% ) showed that these associations remained significant in 815 ( 70.3% ) patients with good - quality retinal images ( data not shown ) . in young type 1 diabetes , we showed that variations in retinal microvascular geometric characteristics were associated with key diabetes - related risk factors , including longer diabetes duration and higher a1c , blood pressure , and cholesterol levels . the principal - specific findings are the following : longer duration of diabetes was associated with larger arteriolar branching angles and increasing deviation of optimality , higher a1c was associated with more tortuous arterioles , increasing sbp level was associated with smaller arteriolar and venular ldr , and increasing cholesterol level was associated with increasing arteriolar ldr and decreasing venular optimality deviation . importantly , we showed that these associations were present even in subjects without mild retinopathy signs , suggesting that early retinal microvascular alternations are present well before the onset of clinical microvascular complications in young patients with type 1 diabetes . most previous studies that have examined retinal microvascular geometric parameters have been conducted in nondiabetic populations , including healthy subjects ( 13 ) , subjects with hypertension ( 11 ) , or those with coronary heart disease ( 10 ) . to the best of our knowledge , there has been no study reporting retinal microvascular geometric characteristics in young type 1 diabetes . our findings that longer duration of diabetes was associated with larger arteriolar branching angle and increasing deviation from optimality of arterioles is biologically plausible and may provide clues to early microvascular alterations in type 1 diabetes . an optimal branching angle is associated with greater efficiency in blood flow with lower energy spent ( 5,7 ) . such efficiency reduces when the branching angle is too large or when the size of daughter vessel is too large or too small relative to its parent vessel ( 5,7 ) . ( 5 ) have shown that departure from normal or increased deviation from optimality could result in increased workload and energy loss in maintaining the blood circulation . branching angle and junctional exponent have been found to be impaired in atherosclerosis ( 14 ) , altered blood flow ( 12 ) , and endothelial dysfunction ( 15 ) . moreover , chapman et al . ( 16 ) demonstrated that branching angle was likely to increase in response to less oxygen saturation . thus , our finding of associations of longer diabetes duration with larger branching angle and higher optimality deviation may reflect alterations in blood flow ( 17 ) , endothelial dysfunction ( 18 ) , and attenuation in oxygen saturation ( 16 ) . the sex difference in arteriolar branching angle , with female subjects having larger arteriolar branching angles than male subjects , could also explain our earlier observations that female patients with type 1 diabetes have greater risk for diabetic microvascular complications compared with male patients of the same age ( 1,3 ) . diabetes is known to be associated with increased shear stress and impaired microvascular endothelium ( 2 ) . previously , increased a1c level had been shown to have a role in reduced endothelial function in people with type 1 diabetes ( 19 ) . increased vessel tortuosity has been documented to be related to increased angiogenesis ( 20 ) and endothelial dysfunction ( 10 ) . we did not find a linear association between a1c levels and arteriolar tortuosity , but we observed a significant difference in tortuosity between patients with a1c 8.5 vs. 8.5% . there have been studies showing that different cutoffs of a1c levels identify people with diabetes at high risk of microvascular injury ( 21,22 ) , although there is no universal consensus on these cutoffs ( 22 ) . we have previously reported that wider arteriolar caliber is associated with an increased risk of diabetic retinopathy in both type 1 and type 2 diabetes ( 3,23 ) . thus , the finding of an association of higher blood pressure levels with increasing arteriolar ldr ( reflecting wider arteriolar caliber ) is consistent with the influence of diabetes and blood pressure on autoregulatory processes in small blood vessels . there have been few studies on cholesterol levels on microvascular structure , with most previous studies reporting associations between cholesterol level and retinal microvascular changes in nondiabetic populations showing inconsistent results ( 24,25 ) . therefore , our findings that higher total cholesterol levels was associated with changes in arteriolar ldr and venular optimality deviation suggests that lipids may also have an influence on microvasculature in young type 1 diabetes . first , our study includes a large cohort of young patients with type 1 diabetes , with a participation rate of > 80% . second , our study included an objective quantitative measurement of the geometry of retinal microvasculature using computer programs . the cross - sectional nature of our study implies that longitudinal studies are needed to determine the temporal sequence of these associations . a number of images ( 18.6% ) were ungradable and thus excluded from the analysis . however , baseline characteristics and diabetes - related risk factors were largely similar in participants with and without gradable images . therefore , exclusion of participants with ungradeable images did not substantially influence our findings . in addition , there are several potential sources of measurement errors in assessing branching / bifurcation angles , particularly in subjects with poor retinal image quality , given that the computer software needs some manual intervention . however , as the measurement errors are random , these study findings are unlikely to be altered substantially by random errors . in summary , our study demonstrated that in young type 1 diabetes , subtle alterations in retinal microvascular geometric parameters are associated with key diabetes - related characteristics , including duration of diabetes and a1c and blood pressure levels . these findings were present in those without any signs of retinopathy , suggesting an effect of these risk factors on the microcirculation prior to development of clinical complications . some of the observed associations are related to known effects of diabetes on microvasculature ; for other associations , the underlying mechanisms remain to be determined . nonetheless , these data support the concept that retinal microvascular geometric changes might represent a novel marker indicative of early diabetes - related microvascular injury in patients with type 1 diabetes . further studies are warrant to determine if these early retinal microvascular geometric alterations can predict the subsequent development of overt microvascular complications .

objectiveto describe retinal microvascular geometric parameters in young patients with type 1 diabetes.research design and methodspatients with type 1 diabetes ( aged 1220 years ) had clinical assessments and retinal photography following standardized protocol at a tertiary - care hospital in sydney . retinal microvascular geometry , including arteriolar and venular tortuosity , branching angles , optimality deviation , and length - to - diameter ratio ( ldr ) , were measured from digitized photographs . associations of these geometric characteristics with diabetes duration , a1c level , systolic blood pressure ( sbp ) , and other risk factors were assessed.resultsof 1,159 patients enrolled , 944 ( 81.4% ) had gradable photographs and 170 ( 14.7% ) had retinopathy . older age was associated with decreased arteriolar ( p = 0.024 ) and venular ( p = 0.002 ) tortuosity , and female subjects had larger arteriolar branching angle than male subjects ( p = 0.03 ) . after adjusting for age and sex , longer diabetes duration was associated with larger arteriolar branching angle ( p 0.001 ) and increased arteriolar optimality deviation ( p = 0.018 ) , higher a1c was associated with increased arteriolar tortuosity ( > 8.5 vs. 8.5% , p = 0.008 ) , higher sbp was associated with decreased arteriolar ldr ( p = 0.002 ) , and higher total cholesterol levels were associated with increased arteriolar ldr ( p = 0.044 ) and decreased venular optimality deviation ( p = 0.044 ) . these associations remained after controlling for a1c , retinal vessel caliber , and retinopathy status and were seen in subjects without retinopathy.conclusionskey diabetes - related factors affect retinal microvascular geometry in young type 1 diabetes , even in those without evidence of retinopathy . these early retinal alterations may be markers of diabetes microvascular complications .

RESEARCH DESIGN AND METHODS Risk factors assessment Retinal photography and retinal image analysis Statistical analysis RESULTS CONCLUSIONS Supplementary Material

neural tube defects ( ntds ) , including spina bifida , anencephaly , and encephalocele , are among the most common birth defects and cause morbidity and mortality among fetuses and babies . approximately one third of 4,000 pregnancies with ntds end with spontaneous or induced abortion , so there are about 2,500 live births with ntd annually in the us . there is a significant geographical variation in the rate of ntds . in the years 1999 - 2000 there were at least 394 cases of isolated ntd in israel , with a nearly fourfold incidence among non - jews compared to jews . the embryonic basis of ntd is not fully understood , but a common abnormality is lack of closure of the neural tube . the normal development and closure of the neural tube are completed by the 25th day after fertilization . the mortality rate among newborns with spina bifida is 10% in the united states , but nearly 100% in china . among surviving babies there is a high rate of neurological complications such as paralysis , hydrocephalus , arnold chiari type ii and syringomyelia . the known risk factors for the development of ntd are low socioeconomical class , maternal diabetes , antiepileptic drugs , fever in the early stage of pregnancy , and obesity . in families with a history of ntd , a study among 130,142 chinese women between 19931995 demonstrated a reduction in the risk for ntd of up to 85% in women who took 0.4 mg / day of folic acid prior to conception and during the first trimester and other studies have provided additional evidence of this beneficial effect . folic acid is absorbed in the proximal part of the small intestine and stored in the liver and csf . the mean dietary intake of folic acid is about 0.1 - 0.2 mg a day . folic acid is present in food products such as liver , kidney , nuts , yeast , and green vegetables . it is reduced in the body to tetrahydrofolate , a coenzyme for many metabolic reactions including dna synthesis and amino acid conversion . the recommended daily dose of folic acid to prevent megaloblastic acid in pregnancy is 0.20.5 mg / day . the israel ministry of heath recommends a daily intake of folic acid of 0.4 mg / day three months before conception and throughout the first trimester . women who previously conceived an infant with ntd should take 0.4 mg / day of folic acid throughout fertility and 4 - 5 mg / day beginning one month before conception and throughout the first trimester . in the present study we assessed the consumption of folic acid for the prevention of ntds among jewish and muslim bedouin women in southern israel and analyzed possible correlations with the recommendations of family physicians and gynecologists . the bedouin sector in southern israel is of a very low socioeconomic status , with many living in small stone houses , wooden or tin huts , or even tents . a considerable percentage of this population is supported by welfare and has only a few years of formal education . the study population included 65 pregnant jewish women from the city of beer - sheva , 64 pregnant bedouin women from the negev desert region of southern israel , 39 gynecologists ( specialists and residents ) from the soroka university medical center in beer - sheva and 60 family physicians ( specialists and residents ) from the negev region . the helsinki committee of the soroka medical center in beer - sheva and the israel ministry of health approved the study . the interviews in the jewish sector were held in three clinics in beer - sheva that represent populations of different socioeconomic status , as determined by the israel ministry of health . the pregnant women were interviewed personally by the first author ( ef ) upon arrival for routine check - up at the mother and child health clinics after consenting to be interviewed and after it was clear that they had not been interviewed previously . women visiting the clinic for the first time and women with a history of congenital anomaly in a previous pregnancy were excluded . the interviews in the bedouin sector were held at a clinic in a bedouin village and in a beer - sheva clinic that serves bedouins living in rural areas outside established villages . in those clinics we used a questionnaire that was translated into arabic with the aid of an interpreter . the doctors completed the questionnaires at work or while attending continuing medical education courses at the university . an anonymous questionnaire that included questions on demographic background , knowledge and use of folic acid was administered to the pregnant women . a second anonymous questionnaire that included questions on demographic background , knowledge , attitudes , and recommendations regarding folic acid use the study data were organized with the excel software program and processed using the ssps statistical package . group differences were compared by the test and statistical significance was set at p < .05 . the mean maternal age among 65 jewish women was 28.6 4.9 years ( range 2039 ) and 27.9 5.6 among bedouin women ( range 1740 years ) . the women from the jewish sector were significantly more educated than the women from the bedouin sector . fifty - one jewish women ( 78.5% ) reported taking folic acid during pregnancy , but only seven ( 10.8% ) began prior to conception . sixty bedouin women ( 93.75% ) reported taking folic acid during pregnancy , but only four ( 6.25% ) began prior to conception ( p < .05 ) ( table 2 ) . only five jewish women ( 7.7% ) and two bedouin women ( 3.1% ) began taking folic acid three months before conception as recommended by the israel ministry of health . sixty - one jewish women ( 94% ) and all of the bedouin women said that they had received these recommendations or knew about them . seven of the ten jewish women and three of the four bedouin women who refrained from taking folic acid cited as the reason previous side effects such as nausea , weakness and dizziness that they attributed to it . twenty - eight jewish women ( 43.1% ) 18 bedouin women ( 28.1% ) believed that folic acid could cause side effects . twenty three jewish women ( 35.4% ) and four bedouin women ( 6.3% ) knew that folic acid could prevent ntds . twenty of the bedouin women knew that taking folic acid could help the fetus , but could not define specific health benefits . only two jewish women ( 3.1% ) and 10 bedouin women ( 15.6% ) had received and read the ministry of health publication on prevention of ntds , which is available at the mother and child clinics . the study was conducted among 60 family physicians ( 54% women , mean age 38.9 7.6 years , range 2956 ) and 39 gynecologists ( 26% women , mean age 39.5 8.5 years , range 2959 ) . more gynecologists than family physicians were department or unit heads , while more family physicians were still in various stages of professional training ( residents ) . thirty - three gynecologists ( 87.9% ) and 36 family physicians ( 60% ) claim to always recommend folic acid on preconception examination . all the gynecologists and 55 family physicians ( 92% ) reported that they almost always or always recommend folic acid supplementation at this stage ( p < .05 ) . thirty - three gynecologists ( 84% ) and 44 family physicians ( 73% ) always recommend folic acid supplementation during pregnancy . only 35 of 56 family physicians ( 62.5% ) and 28 of 39 gynecologists ( 71.8% ) recommended the correct daily dose of 0.4 mg . eight family physicians ( 32.1% ) and nine gynecologists ( 21.3% ) recommended a high daily dose of 5 mg . seventeen of 37 gynecologists ( 45.9% ) and 32 of 57 family physicians ( 56.1% ) believe that more than 30% of the women actually begin to take folic acid prior to conception . folic acid supplementation before and during pregnancy is an inexpensive , simple strategy that can significantly reduce the incidence of ntd , a very severe malformation . the average lifetime cost of a baby with ntd is estimated at about $ 294,000 . folic acid treatment before and during pregnancy is recommended by the israel and the united states ministries of health and by the american academy of pediatrics [ 2 , 9 ] . the results of the present study demonstrate that despite the clear recommendations , only 10.8% of jewish women and 6.25% of bedouin women take folic acid prior to conception , although 78.5% of the jewish women and 93.75% of the bedouin women said they took folic acid at some time during pregnancy . the issue of folic acid supplementation prior to conception has been studied in many countries . in british columbia 49.4% of 1,004 took folic acid prior to conception . in puerto rico 31.5% of 479 women took folic acid appropriately . in ireland 16% of 300 women took folic acid prior to conception . in a previous israeli study conducted in 1999 only 2.8% of 920 women took folic acid before conception . in the present study we found that although more bedouin women take folic acid during pregnancy than jewish women , fewer bedouin women begin taking it prior to conception . since a relatively high rate of jewish and bedouin women take folic acid during pregnancy , even if inappropriately , the potential exists for better adherence to recommendations with a concomitant reduction in the rate of ntds . the rate of ntds is almost fourfold among bedouins and other non - jewish populations compared to jews the reasons for this finding include consanguineous marriage among bedouins , differences in nutrition , and religious prohibition of abortions . special intervention plans should be developed for each population based on its cultural and socioeconomic characteristics . a minority of women who did not take folic acid during pregnancy despite recommendations cited fear of side effects that they attributed to its use during previous pregnancies . even the high dose of folic acid ( 5 mg ) prescribed by 32.1% of the family physicians and 23.1% of gynecologists is unlikely to explain the reported side effects , since these side effects are rare even at this higher dose . it is possible that some of the women used this explanation as an excuse because they were embarrassed that they did not take folic acid . there is a significant difference between doctors ' reports and the actual degree to which women took folic acid prior to conception . all the gynecologists and 92% of the family physicians in the study said that they always / almost always recommend taking folic acid prior to conception , but only 10.8% of the jewish women and 6.25% of the bedouin women reported taking folic acid at this stage . on the other hand , there is correspondence between the doctors ' reports of recommendations and the pregnant women 's reports of folic acid use during pregnancy . despite the study 's small sample size since folic acid supplementation is the only way to provide 4 - 5 mg of folic acid daily it is important to increase the public 's awareness of this requirement . an intervention plan should include three basic strategies : diet , folic acid supplementation , and food fortification . according to a survey conducted in the year 2000 the mean daily dietary intake of folic acid is 213.8 181.8 micrograms among jewish women and 302.0 226.9 micrograms among bedouin women ( unpublished data ) . this difference may be due to the relatively high intake of fresh vegetables by bedouins . our study demonstrates that only a small minority of the women received and read the ministry of health 's publication on this issue . other studies have reported that popular newspapers and the electronic media are more effective than printed material disseminated by health authorities . it is important to train health system workers in the community to recommend folic acid before , and not only after , conception . it is important for physicians to encourage a preconception examination in which counseling and recommendations for folic acid supplementation are provided . special attention should be given to family physicians since , according to the results of the present study , only 60% always recommend folic acid prior to conception .

background and objective . daily folic acid intake , prior to conception and in early pregnancy , significantly reduces neural tube defects ( ntds ) . we compared folic acid consumption among jewish and bedouin women and the recommendations of family physicians and gynecologists . methods . we compared 64 muslim bedouin women and 65 jewish women . we also compared 39 gynecologists and 60 family physicians . results . fifty - one jewish women ( 78.5% ) took folic acid during pregnancy , but only seven ( 10.8% ) before conception . sixty bedouin women ( 93.75% ) took folic acid during pregnancy , but only four ( 6.25% ) before conception ( p < .05 ) . five jewish women ( 7.7% ) and two bedouin women ( 3.1% ) took folic acid three months before conception . thirty - three gynecologists ( 87% ) recommend preconception folic acid compared with thirty - six family physicians ( 60% ) ( p < .05 ) . conclusions . the majority of women use folic acid during pregnancy , but only few do so to prevent ntds . there is a significant difference between doctors ' recommendations and actual practice .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions

stanford type a acute aortic dissection ( aad ) is a challenge due to high mortality risk in acute phase . during the last decades , thanks to diagnostic and intensive care improvements , evolving surgical techniques , and perioperative management , the early mortality rate has progressively decreased , as confirmed in a recent italian registry . more attention has recently raised in reducing the incidence of late complications and avoiding re - operations . aad is often associated with a variable degree of concomitant aortic valve regurgitation ( ar ) ; emergency surgery has to also address the aortic valve function . the choice between either conservative approach or replacement of the aortic valve is crucial and basically relies on the degree of aortic root involvement , aortic valve morphology , and the underlying mechanism of ar . effective aortic valve repair results in the maintenance of proper hemodynamics , avoiding the consequences of prosthetic valve such as endocarditis , thromboembolism , and chronic anticoagulation . on the other hand , aortic valve preservation exposes patients to a theoretically increased risk of aortic root late complications ; besides late root aneurysms or pseudoaneurysms , recurrence of ar is the most frequent cause of re - operation . we performed a clinical and echocardiographic retrospective study in patients operated on an emergency basis for aad with the preservation of aortic native valve to evaluate the overall survival , the long - term results of aortic valve conservative surgery , and the determinants of recurrent ar . after obtaining approval by the local ethical committee , we retrospectively collected data on fifty patients [ median age : 63 years and interquartile range ( iqr ) : 5372 ; table 1 ] who underwent surgical operation for aad , between 2000 and 2011 , and discharged alive . bav = bicuspid aortic valve all operations were performed by trained staff cardiovascular surgeons , and all patients underwent conservative aortic valve surgery . after the induction of anesthesia , the surgery was preceded by transesophageal echocardiography ( tee ) to assess preoperative ar and the anatomy of the aortic root . myocardial protection was achieved by cold blood retrograde cardioplegia , and neurological protection was improved by selective anterograde cerebral perfusion . with regard to operative techniques , the patient was treated with different surgical operations : twenty - six patients ( 52% ) underwent ascending aorta replacement alone with prosthetic graft , with proximal anastomosis at the sinotubular junction ; inclusion criteria were a normal aortic root and a morphologically normal aortic valve , not prolapsingnineteen patients ( 38% ) with dissected aortic root but apparently normal aortic valve underwent ascending aorta replacement and reconstruction of the diseased sinuses by biological glue and concomitant resuspension of prolapsing aortic cuspfour patients ( 8% ) with most severe dissection of one or more valsalva sinuses but apparently normal aortic valve underwent replacement of pathological sinuses of valsalva , with a specifically tailored vascular graft to replace the severely affected sinusesone patient ( 2% ) with localized dissection of atherosclerotic ascending aorta underwent adhesion of the aortic wall layers with biological glue , without replacement of ascending aorta . twenty - six patients ( 52% ) underwent ascending aorta replacement alone with prosthetic graft , with proximal anastomosis at the sinotubular junction ; inclusion criteria were a normal aortic root and a morphologically normal aortic valve , not prolapsing nineteen patients ( 38% ) with dissected aortic root but apparently normal aortic valve underwent ascending aorta replacement and reconstruction of the diseased sinuses by biological glue and concomitant resuspension of prolapsing aortic cusp four patients ( 8% ) with most severe dissection of one or more valsalva sinuses but apparently normal aortic valve underwent replacement of pathological sinuses of valsalva , with a specifically tailored vascular graft to replace the severely affected sinuses one patient ( 2% ) with localized dissection of atherosclerotic ascending aorta underwent adhesion of the aortic wall layers with biological glue , without replacement of ascending aorta . echocardiographic follow - up was completed in june 2014 , at a median follow - up time of 50.5 months ( iqr : 25.282.5 , at a maximum of 167 months ) ; all patients had an echocardiographic assessment at least after 3 months after discharge . two - dimensional color doppler transthoracic echocardiography ( tte ) was performed , and standard variables were collected according to the jase / eacvi dataset . for patients requiring re - operation , last tte immediately before the operation was considered . data regarding survival and re - operation rate were obtained by the departmental and outpatients clinic database , by phone contacts with referral cardiologist or general practitioners , and by phone call to patient or family . median clinical follow - up time was 55.8 months ( iqr : 17.6 - 93.2 , at a maximum of 167 months ) . data are expressed as mean and standard deviation , or median and iqr ( iqr i - iii ) , when appropriate , or percentage ( % ) . comparison between continuous variables was performed with spearman test ( nonparametric ) , while comparison between groups was tested by mann statistical analysis was computed using spss , version 17.0 for windows ( spss inc . , all operations were performed by trained staff cardiovascular surgeons , and all patients underwent conservative aortic valve surgery . after the induction of anesthesia , the surgery was preceded by transesophageal echocardiography ( tee ) to assess preoperative ar and the anatomy of the aortic root . myocardial protection was achieved by cold blood retrograde cardioplegia , and neurological protection was improved by selective anterograde cerebral perfusion . with regard to operative techniques , the patient was treated with different surgical operations : twenty - six patients ( 52% ) underwent ascending aorta replacement alone with prosthetic graft , with proximal anastomosis at the sinotubular junction ; inclusion criteria were a normal aortic root and a morphologically normal aortic valve , not prolapsingnineteen patients ( 38% ) with dissected aortic root but apparently normal aortic valve underwent ascending aorta replacement and reconstruction of the diseased sinuses by biological glue and concomitant resuspension of prolapsing aortic cuspfour patients ( 8% ) with most severe dissection of one or more valsalva sinuses but apparently normal aortic valve underwent replacement of pathological sinuses of valsalva , with a specifically tailored vascular graft to replace the severely affected sinusesone patient ( 2% ) with localized dissection of atherosclerotic ascending aorta underwent adhesion of the aortic wall layers with biological glue , without replacement of ascending aorta . twenty - six patients ( 52% ) underwent ascending aorta replacement alone with prosthetic graft , with proximal anastomosis at the sinotubular junction ; inclusion criteria were a normal aortic root and a morphologically normal aortic valve , not prolapsing nineteen patients ( 38% ) with dissected aortic root but apparently normal aortic valve underwent ascending aorta replacement and reconstruction of the diseased sinuses by biological glue and concomitant resuspension of prolapsing aortic cusp four patients ( 8% ) with most severe dissection of one or more valsalva sinuses but apparently normal aortic valve underwent replacement of pathological sinuses of valsalva , with a specifically tailored vascular graft to replace the severely affected sinuses one patient ( 2% ) with localized dissection of atherosclerotic ascending aorta underwent adhesion of the aortic wall layers with biological glue , without replacement of ascending aorta . echocardiographic follow - up was completed in june 2014 , at a median follow - up time of 50.5 months ( iqr : 25.282.5 , at a maximum of 167 months ) ; all patients had an echocardiographic assessment at least after 3 months after discharge . two - dimensional color doppler transthoracic echocardiography ( tte ) was performed , and standard variables were collected according to the jase / eacvi dataset . the degree of ar was classified in a 4-grade scale . for patients requiring re - operation , data regarding survival and re - operation rate were obtained by the departmental and outpatients clinic database , by phone contacts with referral cardiologist or general practitioners , and by phone call to patient or family . median clinical follow - up time was 55.8 months ( iqr : 17.6 - 93.2 , at a maximum of 167 months ) . data are expressed as mean and standard deviation , or median and iqr ( iqr i - iii ) , when appropriate , or percentage ( % ) . comparison between continuous variables was performed with spearman test ( nonparametric ) , while comparison between groups was tested by mann statistical analysis was computed using spss , version 17.0 for windows ( spss inc . , late all - causes mortality was 18% [ figure 1 ] ; the median interval time between deaths and surgery for aad was 59 months ( iqr : 47104 ) . survival curve of kaplan meier estimate ( all - causes mortality ) after a median of 25 postoperative months ( iqr : 17 - 89 ) , ten patients ( 20% ) underwent re - operation : five ( 10% ) for distal aortic disease ( arch pseudoaneurysm and thoracic endovascular aortic repair ) , and five patients ( 10% ; after 2 , 5 , 7 , 8 , and 9 years ) due to severe recurrent ar . no patients who undergone root re - operation died . due to the small number of patients in redo - group , we could only provide a descriptive analysis of preoperative ar grade , late ar grade , and late aortic root diameter [ figure 2a and b ] . patients with or without proximal re - operation ( 5 vs. 45 , mean standard deviation ) . box ( a ) descriptive analysis of aortic regurgitation grade ; ar = aortic regurgitation . box ( b ) descriptive analysis of late aortic root diameter late echocardiographic data are reported in table 2 . seventy - seven percent of the patients had a preoperative ar grade <3 ; thus , a non - negligible number of patients with an acute significant ar ( 23% with grade 3 ) at the time of surgery underwent conservative aortic valve surgery , in terms of simple ascending aorta replacement ( 40% ) or resuspension of aortic cusp with commissuroplasty / reconstruction of pathological sinuses of valsalva ( 60% ) . late echocardiographic data ar = aortic regurgitation , ivs = interventricular septum , lvedd = left ventricular end - diastolic diameter , lvedv = left ventricular end - diastolic volume , lvef = left ventricular ejection fraction we evaluated the relationship between recurrent ar at follow - up and two variables : preoperative ar and late aortic root diameter . neither preoperative ar nor late aortic root diameter were the significant predictors of recurrent late ar ( p values of 0.14 and 0.09 , respectively ) . we analyzed our population according to the surgical technique : ascending aorta replacement with or without aortic root procedure and aortic valve resuspension . the median preoperative ar grade was 1 ( 13 ) and 1 ( 12 ) , respectively . late recurrent ar grade was significantly higher among patients who underwent cusp resuspension [ 2.29 1.05 vs. 1.58 1.03 , p = 0.028 ; table 3 ] . late all - causes mortality was 18% [ figure 1 ] ; the median interval time between deaths and surgery for aad was 59 months ( iqr : 47104 ) . survival curve of kaplan meier estimate ( all - causes mortality ) after a median of 25 postoperative months ( iqr : 17 - 89 ) , ten patients ( 20% ) underwent re - operation : five ( 10% ) for distal aortic disease ( arch pseudoaneurysm and thoracic endovascular aortic repair ) , and five patients ( 10% ; after 2 , 5 , 7 , 8 , and 9 years ) due to severe recurrent ar . no patients who undergone root re - operation died . due to the small number of patients in redo - group , we could only provide a descriptive analysis of preoperative ar grade , late ar grade , and late aortic root diameter [ figure 2a and b ] . patients with or without proximal re - operation ( 5 vs. 45 , mean standard deviation ) . box ( a ) descriptive analysis of aortic regurgitation grade ; ar = aortic regurgitation . seventy - seven percent of the patients had a preoperative ar grade <3 ; thus , a non - negligible number of patients with an acute significant ar ( 23% with grade 3 ) at the time of surgery underwent conservative aortic valve surgery , in terms of simple ascending aorta replacement ( 40% ) or resuspension of aortic cusp with commissuroplasty / reconstruction of pathological sinuses of valsalva ( 60% ) . late echocardiographic data ar = aortic regurgitation , ivs = interventricular septum , lvedd = left ventricular end - diastolic diameter , lvedv = left ventricular end - diastolic volume , lvef = left ventricular ejection fraction we evaluated the relationship between recurrent ar at follow - up and two variables : preoperative ar and late aortic root diameter . neither preoperative ar nor late aortic root diameter were the significant predictors of recurrent late ar ( p values of 0.14 and 0.09 , respectively ) . we analyzed our population according to the surgical technique : ascending aorta replacement with or without aortic root procedure and aortic valve resuspension . the median preoperative ar grade was 1 ( 13 ) and 1 ( 12 ) , respectively . late recurrent ar grade was significantly higher among patients who underwent cusp resuspension [ 2.29 1.05 vs. 1.58 1.03 , p = 0.028 ; table 3 ] . emergency surgery of aad is a lifesaving procedure ; the main aims are : first , prevention of acute death and second , prevention of late sequelae . thus , many studies evaluated the predictors of death to determine the optimum surgical strategy . in the present study , all - causes mortality in survivors after discharge was 18% with the longest follow - up reaching almost 14 years . long - term complications after aad surgery include aneurysm of the residual aorta , anastomotic pseudoaneurysm , and recurrent ar after conservative approach to the aortic valve and aortic root . in the treatment of acute ar in this scenario , the decreasing hospital mortality and better long - term survival have potentially increased the sequelae of aad , particularly in terms of late recurrence of ar . from this point of view , the role of both clinical and echocardiographic follow - up is crucial . according to our retrospective analysis , freedom from proximal re - operation for aortic root or valve diseases was 90% ; re - operations themselves showed good results in terms of hospital mortality and morbidity . the main issues related to aortic valve repair in aad is avoiding valve prosthesis versus the risk of re - operation for late recurrent ar . reported a freedom from aortic root re - operation of 89% , 5 years after conservative surgery and mazzucotelli et al . in a recent review observed a linearized late mortality rate of 4.7%/patient - year and a late aortic valve reintervention rate after initial repair of 2.1%/patient - year , with a freedom from aortic valve reintervention of 89% at 5 years . conservative aortic valve procedures are , also in our experience , an appropriate surgical approach with good long - term results . however , we have to consider that these results were obtained in a single center on a selected group of patients for which conservative techniques appeared ultimately appropriated . we focused on the pathogenic mechanisms of late ar , especially on aortic root dimensions measured at late tte . our data interestingly revealed that recurrent ar was not associated with the intrinsic parameters of aortic root , thus not just related to the pathological mechanism causing root dilatation . furthermore , several studies stressed the relevance of preoperative ar on patients outcome : pessotto et al . concluded that preservation of the aortic valve is associated to an increased risk of recurrent ar in patients presenting with moderate - to - severe ar ; yamanaka et al . showed that high - grade preoperative ar improved during the postoperative follow - up after valve preservation with root reconstruction ; on the contrary , bekkers et al . demonstrated that the aortic valve preservation in patients with severe preoperative ar was associated with an increased risk of aortic valve re - operation . in our experience , late ar was not related to preoperative ar obtained by tee , furthermore , a worse outcome in patients with high preoperative ar grade was not confirmed ; between the six patients with preoperative severe ar , conservatively treated , only one patient presented moderate recurrent ar at follow - up and another one presented severe ar due to late endocarditis . on the other hand , late ar was significantly influenced by surgical technique ; patients who underwent the aortic cusp resuspension showed a significantly higher ar at the follow - up compared to those who underwent the replacement of the ascending aorta alone . the changes of the aortic root geometry due to the surgical approach , which is obviously affected by the extent of dissection , seemed to play a role in the development of late ar . it is known that aortic competence depends on the correct balance between the elements of the single unit consisting of the aortic root and cusps ; moreover , this unit can undergo long - term changes if conservative techniques are performed . despite the retrospective nature of our study a supposed bystander is the biological glue which was widely used ; since its introduction , surgical mortality decreased because it reinforced fragile tissues , provided hemostasis , and reduced the time required for surgery . however , its cytotoxicity is unknown and several reports described the disadvantages of using glue such as the high rates of re - operation for redissection and recurrent aortic sinus aneurysm formation , the risk of aortic wall necrosis , and the possible increased difficulty of a surgical procedure at re - operation . if the risk of aortic root redissection is well known , data about the effects of glue on late aortic competence are scarce . von oppell et al . concluded that biological glue used to repair dissected sinuses and combined with resuspension aortic valve is associated with a high recurrence rate of ar on follow - up ; other authors registered the high ar incidence after the use of glue . according to our results , the use of glue in the dissected aorta wall of the root , with a higher amount in patients who underwent aortic cusp resuspension , was associated with the greater incidence of recurrent ar . nevertheless , on the basis of our results , we can not blame the glue as a cause of recurrent ar ; rather , its use in the dissected root may be considered as an expression of extremely severe derangement of the aortic wall of the root , secondary to acute dissection itself . the observation that the aortic root diameter is not a causative mechanism for late ar should carry out focalized researches on prospective tte after surgery and dedicated views to measure the aortic annulus and its ratio with prosthesis / sinotubular junction . the first limitation is the retrospective nature of the study and the absence of dedicated echocardiographic views for annulus measurements in all patients . second , we could not analyze data about other surgical techniques , even more invasive on the aortic root , such as the reconstruction of the sinuses of valsalva or david procedures , due to the small number of patients . finally , an intraoperative quantitative evaluation of biological glue will be necessary to confirm our hypothesis about its role in the development of late ar . the first limitation is the retrospective nature of the study and the absence of dedicated echocardiographic views for annulus measurements in all patients . second , we could not analyze data about other surgical techniques , even more invasive on the aortic root , such as the reconstruction of the sinuses of valsalva or david procedures , due to the small number of patients . finally , an intraoperative quantitative evaluation of biological glue will be necessary to confirm our hypothesis about its role in the development of late ar . simple aortic valve resuspension seems to be effective in the long - term period , with low incidence of late ar and re - operation . preoperative ar and aortic root diameter were not the predictors of late ar , while ar is higher in patients who underwent cusp resuspension compared to the only replacement of the ascending aorta , unmasking the possible impact of greater acute derangement of the aortic root secondary to a more severe aad .

objective : in case of concomitant aortic regurgitation ( ar ) valve , sparing operation is considered the first choice in selected patients . the aim of this retrospective clinical and echocardiographic study was to evaluate the long - term survival results of conservative approach and the determinants of recurrent ar.methods:from 2000 to 2011 , fifty patients ( median : 63 years and interquartile range : 5372 ) underwent an aortic valve - sparing procedure for acute aortic dissection , and discharged alive . the long - term clinical and echocardiographic outcome was analyzed.results:late all - causes mortality was 18% ( nine patients ) at a median follow - up of 55.8 months . ten patients ( 20% ) underwent re - operations , five of them for aortic valve / root recurrent disease ; freedom from proximal re - operation was 90% . two - third of the patients had a preoperative ar grade <3 ; a non - negligible number of patients with acute significant ar ( 23% with grade 3 ) at the time of surgery underwent conservative aortic valve surgery . at a median echocardiographic follow - up of 50.5 months , we found no significant correlation between late recurrent ar and ar grade at the time of surgery and the aortic root diameter . late recurrent ar grade was significantly higher in patients who underwent aortic commissures and cusps resuspension than those who underwent lone ascending aorta replacement ( 2.29 1.05 vs. 1.58 1.03 , p = 0.028).conclusions : preoperative ar and late aortic root diameter were not the predictors of late ar . late ar is higher in patients who underwent aortic commissures and cusps resuspension compared to the only replacement of the ascending aorta .

INTRODUCTION METHODS Surgical techniques Echocardiographic analysis Clinical follow-up Statistical analysis RESULTS Mortality and re-operations Predictors of aortic valve regurgitation DISCUSSION Limitations CONCLUSIONS Financial support and sponsorship Conflicts of interest

epidemiological and observational studies indicate hypertension as a major cause of chronic kidney disease ( ckd ) . in fact , hypertension and ckd are strongly connected because hypertension is both a cause and a consequence of ckd . hypertensive patients with ckd present higher morbidity and mortality rates when compared with those with normal renal function [ 3 , 4 ] . additionally , high blood pressure is a predictor of decline of glomerular filtration rate ( gfr ) , and conversely , adequate blood pressure control contributes to preserve renal function [ 57 ] . vascular changes are commonly observed in ckd patients , including reduced arterial elasticity observed in patients with end - stage renal disease [ 8 , 9 ] . fibroelastic intimal thickening , increased extracellular matrix , enhanced collagen density , and vascular calcification seem to contribute to stiffer arteries in ckd patients [ 10 , 11 ] . it has been demonstrated that vascular stiffness may predict adverse cardiovascular outcomes [ 12 , 13 ] . current gold standard for vascular stiffness evaluation is the pulse wave velocity ( pwv ) which requires complex equipment and therefore is not commonly used in clinical practice . recently , a new parameter named ambulatory arterial stiffness index ( aasi ) was proposed for this evaluation [ 15 , 16 ] . this index is derived from the regression slope of the diastolic on systolic blood pressure , using all of the readings during ambulatory blood pressure monitoring ( abpm ) . it has already been reported that aasi presents good correlation with target organ damage and glomerular filtration rate ( gfr ) in essential hypertension [ 1720 ] . moreover , aasi may also correlate with cardiovascular events and mortality [ 21 , 22 ] . on the other hand , there have been few studies investigating this index in hypertensive patients with different stages of ckd . the aim of our study was to evaluate the aasi in nondiabetic hypertensive patients with ckd and in those with normal renal function . a case - control study involving 60 consecutive patients with primary hypertension was carried out in our institution . hypertensive patients with ckd , aged 40 to 75 years and estimated glomerular filtration rate ( egfr ) < 60 ml / min by the modification of diet in renal disease ( mdrd ) equation , were included ( ckd group ) . other 30 patients matched by age ( 2 years ) and gender with the ckd patients were selected from the hypertension outpatient clinic at the same institution . these patients presented systemic arterial hypertension ( sah group ) and serum creatinine less than 1 mg / dl . exclusion criteria were diabetes mellitus , hypertriglyceridemia ( > 400 mg / dl ) , urinary albumin - to - creatinine ratio ( uacr ) > 1000 mg / g , acute renal failure , renal replacement therapy , regular use of anti - inflammatory drugs , and history of myocardial infarction or cerebrovascular disease in the last 6 months . the local ethics committee has previously approved the study protocol , and all participants gave written informed consent . office blood pressure was obtained using an electronic device ( model hem-705cp , omron healthcare inc . patients were seated for 30 minutes before measurement and refrained from smoking and caffeine ingestion in this period of time . three readings , one minute apart , were done , and the average of these measurements was defined as the patient clinic blood pressure . the patients underwent 24-hour abpm in nondominant arm with spacelabs 90207 monitor ( spacelabs inc . , redmond , wa , usa ) , validated by the british hypertension society and the association for the advancement of medical instrumentation protocol . readings were taken every 20 minutes during the day and every 30 minutes at night . the percentage decline in nocturnal blood pressure was calculated as follows for systolic ( sbp ) and diastolic ( dbp ) blood pressures : percentage decline in nocturnal blood pressure = ( daytime blood pressurenight - time blood pressure ) 100/daytime blood pressure . the aasi was calculated from 1 minus the regression slope of diastolic pressure on systolic blood pressure . fasting venous blood was collected from participants to measure total cholesterol , triglycerides ( tg ) , high - density lipoprotein cholesterol ( hdl - c ) , glucose , creatinine ( enzymatic method ) , and uric acid . the low - density lipoprotein cholesterol ( ldl - c ) level was calculated by the friedewald formula ( 8) . egfr was assessed by modified mdrd equation : egfr = 0.741 175 cr age( 0.742 if female ) . c - reactive protein ( nephelometry , bn ii , siemens ag inc , munich , germany ) and morning urinary spot albumin and creatinine ( nephelometry , immage , beckman coulter inc , fullerton , ca , usa ) were also measured . data are presented as mean standard error of mean ( sem ) . for database management and statistical analyses , we used graphpad prism software , version 5.0 ( graphpad software inc . , ca , usa ) . chi - squared test ( for 2 3 tables ) or fisher 's exact test ( for 2 2 tables ) and student 's t - test were used to compare proportions and means , respectively . pearson 's correlation coefficients were used to explore associations between examined continuous variables with parametric distribution . as expected , hemoglobin was significantly lower and serum creatinine and uric acid were significantly higher in ckd group ( table 1 ) . mean egfr by mdrd in the ckd group was 35.3 2.8 ml / min . c - reactive protein and uacr were significantly higher in ckd group ( table 1 ) . the mean office and ambulatory blood pressure readings were similar in both groups ( table 2 ) , although ckd patients needed to use more antihypertensive drugs ( 2.7 0.2 versus 2.2 0.1 , p = 0.0398 ) to obtain blood pressure control . when ckd was compared to sah group concerning antihypertensive treatment , there was no significant difference for diuretics ( 60% versus 70% ) , angiotensin converting enzyme inhibitors / angiotensin receptor blockers ( 86% versus 77% ) and beta blockers ( 40% versus 37% ) . however , use of calcium channel antagonists was significantly more common in ckd patients ( 47% versus 23% , p < 0.01 ) . the mean nocturnal systolic blood pressure fall was lower than 10% in both groups ( 4.0 1.5% in ckd versus 7.6 1.1% in sah , p = 0.0588 ) . there were 40% of dipper patients , 50% of nondipper , and 10% of reverse dipper in sah group . on the other hand , ckd group presented 30% of dipping pattern , 40% nondipping , and 30% reverse dipping ( figure 1 ) . pulse pressure was not different between ckd and sah groups ( 54 2 versus 50 2 mmhg , p = 0.20 ) . the aasi index was significantly higher in ckd patients when compared with sah group ( 0.45 0.03 versus 0.37 0.02 , p = 0.04 ) . correlation tests showed that aasi was positively related to age ( r = 0.38 , p < 0.01 ) , pulse pressure ( r = 0.43 , p < 0.01 ) and inversely related to nocturnal blood pressure fall ( r = 0.28 , p = 0.03 ) ( figure 2 ) . however , when egfr was analyzed among all patients , those with egfr less than 30 ml / min had higher aasi ( figure 3 ) . the results of our study demonstrate that hypertensive patients with ckd presented a higher aasi when compared to those with normal renal function . supporting the concept that aasi is a marker of arterial stiffness , li et al . moreover , in a cohort of 11,291 patients , dolan et al . showed that aasi carried prognostic information , as it was a predictor of stroke and cardiac death . in a japanese study , kikuya et al . also observed that aasi predicted cardiovascular and stroke mortality over and beyond pulse pressure . demonstrated that aasi is a predictor of cardiovascular morbidity and mortality in 547 patients with resistant hypertension . furthermore , some research groups have already reported that aasi presents good reproducibility , with repeatability coefficients close to 60% [ 26 , 27 ] . criticizers say that this index is dependent on pulse pressure and dipping pattern and do not provide new information [ 28 , 29 ] . schillaci et al . studied 515 untreated hypertensive patients and found that aasi was strongly dependent on the degree of nocturnal blood pressure fall and only weakly related to pulse wave velocity . showed that aasi was positively related to urinary albumin excretion and negatively related to estimated creatinine clearance in a population of 168 patients with recently diagnosed hypertension and without drug treatment . studied 142 hypertensive patients without drug treatment and with serum creatinine less than 1.5 mg / dl and demonstrated that patients with high aasi presented lower gfr . this paper also suggested that aasi was a better predictor of gfr decline than 24 h pulse pressure . in 554 hypertensive patients with and without drug treatment , garcia - garcia et al . observed that aasi correlates with egfr , carotid intima - media thickness , and cornell voltage - duration product . elderly people are predisposed to lose arterial elastic laminae and increase collagen deposits in vascular wall [ 10 , 31 ] . this way , assuming aasi as a marker of vascular stiffness interestingly , ckd patients tend to have stiffer vessels when compared to age- and blood - pressure - matched patients with normal renal function [ 31 , 32 ] . beyond traditional risk factors , such as hypertension and dyslipidemia , uremia seems also play a role to this finding . mineral metabolism alterations and arterial calcification are probably relevant mechanisms [ 10 , 33 ] . this may be one hypothesis to explain why aasi was higher in ckd than in sah group despite similar office and ambulatory blood pressure measurements in the present study . moreover , patients with the lowest egfr ( stage 4 , according to the american national kidney foundation ) presented higher aasi . pulse pressure and nocturnal blood pressure fall are two parameters from abpm that correlate with arterial stiffness [ 34 , 35 ] . . showed that hypertensive patients with nondipper pattern presented stiffer vessels when measured by pulse wave velocity , suggesting a relationship between blunted nocturnal blood pressure fall and reduced arterial elasticity [ 35 , 36 ] . these findings corroborate with our study , since nocturnal blood pressure fall and pulse pressure were correlated with aasi . . however , our data strongly implies the value of aasi as a noninvasive tool for hemodynamic evaluation of ckd patient and reinforces the role of abpm in hypertensive patients with renal dysfunction . increased aasi might be one of the pathophysiological changes observed in ckd patients before the progression to end stage renal disease . more studies are needed to support the clinical usefulness of this parameter , but we propose that the software for abpm analysis should include aasi value as a marker of cardiovascular risk assessment in the near future .

ambulatory arterial stiffness index ( aasi ) is a parameter obtained from ambulatory blood pressure monitoring ( abpm ) that correlates with clinical endpoints . the aim of this study was to compare aasi in nondiabetic hypertensive patients with and without chronic kidney disease ( ckd ) . subjects with systemic arterial hypertension ( sah , n = 30 ) with normal renal function , aged 40 to 75 years , were compared to hypertensive patients with ckd ( n = 30 ) presenting estimated glomerular filtration rate ( egfr ) < 60 ml / min by mdrd formula . abpm was carried out in all patients . in ckd group , egfr was 35.3 2.8 ml / min . the mean 24-hour systolic and diastolic blood pressure ( bp ) was similar in both groups . aasi was significantly higher in ckd group ( 0.45 0.03 versus 0.37 0.02 , p < 0.05 ) , positively correlated to age ( r = 0.38 , p < 0.01 ) and pulse pressure ( r = 0.43 , p < 0.01 ) and negatively correlated to nocturnal bp fall ( r = -0.28 , p = 0.03 ) . these findings indicate the presence of stiffer vessels in ckd hypertensive patients .

1. Introduction 2. Methods 3. Results 4. Discussion

pfeiffer syndrome ( ps ) is a rare autosomal dominant congenital disorder , originally described by pfeiffer in 1964 , and is characterized by an acrocephalic skull , regressed midface , syndactyly of hands and feet , and broad thumbs and big toes , with a wide range of variable severity ( 1 - 3 ) . ps is known to be caused by mutations in exons iiia or iiic of the fibroblast growth factor receptor ( fgfr ) 2 or fgfr 1 gene ( 4 - 7 ) . we report with a review of literature the first neonate in korea with ps who had bicoronal craniosynostosis , bilateral syndactyly of the fingers and toes , and broad thumbs and big toes . a girl was delivered by spontaneous normal vaginal delivery at 38 weeks gestation after an unremarkable pregnancy . she was 3.5 kg in weight ( 25 - 50 percentile ) , the head circumference was 33 cm ( 25 - 50 percentile ) , and the height at birth was 52 cm ( 25 - 50 percentile ) . her mother is a gravid 4 , para 3 , 40 yr - old korean woman . the patient is the 2nd among 3 children and is the only affected child . upon birth , the infant demonstrated bilaterally fused coronal sutures , mild proptosis , and maxillary hypoplasia ( fig . the soft tissues of her 2nd , 3rd and 4th fingers were fused bilaterally to the proximal portion of distal phalanx and her bilateral thumbs were broad and deviated radially ( fig . computed tomogram ( ct ) and magnetic resonance imaging ( mri ) studies of the cranium revealed no other abnormalities except for the presence of craniosynostosis . three - dimensional reconstruction ct showed 3-dimensional virtual images of the bilaterally fused coronal sutures ( fig . 2a ) but that the sagittal and lambdoid suture were open more than normal to compensate for the small intracranial space , and resulting overall in a brachycephalic head shape ( fig . plain radiography of her hands and feet showed no bony fusion between the phalanges ( fig . three - dimensional ct image of choanal air airway showed small and narrow airway of choanal space ( fig . 4 ) . she did not have any visceral abnormality upon ultrasonographic examination of the abdomen and heart . the patient had a decompressive endoscopic tunnel craniectomy at 4 weeks of age , and is planned for second surgical correction of craniosynostosis , followed by additional correction procedure for the finger and craniofacial anomalies at a later date . the exact incidence of ps is unknown , but is expected to be 1 in every 100,000 births in the western population , and approximately 60 cases to date have been reported in the past world - wide literature ( 8 , 9 ) . however , it is rarer in asian population : only several cases were reported in japan ( 10 , 11 ) and no reports as yet in korea . cohen ( 12 ) in 1993 classified this syndrome into 3 clinical subtypes and suggested that these subtypes might not be classified as separate entities , even though these classifications have important diagnostic and prognostic implications . type 2 consists of a cloverleaf skull with pfeiffer hands and feet , together with ankylosis of the elbows . 3 ps , ocular proptosis is severe , and the anterior cranial base is markedly short . early demise is characteristic of both types 2 and 3 , which to date has been reported to occur only as sporadic cases ( 7 , 9 ) . clinical manifestations of this patient appeared to fit into subtype 1 according to cohen 's clinical classification of ps . the etiology for this syndrome is autosomal dominant or fresh mutations in type i conditions , and sporadic in types ii and iii . some cases are linked to mutations at the fgfr 1 gene at chromosome 8p11.22-p12 ( 4 ) . mutations at the fgfr2 gene , which map at chromosome 10q25-q26 , have also been reported recently ( 9 ) . these mutations are related to the old age of parents , especially the father , because these mutations are suspected to confer a selective advantage to survival of sperm ( 13 ) . usually , the parents are not affected and the risk for future children of that couple is minimal . the offspring of a patient with ps have a 50 - 60% chance of inheriting the syndrome due to the dominant characteristics of this gene . ps may involve craniosynostosis that is most often of the coronal and lambdoid , and occasionally sagittal sutures . a severe form of craniosynostosis in the ps is kleebattschdel ( cloverleaf anomaly ) . the abnormal skull shape could result in a small intracranial volume that increases intracranial pressure , which might be commonly presented as headaches or visual changes . some children with ps may have only one or both mid - face problems and craniosynostosis . the most common features of the face are regressed mid - face and shallow orbits , which may be present at birth but sometimes become more evident as the childhood progresses . underdeveloped maxillary bone of the face results in very shallow orbits and exophthalmos or proptosis . the shallow orbits and proptosis may lead to damage of the cornea from dryness , or exposure keratosis . maxillary hypoplasia also results in a small larynx and pharynx behind the nose and mouth . this restricts the passage of air into the trachea and lungs and causes respiratory distress , particularly at night when snoring and snuffling can interrupt sleep . similarly , the passage of food is restricted , and regurgitation may result in aspiration of food into the lungs . ( 14 ) in 1990 described an infant with ps in whom the trachea showed replacement of the cartilaginous rings by a solid cartilaginous plate extending the full length of the trachea and beyond the carina resulting in tracheal stenosis . some other features commonly seen in these patients are visual disturbances related to an imbalance of the muscle that move the eyes and hearing loss due to recurrent ear infections . vallino - napoli ( 15 ) in 1996 reviewed the audiologic and otologic features of 9 patients with ps , ranging in age from 2 to 12 yr . seven patients had conductive hearing loss and 1 had mixed loss ; none had purely sensorineural loss . primary ct findings were stenosis and/or atresia of the external auditory canal , hypoplasia of the middle ear cavity , and an enlarged middle ear cavity . ( 3 ) reported upper airway compromise in three of four patients with ps type 2 , and five of seven patients with ps type 3 , and cited this as the cause of death in two cases . mccarthy et al . ( 16 ) found upper airway anomalies in 5 of 15 patients with ps of unspecified severity , and these were the primary cause of death in two patients . congenital anomalies of the upper airway are clearly an important cause of morbidity and mortality in ps type 2 and 3 but are rare in ps type 1 ( 14 ) . the hands and feet in ps are involved to variable degree ( 17 - 19 ) . . patient may have mild soft tissue webbing of syndactyly between the second , third , and fourth digits of either or both hands and feet . the digits may be short and misshapen . whilst the fusion of the fingers and toes in apert syndrome always involves fusion of the soft tissues of the second , third and fourth digits , this manifestation is similar with ps , but often there is fusion of the bones as well as soft tissues in apert syndrome ( 17 ) . other anomalies in ps could include a cleft palate , choanal atresia , tracheo- and broncho - malacia , cloverleaf skull , fused vertebrae , arnold - chiari malformation , hydrocephalus , and imperforate anus . the above case presented by authors is in accordance with type 1 ps because the infant had a craniosynostosis only of bilateral coronal suture and demonstrated broad and radially deviated bilateral thumbs and big toes that are characteristic of ps , even though she also had syndactyly of the bilateral 2nd , 3rd and 4th fingers only with soft tissue webbing . the diagnosis of the apert syndrome is appropriate rather than ps if a patient has broad thumbs and syndactyly with bony fusion ( 17 ) or syndactyly of the second , third and fourth web spaces ( 19 ) . however , clinical differential diagnosis has been confusing until now in relation with genetic findings because pfeiffer mutation has been reported to occur in a patient with apert syndrome ( 20 ) , and identical mutations in the fgfr2 gene could cause both ps and crouzon syndrome phenotypes ( 21 ) as illustrated by the demonstration of both phenotypes of apert and of ps in experimental mice ( 22 ) . some authors have attempted to revise the classification for apert , carpenter , crouzon , jackson - weiss , pfeiffer and saethre - chotzen syndromes on a genetic basis . because of widely intermingling of phenotypic expressions or genetic sharing of the same mutation , as yet there is no definite evidence that phenotypic expressions correlate accurately with genetic mutations ( 10 , 20 - 24 ) . this is probably because the gene that controls these expressions has not been elucidated to date ( 25 ) . the prognosis depends on the severity of associated anomalies , mainly the severity of the central nervous system compromise . patients with types 2 and 3 usually expire early in infant or childhood even though some may survive with aggressive medical and surgical management ( 5 ) . multiple staged surgeries are the general treatment plan for patients with ps . in the first year of life it is preferred to release the synostotic sutures of the skull to allow adequate cranial volume and thus allowing for brain growth and expansion . in this patient , we performed a decompressive endoscopic craniectomy at 4 weeks of age for decompression of the high intracranial pressure due to coronal synostosis and maxillary hypoplasia . secondary decompressive skull remodeling is planned at 4 - 6 months of age , and additional skull expansion surgery would be repeated as the child grows to about 2 or 3 yr of age . although significant malformation of the fingers and toes are present , usually these function adequately and do not require the surgical correction . however , this patient had soft tissue webbing between the fingers and toes , and thus will require surgical dividing procedure after infancy for future active physical therapy . mid - facial advancement may be performed in order to provide an adequate orbital volume and reduce the exophthalmos , and to correct the dental occlusion to achieve appropriate functional position , but these procedures are not common in patients with ps . this patient also had severe choanal hypoplasia with respiratory distress from maxillary hypoplasia that will be corrected with mid - facial advancement at 5 - 6 yr of age .

pfeiffer syndrome is as rare as apert syndrome in the western population . this condition is very rare in the asian population and has not been previously reported in korea . the authors report with a review of literature the case of a newborn baby with pfeiffer syndrome , manifested by bicoronal craniosynostosis , broad thumbs , and big toes . the infant also had bilateral syndactyly of the fingers and toes , mild proptosis , choanal hypoplasia and maxillary hypoplasia .

INTRODUCTION CASE REPORT DISCUSSION

male landrace pigs ( 19 to 26 kg ) were pre - dosed orally with aspirin and clopidogrel 24 h before surgery , and they were maintained on this dual antiplatelet therapy throughout the study to reduce the risk of in - stent thrombosis . vascular access was obtained by femoral artery cutdown and the insertion of a 6-f transradial sheath ( arrow international , reading , pennsylvania ) . coronary angiography was performed before the deployment of either bms ( gazelle , biosensors europe sa , morges , switzerland ) or biolimus a9 eluting stents ( biomatrix flex , biosensors europe sa ) to achieve a target ratio of stent to artery diameter of 1.2:1 . animals were euthanized after 7 or 28 days . in the murine model , a stainless steel stent ( 5-cell , 2.5 mm 0.8 mm ; strut thickness 0.06 mm ; cambus medical , galway , ireland ) was crimped onto a 1.20-mm 8-mm mini - trek balloon angioplasty catheter ( abbott vascular , abbott park , illinois ) and deployed ( 10 atm for 30 s ) into the thoracic aorta before engraftment into a recipient mouse . mice were allowed to recover in heated chambers for 24 h and were returned to normal housing conditions , where they were maintained on aspirin - supplemented water and a normal chow diet for another 28 days before being killed . murine aortas were harvested from male mice 8 to 12 weeks of age , and vascular smcs were isolated and cultured . bone marrow derived macrophages ( bmdm ) were generated and bone marrow cells isolated from femurs and tibiae of wild - type ( wt ) and mir-21 knockout ( ko ) mice . flow cytometry was performed by using a bd facscanto ii or lsr ii ( bd biosciences , san jose , california ) . global profiling for mirnas in the control unstented arteries and stented porcine coronary arteries was performed with mirna quantification by using real - time polymerase chain reaction . for analysis , fold changes for each mirna were normalized to u6 because this mirna was the most suitable endogenous mirna in porcine tissue . data analysis was performed by using sds software version 2.3 ( applied biosystems , carlsbad , california ) , and the baseline and threshold were automatically set . data were normalized and then analyzed to identify mirnas differentially expressed between the control ( unstented ) arteries and arteries subjected to stenting for 7 or 28 days . visual assessment was used to check for any lack of normality ; because there was no evidence of this , 1-way analysis of variance followed by a tukey multiple comparison test ( for comparison of > 2 groups ) or student t test ( for comparison of 2 groups ) were conducted . for all the quantitative polymerase chain reaction experiments , values are expressed as fold - change . all statistical analyses were conducted by using prism version 4 ( graphpad software , san diego , california ) . comparisons of in vitro smc proliferation and migration were performed by using 2-way analysis of variance and bonferroni post - hoc tests . all animal procedures were performed in accordance with the united kingdom home office guidance on the operation of the animals ( scientific procedures ) act 1986 and institutional ethical approval . the effects of bms and des on isr were assessed in pig coronary arteries by using optical coherence tomography imaging ( figures 1a to 1d ) . the des reduced neointima formation by 33% compared with bms at 28 days ( figure 1a ) , leading to a significantly larger luminal area ( figure 1b ) with no difference in stent expansion ( figure 1c ) . 116 mirnas were differentially regulated in the bms group , with 23 mirnas remaining dysregulated at 28 days . at 7 days , multiple mirnas associated with inflammatory cell infiltration and activation were altered ( online table 1 , online figure 1 ) . of note , mir-21 - 3p was substantially up - regulated in both bms- and des - treated arteries at 7 days , suggesting that the mir-21 stem loop ( i.e. , both lead and passenger strands ) may be important post - stenting . the expression profile of mir-21 - 3p and mir-21 - 5p were validated , and this revealed that mir-21 - 5p was significantly up - regulated in stented arteries at 7 days regardless of stent type and remained up - regulated at 28 days compared with unstented control arteries ( figure 2a ) . mir-21 - 3p was also up - regulated at 7 and 28 days post - stenting in both groups ( figure 2b ) . we then identified the localization of expression of the mir-21 axis in the vessel wall after stenting , focusing on mir-21 - 5p ( because the absolute levels of mir-21 - 3p are lower and below the detection threshold for in situ hybridization ) . in control coronary arteries , mir-21 - 5p was detected within the media ( figure 2c ) . in injured vessels 28 days post - stenting , increased signal intensity was observed in the medial layer and developing neointima , particularly around stent struts . to determine if this expression pattern is maintained in the clinical setting , in situ hybridization was performed in human coronary arteries 14 days post - stenting from tissue obtained from a heart transplant patient . in concordance with the staining pattern in porcine arteries , abundant mir-21 expression was observed in the developing neointima and infiltrating inflammatory cells ( figure 2d , online figure 2 ) . taken together , these findings suggest that mir-21 - 5p and mir-21 - 3p may be important in the development of post - stent pathophysiological responses to injury . hybridization confirmed abundant mir-21 - 5p staining around the stent struts and developing neointima ( figure 3 ) , consistent with the porcine data . morphometric analysis found that mir-21 ko mice stent grafts had a reduced neointimal area , neointimal thickness , and neointima - to - medial ratio compared with wt controls ( 1.37 0.18 [ n = 8 ] vs. 2.11 0.17 [ n = 9 ] ; p < 0.05 ) ( figures 4a and 4b , online table 2 ) . measurements of strut depth were concordant with greater strut depth in wt - stented grafts than in ko - stented grafts . a significant reduction in luminal area in wt mice compared with mir-21 ko mice was also observed ( figures 4c and 4d , online table 2 ) . there were no apparent differences in luminal , media , or total vessel area between wt and mir-21 ko mice at baseline ( before stent injury ) , although an increased sample size would be needed to confirm this observation ( online table 3 ) . furthermore , the total vessel area did not differ significantly at 28 days ( figure 4e ) , and no differences were observed in unstented vessels at baseline ( online table 3 ) . the neointimal lesions from mir-21 ko mice contained significantly less -actin positive smcs ( 28 2.4% [ n = 8 ] vs. 14 3.7% [ n = 9 ] ; p < lesions in wt mice contained 50% more elastin than in ko mice ( figures 5c and 5d , online table 2 ) . no difference in cell proliferation was observed after quantification of cells in the neointima ( figures 5e and 5f , online table 2 ) . we assessed vessel reendothelialization and , importantly , found no significant difference between mir-21 ko and wt mice ( 91.0 3.9% vs. 88.7 2.9% , respectively ) ( figures 5 g and 5h ) . no differences were observed at baseline ( i.e. , unstented vessels ) ( online table 3 ) . to explore the mechanisms responsible for the reduction in neointima formation in mir-21 ko mice , proliferation and wound healing assays were performed . the proliferative response of mir-21 ko smc was dramatically attenuated in response to platelet - derived growth factor ( pdgf ) ( figure 6a ) , as was migration in response to pdgf ( figures 6b and 6c ) . to identify potential targets responsible for these effects , we stimulated aortic smc from mir-21 wt and ko mice with pdgf ( n = 5 ) to elevate mir-21 levels ( 3.03 0.23-fold vs. controls ; p < the messenger rna levels of known mir-21 targets with defined roles in smc proliferation and migration were profiled . the expression of programmed cell death protein 4 ( pdcd4 ) and signal transducer and activator of transcription 3 were significantly reduced in aortic smc from wt mice after pdgf stimulation . these changes were not observed in mir-21 ko cells stimulated with pdgf ( figures 6e and 6f ) ; however , further experiments are required to determine whether repression of these genes is direct or indirect via mir-21 regulation in this setting . mir-21 ko mice contained greater numbers of galactin-3 + ( mac-2 ) macrophages in the neointima compared with wt mice ( 0.79 0.23% vs. 2.54 0.75% ; p < 0.05 ) ( figures 7a and 7b ) and enhanced levels of ym-1positive macrophages , a validated murine marker of the alternatively activated ( m2 ) macrophage ( 1.93 0.54% vs. 0.50 0.21% ; p < 0.01 ) ( figures 7c and 7d ) . thus , loss of mir-21 results in altered inflammatory cell phenotype within injured vessels . to investigate whether these effects were derived from any hematological defect , before cell recruitment to the vessel wall , the populations of immune cells in both bone marrow and blood of wt and mir-21 ko mice were examined ( 16 ) . in bone marrow , the percentage of cluster of differentiation ( cd ) 3 + t cells was significantly reduced in mir-21 ko mice , but neutrophils , monocytes , b cells , cd4 + , and cd8 + t cells did not differ ( online figures 3a and 3b ) . in blood , mir-21 ko mice exhibited a significantly reduced percentage of circulating ly6c+hi monocytes and cd3 + t cells ( figures 7e and 7f ) , potentially indicating a reduced capacity to develop proinflammatory responses . to investigate whether the absence of mir-21 leads to altered macrophage differentiation , bmdms from wt and mir-21 ko mice were generated and treated with either lipopolysaccharide ( lps ) or interleukin ( il)-4 in vitro to induce m1 and m2 polarization , respectively . both lps and il-4 significantly up - regulated the expression of mir-21 - 3p and mir-21 - 5p in wt macrophages ( 3.00 0.23-fold , 4.38 0.91-fold , 4.74 0.13-fold , and 4.88 0.7-fold , respectively ; p < 0.05 ) , indicating that inflammatory mediators can modulate the expression of mir-21 ( figure 8a ) . at baseline , unstimulated ( m0 , nonpolarized ) mir-21 ko macrophages had significantly higher levels of peroxisome proliferator activated receptor ( ppar)- expression ( m2 polarization marker ) than wt cells ( p < 0.01 ) ( figure 8b ) . after activation with lps , expression of nitric oxide synthase ( an m1 marker ) was significantly reduced in mir-21 ko versus wt macrophages ( figure 8c ) . in addition , the ratio of messenger rna expression of nitric oxide synthase , compared with arginase 1 ( an m2 marker ) , was significantly higher in wt than mir-21 ko macrophages treated with lps ( 20.6 6.52-fold vs. 2.7 1.24-fold ; p < 0.001 ) ( figure 8d ) . flow cytometric analysis demonstrated that after activation with lps , cd69 was significantly reduced in mir-21 ko versus wt lps - treated macrophages ( 1.8-fold ; p < 0.05 ) ( figure 8e ) . however , all other markers examined by using flow cytometry ( major histocompatibility complex - ii , cd11c , cd86 , cd206 , and toll - like receptor-2 ) were not significantly different in either lps- or il-4treated wt or mir-21 ko bmdm ( online figure 4 ) . the levels of several proinflammatory mediators were significantly reduced from lps - stimulated macrophages of mir-21 ko compared with wt mice : il-1 , il-1 , il-6 , il-12 , tumor necrosis factor- , and macrophage inflammatory protein-1 ( figure 8f ) . in addition , the il-12/il-10 ratio for lps - stimulated mir-21 macrophages was almost double that of ko macrophages ( 24 vs. 13 ) . to address whether mir-21 ko macrophages had a reduced capacity to infiltrate and migrate into vascular lesions after injury , we studied bmdm migration through matrigel - coated transwell inserts containing 8-m pores ( sigma , united kingdom ) . these experiments showed that both bmdm isolated from wt and mir-21 ko mice had the same capacity to migrate through a matrix membrane in response to monocyte chemoattractant protein-1 ( figure 8 g ) . we report for the first time mirna patterns associated with delayed arterial healing and neointima formation after stenting ( central illustration ) . of particular interest , the stem loop of mir-21 , including lead ( mir-21 - 5p ) and passenger ( mir-21 - 3p ) strands , was up - regulated in both the bms and des groups compared with controls . further experiments highlighted that loss of the mir-21 stem loop in ko mice blocked neointimal formation through effects on smc proliferation and migration , macrophage polarization , and inflammatory activation . a causal network analysis was performed of mir-21 - 3p and mir-21 - 5p targets to determine whether these mirnas affect the inflammatory networks associated with vascular injury . despite a relatively small overlap of predicted genes , pathways associated with tumor necrosis factor- and il-1 were 1 of the top - ranking regulatory networks ( online figure 5 ) ; this finding may be relevant because both of these cytokines play prominent roles in neointima formation ( 17,18 ) . mir-21 ko mice exhibited reduced smc deposition , neointima formation , and an altered inflammatory phenotype , resulting in enhanced levels of anti - inflammatory m2 macrophages in response to vascular injury and stenting , with no effect on endothelial regeneration . subsequent profiling of immune cell populations in the blood of mir-21 ko mice demonstrated reduced numbers of ly6c+hi cells , a cell type that can differentiate into m1 macrophages after tissue infiltration . this finding suggests that mir-21 ko mice have a reduced capacity to develop an m1 inflammatory phenotype in response to injury . we also found reduced total cd3 + t - cell counts in the bone marrow and blood of the mir-21 ko . recently , it has been reported that mir-21 can modulate t - cell responses , including alterations in cytokine production and apoptosis rates ( 1921 ) . t cells are already known to contribute to the inflammatory response after coronary artery stenting ( 22 ) ; thus , the absence of mir-21 in t cells likely contributes to the altered inflammatory responses and reduced in - stent stenosis seen in mir-21 ko . in support of the altered inflammatory responses in vivo , our bmdm experiments revealed that mir-21 ko macrophages contain enhanced baseline levels of ppar- , a well - characterized m2 macrophage marker , and a reduced ratio of nitric oxide synthase / arginase 1 ( m1/m2 markers ) . these findings are particularly relevant in the setting of inflammatory vascular disease because numerous studies suggest that ppar- activation can curtail the inflammatory response . activation of ppar- in human macrophages reduces matrix metallopeptidase 9 activity and inhibits expression of il-1 , il-6 , and tumor necrosis factor- ( 2325 ) . furthermore , several groups have reported that ppar- agonists inhibit atherosclerosis development and reduce inflammatory markers in apolipoprotein e ko mice ( 26,27 ) . furthermore , mir-21 ko macrophages exhibited a reduced capacity to secrete proinflammatory mediators such as il-1 , tumor necrosis factor- , macrophage inflammatory protein-1 , il-6 , and il-12 in response to lps , a substance known to stimulate the inflammatory m1 macrophage phenotype . however , a defective response to lps does not necessarily mean altered polarization and may simply reflect , for example , defective cd14 expression . further phenotyping of surface receptors from bmdm also showed reduced expression of cd69 in mir-21 ko mice . a cd69 deficiency may contribute to reduced inflammatory cytokine secretion , as previous studies have reported that cd69 activation mediates numerous inflammatory processes such as nitric oxide production and release of tumor necrosis factor- from murine macrophages and t cells ( 28,29 ) . thus , loss of mir-21 may accelerate wound healing and resolution of the inflammatory response after vascular injury and stenting , events that could reduce the incidence of late stent thrombosis . it is important to note that we used human arrays in the porcine samples to identify mirnas that would extrapolate to the pathology in the clinical setting . it is possible that a proportion of mirnas may be underrepresented due to the sequence variation or chromosomal locations between pig and human . we identified an almost 20-fold up - regulation of mir-21 - 3p after stenting with bms and des . our subsequent experiments in bmdm demonstrated that both mir-21 - 3p and mir-21 - 5p were up - regulated in response to lps , which induces classical macrophage polarization ( m1 ) . in combination with reduced inflammatory cytokine release from mir-21 ko macrophages , these results suggest that mir-21 - 3p and mir-21 - 5p may both play a pathological role in macrophage activation in response to inflammatory stimuli . we observed that mir-21 plays a prominent role in smc proliferation and migration in response to vascular injury , consistent with other studies ( 30,31 ) . aortic smc isolated from mir-21 ko mice had a reduced capacity to migrate and proliferate in response to pdgf , an important observation because novel des must retain the antiproliferative effect on smc accumulation to maintain their clinical efficacy . these findings are consistent with previous reports demonstrating that pharmacological or genetic knockdown of mir-21 reduces smc proliferation and neointima formation after balloon injury or vein grafting ( 3033 ) . several of these reports suggest that mir-21 mediates a beneficial effect on smc proliferation and neointima , at least in part , via inhibition of phosphatase and tensin homolog . we also found that the levels of pdcd4 were suppressed after pdgf exposure in wt mice . importantly , the levels of pdcd4 were increased in mir-21 ko mice , suggesting that pdcd4 is modulated after exposure to pdgf and plays a role in smc proliferation and migration , effects repressed in mir-21 ko smcs . in concordance with these findings , previous studies suggest that pdcd4 is down - regulated after vascular injury , and overexpression of pdcd4 with adenoviral vectors increases apoptosis and reduces smc proliferation ( 34 ) . although the present study demonstrates that mir-21 play a prominent role in the pathology of in - stent restenosis , it is important to note that these studies are based on data from pre - clinical animal models of restenosis . although our results in mir-21 knockout mice demonstrate that loss of mir-21 reduces in - stent restenosis and inflammatory cell function , it is important to note that these defects are present before vascular injury , these deficiencies may alter the response to injury in these mice . in order to demonstrate that these finding have efficacy in the clinic , further studies are required to demonstrate that pharmacological knockdown of mir-21 or mir-21 targets can inhibit neointimal formation and vessel inflammation from current drug - eluting stent platforms . furthermore , detailed pharmacokinetic profiling would be needed to demonstrate an effect elution profile of antimir-21 therapy , without any off - target effect . although the present study demonstrates that mir-21 play a prominent role in the pathology of in - stent restenosis , it is important to note that these studies are based on data from pre - clinical animal models of restenosis . although our results in mir-21 knockout mice demonstrate that loss of mir-21 reduces in - stent restenosis and inflammatory cell function , it is important to note that these defects are present before vascular injury , these deficiencies may alter the response to injury in these mice . in order to demonstrate that these finding have efficacy in the clinic , further studies are required to demonstrate that pharmacological knockdown of mir-21 or mir-21 targets can inhibit neointimal formation and vessel inflammation from current drug - eluting stent platforms . furthermore , detailed pharmacokinetic profiling would be needed to demonstrate an effect elution profile of antimir-21 therapy , without any off - target effect . the mir-21 stem loop plays an important role in smc and macrophage activation after vascular injury . our findings in the murine model of isr revealed that loss of mir-21 attenuates neointima formation and macrophage activation , resulting in a less inflammatory phenotype . these findings suggest that mir-21 modulation could enhance wound healing and resolve the inflammatory response , effects that could improve the clinical efficacy of currently available des . the mir-21 axis warrants further investigation as a therapeutic target in the setting of stent - induced inflammatory vascular disease . lineage - restricted knockouts maybe required to unravel the complex role of mir-21 in this setting.perspectivescompetency in patient care : des reduce in - stent restenosis after percutaneous coronary intervention but are associated with a greater risk of stent thrombosis due to delayed arterial healing that is characterized histologically by incomplete reendothelialization and persistent fibrin and inflammatory cell deposition . an array of microrna molecules are involved in the inflammatory processes driving the cellular response to vascular injury , and genetic ko of the mir-21 stem loop attenuates neointimal formation after arterial stenting in mice.translational outlook : future investigations should seek to determine whether local antimir to knockdown mir-21 levels delivered directly from drug - eluting stents could reduce vessel inflammation and neointima formation and reduce the risk of both restenosis and stent thrombosis . competency in patient care : des reduce in - stent restenosis after percutaneous coronary intervention but are associated with a greater risk of stent thrombosis due to delayed arterial healing that is characterized histologically by incomplete reendothelialization and persistent fibrin and inflammatory cell deposition . an array of microrna molecules are involved in the inflammatory processes driving the cellular response to vascular injury , and genetic ko of the mir-21 stem loop attenuates neointimal formation after arterial stenting in mice . translational outlook : future investigations should seek to determine whether local antimir to knockdown mir-21 levels delivered directly from drug - eluting stents could reduce vessel inflammation and neointima formation and reduce the risk of both restenosis and stent thrombosis .

backgrounddrug - eluting stents reduce the incidence of in - stent restenosis , but they result in delayed arterial healing and are associated with a chronic inflammatory response and hypersensitivity reactions . identifying novel interventions to enhance wound healing and reduce the inflammatory response may improve long - term clinical outcomes . micro ribonucleic acids ( mirnas ) are noncoding small ribonucleic acids that play a prominent role in the initiation and resolution of inflammation after vascular injury.objectivesthis study sought to identify mirna regulation and function after implantation of bare - metal and drug - eluting stents.methodspig , mouse , and in vitro models were used to investigate the role of mirna in in - stent restenosis.resultswe documented a subset of inflammatory mirnas activated after stenting in pigs , including the mir-21 stem loop mirnas . genetic ablation of the mir-21 stem loop attenuated neointimal formation in mice post - stenting . this occurred via enhanced levels of anti - inflammatory m2 macrophages coupled with an impaired sensitivity of smooth muscle cells to respond to vascular activation.conclusionsmir-21 plays a prominent role in promoting vascular inflammation and remodeling after stent injury . mirna - mediated modulation of the inflammatory response post - stenting may have therapeutic potential to accelerate wound healing and enhance the clinical efficacy of stenting .

Methods Results Discussion Study limitations Conclusions Appendix

the genetic events and their relationship to the complex interaction with the microenvironment transforming normal melanocytes into melanoma are under intensive investigation . in the last decade melanoma was dissected into several molecular subgroups on the basis of genomic alterations , including mutations , deletions and amplifications , in addition to clinical features . first , up to 50% of melanomas derived from the skin without chronic sun damage ( intermittently exposed to ultraviolet ( uv ) ) contain mutations in the gene encoding the serine threonine protein kinase braf . braf together with araf and craf activates a second protein known as mitogen - activated protein kinase kinase ( mek ) , which in turn activates extracellular signal - regulated kinase ( erk ) . most of the nras mutated melanomas are superficial , spreading melanomas ( intermittently exposed to uv ) . however , nras seems also to be involved in melanomas deriving from giant congenital nevi . a recently published model for congenital nevi used a melanoma mouse model over - expressing nras under the control of a tyrosinase promoter in combination with loss of ink4a . finally , minor percentages have activating mutations in the kit gene , most common in mucosal melanomas derived from the genital regions or mutations in gna11 or gnaq genes in uveal melanomas . some of the targetable mutations in the kit gene are also found in acral and other mucosal ( for example , penile or anal ) melanomas but with lower frequency . the kit receptor protein tyrosine kinase is a transmembrane protein consisting of extracellular and intracellular domains . most kit mutations are located in exon 11 , which codes for the juxtamembrane domain , and in exon 13 , which codes for a kinase domain . recently , deep exome sequencing shed further light on the genomic landscape of melanoma . both publications impressively demonstrated that uv light is responsible for most mutations in melanomas derived from uv - exposed skin . hodis et al . reported that more than 80% of all non - silent coding mutations are uvb - dependent . in addition , both papers provide evidence that rac1 might be another driver mutation for melanoma in addition to braf and nras . rac1 is a member of the rho family of gptases and therefore shares some features with the ras oncogenes . these molecules might contribute as a feedback mechanism during the activation of signalling pathways , and defects in their function may contribute to tumour initiation and progression . . it will be crucial to identify driver mutations that are promising targets for therapy in this huge landscape of genetic alterations . there are also relevant mutations in non - coding dna sequences such as promoter regions . huang et al . described two independent mutations in the promoter of telomerase reverse transcriptase ( tert ) . keeping these data in mind , we can expect many more surprising discoveries using these powerful techniques in the near future . the best - validated targeted drugs in melanoma are the selective braf inhibitors vemurafenib ( plx4032 , zelboraf ) and dabrafenib ( gsk2118436 , tafinlar ) as well as the lgx818 ( novartis ) compound that appears to have the highest affinity for the catalytic domain of the kinase . all of them are relatively selective for their intended target v600e braf , with little cross - reactivity for wild - type braf and craf . a few other kinases are inhibited with 10- to 100-fold of the concentration needed to inhibit v600 braf . these molecules selectively inhibit the growth of cells that harbour a v600 braf mutation . in phase i clinical trials , where patients were selectively enrolled on the basis of the presence of a tumour braf or braf mutation , vemurafenib and dabrafenib both demonstrated evidence of tumour regression early in the course of therapy in the majority of patients [ 1113 ] . subsequent phase ii and iii trials designed to evaluate overall disease control and survival in comparison with standard chemotherapy have documented the durability of response in larger cohorts . in a phase ii trial , vemurafenib produced objective responses in 53% of 132 patients with metastatic melanoma harbouring a braf or braf mutation . the median duration of response was 6.7 months . in a phase iii trial with single - agent dacarbazine as the control arm , overall survival ( os ) was significantly improved amongst 337 patients with braf mutant metastatic melanoma receiving vemurafenib compared with 338 patients who received dacarbazine ( hazard ratio ( hr ) 037 ; 95% confidence interval ( ci ) : 0.260.55 ; p < 0.001 ) . progression - free survival ( pfs ) was also significantly improved ( hazard ratio 0.26 ; 95% ci : 0.200.33 ; p < 0001 ) and the response rate was superior in the vemurafenib arm ( 48% objective response rate versus 5% ; p the benefit of vemurafenib was maintained in an updated overall survival ( os ) analysis with approximately 10 months median follow - up , as demonstrated by the median os with vemurafenib of 13.2 months compared with 9.6 months with dacarbazine and a hazard ratio ( hr ) for death of 0.62 ( 95% ci 0.490.77 ) in favour of vemurafenib . these data led to approval of vemurafenib in the united states ( us ) , european community and switzerland . dabrafenib was compared with dacarbazine ( random ratio 3:1 ) in a phase iii trial in patients with previously untreated stage iv or unresectable stage iii melanoma harbouring the brafv600 mutation . median pfs was 5.1 months for dabrafenib ( 187 patients ) and 2.7 months for dacarbazine ( 63 patients ) , with an hr of 0.30 ( 95% ci : 0.180.51 ; p < 0.0001 ) . this drug was recently approved by the food and drug administration ( fda ) in the us . in summary , vemurafenib and dabrafenib have both demonstrated impressive clinical efficacy with response rates in the region of 50% in v600 braf mutated advanced melanoma . although the response duration is highly variable , as shown by these phase ii and phase iii trials , these results are a breakthrough in melanoma treatment . furthermore , multiple in vitro studies have demonstrated that mutated braf signalling is mediated via mek and erk . thus , selective mek inhibitors have also shown efficacy in patients with braf mutant metastatic melanoma . selumetinib was the first allosteric , selective mek inhibitor to be evaluated in a phase ii clinical trial in patients with metastatic melanoma . this agent produced an objective response rate in patients with braf mutant tumours , whereas no response was observed in wild - type tumours , reinforcing the importance of selecting a specific patient population . the addition of selumetinib to dacarbazine has resulted in prolonged pfs in braf mutated metastatic melanoma and was the first agent to show clinical activity in uveal melanoma when compared to temozolomide . it demonstrated a reasonable objective response rate and an improved survival compared to chemotherapy in braf mutant melanoma . the median pfs was close to 5 months using the mek inhibitor in comparison to 1.5 months in the chemotherapy group . after 6 months , there was an improvement in the overall survival rate in the trametinib group of 81% ( versus 67% in the chemotherapy group ) . trametinib ( mekinist ) was recently approved by the fda for the treatment of patients with unresectable or metastatic melanoma with braf v600e or v600k mutation . the combination treatment with dabrafenib and trametinib was analysed with different dosages in 247 braf v600 mutated melanoma patients . median pfs was significantly improved at 9.4 months for the patients treated with 150 mg dabrafenib twice daily and 2 mg trametinib daily , as compared with 5.8 months for the patients treated with dabrafenib alone ( hr for progression or death , 0.39 ; 95%ci , 0.250.62 ; p < 0.001 ) . the rate of complete or partial response in the combination group was 76% ( compared with 54% with monotherapy ( p = 0.03 ) . in other words , the combination of braf and mek inhibitors results in an increased response rate and prolonged pfs . today , there are several phase iii clinical trials that compare the response rates and the pfs in braf mutated patients for monotherapy with a braf inhibitor versus combination of braf and mek inhibitors . have investigated the erk - dependent feedback mechanism during braf inhibition using a selective inhibitor . they could clearly show that a braf inhibitor blocks raf monomers , resulting in raf dimer formation . the addition of a mek inhibitor can overcome this problem and enhance the inhibition of the pathway and antitumour efficacy . in vitro investigations using nras mutant melanoma cell lines a recent clinical trial using the mek1/2 inhibitor mek 162 in a phase ii clinical trial has confirmed that advanced nras mutant metastatic melanoma can be successfully treated in patients . a response rate of approximately 25% was found , with a pfs similar to that observed using mek inhibitors in braf mutant metastatic disease . other mek inhibitors have been investigated in nras mutated melanomas with some promising results . until recently , most clinical trials investigating immunomodulation , chemotherapy or targeted therapy have excluded patients with brain metastasis due to concerns about drug penetration through the blood brain barrier and symptoms such as intracranial bleeding resulting in life - threatening consequences . lately , the urgent need for medical treatment of this patient population led to a trend change . there are recent data using the anti - ctla-4 antibody ipilimumab at a dose of 10 mg / kg every 3 weeks for four cycles in melanoma patients with brain metastasis , with a response rate of 16% in asymptomatic and 5% in symptomatic patients . a recently reported trial on dabrafenib in asymptomatic patients with braf - mutated melanoma and at least one measurable brain metastasis between 5 mm and 40 mm in diameter has further demonstrated clinical activity . moreover , in a pilot study of 24 symptomatic , very advanced melanoma patients with brain metastasis and harbouring a braf v600 mutation were treated safely and effectively with vemurafenib , improving patients performance status and quality of life . further clinical trials including combined therapies with other inhibitors , with immunotherapy and with stereotactic radiosurgery are needed in the near future . both braf and mek inhibitors have a very peculiar side - effect profile . as recently reported , braf inhibitors are characterised by activating germline mutations of ras and lead to cutaneous side effects recently defined as rasopathic . they involve both epidermis and adnexa and include inflammatory disorders such as maculopapular exanthema , follicular rash or pruritus , hair and nail changes , as well as keratinocytic proliferations such as keratosis pilaris , palmoplantar hyperkeratosis , acanthopapilloma , keratoacanthoma and squamous - cell carcinoma . , vemurafenib causes an important uva - dependent phototoxicity that needs adequate uv protection . its most common adverse events include skin - related toxic effects , fever , fatigue , arthralgia , and headache . mek inhibitors can cause papulopustular rashes , xerosis cutis ( often associated with fissured finger tips ) , diarrhoea , nausea , vomiting , fatigue and blurred vision . moreover , self - limiting retinopathy - like dose - dependent retinal disorders with early onset have been described . however , a close monitoring of the retina with a specific mark on sub - retinal exudates is highly recommended . the cutaneous side effects during mek inhibition are similar to those observed with epithelial growth factor receptor ( egfr ) inhibitors . notably , in a recently conducted trial , cutaneous adverse events were observed in over 85% of the patients . thus , regular skin examination and management by experienced dermatologists , as well as continuous prophylactic photo - protection , including a uva optimised sun screen , are mandatory . the combination of braf and mek inhibitors interestingly seems to reduce the common cutaneous side effects . the impressive progress observed with the use of kinase inhibitors is unfortunately limited by the development of resistance that is observed after 6 months on average using braf inhibitor monotherapy and after 910 months using braf there is intensive research ongoing to understand the mechanisms behind this clinically very relevant phenomenon . it remains unclear which resistance mechanisms are the most frequent in vivo in humans . a recent study using human melanoma xenografts in a nude mouse model has shown that melanoma cells can transcriptionally up - regulate the braf molecule in order to compensate the inhibition by vemurafenib . if vemurafenib is removed from the system , there is an over - stimulation of the pathway resulting in decreased proliferation , probably related to oncogene - driven senescence . as a consequence this observation is interesting and needs to be further investigated in the clinical setting in the near future . in contrast to braf mutated melanoma , the kinase inhibitor imatinib has proven efficacy in patients with advanced melanoma harbouring kit mutations . kit mutations are found at low frequencies ( 10% ) in melanomas arising from mucosal or acral lentiginous surfaces . as the vast majority of patients with metastatic melanoma suffer from primary tumours on glabrous skin ( trunk , extremities , and head / neck ) , durable responses were observed in 16% of a 51-patient cohort with either mutations or amplifications in kit . in a phase ii trial , in which 43 patients with kit mutations or amplification were enrolled , 23% of patients had objective responses . in both studies , certain mutations in exon 11 and 13 of c - kit ( particularly l576p mutation in exon 11 ) were associated with the highest response rate . in addition nilotinib , a tyrosine kinase inhibitor used in imatinib - resistant chronic myelogenous leukaemia ( cml ) , seems another promising agent in the treatment of kit mutated metastatic melanoma and is currently under clinical investigation . thus , sensitivity to kit inhibition exists in metastatic melanoma but it is confined to a subset of this already small subpopulation of patients . after decades of standstill , progress in understanding the biology of melanomas has resulted in powerful targeted therapies with impact on progression - free and overall survival . ongoing research is focused on resistance mechanisms and strategies to overcome them . in order to further improve the outcome in this still poor - prognosis population professor dummer receives research funding from astra zeneca , novartis , cephalon , merck sharp & dhome , transgene , bristol - myers squibb , roche , glaxosmithkline , and bayer , and has a consultant or advisory board relationship with astra zeneca , novartis , cephalon , merck sharp & dhome , transgene , genta , bayer , roche , bristol - myers squibb , glaxosmithkline , and spirig .

melanoma today is considered as a spectrum of melanocytic malignancies characterised by clinical and molecular features , including targetable mutations in several kinases such as braf or c - kit . the successful development of therapies targeting these mutations has resulted in new specific treatment options . these include vemurafenib , dabrafenib , trametinib , imatinib and other kinase inhibitors that are selected when the respective mutation is present.the braf inhibitor vemurafenib has resulted in improved survival in patients with braf - mutated advanced melanoma . dabrafenib has shown similar efficacy . the mek inhibitor trametinib also improved overall survival . in addition , the mek inhibitor mek 162 was investigated in a phase ii clinical trial and showed promising efficacy in terms of response rate and progression - free survival ( pfs ) in nras - mutated melanomas . after this first success in the treatment of advanced melanoma , there is expectation that combinations of kinase inhibitors will additionally improve overall survival rates and pfs in advanced melanoma .

Introduction Molecular dissection of melanoma Breakthrough with kinase inhibitor therapy in melanoma subgroups Conflict of interest statement

enantioseparation ( es ) and enantiodiscrimination ( ed ) are , therefore , mandatory steps in the elucidation of enantiospecific biological functions on the molecular level . interestingly , ed with isotropic solution nmr was first demonstrated by pirkle,1 who also developed enantioseparating chromatographic phases.2 however , since these times , the two areas have evolved increasingly independently . although nmr studies are used to elucidate structural aspects of es,3a the role of enantioselective chromatography as a versatile tool in the understanding of ed obtained in nmr has been largely neglected . since the early report of pirkle and pochapsky in 1986,3b isotropic solutionnmr studies have been used to investigate structural aspects of chromatographic enantiorecognition processes.3c,3d for this purpose , chemical shifts and nuclear overhauser effects ( noes ) were largely used . one recent approach for ed in nmr is the use of chiral anisotropic media.4 in general , anisotropic achiral media permit the measurement of nmr parameters that require a partial alignment of the solute ( e.g. residual dipolar couplings or chemicalshift anisotropies ) . currently , a number of new alignment media that have been developed are becoming very important in the structural analysis of smalltolarge molecules.5 crosslinked acrylamidebased copolymers introduced in 20006a have been further developed into an alignment medium compatible with a wide range of solvents including dmso.6b recently , ed was reached by introducing a chiral , negatively charged monomer : ( r)aphes [ ( 2acrylamide)2phenylethanesulfonic acid , scheme s1 a].7 as expected from work with chiral ionexchange phases,8 ed was only reached for cationic ( protonated basic ) solutes ( erythromefloquine , menthylamine , and strychnine ) . no ed was observed for the neutral compound menthol.7 this approach had been inspired by previous work in the field of enantioselective ( chiral ) liquidphase chromatography . enantioselective chromatographic separation is based on the difference in the retention factor for a pair of enantiomers induced by a small difference in the phasetransfer equilibrium constants , owing to the involvement of diastereomeric associations ( between the chiral solute and the chiral selector ) in the phasetransfer process.8a a special class of stationary phases developed for enantioselective liquidphase chromatography ( chiral ionexchange phases ) employs positively and/or negatively charged immobilized chiral selectors , which can undergo the formation of diastereomeric associates ( ion pairs ) with protonated or deprotonated ( ionisable ) chiral solutes.8b , 8c it is now of interest to investigate whether or not the successful ed observed with a chiral alignment gel used for anisonmr ( nmr spectroscopy under anisotropic conditions ) can be further elucidated by using chromatographic studies employing the same alignment gel as a chiral separation medium in capillary electrochromatography ( cec ) ( figure 1 , and explanations given therein ) . for clarity , this approach should be clearly distinguished from techniques such as nmrce , capillary nmr , and chromatographic nmr spectroscopy.9 left side : schematic capillary / nmr tube cross section used for isotropic gelbased capillary electrochromatography ( cec ; upper part with isotropic mesh openings ) , and nmr under anisotropic conditions ( anisonmr ; lower part with anisotropic mesh openings ) . right side : electrochromatogram obtained by cec ( upper part ) , and twodimensional nmr spectrum with split signals attribtued to anisotropic alignment of the analyte ( lower part ) . ideally , for thermodynamic studies , the separation medium investigated through chromatographic studies should be identical to the alignment medium used for anisonmr . many alignment media ( e.g. bicelles , phages ) , however , are incompatible with cec instrumentation , whereas homogeneous gels are separation media in plate gel electrophoresis ( pge ) , in capillary gel electrophoresis ( cge ) , and in gelbased capillary electrochromatography ( gbcec).10 separation in pge and cge is based on differences in the migration velocity of the solute , induced by differences in the effective electrophoretic mobility of the solutes and the sieving effect . separation in gbcec ( in analogy to chromatography ) , however , is based on differences in the equilibrium constant characterizing the transfer of the solute from the electrokinetic transport zone ( etz , where the velocity of the solute is given by the electrophoretic velocity of this solute and the electroosmotic velocity ) into the interaction zone ( iz , where the velocity of the solute is zero ) . we describe this equilibrium as a distribution process , although the highly swollen gel employed in gbcec is a homogenous phase ( in contrast to the system of two interpenetrating continuous phases characteristic for cec with a monolith ) . gbcec was introduced by fujimoto,10a using charged amphiphilic polyacrylamide gels for the separation of uncharged lowmolecularweight compounds . gbcec with hydrophilic or amphiphilic gels with embedded or immobilized chiral selectors was also successfully applied to the separation of different enantiomers.10c10 g returning to previous results,7 we selected the synthetic antimalaria drug mefloquine ( figure 2 ) with two stereocenters as the test solute.11a in recent years , the correct absolute configurations of the enantiomers of the erythro form , which is administered as the racemate , and of the enantiomers of the threo form , have been subject to debate . they were not reliably established until 20122013.11b11f in contrast to these difficulties , es of the four stereoisomers had been successfully achieved much earlier by using capillary electrophoresis ( ce ) with cyclodextrin derivatives or other native cyclodextrins as chiral selectors,12 and by using highperformance liquid chromatography ( hplc ) either with amylose tris3,5dimethylphenyl carbamatecoated silica gel13 or with a quinidinebased zwitterionic chiral stationary phase ( chiral selector covalently anchored to the chemically modified silica gel).14 structural formulae of a ) ( + ) erythromefloquine [ ( + ) emq ] , b ) ( )erythromefloquine [ ( )emq ] , c ) ( + ) threomefloquine [ ( + ) tmq ] , and d ) ( )threomefloquine [ ( )tmq ] . against this background , in the present work , we study the chromatographic separation of the four stereoisomers of mefloquine [ ( )threomefloquine ( tmq ) and ( )erythromefloquine ( emq ) ] by using gbcec , employing the same polymeric gel as the chiral separation medium as that already successfully taken for the ed of emq through anisonmr . the retention data gained by using gbcec will permit the determination of the ratio of ( distribution ) equilibrium constants . with this ratio , we confirm those parameters that have been calculated in the interpretation of previously reported anisotropic nmr data.7 with all types of gels obtained by varying the solvent composition , a good diastereoselective and enantioselective separation of the four mefloquine stereoisomers was achieved ( figure 3 ) . although the peak of propranolol , taken as a marker of the holdup time ( see the supporting information ) , can be characterized to be symmetric , the peaks for the more retarded stereoisomers of mefloquine show a characteristic ( rightskewed ) triangular broadened shape , which can be attributed to electromigration dispersion resulting from local electric field strength inhomogeneities . separation of the four stereoisomers of mefloquine by gbcec with alignment gel as the separation medium with superimposed scaled trace for ( )propranolol . peak assignment : 1 ) ( )propranolol ; 2 , 3 ) ( )threomefloquine ; 4 ) ( )erythromefloquine ; 5 ) ( + ) erythromefloquine ; mobile phase = methanol / water ( 50:50 , v / v ) buffered with triethylamine / acetic acid , ph*=6.66 , electric conductivity=125 s cm , capillary dimensions=173 mm ( 102 mm)100 m , photometric ingel detection=283 nm , electrokinetic injection=2 kv2 s , separation voltage=2.0 kv , c ( analyte in sample)=0.25 g l ( analyte dissolved in mobile phase ) . we confirmed the suitability of propranolol as a marker of the holdup time by application of a second independent method , which is based on the comparison of retention times obtained for the two enantiomers of emq with two capillaries filled with alignment gel having different effective lengths ( see the supporting information ) . it should be noted that , in gbcec , the holdup time can only be measured correctly if a marker is available that is not significantly retarded and has exactly the same effective electrophoretic mobility as the effective electrophoretic mobility of the analyte in the electrokinetic transport zone . the depicted peak distortions ( figure 3 ) are typical in cec for charged analytes showing a strong interaction with the oppositely charged stationary phase ( ionexchange cec).15 with analogously synthesized monolithic separation capillaries [ employing ( r)aphes as negatively charged comonomer ] synthesized for comparison purposes under phaseseparation conditions ( see the supporting information ) , leftskewed and rightskewed peaks can be obtained , dependent on the separation conditions ( figures s1 a and s1 b ) . under the conditions selected for recording the chromatogram depicted in figure 4 ( see legend ) , peak broadening by electromigration dispersion is absent and a higher peak efficiency is obtained . under these conditions , the method enabled baseline separation of the enantiomers of emq ( figure 4 ) . spiking the sample with ( + ) emq hcl revealed a higher retention factor for ( + ) emq than for ( )emq ( figure s2 ) . repeatability of the data was confirmed by sequential runs ( figure s3s5 ) . for five repeated runs , the relative standard deviations of the observed mobilities are 2.02.3 % ( table s1 ) . this corresponds to a relative confidence range of the mean of 2.52.9 % ( p=0.95 , twotailed test ) . separation of the enantiomers of erythromefloquine by gbcec with alignment gel as the separation medium . peak assignment : 1 ) ( )erythromefloquine ; 2 ) ( + ) erythromefloquine ; mobile phase = methanol buffered with 25 mm acetic acid and 2.66 mm triethylamine , ph*=4.46 , electric conductivity=55 s cm , capillary dimensions=234 mm ( 167 mm)100 m , photometric ingel detection=283 nm , electrokinetic injection=2.5 kv2 s , separation voltage=6.1 kv , c ( analyte in sample)=0.25 g l ( analyte dissolved in mobile phase ) . following two different approaches ( see the supporting information ) , we estimated the chromatographic separation factor ( = ratio of the two retention factors ) for tmq to be 1.08 and for emq to be 1.10 . these two factors correspond to the ratio of the two equilibrium constants related to the transfer from the electrokinetic transport zone to the interaction zone . hence , the energetic differences g for the involved ( distribution ) equilibria are 190 and 230 j mol for tmq and emq , respectively . taking propranolol as a nonretarded marker with an effective electrophoretic mobility similar to that of tmq and emq ( figure 3 and scheme s1 b ) , we obtained the following retention factors : 0.41 and 0.44 for the two enantiomers of tmq , 0.73 for ( )emq , and 0.80 for ( + ) emq . a larger difference in retention factors was obtained for the separation of tmq and emq , which corresponds to g=1.7 kj mol . under isotropic conditions , anisotropic parameters such as dipolar couplings between two nmrsensitive nuclei are averaged to zero.16a socalled alignment media lead to a low degree of orientation and reintroduce observable couplings . by using only a weak alignment , the large dipolar couplings that are in the range of khz are scaled down to values in the range of hz ( residual dipolar couplings).16b with a set of experimentally determined rdcs , an alignment tensor ( a matrix with five independent elements ) can be calculated , which describes the degree and orientation of the alignment . in 2001 , the concept of the generalized degree of order ( gdo ) was introduced to describe the dynamics of protein fragments.17 gdo is a scalar quantity that is calculated from the alignment tensor ( or the order tensor ) through the determination of the euclidian norm of this matrix.16a the gdo can be employed to characterize differences in the strength of two alignments , as it is a quantity that is only dependent on the extent of dynamic averaging , arising from both overall alignment effects and internal motional effects ( the latter being neglected with rigid molecules).16a , 17 for ( )emq [ in the gel prepared with ( r)aphes ] gdo=5.1910 , which is smaller than for ( + ) emq [ gdo=5.8510 ; absolute difference : 0.6610].7 under the assumption that a weaker alignment correlates with a weaker interaction of the solute with the polymer chains of the gel ( being identical with a less stable diastereomeric association ) , the determined gdos correctly predict that ( )emq elutes first in a chromatographic separation . the significance of this result was confirmed by a duplicate determination of gdo for ( )emq.7 gdo1 = 5.2510 and gdo2 = 5.1310 , resulting in a difference of 0.1210 . the confidence range of the arithmetic mean ( p=0.95 , onetailed test ) is 0.3810 , which is smaller than the difference between the values for the two isomers . with the aim to identify fragments of a protein , where motion has an effect on the accuracy of structure determination , tolman et al.17 defined a fragmentspecific internal gdo as the ratio of the observed fragment gdo to the alignment tensor gdo . we propose here the enantioselectivity parameter ( = gdo ratio ) , referring to a nonracemic chiral alignment medium and a specific enantiomer pair . this parameter is quantified by determining the ratio of the ( higher ) gdo for enantiomer 1 to the ( smaller ) gdo for enantiomer 2 ( with 1 ) . in the present case , (emq)=gdo / gdo=1.13 . it is interesting to compare this value to the selectivity factor ( = k / k ) obtained for ( )emq by gbcec , which is 1.10 . both methods agree in terms of the sign [ ( + ) > ( ) ] and magnitude of the quantity describing ed / es , which should hold for cases in which the degree of alignment prevails the enantiospecific difference of the measured anisotropic parameter ( here : rdc ) . based on the retention factors determined by gbcec , it can be now concluded that the ed observed in the present case in anisonmr is directly related to the different degrees of transfer of the solute from the etz into the iz . this degree of transfer is quantified by the molar fraction x iz = n iz/(n it is directly accessible from chromatographic data through x iz = k/(1+k ) . for the different stereoisomers of mefloquine , it represents the fraction of those molecules being associated with the charged moieties of the crosslinked polymer acting as interaction sites . although n total ( = n etz+n iz ) is kept constant , the values for x iz are 0.29 and 0.31 for the two enantiomers of tmq , 0.42 for ( )emq , and 0.44 for ( + ) emq . with all types of gels obtained by varying the solvent composition , a good diastereoselective and enantioselective separation of the four mefloquine stereoisomers was achieved ( figure 3 ) . although the peak of propranolol , taken as a marker of the holdup time ( see the supporting information ) , can be characterized to be symmetric , the peaks for the more retarded stereoisomers of mefloquine show a characteristic ( rightskewed ) triangular broadened shape , which can be attributed to electromigration dispersion resulting from local electric field strength inhomogeneities . separation of the four stereoisomers of mefloquine by gbcec with alignment gel as the separation medium with superimposed scaled trace for ( )propranolol . peak assignment : 1 ) ( )propranolol ; 2 , 3 ) ( )threomefloquine ; 4 ) ( )erythromefloquine ; 5 ) ( + ) erythromefloquine ; mobile phase = methanol / water ( 50:50 , v / v ) buffered with triethylamine / acetic acid , ph*=6.66 , electric conductivity=125 s cm , capillary dimensions=173 mm ( 102 mm)100 m , photometric ingel detection=283 nm , electrokinetic injection=2 kv2 s , separation voltage=2.0 kv , c ( analyte in sample)=0.25 g l ( analyte dissolved in mobile phase ) . we confirmed the suitability of propranolol as a marker of the holdup time by application of a second independent method , which is based on the comparison of retention times obtained for the two enantiomers of emq with two capillaries filled with alignment gel having different effective lengths ( see the supporting information ) . it should be noted that , in gbcec , the holdup time can only be measured correctly if a marker is available that is not significantly retarded and has exactly the same effective electrophoretic mobility as the effective electrophoretic mobility of the analyte in the electrokinetic transport zone . the depicted peak distortions ( figure 3 ) are typical in cec for charged analytes showing a strong interaction with the oppositely charged stationary phase ( ionexchange cec).15 with analogously synthesized monolithic separation capillaries [ employing ( r)aphes as negatively charged comonomer ] synthesized for comparison purposes under phaseseparation conditions ( see the supporting information ) , leftskewed and rightskewed peaks can be obtained , dependent on the separation conditions ( figures s1 a and s1 b ) . under the conditions selected for recording the chromatogram depicted in figure 4 ( see legend ) , peak broadening by electromigration dispersion is absent and a higher peak efficiency is obtained . under these conditions , the method enabled baseline separation of the enantiomers of emq ( figure 4 ) . spiking the sample with ( + ) emq hcl revealed a higher retention factor for ( + ) emq than for ( )emq ( figure s2 ) . repeatability of the data was confirmed by sequential runs ( figure s3s5 ) . for five repeated runs , the relative standard deviations of the observed mobilities are 2.02.3 % ( table s1 ) . this corresponds to a relative confidence range of the mean of 2.52.9 % ( p=0.95 , twotailed test ) . separation of the enantiomers of erythromefloquine by gbcec with alignment gel as the separation medium . peak assignment : 1 ) ( )erythromefloquine ; 2 ) ( + ) erythromefloquine ; mobile phase = methanol buffered with 25 mm acetic acid and 2.66 mm triethylamine , ph*=4.46 , electric conductivity=55 s cm , capillary dimensions=234 mm ( 167 mm)100 m , photometric ingel detection=283 nm , electrokinetic injection=2.5 kv2 s , separation voltage=6.1 kv , c ( analyte in sample)=0.25 g l ( analyte dissolved in mobile phase ) . following two different approaches ( see the supporting information ) , we estimated the chromatographic separation factor ( = ratio of the two retention factors ) for tmq to be 1.08 and for emq to be 1.10 . these two factors correspond to the ratio of the two equilibrium constants related to the transfer from the electrokinetic transport zone to the interaction zone . hence , the energetic differences g for the involved ( distribution ) equilibria are 190 and 230 j mol for tmq and emq , respectively . taking propranolol as a nonretarded marker with an effective electrophoretic mobility similar to that of tmq and emq ( figure 3 and scheme s1 b ) , we obtained the following retention factors : 0.41 and 0.44 for the two enantiomers of tmq , 0.73 for ( )emq , and 0.80 for ( + ) emq . a larger difference in retention factors was obtained for the separation of tmq and emq , which corresponds to g=1.7 kj mol . under isotropic conditions , anisotropic parameters such as dipolar couplings between two nmrsensitive nuclei are averaged to zero.16a socalled alignment media lead to a low degree of orientation and reintroduce observable couplings . by using only a weak alignment , the large dipolar couplings that are in the range of khz are scaled down to values in the range of hz ( residual dipolar couplings).16b with a set of experimentally determined rdcs , an alignment tensor ( a matrix with five independent elements ) can be calculated , which describes the degree and orientation of the alignment . in 2001 , the concept of the generalized degree of order ( gdo ) was introduced to describe the dynamics of protein fragments.17 gdo is a scalar quantity that is calculated from the alignment tensor ( or the order tensor ) through the determination of the euclidian norm of this matrix.16a the gdo can be employed to characterize differences in the strength of two alignments , as it is a quantity that is only dependent on the extent of dynamic averaging , arising from both overall alignment effects and internal motional effects ( the latter being neglected with rigid molecules).16a , 17 for ( )emq [ in the gel prepared with ( r)aphes ] gdo=5.1910 , which is smaller than for ( + ) emq [ gdo=5.8510 ; absolute difference : 0.6610].7 under the assumption that a weaker alignment correlates with a weaker interaction of the solute with the polymer chains of the gel ( being identical with a less stable diastereomeric association ) , the determined gdos correctly predict that ( )emq elutes first in a chromatographic separation . the significance of this result was confirmed by a duplicate determination of gdo for ( )emq.7 gdo1 = 5.2510 and gdo2 = 5.1310 , resulting in a difference of 0.1210 . the confidence range of the arithmetic mean ( p=0.95 , onetailed test ) is 0.3810 , which is smaller than the difference between the values for the two isomers . with the aim to identify fragments of a protein , where motion has an effect on the accuracy of structure determination , tolman et al.17 defined a fragmentspecific internal gdo as the ratio of the observed fragment gdo to the alignment tensor gdo . we propose here the enantioselectivity parameter ( = gdo ratio ) , referring to a nonracemic chiral alignment medium and a specific enantiomer pair . this parameter is quantified by determining the ratio of the ( higher ) gdo for enantiomer 1 to the ( smaller ) gdo for enantiomer 2 ( with 1 ) . in the present case , (emq)=gdo / gdo=1.13 . it is interesting to compare this value to the selectivity factor ( = k / k ) obtained for ( )emq by gbcec , which is 1.10 . both methods agree in terms of the sign [ ( + ) > ( ) ] and magnitude of the quantity describing ed / es , which should hold for cases in which the degree of alignment prevails the enantiospecific difference of the measured anisotropic parameter ( here : rdc ) . based on the retention factors determined by gbcec , it can be now concluded that the ed observed in the present case in anisonmr is directly related to the different degrees of transfer of the solute from the etz into the iz . this degree of transfer is quantified by the molar fraction x iz = n iz/(n etz+n iz ) . it is directly accessible from chromatographic data through x iz = k/(1+k ) . for the different stereoisomers of mefloquine , it represents the fraction of those molecules being associated with the charged moieties of the crosslinked polymer acting as interaction sites . although n total ( = n etz+n iz ) is kept constant , the values for x iz are 0.29 and 0.31 for the two enantiomers of tmq , 0.42 for ( )emq , and 0.44 for ( + ) emq . taking a chiral alignment medium as the chiral separation medium , gbcec separates the four stereoisomers of mefloquine . this separation confirms our previous hypothesis that ed with this gel is based on the formation of localized diastereomeric associates between the charged solute and the oppositely charged moiety of the anisotropically deformed gel.7 consequently , ed is attributed to differences in the equilibrium constants related to associate formation . through gbcec , retention data are obtained that permit the calculation of the degrees of transfer of the solute from the electrokinetic transport zone into the interaction zone and the precise quantification of the gibbs energy difference regarding the two diastereomeric associates involved in the enantioselective separation process . hence , our approach provides physicochemical data that are important in the understanding and optimization of alignment processes.18 the capillary pretreatment procedure and the subsequent in situ synthesis of the polymeric gel are described in detail in the supporting information . briefly , the separation capillaries are treated with a 30 % ( v / v ) solution of 3(trimethoxysilyl ) propylmethacrylate ( bind silane ) in acetone to introduce vinylic anchoring groups to the inner wall of the fused silica capillary . these vinylic anchoring groups enable the covalent attachment of the synthesized gel to the inner capillary wall , which is a prerequisite to obtain homogeneously filled capillaries . subsequently , the chiral monomer ( 2acrylamide)2phenylethanesulfonic acid [ ( r)aphes ] , is copolymerized with n , ndimethylacrylamide ( dmaa ) and n , nmethylenebisacrylamide ( bis ) through a freesolution radical copolymerization ( 15 min at 70 c ) . the preparation procedure of the gel within the capillary is identical ( with respect to concentration of monomers , reaction time , reaction temperature ) to the one used for the measurement of anisonmr parameters with the exception of the absence or presence of a washing step ( removal of nonreacted monomers ) , an additional drying and swelling step ( in the case of anisonmr ) , and the solvent composition.7 although dmso was employed for anisonmr , the solvent is either methanol or methanol / water [ ( 50:50 , v / v ) or ( 25:75 , v / v ) ] in gbcec ( after equilibration ) . previous studies , however , with either methanol or dmso as the solvent demonstrated that the type of solvent has only a minor influence on the determined anisonmr parameters.19 in gbcec , the separation gel is buffered with triethylamine / acetic acid , ph*=4.56.7 , whereas in anisonmr all experiments were performed in unbuffered gels . the influence of the electrolyte concentration on the alignment properties of a very similar achiral polyacrylamide gel [ aphes replaced by 2(acrylamide)2methylpropanesulfonic acid ( amps ) ] was determined by trigomourio et al.20 according to these results , we can assume a negligible influence of the selected buffer concentration ( 2.7 mm ) on alignment properties and retention behavior . ( for details refer to the supporting information).21 a scheme of the apparatus allowing ingel detection at 283 nm is shown in scheme s2 . as a service to our authors and readers , this journal provides supporting information supplied by the authors . such materials are peer reviewed and may be reorganized for online delivery , but are not copyedited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors .

abstractenantiodiscrimination and enantioseparation are two highly important processes in chemistry , often performed by using nmr spectroscopy and chromatography . for a better understanding of the mechanistic details , the same system should be studied by both methods . in addition , isotropic and anisotropic nmr parameters should be obtained , the latter using alignment media so that residual dipolar couplings and chemicalshift anisotropies can be measured . consequently , a chiral alignment medium was used for the first time in chiral gelbased capillary electrochromatography with the four stereoisomers of the antimalaria drug mefloquine as test compounds . chromatographic data verify that enantiodiscrimination obtained with this alignment gel is caused by differences in the equilibrium constants related to associate formation . hence , the chromatographic separation provides physicochemical data that form a basis for the understanding and optimization of alignment processes , and vice versa .

Introduction Results and Discussion Chromatographic Separation Selectivity Factor Generalized Degree of Order Conclusions Experimental Section Supporting information

this prospective , multicenter , open - label , surveillance study was conducted between august 2006 and march 2009 at 26 clinical practice sites in korea . at the time all patients provided voluntary , signed informed consent before the commencement of any study - related procedures . the main inclusion criteria for enrollment were age > 18 years ; a diagnosis of moderate to severe dry eye disease ( based on clinicians ' standard clinical practices ) ; symptomatic dry eye disease ; and nonresponsiveness to conventional treatment such as artificial tear drops , gels , ointments , and punctal plugs . the main exclusion criteria were use of systemic or topical csa in the previous 90 days ; anticipated use of any temporal punctal plugs during the study ; women who were pregnant , planning a pregnancy , or lactating ; end - stage lacrimal disease or dry eye disease caused by destruction of goblet cells ; active ocular infections ; and suspected hypersensitivity to any of the ingredients in the csa formulation . the study comprised a baseline visit and three follow - up visits after 1 , 2 , and 3 months of treatment . irvine , ca , usa ) was applied every 12 hours to each eye as monotherapy or adjunct therapy . new treatments , including any eye drops or punctal plug , were not added after enrollment . at each study visit , patients were examined according to the clinicians ' standard clinical practices . in addition , symptoms of ocular discomfort ( i.e. , stinging / burning , itching , sandiness / grittiness , blurred vision , light sensitivity , pain , and soreness ) , schirmer scores , use of artificial tears , global evaluation of improvement , and adverse events were recorded at each visit . there was an optional measurement of conjunctival staining ( oxford score ) performed at baseline and after three months of treatment . clinicians ' also assessed global evaluation of improvement in symptoms of dry eye disease from baseline at each follow - up visit according to the following categories ; > 90% improvement , 75% to 90% improvement , 25% to 75% improvement , condition unchanged , and condition worsened . patient satisfaction was assessed through the completion of a four - question survey at the final study visit . the exit survey comprised the following questions : how are your chronic dry eye symptoms ? ; with restasis , how does your chronic dry eye condition now affect your normal daily activities ? ; overall , how satisfied are you with restasis ? ; how quickly did restasis start working to relieve your chronic dry eye symptoms ? scoring for each question was based on a scale bar from 0 ( no symptom ) to 10 ( maximum symptom experienced ) . the primary effectiveness outcome measures included changes in symptom scores ( rated on a scale of 0 [ no symptoms ] to 4 [ always have symptoms ] ) and schirmer scores ( mm , with or without anesthesia ) . the secondary effectiveness outcome measures included change in conjunctival staining ( rated on a scale of 0 to 5 ) , use of artificial tears , global evaluation of the treatment , and patient satisfaction with the treatment . all data are summarized using frequency distributions and/or descriptive summary statistics ( mean and standard deviation [ sd ] ) . all statistical analyses included data for the treated eye or the mean data for both eyes ( if patients received treatment for both eyes ) . schirmer scores ( with or without anesthesia ) for all visits were compared using a repeated measures analysis of variance . for all other effectiveness variables , patients with missing baseline values were excluded from the analyses , and missing post - treatment data were inferred by carrying forward the subsequent observation . this prospective , multicenter , open - label , surveillance study was conducted between august 2006 and march 2009 at 26 clinical practice sites in korea . at the time all patients provided voluntary , signed informed consent before the commencement of any study - related procedures . the main inclusion criteria for enrollment were age > 18 years ; a diagnosis of moderate to severe dry eye disease ( based on clinicians ' standard clinical practices ) ; symptomatic dry eye disease ; and nonresponsiveness to conventional treatment such as artificial tear drops , gels , ointments , and punctal plugs . the main exclusion criteria were use of systemic or topical csa in the previous 90 days ; anticipated use of any temporal punctal plugs during the study ; women who were pregnant , planning a pregnancy , or lactating ; end - stage lacrimal disease or dry eye disease caused by destruction of goblet cells ; active ocular infections ; and suspected hypersensitivity to any of the ingredients in the csa formulation . the study comprised a baseline visit and three follow - up visits after 1 , 2 , and 3 months of treatment . irvine , ca , usa ) was applied every 12 hours to each eye as monotherapy or adjunct therapy . new treatments , including any eye drops or punctal plug , were not added after enrollment . at each study visit , patients were examined according to the clinicians ' standard clinical practices . in addition , symptoms of ocular discomfort ( i.e. , stinging / burning , itching , sandiness / grittiness , blurred vision , light sensitivity , pain , and soreness ) , schirmer scores , use of artificial tears , global evaluation of improvement , and adverse events were recorded at each visit . there was an optional measurement of conjunctival staining ( oxford score ) performed at baseline and after three months of treatment . clinicians ' also assessed global evaluation of improvement in symptoms of dry eye disease from baseline at each follow - up visit according to the following categories ; > 90% improvement , 75% to 90% improvement , 25% to 75% improvement , condition unchanged , and condition worsened . patient satisfaction was assessed through the completion of a four - question survey at the final study visit . the exit survey comprised the following questions : how are your chronic dry eye symptoms ? ; with restasis , how does your chronic dry eye condition now affect your normal daily activities ? ; overall , how satisfied are you with restasis ? ; how quickly did restasis start working to relieve your chronic dry eye symptoms ? scoring for each question was based on a scale bar from 0 ( no symptom ) to 10 ( maximum symptom experienced ) . the primary effectiveness outcome measures included changes in symptom scores ( rated on a scale of 0 [ no symptoms ] to 4 [ always have symptoms ] ) and schirmer scores ( mm , with or without anesthesia ) . the secondary effectiveness outcome measures included change in conjunctival staining ( rated on a scale of 0 to 5 ) , use of artificial tears , global evaluation of the treatment , and patient satisfaction with the treatment . all data are summarized using frequency distributions and/or descriptive summary statistics ( mean and standard deviation [ sd ] ) . all statistical analyses included data for the treated eye or the mean data for both eyes ( if patients received treatment for both eyes ) . schirmer scores ( with or without anesthesia ) for all visits were compared using a repeated measures analysis of variance . for all other effectiveness variables , the changes from baseline data were compared using a paired sample t - test . patients with missing baseline values were excluded from the analyses , and missing post - treatment data were inferred by carrying forward the subsequent observation . for all analyses , statistical significance was set at p 0.05 . a total of 392 patients were enrolled in the study and included in the safety and tolerability analysis ; 362 ( 92.3% ) patients completed the study and were included in the effectiveness analysis . the main reasons for study discontinuation were loss to follow - up ( n = 21 patients ) and adverse events ( n = 6 patients ) . patients were predominantly female , and slightly less than half were 50 years of age or older ( table 1 ) . most patients had been diagnosed with moderate to severe dry eye disease for one to five years , and approximately one - quarter had undergone a previous ocular surgery . treatment with csa 0.05% significantly improved symptom , schirmer , and staining scores and reduced the use of artificial tears . there were significant reductions from baseline in all mean ocular symptom scores ( stinging / burning , itching , sandiness / grittiness , blurred vision , light sensitivity , pain or soreness ) after one month of treatment ( p < 0.0001 ) ( table 2 ) and which persisted for up to three months of treatment ( p < 0.0001 ) . there were significant increases from baseline in mean schirmer scores both with and without anesthesia at each study visit ( p < 0.0001 ) ( fig . 1 ) . there were no significant differences between schirmer scores with and without anesthesia . the baseline conjunctival staining score ( oxford score sd ) was 3.2 2.2 , while the corresponding score after three months of treatment was 2.8 2.8 . after three months of treatment , the mean percentage ( sd ) reduction from baseline in conjunctival staining score was -12.8 64.7% ( p < 0.01 ) , and there was a significant reduction from baseline in mean ( sd ) use of artificial tears ( 5.6 5.7 and 4.0 4.5 drops / day for baseline and three months , respectively ; p < 0.0001 ) . according to clinicians ' global evaluations of treatment with csa 0.05% , the majority of patients experienced an at least 25% to 50% improvement at each visit ( fig . a greater percentage of patients experienced a 75% to 90% improvement after three months of treatment than after either one or two months of treatment . dry eye disease worsened in three , five , and two patients after one , two , and three months of treatment , respectively . according to the exit survey findings , patients generally reported that csa 0.05% treatment provided relief from dry eye symptoms , and that their experience with the treatment was positive ( fig . specifically , most patients ( 57.2% ) reported that they had no or mild symptoms of dry eye disease ( score of 0 to 4 out of 10 , 214 / 374 patients ) ( fig . most patients ( 55.2% ) also reported that their dry eye disease had no or little effect on their normal daily living ( scores 0 to 4 out of 10 ; 206 / 373 patients ) ( fig . the majority of patients ( 72.0% ) were satisfied or very satisfied with csa 0.05% treatment ( scores of 5 to 10 out of 10 ; 270 / 375 patients ) ( fig . relief from dry eye symptoms began three to five weeks after commencing csa 0.05% treatment ( 209 / 356 , 58.7% ) ( fig . 3d ) . treatment with csa 0.05% was well - tolerated , and very few patients discontinued the study because of adverse events . the most common adverse events ( incidence > 5% ) were ocular pain ( 43 / 392 patients , 11.0% ) and ocular irritation ( 23 / 392 patients , 5.9% ) . six patients ( 1.5% ) discontinued the study due to adverse events , including three patients ( 0.8% ) with ocular irritation , two patients ( 0.5% ) with ocular pain , and one patient ( 0.3% ) with ocular hyperemia . our study findings suggest that csa 0.05% is an effective and tolerable treatment for moderate to severe dry eye disease in korean clinical practice . we found that treatment with csa 0.05% for three months led to improvements in both objective ( schirmer and conjunctival staining scores ) and subjective ( symptom scores and artificial tear use ) measures of dry eye disease . favorable tolerability was indicated by the very low percentage of patients who discontinued the study , the absence of serious adverse events , and high patient satisfaction with treatment . the results of our study extend the efficacy findings of earlier clinical trials and clinical practice studies of csa 0.05% in korean patients with moderate to severe dry eye disease and support the use of csa 0.05% for treatment of moderate to severe dry eye disease in korean clinical practice . consistent with previous studies , we found that csa 0.05% was an effective treatment for dry eye disease as indicated by changes in symptom , schirmer , and conjunctival staining scores , artificial tear use , and clinicians ' global evaluations of treatment . the statistically significant reductions in symptom scores in our study are similar to those found in phase ii and phase iii clinical trials of csa 0.05% . likewise , the significant improvements in mean schirmer scores in our study are similar to those found in korean patients after short - term ( six to eight weeks ) and longerterm ( up to three months ) csa 0.05% treatment . importantly , our study included a much larger number of patients ( n = 362 ) in the effectiveness analyses compared with those in the two previous clinical practice studies performed in korea ( n = 23 and n = 26 ) . the improvements in schirmer scores ( with and without anesthesia ) , conjunctival staining score , artificial tear use , and the clinicians ' global evaluations of treatment in our study are in agreement with those determined in a randomized controlled trial of csa 0.05% . the significant reduction in artificial tear use in our study is also consistent with findings from a previous study , in which the majority ( > 60% ) of patients reported decreased artificial tear use after 60 days of csa 0.05% treatment . together , these findings confirm that csa 0.05% reduces the symptoms of dry eye disease . therefore , we speculate that csa 0.05% partially resolves the pathophysiological changes that cause dry eye disease . however , additional studies are needed to confirm this conclusion . we found that treatment with csa 0.05% was generally well - tolerated , and that most patient experiences with csa 0.05% were positive . the most common adverse events in our study ( ocular irritation and pain ) are known adverse reactions to csa 0.05% [ 20 - 22 ] . the proportion of patients who discontinued treatment because of adverse events ( 1.5% ) in our study is similar to the proportion ( 2.2% ) in a phase ii trial of csa 0.05% . moreover , the patient - reported experience with csa 0.05% in our study is similar to that in clinical practice studies performed in the united states . our findings regarding patientreported rating of symptoms ( i.e. , no or mild symptoms ) and the effect of dry eye on daily activities after three months of csa 0.05% treatment are similar to those of a clinical practice study in which patients reported a 30% reduction in symptom severity and improvement in their abilities to perform daily activities after two months of treatment with csa 0.05% . likewise , our patientreported satisfaction findings are similar to those of a self - reported compliance study in which compliant patients had a mean satisfaction score of 7.7 ( 0 = not at all satisfied , 10 = very satisfied ) after two months of treatment with csa 0.05% . first , interpretation of our findings is limited by the single - arm , open - label study design . however , the clinical practice setting enhances the external validity of our findings , as does the prospective , multicenter , surveillance design . second , as our patients were not grouped according to the cause of dry eye disease , we were unable to determine which patients may ( or may not ) respond to csa 0.05% treatment . third , we can not exclude the possibility that improvements in dry eye disease were caused by the emulsion vehicle or that subjective improvements were a reflection of patients ' providing socially desirable answers . however , the results of two randomized controlled trials have shown that csa treatment results in significantly greater improvements in dry eye disease than does treatment with the emulsion vehicle . in addition , the fact that there was agreement between clinician and patient evaluations of treatment ( i.e. , treatment resulted in improvement ) would argue against the possibility that patients provided socially desirable answers . finally , we did not assess patient adherence to study medication , document the type of artificial tear use , or use a validated questionnaire for assessing patient satisfaction with treatment . despite these limitations , the use of multiple objective ( i.e. , schirmer and conjunctival staining scores ) and subjective measures ( i.e. , symptom scores and artificial tear use ) has enabled us to provide clinically relevant evidence of the effectiveness of csa 0.05% for treatment of dry eye disease . in conclusion , the effectiveness and tolerability findings of our study support the use of csa 0.05% for the treatment of moderate to severe dry eye disease in the korean clinical practice setting .

purposeto assess the effectiveness and tolerability of cyclosporine ophthalmic emulsion ( csa ) 0.05% in patients with moderate to severe dry eye disease in korea.methodsthis was a prospective , multicenter , open - label , surveillance study of 392 korean patients with moderate to severe dry eye disease who were treated with csa 0.05% for three months . an assessment of effectiveness was performed at baseline , and after 1 , 2 , and 3 months . the primary effectiveness outcomes were changes in ocular symptoms and schirmer score . the secondary effectiveness outcomes were a change in conjunctival staining , use of artificial tears , global evaluation of treatment , and patient satisfaction . the primary safety outcome was the incidence and nature of adverse events.resultsa total of 362 patients completed the study . after three months , all ocular symptom scores were significantly reduced compared to the baseline values , while the schirmer scores were significantly increased relative to baseline ( p < 0.0001 ) . after three months , there were significant reductions from baseline in conjunctival staining ( p < 0.01 ) and use of artificial tears ( p < 0.0001 ) . according to clinicians ' global evaluations , most patients ( > 50% ) experienced at least a 25% to 50% improvement in symptoms from baseline at each follow - up visit . the majority of patients ( 72.0% ) were satisfied with the treatment results , and 57.2% reported having no or mild symptoms after treatment . the most common adverse events were ocular pain ( 11.0%).conclusionsour findings indicate that csa 0.05% is an effective and tolerable treatment for dry eye disease in korean clinical practice .

Materials and Methods Study design and setting Study participants Study protocol Outcome measures Statistical analysis Results Discussion

population growth has increased the demand for food ; rising prosperity has increased the demand for quality food . at the same time , consumers demand convenience foods , since they are becoming increasingly health conscious ; therefore , there is a need to diversify food products . wheat is being used as a staple food for most part of the world , because of its special dough characteristic like cohesiveness and thus being used in the preparation of bread and other wide ranges of products like noodles , soups , pasta , and other foods like biscuits , cookies , cakes , and breakfast cereal . pastas include noodles in various lengths , widths and shapes and varieties that are filled with other ingredients like ravioli and tortellini . pasta is an excellent source of complex carbohydrates , which provide a slow release of energy . unlike simple sugars that offer a quick , yet fleeting boost of energy enriched varieties provide a good source of several essential nutrients , including iron and several b - vitamins . in recent years increase in popularity of pasta products and their increased consumption make it very important for increase in availability of raw materials . durum wheat , being the hardest of wheat 's is used at large scale for pasta production . being commercially expensive to produce due to the limited availability makes it a case of study . the endosperm made up of durum is completely different from other wheat species because the mineral was distributed throughout endosperm and has more carotenoids contents , low protein efficiency ratio , and excellent rheological characteristics which are desirable for making pasta products . however , over the years , numerous studies on alternate methods for production of pasta from different raw materials have been conducted , where alternates for raw materials were used for production focusing on reduced cost and to match similar parameters and to improve nutritional value . limited availability of durum wheat is noticed due to the increase in consumption of pasta products , making it commercially expensive for procurement of raw material . on the other hand however , the availability of durum wheat for production of pasta products is very limited . alternates are thus researched upon , where it is found that the common wheat widely available throughout the world is similar in comparison with durum wheat . the percentages of starch , protein , minerals , lipids , and amino acids are roughly equivalent . common bread wheat occupying 90% of the world total production makes it a cheap and readily available alternate for pasta production , thus saving money and creating ample opportunities to improve nutritional quality . the objective of the present study is to find suitability of t. aestivum milled products ( refined wheat flour , semolina , and whole wheat flour ) in the place of durum semolina for preparation of pasta , thereby reducing the cost of production , maintaining or improving the quality of the product , and then study in detail the rheological properties of the pasta dough , chemical composition , nutritional profile , and quality of developed pasta products . freshly prepared refined wheat flour , whole wheat flour , and semolina were obtained from narasu 's roller flour mills , salem , tamil nadu , india . all reagents and chemicals used are of analytical grade ( ar ) unless otherwise specified . raw materials were analyzed for particle size ( aacc 55 - 30.01 ) , moisture content ( aacc-44 - 15a ) , ash content ( aacc-80 - 01 ) , gluten content ( aacc-38 - 10 ) , and micro - kjeldahl method was used to determine nitrogen contents of pasta samples ( aacc , 2000 ) , sedimentation value ( aacc-56 - 70 ) , farinograph ( aacc-54 - 21 ) , mixograph ( aacc 54 - 40.02 ) , and alveograph characteristics ( aacc-54 - 30a ) using the mentioned standard methodologies . raw materials and water were premixed in a spar mixer at speed 1 ( 60 rpm ) for 10 min to facilitate uniform distribution of water . the premixed dough ( 500 g ) was transferred to a laboratory pasta machine ( la monferrina , model dolly , asti , italy ) . the dough was then extruded through the brass die for pasta type shells in the required size and was dried in sakar drier ( shirsat , mumbai ) at 75c for 4 h. the pasta samples were then allowed to cool at room temperature and then packed in polyethylene covers for storage . cooking loss was determined according to the bureau of indian standards ( bis 1976 ) . twenty - five grams of pasta sample was weighed and put in the 250 ml of boiling water . start timer count is and stirs well to make sure that the pieces are separated . check the piece of pasta after every 30 sec intervals for its hydration and cooking by squeezing the sample . firmness of cooked pasta was measured according to method adopted by krishnan and prabhasankar using a universal texture measuring system ( lloyds instruments , lr-5 k , hampshire , uk ) . a panel consisting of 25 panelists ( n = 25 ) , who were regular eaters of pasta , was employed for the sensory evaluation of pasta samples . briefly , panelists evaluated the randomly coded pasta samples for their colour , appearance , aroma , texture , taste , and overall acceptability . assessors were instructed to cleanse their palate with cold , filtered tap water before tasting each sample . the overall sensory attributes were measured using hedonic scale of 19 where 9 = like extremely , 8 = like very much , 7 = like moderately , 6 = like slightly , 5 = neither like nor dislike , 4 = dislike slightly , 3 = dislike moderately , 2 = dislike very much , and 1 = dislike extremely . all the parameters were carried out in quadruplicates and the means values were reported . the values of surface colour ( l , a and b ) of raw pasta in terms of lightness ( l ) and colour ( + a : red a : green ; + b : yellow ; b : blue ) and e were measured using hunter lab colour measuring system ( colour measuring labscan xe system , usa ) . the cooked pasta samples were freeze - dried using heto freeze dryer ( dw3 , allerod , denmark ) . surface and cross section of freeze - dried samples were mounted on the specimen holder and sputter - coated with gold ( 2 min , 2 mbar ) . finally , each sample was transferred to the microscope where it was observed at 15 kv and a vacuum of 9.75 10 torr . a scanning electron microscope ( leo 435 vp , leo electronic systems , cambridge , uk ) was used to scan the images . the method was followed from aoac method 32.1.17 . in vitro digestibility of starch freeze dried and ground sample ( 50 mg ) was dispersed in 4 ml of sodium acetate buffer ( ph 4.6 , 0.4 m ) containing amyloglucosidase and was incubated in water bath for 30 min at 60c . then , the enzyme was inactivated by placing the tubes in boiling water bath ( 100c ) for 15 min . the tubes were cooled to room temperature and then centrifuged at 5000 rpm for 10 min . supernatant was measured for its glucose content using a glucose oxidase - peroxidase ( god - pod ) kit ( autospan , span diagnostics limited , india ) . absorption was measured at 505 nm , and the glucose concentration was converted into starch content using a 0.9 factor . the particle size distributions of the flour samples were determined with a series of standard sieves , and the results were expressed as a percentage of the sample weight ( table 2 ) . ideally , the majority of semolina particles should fall within a narrow range of particle size range so that pasta dough water uptake will be homogenous . it was observed that the refined wheat flour ( refined wheat flour ) is much finer than the control ( semolina ) . the nonuniformity in the particle size can be attributed to the grinding of the semolina particles , which was carried out by an external minimill grinder . however , the results obtained were good within the requirements for good pasta making quality . whole wheat was ground to a granulation similar to that of the semolina ( table 2 ) . incomplete hydration of semolina or ground whole wheat would result in white specks in the spaghetti . thus , white specks would affect the appearance , mechanical strength , and cooking quality of the spaghetti . proximate analysis of all the raw materials ( refined wheat flour , whole wheat flour , semolina ) used were shown in the table 3 . it is noticed that comb1 ( t. aestivum wheat flour & semolina with additives ) with the presence of additives has moisture of 10.87% is very much higher compared to the control ( 9.8% ) . the other two blends ( comb2 & comb3 ) had acceptable levels of moisture compared to the control as they have the presence of semolina . the proximate composition of all the samples was found to be within limits of pfa and isi standards . similar results of semolina were also reported by where moisture of indian durum varieties varied from 9.0 to 11.5% , ash content varied from 0.79 to 0.86% , and the protein content varied between 12.1 and 15.9% . ash , an index of the mineral content of the flour , is of much relevance , and in that it gives the indication of the grade or the extraction of the flour . this is because of the low level of mineral content present in the endosperm when compared to the outer bran content . the bran layer is rich in ash and protein , so removing bran during milling would lower the protein and ash content . it is noticed that all the comb samples have lower ash content when compared to the control , where comb3 ( semolina ( t. aestivum ) and semolina ( t. durum ) ) ( table 3 ) has 0.596% . comb2 & comb1 samples also have lower ash values , thus improving the quality of the flour . a significant increase in protein content is noticed with the comb samples ( table 2 ) , which could be due to the addition of additives . this is similar to the results reported by prabhasankar et al . for increase in nutritional attributes of pasta samples with the addition of additives . the maximum increase is noticed in comb2 ( refined wheat flour and semolina ( t. durum ) ) which is 15.40% ( table 3 ) when compared to the control which has 12.8% ( table 3 ) of protein . increase is also noted in comb1 at a level of 13.70% , thus imparting better nutritional value . the mixograph is a primary physical dough testing procedure in the us durum wheat - breeding program . figure 1 details the results of the monograms obtained from the pasta samples . comparing the two raw materials used , dough strength is greatly deferred between the samples where figures 1(b ) and 1(c ) were comparatively stronger than the others . this is because of the presence of refined wheat flour , which is generally high in strength compared to semolina . regardless of samples , refined wheat flour had higher peak heights as shown in figures 1(b ) and 1(c ) , but semolina had higher peak width as shown in figures 1(a ) , 1(d ) , and 1(e ) . higher peak proves that the flour is very resistant to extensibility and also has greater mixing ability making it more stable as seen in control ( figure 1(b ) ) . but semolina generally is seen to have a lower peak height ( figure 1(a ) ) , thereby reducing the extensibility of the flour making it better for pasta making . mixogram dough development time ( the time required for the mixogram curve to reach maximum height ) was greater for comb1 had the highest ( 4.26 ) which was made up of refined wheat flour . based on the mixograms obtained , refined wheat flour had higher dough stability when compared to semolina as evidenced by the rapid decline of the mixogram curves after 3.5 min . dough stability indicates the time during which the dough resists mechanical action without undergoing a change in consistency . lack of dough stability or tolerance to over mixing could be related to the dilution of semolina with bran or germ as fewer storage proteins are available to form a gluten matrix . other researchers have reported that dough stability decreases with increase in bran concentration . in spite of all these issues , all the parameters were in accordance was the norms and were able to produce good pasta quality . it is seen from ( figure 1(d ) ) , that the addition of refined wheat flour along with semolina has given the flour additional properties showing a higher peak height when compared to control ( figure 1(a ) ) , thus aiding in better pasta making ability . the results obtained along with the amount of water absorption required to centre the farinogram curve on the 500 bu ( brabender units ) line varied as shown in table 4 . it is well noticed from the results that the samples with a higher concentration of refined wheat flour exhibit stronger dough characteristics ( increased water absorption , dough development time and dough stability ) , that is , refined wheat flour & comb1 in contrast to weak dough development . the whole wheat flour showed higher water absorption 63.9% due to high damages starch , whereas the dough development time and dough stability were lower than those in the whole - wheat flour . the addition of gluten as indicated in samples combs 1 , 2 , and 3 , increased water absorption , dough development time and dough stability was seen . it was also noticed that stability and dough development time are related with each other , and increase in development time caused increased stability , thus leading to stronger dough . samples with maximum wheat bran concentration caused increase in water absorption with whole wheat flour ( whole wheat flour ) having maximum absorption ( 65.9% ) ( table 4 ) which was also noticed by and , lowest for refined wheat flour ( comb1 ) . reported that the differences in water absorption are mainly caused by the greater number of hydroxyl group which exist in the fibre structure and allow more water interaction through hydrogen bonding . alveograph is used to measure the viscoelastic properties ( strength and extensibility ) of the gluten protein that correlate 's well with the firmness and springiness of cooked pasta . gluten strength and tenacity / extensibility ratio pl is a good predictor of cooking quality , thus making it relevant for pasta - making ability . figure 2 results obtained from the alveograph of the various samples of flour . regarding the bread making and pasta characteristics of the flour under examination , the most dramatic effect of additives addition on semolina was observed when the biaxial properties of the wheat dough were assessed in the alveograph . the addition induced a significant modification of the alveograph parameters , where a steady increase of the tenacity ( p ) besides to a significant decrease in dough extensibility ( l ) . overall effect on tenacity and extensibility led to a significant increase of the curve configuration ratio ( pl ) . similar results were obtained by bonet et al . where he noticed the same , with increase in go ( glucose oxidase ) concentration . the same was also noticed in regard to go by . but the only change was seen in the deformation energy ( w ) where a steady decrease was noticed in this case . this could be attributed to the addition of a percent of refined wheat flour as the raw material . as the percent of refined wheat flour reduced , the pl ratio increased which was very similar to that of control . thus leading to better pasta quality . whereas bonet et al . noticed a steady increase as there was no presence of refined wheat flour and also the addition of go cause additional protein cross - links resulting in larger pl values . colour of pasta is a key quality because of the vital impact on the point of sale . in pasta products made with semolina , the higher the value , the more desirable the product . among l , a , and b parameters , the first two are considered more important as colour attributes . hunter colour parameters ( l , a , b ) of raw samples of durum pasta ( control ) the other raw materials used along with the sa and the comb pasta are shown in the table 5 . the higher values are noticed in table 5 , which shows increased levels of pasta quality and the presence of no adulteration in the pasta samples proving high levels of economical advantage as well as appearance . the lightness is seen lower in the all the samples , this may be attributed due to the alteration with different wheat milled products which has a lower lightness index compared to the control , thereby lower carotenoid pigments which contribute to the colour of durum pasta . yellowness in all samples was seen lower than the control , and this is due to the same factors affecting the lightness index . it is noted that the comb3 blend shows the highest lightness , but the comb2 blend gives the highest yellowness when compared to the control . it is also noted that the influence of the additives did not cause any major difference in both indexes . but it is seen that the influence of additives has reduced the lightness but has increased the yellowness . the cooking characteristics of all the raw materials and the trails along with the comb blends compared to the control are presented in figure 3 and the photographs of raw , and cooked samples compared to the control are shown in figure 4 . high - quality pasta has a good cooking resistance and firmness , does not release amount of organic matter into the cooking water , and does not show stickiness . the cooking loss of all the comb samples is much lesser when compared to the control . the lowest cooking loss is seen in ( figure 3 ) where it is 2.88% compared to the control , which is 4.05% . the quality of the cooked pasta can be better explained on the basis of the interactions between starch and gluten whose intensity is strongly dependant on the drying conditions , as reported by many authors . if the coagulated gluten structure lacks compactness and elasticity , the starch granules structure swell up easily during cooking and lose more soluble materials into the cooking water . starch was the main component ( 63.1% ) on dry basis of the cooking liquor when 100% semolina was used in spaghetti and vermicelli samples . high - temperature drying strengthens the gluten matrix , which protects starch granules from rupturing during cooking . furthermore , high temperature drying reduces water permeability and a crake in packaging & arrangement of starch granules , contributing to reduced cooking looses and increased cooked firmness . a reconstitution study indicates that the gluten quality is the major factor determining the cooking quality . this could be very well noticed in the comb samples where gluten was added , thus decreasing the cooking loss . this is also noticed in the comb samples , where the presence of hydroxypropyl - methylcellulose ( hpmc ) added as an additive is noted to increase water solubility and also at high temperatures forms a gel creating a temporary network of hydrocolloid chains , thus maintaining the structure . the main criteria generally accepted to access the overall quality of the cooked pasta are based on the textural evaluation . the work done by the probes to cut the pasta cooked firmness was greater for semolina than refined flour or whole wheat flour as seen in figure 5(a ) . whole wheat pasta was reported to have lower cooked firmness than traditional semolina . when cooked to optimum , firmness was seen the greatest in semolina ( semolina ) obtained from t. aestivum wheat but is deemed not suitable for the consumer as it fails at the other parameters . it is also noticed that the addition of additives has resulted in firmness , which is similar to that of control . this can be explained as gluten present is seen to improve the protein content , thus improving firmness of pasta . good - quality pasta should be al dente ; that is , it should have high degrees of firmness and elasticity . comb3 pasta has moisture values higher than that of control , and at the same time , its firmness showed increased values . this would suggest that the substitution of additives to 100% semolina contributes to structural strength . in this case of comb3 , this hypothesis could also be related to the low values obtained for cooking losses , indicating a well - formed structure from which small amounts of solids are released during cooking . longer cooking times resulted in an increased water absorption that led to moisture migration into the centre of the pasta . hence , we hypothesize that moisture migration into the centre of the pasta diminishes the resistance of the sample to cutting , since the plasticizing action of water increases the mobility of biopolymers chains . another factor that could have affected the texture was the more prolonged exposure to heat while drying that changed the structural conformation of the protein - starch network . this phenomenon may have caused loss of rigidity in the structure with a consequent decrease in firmness . a product , even if it is highly nutritious , but does not taste good , will not be accepted in the society , thus making sensory evaluation very important and a crucial criteria in the formulation of pasta . based on the different properties of importance , it is characterised and formulated as shown in table 6 . it was noticed that the overall score when compared to the control is slightly less compared to all the other samples . refined wheat flour considered being the alternate for semolina as a raw material is considered to be the second control . the main aim of the present study to find a suitable alternate for the control ( durum semolina ) thus needs an overall score to at least at par to be acceptable . it is noticed that additives improved the sensory scores especially the mouth feel character . the increase in mouth feels score ( table 6 ) , which is one of the main attributes for pasta sensory evaluation , may also be attributed to the increased water absorption ( 103.8% , 94% , and 104% ) , respectively , for the comb blends . comb3 mixture of semolina of t. aestivum and t. durum gives the highest overall score , which is very much similar to the control . being very essential for the bowel movement and the digestion in the body , dietary fiber levels are much noted in the present world . remarkable changes in the levels of fiber are noticed in the comb2 and 3 samples , where they are 4.25 and 4.1 , respectively ( figure 5(b ) ) . higher levels of dietary fiber are mostly recommended in the present world , owing to the increase in diet - conscious people and the increased digestive problems faced in today 's society . the link between dietary fiber and human health is well explained by where they explain that higher levels of dietary fiber increase cancer prevention , lower risk of chronic disease and help in diabetes management and prevention . it was also noticed by that incorporation of a combination of blends of wheat bran by - products increased the dietary fiber content . two views of sem showing the cut section and the network of the pasta are presented in figure 6(b ) . these micrographs are all presented in 100x and 3000x , respectively , for all of the samples . micrographs of the control pasta samples show the protein - starch matrix to be well formed , with strong and continuous protein strands entrapping large starch granules . the starch granules within the pasta appear to be slightly swollen and regular in shape and size , perhaps indicating a level of gelatinization during the extrusion process . it is also noticed that the control has smaller pores when compared to the comb blends . this supports the higher cooking loss ( 4.05% ) and lower cooking time ( 6.5 min ) as boiling water can reach the deeper core faster and stay in the cavities . the addition of refined wheat flour to the semolina is seen to disrupt the continuity of the protein matrix . the protein - fiber matrix within the pasta containing a portion of refined wheat flour appears to be less developed than the control resulting in an open appearance with discrete starch granules , which is uncovered and exposed to enzymatic attack . this may be explained by the dilution of the gluten protein , thus showing reduced firmness ( 1.757 n ) and increased water absorption ( 103.8% ) , making it a more elastic structure . the addition of semolina with refined wheat flour shows a protein starch matrix similar to that of the control . this sample is seen to have a gelatinized structure when compared to the other blends , thus forming a more compact structure , which enable reduced water absorption ( 92% ) during cooking . unswollen starch granules can still be found inside the durum wheat , thus reducing the water absorption during cooking due to the layer of gelatinized starch . use of whole semolina shows a higher network of protein - starch matrix , thus resulting in a higher degree of firmness ( 2.43 n ) and improved chewiness . the rate of starch digestion and absorption seems to be a determinant of the metabolic response to a meal . the gi is defined as the postprandial incremental glycemic area after a test meal , expressed as the percentage of the corresponding area an equi - carbohydrate portion of a reference food ( glucose or white bread ) . a recent joint fao / who expert consultation recommended increased consumption of low - glycaemic index ( gi ) foods . there is increasing evidence that a low -glycaemic - index diet can be beneficial in that , it improves metabolic control of hyperlipidaemia in diabetic patients as well as in healthy subjects . the rate and extent of starch digestion instigate a number of physiological functions that have different effects on health , including reduction of the glycemic and insulinaemic responses to a food , hypocholesterolemic effects , and protective effects against colorectal cancer . it is interesting to note that the addition of refined wheat flour as a raw material to the pasta sample increased the starch released in the pasta where pure refined wheat flour as pasta shows the highest level of starch release ( 44.90% ) . this is because as refined wheat flour consists of only the endosperm of the wheat grain , thus removing the nutritious bran and germ content ( wikipedia , refined wheat flour ) . goi et al . , have reported a total starch content of 74 2.24 for spaghetti , but the starch content obtained for the control pasta is approximately half of the value said above . this discrepancy may probably be due to the use of raw pasta for our analysis whereas goi et al . used the cooked samples of the pasta for their analysis . methodological differences in determination of total starch may also be a reason for the discrepancy . there are many factors that may influence the rate of starch digestion , including the nature of starch , the starch - protein interaction , the presence of fiber and antinutrients such as lectins , phytates and enzyme inhibitors and method and time of cooking . the present study revealed that pasta could be made using different wheat - milled products , which are economically viable and also have beneficial nutritional applications . different combinations of mixtures had been experimented to produce viable alternates where different concentrations of refined wheat flour , whole - wheat flour , and semolina of different varieties of wheat were used to prepare pasta . pasta made from refined wheat flour was seen to have reduced cooking loss , colour , firmness and sensory score . pasta made from a combination of t. durum ( 50% ) semolina and t. aestivum ( 50% ) has acceptable levels of all the parameters , but due to increase in financial requirements , it is not suitable . from all results obtained , it was seen that semolina t. durum ( 50% ) mixed with refined wheat flour t. aestivum ( 50% ) can be used as alternate for making pasta for use as a low glycemic index snack product with acceptable physical and sensory properties . the addition of additives to the pasta has made it almost equivalent to the standard of original 100% semolina pasta . acceptable cooking quality parameters were obtained in the pasta samples containing a mixture of both semolina and refined wheat flour , as measured by cooked weight , cooking loss , and so forth during cooking . however the incorporation of additives in the mixture of 50% semolina ( t. durum ) along with 50% refined wheat flour ( t. aestivum ) was finally concluded as the optimal alternate , because of its almostequivalent properties to the 100% t. durum semolina pasta , thus making it a economically nutritious alternate ' resulting as a staple food in developing countries .

this study is aimed to assess the suitability of t. aestivum wheat milled products and its combinations with t. durum semolina with additives such as ascorbic acid , vital gluten and hpmc ( hydroxypropyl methyl cellulose ) for pasta processing quality characteristics such as pasta dough rheology , microstructure , cooking quality , and sensory evaluation . rheological studies showed maximum dough stability in comb1 ( t. aestivum wheat flour and semolina ) . colour and cooking quality of comb2 ( t. durum semolina and t. aestivum wheat flour ) and comb3 ( t. aestivum wheat semolina and t. durum semolina ) were comparable with control . pasting results indicated that t. aestivum semolina gave the lowest onset gelatinization temperature ( 66.9c ) but the highest peak viscosity ( 1.053 bu ) . starch release was maximum in comb1 ( 53.45% ) when compared with control ( 44.9% ) as also proved by microstructure studies . firmness was seen to be slightly high in comb3 ( 2.430 n ) when compared with control ( 2.304 n ) , and sensory evaluations were also in the acceptable range for the same . the present study concludes that comb3 comprising 50% t. durum semolina and 50% t. aestivum refined wheat flour with additives would be optimal alternate for 100% t. durum semolina for production of financially viable pasta .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusion

with the increasing burden of patients with multiple disease states , the drug therapy has grown more complex . the complex therapeutic regimens increase the risk of drug - drug interaction ( ddi ) to a great extent . ddi is said to occur when the effect of one drug is altered by the concurrent administration of other . pharmacokinetic interaction occurs when either of the concurrently administered drugs have potential to alter other 's pattern of absorption , distribution , metabolism and excretion . ddi is said to account for a number of severe adverse drug reactions ( adr ) resulting in hospitalizations and emergency department visits . it is estimated that ddi contribute to about 6 - 30% of all adrs . furthermore , adr due to ddi accounts for about 2.8% of hospital admission every year . they are regarded as a leading cause of deaths world - wide . in nepal , cardiovascular disease furthermore , patients with cardiovascular disorders are even at higher risk of ddi due to the number and types of drug they receive and the influence of heart disease on drug metabolism . the potential of cardiovascular drug in the involvement of ddi is relatively higher as shown in the studies conducted world - wide . a prospective study conducted in one of the teaching hospitals in india indicated that the incidence of potential drug interaction amongst cardiac drugs in hospitalized patients is 30.67% . a study in palestine among patients receiving antihypertensive medications came up with 433 different unique pairs of potential drug interactions among 867 patients . another study from nepal regarding ddi indicated that 53% of the patients admitted to the department of internal medicine experience one or more ddis . yet another study in nepal to evaluate the pattern of ddi amongst diabetic outpatients also found that 47.5% of medications potentially interacting with antidiabetics were cardiovascular drugs . while similar studies are reported to be common world - wide there have been few reports of drug interaction and the factors associated with it among south asian cardiac patients . hence , this study was conducted to evaluate the pattern of potential drug - drug interaction ( pddi ) and to identify the associated risk factors among hospitalized cardiac patients in a tertiary care hospital in nepal . the prospective observational study was undertaken for the period of 4 months from may 1 , 2012 to august 28 at the department of internal medicine of manipal teaching hospital ( mth ) , a tertiary care teaching hospital in western nepal . cardiac patients aged 18 years or older admitted to the cardiac unit of general medicine wards with a hospital stay of at least 24 h and those prescribed two or more drugs were enrolled for the study . patient profile form was used for collecting the socio - demographic variables and medication profile of patients . ddi database system ( micromede 2.0 ) was used to identify and analyze the pattern of potential ddis . micromedex is an electronic database that contains a separate section on ddi known as the drug - reax system . on entering the list of medications , it enlists all the potentially hazardous drug interactions on the basis of severity , onset and documentation status . on the basis of severity micromedex classifies ddi as major , moderate and minor as follows : major : potentially life - threatening ; requires medical intervention to minimize or prevent the serious adverse effects)moderate : results in potential deterioration of patients clinical condition and may require an alteration in therapy.minor : the effects are usually mild and may not require change in therapy . major : potentially life - threatening ; requires medical intervention to minimize or prevent the serious adverse effects ) moderate : results in potential deterioration of patients clinical condition and may require an alteration in therapy . it also classifies potential ddi as excellent , good , fair , poor or unlikely on the basis of documentation status as mentioned follows : excellent : the existence of the drug interaction has been clearly established by the controlled studies.good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking.fair : available documentation is poor.poor : documentation is scant ; however , the possibility of a clinical conflict exists.unlikely : documentation as well as a sound pharmacological basis is lacking . excellent : the existence of the drug interaction has been clearly established by the controlled studies . good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking . poor : documentation is scant ; however , the possibility of a clinical conflict exists . their demographic and medical details were properly documented in the self - designed patient profile form . the medications taken by the patients during their hospital stay were analyzed for possible drug interaction via the electronic database - micromedex 2.0 . there included a number of medicines , length of hospital stay and concurrent illnesses as the risk factors studied . pearson correlation was used to find the association between length of hospital stay and pddi , number of medicines prescribed and pddis and concurrent illness and pddis . all the statistical analysis was carried out with statistical package for social sciences ( ibm corporation ) , version 16.0 considering p < 0.01 as statistically significant . socio - demographic data was obtained from the patients after obtaining their verbal informed consent . the prospective observational study was undertaken for the period of 4 months from may 1 , 2012 to august 28 at the department of internal medicine of manipal teaching hospital ( mth ) , a tertiary care teaching hospital in western nepal . cardiac patients aged 18 years or older admitted to the cardiac unit of general medicine wards with a hospital stay of at least 24 h and those prescribed two or more drugs were enrolled for the study . patient profile form was used for collecting the socio - demographic variables and medication profile of patients . ddi database system ( micromede 2.0 ) was used to identify and analyze the pattern of potential ddis . micromedex is an electronic database that contains a separate section on ddi known as the drug - reax system . on entering the list of medications , it enlists all the potentially hazardous drug interactions on the basis of severity , onset and documentation status . on the basis of severity micromedex classifies ddi as major , moderate and minor as follows : major : potentially life - threatening ; requires medical intervention to minimize or prevent the serious adverse effects)moderate : results in potential deterioration of patients clinical condition and may require an alteration in therapy.minor : the effects are usually mild and may not require change in therapy . major : potentially life - threatening ; requires medical intervention to minimize or prevent the serious adverse effects ) moderate : results in potential deterioration of patients clinical condition and may require an alteration in therapy . it also classifies potential ddi as excellent , good , fair , poor or unlikely on the basis of documentation status as mentioned follows : excellent : the existence of the drug interaction has been clearly established by the controlled studies.good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking.fair : available documentation is poor.poor : documentation is scant ; however , the possibility of a clinical conflict exists.unlikely : documentation as well as a sound pharmacological basis is lacking . excellent : the existence of the drug interaction has been clearly established by the controlled studies . good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking . poor : documentation is scant ; however , the possibility of a clinical conflict exists . their demographic and medical details were properly documented in the self - designed patient profile form . the medications taken by the patients during their hospital stay were analyzed for possible drug interaction via the electronic database - micromedex 2.0 . there included a number of medicines , length of hospital stay and concurrent illnesses as the risk factors studied . pearson correlation was used to find the association between length of hospital stay and pddi , number of medicines prescribed and pddis and concurrent illness and pddis . all the statistical analysis was carried out with statistical package for social sciences ( ibm corporation ) , version 16.0 considering p < 0.01 as statistically significant . socio - demographic data was obtained from the patients after obtaining their verbal informed consent . the prospective observational study was undertaken for the period of 4 months from may 1 , 2012 to august 28 at the department of internal medicine of manipal teaching hospital ( mth ) , a tertiary care teaching hospital in western nepal . cardiac patients aged 18 years or older admitted to the cardiac unit of general medicine wards with a hospital stay of at least 24 h and those prescribed two or more drugs were enrolled for the study . patient profile form was used for collecting the socio - demographic variables and medication profile of patients . ddi database system ( micromede 2.0 ) was used to identify and analyze the pattern of potential ddis . micromedex is an electronic database that contains a separate section on ddi known as the drug - reax system . on entering the list of medications , it enlists all the potentially hazardous drug interactions on the basis of severity , onset and documentation status . on the basis of severity micromedex classifies ddi as major , moderate and minor as follows : major : potentially life - threatening ; requires medical intervention to minimize or prevent the serious adverse effects)moderate : results in potential deterioration of patients clinical condition and may require an alteration in therapy.minor : the effects are usually mild and may not require change in therapy . major : potentially life - threatening ; requires medical intervention to minimize or prevent the serious adverse effects ) moderate : results in potential deterioration of patients clinical condition and may require an alteration in therapy . it also classifies potential ddi as excellent , good , fair , poor or unlikely on the basis of documentation status as mentioned follows : excellent : the existence of the drug interaction has been clearly established by the controlled studies.good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking.fair : available documentation is poor.poor : documentation is scant ; however , the possibility of a clinical conflict exists.unlikely : documentation as well as a sound pharmacological basis is lacking . excellent : the existence of the drug interaction has been clearly established by the controlled studies . good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking . poor : documentation is scant ; however , the possibility of a clinical conflict exists . their demographic and medical details were properly documented in the self - designed patient profile form . the medications taken by the patients during their hospital stay were analyzed for possible drug interaction via the electronic database - micromedex 2.0 . there included a number of medicines , length of hospital stay and concurrent illnesses as the risk factors studied . pearson correlation was used to find the association between length of hospital stay and pddi , number of medicines prescribed and pddis and concurrent illness and pddis . all the statistical analysis was carried out with statistical package for social sciences ( ibm corporation ) , version 16.0 considering p < 0.01 as statistically significant . socio - demographic data was obtained from the patients after obtaining their verbal informed consent . a total of 150 cardiac patients admitted to the cardiac unit of general medicine ward were enrolled . among them , prescriptions of 32 patients were identified which had least one potentially interacting drug combination . the overall incidence rate of potential drug interaction was 21.3% . among 150 study patients , majority were in the age group of 61 - 70 years . male patients ( 91 [ 60.7% ] ) were in higher number compared with females ( 59 [ 39.3% ) ] . majority of patients ( 115 [ 76.7% ] ) had cardiovascular disease as their primary admission disease and rest ( 35 [ 23.3% ] ) had it as concurrent illness . hypertension ( 80 [ 53.3% ] ) was the most common diagnosis in the study population followed by coronary heart disease ( 29 [ 19.33% ] ) [ table 1 ] . hence the average number of medicines prescribed per patient was 6.9 . among the total of 1037 medicines , 464 were cardiovascular drugs . hence cardiovascular medicines constituted 44.8% of total medicines . the length of hospital stay of the patient was found to be 6.16 2.3 days . diagnosis of patients with cardiovascular diseases in a tertiary care hospital in nepal ( n=150 ) cardiovascular medicines prescribed to the patients admitted to a tertiary care hospital in nepal ( n=1037 ) a total of 48 pddis were identified . among the total of 150 patients , majority of patients ( 20 [ 62.5% ] ) were encountered with single interacting combination . this was followed by the patients who encountered two ( 8 [ 25% ] ) and three interactions ( 4 [ 12.5% ] ) . significant proportion of potential drug interactions identified was of moderate severity ( 30 [ 62.5% ] ) while just 18 interacting combinations identified were of major severity ( 37.5% ) . the interacting pairs of major severity along with the potentially hazardous effects and documentation status are enlisted in the table 3 . among 48 pddi identified , 6 ( 12.5% ) had excellent status of documentation , 36 ( 75.5% ) had good documentation status and 6 ( 12.0% ) were of fair documentation status . interactions encountered were analyzed on the basis of mechanism of interaction . in total , 28 ( 58.3% ) pddi was of pharmacokinetic type , 16 ( 33.3% ) were of pharmacodynamic type and remaining 4 ( 8.4% ) were of unknown mechanism . the most common interacting pairs identified in this study were atorvastatin / azithromycin ( 5 [ 10.4% ] ) , enalapril / metformin ( 5 [ 10.4% ] ) , enalapril / potassium chloride ( 5 [ 10.4% ] ) , atorvastatin / clarithromycin ( 4 [ 8.3% ] ) and furosemide / gentamicin ( 3 [ 6.3% ] ) . atorvastatin ( 16 [ 33.3% ] ) was the topmost drug found to be involved in potential ddi followed by enalapril ( 15 [ 31.2% ] ) , digoxin ( 4 [ 8.3% ] ) , furosemide ( 4 [ 8.3% ] ) , clopidogrel ( 3 [ 6.3% ] ) and warfarin ( 3 [ 6.3% ] ) . interacting pairs of major severity with the potentially hazardous effect and documentation status some factors were assessed to determine their association with the likelihood of occurrence of ddis . length of hospital stay , number of medicines and concurrent illness were the factors studied . statistical analysis by pearson correlation co - efficient revealed that there was a significant linear relationship between these factors and the occurrence of ddis [ table 4 ] . among 150 study patients , majority were in the age group of 61 - 70 years . male patients ( 91 [ 60.7% ] ) were in higher number compared with females ( 59 [ 39.3% ) ] . majority of patients ( 115 [ 76.7% ] ) had cardiovascular disease as their primary admission disease and rest ( 35 [ 23.3% ] ) had it as concurrent illness . hypertension ( 80 [ 53.3% ] ) was the most common diagnosis in the study population followed by coronary heart disease ( 29 [ 19.33% ] ) [ table 1 ] . hence the average number of medicines prescribed per patient was 6.9 . among the total of 1037 medicines , 464 were cardiovascular drugs . the length of hospital stay of the patient was found to be 6.16 2.3 days . diagnosis of patients with cardiovascular diseases in a tertiary care hospital in nepal ( n=150 ) cardiovascular medicines prescribed to the patients admitted to a tertiary care hospital in nepal ( n=1037 ) a total of 48 pddis were identified . among the total of 150 patients , majority of patients ( 20 [ 62.5% ] ) were encountered with single interacting combination . this was followed by the patients who encountered two ( 8 [ 25% ] ) and three interactions ( 4 [ 12.5% ] ) . significant proportion of potential drug interactions identified was of moderate severity ( 30 [ 62.5% ] ) while just 18 interacting combinations identified were of major severity ( 37.5% ) . the interacting pairs of major severity along with the potentially hazardous effects and documentation status are enlisted in the table 3 . among 48 pddi identified , 6 ( 12.5% ) had excellent status of documentation , 36 ( 75.5% ) had good documentation status and 6 ( 12.0% ) were of fair documentation status . interactions encountered were analyzed on the basis of mechanism of interaction . in total , 28 ( 58.3% ) pddi was of pharmacokinetic type , 16 ( 33.3% ) were of pharmacodynamic type and remaining 4 ( 8.4% ) were of unknown mechanism . the most common interacting pairs identified in this study were atorvastatin / azithromycin ( 5 [ 10.4% ] ) , enalapril / metformin ( 5 [ 10.4% ] ) , enalapril / potassium chloride ( 5 [ 10.4% ] ) , atorvastatin / clarithromycin ( 4 [ 8.3% ] ) and furosemide / gentamicin ( 3 [ 6.3% ] ) . atorvastatin ( 16 [ 33.3% ] ) was the topmost drug found to be involved in potential ddi followed by enalapril ( 15 [ 31.2% ] ) , digoxin ( 4 [ 8.3% ] ) , furosemide ( 4 [ 8.3% ] ) , clopidogrel ( 3 [ 6.3% ] ) and warfarin ( 3 [ 6.3% ] ) . interacting pairs of major severity with the potentially hazardous effect and documentation status some factors were assessed to determine their association with the likelihood of occurrence of ddis . length of hospital stay , number of medicines and concurrent illness were the factors studied . statistical analysis by pearson correlation co - efficient revealed that there was a significant linear relationship between these factors and the occurrence of ddis [ table 4 ] . among 150 study patients , majority were in the age group of 61 - 70 years . male patients ( 91 [ 60.7% ] ) were in higher number compared with females ( 59 [ 39.3% ) ] . majority of patients ( 115 [ 76.7% ] ) had cardiovascular disease as their primary admission disease and rest ( 35 [ 23.3% ] ) had it as concurrent illness . hypertension ( 80 [ 53.3% ] ) was the most common diagnosis in the study population followed by coronary heart disease ( 29 [ 19.33% ] ) [ table 1 ] . hence the average number of medicines prescribed per patient was 6.9 . among the total of 1037 medicines , 464 were cardiovascular drugs . the length of hospital stay of the patient was found to be 6.16 2.3 days . diagnosis of patients with cardiovascular diseases in a tertiary care hospital in nepal ( n=150 ) cardiovascular medicines prescribed to the patients admitted to a tertiary care hospital in nepal ( n=1037 ) majority of patients ( 20 [ 62.5% ] ) were encountered with single interacting combination . this was followed by the patients who encountered two ( 8 [ 25% ] ) and three interactions ( 4 [ 12.5% ] ) . significant proportion of potential drug interactions identified was of moderate severity ( 30 [ 62.5% ] ) while just 18 interacting combinations identified were of major severity ( 37.5% ) . the interacting pairs of major severity along with the potentially hazardous effects and documentation status are enlisted in the table 3 . among 48 pddi identified , 6 ( 12.5% ) had excellent status of documentation , 36 ( 75.5% ) had good documentation status and 6 ( 12.0% ) were of fair documentation status . interactions encountered were analyzed on the basis of mechanism of interaction . in total , 28 ( 58.3% ) pddi was of pharmacokinetic type , 16 ( 33.3% ) were of pharmacodynamic type and remaining 4 ( 8.4% ) were of unknown mechanism . the most common interacting pairs identified in this study were atorvastatin / azithromycin ( 5 [ 10.4% ] ) , enalapril / metformin ( 5 [ 10.4% ] ) , enalapril / potassium chloride ( 5 [ 10.4% ] ) , atorvastatin / clarithromycin ( 4 [ 8.3% ] ) and furosemide / gentamicin ( 3 [ 6.3% ] ) . atorvastatin ( 16 [ 33.3% ] ) was the topmost drug found to be involved in potential ddi followed by enalapril ( 15 [ 31.2% ] ) , digoxin ( 4 [ 8.3% ] ) , furosemide ( 4 [ 8.3% ] ) , clopidogrel ( 3 [ 6.3% ] ) and warfarin ( 3 [ 6.3% ] ) . some factors were assessed to determine their association with the likelihood of occurrence of ddis . length of hospital stay , number of medicines and concurrent illness were the factors studied . statistical analysis by pearson correlation co - efficient revealed that there was a significant linear relationship between these factors and the occurrence of ddis [ table 4 ] . it may pose a significant health hazard to patients when the risk - benefit ratio of combining interacting drugs is not accurately estimated . it has already been approximated that the effect of drug interactions can range from any minor morbidity to fatal consequences . the present study identified the pattern of pddis among patients admitted to cardiac unit of general medicine ward . the value obtained in the present study is relatively less compared with the study by patel et al . in india who reported an incidence rate of 30.23% . these differences might be because our study took into consideration only the potential drug interactions of major and moderate severity in contrast to the other studies that considered drug interactions of all severity . data regarding the incidence of pddi in cardiac patients is not sufficient but previous studies in general medicine in mth had identified the maximum involvement of cardiovascular medicines in drug interactions . on analyzing the mechanism of drug interaction identified here , pharmacokinetic type of reactions ( 58.3% ) was found in higher number compared to pharmacodynamic type ( 33.3% ) . aparasu et al . who reported 76% of pharmacokinetic and 22% of pharmacodynamic interactions respectively . of the total pddis identified , the interacting combination of moderate severity ( 62.5% ) constituted majority of pddi . the most common interacting pairs identified were atorvastatin / azithromycin , enalapril / metformin , enalapril / potassium chloride , atorvastatin / clarithromycin and furosemide / gentamicin . the pddi involving atorvastatin ( 16 [ 33.3% ] ) was the highest among all followed by enalapril ( 15 [ 31.2% ] ) , digoxin ( 4 [ 8.3% ] ) , furosemide ( 4 [ 8.3% ] ) , clopidogrel ( 3 [ 6.3% ] ) and warfarin ( 3 [ 6.3% ] ) . the values obtained here are quite different from the study in india where patel et al . reported aspirin ( 44.85% ) , heparin ( 42.78% ) , clopidogrel ( 22.16% ) , warfarin ( 11.59% ) , atorvastatin ( 7.22% ) and ramipril ( 6.95% ) as the highest risk drug categories for ddi . there was maximum involvement of atorvastatin ( 16 [ 33.3% ] ) in pddi encountered in this study . this might be because atorvastatin ( 68 [ 14.7% ] ) was one of the most commonly prescribed medicines in the present study . a significant positive linear relationship was found between the length of hospital stay and pddis ( r = 0.63 , p < 0.01 ) . our finding well resembles to the finding by several studies which have also shown that increased incidence of pddi corresponds with an increase in duration of hospital stay . the reason might be that the likelihood of getting the multiple drugs increases with the increased length of hospital stay which in turn will increase the likelihood of pddi . similar positive linear relationship was also found between the number of medicines prescribed and pddi ( r = 0.5 , p < 0.01 ) and with concurrent illness and pddi ( r = 0.62 , p < 0.01 ) . the findings well correlate with the fact that polypharmacy increases the likelihood of ddis to a great extent as shown by several studies . this study shows that cardiac in - patients are at a high risk of hazardous ddi . this emphasizes the need to consider pddi during therapeutic planning , protect patients from consequence of drug interactions . in addition , providing ddi related information to the prescribers and drug interaction alert software to the dispensing pharmacist can play a vital role in minimizing the incidence rate of ddi . in the present study , monitor the occurrences of ddi clinically along with pharmacokinetic estimation of the drug is suggested , especially for moderate to major pddi .

aim : drug - drug interaction ( ddi ) is of major concern in patients with complex therapeutic regimens . the involvement of cardiovascular medicines in drug interaction is even higher . however , reports of ddi between these groups of drugs are few . the study aims to identify the potential ddi among hospitalized cardiac patients . furthermore , we assessed the possible risk factors associated with these interactions.subjects and methods : the type of study prospective observational study was conducted from may 2012 to august 2012 among hospitalized cardiac patients . cardiac patients who were taking at least two drugs and who had a hospital stay of at least 24 h were enrolled . the medications of the patients were analyzed for possible interactions using the standard drug interaction database - micromedex -2 ( thomson reuters ) 2.0.results:from a total of 150 enrolled patients , at least one interacting drug combination was identified among 32 patients . the incidence of potential ddi was 21.3% . a total of 48 potentially hazardous drug interactions were identified . atorvastatin / azithromycin ( 10.4% ) , enalapril / metformin ( 10.4% ) , enalapril / potassium chloride ( 10.4% ) , atorvastatin / clarithromycin ( 8.3% ) and furosemide / gentamicin ( 6.3% ) were the most common interacting pairs . drugs most commonly involved were atorvastatin , enalapril , digoxin , furosemide , clopidogrel and warfarin . majority of interactions were of moderate severity ( 62.5% ) and pharmacokinetic ( 58.3% ) in nature . increased number of medicines , prolonged hospital stays and comorbid conditions were the risk factors found associated with the potential ddi.conclusions:this study highlighted the need of intense monitoring of patients who have identified risk factors to help detect and prevent them from serious health hazards associated with drug interactions .

Introduction Subjects and Methods None Setting and Study Design Study Population Tools Used Operational Modality Statistical Analysis Ethics Results None Characteristics of Study Population Potential Drug Interactions Risk Factors Discussion Conclusion

important risk factors for thoracic actinomycosis are underlying respiratory disorders such as emphysema and chronic bronchitis.different ct patterns can be distinguished in thoracic actinomycosis : parenchymal , bronchiectatic , endobronchial and extrapulmonary.typical ct findings in the parenchymal pattern are a central low density within the parenchymal consolidation and adjacent pleural thickening . important risk factors for thoracic actinomycosis are underlying respiratory disorders such as emphysema and chronic bronchitis . different ct patterns can be distinguished in thoracic actinomycosis : parenchymal , bronchiectatic , endobronchial and extrapulmonary . typical ct findings in the parenchymal pattern are a central low density within the parenchymal consolidation and adjacent pleural thickening . actinomycetes are branching gram - positive anaerobic bacteria belonging to the actinomyceataceae family and are commensal in the human oropharynx , gastrointestinal tract and female genitalia . it is a rare infection that in the past has been reported to occur in 1 in 300,000 people per year . the incidence of all forms of actinomycosis has declined markedly in the last 3 to 4 decades . the infection can involve every organ of the body , and pulmonary actinomycosis is the third most common type after cervicofacial and abdomino - pelvic forms . the pulmonary form of actinomycosis constitutes 15 % of the total burden of disease , although estimates of up to 50 % have been reported . pulmonary actinomycosis has worldwide distribution and has no age or race predilection , although men acquire the disease slightly more often than women ( in an approximately 3:1 ratio ) , and children are rarely affected [ 4 , 5 ] . a higher incidence of pulmonary actinomycosis has been reported in patients with underlying respiratory disorders such as emphysema , chronic bronchitis , bronchiectasis and any condition in which the lung parenchyma are destroyed by a previous infection . alcoholism , poor oral hygiene , dental disease , and facial or dental trauma are important risk factors for the thoracic form . various chronic debilitating diseases and conditions may facilitate respiratory tract infection , including being at risk for aspiration and having diabetes mellitus , neurologic and psychiatric diseases , malnutrition , drug abuse and hiatal hernia [ 1 , 3 ] . unlike in many other unusual granulomatous infectious diseases , the infection rate does not seem to be increased in immunocompromised patients , such as those with acquired immune deficiency syndrome , undergoing chemotherapy , or on steroid or immunosuppressive therapy [ 5 , 6 ] . sulphur granules by gram or histologic staining is the principal method of direct detection of actinomyces . this material contains sulphur granules , colonies of organisms forming an amorphous centre surrounded by a rosette of clubbed filaments . direct bacterial confirmation by culture is difficult because of inadequate anaerobic culture , prior antibiotic therapy or overgrowth of concomitant organisms . thus , culture of expectorated sputum or bronchoscopy aspirates are usually not successful , while fine - needle aspiration , transbronchial biopsy and computed tomography or ultrasound - guided biopsies lead to accurate diagnoses [ 1 , 4 , 7 ] . the clinical manifestation of the disease has changed to a less aggressive form compared to the pre - antibiotic era . the usual presentation is now an indolent , slowly progressive pneumonia with fever , weight loss , cough , sputum and chest pain . if the disease progresses to the proximal airway and vessels , life - threatening complications such as massive haemoptysis or bronchoesophageal fistula may occur . the principal treatment of actinomycosis is long - term use of high - dose intravenous penicillin . however , many recent studies have reported that the short - term treatment is successful and that the traditional intensive , long - term regimen is not necessary [ 8 , 9 ] . in the setting of abscess , surgical resection may be a valid option for patients who do not respond to antibiotics for up to 12 weeks according to a recent study . an awareness of the typical imaging findings of thoracic actinomycosis helps in making an early diagnosis , preventing fatal complications and unwarranted surgery . parenchymal patterns of pulmonary actinomycosis include a peripheral pulmonary nodule , mass or consolidation , all of which may or may not be cavitary or multifocal . typical ct findings are reported as central areas of low attenuation within the consolidation in 6275 % of cases and adjacent pleural thickening in 5073 % ( fig . 1 ) the disease initially manifests as a small , poorly defined , peripheral pulmonary nodule with or without interlobular septal thickening . the pulmonary nodule gradually increases to segmental air - space consolidation , which suggests bronchogenic spread of the disease ( fig . 2 ) . if therapy is not adequate , central areas of low attenuation with a cavity form with the slow progression ( fig . 3 ) . central low - attenuation areas may be multiple and variable in size and show rim - like peripheral enhancement on enhanced ct . in later stages , lung parenchyma may be destroyed and the infection may extend across fissures to a neighbouring pulmonary lobe ( trans - fissural extension ) , the pleura or chest wall , with abscess formation in these areas ( fig . 4 ) [ 10 , 11 , 13].fig . the ct scan in the mediastinal window setting shows a 3-cm mass - like consolidation with central , low - density peripheral rim enhancement in the right lower lobe and adjacent pleural thickening with subpleural fat infiltration ( arrow)fig . 2early stage parenchymal actinomycosis in a 44-year - old man who was a heavy alcohol drinker . the ct image displayed on lung window settings shows focal consolidation surrounded by ill - defined , peripheral pulmonary nodules ( arrows ) , which suggests bronchogenic spread of the disease and interlobular septal thickeningfig . 3parenchymal actinomycosis with abscess formation in a 51-year - old man with cough and dyspnoea who had a history of alcoholism . chest ct scan displayed on a mediastinal setting ( a ) and thin - section ct ( b ) show peripheral consolidation with multiple internal cavities , a central low - attenuation area and adjacent pleural thickening in the right upper lobefig . 4transfissural extension of parenchymal actinomycosis in a 43-year - old man with high fever for 10 days manifesting as chronic necrotising pneumonia . chest ct scan in mediastinal window setting shows multifocal lobar consolidation containing a large low attenuation area in the right lower and middle lobe , which demonstrates extension across the fissure typical parenchymal actinomycosis in a 58-year - old man with haemoptysis for a month . the ct scan in the mediastinal window setting shows a 3-cm mass - like consolidation with central , low - density peripheral rim enhancement in the right lower lobe and adjacent pleural thickening with subpleural fat infiltration ( arrow ) early stage parenchymal actinomycosis in a 44-year - old man who was a heavy alcohol drinker . the ct image displayed on lung window settings shows focal consolidation surrounded by ill - defined , peripheral pulmonary nodules ( arrows ) , which suggests bronchogenic spread of the disease and interlobular septal thickening parenchymal actinomycosis with abscess formation in a 51-year - old man with cough and dyspnoea who had a history of alcoholism . chest ct scan displayed on a mediastinal setting ( a ) and thin - section ct ( b ) show peripheral consolidation with multiple internal cavities , a central low - attenuation area and adjacent pleural thickening in the right upper lobe transfissural extension of parenchymal actinomycosis in a 43-year - old man with high fever for 10 days manifesting as chronic necrotising pneumonia . chest ct scan in mediastinal window setting shows multifocal lobar consolidation containing a large low attenuation area in the right lower and middle lobe , which demonstrates extension across the fissure not uncommonly , the infection presents as a mass , sometimes cavitating , that may mimic lung cancer [ 10 , 14 ] . associated findings are interlobular septal thickening , bronchiectasis , mediastinal lymphadenopathy , adjacent pleural thickening , pleural effusion and empyema [ 10 , 12 , 14 ] . histologically , central low - attenuation areas seen on ct represent microabscesses , which contain actinomycotic or sulphur granules . the peripheral enhancing portion is composed of an outer rim of granulation tissue and fibrosis . the differential diagnosis of the parenchymal type should include tuberculosis , bacterial or fungal necrotising pneumonia , and lung cancer . previous infections resulting in lung destruction such as tuberculosis and bacterial infections predispose patients to actinomycosis ( fig . 5 ) . common co - pathogens for thoracic actinomycosis have been described as actinobacillus actinomycetemcomitans , staphylococci , streptococci , haemophilus spp . and aspergillus [ 16 , 17 ] . the pathogenesis of co - infection is a synergistic effect : oxygen deprivation due to other bacteria creates an anaerobic milieu in which actinomyces thrive ( fig . 6 ) . ct features of the bronchiectatic form include localised areas of bronchiectasis , irregular bronchial wall thickening , and irregular peribronchial consolidation with or without abscess formation . it is sometimes difficult to differentiate cystic bronchiectasis from central low attenuated necrotic areas of the parenchymal type ( fig . chest ct scan displayed on the mediastinal ( a ) and lung ( b ) window setting shows bronchial dilatation with wall thickening and focal consolidation . chest ct scan in lung window setting obtained with an axial scan ( a ) and a coronal reformatted image ( b ) shows focal bronchiectatic changes with intraluminal nodules in the right upper lobe . low - power photomicrography of the surgical specimen from the right upper lobe ( c ) ( original magnification , 20 ; haematoxylin - eosin stain ) demonstrates an actinomyces colony ( star ) within the ectatic bronchus ( arrows ) . medium - power photomicrograph ( d ) ( original magnification , 400 ; gomori methenamine - silver stain ) shows a nodule within bronchiectasis composed of central aggregation of aspergillus ( arrow ) and surrounding actinomyces colonies ( star)fig . 7mixed parenchymal , bronchiectatic and mediastinal actinomycosis in a 69-year - old woman with haemoptysis . a chest ct scan with a modified window setting shows lobar consolidation with cavitary change in the left lower lobe . note the distended oesophagus with a beak - like appearance that extends toward the consolidation of the left lower lobe ( arrow ) . b chest ct scan with the mediastinal window setting at the level below ( a ) shows calcified brocholith ( arrowhead ) within the dilated tubular mucus retained bronchus . c left lower lobectomy specimen showed cystic dilation of thickened bronchi , filled with greyish , fragile and granular material with a foul odour . a connection between the oesophagus and bronchus ( oesophagobronchial fistula , arrow ) was found where the steel rod is pointing ( arrow ) bronchiectatic form of actinomycosis in a 44-year - old man with underlying bronchiectasis . chest ct scan displayed on the mediastinal ( a ) and lung ( b ) window setting shows bronchial dilatation with wall thickening and focal consolidation . note the adjacent pleural thickening in the right lower lobe co - infection of bronchiectatic actinomycosis with aspergillus in a 52-year - old man . chest ct scan in lung window setting obtained with an axial scan ( a ) and a coronal reformatted image ( b ) shows focal bronchiectatic changes with intraluminal nodules in the right upper lobe . low - power photomicrography of the surgical specimen from the right upper lobe ( c ) ( original magnification , 20 ; haematoxylin - eosin stain ) demonstrates an actinomyces colony ( star ) within the ectatic bronchus ( arrows ) . medium - power photomicrograph ( d ) ( original magnification , 400 ; gomori methenamine - silver stain ) shows a nodule within bronchiectasis composed of central aggregation of aspergillus ( arrow ) and surrounding actinomyces colonies ( star ) mixed parenchymal , bronchiectatic and mediastinal actinomycosis in a 69-year - old woman with haemoptysis . a chest ct scan with a modified window setting shows lobar consolidation with cavitary change in the left lower lobe . note the distended oesophagus with a beak - like appearance that extends toward the consolidation of the left lower lobe ( arrow ) . b chest ct scan with the mediastinal window setting at the level below ( a ) shows calcified brocholith ( arrowhead ) within the dilated tubular mucus retained bronchus . c left lower lobectomy specimen showed cystic dilation of thickened bronchi , filled with greyish , fragile and granular material with a foul odour . a connection between the oesophagus and bronchus ( oesophagobronchial fistula , arrow ) was found where the steel rod is pointing ( arrow ) the endobronchial form reflects actinomycosis colonisation of pre - existing obstructive broncholiths or endobronchial foreign bodies , which inflames the adjacent airway and causes distal obstructive pneumonia . broncholiths are formed by erosion of calcified lymph nodes into the airway as a result of a granulomatous process . other causes of broncholithiasis include aspiration of bone or foreign material , erosion by and extrusion of calcified bronchial cartilage plates . endobronchial actinomycosis associated with broncholithiasis manifests on ct as a proximal obstructive calcified endobronchial nodule with distal obstructive pneumonia of the involved lobe or segment ( fig . the calcified foci are mixed with actinomyces microcolonies and the distal pneumonic consolidation seen on ct is composed of acute suppurative inflammation , abscesses or organising pneumonia .fig . a b the axial and coronal chest ct scans in the lung window setting show a small broncholith ( arrow ) obstructing the lumen of the subsegmental bronchus of the anterior segment and distal subsegmental consolidation within the internal necrotic portion . broncholiths were confirmed as granular colonies of actinomyces . calcified mediastinal lymph nodes are also noted , suggesting previous tuberculosis infection ( arrowheads ) . c photographs of the right upper lobectomy specimen show dilatation of central bronchi filled with yellowish - grey granular and fragile material endobronchial actinomycosis with broncholithiasis in a 53-year - old woman . a b the axial and coronal chest ct scans in the lung window setting show a small broncholith ( arrow ) obstructing the lumen of the subsegmental bronchus of the anterior segment and distal subsegmental consolidation within the internal necrotic portion . broncholiths were confirmed as granular colonies of actinomyces . calcified mediastinal lymph nodes are also noted , suggesting previous tuberculosis infection ( arrowheads ) . c photographs of the right upper lobectomy specimen show dilatation of central bronchi filled with yellowish - grey granular and fragile material bronchial infection may occur from aspiration of a foreign body contaminated with actinomyces organisms . this type of infection develops in debilitated patients with poor oral hygiene and in a condition that facilitates foreign body aspiration . reported causes of aspirated foreign bodies resulting in thoracic actinomycosis include teeth , chicken bones , fish bones , grape seeds and beans . ct shows a radio - opaque foreign body with surrounding bronchial thickening and distal obstructive pneumonia . other secondary findings such as consolidation , atelectasis , bronchiectasis and pleural effusion may also be present [ 12 , 19 ] . calcified endobronchial actinomycosis should be differentiated from an aspirated foreign body or de novo calcification . broncholiths are usually associated with evidence of previous tuberculous infection such as calcified hilar and mediastinal lymph nodes , and the calcification conforms to the shape of the bronchial lumen . rare case reports of endobronchial actinomycosis are associated with an endobronchial mass . in these cases , a reddish polypoid friable mass within the bronchi and surrounding inflamed mucosa these endobronchial forms may mimic endobronchial tuberculosis or bronchogenic carcinoma [ 21 , 22 ] . actinomycosis may spread from the lung to the pleura , mediastinum and chest wall , with little regard for anatomic barriers . the organisms produce proteolytic enzymes , and peripheral pneumonia tends to involve the pleura , producing empyema , and invade the chest wall with involvement of bones such as the ribs or vertebrae [ 10 , 23 ] . chest wall involvement is usually preceded by lung infection , but may occur by means of a direct extension from the neck , oesophagus , abdomen or retroperitoneum . chest wall involvement is much less common now than previously reported because antibiotic treatment earlier in the disease course is now the rule . radiological manifestations of chest wall involvement include a soft tissue chest wall mass continuous with pulmonary disease , with or without central low - attenuation , empyema , periosteal proliferation along the ribs , and destruction of ribs or vertebrae ( fig . pulmonary infections , which can produce similar findings of contiguous chest wall invasion , are tuberculosis , blastomycosis , nocardiosis , cryptococcosis and invasive aspergillosis . other differential diagnoses are lymphoma , bronchogenic carcinoma , malignant mesothelioma and rare chest wall tumours .fig . 9actinomycosis involving the chest wall in a 75-year - old woman , manifesting as a palpable mass on the left chest wall . axial ct image shows a heterogeneous mass with central low attenuation and peripheral enhancement on the left chest wall , contiguous with consolidation ( arrow ) in lung parenchyma , and pleural effusion actinomycosis involving the chest wall in a 75-year - old woman , manifesting as a palpable mass on the left chest wall . axial ct image shows a heterogeneous mass with central low attenuation and peripheral enhancement on the left chest wall , contiguous with consolidation ( arrow ) in lung parenchyma , and pleural effusion mediastinal actinomycosis is extremely rare . infection most commonly results from contiguous spread from adjacent lung or may be induced by perforation of the oesophagus , chest trauma , thoracic surgery , tracheobronchial perforation or , rarely , hematogenous dissemination . reported findings are pericardial effusion with or without pleural effusion , a pericardial mass , a mediastinal mass resulting in various complications such as superior vena cava syndrome , pancoast syndrome or an oesophagotracheal fistula ( figs . 7 and 10 ) [ 2528 ] . actinomycotic bacteraemia is very uncommon , but arises more frequently from pulmonary foci than from any other form ; it may give rise to metastatic abscesses in other viscera , the skeleton or soft tissues.fig . 10actinomycosis with oesophagobronchial fistula in a 44-year - old woman who complained of a repetitive cough with food intake . a barium oesophagography depicts the fistula tract ( arrowheads ) between the oesophagus and right bronchus intermedius . b chest ct scan shows a segmental consolidation and bronchiectasis containing a broncholith ( arrow ) in the right lower lobe of the superior segment . consolidation extends to the mediastinum and oesophagus actinomycosis with oesophagobronchial fistula in a 44-year - old woman who complained of a repetitive cough with food intake . a barium oesophagography depicts the fistula tract ( arrowheads ) between the oesophagus and right bronchus intermedius . b chest ct scan shows a segmental consolidation and bronchiectasis containing a broncholith ( arrow ) in the right lower lobe of the superior segment . the prognosis is excellent with low mortality when the disease is recognised early and appropriate treatment is given . thus , actinomycosis should be considered as part of the differential diagnosis when confronted with chronic unresolving pneumonia that extends across anatomic barriers or manifests as an endobronchial lesion .

backgroundthoracic actinomycosis is an uncommon , chronic suppurative bacterial infection caused by actinomyces species , especially actinomyces israelii.methodsit is usually seen in immunocompetent patients with respiratory disorders , poor oral hygiene , alcoholism and chronic debilitating diseases.resultswe illustrate the radiological manifestations of thoracic actinomycoses in various involved areas in the thorax.conclusionthoracic actinomycosis can be radiologically divided into the parenchymal type , the airway type including bronchiectasis , the endobronchial form , and the mediastinum or chest wall involvement type.teaching pointsimportant risk factors for thoracic actinomycosis are underlying respiratory disorders such as emphysema and chronic bronchitis.different ct patterns can be distinguished in thoracic actinomycosis : parenchymal , bronchiectatic , endobronchial and extrapulmonary.typical ct findings in the parenchymal pattern are a central low density within the parenchymal consolidation and adjacent pleural thickening .

None Introduction Parenchymal actinomycosis Bronchiectatic actinomycosis Endobronchial actinomycosis Extrapulmonary spread Conclusions

the anatomy of the scapular neck may have significant implications for rtsa design , surgical planning , and reduction of associated complications . reverse total shoulder arthroplasty ( rtsa ) is a popular treatment for rotator cuff tear arthropathy and other painful shoulder conditions in which the rotator cuff is deficient . scapular notching is an important complication that occurs in up to 96% of patients and has been correlated with poorer clinical outcomes , chronic inflammation , osteolysis , and early prosthesis failure . the exact cause of scapular notching and methods for preventing this complication are poorly understood . recently , investigative work has focused on delineating the various implant related factors influencing the incidence of scapular notching . earlier studies have demonstrated anatomical variations in the anatomy of patient scapulae and the influence that patient anatomy has on the development of scapular notching . these studies have demonstrated that there is no one - size - fits - all approach to rtsa that will provide an optimal impingement free range of motion . scapular neck length ( snl ) or the lateral offset of the humerus from the glenoid , has been shown to have a large influence over the rate of scapular notching . have shown in numerous studies , both clinically and in the laboratory that lateralization of the humerus dramatically reduces the rate of scapular notching . recently , paisley et al . have demonstrated significant variability in snl in a small series of patients . however , there are currently no studies that have characterized the variations in snl in a large body of human specimens . the purpose of this study is to characterize the snl in a large body of patient scapulae using the haman - todd osteological collection , one of the largest osteological collections of complete human skeletons in the world . we hypothesize that the ability to understand the variations in snl across a population of patients will enable us to minimize scapular notching by incorporating snl measurements into the reconstruction plan of patients undergoing rtsa 's . our secondary hypothesis is that measurements of the snl and scapular neck angle ( sna ) , using defined anatomic landmarks , would be reproducible between investigators , and that these measurements would differ considerably between individuals . for the purposes of this study , we obtained 442 dry human scapulae from the hamann - todd osteological collection , which is housed in the cleveland museum of natural history ( cleveland , oh ) . this collection contains over 3000 complete disarticulated human skeletons that were preserved and prepared by case western reserve university school of medicine faculty from unclaimed remains at the cleveland city morgue between 1912 and 1938 . inclusion criteria included adult human skeletons , age 18 - 65 , with intact bilateral scapulae . exclusion criteria included pediatric skeletons , those with damaged or missing scapulae and those skeletons without a bilateral set . digital photographs of scapulae using a standardized posteroanterior view were obtained , which was chosen based on work by handling et al . who described the insertion of the long head of the triceps on the posterior lateral portion of the scapula at the infraglenoid tubercle . for the purposes of photography and measurement , the scapula was positioned at a standard distance from the camera lense . specifically the scapula was positioned along the craniocaudal axis to allow the glenoid fossa anterior and posterior margins to be flush and parallel to the camera . the scapula was positioned along the medial - to - lateral axis so that the entire glenoid fossa could be visualized free of obstruction from the acromion . snl was defined as the distance from the most lateral portion of the infraglenoid tubercle to the most inferolateral portion of the glenoid fossa [ figure 1 ] . the infraglenoid tubercle was consistently found in a location that could be alternatively located by following the topographical change at the neck - body junction that coursed inferiorly from the spinoglenoid notch to the most lateral extent of the infraglenoid tubercle [ figure 2 ] . this was used as an additional criterion for location of the tubercle if its location was not immediately obvious . the sna , as defined by simovitch et al . , was measured as the intersection of a line parallel to the face of the glenoid and a second line between the inferior glenoid rim and a point 1 cm along the inferior border of the scapula . length determined from lateral edge of infraglenoid tubercle to lateral edge of inferior glenoid measurement of scapular neck length assisted by topographical change noted from spinoglenoid notch to lateral border of infraglendoid tubercle as indicated by red line the mean , standard deviation , and ranges for snl and sna were calculated and compared between groups based on sex and race using a student 's t - test . a single examiner performed all digital measurements using standard imagej nih type software . interobserver and intraobserver reliability for scapular neck measurements were performed by four of the authors in order to determine the validity of our measurement protocol using the intraclass correlation coefficient . our study population consisted of 221 cadaveric specimens ( 188 male , 33 female ) of mean age 45.1 years at the time of death . 165 specimens were caucasian , and 56 were african - american [ table 1 ] . the scapular neck could be measured bilaterally in all specimens , for a total of 442 scapulae measured . the mean snl for our study cohort was 10.6 mm 3.3 mm ( range 3.7 - 24.3 mm ) . the mean snl for the left scapula was 10.4 mm 3.4 mm , and the mean snl for the right scapula was 10.7 mm 3.5 mm ( p < 0.36 ) . males had a slightly larger snl ( 10.8 mm 3.3 mm ) than females ( 10.1 mm 3.2 mm ) , which was not statistically significant ( p < 0.12 ) . caucasians had a larger snl ( 10.9 mm 3.3 mm ) than african - americans ( 10.0 mm 0.32 ) that was statistically significant ( p < 0.01 ) . intra - observer reliability for the measurement of snl was 0.973 , and inter - observer reliability was 0.856 ( 95% confidence interval [ ci ] = 0.716 - 0.937 ) . demographics of specimens the mean sna was 106.7 11.0 , and ranged from 76.9 to 139.4. the mean sna for the left scapula was 107.1 10.3 , and the mean sna for the right scapula was 106.3 11.5 ( p < 0.29 ) . males demonstrated a mean sna of 106.5 10.7 , and the mean sna for females was 107.3 12.6 ( p < 0.59 ) . the mean sna for caucasians was 107.2 11.1 , and the mean for african - americans was 105.3 10.7 ( p < 0.10 ) . interobserver reliability for the measurement of sna was 0.682 ( 95% ci = 0.348 - 0.861 ) , and the intraobserver reliability was 0.681 ( 95% ci = 0.363 - 0.859 ) . no significant correlation was found between snl and sna ( pearson correlation coefficient = 0.018 ) ( p = 0.702 ) [ figure 3 ] . no significant correlation between scapular neck angle ( deg , x - axis ) and scapular neck length ( cm , y - axis ) we have performed the largest anatomic study of scapular neck morphology using 442 dry cadaveric specimens from an osteological collection , and we report the mean and ranges for the snl and sna with respect to sex and race . our hypothesis that these measurements would be reproducible between investigators was confirmed , as was our hypothesis that significant differences in snl and sna would be identified . the relationship between race and sex demonstrated that the snl was insignificantly larger in males and significantly larger in the caucasian population . however , the absolute value of these race and sex differences were quite small , making this finding difficult to interpret , and creating a need for the future follow up studies . the anatomy of the scapula has long been of interest to shoulder surgeons , but few anatomic studies have been performed in order to identify differences within the population that may impact the treatment of shoulder pathology . multiple studies have discussed the wide variation in glenoid morphology , especially as this relates to traumatic and degenerative disease . however , although snl and sna have both been implicated as contributing to complications and functional outcomes after reverse shoulder arthroplasty , ours is the first study to define the population means and ranges for these parameters . previously measured the snl of cadaveric specimens using a reference from the most inferolateral edge of the glenoid fossa to the most lateral extent of the posterior column to define the snl . however , our analysis of the scapula demonstrated significant variability in determining the lateral extent of the posterior column . our study instead demonstrated a consistent finding of the infraglenoid tubercle from the posterior view , and we propose this functional snl should be defined as the portion of the lateral border of the scapula from the most lateral edge of the infraglenoid tubercle to the most inferolateral edge of the glenoid fossa [ figure 1 ] . as stated by middernacht et al . , the majority of scapular notching occurs in the first cm of the scapular neck which appears to be related to the position of the infraglenoid tubercle . a far lateral infraglenoid tubercle implies a shorter functional snl and an increased propensity for scapular notching . our study has established population norms for snl and sna using a large sample size , and based on the wide variation in these parameters , greater customization in rtsa design may be necessary . we found snl to be highly variable among specimens , with a minimum of 3.7 mm and a maximum of 24.3 mm , which represents more than a 6-fold difference in snl within our population [ figure 4 ] . the variability of sna within our population was also high , with a large range of 76.9 - 139.4. in the setting of rtsa , previous studies have postulated that acute angles , or superior tilting of the glenosphere , can lead to increased scapular notching and early prosthesis failure while large angles , or inferior tilting of the glenosphere may be protective against scapular notching . one can propose that patients with a short snl , an acute sna or a combination there of may be prone to scapular notching . it can also be hypothesized that using a lateralized rtsa design in a patient with a long scapular neck may excessively lateralize the scapulohumeral articulation , leading to increased risk of acromial stress fracture . comparatively , for patients with a short snl , others have lateralized the center of rotation using bone graft or a lateralized rtsa design , which may lead to greater external rotation , improved deltoid contour , and diminished notching . it is likely that the snl and the sna plays a large role in scapular notching , and should be taken into account when planning for rtsa . greater customization of rtsa implants would help surgeons better fit patients based on their native anatomy , which may improve clinical outcomes and reduce complications . short versus long necked scapula limitations of the present study relate to its design using cadaveric specimens , which limits the conclusions that may be drawn regarding the function of these shoulders in daily life . it is unknown whether any of these persons may have had shoulder pain or other issues related to their anatomy . in addition , our study population contained only african - american and caucasian specimens , which limits the conclusions that may be drawn regarding other ethnicities . future studies may analyze the impact of snl and sna on scapular notching , range of motion , and functional outcomes using different implant designs , as well as develop optimal imaging techniques to better assess preoperative scapular anatomy for use in surgical planning . a wide range of snl s and snas were observed in our study population , and only small differences were noted based on sex and race . the anatomy of the scapular neck may have significant implications for rtsa design and surgical planning , and greater attention to matching a patient 's native anatomy may ultimately reduce the incidence of complications such as scapular notching .

purpose : reverse total shoulder arthroplasty ( rtsa ) may be used to treat a variety of pathologic shoulder conditions , but complications such as scapular notching continue raise concerns . variable anatomy surrounding the glenoid may have implications for future rtsa design , but at present the anatomy of the scapular neck in the human population has not been clearly defined.materials and methods:442 human scapulae from the hamann - todd osteological collection were measured for scapular neck length ( snl ) and scapular neck angle ( sna ) . snl was defined as the distance from the most lateral portion of the infraglenoid tubercle to the most inferolateral portion of the glenoid fossa . the sna was measured according to gerber et al . previously . the mean , standard deviation and ranges for snl and sna were calculated and compared based on sex and race , and interobserver variability was calculated.results:the mean snl was 1.06 cm 0.33 cm ( 0.37 - 2.43 cm ) . males demonstrated a larger snl ( 1.08 cm 0.33 cm ) than females ( 1.01 cm 0.32 cm ) ( p < 0.12 ) , and caucasians ( 1.09 cm 0.33 cm ) demonstrated a significantly larger snl than african - americans ( 1.00 cm 0.32 ) ( p < 0.01 ) . the mean sna was 106.7 11.0 ( 76.9 - 139.4 ) . no significant correlation was found between snl and sna ( pearson correlation coefficient = 0.018 ) ( p < 0.702).conclusion : scapular neck length and sna vary widely within the population but there appears to be a tendency towards increased snl in males and caucasians.clinical relevance : the anatomy of the scapular neck may have significant implications for rtsa design , surgical planning , and reduction of associated complications .

Clinical Relevance: INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

no clear etiology has been associated with most ependymomas to date . unlike many other cancers for which existing familial cancer syndromes provided important clues for our initial understanding of tumorigenic mechanisms , there are few known familial ependymoma syndromes . we do know , however , that there is increased incidence of spinal intramedullary ependymomas in patients with neurofibromatosis type 2 ( nf2),.the nf2 gene is located on chromosome 22q , which is frequently lost in patients with spinal ependymomas. however , many of these tumors , especially those that occur intracranially , do not harbor nf2 mutations . thus , despite that the nf2 gene may be important in the formation of some spinal ependymomas , it is probably not the critical tumor suppressor gene on chromosome 22q that is involved in sporadic intracranial ependymoma tumorigenesis. ependymoma has also been reported in patients with li - fraumeni syndrome , i.e. germline mutation of the tp53 tumor suppressor gene , but such occurrences as well as somatic mutations of tp53 in sporadic ependymomas are rare , thus diminishing the role of p53 in ependymoma tumorigenesis . there has been one report of a patient with turcot syndrome , i.e. germline mutation of the adenomatous polyposis coli ( apc ) gene , whose loss of function activates the wnt pathway and predisposes the patient to colorectal cancer , who developed multiple ependymomas located intracranially and spinally , . both intracranial and spinal ependymomas have also been observed in patients with the multiple endocrine neoplasia type i ( men1 ) syndrome. however , the role of the apc gene / wnt signaling activation and that of men1 in sporadic ependymoma tumorigenesis remain unknown . furthermore , there are a few families with increased ependymoma incidence but without any currently known familial cancer syndromes. two such families have loss of 22q but lack nf2 mutation , further suggesting the presence of another crucial tumor suppressor gene at that chromosomal region , . dna sequences similar to sv40 virus and the virus - encoded large t - antigen have also been found in some ependymomas. furthermore , ependymoma can be induced in rodents through intracerebral inoculation of the sv40 virus , . nevertheless , several studies have disqualified the sv40 tumor virus as a causative agent of ependymoma. the strongest opposing argument is based on epidemiologic studies that showed no increase in the incidence of ependymoma and other cancers in the years following the massive introduction of sv40-contaminated polio vaccines into the human population , . to date , knowledge of whether sv40 virus contributes to ependymoma tumorigenesis remains unknown ; and if it does , the oncogenic pathways on which it acts remain to be elucidated . over the years , cytogenetic studies using karyotyping and comparative genomic hybridization ( cgh ) have reported numerous broad chromosomal abnormalities in ependymoma . results varied considerably among early studies largely due to their small sample sizes and the variations among sample sets in terms of patient age and anatomical tumor location . in fact , increasing evidence supports that ependymomas are heterogeneous and can be classified as distinct disease subtypes based on patient age , anatomical tumor location , and genetic alterations , , . frequently observed genomic anomalies in pediatric ependymomas include loss of chromosomes 1p , 2 , 3 , 6/6q , 9p , 13q , 17 , and 22 as well as gains of 1q , 5 , 7 , 8 , 9 , 11 , 18 , and 20 , with the gain of 1q occurring in over 20% of cases being the most common . in adult ependymomas , chromosomes 6 , 10 , 13q , 14q , 16 , and 22/22q are frequently lost whereas chromosomes 2 , 5 , 7 , 9 , 12 , 18 , and x are gained , with gains of 7 and 9 and loss of 22q being the most frequently observed , though with each occurring in only 30% of cases or less . location - specific genomic anomalies observed in intracranial versus spinal ependymomas roughly correspond to those seen in children versus adults , as most pediatric ependymomas occur intracranially whereas adult cases predominantly occur within the spinal cord . aside from genomic gains and losses , cytogenetic studies have also identified translocations within the ependymoma genome , often involving chromosomes 1 , 11 , and 22. adult ependymomas have been observed to display more frequent and broader chromosomal aberrations than pediatric tumors . based on a meta - analysis of all cgh studies performed on more than 300 primary ependymomas , kilday et al . calculated that there are on average 7.5 and 3.8 genomic anomalies per adult and pediatric tumor , respectively . this finding is reinforced given that over 40% of pediatric ependymomas exhibit balanced genetic profiles , whereas a balanced genomic profile is observed in less than 10% of adult cases , , . interestingly , the large number of genomic aberrations often seen in adult spinal ependymomas is associated with tumors of lower histological grades and favorable patient outcome , , . furthermore , according to the cgh analysis done by dyer et al . on pediatric intracranial ependymomas , tumors can be subdivided into three distinct subgroups based on the number of chromosomal anomalies detected per tumor . tumors with a balanced genetic profile make up the balanced group , which is significantly associated with an infant age at diagnosis . the second structural group shows few and mainly partial genomic imbalances . lastly , the third numerical group exhibits 13 or more primarily whole chromosome imbalances similar to those often seen in adult ependymomas . these subdivisions are significantly associated with prognosis , with the numerical group demonstrating the best patient outcome and the structural group doing the worst . consistent with this observation , almost all recurrent ependymomas exhibit genetic profiles characteristic of the structural group , , . despite the identification of the aforementioned common genomic gains and losses in ependymomas and their cytogenetic profile - based stratification , few insights into the oncogenes , tumor suppressors , and molecular pathways responsible for the development of ependymoma could be obtained from these findings . furthermore , specific genetic events could not be identified as prognostic markers for this disease at only chromosome - level resolution . these chromosome - level aberrations are broad and typically span numerous genes , making it difficult to discriminate driver genetic events from passenger events . recently , array cgh ( acgh ) has been adopted by the research community to fine - map copy number variations in cancer at much higher resolutions . the list of genes within the common regions of amplification or deletion identified using acgh can be further narrowed through correlation with their expression levels . this permits the discovery of candidate driver genes for ependymoma development , with putative oncogenes and tumor suppressor genes exhibiting copy number - driven expression . indeed , the advances in microarray and next generation sequencing technologies have permitted the examination of ependymoma genetics in terms of copy number variations and gene expression levels in much greater detail . irrespective of anatomical tumor location or patient age , monosomy 22 and allelic losses on chromosome 22q have been found in numerous studies to be the most common genetic abnormalities in sporadic ependymoma , with frequencies ranging from 26 % to 71% , . initial quests for tumor suppressor genes present on 22q focused on nf2 located at 22q12 ; however , nf2 mutation is not associated with the majority of ependymoma cases.another potential tumor suppressor gene is hsnf5/ini1 at 22q11.23 . kraus et al . found no mutations or homozygous deletions of this gene in a series of 53 ependymomas , and this gene has not been shown to be silenced by dna promoter methylation . mapping of deletions and translocation breakpoints on 22q using high - resolution techniques revealed 22pter22q11.2 , 22q11 , 22q11.2112.2 , and 22q13.113.3 to be the hotspots where the elusive tumor suppressor gene is likely to be found,. within the frequently deleted region 22q12.3q13.33 , karakoula et al . found rac2 and c22orf2 to be deleted in 38% and 32% of the 47 pediatric intracranial ependymomas analyzed , respectively . in over 60% of these ependymomas , c22orf2 was found to be transcriptionally inactive , indicating its potential importance in the development of pediatric intracranial ependymomas . loss of rac2 , on the other hand , was shown to be a prognostic factor significantly associated with shorter overall survival in patients younger than 2 years . using gene expression microarray technology , suarez - merino et al . found the transcripts of four genes mapping to 22q12.322q13.33 , namely c22orf2 , as identified by karakoula et al . mentioned above , fbx7 , cbx7 , and sbf1 , to be under - expressed in pediatric ependymomas as compared to normal brain controls . allelic loss of one of these genes , cbx7 located at 22q13.1 , could be detected in 55% of ependymoma cases . interestingly , cbx7 controls cellular lifespan through regulating both the p16/rb and the arf / p53 pathways . the role of these pathways in ependymoma is unclear , though their deregulation is central to many types of cancer , including gliomas . furthermore , deletion and hypermethylation of cdkn2a / p16 at 9q21.3 and rb at 13q14.2 have been reported in ependymomas. in the same study by suarez - merino et al . schip-1 is known to interact with the nf2 gene product merlin , and their interaction is regulated by conformational changes in merlin induced by post - translational modifications , alternative splicing , or mutations . furthermore , by integrating the genomic and expression profiles of 24 primary intracranial ependymomas , modena et al . identified down - regulation of the sult4a1 gene located at 22q13.3 . in pediatric intracranial ependymomas , importantly , this genetic aberration is preferentially associated with tumors in the posterior fossa location in children and with anaplastic histological features . it is also a significant predictor of tumor aggressiveness and poor patient outcome,,,,,. interestingly , 1q gain is occasionally the only observable alteration , with few other chromosome imbalances detected in ependymomas , , , ; yet , in some cases , it marks tumor recurrence , . this suggests the presence of genes located on 1q that may be involved in the initiation , progression , and/or therapeutic resistance of ependymoma . thus , efforts have been made to determine the critical region on chromosome 1q for the identification of these crucial genes . reported a minimal overlapping region with high - copy amplification at 1q2431 in pediatric ependymomas . subsequently , an acgh study done on 49 sporadic intracranial ependymomas by mendrzyk et al . identified two commonly gained regions on 1q , one at 1q21.323.1 and another at 1q31.131.3 . they also found that gains of 1q21.132.1 were correlated with tumor recurrence and identified the gain of 1q25 as an independent prognostic marker for significantly lower recurrence - free or overall survival rate . additionally , they identified dusp12 , found to be over - expressed in all their tested samples , as a candidate gene located at 1q23.3 . the mrna level of dusp12 correlates with that of cyclin d1 throughout the cell cycle , suggesting its role in regulating cell division and potentially in neoplastic transformation , . dusp12 was also found to be important for cell survival in response to heat - shock - induced cell death , which further supports its proposed oncogenic function . gene expression analyses correlated with copy number variations have since uncovered additional candidate oncogenes located within 1q2132 , including laminin , prelp , hspa6 , gac1 , chi3l1 , tpr , jtb , shc1 , and s100a10 and other s100 family members , , , , . among these , gac1 amplification - driven over - expression has also been implicated in the pathogenesis of other malignant gliomas , suggesting its likely importance in ependymoma development . in addition to chromosome 22q loss and 1q gain , other commonly identified chromosomal aberrations include deletion of chromosomes 6q and 9 and gain of chromosome 7 , notably the region from 7q11.2322.1 , which is associated almost exclusively with spinal ependymomas , , . candidate oncogenes proposed by analyzing recurrent gains on chromosome 7 include egfr ( epidermal growth factor receptor ) at 7p11.2 , twist1 and hdac9 at 7p21.1 , and arhgef5 at 7q34 , egfr in particular exhibits frequent gains and high - level amplifications in intracranial ependymomas , and its over - expression predicts poor patient outcome . loss of chromosome 6q is found mostly in infratentorial tumors , whereas deletions on chromosome 9 occur more frequently in supratentorial tumors , , , . with microsatellite analysis , loh hotspots on chromosome 6 were determined to be 6q1516 , 6q2122.1 , and 6q24.325.3 , which were further limited to 6q24.3 and 6q25.225.3 , . locus 6q25.3 , containing the snx9 and synj2 genes , was found to be the most frequently deleted . however , loss of 6q25.3 was a favorable prognostic marker for overall survival of patients with anaplastic intracranial ependymomas , as the deletion of the snx9 and synj2 genes , which are known to regulate cell migration and invasion , could inhibit tumor progression . furthermore , the polyamine biosynthesis gene amd1 and the cyclin - dependent kinase cdk11 , both located at 6q21 , as well as the tumor suppressor gene sash1 at 6q24.3 were found to be under - expressed by suarez - merino et al . which is also frequently deleted in patients with ependymomas , homozygous deletion spanning the cdkn2a locus at 9q21.3 has been detected and is a characteristic of anaplastic supratentorial tumors , the molecular staging system developed by korshunov et al . highlighted that cdkn2a deletion together with young age at diagnosis and gain of 1q comprise the most reliable independent indicators of unfavorable patient outcome . in contrast , gains of chromosomes 9 , 15q , and 18 and loss of chromosome 6 are features indicating excellent chance of survival . furthermore , detection of the expression of p14 protein by immunohistochemistry in 103 intracranial ependymomas revealed that decreased p14 expression is associated with tumor aggressiveness in terms of higher tumor grade , elevated growth fraction , and p53 protein accumulation . closely examined the aberrations on chromosome 9 in both adult and pediatric ependymomas and identified 9p21.122.3 and 9q31.333.2 to be the potential tumor suppressor genes located within 9q31.333.2 include dbc1 , which is frequently deleted in bladder cancer and also exhibits markedly reduced mrna expression in gliomas , ; dec1 , whose down - regulation driven by copy number loss is frequently seen in esophageal cancer and contributes to tumor cell motility , ; lpar1 , which is known to mediate cell proliferation , differentiation , and migration among other functions ; and txn , which inhibits apoptosis and enhances drug resistance in cancer cells , . other frequently occurring regions of genomic imbalances have been revealed by profiling the ependymoma genome at high resolution,,,,, and are summarized in table 1 . among these imbalances , the combined presence of 6p22-pter and 13q14.3-qter losses predicted significantly reduced survival in intracranial pediatric ependymomas . puget et al . found that gains of 1q and 9qter and loss of 6q occurred more often in recurrent tumors . interestingly , the specific 9qter region linked to tumor recurrence is associated with posterior fossa ependymomas , whereas chromosome 9 deletion is usually associated with supratentorial ependymomas . candidate oncogenes and tumor suppressor genes proposed based on these copy number variation hotspots,,, are listed in table 2 . among the putative oncogenes in ependymoma are notch1 , notch4 , and jag1 , which are two of the membrane receptors and one of the ligands , respectively , of the notch signaling pathway , suggesting the involvement of notch signaling in ependymoma tumorigenesis . furthermore , recurrent gains at 5p15.33 , which includes the human telomerase reverse transcriptase ( htert ) gene , were validated by immunohistochemistry . elevated htert expression has been shown to be associated with ependymoma progression and recurrence and is currently the most important predictor of survival for pediatric intracranial ependymomas independent of other clinicopathologic prognostic features,. furthermore , htert expression relates with telomerase activity . recently , wong et al . proposed telomerase inhibition as a novel therapy for ependymoma after demonstrating its effects on reducing ependymoma cell viability by increasing dna damage , decreasing proliferation , and increasing apoptosis . in addition to fine - mapping genomic aberrations to identify candidate genes involved in ependymoma development , profiling studies have also been used to divide ependymomas into distinct subgroups that correlate with tumor location . categorized these tumors into three molecularly distinct subgroups that correlate with the anatomical location of the tumor , namely the supratentorial region , the posterior fossa , or the spine . although ependymomas from these different anatomical regions are histologically indistinguishable , they are in fact molecularly distinct diseases that should be separately examined to determine the genetic events involved in tumorigenesis and progression , as well as prognostic factors and patient outcome . according to the results of their acgh experiment , taylor et al . found that cdkn2a deletion occurred in > 90% of supratentorial ependymomas but was rare in tumors from other regions of the central nervous system ( cns ) . furthermore , posterior fossa ependymomas could be further classified into three subgroups : tumors harboring multiple concurrent dna amplifications , tumors with chromosome 1q gain , and tumors exhibiting a balanced genomic profile . although acgh analyses have considerably advanced our understanding of the genetic events in ependymoma tumorigenesis , almost half of ependymomas present a balanced acgh profile , making it imperative to interrogate alternative mechanisms of gene regulation . epigenetics in the form of promoter dna ( cpg ) hypermethylation is an important route by which transcriptional inactivation can be achieved and , as in other cancers , likely plays a significant role in silencing tumor suppressor genes involved in ependymoma development . unfortunately , epigenetic studies on ependymoma have been limited to candidate gene approaches , with the genes in question selected based on their roles as tumor suppressor genes and methylation status in other malignancies . therefore investigated the methylation status of the hypermethylated in cancer 1 ( hic-1 ) putative tumor suppressor gene , which exhibits hypermethylation and loss of expression in various tumors such as medulloblastoma and gliomas . they detected hic-1 hypermethylation and down - regulation in 83% and 81% of ependymomas , respectively , and found that hic-1 hypermethylation was significantly correlated with nonspinal localization and pediatric age . the ras association domain family 1 isoform a ( rassf1a ) gene has also been found , independent of clinical and histological subtype , to be frequently silenced by methylation in ependymoma , with an incidence of 86% . rassf1a is a recently well - recognized tumor suppressor gene whose inactivation through promoter methylation is implicated in the development of many human cancers . rna interference experiments have shown that down - regulation of rassf1a , an effector of ras , results in loss of cell cycle control , enhanced genetic instability and cell motility , and resistance to k - ras and tumor necrosis factor ( tnf)-induced apoptosis . identified the trail apoptosis pathway - related genes casp8 , tfrsf10c , tfrsf10d , and tnfrsf10c to be methylated in ependymoma , with incidences of 30% , 9.5% , 36.4% , and 9.5% , respectively . other commonly methylated genes in ependymoma include dapk , thbs1 , timp3 , tp73 , mgmt , gstp1 , cdkn2a , fhit , rarb , blu , and mcj , with incidence ranging from 10% to 57% , , , , . gene expression profiling employs microarray technology to capture gene expression levels of thousands of genes simultaneously . integration of gene expression with copy number data allows one to determine the genes demonstrating copy number - driven expression as putative oncogenes and tumor suppressor genes . moreover , by applying ontological analysis on the gene expression profiles , it is possible to uncover those aberrant cellular processes and pathways that contribute to ependymoma . using microarray - based gene expression profiling to compare ependymoma with normal brain controls , suarez - merino et al . identified 112 abnormally expressed genes in ependymoma . genes with increased expression included the oncogene wnt5a , tp53 homologue tp63 , and several cell cycle , proliferation , adhesion , and extracellular matrix genes such as the transcription factor zic1 , the angiogenesis factor vegf , and fibronectin 1 ( fn1 ) . other putative oncogenes identified in this study that have been implicated in other cancers are col4a1 , ibp2 , hox7 , wee1 , and gac1 . genes that were found to be down - regulated included the a / f2-interacting gene schip-1 , the apc - associated gene eb1 , and genes that are involved in vesicle trafficking and recycling such as npc1 , rab40b , tj2 , and sh3gl3 . consistent with ependymoma subgroups based on acgh profiles , ependymoma gene expression profiles are significantly associated with tumor location , patient age at disease onset , grade , and retrospective risk for relapse , , , . found that supratentorial ependymomas expressed markedly elevated levels of members of the ephb - ephrin ( ephb2/3/4 and ephrin a3/4 ) and notch ( jagged 1/2 ) signaling pathways , as well as genes involved in cell cycle regulation ( cyclin b2/d1/g2 , cdk2/4 , and cdkn1c/2c ) . on the other hand , the highly expressed genes that distinguished posterior fossa ependymomas were inhibitors of differentiation ( id1/2/4 ) and members of the aquaporin family ( aqp1/3/4 ) . spinal ependymomas are characterized by the up - regulation of multiple homeobox ( hox ) family members ( hoxa7/9 , hoxb6/7 , and hoxc6/10 ) and insulin - like growth factor 1 ( igf1 ) . subsequently , gene expression profiling studies performed by modena et al . and palm et al . confirmed that intracranial ependymomas are indeed characterized by high expression levels of genes involved in notch signaling and that spinal ependymomas are defined by over - expression of numerous hox genes . additionally , up - regulation of the sonic hedgehog ( shh ) and bone morphogenetic protein ( bmp ) pathway members were also evident in intracranial ependymomas , . deregulated notch signaling , which is crucial for neural development , is believed to play a significant role in ependymoma tumorigenesis , especially at the supratentorial location , since oncogenesis is thought to mirror normal development gone awry . in addition to over - expression of the notch ligands jagged 1/2 shown by taylor et al . , there is consistent up - regulation of the notch receptors ( notch1/2 ) , ligands ( jagged 1/2 and dll1/3 ) , and target genes ( hes1/5 , hey2 , c - myc , and erbb2 ) , whereas fbxw7 , the major repressor of the notch pathway , is consistently down - regulated , , , . in an early study , missense mutations of notch1 , either in the heterodimerization domain c or the transactivation domain , were detected in 8.3% of pediatric intracranial ependymomas from the posterior fossa , thus making notch1 the first oncogene found to be mutated in ependymomas . these mutations cause the notch1 receptor to be constitutively active in a ligand - independent manner . moreover , inhibition of notch signaling with -secretase inhibitor gsi - ix impaired ependymoma primary cell culture growth . gilbertson et al . further demonstrated that high - level expression of erbb receptors ( erbb2/4 ) , which are direct targets of notch signaling , could be found in over 75% of pediatric ependymomas and were significantly correlated to tumor proliferative activity as measured by the ki-67 labeling index . functional studies proved that activating erbb receptor signaling in short - term ependymoma cell cultures resulted in akt phosphorylation and cell proliferation , which could be effectively blocked in a dose - dependent manner with an inhibitor of erbb2 tyrosine kinase activity . coincidentally , we have recently learned that the sv40 virus , which was thought to be a causative agent for ependymoma , can in fact induce oncogenic transformation of human mesothelial cells through direct induction of notch1 over - expression . currently , neither cell lines nor animal models are available to elucidate the sequential events in ependymoma development . thus , researchers have compared the gene expression profiles of low grade versus high grade ependymomas and primary tumors versus recurrent tumors to better understand the molecular genetics of ependymoma progression . palm et al . revealed that who grade 3 anaplastic ependymomas differed from grade 2 tumors by the over - expression of genes implicated in wnt/-catenin signaling activation , cell cycle regulation / cell proliferation ( cyclin - dependent kinases cdk2/4 , cell division cycle proteins cdc25a/25b/25c/2 , and minimal chromosome maintenance proteins mcm2/3/5/6/7 ) , apoptosis ( tumor necrosis factor super family members tnfrsf11a/21 and caspases casp1/4 ) , angiogenesis ( vegf , vegfr2 , vegfb , tnip2 , and doc2 ) , and remodeling of adherens junctions through e - cadherin destruction ( met , mn23h1 , caveolin , rab5/7 gtpases ) , as well as up - regulation of the transcription factors e2f1 and dp1 ( tfdp1 ) . wnt signaling activation in grade 3 ependymomas is indicated by the over - expression of wnt ligand ( wnt11 ) , frizzled receptors ( fzd2/5/8 ) , and dishevelled genes ( dvl2/3 ) . furthermore , increased expression was detected for -catenin ( ctnnb1 ) and its associated transcription factor tcf3 and the wnt target genes birc5 , ccnd1 , fosl1 , c - myc , and tp53 . similarly , to comprehend the molecular mechanisms underlying ependymoma recurrence , peyre et al . performed a dual - color gene expression microarray analysis on 17 tumors at diagnosis co - hybridized with 27 corresponding tumors at first or subsequent relapses they identified 87 genes collectively as the expression signature of ependymoma recurrence . like the gene expression characteristics of high grade ependymomas noted by palm et al . , the signature of ependymoma recurrence was also marked by wnt pathway activation with over - expression of sfrp1 , sfrp2 , fzd2 , fzd8 , and wnt10b . other frequently over - expressed genes in recurrent ependymomas included cd133 , members of the notch signaling pathway , and genes involved in the kinetochore ( kif14 , kif11 , kif1c , kif2c , prc1 , bub1b , zwint , aspm , kntc2 , and cenpf ) . the genes that were significantly down - regulated were metallothionein genes ( mt1l , mt1 g , mt1e , mt1x , mt1b , mt2a , mts ) , with reduced expression in up to 80% of recurrences , and genes involved in the immune system ( cxcl5 , cx3cl1 , traf3ip2 , itgbl1 , serping1 , ift20 , entpd3 , hp , and hpr ) . the importance of immune function in hindering ependymoma progression and recurrence was also recognized through the study by donson et al .. their ontological analysis on gene expression profiles from pediatric ependymomas correlated with clinical outcome revealed that the up - regulation of immune function - related genes was associated with non - recurrent ependymomas and a longer time to progression in recurrent ependymomas . in addition , increased infiltration of cd4 t cells were observed by immunohistochemistry in non - recurrent ependymoma samples . furthermore , like the primary ependymomas which can be subgrouped based on location , peyre et al . found that supratentorial versus infratentorial ependymomas showed distinct changes in expression profile at recurrence . recurrent supratentorial ependymomas were characterized by the up - regulation of genes related to cytoskeleton organization ( gelsolin , sema5a , contactin-1 , sarcoglycan , villin - like , scinderin ) and extracellular matrix - cell interactions ( gliomedin , extl1 , galectin-9 , desmuslin , tetranectin , versican , col21a1 , col16a1 , cxcl12 ) , which are functionally involved in the mesenchymal transition . infratentorial ependymoma recurrences , on the other hand , were associated with over - expression of ribosomal protein genes , which are markers of oncogenic transformation in many human tumor types , . one of the key questions to answer in the field of cancer research is to determine the normal cell type that gives rise to a particular malignancy . this is a crucial step towards functionally identifying the successive oncogenic events leading to tumor onset and progression , which would be indispensable for developing targeted therapies and finding keys to prevention . growing evidence suggests that tumor subgroups may arise due to deregulation of cell signaling pathways involved in normal development of different precursor cell populations . thus , the unique gene expression signatures of ependymoma subgroups might provide insight into their cells of origin . indeed , taylor et al . showed that the signature genes which characterize supratentorial , posterior fossa , and spinal ependymomas are expressed in the matching regions in the developing cns of embryonic mice . moreover , many of these signature genes are members of signaling pathways that modulate neural precursor cell proliferation and differentiation in the corresponding regions of the cns , . this confirmed the hypothesis that subgroups of ependymoma either maintain or recapitulate the developmental expression profiles of anatomically restricted progenitor cells , which were then identified to be radial glial cells ( rgcs).taylor et al . further demonstrated that rgcs are likely the cells of origin for ependymoma by isolating a rare population of self - renewing and multipotent cancer stem cells from fresh samples of ependymoma . these cancer stem cells exhibited bipolar morphology resembling rgcs , expressed the rgc immunophenotype cd133/ nestin/ rc2/brain lipid - binding protein ( blbp ) , and were both required and sufficient to recapitulate the original tumor when transplanted into immunocompromised mice . rgcs are a pivotal cell type in the developing cns of all vertebrates and are a specific group of neural stem cells . they serve as ubiquitous precursors that generate neurons and glia , as guide cells for subsequent neuronal migration , and as key elements in patterning and region - specific differentiation of the cns . studies have also shown that ependymal cells , from which ependymoma arises , are derived from rgcs during embryogenesis . genetic mutations in rgcs may therefore lead to their transformation into cancer stem cells of pediatric ependymomas , . since supratentorial ependymomas are characterized by elevated expression of members of the notch and ephb - ephrin signaling pathways , it is likely that over - activation of these pathways may induce neoplastic transformation of rgcs in the cerebral subventricular zone . likewise , up - regulation of the hox family of transcription factors may be involved in spinal ependymoma development by transforming rgcs in the spinal region . furthermore , there is evidence that rg - like cells are present not only during development but also persist in the adult cns , specifically in the subventricular zone and the spinal cord . thus , these rg - like cells may serve as the cells of origin for adult ependymomas. recently , johnson et al . catalogued dna copy number alterations among 204 tumor samples , which is the largest cohort of ependymomas ever examined at the highest resolution . they further generated mrna and microrna expression profiles for 83 and 64 of these tumors , respectively . these profiles segregated ependymomas by cns location and unmasked additional subgroups among supratentorial , posterior fossa , and spinal ependymomas . to test that distinct subgroups of ependymoma might arise due to oncogenic transformation of regionally and developmentally restricted populations of rgcs by characteristic genetic mutations , the gene expression profile of a subset of human supratentorial ependymomas was matched with that of embryonic cerebral rgcs taken from ink4a / arf ( cdkn2a)-null mice , as the cdkn2a locus is frequently deleted from human supratentorial ependymomas . these embryonic cerebral ink4a / arf ( cdkn2a)-null rgcs were first transduced with ephb2 , which has been shown to be focally amplified in a subgroup - specific manner and to exhibit copy number - driven over - expression in supratentorial ependymomas , and were subsequently implanted into the cerebrum of immunocompromised mice . this established the first highly penetrant ( over 70% incidence ) murine allograft model of supratentorial ependymoma that accurately recapitulates the histological features and gene expression profile of the human tumor . comparative gene expression analysis of matched mouse and human tumors revealed deregulation of genes in neural differentiation and maintenance , particularly ion transport and synaptogenesis , thus highlighting the importance of these events in the formation of this ependymoma subgroup . thus , this study provided functional confirmation that ependymoma variants indeed arise from their matched populations of rgcs transformed with the subgroup - specific mutations . over the past decade , research has significantly advanced our knowledge on the molecular genetics of ependymoma . early cytogenetic studies identified broad chromosomal gains and losses , with loss of 22q being the most common . nf2 is recognized as a putative tumor suppressor gene in spinal ependymomas based on mutational analysis and increased incidence of ependymoma in patients with nf2 familial syndrome . it is , however , rarely mutated in pediatric intracranial ependymomas , for which much effort is still being made in identifying the elusive tumor suppressor gene(s ) on chromosome 22q . other common genomic imbalances include gain of 1q and losses of 6q and 9 in intracranial ependymomas , and gain of 7 in spinal ependymomas , among many others . with the advent of acgh technology permitting the identification of genomic imbalances at much greater resolution , it became possible to uncover putative oncogenes and tumor suppressor genes , such as htert and cdkn2a , respectively , by testing candidates found in focal regions of amplifications and deletions for copy number - driven expression . importantly , over the years , ependymoma tumor heterogeneity has become progressively more appreciated at the genetic level and can be subgrouped based on chromosomal abnormalities , acgh , and , recently , gene expression profiles . it is now generally recognized that ependymomas from different regions of the cns , i.e. the supratentorium , posterior fossa , and the spinal cord , are genetically distinct diseases marked by unique gene expression signatures , indicating the deregulation of different developmental pathways involved in tumorigenesis . supratentorial ependymomas are characterized by notch and ephb - ephrin signaling , whereas spinal ependymomas show specific over - expression of hox family transcription factors . furthermore , comparing the expression profiles of ependymomas at first diagnosis versus at relapse and at low grade versus high grade revealed that ependymoma recurrence and progression likely result from the up - regulation of wnt signaling and down - regulation of immune function - related genes . recently , rcgs at various locations throughout the cns have been identified to be the cells of origin for the corresponding ependymoma subgroups , as illustrated in figure 1 . the notion that subgroups of ependymoma arise from regionally and developmentally distinct rgcs that have undergone transformation by subgroup - specific genetic mutations was further confirmed functionally in the case of supratentorial ependymomas . despite these achievements in ependymoma research , greater progress is still urgently needed if we are to realize the ultimate goal of improving clinical outcome for patients . with newly developed microarray platforms able to detect copy number changes and gene expressions at even higher resolution , next - generation sequencing technologies and high - throughput techniques for unbiased epigenetic profiling posterior fossa ependymomas in particular deserve our attention , as they frequently occur in children of very young age , and complete surgical resection is often difficult to achieve owing to the involvement of multiple cranial nerves and branches of the vertebrobasilar arterial system at this location . in addition , up to half of posterior fossa ependymomas present a balanced genomic profile , making the identification of genetic events contributing to their tumorigenesis especially challenging . consequently , it is important to examine the genetics of posterior fossa ependymomas at a greater resolution to identify very focal amplifications and deletions , as well as to concentrate on decoding its epigenome . candidate oncogenes and tumor suppressor genes discovered to date should be promptly assessed for their diagnostic and therapeutic potential , with the aim to effectively translate our knowledge from laboratory to clinic . at present , however , ependymoma research is severely hampered by the lack of in vitro and in vivo systems to functionally examine the genetic events identified through acgh and gene expression studies that potentially contribute to ependymoma development . indeed , the identification of rgcs as the cells of origin for ependymoma was a significant breakthrough towards mapping out the pathogenic mechanisms of ependymoma . similar to what has been done for one subset of supratentorial ependymomas , the next step will be to identify the distinct populations of rgcs for all ependymoma variants and functionally determine the subgroup - specific driver mutations necessary for transforming corresponding rgcs to ependymoma . this approach will allow us to functionally identify the key genetic events involved in the initiation and progression of all ependymoma subgroups , as well as to model these subgroups in vitro and in vivo . unlike end - stage tumor samples which provide little information on the chronology and relative importance of the uncovered genetic events in the process of ependymoma pathogenesis , these functional models will be instrumental in deciphering the pathogenic mechanisms of the ependymoma subgroups , as well as in uncovering and verifying potential targets for therapy .

brain tumors are the leading cause of cancer death in children , with ependymoma being the third most common and posing a significant clinical burden . its mechanism of pathogenesis , reliable prognostic indicators , and effective treatments other than surgical resection have all remained elusive . until recently , ependymoma research was hindered by the small number of tumors available for study , low resolution of cytogenetic techniques , and lack of cell lines and animal models . ependymoma heterogeneity , which manifests as variations in tumor location , patient age , histological grade , and clinical behavior , together with the observation of a balanced genomic profile in up to 50% of cases , presents additional challenges in understanding the development and progression of this disease . despite these difficulties , we have made significant headway in the past decade in identifying the genetic alterations and pathways involved in ependymoma tumorigenesis through collaborative efforts and the application of microarray - based genetic ( copy number ) and transcriptome profiling platforms . genetic characterization of ependymoma unraveled distinct mrna - defined subclasses and led to the identification of radial glial cells as its cell type of origin . this review summarizes our current knowledge in the molecular genetics of ependymoma and proposes future research directions necessary to further advance this field .

Familial Syndromes and Risk Factors Cytogenetic Abnormalities Molecular Genetic Aberrations Epigenetics Gene Expression Profiles Cells of Origin of Ependymoma Conclusion and Future Directions

circadian rhythms are the regular ; near 24 hs rest / wake patterns seen in normal individuals . there is a progressive deterioration of circadian rhythms with aging . these include changes in the sleep wake cycle manifested by reductions in sleep quality and impairment in cognitive performance [ 1 , 2 ] . furthermore , exaggeration of age - related changes is seen in alzheimer 's disease ( ad ) affecting as many as a quarter of patients during some stage of their illness . few sleep questionnaires - based comparison studies have shown increased sleep disturbances in vascular cognitive disturbances compared to ad patients . day - time agitation , night - time insomnia , and restlessness are among the common behavioral changes observed in ad . nocturnal sleep disturbance is often accompanied by day - time napping , frequently in direct association with the extent of dementia . circadian rhythm disturbances ( crds ) in ad often present dramatically and are among the major triggers for institutionalization [ 5 , 6 ] . they are also associated with shorter survival in long - term care residents [ 7 , 8 ] and are a cause of physical and psychological burden for caregivers . the main reason for the alteration in sleep - wake cycle is related to alterations in the suprachiasmatic nucleus ( scn ) and melatonin secretion . though not clear , genetic risk factors such as in ad patients who are negative for the apoe-4 allele have also been implicated in the development of sleep problems the scn of the anterior hypothalamus is the master clock controlling circadian rhythms in mammals . the circadian influences of scn neurons are distributed through different target organs by efferent neural and humoral signals particularly circulating melatonin . the nucleus controls melatonin secretion via a multisynaptic pathway producing changes in the biological clock , core body temperature , and sleep . the activity of the scn is regulated by environmental signals in a 24-hour cycle and runs autonomously . it resets on a daily basis via light inputs from the retina during the day and melatonin secretion during the night . these inputs are conveyed through the retinohypothalamic tract ( rht ) resulting in oscillation of scn and melatonin secretion [ 1315 ] . these cells express pituitary adenylate cyclase - activating peptide ( pacap ) which elicits phase shift signals in the scn . there is also a serotonergic input from the midbrain raphe nuclei providing an arousal - dependent influence that can reset the circadian clock and modulate the photic synchronization of the scn [ 16 , 17 ] . the scn contains gamma - aminobutyric acid ( gaba ) and arginine vasopressin ( avp ) neurons that send a direct inhibitory projection to the nucleus of the paraventicular nucleus of the hypothalamus [ 1820 ] . circulating melatonin acts via mt1 and mt2 receptors and inhibits the firing of the scn . as mentioned above , alterations in the scn and melatonin secretion are assumed to be the main reason for crd in ad . further , there is loss of expression of avp and mt1 receptors in the scn . these in turn result in a reduced production of melatonin and disappearance of normal melatonin rhythm [ 21 , 22 ] . the functional disruption of the scn is even observed in the early stages of ad . the changes in the mt1 receptor explain why large studies have failed to show improvement in sleep - wake cycle after melatonin administration in ad patients . certain sleep changes in ad seem to represent an exaggeration of changes that appear with normal aging . ad patients spend an increased amount of time in stage 1 sleep with increased number and duration of awakenings , compared to age - matched non - ad controls [ 24 , 25 ] . with disease progression , it is also very difficult to separate eeg features of stage two sleep from stage 1 sleep . they are also of lower amplitude , shorter duration , and much less numerous [ 26 , 27 ] . the proportion of nonrapid eye movement ( nrem ) sleep increases with further disappearance of the true delta wave of slow wave sleep ( sws ) [ 2428 ] . the percentage of time spent in rem sleep , which remains stable with normal aging , is reduced in patients with ad . a decrease in the mean rem sleep episode duration and rem sleep percentage can be due to degeneration of the nucleus basalis of meynert . the nucleus normally exerts an inhibitory influence on the nucleus reticularis of the thalamus , the rhythm generator responsible for nrem sleep . in addition , many subcortical structures such as the basal forebrain , distal and superior raphe nucleus , and the reticular formation of the pons and medulla seem to be involved in the initiation of sleep and oscillation between rem and non - rem states . all of these structures may potentially be damaged by the degenerative changes which are part of ad . their deterioration may explain many of the sleep architecture and rhythm changes in ad ( see table 1 ) . in accordance with the endogenous circadian rhythm , melatonin also promotes sleep by lowering core body temperature via peripheral vascular dilatation . lowest body temperature occurs 1 - 2 hours before awakening , generally between 4 and 5 am . core body temperature and melatonin are the two primary and measurable markers of the circadian cycle . the core body temperature rhythm is generally accepted as yielding the best estimate of endogenous circadian amplitude and endogenous circadian phase in human subjects . however , studies of circadian core body temperature rhythms in human subjects are complicated by the influence of many confounding variables ( e.g. , light , posture , meals , and social interaction ) [ 31 , 32 ] . keeping these different variables constant while measuring core body temperature , studies examining differences and similarities in circadian disturbances in groups of normal elderly and patients with probable ad , as compared with a comparison group of young , normal volunteers have shown a reduction in endogenous circadian amplitude ( eca ) and a delay in endogenous circadian phase ( ecp ) of core body temperature in patients with probable ad [ 33 , 34 ] . studies have demonstrated that normal aging is accompanied by a 40% decrease in endogenous circadian amplitude . related studies showing a 50% reduction in amplitude in the ad group , as compared with normal young subjects could be interpreted as exaggerated or abnormal aging , rather than any discrete effect attributable to ad . insomnia , nocturnal behavioral changes , and excessive daytime sleepiness , probably as a consequence of disrupted nocturnal sleep , are the common ad symptoms related to crd . the actingout of rem sleep , often described as rem behavior disorder , is also briefly described here . though it may be primarily related to the underlying neurodegenerative changes producing crd described above , other differential diagnostic issues need to be ruled out . for example , breathing problems during sleep , such as sleep apnea , with subsequent confusional arousal may be a possibility . a treatment trial with a continuous positive airway pressure ( cpap ) breathing device has been reported to improve insomnia and even cognitive function in some patients . insomnia may also be secondary to restless legs syndrome ( rls ) , where patients feel a need to pace in the evening or night - time hours . several studies have shown that the severity of leg discomfort follows a circadian rhythm , reaching a peak after midnight . most patients report difficulty falling asleep or waking up shortly after sleep onset with unpleasant leg sensations . rls , unlike the well - known mechanism of crd , is mainly due to striatal dopamine depletion . another common cause of insomnia especially in institutionalized or hospitalized ad patients may be secondary to noise in the environment and frequent room checks by staff . simple measures such as treating urinary tract infections and pain , relieving fecal impaction , and reviewing the patient 's current medications to determine if they may be promoting wakefulness or frequent awakenings can result in drastic improvements of sleep and nocturnal agitation . macular degeneration and optic nerve degeneration are common conditions seen in ad which limit light input , further complicating circadian rhythms . in patients with ad , other psychiatric comorbidities , and adding to sleep difficulties can be the excessive intake of stimulants such as coffee , tea , or caffeine containing soft drinks . theophylline , commonly used for broncho - pulmonary disease , is also a frequent cause of insomnia . the cholinesterase inhibitors which are commonly prescribed in ad , and in particular donepezil , have been shown to occasionally cause insomnia [ 41 , 42 ] . excessive daytime sleepiness ( hypersomnia ) is related to changes either in nrem ( idiopathic hypersomnia ) , rem sleep ( narcolepsy without cataplexy ) or could be central in origin ( narcolepsy with cataplexy ) . narcolepsy is seldom described as a degenerative disease of the hypocreatin - containing neurons in the lateral hypothalamus . it may be accompanied by cataplexy or sudden loss of muscle tone , vivid hallucinations , and brief periods of total paralysis and rem sleep at inappropriate times . patients experience sudden urge to sleep which usually lasts for few seconds or minutes . a mutation in chromosome 6 ( controls the human leukocyte antigen ) narcolepsy is usually seen in early adulthood and it is very uncommon in elderly patients with ad . in elderly , hypersomnia is more frequently seen in patients with parkinson disease ( pd ) , lewy body dementia ( dlb ) , or parkinson disease dementia ( pdd ) than in ad . a recently discovered hypothalamic wake - stimulating neuropeptide , hypocretin-1 , has been hypothesized to be involved in consolidation of wakefulness . lower hypocretin-1 levels may be permissive for increased wake fragmentation in ad resulting in daytime sleepiness . stimulation of the hypocretin system may be a therapeutic means for improving daytime wakefulness in ad and in turn it helps sleep consolidation . in ad patients , treatments of insomnia or medications used to control agitation at night are usually the most important causes of excessive daytime sleepiness ( hypersomnia ) . in addition , the use of anticonvulsants and strongly anti cholinergic drugs such as antihistamines , antidiarrheals , and drugs used to reduce bladder irritability can cause daytime sedation and sleepiness . lack of engaging activities can also cause boredom and daytime sleepiness . at times , caregivers may promote daytime sleeping to ease their caregiving burden . these in turn may result in night - time awakenings , further worsening the crd [ 4749 ] . sundowning refers to agitation or a delirium - like state usually occurring late - afternoon / early evening or at night . individual behavioral components of the condition which is frequently termed sundowning include loud vocalization , wandering , physical aggression , combativeness , maladaptive physical behaviors , and overall agitation [ 5055 ] . these behaviors usually start at around 4 pm and last as late as 11 pm . however , some studies have reported the peak time of agitation as being in the afternoon , at approximately 2:30 pm , rather than in the early evening . the tendency for agitation in ad to occur at night suggests that some changes in sleep and wakefulness in the ad patient ( chronobiologic changes ) may reflect alterations in the body 's ability to regulate the timing of certain physiological events . common factors which cause late day confusion or sundowning include exhaustion , reduced lighting , and increased shadows with inability to separate dream from reality when sleeping . agitation usually worsens when patients are transferred to a new environment or a new nursing home or when there is a change or shifting of caregivers . sundowning typically peaks in the middle stage of the disease and then diminishes as ad progresses . rbd is classified as either idiopathic or secondary to some identifiable causes . in most cases , it is due to neurodegenerative diseases . narcoleptic patients may sometimes experience rbd due to degeneration of hypocreatin - containing hypothalamic neurons . it may also be induced by some medications ( e.g. , sedative hypnotics , tricyclic antidepressants , anticholinergics , selective serotonin reuptake inhibitors ) . two case reports of rivastagmine-(cholinesterase inhibitor- ) induced rbd have been shown in ad patients . there is a high level of tonic and phasic muscle activity seen most typically in rem sleep . this is suggested by electromyographic ( emg ) activity recorded from facial and limb muscles during sleep . this condition can be seen in ad patients ' comorbid with synucleiopathies such as parkinsons disease and/or diffuse lewy body dementia . patients with rem sleep behavior disorder may sustain injuries to themselves or their bed partner . rem sleep behavior disorder should be distinguished from nocturnal paroxysmal dystonia ( npd ) which is characterized by abnormal night - time movement of the trunk and head with alternating flexing and extension of the limbs . npd is often considered to represent nocturnal frontal lobe epilepsy typically accompanied by abnormal slow wave and spike activity with paroxysmal intrusions in nrem , rather than rem sleep . it is usually treated with anticonvulsants such as carbamazepine [ 6264 ] , unlike rem behavior disorder which is usually treated with clonazepam . there is empirical evidence to support the use of behavioral and environmental strategies in alleviating sleep problems and night - time behavioral disturbances in patients with ad . however , one needs to keep in mind that certain medications used for this purpose ( e.g. , benzodiazepines ) can worsen cognitive deficits and obstructive sleep apnea syndrome ( a condition commonly associated with ad ) . in addition , some medications may also induce daytime sleepiness , resulting in night - time wakefulness . there are simple and effective interventions which should initially be tried to treat crds in ad patients . these include a regular activity schedule ( e.g. , serving dinner early ) , bed time routines , decreasing noise and night - time exposure to room level light , and limiting caffeine and alcohol intake . outpatients and long - term care residents with ad may spend too much time in bed and not enough time engaging in physical activities . mental , physical , and social activities to avoid boredom and daytime napping may be useful . carvalho - bos et al . used two - week actigraphic estimates of sleep - wake rhythm and investigated its association with a range of functional domains in 787 demented elderly women . they found that nocturnal restlessness is related to impairment in daily function and social interaction . typically , long - term care residents with ad are exposed to only a few minutes of bright light each day , even less than patients living in the community . because light exposure is the strongest known zeitgeber ( time cue ) in humans , lack of daytime light exposure may contribute to circadian dysregulation [ 67 , 68 ] . in one randomized controlled trial , 36 community - dwelling patients with ad were studied using a sleep survey and wrist actigraphy . cases were randomized to daily exercise plus 60 minutes daily of bright light therapy ( blt ) versus controls for 2 months . there was a significant reduction in the number of night - time awakenings , total time awake at night , and depression ( p patients were also followed for the next 6 months and benefits were maintained [ 69 , 70 ] . in institutionalized patients , blt has been shown to reduce daytime nap duration by 30 minutes during the treatment period ( p < .004 ) [ 7072 ] . however , the effect in another study was not sustained after the cessation of light therapy [ 69 , 73 ] . another study has demonstrated modest benefits of bright light for two weeks on actigraphic measurements of activity [ 7476 ] . a long - term ( 5 years ) double blind , placebo - controlled randomized trial performed on 189 residents of 12 group care facilities in netherlands has shown another modest benefit of bright light therapy in improving some of the cognitive and noncognitive symptoms of dementia . this effect is more attenuated when combined with melatonin administration [ 77 , 78 ] . in summary , regular exposure to bright light may be effective for day / night reversal problems [ 73 , 7981 ] . if the patient has rbd , removing potentially dangerous items from the bedroom and placing a soft mattress next to the bed may also help to ensure safety . clinical experience - based data also suggest that some ad patients with obstructive sleep apnea syndrome , treated with cpap , may benefit from improved sleep consolidation [ 8284 ] . it is recommended that behavioral and environmental techniques should be tried first before resorting to pharmacologic approaches . there are various medications to treat insomnia , behavioral dyscontrol , and nocturnal agitation / wandering , excessive daytime sleepiness , rem sleep behavior disorder , and restless legs syndrome . medications should always be combined with environmental / behavioral modifications . the choice of agent to use and dosing must be individualized . it is also the responsibility of the clinician to consider potential side effects and drug interactions . insomnia is a common problem which if left untreated / undertreated may exacerbate the cognitive and non cognitive symptoms of ad . commonly used medications to promote sleep include trazodone , zolpidem , sedating antidepressants such as mirtazapine , less commonly , low doses of sedating atypical antipsychotics such as quetiapine , and chloral hydrate . ( refer to table 2 for dosing ) . in considering the use of antipsychotics in the ad population , the fda - issued warnings about increased risks of mortality , stroke , and stroke - like events need to be considered as part of risk / benefit calculations . as much as possible it is best to avoid benzodiazepines as they may worsen cognitive functions in ad patients . nocturnal agitation / wandering , physical and verbal aggression , and hallucinations can also be treated pharmacologically after ruling out other medical and environmental causes and a trial of behavioral techniques . atypical antipsychotics [ 8587 ] , anticonvulsants , short - acting benzodiazepines such as lorazepam or longer - acting agents ( e.g. , clonazepam ) , and trazodone may be effective measures . cholinesterase inhibitors such as donepezil , rivastigmine , and galantamine may improve nocturnal behaviors over time [ 88 , 89 ] . because of the dependence of rem sleep on the integrity of the cholinergic system , theoretically , one may predict increased rem sleep in cholinesterase inhibitor - treated ad patients . however , cholinesterase inhibitors , especially donepezil , are also known to increase vivid dreams which subsequently may result in further disturbance in sleep [ 92 , 93 ] . a double - blind placebo - controlled trial done by grossberg et al . has shown that ad patients who received memantine performed better on behavioral outcomes compared to placebo . clonazepam , started at a low dose of 0.25 mg at night , is proven to be effective in the treatment of rem sleep behavior disorders . melatonin [ 95 , 96 ] or pramipexole can also be used in such cases although they are not as effective as clonazepam . although their use at higher doses is limited because of their potential for cardiovascular side effects in elderly , lower doses of methylphenidate or modafinil can be used if excessive daytime sleepiness is a problem . as usual , the risk / benefit ratio of these agents needs to be considered and discussed with patient / family before initiating these medications . it is also worth noting that stimulants may worsen some features of sundowning , restless leg syndrome is a common condition which is not uncommonly seen in ad patients and which can further complicate crd . it is always important to rule out other potentially treatable comorbidities such as depression and delirium . caregiver understanding of the nature of ad and the implementation of environmental and behavioral techniques can reduce both patient and caregiver distress and may decrease hospital readmissions and delay institutionalization . we are beginning to understand the neuroanatomic , neurochemical , and endocrine underpinnings of crds in ad and their impact on sleep / wake disturbances as well as common behavioral abnormalities . at present , there is no definitive treatment to address the neurodegenerative changes underlying crds in ad . however , current therapies , whether behavioral / environmental or pharmacotherapeutic , can provide symptomatic benefits for patients and relieve stress on their family or professional caregivers . large controlled studies are needed to evaluate the effects of treatments such as blt and engaging daytime activities in the reduction of crds and resultant sleep / wake cycle and behavioral disturbances in ad patients .

circadian rhythm disturbances ( crds ) affect as many as a quarter of alzheimer 's disease ( ad ) patients during some stage of their illness . alterations in the suprachiasmatic nucleus and melatonin secretion are the major factors linked with the cause of crds . as a result , the normal physiology of sleep , the biological clock , and core body temperature are affected . this paper systematically discusses some of the causative factors , typical symptoms , and treatment options for crds in patients with ad . this paper also emphasizes the implementation of behavioral and environmental therapies before embarking on medications to treat crds . pharmacotherapeutic options are summarized to provide symptomatic benefits for the patient and relieve stress on their families and professional care providers . as of today , there are few studies relative to crds in ad . large randomized trials are warranted to evaluate the effects of treatments such as bright light therapy and engaging activities in the reduction of crds in ad patients .

1. Introduction 2. What does Cause CRDs? 3. Common AD Symptoms Related to CRDs 4. Treatment Approaches 5. Recommendations/Conclusions

generalized vitiligo ( gv ) is a complex disorder in which acquired , progressive , multifocal loss of pigmentation of skin and hair results from loss of melanocytes from the affected areas ( taeb and picardo , 2009 ; birlea et al . , 2011b ) . while the striking phenotype of gv has been recognized for thousands of years ( nordlund et al . , 2006 ) , its underlying pathobiology has remained largely unknown , despite the suggestion of numerous theories ( boissy and spritz , 2009 ; picardo and taeb , 2010 ) . some of the earliest attempts to understand the biology of gv considered its possible genetic basis , in the hope that understanding the genetics would lead to insights into basic pathogenetic mechanisms of disease ( stttgen , 1950 ; teindel , 1950 ; lerner , 1959 ) . over the next four decades , various types of genetic studies aimed to discover genes involved in mediating gv susceptibility , with limited success . however , recently genomewide genetic association studies have succeeded in identifying a veritable cornucopia of gv susceptibility genes , encoding components of biological networks that largely regulate elements of the immune system and their targeting and destruction of melanocytes . probably the earliest evidence relating to the genetic basis of vitiligo was the 1855 description by addison , of a patient with idiopathic adrenal insufficiency ( now known as addison s disease ) , gv , and pernicious anemia ( addison , 1855 ) . over the next century numerous case reports described co - occurrence of gv and various other autoimmune diseases , and in 1908 claude suggested the likelihood of a causal connection among autoimmune diseases that tended to occur concomitantly ( claude , 1908 ) . in 1926 schmidt codified the concomitant occurrence of addison s disease and autoimmune thyroid disease , in what came to be called schmidt syndrome ( schmidt , 1926 ) , and in1980 neufeld and blizzard re - classified the so - called autoimmune polyglandular syndromes ( aps ) , denoting a rare autosomal recessive form ( sometimes including gv ) as aps-1 , denoting schmidt syndrome ( sometimes including gv ) as aps-2 , denoting concomitant occurrence of autoimmune thyroid disease with at least one other autoimmune disease ( again , often including gv ) as aps-3 , and denoting all other combinations of autoimmune diseases as aps-4 ( neufeld and blizzard , 1980 ) . while there have been a number of subsequent attempts to refine the aps classification , except for aps-1 ( now renamed apeced ; autoimmune polyendocrinopathy , candidiasis , ectodermal dystrophy , resulting from mutations in the aire gene ) , this system is no longer widely used . instead , it has become clear that multiple autoimmune disease is more complex , and that gv is part of a more general autoimmune disease diathesis . indeed , the frequencies of many other autoimmune diseases , particularly including autoimmune thyroid disease ( hashimoto s thyroiditis and graves disease ) , adult - onset type 1 diabetes mellitus , rheumatoid arthritis , psoriasis , pernicious anemia , addison s disease , and systemic lupus erythematosus ( sle ) , are elevated among gv patients ( alkhateeb et al . , 2003 ; laberge et al . , 2005 ; sun et al . , 2006 ) . furthermore , the frequencies of these same autoimmune diseases are elevated among the first - degree relatives of gv patients , even those who do not have gv ( alkhateeb et al . , 2003 ; laberge et al . , 2005 ) . this has led to the belief that general susceptibility to autoimmunity is a complex trait involving various shared susceptibility genes , while other genes and exposure to unknown environmental triggers determine the occurrence of gv and other specific autoimmune diseases in individual patients ( spritz , 2008 ) . in addition , genetic epidemiological studies have addressed the likely mode of inheritance of gv . while there had been a number of previous case reports describing the occurrence of gv in occasional relatives , perhaps the first to address the inheritance of gv specifically were stttgen ( 1950 ) and teindel ( 1950 ) , who described multigenerational families with multiple cases of gv ( multiplex families ) and other autoimmune diseases , concluding that autosomal dominant inheritance seemed likely . based on a survey of 128 vitiligo patients , lerner ( 1959 ) noted that 38% reported affected relatives , and concluded the disease is often transmitted as a dominant trait . however , while many subsequent patient surveys around the world confirmed frequent clustering of vitiligo cases within families ( e.g. mehta et al . , 1973 ; howitz et al . , 1977 ; obe , 1984 ; das et al . , 1985 ; bhatia et al . , 1992 ; majumder et al . , 1993 ; kim et al . , 1998 ; , 2003 ; sun et al . , 2006 ) , a single - locus mendelian pattern of inheritance was not supported , and most favored a polygenic , multifactorial model involving multiple genes and also environmental risk factors , what is now termed a complex trait . an major environmental component in gv is also indicated by its limited concordance in monozygotic twins ; among 22 such twin - pairs , alkhateeb et al . ( 2003 ) reported a concordance of only 23% , underscoring the importance of environmental triggers in disease pathogenesis . the earliest analyses testing the involvement of specific genes in the pathogenesis of gv were candidate gene association studies , of hla ( retornaz et al . , 1976 ) , abo ( kareemullah et al . , 1977 ) , and other blood groups ( wasfi et al . , 1980 ) , and positive candidate gene associations with gv have been reported subsequently ( reviewed in birlea et al . , 2011a ) . however , candidate gene association studies of complex diseases have generally proven to be highly subject to false - positive artifacts , principally due to occult ethnic differences between cases and controls ( population stratification ) and inadequate correction for multiple - testing ( hirschhorn et al . , 2002 ; freedman et al . , 2004 ) , and thus have fallen into general disfavor . except for hla and ptpn22 , most candidate gene associations reported for gv have not been confirmed by subsequent studies ( reviewed in birlea et al . , 2011a ) . genomewide linkage and association studies are not biased by a priori biological hypotheses and are less subject to methodological pitfalls than are candidate gene approaches . genetic linkage studies are best suited to detecting relatively rare disease susceptibility alleles with large effects , as in multiplex families . while genetic linkage studies are typically carried out genomewide , in fact the first linkage analysis reported for gv was a negative study , testing linkage of a candidate gene , mitf ( tripathi et al . , 1999 ) . the first direct genomewide linkage study of gv , in a unique large caucasian kindred with apparent autosomal dominant inheritance with incomplete penetrance , detected linkage in chromosome 1p31.3p32.2 ( ais1 ; alkhateeb et al . , 2001 ) . subsequent dna sequence analysis of genes in the chromosome 1p linkage interval identified a transcriptional regulatory variant of foxd3 , which encodes forkhead box d3 ) , a key regulator of melanoblast lineage differentiation and development ( alkhateeb et al . , 2005 ) . genomewide linkage studies of many other caucasian multiplex gv families identified additional linkage signals on chromosomes 7 , 8 , 9 , 11 , 13 , 17 , 19 , and 22 ( fain et al . the strongest of these signals , on chromosome 17p13 , coincided with slev1 , a linkage signal for sle detected in families that also included at least one relative with gv ( nath et al . , 2001 ) or other autoimmune diseases ( johansson et al . , 2004 ) . targeted family - based genetic association analysis of snps spanning the chromosome 17p linkage interval identified the corresponding gene as nalp1 ( now renamed nlrp1 ) , encoding nacht , lrr , and pyd domains - containing protein 1 ( jin et al . , 2007a ) , a key regulator of the innate immune system that apparently acts as a sentinel for bacterial infection in the skin ( lamkanfi and dixit , 2009 ) . several studies have subsequently confirmed association of nlrp1 with gv , ( jin et al . , 2007b ; alkhateeb and qarqaz , 2010 ) , and also with type 1 diabetes ( magitta et al . , 2008 ) , addison s disease ( magitta et al . , 2008 ; zurawek et al . , 2010 ) , celiac disease ( pontillo et al . , 2011 ) , systemic sclerosis ( dieud et al . , 2010 ) , and perhaps inflammatory bowel disease ( de iudicibus et al . , 2011 ) . parallel genetic linkage studies of gv in han chinese detected linkage signals on chromosomes 1 , 4 , 6 , 14 , and 22 ( chen et al . , 2005 ; liang et al . these linkage signals generally did not correspond to those detected in caucasian families , suggesting that different genes might underlie gv susceptibility in these different populations . by candidate gene association analysis of several genes located within the chromosome 22q12.112.3 linkage peak , ren et al . ( 2009 ) subsequently identified xbp1 , encoding transcription factor x - box binding protein 1 , which activates mhc class ii gene expression , regulates plasma cell differentiation , and mediates inflammatory response to endoplasmic reticulum stress . additional support for involvement of xbp1 in the pathogenesis of gv came from a subsequent genomewide association study of caucasians ( birlea et al . , 2011a ) , and xbp1 has been independently associated with genetic risk of crohn s disease ( kaser et al . , 2008 ) . genomewide association studies ( gwas ) are best suited to detecting relatively common disease susceptibility alleles with modest effects , as may be most relevant to typical singleton cases of a complex disease such as gv . of particular importance , gwas can adequately correct for both population stratification and multiple - testing ( hirschhorn et al . , 2002 ; freedman et al . , 2004 ) , and so are currently considered the gold standard for identifying complex trait susceptibility genes . the first gv gwas ( birlea et al . , 2010 ) involved a special population , an isolated village with a very high prevalence of gv located in the mountains of northwestern romania ( birlea et al . , 2008 ) . this analysis detected gv association with snps in distal chromosome 6q27 , in the vicinity of iddm8 , a linkage and association signal for type i diabetes mellitus and rheumatoid arthritis . a subsequent , much larger gwas in caucasians detected at least 13 different gv susceptibility loci ( jin et al . , 2010a , b ; table 1 ) , and confirmed three others that had been reported in previous candidate gene studies ( birlea et al . , 2011a ) . a parallel gv gwas in chinese ( quan et al . , 2010 ) detected two association signals , one or both of which were among those detected in caucasians ( table 1 ) . in both the caucasian and chinese studies , strong gv association signals were detected in the major histocompatibility complex ( mhc ) on chromosome 6p21.3 . in caucasians , independent associations were detected in both the mhc class i and class ii gene regions . in the class i region , the major association signal was in the vicinity of hla - a , in strong linkage disequilibrium with hla - a*02 . in the class ii region , the major association signal was located between hla - drb1 and hla - dqa1 , in moderate linkage disequilibrium with hla - drb1 * 04 ( jin et al . , 2010a ) . these results are thus consistent with previous reports of association of gv with both hla - a*02 ( liu et al . , 2007 ) and hla - drb1 * . however , in chinese , the major mhc association signal was in the class iii gene region , with some evidence for additional association in the class ii gene region ( quan et al . , 2010 ) . perhaps more important , these two large - scale gv gwass also detected association with a number of non - mhc loci , almost all of which encode immunoregulatory proteins . the caucasian gwas ( jin et al . , 2010a , b ) detected associations with at least ten non - mhc loci : tyr ( tyrosinase ) , ptpn22 ( lymphoid - specific protein tyrosine phosphatase nonreceptor type 22 ) , rere ( arginine - glutamic acid dipeptide [ re ] repeats protein ; atrophin - like protein 1 ) , foxp1 ( forkhead box p1 ) , lpp ( lim domain - containing preferred translocation partner in lipoma ) , il2ra ( interleukin-2-receptor alpha chain ) , gzmb ( granzyme b ) , ubash3a ( ubiquitin - associated and sh3 domain - containing a ) , c1qtnf6 ( c1q and tumor necrosis factor - related protein 6 ) , and ccr6 ( c - c chemokine receptor type 6 ) . this last gene is located very close to the 6q27 association signal detected in the romanian village ( birlea et al . , 2010 ) , and was the only non - mhc locus detected in the chinese gwas ( quan et al . , 2010 ) . the caucasian gwas also provided evidence of association of gv with additional loci previously suggested as candidate genes for gv : xbp1 ( see above ) , foxp3 ( forkhead box p3 ) , tslp ( thymic stromal lymphoprotein ) , and ctla4 ( cytotoxic t - lymphocyte antigen 4 ) , though as in previous studies association of gv with ctla4 appeared to reflect primary association with other concomitant autoimmune diseases in the gv cases , rather than with gv itself ( birlea et al . , 2011a ) . among the most illuminating associations with gv are those of mhc class i and tyr , which together highlight one of the pathways by which the immune system sees and thus targets melanocytes . the mhc class i association in caucasians with gv is with snp rs12206499 , which tags hla - a*02 ( predominantly hla - a*02:01 ) , while the tyr association with gv is with the major ( r ; arg ) allele of the r402q polymorphism ( snp rs1126809 ) that is relatively common among caucasians ( minor allele frequency 0.22 to 0.40 ) but is rare in other populations ( tripathi et al . , 1991 ) . in contrast , the minor ( q ; gln ) allele of the tyr r402q polymorphism is associated with susceptibility to malignant melanoma ( gudbjartsson et al . , 2008 ; bishop et al . , 2009 ) , while hla - a*02 is associated with relatively favorable response to melanoma immunotherapy ( mitchell et al . , tyrosinase is a major gv autoantigen presented to the immune system on the surface of melanocytes and melanoma cells by hla class i molecules , principally hla - a*02 . one of the important epitopes presented by hla - a*02 is a modified tyrosinase nonapeptide , ymdgtmsqv ( skipper et al . , 1996 ) . however , the variant 402q form of tyrosinase is hypoglycosylated ( toyofuku et al . , 2001 ) , which in turn would prevent the modification that is essential for its antigenic presentation by hla - a*02 . as the result , tyrosinase-402r likely makes a quantitatively greater contribution than tyrosinase-402q to presentation of antigenic tyrosinase by hla - a*02 . together , these findings indicate an apparent inverse relationship between genetic susceptibility to gv and genetic susceptibility to malignant melanoma as mediated by tyr and hla - a*02 ( jin et al . , 2010a ) , suggesting that gv may represent a dysregulated mechanism of immune surveillance against malignant melanoma , with tyr r402q modulating immune recognition of melanocytes by modulating availability of tyrosinase peptide for presentation by hla - a*02 ( spritz , 2010 ) . in addition to primary searches for genes that influence disease susceptibility per se , genomewide approaches can also be used to identify genes that influence the natural history of disease . such an approach has recently been taken to identify a locus that contributes to gv age of onset ( jin et al . , these authors reanalyzed their previous gv gwas dataset , considering only the affected individuals , in whom gv age of onset was assessed as a quantitative trait . this analysis identified a major gv age of onset locus in the mhc class ii region , apparently reflecting the same locus that was associated with gv susceptibility ( jin et al . , 2010a ) , while none of the other loci that had been associated with gv susceptibility per se were associated with age of onset . these results suggest that some loci likely mediate gv susceptibility per se , whereas other loci , particularly variation in the mhc class ii region , might mediate response to environmental triggers encountered over the course of life , thereby influencing age of disease onset . for gv as for many other complex diseases , application of genomewide approaches , particularly gwas , has resulted in rapid progress in identifying true disease susceptibility genes , while most genes suggested as a priori biologically - based candidates have remained unconfirmed . accordingly , for gv as for many other diseases , we have entered a new era of understanding the true underlying pathobiology , which in the case of gv appears to be predominantly autoimmune . furthermore , many of the genes that are genetically associated with gv are also genetically associated with some of the other autoimmune diseases with which gv is epidemiologically associated , confirming the longstanding hypothesis that these epidemiological associations reflect underlying shared causal elements ( claude , 1908 ) . the studies carried out so far have identified a plethora of new biological pathways that constitute potential targets for therapeutic intervention and perhaps even disease prevention . however , these findings only scratch the surface , accounting for a relatively small portion of total disease risk . larger genomewide studies can be expected to identify even more gv susceptibility genes and thus shed even more light on the nature of the disease , and perhaps even provide clues to environmental triggers . moreover , for only a few of the gv susceptibility genes found thus far have the corresponding underlying causal variants been identified ; this will require extensive dna sequencing of large numbers of gv patients , careful bioinformatics analysis , and targeted functional studies to assess the effects of specific variants , both individually and in combination . nevertheless , the way forward is now clear , with many doors opening to new understanding and new opportunities for treating patients with gv .

generalized vitiligo ( gv ) is a complex disease in which patchy depigmentation results from autoimmune loss of melanocytes from affected regions . genetic analyses of gv span six decades , with the goal of understanding biological mechanisms and elucidating pathways that underlie the disease . the earliest studies attempted to describe the mode of inheritance and genetic epidemiology . early genetic association studies of biological candidate genes resulted in some successes , principally hla and ptpn22 , but in hindsight many such reports now seem to be false - positives . later genomewide linkage studies of multiplex gv families identified nlrp1 and xbp1 , which appear to be valid gv susceptibility genes that control key aspects of immune regulation . recently , the application of genomewide association studies to analysis of gv has produced a rich yield of validated gv susceptibility genes that encode components of biological pathways reaching from immune cells to the melanocyte . these genes and pathways provide insights into underlying pathogenetic mechanisms and potential triggers of gv , establish relationships to other autoimmune diseases , and may provide clues to potential new approaches to gv treatment and perhaps even prevention . these results thus validate the hopes and efforts of the early investigators who first attempted to comprehend the genetic basis of vitiligo .

INTRODUCTION EPIDEMIOLOGICAL STUDIES CANDIDATE GENE ASSOCIATION STUDIES GENOMEWIDE LINKAGE STUDIES GENOMEWIDE ASSOCIATION STUDIES PERSPECTIVE

the increasing prevalence of advanced chronic kidney disease [ ackd ; estimated glomerular filtration rate ( egfr ) < 30 ml / min/1.73 m ] has led to increasing morbidity mortality and socio - economic costs . implementation of adequate infrastructure to care for this population will decrease cardiovascular morbidity mortality , delay the need for renal replacement therapy ( rrt ) and help prepare for rrt . however , there are several pitfalls that can be addressed to improve outcomes and optimize the use of current resources : inadequate medical training for nephrological referral and rrt selection internal medicine residents have widely differing perceptions of indications for nephrology referral : 10% of residents do not refer patients with ackd and 20% refer only with an egfr < 15 ml / min . limited medical training in peritoneal dialysis ( pd ) or transplantation may also contribute to individual or centre biases . in a spanish survey , over half of nephrology residents and their mentors believed that their training in pd was insufficient and 60% had never seen a pd catheter being inserted .late referral to nephrologists . early referral to the nephrologist is usually understood as at least a 3-month contact with the nephrology unit prior to initiating rrt . however , at least 1 year is required to provide ckd education and to optimize preparation for rrt . there are wide differences between different centres and countries in late referrals . in spain , italy and france , recent data show that 2025% of patients experienced late referrals , while higher figures were reported for america and other european and asian countries [ 816].inadequate patient education for choice of rrt modality . early diagnosis of ckd and early referral to the nephrologist are both necessary but insufficient to guarantee an informed choice of rrt technique . patient education and preparation for rrt requires adequate time ( 724 h per patient ) and frequent sessions . although more than 90% of nephrologists inform patients about different rrt options , most of these practitioners lack a standardized procedure . in most cases , time spent informing about rrt techniques lasted 560 min , but some patients were not informed and more time was devoted to haemodialysis ( hd ) than to pd or pre - emptive transplantation . in spain , only one - third of the centres use an informed consent document before starting rrt ( unpublished data).lack of multidisciplinary ackd / low - clearance / pre - dialysis clinics . multidisciplinary ackd clinics require at least one nephrologist and a nurse to guarantee patient education , information , training , care and psychosocial support , as well as research support [ 1927 ] . other specialists such as dietitians , social workers and psychologists provide very valuable input . centres with specialized ackd clinics offer more complete information and care , devote more time to overall patient / family educational training and achieve a higher percentage of programmed rrt initiation despite a shorter overall monitoring period than conventional single - specialist practices [ 1923,28,29 ] . however , such infrastructures are scarce and are frequently insufficiently staffed [ 712,19,30,31 ] . interviews with representatives from 60 spanish hospitals have revealed that 35% of hospitals have multidisciplinary monographic ackd clinics , 40% have conventional monographic ackd clinics and 25% lack these clinics . in the united kingdom , very few units had a full complement of the recommended multidisciplinary renal team .lack of programmed rrt initiation . there is a wide variability between countries , regions and centres in non - planned dialysis start times despite prior nephrological follow - up . failure of nephrological follow - up to prevent non - planned rrt initiation may be partially ascribed to events that deteriorate renal function , loss to follow - up or other medical reasons [ 20,21,28,3436 ] . however , one key modifiable factor has been the failure to achieve a timely permanent rrt access because of deficient nephrology surgery interdepartmental coordination . up to 60% of spanish rrt patients were not offered optimal ackd care , which was defined as adequate education on ckd and rrt options , starting rrt in a programmed fashion and care provided by a nephrologist . internal medicine residents have widely differing perceptions of indications for nephrology referral : 10% of residents do not refer patients with ackd and 20% refer only with an egfr < 15 ml / min . limited medical training in peritoneal dialysis ( pd ) or transplantation may also contribute to individual or centre biases . in a spanish survey , over half of nephrology residents and their mentors believed that their training in pd was insufficient and 60% had never seen a pd catheter being inserted . early referral to the nephrologist is usually understood as at least a 3-month contact with the nephrology unit prior to initiating rrt . however , at least 1 year is required to provide ckd education and to optimize preparation for rrt . there are wide differences between different centres and countries in late referrals . in spain , italy and france , recent data show that 2025% of patients experienced late referrals , while higher figures were reported for america and other european and asian countries [ 816 ] . . early diagnosis of ckd and early referral to the nephrologist are both necessary but insufficient to guarantee an informed choice of rrt technique . patient education and preparation for rrt requires adequate time ( 724 h per patient ) and frequent sessions . although more than 90% of nephrologists inform patients about different rrt options , most of these practitioners lack a standardized procedure . in most cases , time spent informing about rrt techniques lasted 560 min , but some patients were not informed and more time was devoted to haemodialysis ( hd ) than to pd or pre - emptive transplantation . in spain , only one - third of the centres use an informed consent document before starting rrt ( unpublished data ) . multidisciplinary ackd clinics require at least one nephrologist and a nurse to guarantee patient education , information , training , care and psychosocial support , as well as research support [ 1927 ] . other specialists such as dietitians , social workers and psychologists provide very valuable input . centres with specialized ackd clinics offer more complete information and care , devote more time to overall patient / family educational training and achieve a higher percentage of programmed rrt initiation despite a shorter overall monitoring period than conventional single - specialist practices [ 1923,28,29 ] . however , such infrastructures are scarce and are frequently insufficiently staffed [ 712,19,30,31 ] . interviews with representatives from 60 spanish hospitals have revealed that 35% of hospitals have multidisciplinary monographic ackd clinics , 40% have conventional monographic ackd clinics and 25% lack these clinics . in the united kingdom , there is a wide variability between countries , regions and centres in non - planned dialysis start times despite prior nephrological follow - up . failure of nephrological follow - up to prevent non - planned rrt initiation may be partially ascribed to events that deteriorate renal function , loss to follow - up or other medical reasons [ 20,21,28,3436 ] . however , one key modifiable factor has been the failure to achieve a timely permanent rrt access because of deficient nephrology surgery interdepartmental coordination . up to 60% of spanish rrt patients were not offered optimal ackd care , which was defined as adequate education on ckd and rrt options , starting rrt in a programmed fashion and care provided by a nephrologist . they offer similar survival rates , except for diabetic female patients over 65 years of age according to the us registry [ 3740 ] . over 70% of patients are potential candidates for hd or pd because they lack medical contraindications [ 4146 ] . nephrologists believe that 3040% of patients are able to receive home rrt . in the absence of medical contraindications or pre - emptive transplantation , pd should be considered as the first rrt option in the large majority of patients : it provides good outcomes , a better preservation of residual renal function and vascular capital , it may speed up delayed graft function , it improves graft patient survival and is cheaper . however , the incidence of pd is much lower than hd , suggesting an influence of non - medical factors in the selection of rrt modality . there is a large geographical variability in the incidence and prevalence of pd and pre - emptive transplantation that has not been justified by clinical criteria . this variability has also been observed within countries and within centres in the same city . in spain , the incidence of pd ranges from 20 to 25% in madrid , galicia and the basque country to almost zero in aragon . the early registration of patients on transplant waiting lists is also highly variable among centres and countries . late referral inevitably lowers the possibility of receiving education on rrt techniques , choosing rrt modality and receiving pre - emptive transplantation or transplantation within 1 year . hd was found to be both the initial and chronic rrt technique in 95% of patients with non - programmed start of rrt ; these patients were never educated in rrt techniques despite nephrological monitoring and late referrals . by contrast , the incidence of pd was maximal ( 30% ) in patients with programmed rrt starts and who were educated about rrt modalities participation of nurses in the education process may increase the incidence of home rrt modalities . in surveys carried out by the patient advocacy group alcer among patients with non - planned dialysis starts , one - third was unaware that they could choose their dialysis technique , 60% never chose the technique and most were unaware of alternatives to hd . in patients with non - programmed dialysis starts , only 6% of hospitals informed about rrt options . however , balanced information backed up by decision - making tools resulted in the choice of home techniques in 38% of non - planned dialysis start patients . there is increasing evidence that late referrals and lack of multidisciplinary ackd clinics negatively impact outcomes [ 17,19,2123,35,5661 ] . late referral is associated with increased mortality and morbidity and deprives the patient of treatment to prevent or delay ckd progression . multidisciplinary ackd clinics may have a direct impact on patient survival , morbidity and hospitalization , permanent rrt access rates at rrt start and health - related quality of life ( qol ) . the absence of free choice for rrt options or permanent vascular or peritoneal access at dialysis initiation impairs vital prognosis [ 21,28,34,6265 ] . ackd patient care consumes double the amount of resources than rrt because of the higher prevalence , higher rates , longer duration of comorbidity - driven hospitalizations and high cost of treatments . efficacy will probably translate into more efficient care , since hospitalizations , hd treatment through catheters and earlier or emergency rrt all increase costs . studies suggest that early referral to nephrologists may be associated with better outcomes and lower costs , but further cost efficacy research is required . a recent evaluation in spain analysing all types of rrt showed that there are more cost - effective scenarios than the current situation , such as modifying the percentage of planned dialysis start ups to 75% , increasing pd incidence to 30% or a combination of both . they offer similar survival rates , except for diabetic female patients over 65 years of age according to the us registry [ 3740 ] . over 70% of patients are potential candidates for hd or pd because they lack medical contraindications [ 4146 ] . nephrologists believe that 3040% of patients are able to receive home rrt . in the absence of medical contraindications or pre - emptive transplantation , pd should be considered as the first rrt option in the large majority of patients : it provides good outcomes , a better preservation of residual renal function and vascular capital , it may speed up delayed graft function , it improves graft patient survival and is cheaper . however , the incidence of pd is much lower than hd , suggesting an influence of non - medical factors in the selection of rrt modality . there is a large geographical variability in the incidence and prevalence of pd and pre - emptive transplantation that has not been justified by clinical criteria . this variability has also been observed within countries and within centres in the same city . in spain , the incidence of pd ranges from 20 to 25% in madrid , galicia and the basque country to almost zero in aragon . the early registration of patients on transplant waiting lists is also highly variable among centres and countries . late referral inevitably lowers the possibility of receiving education on rrt techniques , choosing rrt modality and receiving pre - emptive transplantation or transplantation within 1 year . hd was found to be both the initial and chronic rrt technique in 95% of patients with non - programmed start of rrt ; these patients were never educated in rrt techniques despite nephrological monitoring and late referrals . by contrast , the incidence of pd was maximal ( 30% ) in patients with programmed rrt starts and who were educated about rrt modalities . participation of nurses in the education process may increase the incidence of home rrt modalities . in surveys carried out by the patient advocacy group alcer among patients with non - planned dialysis starts , one - third was unaware that they could choose their dialysis technique , 60% never chose the technique and most were unaware of alternatives to hd . in patients with non - programmed dialysis starts , only 6% of hospitals informed about rrt options . however , balanced information backed up by decision - making tools resulted in the choice of home techniques in 38% of non - planned dialysis start patients . there is increasing evidence that late referrals and lack of multidisciplinary ackd clinics negatively impact outcomes [ 17,19,2123,35,5661 ] . late referral is associated with increased mortality and morbidity and deprives the patient of treatment to prevent or delay ckd progression . multidisciplinary ackd clinics may have a direct impact on patient survival , morbidity and hospitalization , permanent rrt access rates at rrt start and health - related quality of life ( qol ) . the absence of free choice for rrt options or permanent vascular or peritoneal access at dialysis initiation impairs vital prognosis [ 21,28,34,6265 ] . ackd patient care consumes double the amount of resources than rrt because of the higher prevalence , higher rates , longer duration of comorbidity - driven hospitalizations and high cost of treatments . efficacy will probably translate into more efficient care , since hospitalizations , hd treatment through catheters and earlier or emergency rrt all increase costs . studies suggest that early referral to nephrologists may be associated with better outcomes and lower costs , but further cost efficacy research is required . a recent evaluation in spain analysing all types of rrt showed that there are more cost - effective scenarios than the current situation , such as modifying the percentage of planned dialysis start ups to 75% , increasing pd incidence to 30% or a combination of both . current incomplete data suggest that deficiencies in ackd care are widespread , but that there are important differences among centres , regions and countries that raise questions about unequal access to healthcare . the following proposals may help to improve the current situation : define the scope of the problem . current information is piecemeal and has been obtained with very different methodologies and definitions . a europe - wide map of ackd care and a registry of ckd stage 4 patients are required . without this information , all efforts to claim cost - effectiveness of ackd care and early referral will remain inaccurate and vague .identify and correct country- , region- or centre - specific local factors that contribute to the current situation . as an example , nephrology outreach services may improve referral patterns .define and implement clear standards to limit potential biases of individual nephrologists or centres . quality indicators allow for monitoring and for establishing areas for improvement and assessing the impact of ackd clinics . a set of ackd care quality indicators is currently being validated but should be universalized and implemented.optimize medical education . educational efforts during residency training to raise awareness and benefits of early referral and to guarantee adequate , high - quality exposure to pd and transplantation may improve ckd management.provide adequate patient education . standardization of the information education process for the election , and not selection , of rrt technique may reduce inter - centre variability in the use of rrt techniques by allowing informed patients to choose the best possible rrt modality , according to medical criteria and individual preferences . there is no consensus on the best method to provide education to patients and families . group is a set of people who have common aims , who interact and play different roles , aware of forming part of a group and who set up relationships of interdependence . in ckd , the aims of group therapy should be psychosocial adaptation ( attitude change ) to the disease , prevention of risky behaviour ( dietary education ) and training ( education in ckd and rrt techniques ) . a pilot experience was performed that involved groups of 10 ackd patients , relatives / friends , healthcare staff ( nurse , assistant and nephrologist ) and volunteer rrt patients who met six times in 6 months . group therapy improved total and health - related qol , the understanding of kidney disease and anxiety . furthermore , 52% of participants chose pd , compared to 28% of same - centre non - participant patients . education of elderly patients is particularly challenging due to cognitive impairment and mood swings and family participation is encouraged .involve the stakeholders . it is the ultimate responsibility of professional societies and healthcare administrations to promote and provide the infrastructures which allow adequate information and education on ckd and rrt options to thus facilitate free informed choice of rrt modality . the right to adequate information that allows free choice of rrt technique was established in the spanish law basic regulatory act on patient autonomy 41/2002 and similar european legislation . healthcare staff select the medical and physical / psychological factors that best fit each rrt modality , but informed patients and families choose the modality . current guidelines highlight aspects such as the need for early referral to a multidisciplinary team and for documentation of balanced information by an informed consent form encompassing information about rrt techniques , choice of rrt technique and vascular or peritoneal access . social support to guarantee choice of home rrt in patients with loss of functional autonomy should also be provided . a europe - wide map of ackd care and a registry of ckd stage 4 patients are required . without this information , all efforts to claim cost - effectiveness of ackd care and early referral will remain inaccurate and vague . identify and correct country- , region- or centre - specific local factors that contribute to the current situation . as an example , nephrology outreach services may improve referral patterns . define and implement clear standards to limit potential biases of individual nephrologists or centres . quality indicators allow for monitoring and for establishing areas for improvement and assessing the impact of ackd clinics . a set of ackd care quality indicators is currently being validated but should be universalized and implemented . educational efforts during residency training to raise awareness and benefits of early referral and to guarantee adequate , high - quality exposure to pd and transplantation may improve ckd management . standardization of the information education process for the election , and not selection , of rrt technique may reduce inter - centre variability in the use of rrt techniques by allowing informed patients to choose the best possible rrt modality , according to medical criteria and individual preferences . there is no consensus on the best method to provide education to patients and families . group is a set of people who have common aims , who interact and play different roles , aware of forming part of a group and who set up relationships of interdependence . in ckd , the aims of group therapy should be psychosocial adaptation ( attitude change ) to the disease , prevention of risky behaviour ( dietary education ) and training ( education in ckd and rrt techniques ) . a pilot experience was performed that involved groups of 10 ackd patients , relatives / friends , healthcare staff ( nurse , assistant and nephrologist ) and volunteer rrt patients who met six times in 6 months . group therapy improved total and health - related qol , the understanding of kidney disease and anxiety . furthermore , 52% of participants chose pd , compared to 28% of same - centre non - participant patients . education of elderly patients is particularly challenging due to cognitive impairment and mood swings and family participation is encouraged . it is the ultimate responsibility of professional societies and healthcare administrations to promote and provide the infrastructures which allow adequate information and education on ckd and rrt options to thus facilitate free informed choice of rrt modality . the right to adequate information that allows free choice of rrt technique was established in the spanish law basic regulatory act on patient autonomy 41/2002 and similar european legislation . healthcare staff select the medical and physical / psychological factors that best fit each rrt modality , but informed patients and families choose the modality . current guidelines highlight aspects such as the need for early referral to a multidisciplinary team and for documentation of balanced information by an informed consent form encompassing information about rrt techniques , choice of rrt technique and vascular or peritoneal access . social support to guarantee choice of home rrt in patients with loss of functional autonomy should also be provided . there is increasing evidence that early referral to nephrologists , especially in the context of multidisciplinary ackd units , facilitates access to balanced and complete information , provides effective and efficient comprehensive care for patients and family members , slows the progression of ckd , decreases comorbidity and ckd complications , promotes informed choice of rrt and prepares for self - care home - based rrt if required .

there is a long distance between the actual worldwide reality in advanced chronic kidney disease care and the desire of how these patients should be managed to decrease cardiovascular and general morbidity and mortality . implementation of adequate infrastructures may improve clinical outcomes and increase the use of home renal replacement therapies ( rrt ) . current pitfalls should be addressed to optimise care : inadequate medical training for nephrological referral and rrt selection , late referral to nephrologists , inadequate patient education for choice of rrt modality , lack of multidisciplinary advanced kidney disease clinics and lack of programmed rrt initiation . these deficiencies generate unintended consequences , such as inequality of care and limitations in patient education and selection - choice for rrt technique with limited use of peritoneal dialysis . multidisciplinary advanced kidney disease clinics may have a direct impact on patient survival , morbidity and quality of life . there is a common need to reduce health care costs and scenarios increasing pd incidence show better efficiency . the following proposals may help to improve the current situation : defining the scope of the problem , disseminating guidelines with specific targets and quality indicators , optimising medical speciality training , providing adequate patient education , specially through the use of general decision making tools that will allow patients to choose the best possible rrt in accordance with their values , preferences and medical advice , increasing planned dialysis starts and involving all stakeholders in the process .

Current status and pitfalls in pre-dialysis care Consequences of the existing deficiencies Inequality of care and limitations in patient education and selectionchoice for RRT technique Impact on outcomes Efficiency of care Proposals for improvement Conclusions

most clinical presentations of the disease are as a result of a critical narrowing of arterial lumen or a sudden thrombotic occlusion caused by the rupture of atherosclerotic plaque . unfortunately prediction of such clinically adverse events is difficult by clinical judgement or symptoms alone.1)2)3 ) comprehensive approach of coronary atherosclerotic plaque including qualitative and quantitative analysis is necessary for the accurate lesion assessment and treatment planning as well as for the prediction of future outcome ( fig . 1).4)5 ) conventional invasive coronary angiography ( cag ) has been regarded as the gold standard for coronary artery disease but has limitation for assessing the extent of mild to moderate coronary atherosclerosis.6 ) intravascular ultrasound ( ivus ) or optical coherence tomography ( oct ) are needed to quantitate the burden of atherosclerosis . coronary computed tomography angiography ( ccta ) can be considered as a non - invasive method that may substitute cag and ivus.7)8 ) ccta is still limited by spatial and temporal resolution but enables non - invasive patient - friendly assessment of the extent of coronary atherosclerosis.9 ) therefore it is expected that ccta will be useful in overcoming limitations of the current diagnosis and treatment of coronary heart disease as shown below . ccta has emerged as a new noninvasive tool for screening of coronary artery disease in place of conventional stress imag - ing or electrocardiography.10)11 ) therapeutic strategy of coronary artery disease depends on the extent and severity of coronary artery disease . ccta can visualize the extent of severity of coronary artery disease with good sensitivity and excellent specificity.12)13 ) the performance of ccta for plaque detection has been validated by ivus and oct.7)14 ) unlike stress imaging or invasive cag , ccta can detect a wide range of coronary artery disease from mild to severe disease.15)16 ) it is difficult to predict future cardiac events through symptoms or clinical risk factors.17)18 ) clinical studies have shown that one - third of acute myocardial infarctions are asymptomatic prior to the event.19)20)21 ) most chronic total occlusions have infarct scar which is evidence of prior myocardial infarction but only half of them had a history of angina symptoms.22)23 ) a ccta is suitable to identify anatomical coronary stenosis , including calcified and non - calcified plaques , measure the atherosclerotic plaque burden and its response to treatment , and differentiate stable plaques from those that tend to rupture.24 ) positive remodeling , low density ( < 30 hounsfield units ) plaque , napkin - ring sign and spotty or micro - calcification detected by ccta has been shown to have vulnerable plaque characteristics ( fig . 2).24)25)26)27 ) these non - invasive findings have been validated by invasive ivus and oct studies.1)26)27 ) moreover , non - contrast coronary calcium ct scan is also useful to evaluate the risk of coronary artery disease using a coronary calcium score and it is well demonstrated to be related to long - term prognosis.28 ) accordingly , ccta can detect the presence of a coronary atherosclerosis most accurately and is one of the best non - invasive imaging modality to predict the clinical outcome of coronary artery disease as well.29 ) the treatment goal of coronary artery disease is the improvement of survival as well as relief of symptom . revascularization by percutaneous coronary intervention or bypass surgery has relieved symptom but has been questioned for improved long - term outcome.30)31 ) refinement of the need of revascularization strategy would be required to achieve improved clinical outcome after revascularization procedure including percutaneous coronary intervention or bypass surgery . the followings are proposed treatment strategy of coronary artery disease based on the ccta . first is to detect coronary atherosclerosis using ccta . ccta has the highest diagnostic power to find coronary atherosclerosis among non - invasive imaging modalities and hence very high negative predictive value.1)16 ) second is to assess myocardial ischemia non - invasively with ccta . the presence of myocardial ischemia is the most important factor for making a prognosis and for the decision to perform a revascularization.31 ) however less than half of coronary arteries with a diameter stenosis 50% cause myocardial ischemia in most clinical studies.31)32)33 ) it would be greatly preferable to find the functional significance of a stenosis as well as the presence of a stenosis by single ccta test . myocardial computed tomography perfusion imaging could be utilized in clinical practice.34)35)36 ) computational fractional flow reserve based on computed fluid dynamics analysis37)38 ) and contrast gradient analysis39)40 ) are being investigated.41)42)43 ) third , it would be important to evaluate the amount of ischemic myocardial mass . several studies have investigated the relationship between the angiographic severity of stenosis and the amount of perfused myocardial tissue . duke jeopardy score,44)45 ) myocardial jeopardy index,46 ) and alberta provincial project for outcome assessment in coronary heart disease ( approach ) lesion score44)47 ) have been used to evaluate the burden of myocardium - at - risk based on angiographic extent of stenosis . however these scoring systems are limited by semi - quantitative grading of the severity of coronary artery disease and myocardium - at - risk . detailed anatomical variations of individual vessels , myocardial mass , and location of stenosis is not taken account in these semi - quantitative scores . nuclear perfusion studies have shown that the clinical benefit of revascularization became evident when the ischemic burden exceeds 12% to 15% of total myocardial mass.48)49)50 ) ccta enables exact quantitative assessment of the burden of myocardium - at - risk and the extent of coronary artery disease ( fig . ccta can find coronary artery disease , determine the significance of coronary artery disease , and guide treatment strategy including revascularization . now it is time to validate and investigate the role of ccta in clinical practice and the impact of ccta on the clinical outcome .

coronary computed tomography angiography ( ccta ) has high negative predictive power for detecting coronary artery disease . however ccta is limited by moderate positive predictive power in the detection of myocardial ischemia . this is not unexpected because the diameter of a stenosis is a poor indicator of myocardial ischemia and discrepancy between the severity of stenosis and noninvasive tests is not uncommon . the value of stenosis for predicting future development of acute coronary syndrome represented by plaque rupture has been questioned . ccta identifies the characteristics of high - risk plaque including positive remodeling , low density plaque and spotty or micro - calcification . also , additional evaluation of myocardial ischemia using computational flow dynamics , and luminal attenuation gradient are expected to increase both diagnostic performance for hemodynamically significant stenosis and the predictive power for future cardiovascular risk . technical advances in ccta would enable evaluation of both coronary artery stenosis and myocardial ischemia simultaneously with high predictive performance , and would improve vastly the clinical value of ccta .

Introduction Demonstrating the Presence of Coronary Artery Disease Prediction of Future Coronary Heart Disease Incidents Roles of CCTA on Determining the Treatment Policy of Coronary Artery Disease Conclusions

hemoglobin is a remarkable molecular machine that uses motion and small structural changes to regulate its action . once the first heme binds oxygen , it introduces small changes in the structure of the corresponding protein chain . the main function of red blood cell is transfer of o2 from lungs to tissue and transfer of co2 from tissue to lungs . glassy carbon derives its name from exhibiting fracture behavior similar to glass , from having a disordered structure over large dimensions ( although it contains a graphitic microcrystalline structure ) , and because it is a hard shiny material , capable of high polish [ 35 ] . glassy carbon is particularly useful in electrochemical applications because of its low electrical resistivity , impermeability to gases , high chemical resistance , and because it has the widest potential range observed for carbon electrode . glassy carbon was first prepared by yamada and sato in 1962 by the hight emperature pyrolysis of phenolic resin , and later by davison who used cellulose as the starting material [ 6 , 7 ] . they concluded that glassy carbon consists of long microfibrils that twist , bend , and interlock to form interfibrillar bonds , and that these microfibrils are randomly oriented . because of the desire to impart selectivity to electrochemical reactions and to control electron transfer kinetics , several investigators have utilized adsorption or covalent bonding of catalysts to the glassy carbon surface . the intent of these modifications is to control the interaction of molecules and ions with the electrode surface . small fibers in the submicron range , in comparison with larger ones , are well known to provide better filter efficiency at the same pressure drop in the interception and inertial impaction regimes [ 10 , 11 ] . while smaller fiber size leads to higher - pressure drop , interception and inertial impaction efficiencies will increase faster , more than compensating for the pressure drop increase . thus , in the particle size of interest , that is , from submicron and up , better filter efficiency can be achieved at the same pressure drop , or conversely , the same filter efficiency at lower pressure drop can be achieved with smaller fiber sizes . polymeric nanofibers can be made using the electrospinning process , which has been described in the literature and in patents . electrospinning uses an electric field to draw a polymer solution from the tip of a capillary to a collector . a voltage is applied to the polymer solution , which causes a jet of the solution to be drawn toward a grounded collector . the fine jets dry to form polymeric fibers , which can be collected on a web . the electrospinning process has been documented using a variety of fiber - forming polymers [ 15 , 16 ] . by choosing a suitable polymer and solvent system , nanofibers with diameters in the range of 402000 nm can be made . pvp is a kind of polymers which is able to adsorb through many points of the molecule on the surface of steel , copper , and zinc and reduce the corrosion rate of the material [ 1719 ] . pvps are considered as food additives and , hence , their addition will not raise health concerns for treating ro water . however , the effect of the molecular weight of polyvinylpyrrolidone , its concentration , and temperature on the corrosion properties of 316 l stainless steel in reverse osmosis water were not evaluated . electrospinning is a process of applying a high - voltage electric field ( several to tens of kilovolts ) to generate electrically charged jets from polymer solutions or melts and further to produce polymer ( nano ) fibers . this technique is quite similar with the commercial process for drawing microscale fibers ; however , it is more suitable for generating nanofibers , because the elongation can be accomplished by a contactless scheme through the application of an external electric field . generally , generation of porous surface on a bulk electrospinning nanofiber can be realized through two different ways . the first one is based on the selective removal of a component from nanofibers made of a composite or blend material , while the other one involved the use of phase separation of different polymers during electrospinning under the application of proper sinning parameters . for instance , in pla / pvp electrospinning nanofibers , more porosity can be generated when the two materials are loaded in equal amounts comparing to the 11 corresponding products by different proportion of pla / pvp . it can be attributed to the rapid - phase separation and solidification in the spinning jet [ 1417 ] . pla is contraction name of polylactic acid , and it is a thermoplastic polymer made from lactic acid and has mainly been used for biodegradable products , such as plastic bags and planting cups . it was found that the average diameter of the pla / pvp / nanofibers lead fibers became larger , and the morphology of the fibers became finer with the content of pla increasing . the formation of pores is also affected by the solvent vapor pressure and the humidity in atmosphere . the cooling effect that comes from rapid evaporation of a highly volatile solvent might induce the polymers to separate into different phases in liquid jet . because of evaporative cooling and condensation , water droplets could also be formed within the fibers to promote the formation of porous nanofibers [ 2022 ] . the electrospun nanofibers exhibit several unique features , which enable the prevalent utilization of them . because electrospinning is a continuous process without any contact force for elongation , the fibers can be as long as several kilometers and can be further assembled into a 3d mat with porous structure . at the same time , electrospun fibers can have a thinner diameter and surface - to - volume ratio , due to the presence of porous structure . in addition , due to the simple fabrication process and the diversity of suitable materials , the electrospinning technique and its resultant nanofiber product have attracted increasing attention . these properties potentiate the use of the electrospun nanofibers in various applications such as reinforced composites , nanofiber - based membranes , nanofiber - based support for enzyme , and catalyst [ 2325 ] . manganese(ii ) nitrate tetrahydrate , and poly(vinylpyrrolidone ) ( pvp , mw = 1,300,000 ) were purchased from sigma . 0.1 m phosphate buffer solutions with various ph values were prepared by mixing stock standard solutions of na2hpo4 and nah2po4 and adjusting the ph values with naoh and h3po4 solution . all solutions used in the experiments were prepared with deionized water generated by a barnstead water system . 44 wt% mn(no3)2 , 11 wt% agno3 , and 44 wt% pvp were dissolved in dmf . the solution was kept under magnetic stirring for 2 h and then loaded into a plastic syringe equipped with a 23-gauge needle made of stainless steel . electrospinning process was conducted at an applied voltage of 20 kv with a feeding rate of 0.3 ml / h and a collection distance of 15 cm . the nanofibers were collected on aluminum foil and then calcined under air atmosphere at 500c for 3 h for the degradation of pvp and the decomposition of mn(no3)2 and agno3 : ( 1)4mn(no3)2mn2o3 + 8no2+ o22agno32ag+2no2+ o2. electrospinning is a process of applying a high - voltage electric field ( several to tens of kilovolts ) to generate electrically charged jets from polymer solutions or melts and further to produce polymer ( nano ) fibers . so , in this study , we used from this method to produce mn2o3-ag nanofibers . the most commonly used carbon - based electrode in the analytical laboratory is glassy carbon ( gc ) . it is made by pyrolyzing a carbon polymer , under carefully controlled conditions , to a high temperature like 2000c . an intertwining ribbon - like material results with retention of high conductivity , hardness , and inertness . glassy carbon electrode ( gce , dia . 3 mm ) was polished with 1 m and 0.05 m alumina slurries sequentially and then rinsed with di water . after that , the electrode was sonicated in deionized water and finally dried under ambient conditions . to prepare the modified gce , the mn2o3-ag nanofibers / glassy carbon electrode was placed into a fresh pbs including 5 mg ml hb ( ph 7.0 , 3 to 5c ) for 8 h. at the end , the modified electrode was washed in deionized water and placed in pbs ( ph 7.0 ) at a refrigerator ( 3 to 5c ) , before being employed in the electrochemical measurements as the working electrode . a jeol 6335f field - emission scanning electron microscope ( sem ) was used to examine the morphology and the size of the as - prepared nanofibers . more detailed morphology and selected area electron diffraction ( saed ) patterns of mn2o3-ag nanofibers were obtained with a tecnai t12 transmission electron microscope ( tem ) operated at 120 kv . xrd pattern was obtained with oxford diffraction xcaliburtm px ultra with onyx detector to study the crystal structure of mn2o3-ag nanofibers measurements which were carried out with a potentiostat / galvanostat ( model 263a , eg&g , usa ) using a single compartment voltammetric cell , equipped with a platinum rod auxiliary electrode . all experiments were conducted using a three - electrode electrochemical cell ( 10 ml volume with a working volume of 5 ml ) , with a working electrode , an ag / agcl reference electrode , and a platinum wire counter electrode . electrochemical measurements were carried out with a potentiostat / galvanostat ( model 263a , eg&g , usa ) using a single compartment voltammetric cell , equipped with a platinum rod auxiliary electrode . figure 2(a ) presents a typical sem image of electrospun precursory pvp - mn(no3)2-agno3 nanofibers . pvp was used for characterization of mn2o3-ag nanofibers , because pvp binds to polar molecules exceptionally well , owing to its polarity . this has led to its application in coatings for increase of photo quality in microscopic studies . when dry , it is a light flaky powder , which readily absorbs up to 40% of its weight in atmospheric water . in solution pvp is a branched polymer , that is , its structure is more complicated than linear polymer , but it too is in a two - dimensional plane . moreover , because mn2o3-ag nanofibers have polar groups too , a good bind was created between this nanofiber and pvp . after calcination , the as - prepared mn2o3-ag composite nanofibers ( figure 2(b ) ) exhibit a porous network structure , and their surfaces are no longer as smooth as the precursory nanofibers . such feature endows the nanofibers with high surface - to - volume ratio , which could provide not only a large surface area for hb loading but also a large interface for direct electron transfer of hb . as a comparison , the nanofibers prepared by single metal salt ( mn(no3)2 ) with pvp and its calcined product ( mn2o nanofibers ) are presented in figures 2(c ) and 2(d ) , respectively . the xrd spectrum of mn2o3-ag nanofibers matches the combination of the standard spectrum of jcpds 41 - 1442 ( mn2o3 ) and jcpds 04 - 0783 ( ag ) . the formation of face - centered cubic crystalline mn2o3 is revealed by the diffraction peaks at 2 values of 32.951 , 38.234 , 45.178 , 49.347 , 55.189 , and 65.806 , corresponding to ( 111 ) , ( 200 ) , ( 220 ) , ( 311 ) , ( 222 ) , and ( 400 ) crystal planes , respectively , while the diffraction peaks at 2 values of 38.116 , 44.277 , and 64.426 , which correspond to ( 111 ) , ( 200 ) , and ( 220 ) crystal planes , respectively , indicate the formation of cubic crystalline ag . the cyclic voltammograms ( cvs ) of different modified electrodes were obtained in 0.1 m pbs with ph 7.0 . compared with bare gc electrode , the background current of mn2o3-ag - nanofibers - modified electrode is apparently larger , which indicates that the effective electrode surface area is significantly enhanced by use of mn2o3-ag nanofibers to modify the electrode . however , the cvs of hb / mn2o3-ag nanofibers / gc electrode give a pair of well - defined redox peaks at 75 mv at scan rate of 100 mv / s , characteristic of heme fe(iii)/fe(ii ) redox couples of hb , suggesting that direct electron transfer has been achieved between hb and modified electrode . the electron transfer of the proteins , at the bare electrodes is very slow so that the redox peak of proteins can usually be observed . figure 4(a ) shows a cyclic voltammogram ( cv ) of the bare glassy carbon electrode . figure 4(b ) shows a cyclic voltammogram of hb / mn2o3-ag nanofibers / glassy carbon electrode in 0.1 m phosphate buffer at ph 7.0 . the hb showed quasireversible electrochemical behavior with a formal potential of 49 mv ( versus ag / agcl ) ; cathodic and anodic peaks were not observed using the bare graphite electrode . this shows that mn2o3-ag nanofibers act as a facilitator of electron transfer from the redox species of hb to the electrode surface and vice versa . these results are in line with the previous work that explains the behavior of nanoparticles as the facilitators of electron transfer . in figure 4(a ) , one sees cyclic voltammogram for bare and modified glassy carbon electrode in 50 , 100 , 200 , 300 , 400 , and 500 scan rates . these data showed that the mn2o3-ag nanofibers were successfully assembled on the glassy carbon electrode surface . the redox peak currents increase linearly with the square root of the scan rate . with an increasing scan rate , the anodic peak potential of adsorbed hb shifted to a more positive value , while the cathodic peak current shifted in a negative direction . the redox peak currents were proportional to the scan rate ( figure 5 ) ; thus , the electrode reaction was typical of the surface - controlled quasireversible process . one can see in figure 5(b ) the dependence of the anodic and cathodic peak currents on the scan rates that the red line is cathodic and the blue line is anodic . the plot of cathodic peak potentials versus the logarithm of scan rates gave a charge transfer coefficient of 0.52 . for scan rates from 50 to 500 mv / s , the electron transfer rate constant k s was estimated to be ( 1.9 ) s. these statements discussed below indicate that hb is strongly adsorbed onto the surface of modified electrode . as could be seen in figure 5(c ) , in the range from 200 to 500 mv s , the catholic peak potential ( e pc ) changed linearly versus lnv with a linear regression equation of y = 0.2425x 0.4254 , r = 0.945 , according to the following equation [ 2628 ] , ( 2)ep = e+rtnfrtnflnv , where is the cathodic electron transfer coefficient , n is the number of electrons , r , t , and f are gas , temperature , and faraday constant , respectively ( r = 8.314 j molk , f = 96493 c / mol , t = 298 k ) , and n is calculated to be 0.52 . given 0.3 < < 0.7 in general , it could be concluded that n = 1 and = 0.52 . from the width of the peak at midheight and low scan rate , therefore , the redox reaction between hb and modified graphite electrode is a single electron transfer process . in order to calculate the value of apparent heterogeneous electron transfer rate constant ( k s ) , the following equation was used [ 2628 ] : ( 3)logks=log(1)+(1)loglog(rtnfv ) (1)nfep2.3rt . the k s was calculated to be 1.90 s. moreover , this k s indicates that transfer of electron is good and fast in hb - modified glassy carbon electrode . the designed biosensor is introduced as a hydrogen biosensor because it has oxidation and reduction reactions in hb ; when fe changes to fe , the oxidation reaction occurred , and when fe changes to fe , the reduction reaction occurred . the electrocatalytic reactivity of hb / mn2o3-ag nanofibers / gc electrode toward h2o2 was investigated by cv . figure 6 displays the cyclic voltammograms obtained for the hydrogen peroxide biosensor in pbs ( ph 7.0 ) containing varied concentration of h2o2 in the absence of oxygen . the catalytic reduction of hydrogen peroxide at the biosensor can be seen clearly in figure 5 . with the addition of h2o2 , the reduction peak current increases obviously , while the oxidation peak current decreases ( figures 6(a ) and 6(b ) ) , indicating a typical electrocatalytic reduction process of h2o2 . however , no similar cathodic peak corresponding to the reduction of h2o2 can be observed at bare gc , mn2o3-ag nanofibers / gc electrode under the same condition , so it can be concluded that hb immobilized on mn2o3-ag nanofibers / gc electrode shows good catalytic activity toward hydrogen peroxide . the sensor was found to have sensitivity in the range of 20 to 700 m based upon the mean of the slope found from the points on the response curve . as can be observed , the sensor response shows good linearity in this range . the correlation factor , r , was found to be 0.9962 . in order to obtain an efficient biosensor for h2o2 , the influence of ph and applied potential on the response of hb / mn2o3-ag nanofibers / gc electrode were investigated . the change of chronoamperometric current with the ph under constant hydrogen peroxide concentration ( 50.0 m ) is shown in figure 7 . this designed biosensor possesses good stability and reproducibility and achieves 95% of the steady - state current in less than 5 s. a new biosensor for h2o2 was prepared based on hb / mn2o3-ag nanofibers / glassy carbon electrode . hb retained well in mn2o3-ag nanofibers / gce , which combined the utilities of mn2o3-ag nanofibers facilitating the electron transfer . at hb / mn2o3-ag nanofibers / gce , the cyclic voltammogram exhibits a pair of redox peaks corresponding to a surface - controlled electrode process with a single - proton transfer .

we investigated the electrochemical behavior of hemoglobin by glassy carbon electrode modified with mn2o3-ag nanofibers . the mn2o3-ag nanofibers were used as facilitator electron transfer between hb and glassy - carbon - modified electrode . the mn2o3-ag nanofibers are studied by scanning electron microscopy ( sem ) and transmission electron microscopy ( tem ) . the hemoglobin showed a quasireversible electrochemical redox behavior with a formal potential of 49 mv ( versus ag / agcl ) in 0.1 m potassium phosphate buffer solution at ph 7.0 . the designed biosensor possesses good stability and reproducibility and achieves 95% of the steady - state current in less than five seconds .

1. Introduction 2. Material and Methods 3. Results and Discussion 4. Conclusion

diabetes and the metabolic derangements typical of diabetes are associated with poor prognosis in cancer [ 111 ] . in perhaps the most comprehensive study to date ; barone and colleagues performed a systematic review and meta - analysis of the literature and found that preexisting diabetes was associated with statistically significant increases of 41% for all - cause mortality , across multiple tumor types , and 76% , 61% , and 32% in endometrial , breast , and colorectal cancer , respectively . poor prognosis may be influenced through biological mechanisms related to hyperglycemia , hyperinsulinemia , and inflammation , which result in tumor cell proliferation and metastases [ 35 , 12 ] . other factors include less aggressive cancer treatment due to diabetes - related comorbidity , poorer response to cancer treatment [ 7 , 11 ] , presentation with later - stage cancer due to suboptimal cancer screening practices and other preventive health - seeking behavior , and that diagnosis of cancer may distract both the patient and the health care team from appropriate management of glycemia , blood pressure , and lipids . factors thought to play a role in observed associations between preexisting diabetes and mortality could be exacerbated in undiagnosed diabetes , but evidence supporting this hypothesis is scarce . data from the second national health and nutrition examination survey ( nhanes ) do suggest that cancer mortality in patients with undiagnosed diabetes may be higher than in those with previously diagnosed diabetes , where undiagnosed diabetes was identified by oral glucose tolerance testing . however , this study was conducted in the general population making the risk of cancer mortality a function of both the risk of developing cancer and the subsequent risk of death due to cancer . also , the two diabetes groups were not compared directly , and differences in mortality compared to a reference group with normal glucose tolerance , while suggestive of an adverse impact , failed to reach statistical significance . data on the incidence of and risk factors for undiagnosed diabetes in cancer also are scarce . while a recent paper reports that detection of many chronic conditions including diabetes increases around the time of breast cancer diagnosis , risk factors for the detection of these conditions were not examined in detail . several studies have examined factors associated with cancer stage at diagnosis , an important predictor of cancer mortality , focusing on demographic and socioeconomic characteristics and patterns of prior health system contact [ 1619 ] . these show that more contact with a primary care physician and/or medical specialist , greater use of general preventive and cancer screening services , and more contact with the health care system as measured by level of comorbidity ascertained through medical claims , all are associated with earlier - stage cancer at diagnosis . in this study , we sought to describe the incidence and risk factors for diabetes that is undiagnosed until cancer . we elected to focus on prior health system contact , comorbidity , race , and socioeconomic status as risk factors since there is evidence that all of these are associated with cancer stage at diagnosis [ 1619 ] , which is another important prognostic factor for cancer outcomes . the source of data for this study was the national cancer institute 's ( nci ) surveillance , epidemiology , and end results ( seer ) cancer registry linked to medicare claims . presently , seer contains cancer incidence and survival data from 17 population - based cancer registries throughout the united states covering approximately 28% of the population . in seer - medicare , cancer registry data are linked to medicare enrollment and claims data , which are available for 93% of those aged 65 years in the seer registry . patients meeting all of the following criteria were included : they were diagnosed with breast , colorectal , lung , or prostate cancer , the four most common types in the elderly , between january 1 , 1999 , and december 31 , 2002 . this was their first and only cancer diagnosed , and they had at least 24 months of medicare part a ( hospital ) and part b ( outpatient ) fee - for - service coverage prior to the diagnosis of cancer . patients were excluded for the following reasons : male breast cancer , cancer diagnosis made by death certificate or autopsy , death within the first month following diagnosis , missing or unknown cancer stage at diagnosis , or in situ lung or prostate cancer ( due to small numbers of patients ) . patients with preexisting diabetes diagnosed between 24 and 4 months ( inclusive ) before cancer initially were included in the study to calculate the proportion of all diabetes cases undiagnosed until cancer ( i.e. , using a denominator of preexisting plus undiagnosed until cancer ) . however , patients with preexisting diabetes were then excluded from all the analyses of risk factors for undiagnosed diabetes . diabetes was defined as the presence of one of the following international classification of diseases , 9th revision , clinical modification diagnosis codes in one inpatient medicare claim or in two outpatient claims at least 30 days apart : 250.xx for diabetes and complications , 357.2x for polyneuropathy in diabetes , 362.0x for diabetic retinopathy , and 366.41 for diabetic cataract . laboratory claims were excluded to reduce the likelihood of misclassifying those patients only undergoing diagnostic evaluation for suspected diabetes as actual diabetes cases . patients were followed from 24 months prior to the diagnosis of cancer until 3 months after diagnosis ( overall follow - up : 27 months ) . the observation period was divided into two consecutive periods : 24 to 4 months prior to cancer diagnosis ( the 21-month look - back period ) and 3 months prior to 3 months after diagnosis or until death , whichever came first ( the 6-month incidence period ) . the first day of the seer month of diagnosis was assigned as the day of diagnosis . the look - back period was used to identify preexisting ( prevalent ) diabetes and to construct measures of prior health system contact . diabetes that was not reported in the claims until the time around cancer diagnosis was considered to be undiagnosed diabetes . it was defined as having a first diagnosis of diabetes between 3 months before and 3 months after cancer diagnosis . the same claims - based algorithm used to identify and subsequently exclude patients with preexisting diabetes during the look - back period ( 24 to 4 months before cancer diagnosis ) also was used to identify undiagnosed diabetes . patients were described according to their demographic , clinical , and socioeconomic characteristics . requiring eligible patients to have at least two years of medicare enrollment prior to diagnosis meant that the minimum age in the cohort was 67 years . stage at cancer diagnosis was based on the seer - modified american joint committee on cancer ( ajcc ) stage variable . in seer , socioeconomic information , including measures of poverty and education , is reported at the census tract level . we constructed two measures of prior health system contact during the look - back period , based on literature describing associations between prior health system contact and stage at cancer diagnosis [ 16 , 17 ] . first , we constructed a physician contact index that classified patients according to the types of ambulatory care visits they received during the look - back period . we searched medicare claims for healthcare common procedure coding system codes indicating physician outpatient visits and used the associated physician specialty code to classify each visit as primary care physician , medical specialist , or other specialist . other specialists included general surgeons , ophthalmologists , orthopedic surgeons , and other surgical specialists . patients were then classified as having had ( a ) primary care physician but no medical specialist ( with or without other specialist ) visits , ( b ) medical specialist but with no primary care physician ( with or without other specialist ) visits , ( c ) both primary care physician and medical specialist visit , ( d ) other specialist but no primary care physician or medical specialist visits , or ( e ) no prior visits . second , we constructed an index of preventive services based on the one developed by gornick et al . , consisting of mammography , screening for colorectal cancer , prostate - specific antigen test , papanicolaou test , screening for glaucoma , influenza immunization , and pneumonia immunization . the presence of one or more claims for each type of service was coded as 1 for that service , and individual scores were combined in an index consisting of 0 , 1 , or 2 . comorbidity is a predictor of breast cancer stage at diagnosis , and when comorbidity is identified from medical claims , it can also be considered an indirect indicator of increased health system contact . to account for this facet of health system contact , we calculated an nci comorbidity index score for each patient [ 2232 ] . we calculated the proportion of all diabetes patients diagnosed during the entire 27-month observation period who were diagnosed during the 6-month incidence period . we performed four multivariate logistic regression analyses , one for each type of cancer , to examine race / ethnicity , socioeconomic factors , and patterns of prior health system contact associated with undiagnosed diabetes all stratified by type of cancer . all models included age , gender ( colorectal and lung only ) , race / ethnicity , year of diagnosis , education , poverty , and geographic area . all three measures of prior health system contact ( physician contact index , index of preventive services , and nci comorbidity index ) were included in each of the four models . however , the three measures of prior health system contact also were assessed in separate models to evaluate the levels of multicollinearity among them . initially , 184,336 patients with breast , colorectal , lung , and prostate cancer were considered for inclusion in the study . of these , 11,426 ( 6.2% ) were excluded due to missing / unknown cancer stage ( all cancer types ) or in situ stage ( lung and prostate cancer only , as stated in section 2 ) . of the remaining 172,910 , an additional 18,218 ( 10.5% ) were excluded prior to the analysis of risk factors for undiagnosed diabetes because they had preexisting diabetes diagnosed during the look - back period : breast 3,850/40,062 ( 9.6% ) , colorectal 6,029/39,034 ( 15.4% ) , lung 4,861/40,622 ( 12.0% ) , and prostate 3,478/53,182 ( 6.5% ) . therefore , 154,692 met all the inclusion and exclusion criteria for the analysis of risk factors associated with undiagnosed diabetes ( table 1 ) . overall , the mean age was 76.3 years : 84.7% were non - hispanic white , 31.6% came from a census tract with > 12% poverty , and 56.0% were from a large metropolitan area . among the measures of prior health system contact , 65.3% had an nci comorbidity index score equal to 0 , 78.1% had at least one preventive service during the look - back period , and 56.5% had visits to both a primary care physician and a medical specialist ( table 2 ) . this differed by cancer type as 69.5% and 69.0% of breast cancer and prostate cancer patients , respectively , had nci comorbidity index scores of 0 compared to 53.9% of lung cancer patients . breast ( 19.2% ) and prostate ( 19.6% ) cancer patients were more likely to have had no preventative service visits compared to colorectal ( 25.2% ) or lung ( 24.8% ) cancer patients . women with breast cancer ( 60.4% ) were more likely to have visited both a primary care physician and medical specialist than were other patients in this study ( 53.3% to 57.2% ) . when viewed over the entire study period ( the incidence and the prevalence look - back periods combined ) , undiagnosed diabetes accounted for 12.8% ( 2,678/20,896 ) of all the diabetes cases : 8.8% of all diabetes in breast , 13.0% of all diabetes in colorectal , 16.8% of all diabetes in lung , and 10.8% of all diabetes in prostate cancer ( not shown in the tables ) . in general , the incidence of undiagnosed diabetes in the 6-month period around the cancer diagnosis was similar across age groups but was higher in those of nonwhite race / ethnicity , those diagnosed with advanced stage cancer , those living in a census tract with a lower proportion college educated , and those living in a census tract with more poverty . the incidence of undiagnosed diabetes was inversely related to the number of preventive services ( figure 1 ) . it was also lower in patients who had visits to a primary care physician and/or a medical specialist . rates were highest among those with no outpatient physician care during the look - back period ( 2.2% in prostate , 5.5% in colorectal , 4.8% lung , and 3.3% breast cancer ) . in multivariate analyses that included all three measures of prior health system contact in the same model , the adjusted odds of undiagnosed diabetes were statistically significantly lower for those with 1 or 2 preventive services ( compared to none ) in breast , colorectal , and prostate ( 2 only ) but not in colorectal and lung cancer ( table 4 ) . the adjusted odds of undiagnosed diabetes also were lower for those with primary care and/or medical specialist care prior to cancer . in general , nonwhite race / ethnicity was associated with increased adjusted odds of undiagnosed diabetes , as was living in a census tract with a lower percent college educated and a higher percent in poverty ( table 4 ) . overall , effect sizes for measures of prior health system contact were larger in models that included only one measure per model ( table 5 ) . in this study , we described the epidemiology of undiagnosed diabetes in a large cohort of elderly cancer patients in the united states . our findings show that undiagnosed diabetes accounted for almost 13% of all diabetes cases identified in an older cohort of patients diagnosed with cancer . this proportion is lower than the one obtained from the general population sampled in nhanes , in which participants underwent an oral glucose tolerance test and the results were compared to self - reported history of diabetes to classify patients as previously diagnosed or undiagnosed . in that study , compared to the group with normal glucose tolerance , those with undiagnosed diabetes were more likely to be nonwhite race / ethnicity and to have less than a high school education . in this regard , our findings were similar in this cohort of elderly cancer patients , all of whom had at least two years of health insurance prior to cancer diagnosis . furthermore , we found that the highest rates of undiagnosed diabetes were observed in those with limited health system contact prior to cancer and in those with advanced - stage cancer . previous research shows that limited health system contact is associated with advanced - stage cancer at diagnosis [ 1618 ] . however , the fact that limited health system contact is associated with both advanced stage and undiagnosed diabetes does not rule out other mechanisms , for example , biological , linking undiagnosed diabetes directly to cancer stage . in addition , in unadjusted models , and several of the adjusted models , both higher levels of poverty and lower levels of education were associated with a greater likelihood of diabetes being diagnosed in the period around cancer diagnosis . the fact that models with all risk factors show some attenuation of the socioeconomic factors suggests that interaction with the healthcare system and socioeconomic status are confounded . plausible mechanisms include exacerbated biological effects [ 35 ] and the added burden on the health care team of managing a previously undiagnosed condition , which could impact treatment selection and response [ 7 , 11 ] . nevertheless , our findings indicate that those already at the greatest risk of poor cancer outcomes due to advanced stage also are most likely to bear any additional adverse prognostic burden of undiagnosed diabetes . the approach to identifying undiagnosed diabetes entailed first dividing an observation period from 24 months before to 3 months after cancer diagnosis into a look - back period and an incidence period , then using a claims - based algorithm to identify diabetes in each period , and finally excluding those with preexisting diabetes from the incidence risk set . the claims - based algorithm we used has a sensitivity of 70% . also , in our study , the overall prevalence of diabetes during the observation period ( look - back and incidence periods combined ) was lower than that previously reported in the literature based , for instance , on hospital medical records review . consequently , it is likely that we have also underestimated the incidence of undiagnosed diabetes . also , to preserve a large sample and include patients as close to the minimum age for medicare eligibility ( 65 years ) as possible , we established the beginning of the look - back period at 24 months before cancer diagnosis , thereby making the minimum age at cancer diagnosis 67 years in this cohort . many of these patients had a claims history beginning more than 24 months before cancer . however , we elected not to use these in identifying preexisting diabetes because this may have indirectly biased the association between patient age at cancer diagnosis and the incidence of undiagnosed diabetes . extending the look - back period farther back in time could have resulted in detecting and excluding more cases of preexisting diabetes and possibly also reducing misclassification of preexisting diabetes as undiagnosed cases during the incidence period , but that would have resulted in smaller sample sizes and the exclusion of younger patients . furthermore , following precedent , we have described diabetes first detected during the incidence period as undiagnosed , which implies that it was present but undetected prior to that . however , simply by chance , it is likely that some of these cases became diabetic during the incidence period . therefore , in this study , we may have underestimated the magnitude and statistical significance of associations between prior health system contact and undiagnosed diabetes . this study was conducted prior to the implementation of the medicare modernization act ( mma ) , which introduced new coverage for diabetes and other screening services in 2005 . introduction of these services is designed to improve early detection of diabetes and other important conditions . therefore , rates of undiagnosed diabetes could have changed due to the implementation of the mma . in addition to affecting the incidence of undiagnosed diabetes , the mma would impact services included in the preventive services measure of prior health system contact . since some of the new services impact diabetes , it is possible that associations between level of preventive services use and undiagnosed diabetes would become stronger as a result but that the overall incidence of undiagnosed diabetes would have declined through improved coverage of preventive services . limitations notwithstanding , our findings indicate that undiagnosed diabetes is relatively common in selected subgroups of cancer patients , such as those with limited prior health system contact and advanced cancer stage , and that poverty and lower educational attainment may contribute directly , as well as through limited interaction with the health system . also , those already at the greatest risk of poor cancer outcomes due to advanced stage also are most likely to bear any additional adverse prognostic burden of undiagnosed diabetes . possible explanations for the association between undiagnosed diabetes and advanced stage include biological mechanisms and/or shared risk factors . as the incidence of diabetes continues to rise , understanding the relationship between undiagnosed diabetes and cancer outcomes may help inform treatment decisions in the management of these patients .

our study describes the incidence and risk factors for undiagnosed diabetes in elderly cancer patients . using surveillance , epidemiology , and end results - medicare data , we followed patients with breast , colorectal , lung , or prostate cancer from 24 months before to 3 months after cancer diagnosis . medicare claims were used to exclude patients with diabetes 24 to 4 months before cancer ( look - back period ) , identify those with diabetes undiagnosed until cancer , and construct indicators of preventive services , physician contact , and comorbidity during the look - back period . logistic regression analyses were performed to identify factors associated with undiagnosed diabetes . overall , 2,678 patients had diabetes undiagnosed until cancer . rates were the highest in patients with both advanced - stage cancer and low prior primary care / medical specialist contact ( breast 8.2% , colorectal 5.9% , lung 4.4% ) . nonwhite race / ethnicity , living in a census tract with a higher percent of the population in poverty and a lower percent college educated , lower prior preventive services use , and lack of primary care and/or medical specialist care prior to cancer all were associated with higher ( p 0.05 ) adjusted odds of undiagnosed diabetes . undiagnosed diabetes is relatively common in selected subgroups of cancer patients , including those already at high risk of poor outcomes due to advanced cancer stage .

1. Introduction 2. Methods 3. Results 4. Conclusions

ramadan is a holy month in the islamic calendar during which muslims all over the world observe a fast between dawn and sunset . during ramadan , muslims who are ill or have health conditions that may deteriorate upon fasting , including patients with diabetes , are exempt . however , fasting during ramadan is highly regarded as a form of religious practice , and thus many devoted muslim patients insist on fasting despite being advised not to by their healthcare providers . it has been estimated that 4050 million individuals with diabetes fast during the month of ramadan . these changes could potentially induce metabolic alterations in both healthy and diseased muslims [ 3 , 4 ] . despite taking fewer meals , this practice is usually compensated by ingesting large amounts of sugary food and drinks that are high in carbohydrates and fats , especially when breaking the fast . the overall calorie consumption of individuals with type 2 diabetes has been reported to increase during ramadan [ 6 , 7 ] . moreover , the doses of antidiabetic agents are often adjusted at this time to reflect the change in lifestyle during ramadan . one study showed that , among diabetic patients whose doses of oral hypoglycemic agents ( oha ) were modified during ramadan , 58% had changed the timings of the administration of their medications . during ramadan , it has also been reported that physical activities , especially exercising , tend to decrease from a fear of feeling too weak . patients with diabetes have been advised by healthcare providers to avoid excessive physical activity during ramadan , because the practice of fasting may increase the risk of hypoglycemia , especially a few hours before the sunset meal . however , one study reported that appropriate levels of physical activity during fasting did not interfere with tolerance to physical exercise . furthermore , it has also been postulated that the act of fasting may increase the risk of poor glycemic control , which raises questions about the safety of ramadan fasting in patients with diabetes . during ramadan fasting , the decrease in blood glucose levels triggers compensatory mechanisms in the body of healthy individuals which cause a reduction in insulin secretion or the breakdown of stored glycogen to prevent hypoglycemia . however , in patients with diabetes , this regulation is compromised due to either a dysfunction of insulin secretion or sensitivity or occasionally both . one epidemiological study reported that the risk of severe hypoglycemia increased sevenfold during the month of ramadan in patients with diabetes . however , another study reported that ramadan fasting was safe and did not significantly increase the incidence of hypoglycemic events . the primary objective of this prospective study was to evaluate the effect of fasting during ramadan on the glycemic control of muslim patients with type 2 diabetes by examining the changes in levels of hba1c before , during , and after ramadan . in addition , the incidence of hypoglycemia and changes in glycemic control in relation to the adjustment of doses of antidiabetic agents , diet , and physical activity during ramadan were examined . this was a prospective study conducted in an outpatient endocrine clinic located within a tertiary hospital in singapore between june and november 2012 ; ramadan took place from july 21 to august 18 , 2012 . in singapore , muslims account for 14.7% of the population , and , during ramadan , muslims fast for 30 days . the study , which was approved by the institutional review board , comprised of data collection from the institutional database and the administration of a set of questionnaires by trained interviewers . eligible patients were screened and approached to determine their interest in participating in this study a month before ramadan . upon signing the consent form , a set of questionnaires all muslim patients over 21 years of age with type 2 diabetes who fasted for at least 10 days during the month of ramadan were included in this study . the patients who reported having been hospitalized for diabetic ketoacidosis or severe hypoglycemia a month prior to ramadan or those who received short - term corticosteroid therapy were excluded from the study . in addition , the patients who were unable to comprehend the questionnaire were excluded from the study . the questionnaire used in this study the questionnaire comprised a sociodemographic section , which included age , gender , level of education , employment status , body mass index , health status , and duration of diabetes , and a section on the types of antidiabetic agents used and dosing regimens , the patients ' perception of their daily physical activities , and their dietary patterns . for the questions relating to diet and physical activity , changes in these parameters during ramadan were self - reported by the patients based on a response of more , less , or unchanged . minor amendments were made to questions related to sociodemographics and diet to suit the local context . the questionnaire was translated into the malay language commonly spoken by the muslim population in singapore . questionnaires were administered to patients in their preferred language by the trained , bilingual interviewers and took approximately 30 minutes to complete . outcome measures , such as hba1c , and responses to the questionnaire were collected before , during , and after ramadan . hba1c is a surrogate marker , commonly used in numerous studies related to ramadan fasting , which reflects glycemic control in muslim patients ; it gives a more reliable reflection of the average plasma glucose concentration over 812 weeks . in this study minor hypoglycemia was defined as an event that can be self - managed by patients irrespective of the severity of the symptoms , while severe hypoglycemia was defined as an event that requires the assistance of a third party to effect treatment . the study patients were also asked if they had experienced any symptoms of hypoglycemia during ramadan fasting . the data are presented as means standard deviations ( sd ) for continuous variables and as percentages for categorical variables . the outcome measure , hba1c , was analyzed between two time periods using a linear mixed model ; this was followed by pairwise comparisons with bonferroni adjustment . all of the analyses were adjusted for age , gender , bmi , level of education , total comorbidities , duration of diabetes , and employment and health status . after multiplication by the number of repeated analyses where appropriate , a two - tailed p < 0.05 was considered to be significant . the computations were performed using spss for windows , version 19.0 ( spss inc . , of the 251 patients recruited , 98 ( 39% ) were excluded from the analysis due to incomplete responses . at the completion of the study , 153 patients with an average of 26 fasting days had been followed up throughout the entire study to give a response rate of 61% . their mean age was 56.7 9.1 years and 62.7% of the participants were women ( n = 96 ) ( table 1 ) . a large majority ( n = 81 ; 52.9% ) of the study subjects had at least a 12th grade education however , more than half of the participants were unemployed ( n = 90 ; 58.8% ) . with regard to diabetes management , 150 ( 98% ) were taking antidiabetic agents of which 72 ( 47.1% ) were on a combination regimen of oha and insulin . metformin was the most commonly prescribed oha ( n = 119 ; 92.2% ) , followed by sulfonylurea ( n = 70 ; 54.3% ) and dipeptidyl peptidase 4 inhibitors ( n = 11 ; 8.5% ) . the majority were given basal insulin concurrently with mealtime insulin ( n = 61 ; 65.6% ) while the remainder were either given basal insulin ( n = 31 ; 33.3% ) or mealtime insulin alone ( n = 1 ; 1.1% ) ( table 2 ) . mean hba1c improved from 8.9 2.0% before ramadan to 8.6 1.8% during ramadan ( p < 0.05 ) . however , this improvement was not sustained after ramadan . an increase of 0.2% in mean hba1c was observed between ramadan and after ramadan ( p > 0.05 ) ( figure 1 ) . during ramadan , the most common method of dose adjustment made by the oha users was a reduction in total daily dose ( n = 55 ; 73.3% ) . the other methods of dose adjustment were cessation of oha ( n = 9 ; 12.0% ) , increased night - time dose alone ( n = 5 ; 6.7% ) , increased total daily dose ( n = 3 ; 4.0% ) , and additional oha ( n = 3 ; 4.0% ) . in this study , the three most commonly adjusted ohas were metformin , glipizide and its equivalents [ 19 , 20 ] , and sitagliptin with average dose reductions of 810.4 632.2 mg , 4.5 6.9 mg , and 16.7 57.7 mg , respectively , during ramadan . among the insulin users , the total daily insulin dose was reduced by 39 ( 60.9% ) patients and stopped by five ( 7.8% ) patients . of the remaining patients who adjusted their insulin therapy , two ( 3.1% ) took additional insulin , six ( 9.4% ) increased their total daily dose , and 12 ( 18.8% ) increased their night - time insulin dose alone . overall , the average dose adjustments of insulin during ramadan for mealtime and basal insulin were 11.8 13.6 units and 8.1 10.4 units , respectively . an improvement in mean hba1c from 9.2 1.9% before ramadan to 8.7 1.6% during ramadan was observed in patients who made dose adjustments during ramadan ( n = 104 ; p < 0.001 ) . in patients who did not make any dose adjustments during ramadan , no significant improvement in hba1c was observed ( n = 49 ; p > 0.05 ) ( figure 2 ) . during ramadan , 99 ( 64.7% ) participants reported a reduction in dietary intake , 45 ( 29.4% ) reported an unchanged dietary intake , and nine ( 5.9% ) reported an increased dietary intake . with regard to physical activity , the majority ( n = 96 ; 62.7% ) reported an unchanged level of activity while 49 ( 32% ) reported less activity and eight ( 5.2% ) increased their level of activity during ramadan . these perceived patterns of diet and physical activity were , however , found to have no significant association with the improvements in mean hba1c during ramadan fasting ( p > 0.05 ) . in this study , 39 ( 25.5% ) patients reported having experienced minor hypoglycemia during ramadan and of which 17 ( 43.6% ) were on combination regimen of oha and insulin , 14 ( 35.9% ) were on oha alone , and 8 ( 20.5% ) were on insulin alone . commonly reported hypoglycemic symptoms included dizziness , tremors , increased sweating , extreme hunger , increased heart rate or palpitation , and increased frequency of headaches , nausea , confusion , and mood changes . this was the first prospective study to observe the changes in glycemia before , during , and after ramadan fasting and also provided a comprehensive understanding on the effects of lifestyle modification and dose adjustment on glycemia during ramadan fasting . in this study , a significant reduction of 0.3% in mean hba1c was observed during ramadan fasting . although an improvement in glycemia was found , a 25.5% rate of minor hypoglycemia occurred in these patients . while any form of hypoglycemic event is undesirable in the management of diabetes , this rate is nevertheless lower than or comparable with other studies conducted in nonfasting patients with type 2 diabetes . a cross - sectional study by miller et al . reported hypoglycemia rates of 12% for patients on diet alone , 16% for patients on oha , and 30% for patients on insulin . some studies have shown that people who received adjustments to antidiabetic drug doses during ramadan achieved better glucose control [ 22 , 23 ] . a study reported by mafauzy showed that those with adjustments to their dose of oha experienced a larger reduction in their level of fructosamine ( 21.9 4.8 mol / l ) than those with no adjustment to their oha dose ( 6.7 5.6 mol / l ) during ramadan ( p = 0.01 ) . in another study by hui et al . , patients who were switched to faster - acting insulin had an improvement of 0.48% in hba1c ( p = 0.0001 ) while those who were maintained on the same insulin regimen had an improvement of only 0.28% in hba1c ( p = 0.007 ) during ramadan . similarly , this study also showed a larger reduction in hba1c among those with adjustments to their dose of antidiabetic agents . indeed , the attempt to mimic the physiological secretion of insulin by adjusting the dose during ramadan may have contributed to the improvement in glycemia . in our study , diet and physical activity were not found to be related to a reduction in hba1c during ramadan . this lack of an association could be explained by examining the pattern of dietary intake and the level of physical activity in our study population . during ramadan , the majority of patients reported that their dietary intakes were reduced but this surprisingly did not contribute to a reduction in glycemia . one possible reason could be that these patients had perceived a reduction in their dietary intakes due to the reduction in meal frequency but that their daily caloric intakes may have remained the same throughout ramadan . a study conducted by haouari - oukerro et al . showed that overall caloric consumption remained unchanged even though the number of meals was reduced to twice daily during the fasting month . in addition , most of our patients did not report any change in their physical activities during ramadan ; hence , the effect of physical activity on glycemia may be very small in this population . furthermore , our findings were also observed in several studies in which glycemic control was not associated with physical activity [ 25 , 26 ] . first , it relied on patient self - reporting for certain information such as dietary pattern , physical activity , the incidence of hypoglycemia , and the management of diabetes . recall bias may have affected the quality of our data but , whenever possible , we also compared the patients ' responses with objective data found in patient case notes and the clinical database . second , we had to exclude 39% of the patients recruited due to our stringent criteria of accepting only questionnaires with a 100% response rate . however , based on the drop - out rate of 3244.2% of similar studies [ 2729 ] , our response rate appears to be reasonable . future studies should compare various types of oha in relation to their risks for hypoglycemic events during ramadan fasting . this will allow patients and clinicians to closely monitor antidiabetic agents that may be more likely to contribute to hypoglycemic symptoms during ramadan . in addition , the quality of pre - ramadan structured education on diet , physical activity , and dose adjustment of medication should also be evaluated to ensure effective and safe diabetes management and positive outcomes . ramadan fasting was observed to improve glycemic control among our patient population with minimal hypoglycemic events . the improvement in hba1c was more notable in patients whose medications were adjusted in an attempt to mimic the physiological secretion of insulin throughout the fasting period . during ramadan , clinicians should individualize the dosing of antidiabetic agents especially in patients who intend to fast .

objectives . this study aimed to examine the effect of ramadan fasting on hba1c in muslim patients with type 2 diabetes . the incidence of hypoglycemia and glycemic changes in relation to the adjustment of doses of antidiabetic agents , diet , and physical activity during ramadan was also evaluated . methods . this was a prospective study conducted in an outpatient endocrine clinic . a set of questionnaires was administered to muslim patients with diabetes who fasted for 10 days . those who were hospitalized for diabetic ketoacidosis or severe hypoglycemia a month prior to ramadan or were given short - term corticosteroid therapy were excluded . the patients ' responses and clinical outcomes from the clinic database were collected before , during , and after ramadan . results . a total of 153 participants completed the study . the mean hba1c improved from 8.9% before ramadan to 8.6% during ramadan ( p < 0.05 ) . although diet and physical activity did not contribute to changes in glycemia , a significant improvement in hba1c was observed in patients who had adjustments made to their doses of antidiabetic agents during ramadan ( p < 0.001 ) . in addition , their rate of hypoglycemia was minimal . conclusions . ramadan fasting appeared to improve glycemic control , especially in those whose doses of antidiabetic agents were adjusted during ramadan .

1. Introduction 2. Methods 3. Results 4. Discussions 5. Conclusions

fifteen type 1 diabetic patients with proliferative retinopathy , an indicator of microangiopathy resulting from chronic hyperglycemia ( type 1 diabetes ) , 29 type 1 diabetic patients without manifest microvascular complications ( type 1 diabetes ) , and 26 healthy control subjects matched for sex and education level enrolled in this study . participants were recruited from the departments of endocrinology and ophthalmology of the vu university medical center , amsterdam , the netherlands ( 50 patients ) , the department of internal medicine , groene hart hospital , gouda , the netherlands ( 10 patients ) , and by advertisements in diabetes magazines and a national newspaper ( 10 patients ) . inclusion criteria were age range 1855 years , right - handedness , for type 1 diabetic patients a disease duration of at least 10 years , proliferative retinopathy as described below , or no signs of microvascular complications . participants were excluded if they had a bmi above 35 kg / m , current use of drugs affecting cerebral functioning , alcohol abuse ( more than 20 g of alcohol per day ) , psychiatric disorders , anemia , thyroid dysfunction , use of glucocorticoids , hepatitis , stroke , severe head trauma , epilepsy , pregnancy , or poor visual acuity below 0.3 . fundus photography ( topcon nw 100 , capelle aan den ijssel , the netherlands ) was performed to screen for retinopathy . for each eye , one photograph with the macula in the center and one with the optic disc in the center were taken ( e.v.d . ) . photographs were rated by an ophthalmologist ( a.m. ) according to the european diabetes ( eurodiab ) classification ( 20 ) . only those participants with either an eurodiab classification score of 0 ( no retinopathy ) or of 4 or 5 ( proliferative retinopathy or lasercoagulation ) twenty - four hour urine collections were performed to assess albumin - to - creatinine ratio ( acr ) . type 1 diabetes patients had normoalbuminuria ( acr < 2.5 mg / mmol in men and < 3.5 mg / mmol in women ) and no neuropathy . hypertension was defined as a systolic blood pressure of 140 mmhg or above , a diastolic blood pressure of 90 mmhg or above , or the use of antihypertensive drugs . the study protocol was approved by the medical ethics committee of the vu university medical center , and written informed consent was obtained from all participants . before eligible participants received written information on the study , their medical records were screened using the aforementioned criteria . those willing to participate were additionally interviewed over the telephone to collect background information including educational level , using a dutch scoring system ranging from 1 to 8 . one indicates unfinished primary school and 8 indicates a completed university study at masters level . as depression may affect cognitive performance , depressive symptoms were assessed using the center for epidemiological studies scale for depression ( ces - d ) . eligible participants were invited for the first visit . during this visit , which took place in the afternoon , fundus photography was performed , blood samples were collected , and meg registration was performed . approximately 6 weeks after the first visit participants returned for neuropsychological assessment during a morning visit . all visits took place at the vu university medical center . to rule out confounding as a result of extreme blood glucose levels , levels were checked in the type 1 diabetes participants before the start of the neuropsychological assessment and meg registration and had to be between 4 and 15 , patients were instructed to inject 2 units of the participants ' current rapid - acting insulin analog when blood glucose levels were between 15 and 20 mmol / l ( 270362 mg / dl ) and 4 units when glucose levels exceeded 20 mmol / l ( 362 mg / dl ) . if hyperglycemia ( i.e. , blood glucose 15 mmol / l ) would persist , participants were instructed to inject another 2 units of insulin ; if hypoglycemia would persist , participants additionally had to eat 20 g of carbohydrates . two type 1 diabetes participants were hyperglycemic before start of the neuropsychological assessment and one before meg acquisition . proper glycemic status was restored for all patients after the first step of the above - mentioned protocol . data of the one participant who experienced hypoglycemia symptoms during neuropsychological testing were included in all analyses , as this did not change analysis outcomes . based on earlier type 1 diabetes cognitive research ( 5,21 ) , as well as clinical neuropsychological practice ( 22 ) , a battery of cognitive tests was chosen to measure potential differences in five major cognitive domains : memory , information processing speed , executive functions , attention , and motor speed . domains were based on an earlier principal component analysis using varimax rotation with kaiser normalization in a large group of healthy subjects and adjusted according to earlier research ( 5,21,23 ) . the domain memory was assessed by the dutch version of the rey auditory verbal learning test ( ralvt ) ( 24 ) , wechsler adult intelligence scale3rd edition revised ( wais - iii - r ) digit span forward and backward ( 25 ) , and the wais - iii - r symbol substitution incidental learning test ( 25 ) . information processing speed was created using the wais - iii - r symbol substitution test ( 25 ) , the stroop color - word test parts 1 and 2 ( 26 ) , the concept shifting task ( cst ) parts a and b ( 27 ) , the simple auditory and visual reaction time tests ( 28 ) , and the computerized visual searching task ( cvst ) ( 28 ) . assessment of the domain executive functions was conducted using the stroop color - word test part 3 ( 26 ) , the cst part c ( 27 ) , the d2-test total errors ( 29 ) , the wisconsin cart sorting test ( 30 ) , and the category word fluency task ( 31 ) . the domain attention was assessed using the d2-test range , with total correct answers and total span ( 29 ) . motor speed consisted of the tapping test ( 28 ) and the cst part 0 , administered three times ( 27 ) . general cognitive ability was constructed by averaging the above mentioned five domains . to enhance comparability and allow construction of these domains , z - scores for every test were created based on the means sd of the healthy control group . meg data were obtained using a 151-channel whole - head meg system ( ctf systems ; port coquitlam , bc , canada ) , while participants were in a supine position in a magnetically shielded room ( vacuumschmelze , hanau , germany ) . a third - order software gradient ( 32 ) was used with a recording passband of 0.25125 hz and a sample frequency of 625 hz . magnetic fields were recorded for 2 min in an eyes - open , 5 min in an eyes - closed , 10 min in a task , and then 3 min in another eyes - closed condition . total acquisition time was 20 min . at the beginning , middle , and end of each recording , the head position relative to the coordinate system of the helmet was determined by leading small alternating currents through three head position coils attached to the left and right preauricular points and the nasion on the subjects head . changes in head position of 1.5 cm during a recording condition were accepted . if head position changed more than 1.5 cm , recordings had to be repeated . from the acquired meg data , information about functional connectivity was calculated by means of a mathematical construct , the synchronization likelihood . for a technical description of synchronization likelihood see stam and van dijk ( 33 ) and montez et al . , interactions between two neural networks , for instance the frontal ( x ) and temporal ( y ) networks , are of interest . meg data are recorded for all sensors surrounding the head , including those in the frontal and temporal areas . these signals represent the time series xi and yi from the frontal ( x ) and temporal ( y ) areas ( fig . it is assumed that x and y more strongly interact when xi and yi more it has been shown , however , that x and y can also interact when xi and yi do not resemble each other ( square with the dashed line in fig . parameter settings used for computation of synchronization likelihood are based on the frequency content of the data ( for parameter settings see montez et al . ) . synchronization likelihood can range from pref ( low synchronization ) to one ( complete synchronization ) . pref is a value close to zero ( zero indicates no synchronization , which is not possible ) and was set on 0.01 for all frequency bands . the square with the continuous line indicates a time series showing high resemblance between both networks . the square with the dashed line is an example of a time series showing low resemblance . a : network x ( frontal ) . ten channels were deleted as their signals were distorted in some participants . this had no influence on the calculations . offline , recordings of the first eyes - closed condition were transformed into ascii - files and imported into the digeegxp software ( c.j . stam , vu university medical center , amsterdam , the netherlands ) . for each participant five artifact - free epochs ( 6.25 s ) of 4,096 samples were selected by two authors ( e.v.d . and total data used were 31.25 s. with digeegxp synchronization likelihood was calculated ( 33 ) . synchronization likelihood was calculated between signals recorded at all possible sensor pairs for ( 0.54 hz ) , ( 48 hz ) , lower ( 810 hz ) , upper ( 1013 hz ) , ( 1330 hz ) , lower ( 3045 hz ) , and upper ( 5580 hz ) frequency bands . subsequently , meg sensors were clustered according to their anatomical location , respectively , right and left frontal , central , parietal , temporal , and occipital . average synchronization likelihood values were then obtained for short - distance , intrahemispheric long - distance , and interhemispheric long - distance groups . short - distance group consisted of 10 areas , left and right hemisphere central , frontal , parietal , occipital , and temporal regions . for each of these 10 areas , the synchronization likelihood between all possible pairs of sensors belonging to that area was averaged . average synchronization likelihood for long distances was obtained by averaging all pair wise synchronization likelihood values of sensors belonging to two different areas . intrahemispheric long - distance group consisted of eight pairs , left and right hemisphere frontoparietal , frontotemporal , parietoocciptal , and temporooccipital . interhemispheric long - distance group consisted of five pairs , left to right hemisphere central , parietal , temporal , occipital , and frontal ( 12,13 ) . now , synchronization likelihood resembles the likelihood that the data obtained from all sensors in the above - defined short - distance and long - distance groups are synchronized . schematic brain , with left and right frontal ( lf and rf , respectively ) , central ( lc and rc , respectively ) , parietal ( rp and lp , respectively ) , occipital ( lo and ro , respectively ) , and temporal ( lt and rt , respectively ) areas indicated as abbreviations . a and b : long - distance intrahemispheric pathways . eight long - distance intrahemispheric pathways ( a ) and five long - distance interhemispheric pathways ( b ) . differences between groups for demographical and medical variables were calculated using student 's t test for independent samples or one - way anova with tukey post hoc test ( for continuous variables ) and test ( for categorical variables ) . to determine group differences in neuropsychological performance mancova was used with all cognitive domains as dependent variables and group as independent variable . in case of a group difference on a domain , post synchronization likelihood data were normalized by means of a transformation ln10(x/[1-x ] ) ( 35 ) to allow the use of parametric statistical tests . to minimize statistical tests , for each meg frequency band ancova with repeated measures was used to determine group differences . for each frequency band , three ancovas with repeated measures repeated measures for intrahemispheric connections consisted of the 8 above - mentioned levels , interhemispheric connections of 5 levels , and local hemispheric connections of 10 levels . in case of significant interaction ( p values of greenhouse - geisser correction for degrees of freedom ) or main effects of group with frequency band , post hoc mancova analysis was used to determine effects of group and spatial location . to determine associations between cognitive functioning and synchronization likelihood , a procedure proposed by stoffers et al . and used in other meg research was implemented ( 11,12 ) . to reduce multiple comparisons , cognitive domains were added as covariates to the above - mentioned ancova with repeated measures method . effects of group on changing cognitive domains and meg regions were determined using mancova tests . positive or negative associations were further calculated using regression analysis . to correct for possible confounding influences , all statistical tests were corrected for age , sex , hypertension , neuropathy , bmi , education level , and depressive symptoms . partial is reported as a proportion of the total variance explained of the determinant by the independent factor corrected for the used covariates . all statistical analyses were performed using spss for windows version 15.0 ( spss , chicago , il ) . before eligible participants received written information on the study , their medical records were screened using the aforementioned criteria . those willing to participate were additionally interviewed over the telephone to collect background information including educational level , using a dutch scoring system ranging from 1 to 8 . one indicates unfinished primary school and 8 indicates a completed university study at masters level . as depression may affect cognitive performance , depressive symptoms were assessed using the center for epidemiological studies scale for depression ( ces - d ) . eligible participants were invited for the first visit . during this visit , which took place in the afternoon , fundus photography was performed , blood samples were collected , and meg registration was performed . approximately 6 weeks after the first visit participants returned for neuropsychological assessment during a morning visit . all visits took place at the vu university medical center . to rule out confounding as a result of extreme blood glucose levels , levels were checked in the type 1 diabetes participants before the start of the neuropsychological assessment and meg registration and had to be between 4 and 15 , patients were instructed to inject 2 units of the participants ' current rapid - acting insulin analog when blood glucose levels were between 15 and 20 mmol / l ( 270362 mg / dl ) and 4 units when glucose levels exceeded 20 mmol / l ( 362 mg / dl ) . glycemic status was evaluated after 30 min . if hyperglycemia ( i.e. , blood glucose 15 mmol / l ) would persist , participants were instructed to inject another 2 units of insulin ; if hypoglycemia would persist , participants additionally had to eat 20 g of carbohydrates . two type 1 diabetes participants were hyperglycemic before start of the neuropsychological assessment and one before meg acquisition . proper glycemic status was restored for all patients after the first step of the above - mentioned protocol . data of the one participant who experienced hypoglycemia symptoms during neuropsychological testing were included in all analyses , as this did not change analysis outcomes . based on earlier type 1 diabetes cognitive research ( 5,21 ) , as well as clinical neuropsychological practice ( 22 ) , a battery of cognitive tests was chosen to measure potential differences in five major cognitive domains : memory , information processing speed , executive functions , attention , and motor speed . domains were based on an earlier principal component analysis using varimax rotation with kaiser normalization in a large group of healthy subjects and adjusted according to earlier research ( 5,21,23 ) . the domain memory was assessed by the dutch version of the rey auditory verbal learning test ( ralvt ) ( 24 ) , wechsler adult intelligence scale3rd edition revised ( wais - iii - r ) digit span forward and backward ( 25 ) , and the wais - iii - r symbol substitution incidental learning test ( 25 ) . information processing speed was created using the wais - iii - r symbol substitution test ( 25 ) , the stroop color - word test parts 1 and 2 ( 26 ) , the concept shifting task ( cst ) parts a and b ( 27 ) , the simple auditory and visual reaction time tests ( 28 ) , and the computerized visual searching task ( cvst ) ( 28 ) . assessment of the domain executive functions was conducted using the stroop color - word test part 3 ( 26 ) , the cst part c ( 27 ) , the d2-test total errors ( 29 ) , the wisconsin cart sorting test ( 30 ) , and the category word fluency task ( 31 ) . the domain attention was assessed using the d2-test range , with total correct answers and total span ( 29 ) . motor speed consisted of the tapping test ( 28 ) and the cst part 0 , administered three times ( 27 ) . general cognitive ability was constructed by averaging the above mentioned five domains . to enhance comparability and allow construction of these domains , z - scores for every test were created based on the means sd of the healthy control group . meg data were obtained using a 151-channel whole - head meg system ( ctf systems ; port coquitlam , bc , canada ) , while participants were in a supine position in a magnetically shielded room ( vacuumschmelze , hanau , germany ) . a third - order software gradient ( 32 ) was used with a recording passband of 0.25125 hz and a sample frequency of 625 hz . magnetic fields were recorded for 2 min in an eyes - open , 5 min in an eyes - closed , 10 min in a task , and then 3 min in another eyes - closed condition . middle , and end of each recording , the head position relative to the coordinate system of the helmet was determined by leading small alternating currents through three head position coils attached to the left and right preauricular points and the nasion on the subjects head . changes in head position of 1.5 cm during a recording condition were accepted . if head position changed more than 1.5 cm , recordings had to be repeated . from the acquired meg data , information about functional connectivity was calculated by means of a mathematical construct , the synchronization likelihood . for a technical description of synchronization likelihood see stam and van dijk ( 33 ) and montez et al . ( 34 ) . in short , interactions between two neural networks , for instance the frontal ( x ) and temporal ( y ) networks , are of interest . meg data are recorded for all sensors surrounding the head , including those in the frontal and temporal areas . these signals represent the time series xi and yi from the frontal ( x ) and temporal ( y ) areas ( fig . it is assumed that x and y more strongly interact when xi and yi more it has been shown , however , that x and y can also interact when xi and yi do not resemble each other ( square with the dashed line in fig . parameter settings used for computation of synchronization likelihood are based on the frequency content of the data ( for parameter settings see montez et al . ) . synchronization likelihood can range from pref ( low synchronization ) to one ( complete synchronization ) . pref is a value close to zero ( zero indicates no synchronization , which is not possible ) and was set on 0.01 for all frequency bands . the square with the continuous line indicates a time series showing high resemblance between both networks . the square with the dashed line is an example of a time series showing low resemblance . ten channels were deleted as their signals were distorted in some participants . this had no influence on the calculations . offline , recordings of the first eyes - closed condition were transformed into ascii - files and imported into the digeegxp software ( c.j . stam , vu university medical center , amsterdam , the netherlands ) . for each participant five artifact - free epochs ( 6.25 s ) of 4,096 samples were selected by two authors ( e.v.d . and total data used were 31.25 s. with digeegxp synchronization likelihood was calculated ( 33 ) . synchronization likelihood was calculated between signals recorded at all possible sensor pairs for ( 0.54 hz ) , ( 48 hz ) , lower ( 810 hz ) , upper ( 1013 hz ) , ( 1330 hz ) , lower ( 3045 hz ) , and upper ( 5580 hz ) frequency bands . subsequently , meg sensors were clustered according to their anatomical location , respectively , right and left frontal , central , parietal , temporal , and occipital . average synchronization likelihood values were then obtained for short - distance , intrahemispheric long - distance , and interhemispheric long - distance groups . short - distance group consisted of 10 areas , left and right hemisphere central , frontal , parietal , occipital , and temporal regions . for each of these 10 areas , the synchronization likelihood between all possible pairs of sensors belonging to that area was averaged . average synchronization likelihood for long distances was obtained by averaging all pair wise synchronization likelihood values of sensors belonging to two different areas . intrahemispheric long - distance group consisted of eight pairs , left and right hemisphere frontoparietal , frontotemporal , parietoocciptal , and temporooccipital . interhemispheric long - distance group consisted of five pairs , left to right hemisphere central , parietal , temporal , occipital , and frontal ( 12,13 ) . now , synchronization likelihood resembles the likelihood that the data obtained from all sensors in the above - defined short - distance and long - distance groups are synchronized . schematic brain , with left and right frontal ( lf and rf , respectively ) , central ( lc and rc , respectively ) , parietal ( rp and lp , respectively ) , occipital ( lo and ro , respectively ) , and temporal ( lt and rt , respectively ) areas indicated as abbreviations . a and b : long - distance intrahemispheric pathways . eight long - distance intrahemispheric pathways ( a ) and five long - distance interhemispheric pathways ( b ) . differences between groups for demographical and medical variables were calculated using student 's t test for independent samples or one - way anova with tukey post hoc test ( for continuous variables ) and test ( for categorical variables ) . to determine group differences in neuropsychological performance mancova was used with all cognitive domains as dependent variables and group as independent variable . in case of a group difference on a domain , post synchronization likelihood data were normalized by means of a transformation ln10(x/[1-x ] ) ( 35 ) to allow the use of parametric statistical tests . to minimize statistical tests , for each meg frequency band ancova with repeated measures was used to determine group differences . for each frequency band , three ancovas with repeated measures were performed for intra- , inter- , and local hemispheric connections . repeated measures for intrahemispheric connections consisted of the 8 above - mentioned levels , interhemispheric connections of 5 levels , and local hemispheric connections of 10 levels . in case of significant interaction ( p values of greenhouse - geisser correction for degrees of freedom ) or main effects of group with frequency band , post hoc mancova analysis was used to determine effects of group and spatial location . to determine associations between cognitive functioning and synchronization likelihood , a procedure proposed by stoffers et al . and used in other meg research was implemented ( 11,12 ) . to reduce multiple comparisons , cognitive domains were added as covariates to the above - mentioned ancova with repeated measures method . effects of group on changing cognitive domains and meg regions were determined using mancova tests . positive or negative associations were further calculated using regression analysis . to correct for possible confounding influences , all statistical tests were corrected for age , sex , hypertension , neuropathy , bmi , education level , and depressive symptoms . partial is reported as a proportion of the total variance explained of the determinant by the independent factor corrected for the used covariates . all statistical analyses were performed using spss for windows version 15.0 ( spss , chicago , il ) . the control group was significantly younger than the type 1 diabetes patients and had a significantly lower mean score of depressive symptoms . type 1 diabetes patients had a significantly lower age of disease onset and longer disease duration . * education level measured with a dutch scoring system from 1 to 8 , with 1 representing unfinished primary school and 8 representing university at masters level . risk of major depression was defined as a ces - d score of 16 or above . hypertension was defined as a systolic blood pressure of 140 mmhg or above , a diastolic blood pressure of 90 mmhg or above , or use of antihypertensive drugs . early disease onset is defined as onset below 7 years of age . * * averaged amount of self - reported severe hypoglycemic events per individual . severe hypoglycemic events are those events for which the participants need others ' assistance to cope with the effects of low blood glucose , loss of consciousness , or seizure . nephropathy was based on a 24-h urine sample and defined as an acr of 2.5 or above for men and 3.5 for women . compared with control subjects , both patient groups had a significantly lower information processing speed ( type 1 diabetes : p = 0.003 , = 0.242 ; type 1 diabetes : p = 0.009 , = 0.135 ) and motor speed ( type 1 diabetes : p < 0.001 , = 0.311 ; type 1 diabetes : p = 0.014 , = 0.122 ) as well as general cognitive ability ( type 1 diabetes : p = 0.007 , = 0.198 ; type 1 diabetes : p = 0.004 , = 0.165 ) . z values were created based on the means sd of the healthy control subjects . asterisk indicates significant decline for type 1 diabetes and type 1 diabetes patients compared with healthy control subjects . meg results are shown in table 2 ( with p values and ) and visually represented in fig . significant interaction effects with group were found for the band local ( p = 0.001 ) , lower band local ( p = 0.004 ) , upper band local ( p = 0.008 ) , band interhemispheric ( p = 0.043 ) , and band local ( p = 0.020 ) . significant main effects of group were found for the lower band intrahemispheric ( p = 0.028 ) , lower band local hemispheric ( p = 0.005 ) , and upper band local ( p = 0.042 ) . post hoc mancova analysis revealed significantly higher synchronization likelihood in the lower right parietooccipital pathway for the type 1 diabetes compared with the healthy control subjects . comparing type 1 diabetes patients with control subjects revealed significantly lower synchronization likelihood in the and lower left and right central and parietal areas , in the upper left frontal and right frontal , central , and parietal areas , and in the interparietal , left parietal and right central , and parietal areas . comparisons of type 1 diabetes with type 1 diabetes patients yielded lower synchronization likelihood scores for type 1 diabetes patients in the left and right central and parietal areas , in the lower band left frontotemporal , left parietooccipital , right parietooccipital , and right temporooccipital pathways , in the left frontal and left and right central and parietal areas , upper left parietal and right central and parietal areas and in the -right parietal area . raw synchronization likelihood values for significant group differences data are given as mean synchronization likelihood values per group sd . p value and ( percentage of total variance explained by the determinant ) are given . black and gray indicate lower synchronization likelihood ; black dashed arrows indicate higher synchronization likelihood . gray circles indicate lower short - distance local hemispheric differences with p < 0.05 ; black circles indicate lower short - distance local hemispheric differences with p < 0.01 ; gray arrows indicate either lower long - distance intra- or interhemispheric differences with p < 0.05 ; black arrows indicate either lower long - distance intra- or interhemispheric differences with p < 0.01 ; black dashed arrow indicates higher long - distance intrahemispheric differences with p < 0.05 . to summarize , type 1 diabetes patients showed lower synchronization likelihood in both higher and lower frequency bands compared with both type 1 diabetes patients and control subjects . type 1 diabetes patients showed increased synchronization likelihood in the lower band compared with control subjects . no statistically significant differences were found when the combined patient groups were compared with healthy control subjects . on the contrary , a significant difference in the intrahemispheric region was observed ( supplementary table 1a and b , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0425/dc1 ) . to test for associations between functional connectivity in different meg frequency bands and cognition , meg statistics were repeated with cognitive domains added as additional covariates for both type 1 diabetes groups . in type 1 diabetes patients significant associations were found between the domain of executive functions and intrahemispheric ( p = 0.041 ) , interhemispheric ( p = 0.008 ) , and local ( p = 0.043 ) frequency band . furthermore , interhemispheric was related to the domains of memory ( p = 0.030 ) , information processing speed ( p = 0.040 ) , and motor speed ( p = 0.014 ) . the lower local frequency band was related to attention ( p = 0.047 ) . for executive functions only , significant relations with anatomical areas were found . in the type 1 diabetes patients significant relations were found between attention and intrahemispheric ( p = 0.048 ) , upper intrahemispheric ( p = 0.013 ) , and interhemispheric ( p = 0.043 ) . motor speed was associated with local ( p = 0.028 ) and local ( p = 0.036 ) . lastly , intrahemispheric was associated with information processing speed ( p = 0.040 ) . subsequently , regression analysis was used to determine positive or negative relations between synchronization likelihood and cognition . in both groups , associations between cognition and meg frequency bands for type 1 diabetes and type 1 diabetes patients the control group was significantly younger than the type 1 diabetes patients and had a significantly lower mean score of depressive symptoms . type 1 diabetes patients had a significantly lower age of disease onset and longer disease duration . * education level measured with a dutch scoring system from 1 to 8 , with 1 representing unfinished primary school and 8 representing university at masters level . risk of major depression was defined as a ces - d score of 16 or above . hypertension was defined as a systolic blood pressure of 140 mmhg or above , a diastolic blood pressure of 90 mmhg or above , or use of antihypertensive drugs . early disease onset is defined as onset below 7 years of age . * * averaged amount of self - reported severe hypoglycemic events per individual . severe hypoglycemic events are those events for which the participants need others ' assistance to cope with the effects of low blood glucose , loss of consciousness , or seizure . nephropathy was based on a 24-h urine sample and defined as an acr of 2.5 or above for men and 3.5 for women . compared with control subjects , both patient groups had a significantly lower information processing speed ( type 1 diabetes : p = 0.003 , = 0.242 ; type 1 diabetes : p = 0.009 , = 0.135 ) and motor speed ( type 1 diabetes : p < 0.001 , = 0.311 ; type 1 diabetes : p = 0.014 , = 0.122 ) as well as general cognitive ability ( type 1 diabetes : p = 0.007 , = 0.198 ; type 1 diabetes : p = 0.004 , = 0.165 ) . z values were created based on the means sd of the healthy control subjects . asterisk indicates significant decline for type 1 diabetes and type 1 diabetes patients compared with healthy control subjects . meg results are shown in table 2 ( with p values and ) and visually represented in fig . significant interaction effects with group were found for the band local ( p = 0.001 ) , lower band local ( p = 0.004 ) , upper band local ( p = 0.008 ) , band interhemispheric ( p = 0.043 ) , and band local ( p = 0.020 ) . significant main effects of group were found for the lower band intrahemispheric ( p = 0.028 ) , lower band local hemispheric ( p = 0.005 ) , and upper band local ( p = 0.042 ) . post hoc mancova analysis revealed significantly higher synchronization likelihood in the lower right parietooccipital pathway for the type 1 diabetes compared with the healthy control subjects . comparing type 1 diabetes patients with control subjects revealed significantly lower synchronization likelihood in the and lower left and right central and parietal areas , in the upper left frontal and right frontal , central , and parietal areas , and in the interparietal , left parietal and right central , and parietal areas . comparisons of type 1 diabetes with type 1 diabetes patients yielded lower synchronization likelihood scores for type 1 diabetes patients in the left and right central and parietal areas , in the lower band left frontotemporal , left parietooccipital , right parietooccipital , and right temporooccipital pathways , in the left frontal and left and right central and parietal areas , upper left parietal and right central and parietal areas and in the -right parietal area . raw synchronization likelihood values for significant group differences data are given as mean synchronization likelihood values per group sd . p value and ( percentage of total variance explained by the determinant ) are given . black and gray indicate lower synchronization likelihood ; black dashed arrows indicate higher synchronization likelihood . gray circles indicate lower short - distance local hemispheric differences with p < 0.05 ; black circles indicate lower short - distance local hemispheric differences with p < 0.01 ; gray arrows indicate either lower long - distance intra- or interhemispheric differences with p < 0.05 ; black arrows indicate either lower long - distance intra- or interhemispheric differences with p < 0.01 ; black dashed arrow indicates higher long - distance intrahemispheric differences with p < 0.05 . to summarize , type 1 diabetes patients showed lower synchronization likelihood in both higher and lower frequency bands compared with both type 1 diabetes patients and control subjects . type 1 diabetes patients showed increased synchronization likelihood in the lower band compared with control subjects . no statistically significant differences were found when the combined patient groups were compared with healthy control subjects . on the contrary , a significant difference in the intrahemispheric region was observed ( supplementary table 1a and b , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0425/dc1 ) . to test for associations between functional connectivity in different meg frequency bands and cognition , meg statistics were repeated with cognitive domains added as additional covariates for both type 1 diabetes groups . in type 1 diabetes patients significant associations were found between the domain of executive functions and intrahemispheric ( p = 0.041 ) , interhemispheric ( p = 0.008 ) , and local ( p = 0.043 ) frequency band . furthermore , interhemispheric was related to the domains of memory ( p = 0.030 ) , information processing speed ( p = 0.040 ) , and motor speed ( p = 0.014 ) . the lower local frequency band was related to attention ( p = 0.047 ) . for executive functions diabetes patients significant relations were found between attention and intrahemispheric ( p = 0.048 ) , upper intrahemispheric ( p = 0.013 ) , and interhemispheric ( p = 0.043 ) . motor speed was associated with local ( p = 0.028 ) and local ( p = 0.036 ) . lastly , intrahemispheric was associated with information processing speed ( p = 0.040 ) . subsequently , regression analysis was used to determine positive or negative relations between synchronization likelihood and cognition . in both groups , associations between cognition and meg frequency bands for type 1 diabetes and type 1 diabetes patients this is , to the best of our knowledge , the first study using meg to assess functional brain connectivity in type 1 diabetes . we showed that , compared with sex- and education - matched type 1 diabetes patients and control subjects , type 1 diabetes had decreased functional connectivity . this decrease was most profound in the left and right central and parietal areas in the , lower and upper , and frequency bands . moreover , some intra- and interhemispheric differences were found in the lower and bands . in contrast , an increase in functional connectivity was found in the lower band when comparing the type 1 diabetes patients with control subjects . several frequency bands were positively related to cognitive domains , indicating these domains to be dependent of functional connectivity . on cognitive assessment , type 1 diabetic patients with and those without proliferative retinopathy performed significantly poorer on information processing speed , motor speed , and general cognitive ability . results are in line with the hypothesis that type 1 diabetes patients show cognitive and cerebral changes . when both patient groups were pooled and compared with control subjects , cognitive results did not change ; however , all differences in meg measurements , but those in the intrahemispheric band , lost statistical significance ( supplementary table 1a and b , available in an online appendix ) . this indicates that type 1 diabetes patients largely resemble the control subjects regarding functional connectivity . given the novelty of the technique used , caution is required when it comes to the interpretation of our connectivity findings . as there is currently only one study available that uses our method in metabolic disease in female obese adolescents , significant increases in functional connectivity in and frequency bands were found ( 13 ) , possibly because of an increase in white matter , which has been reported in mri studies of obese adolescents . in contrast , in multiple sclerosis a decrease in functional connectivity was reported ( 37 ) . this might be related to a loss of white matter , one of the core features of the disease . following this line of reasoning , our results for the type 1 diabetes patients could be a consequence of white matter loss , as has been found earlier in type 1 diabetes patients than type 1 diabetes patients and healthy control subjects ( 5 ) . conversely , the increase in functional connectivity in type 1 diabetes patients might reflect a compensatory mechanism , which fails in type 1 diabetes patients . because of large interindividual differences in cognitive performance , this compensation was not observed in cognitive functioning . therefore , larger samples are needed to enable the detection of significant differences between type 1 diabetes and type 1 diabetes patients . importantly , we found a significant positive relationship between cognitive functioning and functional connectivity , suggesting changes in functional connectivity are involved in cognitive dysfunction in type 1 diabetic patients . our data support the hypothesis that cerebral changes are related to the presence of microvascular complications . however , we also found indications that cerebral and cognitive changes may be present before microvascular complications become apparent . it could be hypothesized that chronic hyperglycemia , even in the absence of clinically detectable microvascular complications , can negatively affect cognitive functioning and cerebral communication . it is known that hyperglycemia as such leads to a cascade of changes , including increases in formation of reactive oxygen species and advanced glycation end products ( 38,39 ) , as well as activation of the hypothalamic - pituitary - adrenal axis ( 40 ) , which could have an effect on the brain . effects of hypoglycemia , particularly of asymptomatic hypoglycemic episodes because of hypoglycemia unawareness , can not be completely ruled out as a contributor . this did not change the results ( supplementary table 2a c , available in an online appendix ) . another possible contributor to the altered functional connectivity and cognitive functions observed in type 1 diabetic patients may be cerebrovascular , that is , macrovascular disease , the subclinical form of which can be estimated by ultrasound measurements of the carotid intima media thickness , which is known to be increased in asymptomatic type 1 diabetic patients relative to their nondiabetic peers ( 41 ) . furthermore , early diabetes onset , defined as disease onset before 7 years of age , has been shown previously to negatively affect intellectual and cognitive performance and cerebral structure ( 42,43 ) furthermore , the cognitive tests chosen had the highest sensitivity to detect cognitive changes . however , these tests may not be sufficiently sensitive to detect very subtle changes in type 1 diabetes associated cognitive decline . second , age of disease onset and subsequently disease duration differ between the patient groups . although the percentages of early disease onset do not statistically differ between groups , for the type 1 diabetes patients age of onset reflects childhood and early adolescence , whereas for the type 1 diabetes patients it reflects late adolescence and early adulthood . in a subsequent analysis we compared both patient groups , additionally correcting for age of diabetes onset and diabetes duration ( see supplementary table 3a c , available in an online appendix ) . taking into account the risk of emerging power problems when adding two more covariates to this small sample , these adjustments did not essentially change the conclusions regarding cognitive functioning and functional connectivity . disease duration might be less of a confounder here as the type 1 diabetes patients had an average disease duration of almost 20 years , which is sufficient for complications to develop . as stated before , an increase in functional connectivity although the possibility of a statistical aberration can not be completely ruled out , synchronization likelihood tended to be higher for 20 of the 23 anatomical locations of the lower band in type 1 diabetes patients than control subjects ( supplementary table 4 , available in an online appendix ) . moreover , we additionally repeated the post hoc mancova for the lower band correcting for multiple comparisons . as both age and depressive symptoms differed between groups , we statistically corrected for both variables in all analyses . supplementary table 5 , available in an online appendix , summarizes the relation between age and depressive symptoms and cognitive and meg variables . third , a1c values over the time period preceding meg recording and neuropsychological assessment unfortunately were not available . in conclusion , this study showed that changes in functional brain connectivity and cognitive function exist in type 1 diabetic patients with and without microvascular complications . longitudinal research in larger samples is required to determine the predictive clinical value of our findings for the progression of cognitive deterioration in type 1 diabetic patients as well as its potential to serve as clinical outcome parameter .

objectivehyperglycemia - associated microvascular disease may underlie changes in cerebral functioning and cognitive performance in patients with type 1 diabetes . functional connectivity , an indicator of functional interactions and information exchange between brain regions , provides a measure of cerebral functioning . this study addresses functional connectivity and cognition in type 1 diabetic patients with and without proliferative retinopathy , relative to healthy control subjects , using magnetoencephalography.research design and methodsfluctuations in magnetic field at scalp for , , lower and upper , , and lower and upper frequency bands were measured using magnetoencephalography . synchronization likelihood , a measure of functional connectivity , was computed . using neuropsychological tests , cognitive functioning was assessed and its associations with functional connectivity were determined.resultscompared with control subjects , type 1 diabetic patients performed poorer on general cognitive ability , information processing speed , and motor speed , irrespective of their microvascular complication status . functional connectivity , however , was lowest for type 1 diabetic patients with retinopathy , compared with type 1 diabetic patients without microvascular complications and control subjects , whereas type 1 diabetic patients without microvascular complications showed an increase relative to control subjects . positive associations were found between functional connectivity and executive functioning , memory , information processing speed , motor speed , and attention.conclusionscompared with healthy control subjects , functional connectivity and cognition differed in type 1 diabetic patients irrespective of microvascular complication status , indicating that chronic hyperglycemia , among other factors , may negatively affect brain functioning even before microvascular damage becomes manifest . the association found between synchronization likelihood and cognition suggests functional connectivity plays a significant role in cognitive functioning .

RESEARCH DESIGN AND METHODS Study design. Neuropsychological assessment. MEG protocol. Synchronization likelihood. Data analysis. Statistical analysis. RESULTS Patient characteristics. Neuropsychological assessment. MEG measurements. Correlation between MEG and cognitive performance. DISCUSSION

psychiatry today is at a juncture where a new , strong biological dimension is being added to an already existing psychosocial approach.this is a welcome change , as it gives more evidence - based approaches and a scientific outlook.however , that does n't mean the psychosocial model is no more relevant.psychiatrists of today should be comfortable with both approaches , as they are mutually complementary . psychiatry today is at a juncture where a new , strong biological dimension is being added to an already existing psychosocial approach . this is a welcome change , as it gives more evidence - based approaches and a scientific outlook . psychiatrists of today should be comfortable with both approaches , as they are mutually complementary . with advances in biological research and greater inclination of psychiatrists towards psychopharmacology , will psychiatry lose its psychosocialdimension?how can biological breakthroughs become a boon rather than a bane for those who advocate a psychosocial approach?will these two apparently antagonistic approaches work together in a complementary manner to make psychiatry a more holistic speciality?how should a psychiatrist , working in the clinic and the community , incorporate these two approaches in a judicious manner in his day - to - day practice ? with advances in biological research and greater inclination of psychiatrists towards psychopharmacology , will psychiatry lose its psychosocial how can biological breakthroughs become a boon rather than a bane for those who advocate a psychosocial approach ? will these two apparently antagonistic approaches work together in a complementary manner to make psychiatry a more holistic speciality ? how should a psychiatrist , working in the clinic and the community , incorporate these two approaches in a judicious manner in his day - to - day practice ? bheemsain tekkalaki md finished his post - graduation in psychiatry from king george medical university lucknow , in the year 2013 . he is currently working as assistant professor of psychiatry at navodaya medical college , raichur , karnataka , india . his areas of interest are cognitive behavioural therapy , consultation liaison psychiatry and culture and psychiatry . adarsh tripathi md is currently working as an assistant professor of psychiatry at the king george 's medical university , lucknow , india . he has more than 30 national and international publications and four book chapters to his credit and has worked in several international and national researches of repute . his areas of interest include addictions , bipolar disorder , cognition , human sexuality , adult adhd and psychopharmacology . late jitendra kumar trivedi was a professor in the department of psychiatry , king george medical university , lucknow , india . he was the fellow member of the american psychiatric association since 2007 , and nominated honorary member of the world psychiatric association in 2008 , and fellow of the national academy of medical sciences . he was a principal investigator for more than 25 multinational clinical trials as well as icmr and who sponsored projects . he had around 260 publications in national and international journals as well as chapters in books . he had been an editor , in the indian journal of psychiatry , president of the indian psychiatric society , and a zonal representative for southern asia - zone - xvi of the world psychiatric association . at the time of his demise , he was president - elect of indian association for social psychiatry , member of executive council of preventive psychiatry section of world psychiatric association and chairman- preventive psychiatry section of world association of social psychiatry .

human behaviour , emotions , and cognition are complex to understand and explain . it is even more difficult to understand the basis for abnormal behaviour , disturbed emotions , and impaired cognitions , something mental health professionals are trying for long . in these pursuits , psychiatry has traversed through eras of humours , witchcraft , spirits , psychoanalysis , and gradually deviated from other medical specialities . now , with recent biological breakthroughs like advances in psychopharmacology , neuroimaging and genetics , increasingly more emphasis is being given to the biological model of psychiatric disorders.these new biological models have given a more scientific appearance to the speciality . it has also revolutionised the management strategies and outcome of many psychiatric disorders . however , this rapid development in biological understanding of psychiatry also leads to a new wave of reductionism . in an attempt to deduce everything in terms of neurons , neurochemicals , and genes , can we neglect psychosocial aspects of mental health ? patients personality , expectations , motives , family background , sociocultural backgrounds continue to affect mental health no matter how much biological psychiatry gets.biological and psychosocial approaches are not mutually exclusive but complementary . integrating them harmoniously is the skill psychiatry demands for comprehensive understanding of mental and behavioural disorders .

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ductal carcinoma in situ ( dcis ) is characterized by proliferation of malignant cells confined by the basement membranes of ductal structures , without evidence of extraductal invasion . dcis comprises different subtypes with heterogeneous proliferative disease processes and varies in architecture , imaging features , and clinical outcome . although the progression of dcis is not fully understood , it is known that untreated dcis is likely to progress to invasive cancer within 10 years of diagnosis . therefore , dcis is considered a preinvasive form of invasive breast cancer that requires immediate treatment [ 2 , 3 ] . with the widespread use of screening mammography , dcis now accounts for 15%20% of all newly detected breast cancer , with the trend still increasing . in mammography , although mammography has been the mainstay for diagnosis of dcis , it has limitations in defining the extension and the margin , especially in patients without microcalcifications or in those with dense breasts or breast implants . furthermore , there is a tendency to underestimate the tumor size of dcis on mammography [ 57 ] . positive surgical margin is known as an independent risk factor for local recurrence [ 810 ] . dynamic contrast - enhanced mri ( dce - mri ) has been proven more sensitive than mammography for detecting breast cancer in young women with a high risk of developing breast cancer [ 1113 ] . in many instances , dce - mri can reveal early stage breast cancer , including dcis and dcis with small invasive carcinomas , which are mammographically , sonographically , and clinically occult . in addition to screening , breast mr can also be used for staging purposes to better characterize the disease extent and the presence of multifocal multicentric lesions , which can impact on treatment planning and the subsequent management . imaging characterization of patients with mixed cohort of pure dcis and dcis with invasive components [ 1417 ] and patients with merely pure dcis [ 1825 ] on dce - mri has been reported before . in a study of 167 patients , kuhl et al . found that mammography only diagnosed 56% pure dcis preoperatively , while mri achieved a diagnostic rate of 92% . for 89 high - grade lesions , mri detected 87 lesions ( 98% ) while mammography only diagnosed 52% of lesions . nevertheless , consensus regarding how mri can be used to aid in management of pure dcis has not been reached yet . given the recent evidence that additional diseases diagnosed by preoperative mri often led to more aggressive treatment yet did not improve the treatment outcome [ 26 , 27 ] , continuing exploration of the role of breast mri for the diagnosis of dcis is needed . the purpose of this retrospective study was to characterize the morphology and enhancement kinetic pattern of pure dcis on dce - mri . since high - grade dcis is associated with a greater risk of local recurrence and progression to invasive breast cancer [ 2830 ] , it would be very helpful if the high - grade and low - grade dcis could be differentiated on imaging . in this paper , the mri features of dcis between high - grade ( grade iii ) and non - high - grade ( including low grade i and intermediate grade ii ) dcis based on the van nuys classification were compared . a total of 34 consecutive patients with histological - proven pure dcis ( without any microinvasion ) were identified from our breast mri database collected from 2002 to 2006 . these patients came to our center to participate in a breast mri research study due to suspicious lesions found in mammography , sonography , or physical examination . the medical records , breast mri images , and mammogram reports ( if available ) were reviewed retrospectively . among these 34 patients , three patients had received surgical excision biopsy before mr imaging , and they were excluded in the analysis . the final diagnosis of pure dcis was obtained from pathological examination of mastectomy ( n = 21 ) and lumpectomy ( n = 10 ) specimens . this study was approved by the institutional review board , and informed consents were obtained from all patients . the mri study was performed using a 1.5 t phillips eclipse mr scanner with a standard bilateral breast coil ( philips medical systems , cleveland , ohio ) . the imaging protocol consisted of high - resolution precontrast imaging and dynamic contrast - enhanced imaging . after setting the iv line , then a sagittal view unilateral t1-weighted precontrast images were acquired from the breast of concern , using a spin echo pulse sequence with tr = 1000 ms , te = 12 ms , and fov = 20 cm , and matrix size = 256 256 . following this , a 3d spgr ( rf - fast ) pulse sequence with 16 frames ( repetitions ) was prescribed for bilateral dynamic imaging . thirty - two axial slices with 4 mm thickness were used to cover both breasts . the imaging parameters were tr = 8.1 ms , te = 4.0 ms , flip angle = 20 , matrix size = 256 128 , fov = 3238 cm . the sequence was repeated 16 times for dynamic acquisitions , four precontrast , and 12 postcontrast sets . the contrast agent ( omniscan , 1 cc/10 lbs body weight ) was manually injected at the beginning of the 5th acquisition and was timed to finish in 12 seconds to make the bolus length consistent for all patients . immediately following the contrast , 10 cc saline the subtraction images at 1-minute postinjection were generated by subtracting the precontrast images acquired in frame number 3 from the postcontrast - enhanced images acquired in frame number 6 . the maximum intensity projections ( mips ) were also generated from the subtraction images to help identify the lesion . the enhancement kinetic was analyzed from manually selected roi ( region of interest ) based on the subtraction images at 1-minute post - injection . the enhanced tumor area was outlined on each imaging slice covering the lesion by an experienced breast radiologist ; then a mean signal intensity time course from all 16 time frames was obtained . the percent enhancement time course was calculated by first subtracting the mean precontrast signal intensity ( mean of first 4 frames ) from each of the subsequent 12 postcontrast signal intensities and then normalized by the mean precontrast signal intensity 100% . morphological appearances and enhancement kinetic features of lesions shown on mri were categorized according to the acr breast imaging reporting and data system ( bi - rads ) breast mri lexicon . the features were evaluated by 2 radiologists ( with 4 and 5 years of experience in reading breast mri ) separately . for cases with different results , based on the lesion size , the mass type can be further separated into single focus / multiple foci ( < 5 mm ) and mass ( 5 mm ) . then for mass lesions other characteristics such as shape , margin , and internal enhancement patterns were evaluated . the nonmass - like lesions were described by the type of enhancements ( diffuse , regional , segmental , focal , ductal , linear ) and the internal enhancement patterns ( punctate / stippled , clumped , and heterogeneous ) . the tumor size was measured as the longest diameter on the maximum intensity projection ( mip ) of subtraction image . the enhancement kinetics was divided into two phases : the initial enhancement phase , defined as enhancement patterns within the first 2 minutes or before the curve starts to change , and the delayed phase , defined as enhancement pattern after 2 minutes or after the curve starts to change . when the enhancement kinetics showed a rapid initial enhancement followed by washout or reaching to a plateau in the delayed phase , it was determined as suspicious of malignancy . all cases are pure dcis without presence of microinvasion . the pathological diagnosis was evaluated based on the van nuys system . dcis was classified according to the nuclear grade ( non - high grade versus high grade ) and the morphologic subtype ( comedo and noncomedo ) . grade - i ( low grade ) and grade - ii ( intermediate grade ) were categorized as non - high grade , and grade - iii was the high - grade . for statistical analysis , fisher 's exact test was used to examine the significant difference between non - high grade dcis and high grade dcis . according to the nuclear grading system , 2 patients had grade - i , 16 patients had grade - ii , and 13 patients had grade - iii , all together 18 non - high grade and 13 high grade . the mean age for non - high grade dcis was 52 years old and 57 years old for high - grade dcis ( p = .27 ) . the comedo type was noted in 12 patients ( 7 high grade and 5 non - high grade ) , and the noncomedo type was noted in 19 patients ( 6 high grade and 13 non - high grade ) ( table 1 ) . there was no significant difference in the histological comedo or noncomedo subtype between the non - high grade and high grade dcis ( p > .05 ) . two cases of high - grade dcis with noncomedo morphology were not detected by mri and were false negative diagnosis . the imaging features of these 29 cases are summarized in table 2 , and 5 case examples are shown in figures 15 . among these 29 cases , mass lesions were seen in 15 cases ( 52% ) including 12 masses and 3 focus / foci lesions and nonmass - like lesions in 14 cases ( 48% ) . two cases were multifoci ( figures 2 and 5 ) , and the size was not measured . for the remaining cases , the size ranged from 0.4 to 5 cm with the mean of 1.9 1.2 cm . the mean size was 1.6 1.2 cm for non - high grade dcis and 2.2 1.3 cm for high - grade dcis , not significantly different ( p = .21 ) . for the two false negative cases , in mammography , one case was occult , and the other was undetermined ( i.e. , needed additional imaging evaluation ) . the mammographically occult case received ultrasound exam and found a suspicious hypoechoic mass in the left breast . after needle biopsy confirming malignancy , the patient received lumpectomy , and a pure dcis of 1.4 cm was found . this patient was diagnosed with dcis in the right breast 2 years ago and had already received right mastectomy . she decided to receive left mastectomy , and a pure high grade dcis of 1.1 cm was found . among the 15 mass lesions , 9 cases were non - high grade , and 6 were high grade . for the 14 nonmass - like lesions , 9 lesions were non - high grade , and 5 cases were high grade ( p = .26 ) . the shape , margin , and internal enhancement patterns were evaluated in 12 mass lesions that were 5 mm . the most frequently seen features were irregular shape ( 50% ) , spiculated / irregular margin ( 92% ) , and heterogeneous enhancement ( 67% ) ( table 3 ) . there was no significant difference in the mr morphology pattern between the non - high - grade and high - grade mass type dcis lesions . three case examples are shown in figure 1 ( low grade ) , figure 3 ( intermediate grade ) , and figure 4 ( high grade ) . of 14 nonmass lesions , 4 lesions showed regional enhancement ( 28% ) , 3 showed ductal ( 22% ) , and 3 showed focal ( 22% ) enhancements . internal enhancement pattern was dominated by the clumped pattern ( figure 5 , high grade ) ( 9/14 , 64% ) followed by the heterogeneous enhancement pattern ( 4/14 , 29% ) ( table 4 ) . two grade - ii lesions had severe motion artifacts due to patient 's movement during the delayed phase , and the enhancement kinetic curves could not be reliably measured . the early postcontrast subtraction images had good quality , and the morphological features of these two lesions could be evaluated . twenty - one lesions ( 21/27 , 78% ) showed the suspicious malignant type enhancement kinetics with a rapid initial enhancement followed by plateau ( figures 1 and 5 ) or washout ( figures 24 ) , including 1/2 grade - i lesion , 12/14 grade - ii lesions , and 8/11 grade - iii lesions . therefore , no significant differences were found between enhancement kinetics of high - grade and non - high - grade dcis ( p = .38 ) . among the other 8 enhanced lesions that showed benign type enhancement kinetic curves or undetermined kinetic curves , 6 lesions showed malignant morphological features . therefore , of the total 31 cases , mri diagnosed 27 as suspicious of malignancy , with the sensitivity of 87% ( 27/31 ) . correlation between screening mammogram and mr imaging was performed in 22 patients whose mammography reports were available . sixteen lesions ( 16/22 , 73% ) were classified as bi - rads 4 or 5 , as suspicious or five of 22 patients were classified as bi - rads category 1 ( normal mammogram ) and one patient as bi - rads category-0 ( need additional imaging evaluation ) . on mri , four of 5 patients with normal mammogram were correctly diagnosed as suspicious of malignancy , showing malignant type enhancement kinetics ( rapid enhancement followed by washout or plateau ) and/or the morphological appearance ( irregular shape and spiculated / irregular margin ) . mammography may miss dcis that does not present certain suspicious patterns of microcalcifications , and mri may have a complementary role in diagnosis of these lesions . most of the mr imaging studies of dcis analyzed mixed patient cohort of pure dcis and dcis with invasive components . several studies reported the mr kinetic and morphologic appearance of pure dcis correlated with histopathology findings [ 1825 ] . in a study by kuhl et al . , only 56% pure dcis was detected by mammography , while mri could detect more than 90% . in another study of 33 pure dcis patients by vag et al . the sensitivity of mammography and mri was 64% and 88% , respectively . in this study , we detected 29/31 ( 94% ) enhancing lesions and diagnosed 27/31 ( 87% ) as suspicious of malignancy . of the 22 patients whose mammography reports were available , the sensitivity of mammography was 74% ( 16/22 ) . the result was comparable to other published studies [ 15 , 17 , 19 ] . it was found that most false - negative diagnosis of dcis by mri appeared to be non - high - grade , noncomedo - type lesions [ 23 , 32 , 33 ] . in this study , we had two false negative cases of high - grade dcis with noncomedo morphology . one case showed strong background tissue enhancements in both breasts , and the 1.4 cm lesion could not be identified . the other case showed clumped enhancement in the affected breast , and the 1.1 cm dcis could not be identified . nuclear grade has been consistently associated with poor prognosis and local recurrence in dcis [ 26 , 3436 ] , and the combination of nuclear grade and comedo necrosis was reported to correlate with the risk of local recurrence after breast conserving surgery [ 34 , 35 ] . found that high - grade pure dcis missed by mammography can be diagnosed by mri alone . the diagnostic yield of mri for non - high - grade pure dcis is also significantly higher than mammography . in the series reported by vag et al . , all 12 mammographically occult dcis lesions ( three low grade , four intermediate grade , five high grade ) were correctly diagnosed by mri . in our series all these 4 lesions were high grade . with the prognostic significance of early detection for high - grade dcis , this result further strengthens the recommendation of mri for diagnosis of dcis . pure dcis lesions often appear as nonmass clumped enhancement in a regional , ductal , or linear distribution [ 16 , 18 , 21 , 22 ] . reported a high specificity of focal branching enhanced pattern on mri for dcis . as the spatial resolution of mri improves , it is anticipated that the ductal pattern as linear or linear branching may be seen more often , which can be a signature feature on mri suggesting dcis . in our study , the typical morphologic appearance of pure dcis on mri was a mass- or a nonmass - like lesion with heterogeneous or clumped enhancement . although our study showed a higher percentage of mass lesions ( 52% ) ( figures 3 and 4 ) compared to previous publications [ 18 , 22 ] , the classification of mass and nonmass lesions is subjective , and this may lead to variations in the determined percentages . in the study by viehweg et al . , of the 48 enhancing pure dcis lesions , 35 lesions ( 35/48 , 73% ) showed either well - defined ( n = 12 ) or ill - defined focal mass lesions ( n = 23 ) . the percentage will change if some ill - defined mass lesions are reclassified as nonmass lesions . , we did not find significant differences between morphologic patterns of high - grade and non - high - grade dcis . pure dcis does not always exhibit the typical malignant washout kinetic curves and could show persistent and plateau curve types [ 16 , 18 , 21 , 22 ] . reported that the peak enhancement ratio at one minute ( e1 ) for pure dcis was less than invasive cancers and more than benign lesions . studies on the enhancement kinetic characteristics among the different nuclear grades of pure dcis did not show consistent results . some reports have indicated that the kinetic characteristics of low - grade pure dcis lesions are different from those of intermediate- and high - grade lesions [ 18 , 37 ] , whereas other studies have revealed no difference [ 22 , 34 ] . we did not find significant difference between the enhancement kinetics of high - grade and non - high - grade groups . for the size measurement of dcis , different local recurrence rates were found between dcis lesions smaller and larger than 10 mm , thus an accurate size measurement is important . . showed a 8% deviation of mri measured tumor size relative to histopathologic size , compared with 29% for mammography . despite that in general the size measurement is more accurate on mri , false positive findings and tumor size overestimation may occur on mri . reported that cases in which mri overestimated dcis were mostly non - high - grade and noncomedo - type lesions . the false - positive enhancement in most cases is due to coexistence of benign proliferative processes such as fibrocystic changes or atypical ductal hyperplasia [ 16 , 40 ] . several limitations existed in our study , including small case number and retrospective case review . nevertheless , the reported cases were from consecutive patients referred to participate in a breast mri research study , with diagnosis of pure dcis ; therefore , allowing objective analysis as presented in this study . the research study was designed to analyze the dce kinetics , so a protocol with a relatively high temporal resolution ( 42 seconds compared to 2 minutes used in a clinical setting ) was chosen . this could only be accomplished by using a compromised spatial resolution , which would inevitably affect the quality of the image . however , since the tumor size of our subjects was relatively large ( 1.6 1.2 cm for mass lesions and 2.2 1.3 cm for nonmass lesions ) , we believe that the relatively low spatial resolution did not affect our findings on the analysis of lesion morphology . the morphological features of the three focal lesions < 5 mm were not evaluated . the feature about spiculated margin might be affected by the low spatial resolution , and the percentage of lesions with spiculated margin could be higher than the reported 42% ( 5/12 cases ) . therefore , the cases with spiculated and irregular margin were reported together . with improved spatial resolution and reduced slice thickness , it is possible that more subtle morphological features , such as linear or linear branching rather than regional enhancements , can be revealed for improved diagnosis of dcis . in this retrospective analysis , the majority ( 94% ) of pure dcis lesions showed contrast enhancement . mri was more sensitive in detecting pure dcis compared to mammography , hence reinforcing the role of mri in detection and preoperative management of pure dcis . however , we did not observe any significant difference in mr morphologic or kinetic features between non - high - grade and high - grade pure dcis . therefore , our results did not provide strong evidence to support the use of mri in staging of dcis for treatment planning .

to characterize imaging features of pure dcis on dynamic contrast - enhanced mr imaging ( dce - mri ) , 31 consecutive patients ( 37 - 81 years old , mean 56 ) , including 2 grade i , 16 grade ii , and 13 grade iii , were studied . mr images were reviewed retrospectively and the morphological appearances and kinetic features of breast lesions were categorized according to the acr bi - rads breast mri lexicon . dce - mri was a sensitive imaging modality in detecting pure dcis . mr imaging showed enhancing lesions in 29/31 ( 94% ) cases . pure dcis appeared as mass type or non - mass lesions on mri with nearly equal frequency . the 29 mr detected lesions include 15 mass lesions ( 52% ) , and 14 lesions showing non - mass - like lesions ( 48% ) . for the mass lesions , the most frequent presentations were irregular shape ( 50% ) , irregular margin ( 50% ) and heterogeneous enhancement ( 67% ) . for the non - mass - like lesions , the clumped internal enhancement pattern was the dominate feature , seen in 9/14 cases ( 64% ) . regarding enhancement kinetic curve , 21/29 ( 78% ) lesions showed suspicious malignant type kinetics . no significant difference was found in morphology ( p > .05 ) , tumor size ( p = 0.21 ) , and kinetic characteristics ( p = .38 ) between non - high grade ( i+ii ) and high - grade ( iii ) pure dcis .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

studies have shown cognitive deficits ( in particular deficits in information processing speed , concentration , and working memory ) to be present in the early stages of ms [ 24 ] . cognitive impairment has a negative impact on quality of life ( qol ) independent of physical symptoms [ 5 , 6 ] . there exists mounting evidence for neuroplasticity as a mechanism to compensate for accumulating pathology in ms and some tentative evidence that cognitive rehabilitation may be effective in preserving or improving cognitive function in patients with ms [ 79 ] . computer - assisted cognitive rehabilitation has the potential to provide a structured and standardised approach to rehabilitation . rehacom is one particular type of software designed and utilised for treatment of cognitive impairment in a number of disease states such as stroke , brain injury , and psychiatric disorders [ 10 , 11 ] . it has been used in a growing number of trials of cognitive rehabilitation in ms as a more standardised intervention [ 8 , 9 , 12 , 13 ] . the difficulty level of the computerised tasks adapts to an individual 's performance , only increasing in difficulty in response to improving performance . few studies have examined the structural basis of cognitive rehabilitation and longitudinal studies are relatively lacking [ 8 , 14 ] . animal data suggest that myelination is , at least in part , regulated by neuronal activity . it is therefore conceivable that techniques , such as magnetisation transfer ( mt ) , which is sensitive to myelin content , might be sensitive to structural plasticity in ms . in this study we combined neuropsychological assessment , functional mri ( fmri ) , and quantitative magnetisation transfer ( qmt ) imaging to explore whether home - based , computerised cognitive rehabilitation is an effective means of promoting cognitive rehabilitation and whether the structural basis for rehabilitation can be better defined . the primary outcome of the study was measured as any improvement in cognition after the training , while the secondary outcomes included changes in fmri , fatigue , and quality of life assessments . participants . thirty - eight patients with objective evidence of cognitive impairment were invited to participate in this study between february 2014 and february 2015 . all participants signed informed written consent before undergoing testing . inclusion criteria were as follows : ( a ) age between 18 and 65 , ( b ) clinically definite ms , according to the mcdonald criteria , ( c ) expanded disability status scale ( edss ) 6.5 , and ( d ) cognitive impairment defined as scores below the 5th percentile for normative data adjusted for age , sex , and years of formal education on one or more of the bicams tests . patients were excluded if they had a history of significant psychiatric disorders , alcohol or substance abuse , visual acuity less than 6/18 corrected , oscillopsia , or diplopia that would interfere with testing . patients were also excluded if they had a ms relapse , received corticosteroids , or changes made to psychoactive medications within the previous month . the study was approved by the northern ireland research ethics committee . study design . neuropsychological and mri data were obtained at baseline ( time 1 ) , immediately following a 6-week intervention period ( time 2 ) and after an additional 12-week follow - up period ( time 3 ) , during which no additional intervention was administered ( supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/4292585 ) . it was not possible for the cognitive assessments to be completed by a blinded assessor . the mri analysis was conducted by a researcher blind to the patients ' group allocation . cognitive and behavioural assessments . at entry all participants underwent a detailed clinical neurological assessment including edss conducted by an experienced neurologist . patients were screened for cognitive impairment using the brief international cognitive assessment for ms ( bicams ) . bicams is a brief ( 15-minute ) screening tool to identify cognitive impairment in patients with ms and comprises the first five learning trials of the california verbal learning test ii ( cvlt - ii ) , the first three recall trials of brief visuospatial memory test revised ( bvmt - r ) , and the symbol digits modalities test ( sdmt ) . the bicams assessment was conducted by a neurology clinical fellow with almost ten years of clinical experience ( j.c ) . the assessing neurologist was trained in bicams assessing methods by an experienced neuropsychologist ( d.l ) . at baseline participants also completed a number of behavioural and qol assessments including euroqol five - dimension questionnaire ( eq-5d ) , a generic health - related quality of life scale , functional assessment of ms ( fams ) ( a ms specific quality of life scale ) , patient activation measure ( pam-13 ) ( a 13-item generic scale for chronic illness management ) , a measure of patient empowerment in ms , unidimensional self - efficacy scale for ms ( use - ms ) , the hospital anxiety and depression scale ( hads ) , multiple sclerosis neuropsychological questionnaire ( msnq ) self - report ( a patient self - reported measure of cognitive function ) , and the fatigue severity scale . at each subsequent time point participants underwent repeat cognitive assessment using bicams ( same test forms ) as well as repeat behavioural and qol assessments . randomisation was performed using a random number generator and allocations were placed inside sealed folders . the treatment group underwent six weeks of home - based , computer - assisted cognitive rehabilitation using rehacom software ( https://www.fixxl.co.uk/ ) . the control group were asked to watch a series of natural history dvds of corresponding duration and frequency to the rehabilitation sessions performed by the treatment group for six weeks . the need to evaluate mri parameters in studies with active control conditions has been highlighted . treatment sessions consisted of training in three specific modules involving working memory , visuospatial memory , and divided attention . in all tasks the level of difficulty is tailored to the individuals performance and increases automatically but only in line with satisfactory progress . real - time data pertaining to performance , progress , and compliance is transmitted to the investigator over the internet during the intervention period . multiple distractions must be navigated and the speed and direction of the vehicle altered according to road conditions . as the complexity of the task increases , more distractors are introduced with increased multitasking skills required . working memory the working memory task consists of remembering a series of playing cards presented briefly on screen . the participant is then asked to select which cards were presented from a longer series of options including distractor cards . as the complexity of the task increases , participants are asked to remember only cards of a particular value or suit and the number of items to remember increases . higher levels involve having to remember the cards in reverse order . visuospatial memory is a similar task involving various objects presented briefly on screen with the patient asked to remember the object as well as its position in the sequence . as the complexity of the task increases , the following sequences were acquired in an order designed to minimise the potential for fatigue on the fmri task : ( 1 ) dual - echo turbo spin - echo for lesion identification ; ( 2 ) high - resolution t1-weighted magnetisation - prepared rapid - acquisition gradient echo ( mprage ) ; ( 3 ) functional mri with echo - planar imaging ( epi ) acquired during a n - visually presented back task ; ( 4 ) quantitative magnetisation transfer ( qmt ) with balanced steady - state free precession ( bssfp ) . t2 lesion volume was measured at baseline for each participant using the software package jim ( version 3.0 , xinapse systems ltd . , this was adapted from sweet et al . and involved three conditions : 0-back , 1-back , and 2-back tasks . the 0-back condition was designed to act as the baseline condition and would provide the baseline activation for comparison in fmri analysis . the 1-back and 2-back conditions provided increasing working memory demands . the n - back task did not constitute part of the cognitive rehabilitation . all participants were allowed to briefly practice the n - back task under supervision for five minutes prior to the mri scan to ensure comprehension of the task and allow familiarity with it . stimuli were projected onto a mirrored screen inside the mri scanner 45 cm from a participant 's nose . this involved of a series of pseudo - randomised consonants in both upper and lower cases . the stimulus duration was 1000 ms with a between stimulus interval of 2000 ms . 0-back , 1-back , and 2-back tasks were presented in a randomised manner resulting in six blocks per nine - minute run . each block consisted of 126 stimuli , one - third of which were targets . twice as many 0-back tasks were presented as 1-back or 2-back . the primary outcome was cognitive performance as measured by improvement in sdmt , bvmt , and cvlt between groups compared to baseline . the means of continuous variables were compared using the independent samples t - test or the mann all tests were two - tailed ; p - values less than 0.05 were considered significant . outcomes were compared between the two groups using independent samples t - test to compare gain scores for cognitive data between groups . to compare differences between groups for other behavioural and qol data , a 2 3 repeat measures analysis of variance ( anova ) was used with time as the within - subject factor and treatment as the between factor ( active rehabilitation versus control ) . analyses were performed using spss version 21 ( armonk , ny : ibm corp ) . fmri data were analysed using spm8 ( wellcome department of cognitive neurology , ucl , london , http://www.fil.ion.ucl.ac.uk/spm/ ) . for each time series individual epis were then realigned to the first remaining image of the series by rigid - body transformation to correct for involuntary head movements during acquisition before normalisation into a standard anatomical space ( montreal neurological institute [ mni ] ) using linear and nonlinear transformations . finally , images were smoothed with an 8 mm full - width - at - half - maximum ( fwhm ) 3d gaussian kernel . first - level analysis . for each participant , the difference in blood oxygen level - dependent ( bold ) response between the 0-back , 1-back , and 2-back conditions was estimated at every voxel across the whole brain using the general linear model ( glm ) . this produced a series of contrasts representing mean activation during each n - back condition minus the 0-back condition . each contrast obtained at the first - level was entered into a second - level glm to generate summary statistical parametric maps ( spms ) . for between - group analysis of difference between the time points , we used a 3 2 anova flexible factorial design with group ( between - subject ) and time ( within - subject ) as separate factors to examine the main effects on group ( treatment versus control ) , time and the interaction between them to evaluate areas of relative change in activity after cognitive training versus control . the threshold for significance was set at alpha of 0.05 corrected for multiple comparisons ( family - wise error ( fwe ) corrected ) . , the location of local maxima of signal intensity increase is expressed as x , y , and z coordinates in mni space . mt and t1 mapping data from all three sessions were first realigned to subject specific mprage structural images using the spm8 rigid - body registration function . the mprage were then segmented into white matter , grey matter , and csf to yield a parenchymal mask . a t1 map was calculated for all datasets by fitting the theoretical spoiled gradient echo as a function of the flip angle to the signal measured by the 3d flash sequences . mt parameters were obtained by performing a voxelwise nonlinear least squares fitting ( levenberg - marquardt ) to a binary spin bath model for bssfp . the statistical analysis was performed voxelwise in spm8 on the resulting warped and smoothed mt maps . the same glms used for the second - level fmri analysis and described in the previous section were used for estimating the main effects of time and group and the interaction between these two factors . patients ( 70.3% ) had rrms , and 11 patients ( 29.7% ) had spms . the duration of ms from diagnosis to enrolment ranged from 12 months to 40 years ( mean 11.61 years , sd 8.2 yrs ) . 20 patients ( 52.6% ) were on disease modifying therapy at enrolment ( natalizumab n = 6 , beta - interferon n = 7 , fingolimod n = 6 , and teriflunomide n = 1 ) . after randomisation to either computer - assisted cognitive training ( treatment group , n = 19 ) or the active control condition ( n = 19 ) , there were no significant differences in terms of baseline demographics ( table 1 ) or quality of life measures ( supplementary table 3 ) between the two groups . the treatment group had higher baseline cognitive scores on the bicams battery ; however , these did not differ significantly from the control group . the most frequently failed component of the bicams test battery was the sdmt with 33 ( 86.8% ) of participants scoring below the 5th centile , 18 ( 47.4% ) failing the cvlt - ii , and 13 ( 34.2% ) failing the bvmt - r . overall 21 ( 55.2% ) failed one test , 10 ( 26.3% ) failed two tests , and 7 ( 18.4% ) failed all three tests of the bicams test battery . this level of impairment is consistent with other published ms samples on bicams [ 3133 ] . overall , 88.9% of patients ( 16/18 ) in the intervention group completed at least 75% of the prescribed sessions with 66.7% ( 12/18 ) completing all the prescribed sessions . , the treatment group showed a significantly greater improvement in gain scores between baseline and early follow - up ( time 2 ) compared to the control group on the sdmt ( treatment 3.94 ( sd 5.08 ) , controls 0.63 ( sd 3.30 ) , 1.47 to 7.66 , ( 95% ci 1.47 to 7.66 ) , p = 0.005 ) illustrated in figure 1 . similar gain scores in the cvlt and bvmt - r were not significantly different between the groups although the bvmt - r gain scores did approach significance ( p = 0.098 ) . overall , there was an improvement in bicams performance across participants at follow - up . the gain scores between the groups at time 3 compared to baseline were , however , not statistically significantly different . there were no significant differences in qol outcome measures , measures of self - efficacy , or subjective cognitive performance between the two groups ( supplementary table 4 ) . the baseline error rate between the treatment and control groups was low ( 8.64% versus 9.48% , p = 0.814 ) . no significant differences were observed in the error rate during the n - back task between the groups at baseline or at follow - up ( supplementary table 5 ) . the 1-back task was associated with activations involving the dorsolateral prefrontal cortex bilaterally as well as bilateral inferior parietal lobule and insular and cerebellar regions relative to the 0-back contrast . the same regions were activated in the 2-back condition but the spatial extent and magnitude of the responses were greater , particularly over the frontoparietal regions ( supplementary figure 2 and supplementary table 2 ) . time 2 versus time 1 . at time 2 , increased activation was seen in the right temporoparietal regions ( right supramarginal and angular gyri ( p < 0.005fwe corrected at cluster level ( k = 228 ) ) ) in the 1-back in the treatment group relative to controls ( group - by - time interaction ) . 3 significant increases in activation were seen in both the 1-back and 2-back conditions in the treatment group relative to controls . in the 1-back task , increased activation was seen in the left frontal ( p < 0.001fwe corrected at cluster level ( k = 294 ) ) and right temporoparietal regions ( p < 0.012fwe corrected at cluster level ( k = 187 ) ) . in the 2-back task , increases in activation were seen in bilateral prefrontal ( p < 0.013fwe corrected at cluster level ( k = 206 ) ) and right temporoparietal regions ( p < 0.024fwe corrected at cluster level ( k = 178 ) ) ( figure 2 ) . quantitative magnetisation transfer . no significant between - group changes were seen in the qmt at time 2 or time 3 , with respect to time 1 . overall qmt measures showed stability across all participants over the course of the study in measures of all indices . in line with previous work , the main outcome of this study was that 6 weeks of computerised cognitive rehabilitation was associated with improvement in cognitive performance as measured on the sdmt . significant alterations in brain fmri activations during the n - back task were also seen at follow - up . the sdmt improvement in the treatment group was , however , not maintained after cessation of cognitive rehabilitation ( time 3 assessments ) although the functional mri changes were seen to persist at follow - up . the sdmt is among the most sensitive tests of slowed information processing speed in ms [ 34 , 35 ] and may also be a proxy for general cognitive impairment . compared to time 1 , the treatment group showed a significantly greater improvement in gain scores between baseline and early follow - up ( time 2 ) compared to the control group on the sdmt ( p = 0.005 ) . however , the gain scores between the groups at time 3 compared to baseline were not statistically significantly different . . it may be the case that cognitive rehabilitation does indeed result in improved cognitive performance but that maintenance of such improvement requires some form of ongoing intervention in the longer term . only one version of the bicams test battery has been validated in ms and thus was used in this study . reported test - retest coefficients on the bicams tests are excellent , suggesting that practice effects are negligible [ 31 , 32 ] . in addition , the experimental design was to compare two groups with identical testing schedules ; therefore the impact of practice effects on the results is likely to be minimal . qol is a complex construct influenced by a multitude of factors such as employment status , social networks , and perceptions of self - worth and self - efficacy . it is possible that cognitive rehabilitation has a positive impact on a number of these factors but such changes in such factors may take time to manifest as improvements in qol . further longitudinal analysis may be required to investigate this . in order to minimise practice effects associated with repeat testing , participants were not directly trained in the n - back task ; rather it was utilised as an outcome measure of working memory . it was anticipated that if cognitive rehabilitation was effective at improving working memory and attention , then the effects would be reflected on the performance on the n - back task . no differences were seen in the error rate between the groups during the n - back task at follow - up ; however , the error rate was low at baseline in both groups . the n - back fmri paradigm in our study cohort was , however , associated with robust baseline cortical activations ( in particular within the dlpfc and posterior parietal cortex ) in keeping with known working memory networks . a significant group - by - time interaction was seen with the treatment group exhibiting increased activation in the bilateral prefrontal cortex and right temporoparietal regions relative to control group at time 3 ( p < 0.05fwecorr ) . changes in functional activation within these regions within the treatment group are felt to be functionally relevant with respect to cognitive rehabilitation . it has been shown that the prefrontal cortex is critical in the executive control of working memory and has a role in response inhibition [ 38 , 39 ] . effective organisation of working memory may attenuate task difficulty resulting in improved working memory performance . a right hemisphere dominant ventral attentional network consisting of the temporoparietal junction , ventral prefrontal cortex , and anterior insula previous work in ms has indicated that attention may be one of the domains most amenable to rehabilitation . many of the computer - training tasks involve sustained attention and it might be postulated that the increased activation seen in the right temporoparietal region at follow - up in the treatment group is as a result of improved efficiency of this network . interestingly , the evolution of much activity on fmri developed after cessation of the active intervention phase . this solidification of neural networks may extend to areas / networks outside those directly trained and may explain why working memory centres such as the prefrontal cortex were seen to be persistently active after cessation of formal training . debate remains however as to the possible interplay between adaptive and maladaptive responses during functional brain reorganisation . the discrepancy between the apparent lack of clinical difference between the groups at time 3 and the sustained fmri effect at time 3 may reflect the fact that bicams does not adequately measure working memory which is primarily domain utilised during the n - back fmri paradigm . some studies have identified structural changes on diffusion tensor imaging as a result of rehabilitation in the context of physiotherapy [ 42 , 43 ] . we did not detect any structural change on qmt after training . due to the short duration of follow - up , this is not entirely surprising . functional alterations in cortical activity may subsequently modulate brain structure at the microstructural level but such changes in structural brain architecture might only be detectable over the longer term . in contrast to many previous studies , which often rely on one - to - one or outpatient administered cognitive rehabilitation , this study sought to explore whether a home - based approach to cognitive rehabilitation was feasible . a home - based approach to cognitive rehabilitation is significantly less resource intensive and may pave the way to greater access for a greater number of patients to such interventions in the future firstly , the groups were relatively small and there was a dropout of patients mainly in the control group between time 2 and time 3 . there was heterogeneity with regard to the cognitive domains that showed deficits among participants in the study . it is likely therefore that they may not have benefited from the rehabilitation in the same way . as the study was largely exploratory in nature , it utilised an open design and is therefore subject to a number of limitations inherent to this type of design . for pragmatic reasons blinding of the investigating neurologist was not established due to the potential need for interaction between patient and investigator . this does present the potential for observer bias , particularly where repeat testing is required . spm analysis of mri data offers objective , largely automated measures , which are independent of measurement bias . investigator blinding was maintained for any methods such as assessment of white matter lesion volumes that involved manual interpretation . in many respects , the sdmt may provide a proxy for overall cognitive functioning but a more detailed cognitive assessment of the domains directly trained may have provided additional insight into effectiveness of cognitive training . bicams is primarily designed as a screening tool for cognitive impairment in ms assessing a limited number of domains . however , strong ecological validity has been demonstrated in relation to everyday task performance and employment , suggesting that the three domains are strongly predictive of comprehensive real - world performance [ 45 , 46 ] . bicams may not necessarily be sensitive to change over the short - term , although the reported test - retest coefficients are excellent which would suggest sensitivity over this period [ 31 , 32 , 47 ] . the lack of significant change in qmt measurements , however , suggests that the microstructural changes thought to underpin adaptive responses may , at present , be beyond the resolution of even the most advanced mri techniques or not manifest within the timescale of this study . additional follow - up of this cohort is planned to determine what , if any , changes are observed in terms of both cortical activation as measured by fmri and structural changes measurable with qmt . longer - term studies may also provide insight into the true functional impact of cognitive rehabilitation such as maintenance of employment .

aim . to explore the efficacy of home - based , computerised , cognitive rehabilitation in patients with multiple sclerosis using neuropsychological assessment and advanced structural and functional magnetic resonance imaging ( fmri ) . methods . 38 patients with ms and cognitive impairment on the brief international cognitive assessment for ms ( bicams ) were enrolled . patients were randomised to undergo 45 minutes of computerised cognitive rehabilitation using rehacom software ( n = 19 ) three times weekly for six weeks or to a control condition ( n = 19 ) . neuropsychological and mri data were obtained at baseline ( time 1 ) , following the 6-week intervention ( time 2 ) , and after a further twelve weeks ( time 3 ) . cortical activations were explored using fmri and microstructural changes were explored using quantitative magnetisation transfer ( qmt ) imaging . results . the treatment group showed a greater improvement in sdmt gain scores between baseline and time 2 compared to the control group ( p = 0.005 ) . the treatment group exhibited increased activation in the bilateral prefrontal cortex and right temporoparietal regions relative to control group at time 3 ( p < 0.05fwe corrected ) . no significant changes were observed on qmt . conclusion . this study supports the hypothesis that home - based , computerised , cognitive rehabilitation may be effective in improving cognitive performance in patients with ms . clinical trials registration is isrctn54901925 .

1. Introduction 2. Subjects and Method 3. Results 4. Discussion

teratomas in the head and neck account for 29% of all teratomas at birth and teratomas in the oropharynx are extremely rare . here , we present a case of oropharyngeal teratoma that was diagnosed antenatally . a 25-year - old female ( gravida 2 para 1 ) was seen by a radiologist for evaluation of polyhydraminos . ultrasound evaluation of the fetus revealed no significant abnormalities [ figure 1 ] . fetal spine and facial structure were normal . amniotic fluid volume was greater than normal , amniotic fluid index ( afi ) measured 45 cm . some problem in fetal swallowing due to small stomach and polyhydraminos was suspected . a fetal magnetic resonance imaging ( mri ) was done to evaluate cause of acute polyhydramnios and relatively small size of the stomach . ultrasound image of fetal face in sagittal projection shows shadow from the hard palate ( arrow ) obscuring the pharyngeal mass . fetal mri done on 1.5 t philips achieva magnet with t2w turbo spin echo sequences and balanced turbo field echo sequences . sagittal [ figure 2 ] and axial [ figure 3 ] images show well - defined mixed intensity lesion with cystic component measuring about 4.5 3.5 cm in the oropharyngeal region . no obvious sign of invasion of hard palate or nasopharynx was noted [ figure 2 ] . sagittal image of fetal mri at 34 weeks gestation shows well - defined mixed intensity lesion with cystic component measuring about 4.5 3.5 cm in oropharyngeal region ( arrow ) . axial image of fetal mri at 34 weeks of gestation shows well - defined mixed intensity lesion with cystic component measuring about 4.5 3.5 cm in oropharyngeal region ( arrow ) . because of surgery for tumor removal was planned and done on the fourth day after birth . at surgery , well - defined lobulated tumor measuring about 4.5 3.4 cm arising from palate was resected [ figure 4 ] and sent for histopathology examination . after 4 weeks , the baby 's serum alfa - feto - proteins were found to be elevated to 600 ng / ml . parents were advised to consult a medical oncologist , who advised follow - up after 6 months . after 6 months , positron emission tomography - computed tomography [ figure 7 ] was done to rule any recurrence . serum alpha - feto - protein was repeated and found to be 180 ng / ml . medical oncologist advised chemotherapy and radiation but the parents did not agree to this treatment . presently , the boy is 27 months old and doing well without any clinical symptoms . histopathology examination of the surgically removed mass shows mass composed of mature and immature elements of teratoma . easiest immature element to identify is neuroepithelium with columnar cells forming rossettes and tubules ( arrow ) . surgically removed mass stained with hematoxylin and eosin at 45 shows mature cartilaginous tissue ( arrow ) . pet ct image of the child , 6-months posttreatment shows metabolic activity in the tongue and adenoids are physiological . soft palate ( arrow ) , where the original tumor was resected is normal indicating no recurrence of tumor . teratoma is a true neoplasia containing cells of all the three germ lines and represent 25 - 30% of all neonatal tumors . teratomas can occur in any part of the body but are most common in the sacro coccygeal region . teratomas of head and neck region are extremely rare and are most common in the cervical region followed by the oropharyngeal region . oropharyngeal teratomas appear as heterogenous masses with solid and cystic components and are believed to be result of migration and entrapment of mesoderm and endoderm with ectoderm during embryogenesis . sometimes the mass can not be appreciated in usg due to the presence of shadows . in our case fetal mri was helpful in diagnosing and revealing the extent of oropharyngeal involvement , defining tracheal anatomy , and excluding intracranial involvement . mri is more useful to delineate soft tissues and can give a near accurate assessment of oropharyngeal extension . ct was not done antenatally in our case as the tumor was well - defined and limited to oropharynx and also to minimize radiation exposure to our patient . pet ct was done on the baby at 6 months of age to look for recurrence or metastasis . oropharyngeal teratoma can encroach airway and cause respiratory distress , progressive dysphagia , and aspiration pneumonia due to impaired swallowing mechanism . antenatal diagnoses of oropharyngeal teratoma is essential as it can radically change the treatment and allows the medical team to plan for ex utero intrapartum treatment ( exit ) or management during early neonatal period with surgery . exit is a technique designed to allow partial fetal delivery via cesarean section while a safe fetal airway is established . upon delivery of the fetal head , neck , and one or both upper limbs , a stepwise , sequential attempt to secure the neonates airway is performed via laryngoscopy , bronchoscopy , tracheostomy , or even resection of the obstructing mass . the placenta remains in utero to maintain feto - placental circulation and subsequently the continuance of fetal oxygenation . once the newborn 's airway is secured , the umbilical cord is clamped and delivery of the infant completed . this technique was first described in 1990 by zerella and finberg to improve the survival of fetuses with head and neck teratomas and also by levine et al . the use of exit has also proven to be effective for the management of fetal airway obstruction due to a variety of other causes including congenital high airway obstruction syndrome ( chaos ) , intrathoracic masses , and congenital diaphragmatic hernias . mri proved superior to ultrasound in diagnosing and delineating exact tumor location , size , extension , and was crucial in determining timing and mode of delivery . oro - pharyngeal teratoma because of its potential to cause airway obstruction , though rare , should be considered while investigating polyhydramnios . early antenatal diagnosis allows the medical team to plan mode of delivery , management through exit procedure or surgery during the early neonatal period and ultimately leads to better outcome .

oropharyngeal teratomas are extremely rare congenital tumors and have an incidence rate of one in 35,000 - 200,000 newborn babies / neonates . oropharyngeal teratomas may cause life threatening airway obstruction to a newborn . early diagnosis with ultrasound and magnetic resonance imaging ( mri ) is essential to plan management . here , we present a rare case of oropharyngeal true teratoma diagnosed with mri antenatally in a fetus at 34 weeks of gestation in a 25-year - old female who was being evaluated for polyhydraminos . we found mri to be more helpful for antenatal diagnosis , counseling , and management than ultrasonography and computed tomography ( ct ) .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION

neuroblastoma , a pediatric malignancy of the developing sympathetic nervous system , is a multifaceted disease with biological and clinical courses ranging from relentless progression to spontaneous regression or differentiation into benign ganglioneuroma . given these different phenotypes , therapeutic regimens vary between wait - and - see approaches to the most intense multimodal treatment . accurate prediction of the natural clinical course of each individual patient at the time of diagnosis is therefore an essential prerequisite for therapeutic decision - making . clinical variables such as stage of the disease and age of the patient at diagnosis are well established predictors of neuroblastoma outcome . in addition , non - random cytogenetic aberrations have been shown to be associated with clinical courses in neuroblastoma and are increasingly used in risk stratification systems ( reviewed in [ 1 - 3 ] ) . whereas amplification of the oncogene mycn and several other genomic alterations , such as loss of the chromosomal regions 1p and 11q or gain of 17q , have been shown to be strong markers of poor outcome , hyper - diploidy of the tumor cells is associated with a favorable clinical phenotype . however , whereas current risk estimation systems for neuroblastoma mostly succeed in discriminating patients with divergent outcomes , further improvements are required to prevent fatal events in low - risk and intermediate - risk groups and to avoid unnecessary cytotoxic treatment of patients in whom spontaneous regression will occur . the advent of microarray - based comparative genomic hybridization ( array - cgh ) has facilitated the analysis of chromosomal alterations in the cancer genome , providing pangenomic alteration profiles with exceptional spatial resolution in a single experiment . initial array - cgh studies of primary neuroblastomas confirmed the clinical significance of known copy number variations and narrowed down breakpoint regions of non - random chromosome aberrations . in a recent survey , caren et al . investigated 165 primary neuroblastomas using affymetrix 250k single nucleotide polymorphism arrays and compared the survival of patient subgroups defined by genomic alterations . patients with only numerical chromosomal aberrations and no other alteration had a favorable long - term outcome . in contrast , the survival of patients characterized by mycn amplification , loss of 11q or gain of 17q was considerably worse , whereas no death or disease was observed in patients with tumors harboring segmental chromosome alterations other than those previously mentioned . these findings support results from previous studies indicating that a limited number of predictive genomic alterations are sufficient for risk assessment of neuroblastoma patients ( reviewed in ) . , however , indicated that global genomic profiles may add significant prognostic information to current neuroblastoma risk estimation . in this study , the prognostic significance of overall genomic alterations was investigated in a cohort of 493 primary neuroblastomas by bacterial artificial chromosome array - cgh . whereas patients with tumors showing only numerical chromosome aberrations had an excellent survival , those with tumors harboring segmental genomic alterations showed a high risk of relapse and a poor outcome . amplification of mycn was confirmed to be a strong predictor of adverse outcome , but other single genomic alterations , such as loss of 11q or gain of 17q , were overridden by the presence of any kind of segmental alterations in multivariate analyses . another significant difference between these two studies was noticed in the fraction of tumors with only numerical chromosome alterations . in the work of janoueix - lerosey this subgroup comprised 47% of the tumors , whereas it accounted for 28% of the cases in the study of caren et al . . similar to the latter findings , this subgroup constituted 21% of the cases in a preliminary analysis of our array - cgh data . these differences might in part be attributed to distinct compositions of the cohorts under investigation . however , they may also result from the lower spatial resolution of the microarrays used in the study of janoueix - lerosey et al . than in the other surveys , which might have resulted in the detection of a smaller fraction of tumors with small gains or deletions and in the classification of fewer patients into subgroups with segmental aberrations . taken together , although the results of these two comprehensive studies are promising with respect to prognostic classification of neuroblastoma using array - cgh , the clinical significance of global genomic alterations needs to be further evaluated in independent studies and compared with current risk estimation strategies . an inherent disadvantage of array - cgh analysis is its propensity to disregard low - level copy number losses or gains in samples with a high proportion of contaminating stromal cells . this potential bias has been taken into account by janoueix - lerosey et al . by analyzing only samples with a tumor content of at least 60% , whereas the tumor content was not specified in the study of caren et al . . this discrepancy in the experimental set - up may have resulted in a higher fraction of flat genomic profiles ( that is , with no alterations ) in the latter study ( 19% ) as compared with the former study ( 4% ) . this suggestion is supported by the finding of only 2% flat genomic profiles in another study in which a tumor content of 60% had been used for sample selection . because of the rare occurrence of neuroblastomas without any chromosomal alterations , the clinical outcome of these patients has so far remained elusive . nevertheless , the routine application of array - cgh in clinical practice might be considerably limited by the issue of contaminating stromal cells , because defined thresholds of tumor content will a priori exclude a substantial fraction of samples from the analysis . in addition , genomic heterogeneity within a single tumor might be missed by array - cgh analysis . although the frequency and the clinical consequences of genomic heterogeneity in neuroblastoma need to be clarified , it might be advisable to complement array - cgh analyses of neuroblastoma samples with methods for detecting chromosomal aberrations on the single cell level , such as fluorescence in situ hybridization , to evaluate the concordance of the results and to validate the clinical implications in large patient cohorts . as an alternative to the overall genomic pattern as a prognostic marker , several reports have provided compelling evidence that specific gene - expression patterns can predict the natural courses of neuroblastoma patients with unprecedented accuracy [ 11 - 15 ] . these studies have shown that gene - expression - based classifiers can distinguish patients with contrasting clinical courses in almost all prognostic subgroups , including those defined by prognostic genomic makers such as mycn amplification or loss of 11q . a systematic comparison of global genomic and transcriptomic classification results is still lacking , however . the routine application of expression - based prognostic markers in clinical practice might be limited by the instability of mrna in comparison with dna , which will require strict adherence to elaborated standard operating procedures in the processing of tumor samples . in addition , similar to array - cgh approaches , classification results of gene - expression - based predictors might be influenced by the relative amounts of stromal cells in the samples . in contrast to classifications based on genomic alterations , however , the prognostic significance of gene - expression profiles might be conferred by the stromal cells themselves , as has been described in other cancer entities , such as lymphoma or breast cancer . re - evaluation of the gene functions from existing gene - expression classifiers and validation of the predictive accuracy in neuroblastoma cohorts with low tumor contents will reveal the contribution of non - tumorous cells to the prognostic validity of gene - expression - based classifiers in neuroblastoma . because of the strong association of numerical and segmental cytogenetic alterations with patient outcome , it has been suggested that neuroblastoma comprises two distinct clinico - genetic classes . the first type corresponds to patients with favorable outcome and is characterized by mitotic dysfunction leading to whole chromosome gains or losses , whereas the second type corresponds to aggressive disease and is characterized by defects in maintaining genomic stability leading to segmental chromosome alterations . given the prevalence of mycn amplification and loss of 11q in unfavorable neuroblastoma , and the inverse correlation between these aberrations in high - risk neuroblastoma , it has been furthermore hypothesized that the natural behavior of high - risk tumors is mainly conferred by these two aberrations . in the work of caren , this suggestion was substantiated by the finding that patients with mycn amplification and those with loss of 11q differed significantly in both their age at diagnosis and their median survival time . however , whereas the influence of mycn amplification on aggressive growth in neuroblastoma has been mostly proven , the effect of 11q loss on neuroblastoma biology is less clear . in a recent integrative genomics analysis of primary neuroblastoma , it was demonstrated that tumors with loss of 11q make up two distinct biological subgroups that differ in their clinical phenotype as well as in their gene - expression patterns . these results suggest that 11q loss is not a primary determinant of neuroblastoma tumor behavior , indicating that the biology of neuroblastoma is more complex than the association of genomic alterations with patient outcome might suggest . we expect that the emerging application of next - generation sequencing will unravel novel genomic alterations that contribute to the programming of the various neuroblastoma phenotypes , which will lead to a refined molecular classification of this malignancy . the prognostic significance of specific single genomic markers is well established in neuroblastoma , and has led to their implementation in current risk assessment . recent studies suggested that overall genomic profiles may further improve neuroblastoma risk estimation . before routine use in clinical practice , the prognostic impact of global genomic alterations needs to be validated prospectively and compared with current stratification systems . in addition , it needs to be evaluated whether analysis of overall genomic profiles , gene - expression - based classifiers , or the combination of both will contribute most to an improved risk estimation of children with neuroblastoma . in any case , such analysis will require elaborate standard operating procedures to avoid technical pitfalls and defined interpretation guidelines to ensure reliable treatment stratification of each individual patient in future clinical trials . this work was supported by grants from the bundesministerium fr bildung und forschung ( bmbf ) through the national genome research network plus ( ngfnplus , grant 01gs0895 ) and the frdergesellschaft kinderkrebs - neuroblastom - forschung e.v .

specific genomic alterations , such as loss of the chromosomal region 11q or amplification of the oncogene mycn , are well established markers of poor outcome in neuroblastoma . the advent of microarray - based comparative genomic hybridization ( array - cgh ) has enabled the analysis of pangenomic alteration profiles in the cancer genome , offering the possibility of identifying new prognostic markers from complex aberration patterns . results from recent studies examining large primary neuroblastoma cohorts by array - cgh show that global genomic profiles may add significant prognostic information . here , we discuss potential implications for risk estimation of neuroblastoma patients in clinical practice as well as for the understanding of neuroblastoma pathogenesis .

The clinical heterogeneity of neuroblastoma Clinical significance of complex chromosomal alterations in neuroblastoma Biological classification of neuroblastoma by chromosome alterations The future: will genomic profiles have prognostic value in the clinic? Abbreviations Competing interests Authors' contributions Acknowledgements

psoriasis is a common inflammatory skin disease characterized by abnormal keratinocyte proliferation and differentiation , angiogenesis , immune activation , and inflammation . although there is evidence for immune activation with lymphocyte infiltration in the psoriatic epidermis , there also exists evidence of primary keratinocyte defects . in the psoriatic epidermis , proteins that are expressed in the early stage of keratinocyte differentiation such as keratinocyte transglutaminase are expressed earlier and in a greater amount . in contrast , the expression of proteins that are usually expressed in the later stages of kertinocyte differentiation such as filaggrin are downregulated in the epidermis of psoriatic plaques . there is accumulating evidence that proteins that mediate intracellular adhesions are involved in keratinocyte differentiation . one candidate for this is beta - catenin ( -catenin ) a 94-kda protein that participates in intercellular adhesion , -catenin links e - cadherin to the actin cytoskeleton through alpha - catenin . overexpression of the basal layer cadherins in suprabasal epidermis leads to increased cytoplasmic activity of -catenin and increased -catenin transcriptional activation . the role of -catenin in regulating keratinocyte stem cell differentiation and hair follicle morphogenesis has been extensively reported . studied -catenin in human skin tumors and noted that -catenin was occasionally present in both nuclear and cytoplasmic locations in the basal cells , scattered differentiated keratinocytes and luminal cells of the sebaceous glands . to gain an insight into the role of -catenin on the regulation of keratinocytes and further investigate the pathogenesis of psoriasis , we compared the expression of -catenin in normal human and psoriatic skin . this study was carried out in 20 psoriasis vulgaris patients with 10 non - psoriatic subjects as controls . these patients were selected randomly from the dermatology outpatient clinic , faculty of medicine , menoufia university and sheibin el - kom educational hospital during the period from april 2009 to june 2010 . all patients instructed to stop all forms of medication one month prior to optaining a skin biopsy . two skin biopsies were taken - one from the representative psoriatic lesion and the other from nonlesional skin under local xylocaine anesthesia . the diagnosis of psoriasis was based on both clinical and histopathological examination by hematoxylin and eosin stain . the primary antibody was rabbit polyclonal antibody ( 0.1 ml ) ( lab - vision , westinghouse , usa ) . beta - catenin expression was assessed according to : intensity : weak ( + ) moderate ( + + ) or strong expression ( + + + ) cell counting : the number of -catenin positive nuclei and total nuclei in the upper half of the suprabasal region were counted in four individual fields from each biopsy specimen . then the percent positivity was calculated . for further statistical analysis , cases were divided into two groups : suprabasal nuclear -catenin staining < 50% , and > 50%location : membranous or nuclear location of expression . intensity : weak ( + ) moderate ( + + ) or strong expression ( + + + ) cell counting : the number of -catenin positive nuclei and total nuclei in the upper half of the suprabasal region were counted in four individual fields from each biopsy specimen . then the percent positivity was calculated . for further statistical analysis , cases were divided into two groups : suprabasal nuclear -catenin staining < 50% , and > 50% location : membranous or nuclear location of expression . data was collected and statistically analyzed using statistical package ( ibm corporation ) for social sciences version 9 program and statview software ( ibm corporation ) . the following statistical measures were employed : arithmetic mean ( x ) for central tendencypercentage ( % ) . b - analytic statistics : student 's t - test to compare two quantitative variableschi - square test ( ) to compare between qualitative variables p < 0.05 was considered as statistically significant ( s ) . b - analytic statistics : student 's t - test to compare two quantitative variableschi - square test ( ) to compare between qualitative variables p < 0.05 was considered as statistically significant ( s ) . student 's t - test to compare two quantitative variables chi - square test ( ) to compare between qualitative variables p < 0.05 was considered as statistically significant ( s ) . the primary antibody was rabbit polyclonal antibody ( 0.1 ml ) ( lab - vision , westinghouse , usa ) . beta - catenin expression was assessed according to : intensity : weak ( + ) moderate ( + + ) or strong expression ( + + + ) cell counting : the number of -catenin positive nuclei and total nuclei in the upper half of the suprabasal region were counted in four individual fields from each biopsy specimen . then the percent positivity was calculated . for further statistical analysis , cases were divided into two groups : suprabasal nuclear -catenin staining < 50% , and > 50%location : membranous or nuclear location of expression . intensity : weak ( + ) moderate ( + + ) or strong expression ( + + + ) cell counting : the number of -catenin positive nuclei and total nuclei in the upper half of the suprabasal region were counted in four individual fields from each biopsy specimen . then the percent positivity was calculated . for further statistical analysis , cases were divided into two groups : suprabasal nuclear -catenin staining < 50% , and > 50% location : membranous or nuclear location of expression . data was collected and statistically analyzed using statistical package ( ibm corporation ) for social sciences version 9 program and statview software ( ibm corporation ) . the following statistical measures were employed : arithmetic mean ( x ) for central tendencypercentage ( % ) . b - analytic statistics : student 's t - test to compare two quantitative variableschi - square test ( ) to compare between qualitative variables p < 0.05 was considered as statistically significant ( s ) . b - analytic statistics : student 's t - test to compare two quantitative variableschi - square test ( ) to compare between qualitative variables p < 0.05 was considered as statistically significant ( s ) . student 's t - test to compare two quantitative variables chi - square test ( ) to compare between qualitative variables p < 0.05 was considered as statistically significant ( s ) . the age of the patients ranged from 12 to 75 years with a mean age 49.7 15.2 years . among 20 patients suffering from psoriasis , the duration of the disease varied from 6 months to 19 years with a mean of 5.67 0.5 years . in all clinically diagnosed patients , histopathology revealed parakeratosis , absent granular cell layer , munro microabscesses , acanthosis , spongiosis , and papillomatosis in the epidermis . dermal capillary dilatation and inflammatory cells were present in all cases and edema in some cases [ figure 1a and b ] . ( a ) psoriasis lesion with munro microabscess in psoriasis ( h and e , 200 ) . ( b ) psoriasis lesion with epidermal , parakeratosis , acanthosis , spongiosis , absent granular cell layer and dermal capillary dilatation and inflammatory cells ( h and e , 400 ) in the present study , we made a comparative study of the structural organization of the epidermis and in particular the intercellular junctions between lesional and nonlesional skin of psoriasis patients . the presence and distribution of -catenin was analyzed by immunohistochemistry of normal skin , and lesional and nonlesional psoriatic skin . in the normal epidermis -catenin was detected solely on the cell membrane of the basal cells and lower spinous cells [ table 1 , figure 2a ] . this protein showed a continuous well defined distribution at the borders of these cells . in nonlesional psoriatic skin , there were a few suprabasal cells showing strong nuclear -catenin expression [ table 2 , figure 2b ] . in lesional psoriatic skin , there was a positive nuclear suprabasal -catenin expression and negative -catenin expression in parakeratotic cells [ table 2 , figure 3a ] . there was a strong nuclear with membranous -catenin expression in psoriatic skin [ figure 3b ] . at the junction between nonlesional and lesional skin there was membranous -catenin expression in nonlesional skin and both membranous and nuclear -catenin expression in lesional psoriatic skin [ figure 4a ] . in nonlesional psoriatic skin , there was a membranous -catenin expression along with a positive dermal inflammatory component [ figure 4b ] . there was positive nuclear and cytoplasmic -catenin expression in normal hair follicles [ figure 4c ] . comparison between membranous beta - catenin staining expression in control , and lesional and nonlesional skin of psoriasis ( a ) skin of a normal control showing membranous beta - catenin ( -catenin ) staining expression and few suprabasal nuclear stains ( ips , 200 ) . ( b ) few strong nuclear suprabasal -catenin staining expression in nonlesional skin of psoriasis ( immunoperioxedase stain , 200 ) comparison between suprabasal nuclear beta - catenin staining expression in controls , and lesional and nonlesional skin of psoriasis ( a ) nuclear suprabasal beta - catenin ( -catenin ) staining expression in all layers of epidermis except the parakeratotic and cornified layers of a psoriasis lesion ( immunoperioxedase stain , 400 ) . ( b ) strong nuclear with membranous -catenin expression and negative expression in the parakeratotic layer of a psoriasis lesion ( immunoperioxedase stain , 400 ) ( a ) junction between nonlesional ( right ) and lesional ( left ) skin of psoriasis showing only membranous staining ( nonlesional ) while both membranous and nuclear beta - catenin ( -catenin ) expression is seen in lesional skin ( ips , 400 ) . ( b ) membranous -catenin expression together with positive dermal inflammatory components in nonlesional skin of psoriasis ( immunoperioxedase stain , 200 ) . ( c ) nuclear and cytoplasmic -catenin expression in normal hair follicle matrix cell ( immunoperioxedase stain , 400 ) in the present study , we made a comparative study of the structural organization of the epidermis and in particular the intercellular junctions between lesional and nonlesional skin of psoriasis patients . the presence and distribution of -catenin was analyzed by immunohistochemistry of normal skin , and lesional and nonlesional psoriatic skin . in the normal epidermis -catenin was detected solely on the cell membrane of the basal cells and lower spinous cells [ table 1 , figure 2a ] . this protein showed a continuous well defined distribution at the borders of these cells . in nonlesional psoriatic skin , there were a few suprabasal cells showing strong nuclear -catenin expression [ table 2 , figure 2b ] . in lesional psoriatic skin , there was a positive nuclear suprabasal -catenin expression and negative -catenin expression in parakeratotic cells [ table 2 , figure 3a ] . there was a strong nuclear with membranous -catenin expression in psoriatic skin [ figure 3b ] . at the junction between nonlesional and lesional skin there was membranous -catenin expression in nonlesional skin and both membranous and nuclear -catenin expression in lesional psoriatic skin [ figure 4a ] . in nonlesional psoriatic skin , there was a membranous -catenin expression along with a positive dermal inflammatory component [ figure 4b ] . there was positive nuclear and cytoplasmic -catenin expression in normal hair follicles [ figure 4c ] . comparison between membranous beta - catenin staining expression in control , and lesional and nonlesional skin of psoriasis ( a ) skin of a normal control showing membranous beta - catenin ( -catenin ) staining expression and few suprabasal nuclear stains ( ips , 200 ) . ( b ) few strong nuclear suprabasal -catenin staining expression in nonlesional skin of psoriasis ( immunoperioxedase stain , 200 ) comparison between suprabasal nuclear beta - catenin staining expression in controls , and lesional and nonlesional skin of psoriasis ( a ) nuclear suprabasal beta - catenin ( -catenin ) staining expression in all layers of epidermis except the parakeratotic and cornified layers of a psoriasis lesion ( immunoperioxedase stain , 400 ) . ( b ) strong nuclear with membranous -catenin expression and negative expression in the parakeratotic layer of a psoriasis lesion ( immunoperioxedase stain , 400 ) ( a ) junction between nonlesional ( right ) and lesional ( left ) skin of psoriasis showing only membranous staining ( nonlesional ) while both membranous and nuclear beta - catenin ( -catenin ) expression is seen in lesional skin ( ips , 400 ) . ( b ) membranous -catenin expression together with positive dermal inflammatory components in nonlesional skin of psoriasis ( immunoperioxedase stain , 200 ) . ( c ) nuclear and cytoplasmic -catenin expression in normal hair follicle matrix cell ( immunoperioxedase stain , 400 ) catenins are cell - cell adhesion molecules that also maintain intracellular adhesiveness through cytoplasmic catenin proteins that bind the microfilaments to the cytoskeleton . -catenin is not required for keratincoyte proliferation , but has been shown to regulate keratinocyte stem cells and hair follicle morophogenesis . with regard to membranous -catenin staining expression , it was significantly demonstrated in the control group and nonlesional areas in psoriatics as compared to lesional areas in psoriatics . this reflects its role of -catenin as an adhesion molecule in uninvolved and normal skin . nuclear -catenin staining expression was significantly demonstrated in lesional and nonlesional skin of psoriatics as compared to controls . this finding agreed with previous work done by stojadinovic et al . , who studied the role of -catenin in the inhibition of epithelization and wound healing . furthermore in the study done by butz and larue , it was found that -catenin was detected only on the cell membrane of the normal epidermis and normal hair follicles with no nuclear -catenin staining detected . our findings are in agreement with those of hampton et al . , who found increased nuclear -catenin in suprabasal lesional psoriatic epidermis . some nuclear -catenin was also detected in nonlesional psoriatic skin , but much less than in lesional psoriatic skin . the distribution of -catenin in the membrane of normal cells more than in the nuclear location may be due to its role in the establishment and regulation of adherens junctions by interaction with e - cadherin through alpha catenin with the actin cytoskeleton . furthermore , some studies suggest that -catenin may be translocated from the nucleus back to the cell membrane , presumably through a soluble , cytosolic intermediate state . watt and collins , found the same result in their study of the role of nuclear -catenin in colorectal tumors . watabe and miyazono , studied nuclear localization of -catenin in the hair matrix cells and differentiated keratinocytes . zhou et al . , showed a significant increase in nuclear -catenin within the suprabasal layers in the involved psoriatic epidermis . also , zhou et al . , observed a marked decrease in membranous -catenin at the same level as increased in the nuclear -catenin . they suggested that in involved suprabasal psoriatic epidermis , an excess amount of free unphosphrylated -catenin enters the cytoplasm and subsequently translocates to the nucleus . zhou et al . , showed in their study that there was no significant increase in the expression of -catenin in both membranous and nuclear locations in nonlesional suprabasal cells of psoriatics . normal skin and the uninvolved psoriatic epidermis might transcriptionally active -catenin released into the cytoplasm that would likely be rapidly targeted for ubiquitination to prevent abnormal signaling events . this leads to little nuclear -catenin in uninvolved or normal suprabasal epidermis . in a study done by li et al . , who studied the expression of e - cadherin and -catenin in the lesional skin of patients with active psoriasis , there was downregulation of -catenin expression , which was found in the granular layer and basal layer of the psoriatic lesions only . in normal control group -catenin was mainly demonstrated as membranous pattern of expression ; this reflects its role as an adhesion molecule . the downregulation of membranous -catenin expression in lesional psoriatic skin suggests that it could be a useful phenotypic marker for the hyperprolifration of keratinocytes in psoriasis . moreover , the mild downregulation of membranous -catenin expression in nonlesional psoriatic skin may provide clues about impending structural abnormalities in the pathogenesis of psoriasis , providing an early diagnostic indication for evolution to a generalized form of the disease . nuclear -catenin expression was not found in the control group but demonstrated in lesional and moderately in nonlesional skin of psoriatics reflecting its role in kerationcyte proliferation . the nuclear and cytoplasmic expression of -catenin in normal hair follicle provides evidence suggesting that it plays an important role in keratinocyte differentiation .

background : psoriasis is a common inflammatory skin disease characterized by abnormal keratinocyte proliferation and differentiation . beta - catenin participates in intercellular adhesion . catenins are proteins found in complexes with cadherin cell adhesion molecules of cells . the role of catenin in regulating keratinocyte stem cell differentiation and hair follicle morphogenesis has been extensively reported.aims and objectives : is to study -catenin expression in lesional and non - lesional psoriatic skin to throw light upon its possible role in the pathogenesis of psoriasis.materials and methods : biopsies were taken from 20 patients with psoriasis vulgaris and from 10 normal controls . the distribution of beta catenin was investigated using polycolonal rabbits b - catenin antibody-1 by immunohistochemical method.results:in this study membranous -catenin expression was significantly demonstrated in the control group then the non - lesional areas in comparison to the lesional areas ( p < 0.001 ) . nuclear -catenin staining expression was significantly more demonstrated in lesional and non - lesional areas in comparison to the control cases ( p < 0.001).conclusions : the down regulation of membranous -catenin expression in lesional psoriatic skin might reflect a useful phenotypic marker of hyperprolifration of keratinocytes in psoriasis . moreover , the mild down regulation of membranous -catenin expression in non lesional psoriatic skin may provide clues about incipient structural abnormalities in the pathogenesis of psoriasis , providing an early diagnostic indicator for evolution to a generalized form of the disease . nuclear -catenin expression was not found in the control group but was demonstrated in lesional and moderately in non - lesional reflecting its role in kerationcyte proliferation .

INTRODUCTION MATERIALS AND METHODS Immunohistopathological Interpretation of beta-catenin expression Statistical analysis RESULTS Immunohistochemical results of beta-catenin staining expression DISCUSSION CONCLUSION

in the previous issue of critical care , o'dwyer and colleagues report the results of a study in 47 patients with septic shock . asymmetric dimethylarginine ( adma ) levels were elevated in these patients at the time of admission to the intensive care unit ( icu ) as compared with a group of 10 healthy controls . adma levels in septic patients directly correlated with the sequential organ failure assessment scores , with the degree of acidaemia and lactaemia , and with vasopressor requirements . interestingly enough , adma levels were higher in patients requiring vasoactive infusions than in those not requiring vasoactive infusions . nonsurvivors tended to have higher adma plasma levels on days 1 and 7 of icu treatment as compared with survivors , although this trend was insignificant in the small patient group . the authors also demonstrated that a genetic polymorphism in the promoter region of the dimethylarginine dimethylaminohydrolase ( ddah ) ii gene was significantly associated with adma levels in these patients , suggesting that the genetically anchored dysfunction of the enzyme that metabolises adma may importantly regulate adma levels . standard therapy for patients with sepsis includes icu admission , careful selection of antibiotic therapy , and , if needed , haemodynamic and ventilatory support . despite the use of these intensive therapies , nitric oxide ( no ) is referred to as a key mediator of vasodilatation and catecholamine resistance in septic shock . whereas in physiological conditions no is mainly formed in the endothelium at low rates by the activity of the constitutive , endothelial isoform of the enzyme no synthase , inflammatory stimuli such as bacterial lipopolysaccharides and cytokines released during sepsis result in a strong upregulation of an inducible isofom of no synthase ( inos ) . this isoform , once upregulated , releases huge amounts of no during prolonged time intervals . the inos - derived no contributes to pathogen elimination . no - induced vasodilatation , however , also contributes to widespread vascular loss of tone and has been implicated in the cardiovascular failure in septic shock . there has been controversy about the pathophysiological roles of inos and no in the development of septic shock after a phase iii trial with n - monomethyl - l - arginine , a nonselective no synthase inhibitor , resulted in excess mortality . indeed , experimental studies have suggested that inos - derived no plays an important role in host defence and organ protection . during the past decade knowledge has accumulated of endogenous compounds that inhibit no synthase activity , and thereby regulate no - dependent vascular function . the major molecule endogenously present in the circulation at concentrations sufficiently high to exert inhibitory effects on no synthesis in vivo is adma . a structural analogue of n - monomethyl - l - arginine , the long - known no synthase inhibitor , and of l - arginine adma levels have been shown to regulate no generation , endothelial function , and vascular resistance in animal models and in humans ( for recent reviews , see [ 8 - 10 ] ) . moreover , adma has evolved from prospective clinical trials as a novel cardiovascular risk marker . previous evidence has linked elevated adma levels to reduced kidney and liver function and to multiple organ failure in icu patients . moreover , prospective clinical data point to the fact that adma may be a superior predictive marker of icu death . we have recently shown that adma levels are prospectively associated with organ dysfunction and with the postoperative complication rate in patients undergoing major elective surgery ( unpublished data ) . in the battle between the invading pathogen and the host organism , no generation has to be kept in a delicate balance between ' live ' ( keeping no within boundaries for cardiovascular homeostasis ) and ' let die ' ( upregulating no to a sufficient degree for pathogen defence ) . here , upregulation of adma levels may be another long ignored but important regulator . upregulation of inos leads to the release of huge amounts of no and superoxide , which , by forming peroxynitrite , can nitrosylate proteins and thereby damage tissues . s - nitrosylation of ddah ii has been shown to downregulate this enzyme 's activity , leading to accumulation of adma . this may explain why adma levels were actually higher in patients with more vasopressor need in the study by o'dwyer and colleagues : vasopressor need identified patients with greater inos induction , which , in turn , may have resulted in ddah nitrosylation and thereby inactivation which finally resulted in higher adma levels that blocked the activities of both constitutive and inducible no synthases ( figure 2 ) . alternatively , compromised kidney and liver function secondary to tissue malperfusion may have led to reduced metabolic clearance of adma by ddahs . the study did not , however , report any data to support or refute any of these hypotheses . adma may interfere , by nonspecific inhibition of no synthases , with critical physiological functions , eventually giving rise to a cascade of organ dysfunction that may be fatal to the critically ill patient . with adma as a novel player in the game , understanding the role of no in the ' live and let die ' issue of septic shock has become even more complex . measuring adma levels should certainly be considered in future studies on no in critically ill patients . adma = asymmetric dimethylarginine ; ddah = dimethylarginine dimethylaminohydrolase ; icu = intensive care unit ; inos = inducible no synthase ; no = nitric oxide . rhb is named as an inventor on patents relating to endogenous inhibitors of the nitric oxide pathway and receives royalties from licenses thereof . structural formulae . schematic drawings of the structural formula of ( a ) l - arginine , the natural substrate of nitric oxide ( no ) synthase , ( b ) n - monomethyl - l - arginine , the no synthase inhibitor used in clinical trials , and ( c ) asymmetric dimethylarginine , the endogenous inhibitor of no synthases . the circle in ( a ) indicates the terminal guanidino nitrogen group , where no is cleaved by no synthase . potential interrelation of nitric oxide generation and the pathophysiology of sepsis . potential interrelation of nitric oxide ( no ) generation by inducible nitric oxide synthase ( inos ) and by asymmetric dimethylarginine ( adma ) and the pathophysiology of sepsis .

nitric oxide ( no ) is an important mediator of host defence and of vascular tone . in septic shock , upregulation of inducible no synthase leads to the production of vast amounts of no , which contribute to pathogen elimination but also to inappropriate vasodilation and to loss of vascular resistance . asymmetric dimethylarginine ( adma ) is an endogenous inhibitor of no synthases shown to contribute to the regulation of vascular tone . adma was recently identified as a marker of organ dysfunction and mortality in intensive care patients and as a novel cardiovascular risk factor . in the present issue of critical care , a study by o'dwyer and colleagues identifies adma as a potential regulator of no production in septic shock . being an inhibitor of no production , adma may at least partly counteract pathological hypotension , but at the same time may impair the no - dependent host defence . a mechanism is proposed by which the interplay between adma and inducible no synthase activity is mediated . adma levels should be determined in future studies evaluating the regulation of no in the intensive care setting .

None Abbreviations Competing interests Figures and Tables

a paradoxical embolism ( pde ) is defined as a systemic arterial embolism that enters from the venous thrombi into the arterial circulatory system through a right - to - left shunt . the most common right - to - left shunt , associated with pdes , is patent foramen ovale ( pfo ) , which has been reported as an important cause of cryptogenic strokes.1 - 3 ) in addition to the isolated cryptogenic strokes , related to pfos , the coexistence of pulmonary thromboembolisms ( pte ) and deep vein thromboses ( dvt ) have also been reported,4 - 6 ) as rare cases of arterial systemic embolic involvement through pfo.7 - 9 ) here , we report a rare case of cryptogenic stroke , caused by a pde through pfo , which is complicated with massive pte , dvt , and renal infarctions . a 52-year - old woman was transferred to our emergency room ( er ) with a 10-hour history of left - side weakness , and a sudden onset of slurred speech , with a clear mental state . her previous medical , medication , and familial histories were unremarkable , and she was a non - smoker . upon arrival in the er , she was hemodynamically stable and her electrocardiogram showed a normal sinus rhythm . however , a magnetic resonance diffusion image of her brain revealed an infarction of the right middle cerebral artery territory ( fig . 1a ) , and a magnetic resonance angiogram of the cerebral and carotid arteries showed a total obstruction of the genu branch of the right middle cerebral artery ( fig . she was immediately admitted to the neurologic intensive care unit , with a diagnosis of acute cerebral infarction . further studies were then ordered , since the etiology of the cerebral infarction was unclear . to evaluate the intracardiac source of the embolism , a transthoracic echocardiogram ( tte ) and transesophageal echocardiogram ( tee ) were performed after 1 week . the tte revealed no evidence of embolism ; however , the tee revealed separation of the septum primum from the septum secundum that constitutes a pfo , up to 6 mm , according to the pressure variance ( fig . 2a ) , and an agitated saline injection test was used to identify the right - to - left microbubble shunting , during a valsalva maneuver used to confirm the pfo presence ( fig . 2b ) . based on these tests , we subsequently recommended an anticoagulation therapy based on warfarinization . the day after these initial tests , she then displayed severe hypoxemia and hypotension , and was immediately intubated and initiated the ventilator care . a computed tomography ( ct ) image of her chest was taken after starting the ventilator care , which showed massive thromboembolisms in the right pulmonary artery and left pulmonary segmental artery ( fig . 3a ) , indicating an acute pte . because she was in a hemodynamically compromised state , we considered the use of thrombolytic therapy , in spite of the general contraindication of a recent cerebral infarction . after the discussion with a neurologist , we decided to treat her with a half dosage of recombinant tissue plasminogen activator { rtpa , actylase ( boehringer ingelheim , ingelheim , germany ) ; 45 mg total } in order to treat the massive pte . within 2 - 3 hours after an intravenous infusion of the thrombolytic agent , hypoxemia and hypotension symptoms returned to a normal state . after stabilization of her vital signs , we checked for the presence of dvt ; the ct venogram of her lower extremities showed thrombi on her left iliac vein and both peroneal veins ( fig . 4a and b ) . on the arterial phase of this ct , previous thrombi in the right main pulmonary and left segmental arteries were resolved ( fig . 3b ) . in addition , there were newly developed multiple wedge - shaped hypoperfusion lesions in both kidneys , compatible with an acute renal infarction ( fig . 4c ) . a work - up of vasculopathies , hypercoagulable states , and hematologic disorders proved unremarkable . as a result of this treatment plan , the patient was discharged 20 days after admission , and has since been visiting the out - patient department on a regular basis without recurrence of these ischemic events . because there is considerable evidence suggesting that pfo is highly associated with cryptogenic strokes , pfo detection during the evaluation of a patient having a cryptogenic stroke is not surprising . but , when the pfo is detected in a patient having a cryptogenic stroke , the presence of dvt and pte always have to be a matter of concern . in our case , dvt was detected after the event of a massive pte , without previous definitive symptoms or signs of dvt . as such , we are not able to explain precisely whether these dvt and subsequent pte were present before the stroke , or produced during her admission and immobilization . however , searching for the coexistence of pte or dvt and their prophylaxis with anticoagulation could be a more important process . in literature , clergeau et al.4 ) demonstrated that pfo is an independent predictor of the silent brain ischemia in patients with pte , and concluded that patients having cryptogenic stroke and pfo may require a systematic search for silent thromboembolisms . tanislav et al.10 ) reported that the silent ptes frequently occur in patients with cryptogenic strokes and pfos . also , the paradoxical embolism from large veins in ischemic stroke study addressed direct evidence of the increased frequency of dvt , among patients with cryptogenic strokes and pfos.6 ) this patient in our case had a recent stroke and experienced a hemodynamically compromised state , due to a massive pte . in this situation , we had difficulty in making a therapeutic decision between the beneficial effects of thrombolysis on the hemodynamic instability of the massive pte , and the potentially catastrophic effect of a cerebral hemorrhage . as such , after the discussion with a neurologist , we administrated a half dose of rtpa , and this modified thrombolytic therapy enabled the patient to make a good recovery from hemodynamic instability . in literature , there are number of case reports presenting similar situations.5)11 ) however , our case was notably different from these cases , both in terms of the timing of the stroke and the treatment modality . therefore , other therapeutic options , including surgical or catheter embolectomy , must be considered when there are contraindications for systemic thrombolysis , like in our case.12 ) however , because there was a lack of experience of invasive embolectomy in our center , and these kinds of modalities are traditionally chosen in case of failure of systemic thrombolysis , we chose a systemic thrombolytic therapy . also , there is no strong evidence to support a particular therapeutic option under the specific conditions of a stroke on a pfo , associated with pte . thus , every treatment modality ranging from a more conservative approach with anticoagulation alone to a more aggressive approach with invasive thrombectomy should be considered on a case - by - case basis . in this case , there was an acute renal infarction , detected on the arterial phase of the ct venogram , suspected to be a result of a pde ; this renal infarction was not detected on the first ct angiogram when the pulmonary embolism was diagnosed . the mechanism of the renal infarction in our case was considered that transiently increased right arterial hypertension , due to acute pulmonary embolism induced continuous opening of pfo and permitted a passage of the thrombi in deep vein into the arterial system.11 ) it should also be noted that though there have been several case reports of systemic pdes with pte associated with pfo,7)8)13 ) cases of non - cerebral , systemic pdes , associated with pfo , are relatively lower than for cerebral pdes . one report showed that only 2.9% of patients with pfo presented with a presumptive diagnosis of systemic embolism,14 ) though when patients with major pulmonary embolism have pfo , the incidence of peripheral artery embolisms , can significantly be increased ( up to 15%).15 ) we are following up this patient with anticoagulation using warfarin , and this patient has not shown recurrent cerebral ischemia or thrombotic events . however , the closure of pfo is considered to eliminate the risk of pde , and to no longer require the use of warfarin . also , many studies have shown the favorable outcomes of percutaneous device closure of pfo.16)17 ) so , we plan to recommend a percutaneous pfo closure to this patient , after confirming the nonexistence of dvt for several months . in conclusion , this report describes a rare case of cryptogenic stroke , associated with pfo and coexisting pte , dvt , and renal infarctions . when pfo is suspected as a cause of the cryptogenic stroke , it is considered here that seeking the presence of pte , dvt , and other systemic arterial embolisms , using an imaging modality is useful for the prediction and prevention of adverse outcomes .

a paradoxical embolism is defined as a systemic arterial embolism requiring the passage of a venous thrombus into the arterial circulatory system through a right - to - left shunt , and is commonly related to patent foramen ovale ( pfo ) . however , coexisting pulmonary embolisms , deep vein thromboses ( dvt ) , and multipe systemic arterial embolisms , associated with pfo , are rare . here , we report a patient who had a cryptogenic ischemic stroke , associated with pfo , which is complicated with a massive pulmonary thromboembolism , dvt , and renal infarctions , and subsequently , the patient was treated using a thrombolytic therapy .

Introduction Case Discussion

data on the pharmacology of darunavir / ritonavir during pregnancy are limited to case reports 18 , few of which examined pharmacokinetics 13,57,9 . the effects of pregnancy on the pharmacokinetics of darunavir remain unclear ; therefore , further pharmacokinetic studies are needed . maternal physiological changes during pregnancy ( e.g. blood volume expansion , increased glomerular filtration rate and alterations in hepatic metabolism ) may result in altered pharmacokinetics 10 . data support the conclusion that the pharmacokinetic parameters of hiv protease inhibitors ( pis ) are changed during pregnancy , leading to lower exposure in pregnant women 3,5,6,11,12 . studying darunavir / ritonavir in hiv - infected pregnant women may provide insights into population - specific pharmacokinetics that could inform treatment strategies and dosing recommendations . this nonrandomized study was designed to investigate the pharmacokinetic profile , antiviral activity and safety of darunavir / ritonavir [ 600/100 mg twice daily ( bid ) or 800/100 mg once daily ( qd ) ] , etravirine ( 200 mg bid ) and rilpivirine ( 25 mg qd ) in hiv - infected pregnant women . here we report only on the group that received darunavir / ritonavir 600/100 mg bid . hiv-1-infected pregnant women , aged 18 years or older , with pregnancies between 18 and 26 weeks of gestation were included in this multicentre , single - arm , open - label trial . subjects had to be on the study drug before screening and enrolment , and had to undergo an obstetric examination and have a normal level ii ultrasound . the study was approved by a centralized ethics committee [ western institutional review board ( irb ) ] or a site - specific irb . subjects with any obstetric complications , any neurological conditions requiring medication or any active disease that may have compromised patient safety or study outcomes were excluded from the study . the primary objective was to assess the effect of pregnancy on the pharmacokinetics of darunavir / ritonavir administered bid during the second and third trimesters of gestation and postpartum . the secondary objectives were to document antiviral activity , safety and tolerability of darunavir / ritonavir - based regimens during pregnancy and postpartum , to compare darunavir / ritonavir concentrations between maternal serum and cord blood at delivery and to assess the outcomes for infants of women treated with darunavir / ritonavir bid during pregnancy . intensive pharmacokinetic samplings over 12 h were performed at three study visits : during the second trimester ( 2428 weeks ) , during the third trimester ( 3438 weeks ) and 612 weeks postpartum . eight blood draws were performed during each visit : predose and 1 , 2 , 3 , 4 , 6 , 9 and 12 h postdose . darunavir ( total and unbound ) and ritonavir ( total ) plasma concentrations were measured . cord blood and maternal plasma concentrations of total darunavir and ritonavir were also measured on the day of delivery . total darunavir and ritonavir plasma concentrations were determined using a previously validated high - performance liquid chromatography tandem mass spectrometry assay with a lower limit of quantification of 5.00 ng / ml for both compounds 13 . the unbound fraction of darunavir was calculated as the ratio of the unbound concentration in the filtrate to the total concentration in the plasma before centrifugation . human serum albumin and 1-acid glycoprotein ( aag ) were measured because darunavir is highly protein bound and the concentration of these proteins usually decreases during pregnancy as a result of haemodilution 14 . infants ' hiv-1 statuses were determined by polymerase chain reaction testing , reported within 16 weeks postpartum . total and unbound pharmacokinetic parameters were derived from the plasma concentration actual time data using noncompartmental analysis ( winnonlin professional 4.1 ; pharsight , mountain view , ca ) . the minimum and maximum plasma concentrations ( cmin and cmax , respectively ) along with time to reach cmax ( tmax ) were obtained by inspection of the plasma concentration time profiles . the area under the plasma concentration time curve from 0 to 12 h ( auc12h ) was determined using the linear trapezoidal rule . the primary pharmacokinetic parameters used in the statistical analysis were auc12h , cmin and cmax on the logarithmic scale . the least squares means ( lsms ) of the primary parameters for each treatment were estimated with a linear mixed - effects model , controlling for period as a fixed effect and subject as a random effect . a 90% confidence interval ( ci ) both the difference between the lsms and the 90% ci were retransformed to the original scale . safety and antiviral activity were summarized using descriptive statistics ( sas / stat version 9.2 ; sas institute inc . , cary , nc ) . hiv-1-infected pregnant women , aged 18 years or older , with pregnancies between 18 and 26 weeks of gestation were included in this multicentre , single - arm , open - label trial . subjects had to be on the study drug before screening and enrolment , and had to undergo an obstetric examination and have a normal level ii ultrasound . the study was approved by a centralized ethics committee [ western institutional review board ( irb ) ] or a site - specific irb . subjects with any obstetric complications , any neurological conditions requiring medication or any active disease that may have compromised patient safety or study outcomes were excluded from the study . the primary objective was to assess the effect of pregnancy on the pharmacokinetics of darunavir / ritonavir administered bid during the second and third trimesters of gestation and postpartum . the secondary objectives were to document antiviral activity , safety and tolerability of darunavir / ritonavir - based regimens during pregnancy and postpartum , to compare darunavir / ritonavir concentrations between maternal serum and cord blood at delivery and to assess the outcomes for infants of women treated with darunavir / ritonavir bid during pregnancy . intensive pharmacokinetic samplings over 12 h were performed at three study visits : during the second trimester ( 2428 weeks ) , during the third trimester ( 3438 weeks ) and 612 weeks postpartum . eight blood draws were performed during each visit : predose and 1 , 2 , 3 , 4 , 6 , 9 and 12 h postdose . darunavir ( total and unbound ) and ritonavir ( total ) plasma concentrations were measured . cord blood and maternal plasma concentrations of total darunavir and ritonavir were also measured on the day of delivery . total darunavir and ritonavir plasma concentrations were determined using a previously validated high - performance liquid chromatography tandem mass spectrometry assay with a lower limit of quantification of 5.00 ng / ml for both compounds 13 . the unbound fraction of darunavir was calculated as the ratio of the unbound concentration in the filtrate to the total concentration in the plasma before centrifugation . human serum albumin and 1-acid glycoprotein ( aag ) were measured because darunavir is highly protein bound and the concentration of these proteins usually decreases during pregnancy as a result of haemodilution 14 . infants ' hiv-1 statuses were determined by polymerase chain reaction testing , reported within 16 weeks postpartum . total and unbound pharmacokinetic parameters were derived from the plasma concentration actual time data using noncompartmental analysis ( winnonlin professional 4.1 ; pharsight , mountain view , ca ) . the minimum and maximum plasma concentrations ( cmin and cmax , respectively ) along with time to reach cmax ( tmax ) were obtained by inspection of the plasma concentration time profiles . the area under the plasma concentration time curve from 0 to 12 h ( auc12h ) was determined using the linear trapezoidal rule . the primary pharmacokinetic parameters used in the statistical analysis were auc12h , cmin and cmax on the logarithmic scale . the least squares means ( lsms ) of the primary parameters for each treatment were estimated with a linear mixed - effects model , controlling for period as a fixed effect and subject as a random effect . a 90% confidence interval ( ci ) both the difference between the lsms and the 90% ci were retransformed to the original scale . safety and antiviral activity were summarized using descriptive statistics ( sas / stat version 9.2 ; sas institute inc . , cary , nc ) . sixteen women were enrolled in this part of the trial and received darunavir / ritonavir 600/100 mg bid ( supplementary table s1 ) . the median age was 24 years ( range 1835 years ) , and 63% were black or african american . most ( 80% ) subjects had a cd4 count 350 cells/l , 33% had a viral load < 50 hiv-1 rna copies / ml , 53% had a viral load between 50 and 400 copies / ml and 14% had a viral load 400 copies / ml . sixty - three per cent were previously treated with one protease inhibitor ( pi)-based regimen . of 16 subjects , 14 had at least one intensive pharmacokinetic visit and were included in the pharmacokinetic analysis , and 12 completed the treatment phase of the study . a total of five subjects discontinued ( four during treatment before delivery ; one during follow - up ) ( table s1 ) . two subjects discontinued because of adverse events ( aes ) : one for increased transaminases related to study drugs and one for unrelated premature delivery ( premature delivery led to an inability to fulfill protocol requirements as the patient missed the postpartum visit ) . two subjects discontinued because of virological failure related to suboptimal adherence as documented by the principal investigator [ one patient reported 87.5% adherence ( auc12h 69 970 ng / h / ml ; cmin 4140 ng / ml ) in the 4 days prior to the second trimester visit and 75% adherence in the 4 days prior to the third trimester visit ( no pharmacokinetic data were available ) , and the other patient reported 62.5% adherence in the 4 days prior to the second trimester visit ; third trimester adherence rate and pharmacokinetic data were not available ] . one subject was ineligible to continue the trial because of a failed drug screening . mean total and unbound darunavir plasma concentrations were higher during the postpartum period compared with the second and third trimesters of pregnancy ( fig . mean ( standard deviation ) plasma concentration time curves for total darunavir ( a ) , unbound darunavir ( b ) and total ritonavir ( c ) after administration of darunavir / ritonavir 600/100 mg twice daily ( bid ) , during the second and third trimesters and postpartum . data for unbound darunavir were not available for all patients as not all plasma samples were available for plasma protein binding analysis . the maximum plasma concentration of total darunavir during the second and third trimesters was 28 and 19% lower , respectively , vs. postpartum based on the lsm ratios . total darunavir cmin was on average 43 and 86% higher during the second and third trimesters , respectively , vs. postpartum . total darunavir auc12h during pregnancy was 24 and 17% lower in the second and third trimesters , respectively , vs. postpartum . unbound darunavir cmax was 22 and 18% lower during the second and third trimesters , respectively , vs. postpartum . the median tmax of unbound darunavir was approximately 3 h during pregnancy and 2 h postpartum . unbound darunavir cmin was 10 and 14% higher during the second and third trimesters , respectively , vs. postpartum . unbound darunavir auc12h was 8 and 7% lower during the second and third trimesters , respectively , vs. postpartum . the free fraction of darunavir ( ratio of unbound plasma concentration vs. total plasma concentration ) was slightly higher during pregnancy vs. postpartum ( data not shown ) . mean ( standard deviation ) * pharmacokinetic parameters and statistical results for total and unbound darunavir and total ritonavir , after administration of darunavir / ritonavir 600/100 mg twice daily ( bid ) , during the second and third trimesters and postpartum lsm , least squares mean ; ci , confidence interval ; c0h , predose plasma concentration ; nd , not determined ; cmin , minimum plasma concentration ; cmax , maximum plasma concentration ; c12h , plasma concentration at 12 hours ; tmax , time to reach the maximum plasma concentration ; auc12h , area under the plasma concentration time curve over 12 h ; cl / f , apparent clearance . data for unbound darunavir were not available for all patients because of poor sample quality ( protein in the filtrate ) or low sample volume . n = 10 for c0h , n = 9 for cmin , and n = 7 for cmax and tmax . n = 11 for c0h , and n = 8 for cmin , cmax and tmax . n = 9 for cmin , and n = 7 for cmax . n = 12 for cmin and cmax . matched maternal and cord plasma concentrations were available for 10 subjects . excluding one outlier resulting from a possible switch of the samples , the mean [ standard deviation ( sd ) ] total darunavir concentrations were higher in maternal plasma vs. cord plasma [ 2324 ( 1056 ) ng / ml vs. 383 ( 322 ) ng / ml , respectively ] when assessed in nine women and their infants . the median cord : maternal plasma ratio was 0.1455 ( range 0.014070.3621 ) . mean total ritonavir plasma concentrations were higher during the postpartum period compared with the second and third trimesters of pregnancy ( fig . median total ritonavir plasma concentrations peaked 4 h after drug administration during pregnancy and at 6 h in the postpartum period ( table 1 ) . total ritonavir cmax during the second and third trimesters of pregnancy was 34 and 37% lower , respectively , vs. postpartum based on the lsm ratios . total ritonavir cmin was on average 8 and 22% higher during the second and third trimesters , respectively , vs. postpartum . total ritonavir auc12h during pregnancy was 28 and 33% lower in the second and third trimesters , respectively , vs. postpartum . excluding one outlier resulting from a possible switch of the samples , the mean ( sd ) total ritonavir concentrations were higher in maternal plasma vs. cord plasma [ 429 ng / ml vs. 17 ng / ml , respectively ) when assessed in seven women and their infants . the median cord : maternal plasma ratio was 0.1076 ( range 0.044320.114 ) . mean baseline albumin and aag concentrations were 31 and 617 g / l and postpartum concentrations were 38 and 790 g / l , respectively , resulting in a 22 to 28% decrease during pregnancy vs. postpartum ( fig . the response rate ( < 50 copies / ml ) increased significantly from 33% ( five of 15 ) at baseline ( table s1 ) to 73% ( eight of 11 ) and 90% ( nine of 10 ) during the second and third trimesters , respectively , and was 80% ( eight of 10 ) at 12 weeks postpartum ( fig . three patients had detectable viral loads during pregnancy ; two patients had detectable viral load during the second but not the third trimester [ 72 copies / ml in one patient ( 100% reported adherence in both trimesters ) and 123 copies / ml in the other ( 87.5% reported adherence in the second trimester , and 100% reported adherence in the third ) ] . the third patient had detectable viral load during both trimesters ( 813 copies / ml in the second trimester and 59 copies / ml in the third trimester ) , with 100% reported adherence for both trimesters . of these three patients with detectable viral load during pregnancy , two had undetectable viral load in the postpartum and follow - up periods . cd4 count is significantly lower in hiv-1-negative women during pregnancy compared with the 12-week postdelivery period 15 ; therefore , the percentage of cd4 cells was plotted instead of absolute cd4 count . the percentage of cd4 cells increased from 30% at baseline to 35% at 25 weeks postpartum ( fig . all 12 infants born to women who stayed on treatment until delivery were hiv negative . one of these infants was born to a patient who discontinued the study but remained on study treatment during follow - up . the most common aes , occurring with an incidence of 25% , were infections and infestations ( 44% ) , gastrointestinal disorders ( 25% ) and premature labour ( 25% ) . of 12 infants , four were born prematurely ( at weeks 30 , 36 , 36 and 37 ) and no congenital abnormalities occurred . sixteen women were enrolled in this part of the trial and received darunavir / ritonavir 600/100 mg bid ( supplementary table s1 ) . the median age was 24 years ( range 1835 years ) , and 63% were black or african american . most ( 80% ) subjects had a cd4 count 350 cells/l , 33% had a viral load < 50 hiv-1 rna copies / ml , 53% had a viral load between 50 and 400 copies / ml and 14% had a viral load 400 copies / ml . sixty - three per cent were previously treated with one protease inhibitor ( pi)-based regimen . of 16 subjects , 14 had at least one intensive pharmacokinetic visit and were included in the pharmacokinetic analysis , and 12 completed the treatment phase of the study . a total of five subjects discontinued ( four during treatment before delivery ; one during follow - up ) ( table s1 ) . two subjects discontinued because of adverse events ( aes ) : one for increased transaminases related to study drugs and one for unrelated premature delivery ( premature delivery led to an inability to fulfill protocol requirements as the patient missed the postpartum visit ) . two subjects discontinued because of virological failure related to suboptimal adherence as documented by the principal investigator [ one patient reported 87.5% adherence ( auc12h 69 970 ng / h / ml ; cmin 4140 ng / ml ) in the 4 days prior to the second trimester visit and 75% adherence in the 4 days prior to the third trimester visit ( no pharmacokinetic data were available ) , and the other patient reported 62.5% adherence in the 4 days prior to the second trimester visit mean total and unbound darunavir plasma concentrations were higher during the postpartum period compared with the second and third trimesters of pregnancy ( fig . mean ( standard deviation ) plasma concentration time curves for total darunavir ( a ) , unbound darunavir ( b ) and total ritonavir ( c ) after administration of darunavir / ritonavir 600/100 mg twice daily ( bid ) , during the second and third trimesters and postpartum . data for unbound darunavir were not available for all patients as not all plasma samples were available for plasma protein binding analysis . the maximum plasma concentration of total darunavir during the second and third trimesters was 28 and 19% lower , respectively , vs. postpartum based on the lsm ratios . total darunavir cmin was on average 43 and 86% higher during the second and third trimesters , respectively , vs. postpartum . total darunavir auc12h during pregnancy was 24 and 17% lower in the second and third trimesters , respectively , vs. postpartum . unbound darunavir cmax was 22 and 18% lower during the second and third trimesters , respectively , vs. postpartum . the median tmax of unbound darunavir was approximately 3 h during pregnancy and 2 h postpartum . unbound darunavir cmin was 10 and 14% higher during the second and third trimesters , respectively , vs. postpartum . unbound darunavir auc12h was 8 and 7% lower during the second and third trimesters , respectively , vs. postpartum . the free fraction of darunavir ( ratio of unbound plasma concentration vs. total plasma concentration ) was slightly higher during pregnancy vs. postpartum ( data not shown ) . mean ( standard deviation ) * pharmacokinetic parameters and statistical results for total and unbound darunavir and total ritonavir , after administration of darunavir / ritonavir 600/100 mg twice daily ( bid ) , during the second and third trimesters and postpartum lsm , least squares mean ; ci , confidence interval ; c0h , predose plasma concentration ; nd , not determined ; cmin , minimum plasma concentration ; cmax , maximum plasma concentration ; c12h , plasma concentration at 12 hours ; tmax , time to reach the maximum plasma concentration ; auc12h , area under the plasma concentration time curve over 12 h ; cl / f , apparent clearance . data for unbound darunavir were not available for all patients because of poor sample quality ( protein in the filtrate ) or low sample volume . n = 10 for c0h , n = 9 for cmin , and n = 7 for cmax and tmax . n = 11 for c0h , and n = 8 for cmin , cmax and tmax . n = 9 for cmin , and n = 7 for cmax . n = 12 for cmin and cmax . matched maternal and cord plasma concentrations were available for 10 subjects . excluding one outlier resulting from a possible switch of the samples , the mean [ standard deviation ( sd ) ] total darunavir concentrations were higher in maternal plasma vs. cord plasma [ 2324 ( 1056 ) ng / ml vs. 383 ( 322 ) ng / ml , respectively ] when assessed in nine women and their infants . the median cord : maternal plasma ratio was 0.1455 ( range 0.014070.3621 ) . mean total ritonavir plasma concentrations were higher during the postpartum period compared with the second and third trimesters of pregnancy ( fig . median total ritonavir plasma concentrations peaked 4 h after drug administration during pregnancy and at 6 h in the postpartum period ( table 1 ) . total ritonavir cmax during the second and third trimesters of pregnancy was 34 and 37% lower , respectively , vs. postpartum based on the lsm ratios . total ritonavir cmin was on average 8 and 22% higher during the second and third trimesters , respectively , vs. postpartum . total ritonavir auc12h during pregnancy was 28 and 33% lower in the second and third trimesters , respectively , vs. postpartum . excluding one outlier resulting from a possible switch of the samples , the mean ( sd ) total ritonavir concentrations were higher in maternal plasma vs. cord plasma [ 429 ng / ml vs. 17 ng / ml , respectively ) when assessed in seven women and their infants . the median cord : maternal plasma ratio was 0.1076 ( range 0.044320.114 ) . mean baseline albumin and aag concentrations were 31 and 617 g / l and postpartum concentrations were 38 and 790 g / l , respectively , resulting in a 22 to 28% decrease during pregnancy vs. postpartum ( fig . the response rate ( < 50 copies / ml ) increased significantly from 33% ( five of 15 ) at baseline ( table s1 ) to 73% ( eight of 11 ) and 90% ( nine of 10 ) during the second and third trimesters , respectively , and was 80% ( eight of 10 ) at 12 weeks postpartum ( fig three patients had detectable viral loads during pregnancy ; two patients had detectable viral load during the second but not the third trimester [ 72 copies / ml in one patient ( 100% reported adherence in both trimesters ) and 123 copies / ml in the other ( 87.5% reported adherence in the second trimester , and 100% reported adherence in the third ) ] . the third patient had detectable viral load during both trimesters ( 813 copies / ml in the second trimester and 59 copies / ml in the third trimester ) , with 100% reported adherence for both trimesters . of these three patients with detectable viral load during pregnancy , two had undetectable viral load in the postpartum and follow - up periods . cd4 count is significantly lower in hiv-1-negative women during pregnancy compared with the 12-week postdelivery period 15 ; therefore , the percentage of cd4 cells was plotted instead of absolute cd4 count . the percentage of cd4 cells increased from 30% at baseline to 35% at 25 weeks postpartum ( fig . all 12 infants born to women who stayed on treatment until delivery were hiv negative . one of these infants was born to a patient who discontinued the study but remained on study treatment during follow - up . the most common aes , occurring with an incidence of 25% , were infections and infestations ( 44% ) , gastrointestinal disorders ( 25% ) and premature labour ( 25% ) . of 12 infants , four were born prematurely ( at weeks 30 , 36 , 36 and 37 ) and no congenital abnormalities occurred . lower exposures of total darunavir / ritonavir 600/100 mg bid were observed during pregnancy compared with postpartum . total darunavir cmax was 28 and 19% lower , and auc12h was 24 and 17% lower during the second and third trimesters , respectively , vs. postpartum . these data are consistent with published reports demonstrating a decrease in total drug exposure ( auc ) of darunavir / ritonavir 600/100 mg bid during pregnancy ranging from 29 1 to 36% 9 . lower levels of drug exposure during pregnancy vs. postpartum have been observed with other hiv pis 11,12,1619 . however , unlike previous reports , the current study also assessed free plasma darunavir levels , which were higher during pregnancy vs. postpartum . as a result , the difference in unbound cmax and auc12h at postpartum compared with values found during pregnancy was minimal . the small number of patients with pharmacokinetic data for unbound darunavir , six in the second trimester and seven in the third trimester , limited the ability to draw definitive conclusions ; however , we hypothesize that the lower total drug exposure of darunavir during pregnancy might be partially compensated for by higher free drug , resulting from lower protein binding to darunavir . the antiviral activity of darunavir / ritonavir in hiv - infected pregnant women was effective in preventing mother - to - child transmission and in suppressing hiv in pregnant women , consistent with findings in pregnant 1,3,5,9 and nonpregnant hiv - infected individuals 20 . despite lower total pi levels in studies in pregnant women , subjects achieved desired immunovirological responses and protection from transmission of hiv to their infants 3,5,6,9,11,12 . taken together , the findings that unbound concentrations of darunavir remained nearly unchanged during pregnancy relative to postpartum and that clinical responses were maintained suggest that no a priori dose adjustment is needed in darunavir / ritonavir when dosed bid in pregnant women . total ritonavir cmax was 34 and 37% lower , and auc12h was 28 and 33% lower during the second and third trimesters , respectively , vs. postpartum , similar to findings in previously published reports 11 . the levels of darunavir / ritonavir transferred from maternal to cord plasma were low , with median cord : maternal ratios of 0.1455 and 0.1076 , respectively , consistent with other reports demonstrating low transplacental passage of pis 3,7,9 . it is interesting to note that cmin values were on average higher during the second and third trimesters , respectively , vs. postpartum for both darunavir ( 43 and 86% higher , respectively ) and ritonavir ( 8 and 22% higher , respectively ) . plasma concentration values at 12 hours have also been presented in table 1 in order to provide additional context to these data . in this study , darunavir / ritonavir bid treatment was generally well tolerated in hiv - infected pregnant women , with few discontinuations because of aes ( 12% ) . the observed ae profile partially overlaps with that found previously in nonpregnant adults 20 , and it is unclear how many aes may actually have been associated with pregnancy independent of darunavir / ritonavir bid treatment . a limitation of this study may be the use of the 6- to 12-week postpartum levels as the reference . darunavir / ritonavir pharmacokinetic parameters after administration of darunavir / ritonavir 600/100 mg bid as part of an antiretroviral regimen during pregnancy and postpartum were not clinically different compared with historical controls . darunavir / ritonavir 600/100 mg bid administered with other antiretrovirals during pregnancy resulted in consistent unbound ( active ) drug exposure during pregnancy and postpartum , was effective in preventing mother - to - child transmission and in suppressing hiv and was generally well tolerated , with few discontinuations because of aes . data from this small pharmacokinetic study , while not definitive , suggest that darunavir / ritonavir 600/100 mg bid may be a treatment option for pregnant women in need of highly active antiretroviral therapy . additional supporting information may be found in the online version of this article at the publisher 's web - site : fig . s1 individual minimum plasma concentrations of total darunavir ( a ) , unbound darunavir ( b ) and total ritonavir ( c ) after administration of darunavir / ritonavir 600/100 mg twice daily ( bid ) , during the second and third trimesters and postpartum . s2 mean ( standard error ) plasma concentration time curves for albumin ( a ) and 1-acid glycoprotein ( aag ) ( b ) assessed at various time - points throughout the study . fig . s3 antiviral activity expressed as percentage response rate ( a ) ( only patients who completed the study in its entirety are included ) and status of the immune system expressed as median percentage cd4 count ( b ) assessed at various time - points throughout the study .

objectivesantiretroviral therapy during pregnancy is recommended to reduce the risk of mother - to - child transmission of hiv and for maternal care management . physiological changes during pregnancy can affect pharmacokinetics , potentially altering pharmacological activity . we therefore evaluated the pharmacokinetics of twice - daily ( bid ) darunavir in hiv-1-infected pregnant women.methodshiv-1-infected pregnant women receiving an antiretroviral regimen containing darunavir / ritonavir 600/100 mg bid were enrolled in this study . total and unbound darunavir and total ritonavir plasma concentrations were obtained over 12 h during the second and third trimesters and postpartum . total darunavir and ritonavir plasma concentrations were determined using a validated high - performance liquid chromatography tandem mass spectrometry assay and unbound darunavir was determined using 14c - darunavir - fortified plasma . pharmacokinetic parameters were derived using noncompartmental analysis.resultsdata were available for 14 women . the area under the plasma concentration time curve from 0 to 12 h ( auc12h ) for total darunavir was 1724% lower during pregnancy than postpartum . the auc12h for unbound darunavir was minimally reduced during pregnancy vs. postpartum . the minimum plasma concentration ( cmin ) of total and unbound darunavir was on average 4386% and 1014% higher , respectively , during pregnancy vs. postpartum . the antiviral response ( < 50 hiv-1 rna copies / ml ) was 33% at baseline and increased to 7390% during treatment ; the percentage cd4 count increased over time . one serious adverse event was reported ( increased transaminase ) . all 12 infants born to women remaining in the study at delivery were hiv-1-negative ; four of these infants were premature.conclusionstotal darunavir exposure decreased during pregnancy . no clinically relevant change in unbound ( active ) darunavir occurred during pregnancy , suggesting that no dose adjustment is required for darunavir / ritonavir 600/100 mg bid in pregnant women .

Introduction Methods Study design and treatment Objectives Evaluations Statistical analysis Results Population and baseline characteristics Disposition Pharmacokinetics of total and unbound darunavir Pharmacokinetics of ritonavir Albumin and Antiviral activity Safety Discussion Supporting Information

congenital segmental dilatation of ureter is a rare entity in children and characterized by a giant , focal segmental dilatation of mid ureter . we report two children with segmental dilatation of ureters ; one child had ipsilateral megacalycosis and contralateral vesico - ureteric reflux ( vur ) presenting with acute renal failure and sepsis . the other child had unilateral duplex system with segmental dilatation of both upper and lower moiety ureters with evidence of xanthogranulomatous pyelonephritis ( xgp ) in the lower moiety . master bt , 7 years male presented with high fever and scanty , high - colored urine , and lethargy . a large lump his blood urea and serum creatinine values at admission were 229 mg% and 5.4 mg% , respectively . ultrasonography ( usg ) showed bilateral gross hydronephrosis and a large cyst on the left side , possibly a bladder diverticulum . an intravenous pyelography ( ivp ) done elsewhere 2 months earlier had shown right - sided gross hydro - ureteronephrosis on right side and non - functioning left kidney . blood urea and serum creatinine values decreased to 90 mg% and 1.7 mg% , respectively , following bilateral percutaneous nephrostomies . exploration revealed a dilated left upper ureter ( 12 cm long ) opening into a large cyst ( 10 cm 6 cm ) , which was thick - walled and adherent to the bladder wall [ figure 1 ] . a 2 cm long lower ureter was seen from the cyst to the bladder neck . the dilated segment along with the lower ureter was excised , and end to side ureteroureteral anastomosis was done to the contralateral ureter . histology of dilated segment showed damaged epithelium with infiltration of sub - epithelial layers with inflammatory cells . the patient was put on chemoprophylaxis for 6 months ( co - trimoxazole and nalidixic acid alternately every 6 weeks ) and has been asymptomatic for last 4 years . his blood urea and serum creatinine values on last visit were 48 mg% and 1.1 mg% , respectively . normal - looking upper ureter opening into the dilated segment ( case 1 ) a 14-month - old male child presented with abdominal distension and pain of 2 months duration . a thick - walled cystic structure with thick fluid inside ( 4 cm 3 cm ) was noted on right side just above the bladder . ivp showed right - sided hydronephrosis , as the radiologist missed the non - functioning lower moiety . renal biochemical parameters were within normal limits . at operation , right - sided duplex kidney was noted . normal size upper moiety ureter ( 8 cm long ) drains into the dilated segment ( 3 cm 2 cm ) . the small lower moiety and dilated upper ureter ( 4 cm long ) drains into a markedly dilated , thickened ureteral segment ( 6 cm 4 cm ) . the dilated segments were densely adherent to the bladder wall and extended up to the recto - vesical pouch . partial nephroureterectomy ( lower moiety ) was done with excision of the dilated segment of both upper and lower moiety ureters . normal upper moiety ureter was re - implanted into the bladder by leadbetter - politano technique . the patient had uneventful recovery and was doing well till last follow up at 18 months . photomicrograph ( h and e stain 200 ) showing groups of large round to oval cells with eosinophilic cytoplasm and vesicular nuclei mixed with interlacing fibrocollagenous tissue , infiltrating into the sub - epithelial stroma s / o juvenile xanthogranulomata ( case 2 ) master bt , 7 years male presented with high fever and scanty , high - colored urine , and lethargy . a large lump his blood urea and serum creatinine values at admission were 229 mg% and 5.4 mg% , respectively . ultrasonography ( usg ) showed bilateral gross hydronephrosis and a large cyst on the left side , possibly a bladder diverticulum . an intravenous pyelography ( ivp ) done elsewhere 2 months earlier had shown right - sided gross hydro - ureteronephrosis on right side and non - functioning left kidney . blood urea and serum creatinine values decreased to 90 mg% and 1.7 mg% , respectively , following bilateral percutaneous nephrostomies . exploration revealed a dilated left upper ureter ( 12 cm long ) opening into a large cyst ( 10 cm 6 cm ) , which was thick - walled and adherent to the bladder wall [ figure 1 ] . a 2 cm long lower ureter was seen from the cyst to the bladder neck . the dilated segment along with the lower ureter was excised , and end to side ureteroureteral anastomosis was done to the contralateral ureter . histology of dilated segment showed damaged epithelium with infiltration of sub - epithelial layers with inflammatory cells . the patient was put on chemoprophylaxis for 6 months ( co - trimoxazole and nalidixic acid alternately every 6 weeks ) and has been asymptomatic for last 4 years . his blood urea and serum creatinine values on last visit were 48 mg% and 1.1 mg% , respectively . a 14-month - old male child presented with abdominal distension and pain of 2 months duration . a thick - walled cystic structure with thick fluid inside ( 4 cm 3 cm ) was noted on right side just above the bladder . ivp showed right - sided hydronephrosis , as the radiologist missed the non - functioning lower moiety . renal biochemical parameters were within normal limits . at operation , right - sided duplex kidney was noted . normal size upper moiety ureter ( 8 cm long ) drains into the dilated segment ( 3 cm 2 cm ) . the small lower moiety and dilated upper ureter ( 4 cm long ) drains into a markedly dilated , thickened ureteral segment ( 6 cm 4 cm ) . the dilated segments were densely adherent to the bladder wall and extended up to the recto - vesical pouch . partial nephroureterectomy ( lower moiety ) was done with excision of the dilated segment of both upper and lower moiety ureters . normal upper moiety ureter was re - implanted into the bladder by leadbetter - politano technique . the patient had uneventful recovery and was doing well till last follow up at 18 months . photomicrograph ( h and e stain 200 ) showing groups of large round to oval cells with eosinophilic cytoplasm and vesicular nuclei mixed with interlacing fibrocollagenous tissue , infiltrating into the sub - epithelial stroma s / o juvenile xanthogranulomata ( case 2 ) segmental dilatation of ureter is a rare entity , and only 12 case reports are found in the english literature . this condition is characterized by giant , focal segmental ureteral dilatation producing an elongated and distorted ureter . it may be associated with megacalycosis , duplication of collecting system and hypoplastic , dysplastic or non - functioning kidney . they usually have a dysplastic or disorganized muscle coat , lined with columnar epithelial mucosa , rather than the usual transitional epithelium . the associated kidney or the moiety may be hypoplastic or the site of comparatively mild hydronephrosis associated with atrophic renal parenchyma . ramaswamy et al . reported a case of congenital segmental megaureter with sparing of the proximal and distal ureter . the authors suggested that attenuated nexuses and thin myofilaments might be responsible for this entity . soler reported a case of an unilateral multicystic dysplastic kidney and contralateral megacalycosis associated with ipsilateral distal segmental megaureter . pinter speculated abnormal recanalization of the solid ureteral duct during development as a possible cause . xgp is an atypical form of chronic pyelonephritis characterized by diffuse , focal or segmental suppurative destruction of renal parenchyma . urinary tract obstruction , urosepsis , renal ischemia , lymphatic obstruction , and altered immune response may be responsible for xgp . in the rare localized form , the lesion can be confined to one or other pole , as was seen in our second case . its association with duplex kidney is known , but its association with segmental dilatation of ureter has not been reported earlier . the treatment of segmental dilatation of ureter is excision of the diseased segment with end - to - end ureteroureterostomy . a better option is to anastomose the upper ureter end - to - side to the contralateral ureter , as was done in our first case . localized form of the disease , as in our 2 case , is rare , and a partial nephrectomy is ideal . these patients need long - term follow up as secondary amyloidosis has been reported in association with xgp .

segmental dilatation of ureter is a giant , focal segmental ureteral dilatation producing an elongated and distorted ureter . two children presented with this condition , one had ipsilateral megacalycosis and contralateral vesicoureteric reflux . the other had duplication of the kidney . the non - functioning lower moiety showed structure of xanthogranulomatous pyelonephritis .

INTRODUCTION CASE REPORTS Case 1 Case 2 DISCUSSION

platinum coils are inserted into the lumen of the aneurysm , so that a local thrombus is formed around the coils , obliterating the aneurysmal sac.4 ) coil embolization , which is performed through the intravascular space , has an inherent risk of causing thromboembolism . thromboembolic complications occur more frequently in ruptured aneurysm that can not be treated with antiplatelet medication and intra - procedural administration of heparin compared to unruptured cases.8)10 ) such pre - medications ( antiplatelet and anticoagulant administration ) can reduce the occurrence of thromboembolism in treatment of unruptured aneurysm.5 ) for ruptured aneurysmal subarachnoid hemorrhage ( asah ) , coil embolization is mainly performed in unplanned emergency . however , pre - medication of antiplatelet or heparin prior to emergency coil embolization in ruptured asah is under many restrictions in real clinical practice , because it is possible to increase the risk of rebleeding . therefore , coil embolization for ruptured aneurysm may inevitably cause more thromboembolic complications than unruptured aneurysm . also , it is widely assumed that dehydration predispose to thromboembolism after acute stroke.6 ) emergency coil embolization in dehydration state , it may be more dangerous . we assumed that calibrated the dehydration be able to reduce the risk of thromboembolic complication in acute phase coil embolization . contrary to the therapeutic treatment , administering a proper dose of fluid to the patient is very easy and simple . it may correct dehydration , causing no additional complication for the patient , and is expected to reduce thromboembolic complications . we examined patients who were to under emergency coil embolization for asah , assuming that administering a proper dose of fluid to the patients would reduce the risk of thromboembolism without additional complications . from january 2009 to december 2013 , 190 patients with ruptured asah treated by coil embolization at our institution were consecutively enrolled in this study . subarachnoid hemorrhage was confirmed by computed tomography , magnetic resonance image , or cerebrospinal fluid analysis . in general , neither antiplatelet premedication nor systemic heparinization was administered during the procedure , until proper protection of the ruptured aneurysm was achieved ( no contrast filling within the aneurysmal dome ) . after confirming of proper protection of ruptured aneurysm ( no contrast filling in the top of the aneurysm ) , a bolus of 3000 iu of heparin was administered intravenously . and additional 1000 iu bolus of heparin was administered every hour . thromboembolic complication was defined as any event with complete or partial occlusion of arteries , detection of low density or high signal on postoperative ct or mr consistent with patient 's symptoms . in period 1 ( from january 2009 to june 2012 , n = 122 ) , pre - procedural fluid was not supplied . in period 2 ( from july 2012 to december 2013 , n = 68 ) , depending on the state of the patient 's body weight , intravenous fluid was started with infusion of approximately 7 ml / kg of 0.9 percent saline ( minimum 300 to maximum 500 ml ) over 30 minutes . no variation in the treatment protocol was observed between the two periods and the coil embolization was performed by three experienced operators . at least two of these three physicians were engaged together in performance of each procedure . statistical analysis was performed using the spss statistical package ( spss for windows , version 20.0 , chicago , il , usa ) . the chi - square , student t - test and univariate logistic regression were used for comparisons as appropriate . variables with p value < 0.05 in univariate analysis were selected for the multivariable model using multiple logistic regression analysis . statistical analysis was performed using the spss statistical package ( spss for windows , version 20.0 , chicago , il , usa ) . the chi - square , student t - test and univariate logistic regression were used for comparisons as appropriate . variables with p value < 0.05 in univariate analysis were selected for the multivariable model using multiple logistic regression analysis . a total of 190 patients were hospitalized due to asah and underwent coil embolization for five years between january 2009 and december 2013 . of these , 122 patients underwent coil embolization based on the old protocol ( period 1 , january 2009 to june 2012 ) and 68 underwent the procedure based on the new one after the period ( period 2 , july 2009 to december 2013 ) . no difference in the patient 's age , gender , past medical history , glasgow coma scale , world federation of neurosurgical societies grading , size of aneurysm , or procedure time for coil embolization was found between the two periods . ( table 1 ) in period 2 , coil embolization was performed approximately six hours ( 368.99 243.54 minutes ) after the occurrence of symptoms and 417 93.21 ml fluid was administered pre - procedurally . the effectiveness of the coil embolization procedure using a single microcatheter was greater with statistical significance in period 1 ( 87% ; 107/122 ) than in period 2 ( 69.1% , 47% ) ( p = 0.003 ) . no statistically significant difference in the rate of intra - procedural rupture considered to be a complication of coil embolization was found between period 1 ( 11.5% ; 14/122 ) and period 2 ( 14.7% ; 10/68 ) ( p = 0.506 ) . with an additional use of microcathter , thromboembolic complications could be increased . in this study , there are more frequent use of additional microcatheter or balloon and stent in period 2 . however , the frequency of thromboembolism showed a drastic decrease , from 18.0% ( 22/123 ) before the protocol to 4.4% ( 3/67 ) after the protocol , which was also statistically significant ( p = 0.007 , chi - square test ) . a cardio - pulmonary problem considered to be a complication of pre - procedural fluid administration 13 cases improved without sequela and the remaining case required treatment using a ventilator ; however , the ventilator care was required due to pulmonary edema before fluid administration ( table 1 ) . analysis of thromboembolism occurrence found that the neck size , width , and longest length of aneurysm , pre - procedural fluid supply , and procedure time were statistically significant related factors ( univariate logistic regression analysis ) ( table 2 ) . in multivariate analysis , the longest length of aneurysm , fluid supply , and length of procedure time were significant related factors ( prolonged procedure time ( odds ratio [ or ] 1.010 , 95% confidence interval [ ci ] 1.001 - 1.020 , p = 0.035 ) and pre - embolization rehydration ( or 0.261 , 95% ci 0.073 - 0.993 , p = 0.039 ) were independent risk factors for thromboembolic complication in multivariate logistic regression analysis ) ( table 3 ) . pre - embolization rehydration , turned out to be a factor that could be reduce the procedural thromboembolic complications . only a third of asah patients may improve and many such patients can die or become disabled.1)2 ) in ruptured aneurysm , if left untreated , the risk of rebleeding within the first 24 hours is 3 - 4%1 ) ; since rebleeding is the most important factor preventing the patient 's recovery , it is widely accepted that a ruptured aneurysm requires emergency treatment . since the international subarachnoid aneurysm trial research found that coil embolization was not less effective in treatment of ruptured aneurysm than clipping,7 ) it has often been used in treatment of ruptured aneurysm and many researchers have reported positive results from the procedure . however , coil embolization is inherently associated with two serious problems : 1 ) intra - procedural rupture and 2 ) intra - procedural thromboembolism . it can involve these two complications more frequently compared with the unruptured aneurysm.3)8)11)12 ) prospective large - scale research found that coil embolization for asah caused rupture in 3.2% to 7.6% of cases and thromboembolism in 11% to 13.3%.3)8 ) the frequency of thromboembolism is approximately twice as high as that for unruptured aneurysm ( 6.7 - 7.3%).8)10 ) larger aneurysm , a wider aneurismal neck , or use of diverse instruments may lead to higher risk of thromboembolism.9)10 ) pre - procedural ample supply of fluid and antiplatelet preparation , intra - procedural heparin administration , intra - procedural thrombolysis when thrombus occurs , and additional post - procedural anticoagulant or antiplatelet medication are used to reduce the problems in case of coil embolization for unruptured cerebral aneurysm and such treatments are known to reduce the frequency of thromboembolism.5 ) patients with unruptured aneurysm are administered a good dose of fluid to prevent dehydration and two antiplatelet agents for at least five days pre - procedurally , as well as heparin intra - procedurally , thus they may inevitably be at lower risk of thromboembolism than those with asah . for ruptured aneurysm , which is treated with unplanned emergency coil embolization , administration of sufficient pretreatment is difficult and the likelihood of rebleeding may place restrictions on antithrombotic administration , consequently causing more thromboembolic complications . however , administration of antiplatelet or heparin in clinical practice with the aim of reducing the high risk of thromboembolism for ruptured aneurysm is difficult . in cases of unruptured aneurysm , more aggressive strategies could be tried for prevention or removal of thrombus . however , coil embolization in ruptured cases has significant limitation compared to unruptured cases , due to the emergency situation for adequate preparation or the high risk of rebleeding of aneurysm or predilection for bleeding due to the fragile nature of the brain caused by antecedent intracerebral hemorrhage . while some asah patients may visit emergency rooms in tertiary hospitals immediately after occurrence of symptoms , many of them may be transferred to a tertiary hospital from primary and secondary hospitals to undergo coil embolization after aneurysm rupture . for this reason , performance of coil embolization after the condition occurs may take much more time . because most patients are under nothing by mouth during transfer , they are likely to be dehydrated unless provided with an adequate dose of fluid . enhanced computed tomography and digital subtraction angiography using contrast during can aggravate dehydration . performing invasive angiography and coil embolization in those patients without supply of fluid is more likely to cause thromboembolism . in this study it took six hours on average for the patients to be transferred to a hospital and undergo coil embolization ; during this period of time , most of them were prevented from drinking any fluid and did not have a proper intravenous supply of fluid . we found that simply providing patients with a proper dose of fluid pre - procedurally could reduce thromboembolism . because many other factors can cause thromboembolism , it may be difficult to say that this method is sufficient to reduce the frequency of thromboembolism . in addition to the factors of aneurysm itself ( size of aneurysm , size of its neck , etc . ) , as mentioned above , using additional microcatheter or inserting a stent is known to increase the frequency of such complications . we also found that the neck size , width , and longest length of the aneurysm and procedure time were associated with the occurrence of thromboembolism . in this study , however , the frequency of thromboembolism was lower in period 2 , although an additional microcatheter was used more frequently than in period 1 . this result could be suggest that becoming more experienced may lead to technical improvement so that use of additional instruments , such as additional microcatheter and stents , does not increase the frequency of thromboembolism ; however , no variation in the frequency of intra - procedural rupture between the two periods demonstrates that an operator 's skill may be dependent neither on prolonged time nor on experience . in addition , the factors based on the skill of individual operators implementing the procedure can not be neglected . however , since at least two of the three physicians in this study were engaged together in performance of each procedure of coil embolization , it is believed that variation based on the differences among the individual doctors can be excluded . therefore it can be said that the variation in the frequency of thromboembolism between the two periods was significantly affected by the pre - procedural supply of fluid . the most frequent complication of pre - procedural rehydration for ruptured aneurysm was a pulmonary complication . no statistically significant difference was found in the frequency of intra - procedural rupture caused by fluid injection . the most significant finding from this study is that an ample supply of fluid in coil embolization for ruptured aneurysm is likely to reduce the risk of thromboembolism and is unlikely to cause additional complications . it goes without saying that this study has many limitations : first , the patients were gathered prospectively in two separate periods but were not assigned randomly and were analyzed retrospectively ; second , although similar complications of ruptured aneurysm were found between the two groups , the possibility that the accumulation of an operator ' experiences led to lower frequency of thromboembolism can not be completely excluded . and , the authors checked degree of dehydration by subjective patients ' felling of thirsty and the dryness of the tongue . since administering a sufficient dose of fluid according to the degree of pre - procedural dehydration for the patient is very simple in a methodological way and is unlikely to cause additional complications , it is worth trying . nevertheless , since this study has many limitations , as mentioned above , care should be taken in interpreting the results and prospective research should be conducted to confirm them . pre - procedural administration of a sufficient dose of fluid considering the patient 's dehydration reduced the frequency of thromboembolism in cases of emergency coil embolization for ruptured aneurysm , without increasing additional specific complications . while this result has come from retrospective research and care should be taken in interpreting it , the pre - procedural supply of fluid is very easy and simple and can be employed with ease in clinical practice .

objectiveto report effects of the pre - procedural rehydration for reduce thromboembolic complications in acute phase aneurysmal subarachnoid hemorrhage coil embolization.materials and methodsfrom january 2009 to december 2013 , 190 patients with ruptured aneurysmal subarachnoid hemorrhage ( asah ) treated by coil embolization at our institution were consecutively enrolled in this study . in period 1 ( from january 2009 to june 2012 , n = 122 ) , pre - procedural fluid was not supplied . in period 2 ( from july 2012 to december 2013 , n = 68 ) , depending on the state of the patient 's body weight and degree of dehydration , intravenous fluid was started with infusion of approximately 7 ml / kg of 0.9 percent saline ( minimum 300 to maximum 500 ml ) over 30 minutes.resultsa total of 190 patients were hospitalized due to asah and underwent coil embolization for five years between january 2009 and december 2013 . of these , 122 patients underwent coil embolization based on the old protocol before june 2012 ( period 1 ) and 68 underwent the procedure based on the new protocol after the period 2 . neck size , width , maximum diameter of the aneurysm and procedure time were associated with procedure related thromboembolic complications in entire periods ( multivariate analysis , p < 0.05 , in respectively ) . the frequency of thromboembolism showed a drastic decrease in period 2 ( re - hydration period ) , from 18.0% ( 22/123 ) to 4.4% ( 3/67 ) , which was also statistically significant ( p = 0.007 , chi - square test).conclusionpre - procedural administration of a sufficient dose of fluid considering the patient 's dehydration reduced the frequency of thromboembolism in cases of emergency coil embolization for ruptured aneurysm , without increasing additional specific complications .

INTRODUCTION MATERIALS AND METHODS Statistics RESULTS DISCUSSION CONCLUSION

a 50 year old male patient suffered from a systemic febrile sensation , edema , and pain at his first day of admission . the patient had a prior history of disc replacement surgery using an artificial disc between his fifth lumbar vertebra and first sacral vertebra performed approximately 18 months earlier but no other specific findings were found . ten days earlier , the patient visited hospital complaining of symptoms of aggravated low back and both buttock pain after lifting heavy loads . therefore , the patient was diagnosed with spinal facet syndrome , and was treated with fji , which alleviated the pain . the patient took nsaids and muscle relaxants to control the pain that had recurred 4 days before admission . the physical examination at admission revealed severe tenderness at his lumbar vertebral area , but no specific symptoms . the hematologic test results was wbc ( 11.4 10/l ) , esr ( 60 mm / hr ) , crp ( 22.79 mg / dl ) , bun ( 69.7 mg / dl ) , creatinine ( 2.5 mg / dl ) , d - dimer ( 1.01 g / ml ) , fdp ( 5.0 g / ml ) , fibrinogen ( 717 mg / dl ) , and antithrombin iii ( 52% ) . the emergency lumbar spine mri scanning test revealed an epidural abscess at the lower lumbar vertebral area with an abscess in both the left psoas major muscle and paraspinal muscle ( fig . 1 ) . compared to the mri image taken 1 day before receiving the fji , the newly developed abscess was observed by following the facet joints that had developed between the left third and fourth lumbar vertebra ( l3-l4 ) and between the left fourth and fifth lumbar vertebra ( l4-l5 ) . therefore , the patient was diagnosed with an epidural and paraspinal abscess , sepsis and acute renal failure . along with the blood culture test , although percutaneous drainage was performed for the paraspinal abscess on the second day of hospitalization , it was not performed satisfactorily . accordingly , epidural abscess debridement was performed under general anesthesia on the fourth day of hospitalization . a mri of the edema and redness at the extremities revealed diffusible cellulitis at both of his lower extremities and at his right upper extremity along with an abscess at both feet and right elbow . the patient complained of pain around his sternal area , and the computed tomography ( ct ) revealed the development of a septic embolism and the formation of an abscess at the right second intercostal space , which was treated with abscess drainage by mediastinotomy . the surgical findings revealed extensive abscess formation that was linked to the mediastinum by following the right second intercostal space . even after aggressive surgical abscess drainage and intravenous antibiotics injection treatment , the patient 's condition did not improve and the sepsis was not interrupted . the bacterial culture test of the abscess collected from patient 's blood , urine , epidural space , extremities and mediastinum area revealed staphylococcus aureus to be the causative organism . goldthwait first reported the facet joint to be an important cause of low back pain and instability of low back in 1911 . in 1933 , ghormley defined the lumbosacral pain as " facet syndrome " regardless of the presence of sciatica that could developed after sudden rotary strain . generally , facet syndrome is not radiated lower than the knee , but tenderness can be present at the paraspinal muscle , which is characterized as pain that could be aggravated by the extension and rotation of the spine . in 1963 , hirsch et al . reported recurrent pain when saline was injected into a spinal facet joint , which initiated the use of diagnostic fji . recently , the performance of fji and mbb using local anesthesia can alleviate the pain in patients with chronic low back pain . currently , the treatment of a facet joint is the second most commonly performed treatment for low back pain in the united states . although the procedure is frequently performed in such manner , coagulopathy , generalized infection , skin infection at the needle insertion points , hypersensitive reaction and an allergic response to drugs , pregnancy with a possible teratogenic effect by radiation , and the cases of percutaneously inaccessible spinal fusion operation that was performed for the target facet joint can be contraindications to the procedure . bleeding , infection , aggravating pain after the procedure , dural puncture with headache , drug allergy , and pneumothorax could be included as complications that can occur after performing fji . among the infectious complications that can occur after fji , there are 7 case reports of 8 patients [ 4,7 - 12 ] : 1 patient with an epidural abscess , 1 patient with a paraspinal abscess , 2 patients with septic arthritis accompanied with a paraspinal abscess , 2 patients showing the development of meningitis accompanied with paraspinal abscess , 1 patient with an epidural abscess accompanied with a paraspinal abscess , and 1 patient showing development of endocarditis accompanied with paraspinal abscess . the patients who developed infectious complications had received fji several times at more than one site . an epidural abscess has been reported to cause paralysis and sepsis in severe cases resulting in sudden death . however , there are no reports of deaths occurring after fji . in addition , the occurrence of such infectious complications is known to occur more prevalently in patients with diabetes mellitus or patients with immune - deficiency , which caused by alcohol and drug addiction . hence , clinicians should consider the potential for infection when performing fji in such patients . the following causative bacteria have been discovered during the infectious complications after performing fji : staphylococcus aureus in 5 patients , staphylococcus epidermidis in 1 patient , and 2 cases in whom the causative bacteria could not be identified , which was reported to be caused by a pre - treatment using prophylactic antibiotics before performing the blood culture tests . reported that staphylococcus aureus is the most commonly detected bacteria in cases of naturally developed spinal facet joint infections that are spread by the blood stream , and they also suggested that percutaneous drainage ( 85% of success rate ) and a single intravenous antibiotics injection ( 71% of success rate ) as treatments . however , most cases of iatrogenic infections require surgical treatment . orpen and birch recommended 6 to 8 weeks of intravenous antibiotics treatment for the spinal facet joint and paraspinal abscess followed by oral antibiotics treatment for 3 months . among the 8 patients who experienced an infection after receiving fji , it is unclear if 2 patients had received treatment for the infection , but 4 patients received surgical treatment and intravenous antibiotics treatment simultaneously , 1 patient only received surgical abscess drainage without antibiotics treatment and 1 patient only received intravenous antibiotics treatment . despite these aggressive treatments , only 1 of the 6 patients who had received treatment , experience sensory and motor dysfunction . in the current case report , the patient died as a result of sepsis despite the surgical and intravenous antibiotics treatments . all the patients who had experienced infectious complications after receiving fji reported an aggravation of their low back pain or a change in their pain pattern . among the 8 reported patients , 3 patients reported paraspinal tenderness , and 4 patients experienced symptoms of systemic infection including chills , fever and malaise . the hematologic test results revealed an increase in the wbc count , esr , and crp levels , and the radiologic tests including mri showed paraspinal and epidural abscesses with various inflammatory findings . muffoletto et al . reported that mri provides high sensitivity and specificity only 2 days after the development of symptoms , and also added that the use of gadolinium contrast medium can be more helpful in making a diagnosis . a failure of skin disinfection , an absence of sterile techniques , the use of contaminated catheters or needles , and cases of natural hematogenously disseminated infections could be included as possible causes of infectious complications after performing fji . these infectious complications developed despite the use of complete aseptic procedures by practicing complete skin disinfection and using aseptic nerve block needles . as the cases that could be regarded as iatrogenic infections , the symptoms developed within a short time after fji , 2 days to 20 days ( mean 9.75 days ) . the time interval from the development of the first symptoms to the time point when treatment was started is the most important factor for the patient 's prognosis , and immediate diagnosis and rapid treatment are essential . to prevent the occurrence of infectious complications after fji , diagnostic or therapeutic fji need to be performed under a precise diagnosis made from the physical examinations and radiologic tests . it should be noted that performing fji in too many sites or too frequently can increase the likelihood of complications . in addition , the procedure has to be performed aseptically by recognizing the fact that fji could be a cause of fatal infectious complications . since no infectious complications were reported after performing mbb , it will be desirable to perform mbb for patients with a high probability of infection , and studies on the difference in infectious complications between the two procedures are warranted . hematologic tests , including wbc count , ers , and crp tests , should to be performed immediately when infection - suspicious symptoms are noticed , such as aggravated pain or a change in the pain pattern along with a febrile sensation and severe tenderness . mri should be performed as soon as possible if an infection is suspected in the hematologic test results . after making a quick diagnosis , surgical or nonsurgical abscess drainage and immediate antibiotics treatment

facet joints have been shown to be a source of chronic low back pain , and it is generally accepted in clinical practice that diagnostic and therapeutic facet joint injections are the most reliable technique for the treatment of facet joint pain , which is considered to be an easy and safe procedure . serious complications and side effects are uncommon after facet joint injection . however , infectious complications including septic arthritis , epidural abscess , meningitis and endocarditis have been reported following facet joint injections . we report here the first case of death following lumbar facet joint injection due to generalized infection .

Case Report DISCUSSION

hump - nosed viper may account for upto 27 - 70% of all snake bites in sri lanka . although previously considered a moderately venomous , hump - nosed viper bites cause significant morbidity in the wet zone ( hot and humid south western part of the island with an annual rainfall of more than 2,500 mm ) of sri lanka . acute kidney injury ( aki ) necessitating hemodialysis and disseminated intravascular coagulation are known complications of hump - nosed viper bite . the lack of an effective antivenom has contributed to the difficulty experienced by the clinicians during management of hump - nosed viper bites . we describe a patient who developed disseminated intravascular coagulation and prolonged aki following a hump - nosed viper bite . he was successfully managed with hemodialysis and blood product support but he after developed severe hyperkalemia and acidosis due to type iv renal tubular acidosis after discharge from hospital . type iv renal tubular acidosis has not been described previously in patients who had snake bites . a 51-year - old male was admitted to the local hospital after a hump - nosed viper bite on his hand while weeding his garden . since the viper was brought alive by the neighbors , primary care physician was able to make a positive identification of the snake as merrem 's hump - nosed viper ( hypnale hypnale ) . the next day his urine output was reduced and he was transferred to a tertiary care institute for hemodialysis . on admission , he had marked swelling and redness in his right hand with blistering at the bite site . on auscultation of the heart , s1 and s2 were heard in normal intensity with no murmurs . his investigation results were as follows : white cell count , 12,700 ( 80% neutrophils ) ; platelet count , 40,000/l ; hemoglobin , 9.9 g / dl ; international normalized ratio , 1.46 ; activated partial thromboplastin time , 43 s ; serum creatinine , 1,040 mol / l ; na , + 130 mmol / l ; k+ , 5.4 mmol / l ; erythrocyte sedimentation rate , 10 mm / hr ; c - reactive protein , 1 mg / l ; aspartate transaminase , 54 u / l ; alanine transaminase , 42 u / l ; serum bilirubin , 22.7 mol / l ; total protein , 52 g / l ( albumin 33 peripheral blood smear revealed marked red cell fragmentation , polychromasia and reduced number of platelets indicating disseminated intravascular coagulation . serum calcium , 7.7 mg / dl ; serum phosphate , 3.1 mg / dl ; creatine phosphokinase , 122 u / l ; urine microscopy showing 10 - 12 pus cells and 1 - 2 red cells per high power field with no casts . after 12 sessions of hemodialysis , he was dialysis dependent and renal biopsy was performed 8 weeks after the viper bite . the biopsy revealed patchy areas of tubular atrophy with interstitial edema and patchy interstitial chronic inflammation suggesting subacute interstitial nephritis . there was no evidence of acute tubular necrosis possibly because biopsy was performed very late after the onset of aki . ten weeks after the viper bite , the patient 's renal functions and urine output were improved . he was discharged from the hospital to be reviewed in clinic as an outpatient . at the time of discharge , he had a serum creatinine of 1.9 mg / dl and serum potassium of 3.9 mmol / l . eighteen weeks after the initial viper bite , he was admitted with sudden onset bilateral lower limb weakness . arterial ph was 7.31 and pco2 18 mmhg , po2 121 mmhg , and hco3 10.3 mmol / l . mmol / l with sodium 130 mmol / l and chloride 114 mmol / l . he was given intravenous calcium gluconate , nebulized with salbutamol and started on insulin - dextrose infusion until an urgent hemodialysis was arranged . there was no history of use of angiotensin - converting enzyme inhibitors ( acei ) , angiotensin receptor blockers ( arb ) , or diuretics like spironolactone . mmol / l , ph 5.0 , serum osmolality 300 mosm / kg , urine osmolality 380 mosm / kg , and urinary anion gap was 9 mmol / l . presence of hyperchloremic metabolic acidosis and hyperkalemia with normal serum creatinine level was suggestive of type iv renal tubular acidosis . the ability to lower the urine ph to 5.0 , positive urinary anion gap , and the low ttkg ( less than five ) were all supportive evidence for type 4 renal tubular acidosis . patient had a good response to fludrocortisone 100 g twice daily with the correction of hyperkalemia and the metabolic acidosis . mmol / l and he was closely observed for the development of hyperkalemia or acidosis . type 4 renal tubular acidosis is due to failure of the collecting duct to excrete both potassium and protons . this is caused by absolute or functional deficiency of aldosterone or a molecular defect in the relevant transporters . aldosterone is necessary for the maintenance of the lumen negative potential by the reabsorption of na by the epithelial sodium channel on the luminal membrane of the principal cells . this lumen negative potential in turn drives the secretion of potassium and protons into the lumen . this subtype of renal tubular acidosis is usually observed in patients with diabetes mellitus and various forms of tubulointerstitial disease including sickle cell disease , amyloid , monoclonal gammopathy , and nsaid - induced interstitial nephritis . drugs that cause a similar picture include acei , arb spironolactone , heparin , and -blockers . the possible pathogenesis of renal tubular acidosis in this patient could be due to snake venom - mediated tubular damage leading to aldosterone receptor dysfunction or aldosterone resistance . although disseminated intravascular coagulation necessitating blood product support and aki are known complications of hump - nosed viper bite , the development of type 4 renal tubular acidosis was a new finding . this case is clinically informative because it describes a previously unknown complication following viper bite and makes a clinician more vigilant regarding renal function and serum electrolyte levels even after patient has recovered from snake venom - induced aki .

hump - nosed viper bite can cause acute kidney injury ( aki ) and disseminated intravascular coagulation . in some patients , it can cause chronic kidney disease necessitating life - long renal replacement therapy . lack of effective antivenom makes the management of these patients difficult . a 51-year - old sri lankan male was admitted with aki and disseminated intravascular coagulation following a hump - nosed viper bite . he made a complete recovery with blood product support and hemodialysis . renal biopsy was performed as his renal recovery was prolonged which revealed patchy tubular atrophy and interstitial inflammation suggestive of subacute interstitial nephritis . later , he presented with hyperkalemic paralysis and acidosis . a diagnosis of late onset type 4 renal tubular acidosis was made and he responded well to a course of fludrocortisone .

Introduction Case Report Discussion Conclusions

palatal sialolipomas are rare and were identified as a definite entity in 2001 . to our knowledge , less than 10 cases from the hard palate are reported . here , we report a case of hard palate sialolipoma , peculiar by the intratumor malformation - type vascular patterns . a 67-year - old man presented with a median mass of the oral hard palate , noticed since several years . focally , the lesion was surrounded by a fibrous tissue rim mainly at the interface with the oral mucosa ( which was not ulcerated ) . deeply , there were minor salivary gland lobules , interspersed completely or in part by adipocytes . the salivary gland showed focal lymphocytic infiltrate , a focus of fibrous regression with ductal atrophy as well as zones of ductal basal cell hyperplasia and/or large duct dilatation . however , numerous hemorrhagic areas were seen in marginal zones , probably in relationship with intraoperative vascular section / rupture . rare nerves were seen in close association with adipocyte , glandular lobules and large vessels at the periphery of the specimen . smooth muscle actin was expressed in the vessel wall and consisted of a rim of positive cells of varied thickness . endothelial cells were positive for cd31 and negative for d2.40 ( expressed in superficial , pericapsular lymphatics ) . photomicrograph showing lesional tissue consisting mainly of adipose tissue ( asterisk ) covered by an intact epithelium with a fibrous band below the epithelium ( black arrows : vessel walls ) ( h&e stain , x25 ) , ( b ) lesional tissue showing large , ramified vessels with irregularly thickened wall ( black arrows ) and salivary gland lobules ( arrow head ) ( h&e stain , x50 ) , ( c ) areas showing salivary gland lobules ( arrow head ) and adipose tissue ( asterisk ) ( h&e stain , x25 ) , ( d ) high power view showing the salivary gland lobules ( arrow head ) ( h&e stain , x200 ) ( asterisk : adipose tissue ) ( inset : photomicrograph showing adipose tissue and nerves / arrows , h&e stain , x100 ) , ( e ) vessel walls showing papillary projections ( white arrows ) ( h&e stain , x200 ) ( inset : low power view of the vessel 's wall ( h&e stain , x25 ) , ( f ) vessel wall showing positivity for smooth muscle actin ( black arrows ) ( ihc stain , x50 ) . here , we report a case of sialolipoma of the hard palate peculiar by the presence of intratumoral malformation - type vascular patterns . several blood vessels were large , with thickened wall and rare papillary projections or tufts . this latter morphological feature , more frequently reported in vascular malformations than in angiomas when considering the gastrointestinal location , rather suggested a vascular lesion of malformation - type than an associated angioma or an angiolipoma - type lesion . moreover , the presence of nerves favored the hypothesis of vascular malformation type lesions . with regard to hard palate angiolipomas , defined as lipomas with a prominent capillary network , these tumors are rare to our knowledge , reported in two cases : one in a young adult man and one in a girl . they are located laterally on the right or left side , age predilection is mainly between 60 - 70 years ( although a symptom duration of 15 days to 10 years ) and measure more than 4-cm in size . the size of hard palate sialolipomas ( 11.8 cm ) and their age distribution reported in the literature several other adipocyte - abundant lesions are also to be ruled out . a traumatic pseudolipoma was not considered since the evolution was long and reparative signs such as fibrosis and inflammation were limited . the diagnosis of lipomatosis was also not considered due to the presence of capsular tissue neither was an infiltrating lipoma considered , although this lipoma - type can contain large vessels . another hypothesis is that of entrapped vessels in adipose tissue , difficult to sustain for a low - fat location such as the hard palate . the origin of lipomatous lesions of the hard palate , is similar to that of oral lesions and remains incompletely elucidated . lipoblastic embryonic cell nests , metaplasia of muscle cells and fatty degeneration can be incriminated as originary events while trauma ( with or without soft palate tissues dislocation ) , infection , chronic irritation or hormone imbalance can be considered as favoring factors . whether an altered oxygenation as a result of intra - tumor malformative vessel network may have an impact on a limited growth although there was a long evolution period remains to be further elucidated . it should also be taken into consideration that losartan - treated patients show a higher body mass index as compared to placebo - treated patients ( despite randomization ) , although losartan does not affect this index . however , adipose hyperplasia or submucosal adipose involution in the context of overweight / obesity may be considered among the hypotheses for histogenesis of fat lesions of the hard palate . interestingly , a hard palate lipomatous lesion of osteolipoma type is reported to be associated with cleft palate in a 6-year - old boy . cleft palate patients are also reported to rarely show vascular malformations either de novo , reported in pediatric cases or as a manifestation after post surgical treatment , reported in a 25-year - old woman . whether in the present case a subclinical osseous malformation was present remains difficult to be determined due to the lack of computed tomography scan . the microscopic features of the tumor in the present case suggest that malformation - type vascular patterns may occur in sialolipomas of the hard palate . this morphological vascular peculiarity should be acknowledged since it can be a possible source of hemorrhage . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed there are no conflicts of interest . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed

palate sialolipomas are rare . less than 10 cases located in the hard palate are reported to our knowledge . we report a case of hard palate sialolipoma , peculiar by the intratumor vascular patterns . a 67-year - old man presented with a 1.5 cm lesion of the oral hard palate . the lesion was surgically resected . on microscopy , the lesion , partly encapsulated , consisted of a proliferation of mature adipocytes containing normal minor salivary gland tissue and branching intratumoral vessels of varied size with irregularly thickened wall and papillary projections or tufts . the microscopic features of the tumor we report suggest that vascular malformation - like patterns may occur in sialolipomas of the hard palate . this morphological vascular peculiarity should be acknowledged since it may represent source of hemorrhage .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION Declaration of patient consent Financial support and sponsorship Conflicts of interest

in diabetes mellitus , high blood glucose levels and advanced glycosylation end products cause constriction and leakage of retinal vasculature , thickening of the basement membrane , and pericyte apoptosis.14 this damage to the vasculature results in ischemia , promoting a cascade of molecular processes including upregulation of hypoxia inducible factor-1 , which in turn upregulates cytokines and growth factors , including vascular endothelial growth factor ( vegf ) , which further contribute to breakdown of the blood retinal barrier.5,6 analyses of vitreous fluid from patients with diabetic retinopathy and proliferative diabetic retinopathy consistently demonstrate abnormally increased vegf concentrations.7,8 in support of the clinical evidence , studies in animal models indicate a correlative role of vegf in diabetic retinopathy . injection of vegf into primate vitreous fluid induces lesions consistent with diabetic retinopathy.9 neutralizing vegf in diabetic rats inhibits blood retinal barrier breakdown in a dose - dependent manner.10 these findings indicate that vegf is a major contributor to diabetic retinopathy and diabetic macular edema . the rise and ride trials demonstrated that intravitreal injection of ranibizumab 0.3 mg ( lucentis , hoffman - la roche ltd , basel , switzerland ) , a monoclonal antibody against vegf - a , as compared with sham injections , improved all measures of vision assessed in the trials.11 the vivid and vista trials demonstrated that intravitreal aflibercept 2 mg ( eylea , regeneron pharmaceuticals , inc , tarrytown , ny , usa ) therapy resulted in greater gains and fewer losses in visual acuity when compared with laser.12 in the diabetic retinopathy clinical research network s ( drcr.net ) protocol i , treatment with ranibizumab plus prompt or deferred laser improved patient visual acuity , as compared with sham injections and laser.13 these and other studies have established anti - vegf intravitreal injections as the standard of care for the treatment of diabetic macular edema . in addition to improvement of visual acuity and reduction of diabetic macular edema , studies have shown that anti - vegf therapies normalize blood vessels on 7-standard fields ( 7sfs ) color fundus photographs . analyses of photographs obtained in rise and ride demonstrated that patients receiving intravitreal ranibizumab were more likely to have 2-step or 3-step improvement in diabetic retinopathy severity scale score than patients receiving sham injections.14 in vivid and vista , a greater proportion of eyes treated with intravitreal aflibercept had 2-step improvement in diabetic retinopathy severity scale score compared with eyes treated with laser photocoagulation.12 a retrospective analysis of fluorescein angiography ( fa ) obtained in the rise and ride trials demonstrated decreased rate of progression of nonperfusion in treated eyes in patients receiving monthly 0.3 mg injections of ranibizumab , or 0.5 mg injections of ranibizumab , as compared with those patients receiving monthly sham injections ( with crossover to ranibizumab injections).15 while these studies showing reduced progression of nonperfusion are encouraging , there are to our knowledge no prior studies evaluating retinal reperfusion after anti - vegf therapy in diabetic retinopathy . development of ultra - widefield fluorescein angiography ( uwfa ) has expanded visualization of the retina to 200 of the retina from 75 on the 7sf protocol , enabling more thorough assessment of peripheral nonperfusion , neovascularization , and panretinal photocoagulation.16 ultra - widefield imaging has demonstrated that peripheral lesions not visualized with 7sf are clinically significant . a study comparing mydriatic ultra - widefield imaging versus 7sf reported that one - third of lesions detected on ultra - widefield imaging were outside the 7sf.17 the presence of these peripheral lesions , undetectable on 7sf , was associated with a 3.2-fold increased risk of progression of diabetic retinopathy and a 4.7-fold increased risk of progression to proliferative diabetic retinopathy.18 uwfa has an additional advantage over 7sf in that it enables more accurate comparison of perfusion over multiple clinic visits . a single 7sf image is produced from seven individual images that are not captured simultaneously , thus the distribution of fluorescein dye in an individual image differs from the distribution of dye in every other individual image . this difference in the points at which separate parts of the 7sf image were captured decreases the accuracy of a comparison of perfusion in two 7sf images taken at two different clinic visits . uwfa captures more area at any single time point following fluorescein dye injection , allowing for a more true comparison of perfusion over multiple visits . , we therefore hypothesized that 1 ) areas of retinal nonperfusion have the potential to reperfuse following anti - vegf therapy and 2 ) uwfa can be used to identify this normalization of perfusion following anti - vegf treatment for diabetic retinopathy . to assess this hypothesis , we performed qualitative and quantitative analysis of areas of nonperfusion noted on fluorescein angiograms obtained prior to and after treatment with anti - vegf injections . patients were initially identified by screening for those with a diagnosis of diabetes with ophthalmic complications ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] code 250.50 ) along with common proficiency test coded indicating that both a fluorescein angiogram ( 92235 ) and an intravitreal injection ( 67028 ) were performed . the inclusion criteria were : diagnosis of diabetic retinopathy ( nonproliferative diabetic retinopathy or proliferative diabetic retinopathy ) , treatment with at least one anti - vegf injections in the study eye ( ranibizumab 0.3 mg , aflibercept 2 mg , or bevacizumab 1.25 mg ) , and pre- and postinjection uwfa imaging available , with the postinjection uwfa being within 5 months of the last intravitreal anti - vegf injection in the study eye . the exclusion criteria were history of intravitreal anti - vegf therapy before the preinjection uwfa , poor quality of imaging due to media opacity , concurrent nondiabetic retinal vascular disease ( retinal artery occlusion , retinal vein occlusion ) , or absence of retinal nonperfusion on preinjection uwfa . the electronic medical record was reviewed and the following data were collected : age , sex , body mass index ( bmi ) , weight , comorbidities ( hypertension [ ht ] , congestive heart failure [ chf ] ) , hba1c , history of smoking , indication for pre- and postinjection uwfa , dates , names , and indications for all intravitreal injections . uwfa images included in this study had been generated using optos optomap panoramic 200a ( optos plc , dunfermline , scotland ) , captured digitally with the optos v2 vantage review software , and stored as uncompressed tiff files . all patients had provided written informed consent for all fluorescein angiograms performed and for each intravitreal anti - vegf injections performed . this retrospective study was approved by the institutional review board at weill cornell medical college . written informed consent for participation in this study was deemed unnecessary by the review board due to its retrospective nature . pairs of pre- and postinjection uwfa images were graded for reperfusion ( 1= reperfusion present in postinjection image compared side - by - side with preinjection image , 0= no reperfusion ) by 2 independent graders ( aml and ir ) ; both graders were masked to all characteristics of the patients and injections that corresponded to each image . reperfusion was verified by a senior grader ( sk ) . because the graders could not be masked to the identities of the images as pre- or postinjection ( due to obvious identifiers such as abnormal neovascularization in preinjection images ) , reperfusion was confirmed quantitatively using imagej software . during this process each image that was graded positively for reperfusion by the graders was converted in imagej to an 8-bit grayscale image ( 256 levels of gray , with pure black pixels = 0 and pure white pixels = 1 ) . then , a minimum threshold of 35% was set so that pixels < 35% appeared black . this threshold was collectively decided upon by 3 graders and applied consistently to every image . each image the difference in white pixel number between the pre- and postinjection images was calculated in selected areas determined by the graders to have reperfusion . we were unable to quantify pixels over the area of an entire image due to artifacts such as eyelashes , lids , glare , etc . pairs of images that were graded positively for reperfusion by the graders and demonstrated increased pixel count in the selected area were considered to demonstrate reperfusion . forty - nine eyes of 39 patients were retrospectively identified as having a diagnosis of diabetes , having underwent intravitreal anti - vegf injection(s ) , and having received pre- and postinjection uwfa , with the postinjection uwfa occurring less than 5 months after the last anti - vegf injection . thirty eyes of 21 patients were excluded due to nongradable images from media opacities ( vitreous hemorrhage , cataract ) or movement artifact , resulting in 19 eyes of 18 patients with gradable images . of these 19 , 1 eye was excluded because of concurrent acute retinal vein occlusion , and 2 eyes of 2 patients were excluded because of absence of nonperfusion in the preinjection image . a total of 16 eyes of 15 patients were thus included in the study . all patients were injection naive prior to their preinjection image . indication for the preinjection uwfa was standard of care diabetic retinopathy management for all 16 eyes . the uwfa revealed a diagnosis of nonproliferative diabetic retinopathy in 1 eye , proliferative diabetic retinopathy not meeting high - risk criteria in 9 eyes , proliferative diabetic retinopathy meeting high - risk criteria in 3 eyes ( 2 of whom subsequently had panretinal laser photocoagulation and 1 of whom did not ) , a history of proliferative diabetic retinopathy with prior panretinal photocoagulation , and persistent neo - vascularization requiring additional fill - in laser ( which was performed during the study period ) in 2 eyes , and a history of proliferative diabetic retinopathy with prior panretinal photocoagulation and improvement in neovascularization in 1 eye . indications for anti - vegf therapy included treatment of diabetic macular edema in 15 of 16 eyes and treatment of high - risk proliferative diabetic retinopathy in the absence of edema in 1 eye after obtaining informed consent including extensive discussion of the risks and benefits of observation versus panretinal photocoagulation versus intravitreal anti - vegf therapy , the ongoing drcr.net protocol s study and as - of - yet unestablished efficacy in clinical trials of anti - vegf therapy for diabetic retinopathy , and the off - label nature of treatment of proliferative diabetic retinopathy with anti - vegf therapies . the mean time between the pre- and postinjection fa was 337 days ( range : 501,173 ) . indications for postinjection uwfa included annual uwfa in 2 patients ( one with prior fa showing nonproliferative diabetic retinopathy and one with prior uwfa showing regressing proliferative diabetic retinopathy status post panretinal photocoagulation ) , reassessment for progression to high - risk proliferative diabetic retinopathy in the 9 eyes that had previously demonstrated non high - risk proliferative diabetic retinopathy , evaluation for regression of neovascularization after laser in 3 eyes , evaluation of the contralateral eye after clearance of vitreous hemorrhage in 1 eye , and reevaluation of high - risk proliferative diabetic retinopathy in 1 eye that was treated with anti - vegf therapy alone for this condition . twelve out of 16 eyes ( 75% ) demonstrated reperfusion both qualitatively and quantitatively on uwfa following anti - vegf therapy . the 11 patients who reperfused included 7 males and 4 females with a mean age of 60.0 years ( range : 4673 ) at the time that the second image was obtained , mean weight of 87.7 kg , mean bmi of 30.7 , and mean hba1c of 8.7% ( range : 7.214.0 ) ( table 1 ) . this group included 10 patients with a diagnosis of ht , 2 with a diagnosis of chf , 7 never smokers , 1 current smoker , and 3 former smokers . eleven eyes who reperfused received only ranibizumab injections , 1 received only bevacizumab injections , and the mean number of injections per eye was 3 ( range : 16 ; table 2 ) . the images were obtained at a mean of 71.9 seconds ( range : 38110 ) after the fluorescein dye was injected , and the pre- and postimages were obtained at a mean of 4.5 seconds ( range : 028 ) apart . the postinjection image was obtained at a mean of 67 days ( range : 21144 ) after the patient received their last anti - vegf injection and 211 days ( range : 50544 ) from the preinjection uwfa . these 4 patients included 2 males and 2 females with a mean age of 61.5 years ( range : 4771 ) at the time that the second image was obtained ( table 1 ) . there were not enough available data to calculate mean weight , bmi , or hba1c . this group included 2 patients with a diagnosis of ht , 2 with a diagnosis of chf , 3 never smokers , and 1 former smoker . one patients received only ranibizumab injections , 1 received ranibizumab and aflibercept , 2 received bevacizumab injections only , and the mean number of injections was 7.25 ( range : 514 ; table 2 ) . the images were obtained at a mean of 58 seconds ( range : 3891 ) after the fluorescein dye was injected , and the pre- and postimages were obtained at a mean of 15.5 seconds ( range : 131 ) apart . the postinjection image was obtained at a mean of 42 days ( range : 2170 ) after the patient received their last anti - vegf injection and 642 days ( range : 2171173 ) from the preinjection uwfa . in this small retrospective case series , we demonstrate that treatment of diabetic retinopathy with anti - vegf injections has the potential to reverse retinal ischemia as noted on uwfa . these findings suggest that areas of ischemia on uwfa can possess viable , salvageable tissue with the potential to reperfuse . though our study is not designed to demonstrate cause and effect between anti - vegf injections and reperfusion , we believe that injections likely contribute to reperfusion by stabilizing healthier blood vessels . reperfusion of vessels might be one reason that clinical trials have shown reversal of diabetic retinopathy after anti - vegf therapy . the molecular mechanism by which anti - vegf therapies normalize retinal blood vessels remains unknown , but studies of other retinal diseases and extensive studies of anti - vegf effects on tumors might give insight into the role of anti - vegf therapy in the retina . one prior study demonstrated reversal of retinal nonperfusion in eyes treated with anti - vegf agents for retinal vein occlusion . the authors of the study hypothesized that anti - vegf agents interrupt a positive feedback loop in which injury induces vegf signaling , resulting in further injury.19 large clinical trials of cancer treatments have demonstrated that anti - vegf therapy enhances the effect of chemotherapy.2022 additional studies have contrasted the efficacy of anti - vegf combined with chemotherapy with the relative ineffectiveness of anti - vegf monotherapy.23 this contrast led to the hypothesis that anti - vegf stabilizes tumor vessels , thus augmenting chemotherapy delivery . investigations of this hypothesis elucidated specific processes involved in the normalization of vessels treated with anti - vegf , including reversal of hyperpermeability , restoration of pericytes , and normalization of the basement membrane.24,25 normalized basement membrane could serve as a framework for vessel regrowth in the retina as well . in our study , some eyes demonstrated expansive reper - fusion , while other eyes reperfused in only limited areas of the retina , while some eyes did not reperfuse at all . one possibility is that ischemic areas that do not reperfuse following anti - vegf injections might be completely infarcted or might involve living tissue that is not responsive to the given doses of anti - vegf injections . one patient who did not reperfuse had expansive scarring from panretinal photocoagulation that may have prevented reperfusion or detection of reperfusion . our finding that not all patients reperfused is supported by a previous study that showed spontaneous reperfusion in some but not all untreated eyes diagnosed with diabetic retinopathy . this previous study defined reperfusion as either neovascularization or recanalization , and found recanalization in one - third of eyes.26 our study was not powered to compare the demographics of the populations that either reperfused or did not reperfuse , as only 5 patients did not reperfuse . a recent analysis of rise and ride reported decreased rates of progression of nonperfusion in patients treated with ranibizumab.15 while no patients were reported to have reperfused in that analysis , the study was not designed to measure reperfusion . in the rise and ride analyses of retinal nonperfusion , nonperfusion was measured categorically by the number of disk areas ( da ) containing nonperfused regions ( eg , > 0 to < 1 da of nonperfusion versus > 1 to < 2 da , etc ) . this method was not tailored for measuring subtle reperfusion , as an image with > 1 to < 2 da of nonperfusion could partially reperfuse while still demonstrating regions of nonperfusion , albeit smaller regions , in > 1 to < 2 da . in our study , some of our patients reperfused outside the area captured by 7sf fa . in patients who reperfused only outside the area captured by 7sf fa our population of patients who reperfused was fairly similar to patients in rise and rise receiving injections : mean ages in all rise and ride groups receiving injections ranged from 61.762.8 years ( our study : 60.0 years ) , mean bmi 31.332.9 kg / m ( 30.7 kg / m ) , history of smoking 45.6%51.6% ( 36% ) , and mean hba1c 7.6%7.7% ( 8.7%).11 our specific goal of measuring reperfusion and our use of uwfa may be the reason that our study detected reperfusion that was not reported in previous analyses of diabetic retinopathy . less than 6 months prior to the start of our study , protocol t published by the diabetic retinopathy clinical research group demonstrated that patients treated with aflibercept required fewer injections and less laser therapy to achieve greater visual acuity than patients treated with ranibizumab or bevacizumab.27 therefore , it would be of interest to study reperfusion in patients treated only with aflibercept . although the number of patients receiving aflibercept in our clinic increased following the publication of protocol t , these patients have yet to receive a postinjection uwfa and do not qualify for this study . the strengths of this study include the use of uwfa to capture peripheral nonperfusion and reperfusion , and the confirmation of qualitative analysis by retina specialists with quantitative measurements of reperfusion . quantitative analysis prevented bias resulting from recognition of an image as pre- or postinjection and from the assessment of an area of reperfusion as darker or lighter based on the brightness of surrounding vasculature . a limitation of this study is that the images were not all captured at exactly the same time point at exactly the same angle , because the time and angle depended on uncontrolled variables such as the patient s precise position during imaging . this variation between images is to be expected in any retrospective review of fa images , as well as in a physician s analysis of images during a patient encounter . this study is also limited by the inability to confirm that areas of increased fluorescence represent areas of reperfusion . some areas of assumed reperfusion clearly show definite vascular outlines , and these areas can be interpreted most confidently as reperfusion . some areas show increased fluorescence , but outlines of vessels are blurred either because the equipment is not sensitive enough to clearly capture the outlines or because the patient was moving . follow - up studies are needed to demonstrate reperfusion in patients treated with anti - vegf agents and to better understand the characteristics of patients who reperfuse . a future prospective study could elucidate the relationship between treatment regimens , demographic factors , imaging and examination findings , and the potential for reperfusion . whether there is any correlation in reperfusion and effect of the anti - vegf therapy on macular thickness the macular edema in these patients waxed and waned and the number of injections over the study period was similarly variable , while perfusion was only assessed at a single pre- and postinjection standpoint , therefore making it difficult to assess correlations between perfusion and macular thickness . treatment with anti - vegf agents can be associated with reperfusion of areas of nonperfusion . prospective studies evaluating the effect of anti - vegf therapy on perfusion should incorporate correlations to macular thickness response and other imaging features in the study design . future studies could also explore the molecular mechanism of reperfusion and the potential role of different anti - vegf therapies .

purposethe aim of this study is to report peripheral reperfusion of ischemic areas of the retina on ultra - widefield fluorescein angiography ( uwfa ) following anti - vascular endothelial growth factor ( vegf ) intravitreal injections in patients treated for diabetic retinopathy.methodsthis study is a retrospective review of 16 eyes of 15 patients with diabetic retinopathy , who received anti - vegf intravitreal injections and underwent pre- and postinjection uwfa . the main outcome measured was the presence of reperfusion in postinjection uwfa images in areas of the retina that demonstrated nonperfusion in preinjection images . images were analyzed for reperfusion qualitatively and quantitatively by two graders.resultstwelve of 16 eyes ( 75% ) or 11 of 15 patients ( 73.3% ) demonstrated reperfusion following anti - vegf injection . on uwfa , reperfusion was detected both within the field of 7-standard field ( 7sf ) fluorescein angiography and in the periphery outside the 7sf . four of 16 eyes or 4 of 15 patients did not demonstrate reperfusion , one of which had extensive scarring from prior panretinal photocoagulation.conclusionin patients with diabetic retinopathy , treatment with anti - vegf agents can be associated with reperfusion of areas of nonperfusion , as demonstrated by uwfa .

Introduction Methods Results Discussion Conclusion

in an era of advancing technology and evolving practice health practitioners need to update practices continually in order to provide quality healthcare.1,2 radiation therapists ( rts ) are required to maintain skills and knowledge in current practice while adapting to this ongoing change . the challenge of establishing and maintaining consistency of practice in a dynamic radiation therapy department highlights the importance of having defined practice standards and a reliable process for assessing performance . well - defined standards can provide a clear statement of what is important in performance and be used to motivate staff and provide feedback on performance.35 performance appraisal must also provide adequate support and training for development needs.3,6 a tool that encompasses these features will facilitate effective performance appraisal and promote staff satisfaction and professional growth . a radiation therapy performance appraisal framework ( rt - paf ) was developed to promote a culture of performance appraisal that was fair , objective , transparent , based on evidence and focussed on skills development . the focus of this initiative was on radiation therapy planning because of the independent nature of practice and the inherent difficulties in providing feedback on performance . development began with base - grade skill and was designed to complement existing work processes rather than add to them . planning practice was structured such that the one rt was responsible for the ct simulation , dosimetry and plan finalisation of each patient allocated to them . the rt - paf was developed incrementally and implemented on a trial basis so that lessons learned from each stage could inform development of ensuing stages . skills sets for each area of practice were identified and defined by performance indicators ; these were then incorporated into case - based feedback forms . the appraisal process commenced once the rt had been in planning for a minimum of 2 weeks and was conducted over a 4-week period . the rt was given a series of forms to include with every patient case they completed during this period . these forms were used to evaluate dosimetry , ct / simulation and plan evaluation , and were completed as part of the routine peer review process for each area of practice . peer reviewers were chosen as experienced in the scope of the review irrespective of relative seniority to reduce the perceived effect of power relationships . at the completion of the review period , a senior rt collated the completed forms to finalise the feedback . final feedback included additional elements of performance such as professional attitude , time / workload management , commitment to quality and technical communication which were assessed by observation and collaboration with peer reviewers . documented guidelines recommended that feedback be delivered within 1 week of the review period ending to allow time for the rt to respond and negotiate a development plan while still rostered in planning . the goal was to integrate the rt - paf into the routine practice and repeat during each planning rotation ; however this depended on frequency and length of rotation . an integral part of the development process was to provide clearly documented protocols and process guidelines to support consistency in practice . rigour was placed on defining skill levels for each skill set to support a consistency and transparency in the assessment process . the purpose of this study was to evaluate the effectiveness of the rt - paf as experienced by rts . feedback quality was evaluated in terms of timely delivery and whether equal focus was given to identifying strengths and highlighting areas for development . the quality of the process was measured in terms of overall satisfaction , the perception of objectivity and fairness and how effectively the development of skills was enabled . this included identifying any obstacles encountered to meeting these goals and seeking recommendation for improvement . a radiation therapy performance appraisal framework ( rt - paf ) was developed to promote a culture of performance appraisal that was fair , objective , transparent , based on evidence and focussed on skills development . the focus of this initiative was on radiation therapy planning because of the independent nature of practice and the inherent difficulties in providing feedback on performance . development began with base - grade skill and was designed to complement existing work processes rather than add to them . planning practice was structured such that the one rt was responsible for the ct simulation , dosimetry and plan finalisation of each patient allocated to them . the rt - paf was developed incrementally and implemented on a trial basis so that lessons learned from each stage could inform development of ensuing stages . skills sets for each area of practice were identified and defined by performance indicators ; these were then incorporated into case - based feedback forms . the appraisal process commenced once the rt had been in planning for a minimum of 2 weeks and was conducted over a 4-week period . the rt was given a series of forms to include with every patient case they completed during this period . these forms were used to evaluate dosimetry , ct / simulation and plan evaluation , and were completed as part of the routine peer review process for each area of practice . peer reviewers were chosen as experienced in the scope of the review irrespective of relative seniority to reduce the perceived effect of power relationships . at the completion of the review period , a senior rt collated the completed forms to finalise the feedback . final feedback included additional elements of performance such as professional attitude , time / workload management , commitment to quality and technical communication which were assessed by observation and collaboration with peer reviewers . documented guidelines recommended that feedback be delivered within 1 week of the review period ending to allow time for the rt to respond and negotiate a development plan while still rostered in planning . the goal was to integrate the rt - paf into the routine practice and repeat during each planning rotation ; however this depended on frequency and length of rotation . an integral part of the development process was to provide clearly documented protocols and process guidelines to support consistency in practice . rigour was placed on defining skill levels for each skill set to support a consistency and transparency in the assessment process . the purpose of this study was to evaluate the effectiveness of the rt - paf as experienced by rts . feedback quality was evaluated in terms of timely delivery and whether equal focus was given to identifying strengths and highlighting areas for development . the quality of the process was measured in terms of overall satisfaction , the perception of objectivity and fairness and how effectively the development of skills was enabled . this included identifying any obstacles encountered to meeting these goals and seeking recommendation for improvement . in march 2011 , after a 4-year period of development and implementation , ethics approval , reference number hrec/11/qpah/047 , was granted by the hospital 's human research ethics committee to conduct a formal evaluation of the framework . surveys were designed for two groups : the reviewers and the recipients . reviewers comprised any rt who had given feedback and recipients were any rt who had received feedback through this process . reviewers evaluated process quality in terms of the usefulness of guidelines and objectivity of the process , and level of demand imposed by the process . feedback quality was evaluated based on timeliness , confidence in feedback delivery and the ability to respond to development needs . recipients evaluated process quality in terms of the clarity of guidelines and transparency of the process . they also assessed feedback quality in terms of timeliness , effectiveness to provide insight to performance , openness of communication and the response to development needs . all respondents were asked to nominate their appointed level and the role(s ) in which they gave or received feedback . likert scale responses were gathered relating to the feedback and process quality with timeliness and identification of strengths and development needs assessed with simple yes or additional short answer questions gathered qualitative data for triangulation with the quantitative findings and identification of themes . table1 presents the definitions for terminology that were supplied to support consistent interpretation of terms . qualitative analysis of participant responses was performed adopting a reflective bracketing approach to minimise the effect of researcher bias . eligibility to participate in the surveys was determined by involvement in the feedback process in any capacity , which made some rts eligible to complete both . a participant information and consent letter was distributed by email to all rts and followed up with information sessions on the study . surveys were facilitated by surveymonkey ( palo alto , california ) ; a web - based program which allows electronic survey distribution , and confidential information storage and analysis . participation was voluntary and anonymous and consent was implied by the submission of a completed survey . responses were required within 14 days of the surveys being distributed , however clinical circumstances delayed response and the deadline was postponed by a further 14 days . in march 2011 , after a 4-year period of development and implementation , ethics approval , reference number hrec/11/qpah/047 , was granted by the hospital 's human research ethics committee to conduct a formal evaluation of the framework . surveys were designed for two groups : the reviewers and the recipients . reviewers comprised any rt who had given feedback and recipients were any rt who had received feedback through this process . reviewers evaluated process quality in terms of the usefulness of guidelines and objectivity of the process , and level of demand imposed by the process . feedback quality was evaluated based on timeliness , confidence in feedback delivery and the ability to respond to development needs . recipients evaluated process quality in terms of the clarity of guidelines and transparency of the process . they also assessed feedback quality in terms of timeliness , effectiveness to provide insight to performance , openness of communication and the response to development needs . all respondents were asked to nominate their appointed level and the role(s ) in which they gave or received feedback . likert scale responses were gathered relating to the feedback and process quality with timeliness and identification of strengths and development needs assessed with simple yes or additional short answer questions gathered qualitative data for triangulation with the quantitative findings and identification of themes . table1 presents the definitions for terminology that were supplied to support consistent interpretation of terms . qualitative analysis of participant responses was performed adopting a reflective bracketing approach to minimise the effect of researcher bias . eligibility to participate in the surveys was determined by involvement in the feedback process in any capacity , which made some rts eligible to complete both . a participant information and consent letter was distributed by email to all rts and followed up with information sessions on the study . surveys were facilitated by surveymonkey ( palo alto , california ) ; a web - based program which allows electronic survey distribution , and confidential information storage and analysis . participation was voluntary and anonymous and consent was implied by the submission of a completed survey . responses were required within 14 days of the surveys being distributed , however clinical circumstances delayed response and the deadline was postponed by a further 14 days . there were 14 respondents to the recipient survey , and 9 to the peer reviewer survey , indicating response rates of 67% and 88% respectively . figure2 provides an overview of the roles to which feedback was given , with the majority being given to ct simulation . of the recipients , 100% found the process to be satisfying and effective and only one reviewer found the process unsatisfying . figure3 shows the obstacles experienced by recipients to ongoing development and figure4 shows the obstacles identified by reviewers to offering strategies for development . having insufficient time was reported by 100% of reviewers as an obstacle to delivering timely feedback and by 75% as an obstacle to offering development strategies . this was echoed by recipients where over 85% cited reliance on rostering to provide adequate time in the area to implement development strategies . several additional themes were extracted from the qualitative data , including : the effectiveness of the rt - paf , the impact of implementation , and recommendations for ongoing improvement . the goal of this evaluation was to determine how effective the rt - paf was in providing meaningful feedback on performance and guidance for development , how it was experienced by rts , and how it could be further developed . the rt - paf was designed to demonstrate strengths and areas for development to rts with an emphasis on providing support for professional development.7,8 when conducted well , a performance appraisal process can enhance learning , improve practice and encourage professional development.9 in this study although 100% of recipients had received suggestions for development and 78% had been offered development strategies , only 67% of reviewers felt they had offered strategies for development . this supports finlay and maclaren 's supposition that the feedback process ( and not just the content ) could prompt the individual to consider their own ideas for development.9 the experiences of respondents indicate the effectiveness of the rt - paf with comments including : this is an excellent tool and should become a part of day to day practice in order to encourage and empower all staff to achieve their potential.(recipient 1 ) feedback from rts who have undergone the rt - paf has been very positive - invaluable in their professional development and establishes a clear path for their development(reviewer 1 ) learning what my strengths were and where i could improve provided some direction for me to focus on.(recipient 2 ) this is an excellent tool and should become a part of day to day practice in order to encourage and empower all staff to achieve their potential . feedback from rts who have undergone the rt - paf has been very positive - invaluable in their professional development and establishes a clear path for their development learning what my strengths were and where i could improve provided some direction for me to focus on . others reported that they had found reward in the recognition of their work by their peers . it was clear that the process enabled staff to validate the skills of others and motivate them to continue to develop : not only senior rts but more junior rts believe in you and your judgment . also , to demonstrate that one does have the skills to achieve higher levels in the department . it also helps to set realistic goals and know that with commitment these goals can be achieved.(recipient 1 ) not only senior rts but more junior rts believe in you and your judgment . also , to demonstrate that one does have the skills to achieve higher levels in the department . it also helps to set realistic goals and know that with commitment these goals can be achieved . one respondent discussed the importance of reaching their own objectives and career goals and relished the input of a peer to provide guidance and motivation with this . through the rt - paf experience they : could see the potential and value in the process . for staff to develop and realise their potential this process should be mandatory.(recipient 3 ) could see the potential and value in the process . for staff to develop and realise their potential effective feedback should also be delivered in a timely manner to ensure relevance and currency , and that sufficient time is given in the rostered area for it to be acted on . as one recipient found , this was not always achieved : more time is needed in the rostered area in order to develop the areas which need to improve.(recipient 4 ) more time is needed in the rostered area in order to develop the areas which need to improve . recipients indicated that they received feedback within the recommended time frames in 93% of cases , although only 33% of reviewers achieved this . one explanation for this discrepancy could be that the majority of recipients received their feedback from the minority of reviewers who ensured it was delivered on time . comments from respondents identified the primary obstacles as being : the lack of resource for the person coordinating the feedback meant that some sessions were rushed as they had their own workload to complete plus the feedback.(recipient 3 ) the challenge in completing more work / forms / feedback . achieving the right balance can be difficult.(reviewer 2 ) being time poor and having minimum time to spend on feedback.(reviewer 3 ) the lack of resource for the person coordinating the feedback meant that some sessions were rushed as they had their own workload to complete plus the feedback . the challenge in completing more work / forms / feedback . delaying the feedback creates a risk that the feedback will no longer be relevant , or participating staff will be rostered out of the area before having the opportunity for adequate response . feedback that balances affirmation of strength with development plans can build confidence by providing evidence of competence , and contribute to ongoing professional development.10 the rt - paf was designed to encourage this balance and according to all respondents , this goal was achieved , and was rewarding for those involved . useful feedback is specific , highlights areas for improvements , identifies strength , and provides guidance for development.11 for it to be effective , however , there must be trust in the process and this is achieved through promoting a culture of performance appraisal that is fair , objective and transparent.12 objectivity can be enhanced through input from multiple contributors to the feedback13 and comments from reviewers reinforced this . one of the most satisfying aspects of participation , as a reviewer cited , was being in a position to : remove doubts about the quality of some staff 's work and remove subjectivity from assessment , which allows staff to feel more comfortable that what is being said about them is true.(reviewer 2 ) remove doubts about the quality of some staff 's work and remove subjectivity from assessment , which allows staff to feel more comfortable that what is being said about them is true . another assessor felt that the process was : developed to encourage consistency and transparency , which is imperative to the success of the process.(reviewer 4 ) developed to encourage consistency and transparency , which is imperative to the success of the process . some recipients , however , found it to be unsatisfying to : know that every action you do can be commented on in the report.(recipient 5 ) know that every action you do can be commented on in the report . and be under the microscope for a period of time.(recipient 6 ) be under the microscope for a period of time . when asked to rate their confidence in delivering feedback , the majority of reviewers felt confident giving feedback in most circumstances , but less confident delivering difficult feedback . one reviewer who felt uncomfortable delivering feedback commented that : not having received feedback on own performance throughout my career affects my personal confidence level in giving feedback - hard to empathise or know how it feels for recipient.(reviewer 1 ) not having received feedback on own performance throughout my career affects my personal confidence level in giving feedback - hard to empathise or know how it feels for recipient . conversely another felt that participation in this process had helped with the development of what is an increasingly important skill : this process is also a valuable educational tool for more senior staff in compiling and delivering effective and successful feedback.(reviewer 4 ) this process is also a valuable educational tool for more senior staff in compiling and delivering effective and successful feedback . documentation contributes to transparency and accountability in the process , but it also has the potential to compromise confidentiality . regardless of who controls the information , trust is required that it will be handled with professional integrity and used for staff development . the choice to give the recipient control of their information is a choice to empower and respect them as professionals who are committed to personal learning and professional development . the most rewarding aspect of this process for one reviewer was the ability to give ownership of the feedback and professional development to the individual rt . another noticed that giving staff control over their information led to : increased confidence of planners in their own ability and the extension of their skills as a result.(reviewer 4 ) increased confidence of planners in their own ability and the extension of their skills as a result . the most demanding aspect of the process was reported as managing case - specific forms and finalising the feedback . all respondents agreed that appropriate allocation of time , training , and managerial support for the process would increase its effectiveness . a performance review process is often seen as time consuming in the already busy schedules of rts and so the provision of protected time is important to achieve a successful feedback process.10 emphasis was also placed on the need to be empowered with opportunity to follow through on development strategies . one reviewer found that they were : not always in a position to follow through on development needs due to lack of knowledge of rostering for me and the recipient.(reviewer 3 ) not always in a position to follow through on development needs due to lack of knowledge of rostering for me and the recipient . identifying the need for managerial support raises a worthy discussion of the balance between managerial responsibility and personal responsibility in professional development . while the support of management is necessary to empower staff in achieving their development goals , shared responsibility must be emphasised to foster a culture where individuals are supported to take responsibility for their own development . achieving consistency of approach is a challenge in the implementation of any peer review process.10 guidelines were provided to support consistency in managing this process ; however it was acknowledged that reference to them was required in order for that to be effective . suggestions for improving the guidelines included ; defining individual responsibilities , clarifying feedback follow - up and access to mediation . these aspects of a process contribute to the perceptions of justice and impact on whether the feedback is accepted or not . fairness is achieved through full disclosure of the process , allowing input from the recipient before the feedback is finalised , and basing all judgements on evidence.12,14 one recipient supported the value of input by identifying the lack of it : there is no real pathway to discuss with senior rts what aids junior rts in learning and development this may help in developing the process further.(recipient 7 ) there is no real pathway to discuss with senior rts what aids junior rts in learning and development this may help in developing the process further . while both groups felt that transparency and objectivity had largely been achieved , it was suggested that the presence of a third party during discussions and documentation of conversations would improve this in some situations . training was also recommended for reviewers to maintain objectivity and thus improve the quality of the feedback . while adequate attention was given to both areas of strength and development needs , this was weakened by the limited ability to respond to strengths with pathways for progression , and to weaknesses with meaningful development plans . the need to train all staff in the goals of staff empowerment and skills development were also recurrent in comments made by both groups , and highlighted in the comment : it is often difficult to give constructive feedback on areas of weakness if rts are under the impression that this can be used against them in developing a career path . it needs to be reinforced that this is more a tool for development and empowerment and will be used to develop them further.(reviewer 5 ) it is often difficult to give constructive feedback on areas of weakness if rts are under the impression that this can be used against them in developing a career path . it needs to be reinforced that this is more a tool for development and empowerment and will be used to develop them further . one recipient expressed frustration in : being given the feedback and knowing what needed to be worked on , then not having the resources , time and support to set goals and improve in these areas.(recipient 1 ) being given the feedback and knowing what needed to be worked on , then not having the resources , time and support to set goals and improve in these areas . two recipients indicated the need for greater evidence on performance but did not indicate where evidence was lacking . evidence was provided through individual forms , and the management of these was cited as being the most onerous tasks in the process . documenting additional evidence may be ideal , but could add to the complexity of planning practice and the demand on the existing workload . stake12 discusses the inevitable presence of bias in any evaluation , where the desire to see the reviewed process supported or further developed may influence respondents in a particular direction . positive responses to the framework may indicate that those who participated were inclined to support the process and promote its development . researchers may have been at equal risk of such bias , however , as health professionals , rts are challenged to continually reflect on their actions in order to nurture a professional and ethical approach to all aspects of practice . this paradigm is particularly relevant to the concept of bracketing where reflection on all stages of the paf development , implementation and evaluation allowed an objective approach to be maintained . accordingly the professional and academic authors of the piece have drawn on these skills to engage in personal and group reflection on the different project stages . a reflective journal approach to bracketing was proposed by tufford and newman.15 although this research relates to social work , it can be seen that the prevalence of reflection in the radiation therapy profession can enable a more active and group approach to using reflection to support bracketing . this approach was taken in order to gain insight into individual experience of the framework . rts experienced the paf at different stages of development and implementation , which may have influenced both experience and memory of it . this potentially excluded rts who may have participated as peer reviewers to individual aspects of feedback but were not instrumental in finalising it . in hindsight , criteria for eligibility may not have been clear , limiting the respondents to only appointed senior rts . there was additional interest in identifying how many respondents contributed to feedback while acting at a higher level , to determine how this may have impacted on feedback satisfaction and confidence . the rt - paf was designed to demonstrate strengths and areas for development to rts with an emphasis on providing support for professional development.7,8 when conducted well , a performance appraisal process can enhance learning , improve practice and encourage professional development.9 in this study although 100% of recipients had received suggestions for development and 78% had been offered development strategies , only 67% of reviewers felt they had offered strategies for development . this supports finlay and maclaren 's supposition that the feedback process ( and not just the content ) could prompt the individual to consider their own ideas for development.9 the experiences of respondents indicate the effectiveness of the rt - paf with comments including : this is an excellent tool and should become a part of day to day practice in order to encourage and empower all staff to achieve their potential.(recipient 1 ) feedback from rts who have undergone the rt - paf has been very positive - invaluable in their professional development and establishes a clear path for their development(reviewer 1 ) learning what my strengths were and where i could improve provided some direction for me to focus on.(recipient 2 ) this is an excellent tool and should become a part of day to day practice in order to encourage and empower all staff to achieve their potential . feedback from rts who have undergone the rt - paf has been very positive - invaluable in their professional development and establishes a clear path for their development learning what my strengths were and where i could improve provided some direction for me to focus on . others reported that they had found reward in the recognition of their work by their peers . it was clear that the process enabled staff to validate the skills of others and motivate them to continue to develop : not only senior rts but more junior rts believe in you and your judgment . also , to demonstrate that one does have the skills to achieve higher levels in the department . it also helps to set realistic goals and know that with commitment these goals can be achieved.(recipient 1 ) not only senior rts but more junior rts believe in you and your judgment . also , to demonstrate that one does have the skills to achieve higher levels in the department . it also helps to set realistic goals and know that with commitment these goals can be achieved . one respondent discussed the importance of reaching their own objectives and career goals and relished the input of a peer to provide guidance and motivation with this . through the rt - paf experience they : could see the potential and value in the process . for staff to develop and realise their potential this process should be mandatory.(recipient 3 ) could see the potential and value in the process . for staff to develop and realise their potential effective feedback should also be delivered in a timely manner to ensure relevance and currency , and that sufficient time is given in the rostered area for it to be acted on . as one recipient found , this was not always achieved : more time is needed in the rostered area in order to develop the areas which need to improve.(recipient 4 ) more time is needed in the rostered area in order to develop the areas which need to improve . recipients indicated that they received feedback within the recommended time frames in 93% of cases , although only 33% of reviewers achieved this . one explanation for this discrepancy could be that the majority of recipients received their feedback from the minority of reviewers who ensured it was delivered on time . comments from respondents identified the primary obstacles as being : the lack of resource for the person coordinating the feedback meant that some sessions were rushed as they had their own workload to complete plus the feedback.(recipient 3 ) the challenge in completing more work / forms / feedback . achieving the right balance can be difficult.(reviewer 2 ) being time poor and having minimum time to spend on feedback.(reviewer 3 ) the lack of resource for the person coordinating the feedback meant that some sessions were rushed as they had their own workload to complete plus the feedback . the challenge in completing more work / forms / feedback . being time poor and having minimum time to spend on feedback . delaying the feedback creates a risk that the feedback will no longer be relevant , or participating staff will be rostered out of the area before having the opportunity for adequate response . feedback that balances affirmation of strength with development plans can build confidence by providing evidence of competence , and contribute to ongoing professional development.10 the rt - paf was designed to encourage this balance and according to all respondents , this goal was achieved , and was rewarding for those involved . useful feedback is specific , highlights areas for improvements , identifies strength , and provides guidance for development.11 for it to be effective , however , there must be trust in the process and this is achieved through promoting a culture of performance appraisal that is fair , objective and transparent.12 objectivity can be enhanced through input from multiple contributors to the feedback13 and comments from reviewers reinforced this . one of the most satisfying aspects of participation , as a reviewer cited , was being in a position to : remove doubts about the quality of some staff 's work and remove subjectivity from assessment , which allows staff to feel more comfortable that what is being said about them is true.(reviewer 2 ) remove doubts about the quality of some staff 's work and remove subjectivity from assessment , which allows staff to feel more comfortable that what is being said about them is true . another assessor felt that the process was : developed to encourage consistency and transparency , which is imperative to the success of the process.(reviewer 4 ) developed to encourage consistency and transparency , which is imperative to the success of the process . some recipients , however , found it to be unsatisfying to : know that every action you do can be commented on in the report.(recipient 5 ) know that every action you do can be commented on in the report . and be under the microscope for a period of time.(recipient 6 ) be under the microscope for a period of time . when asked to rate their confidence in delivering feedback , the majority of reviewers felt confident giving feedback in most circumstances , but less confident delivering difficult feedback . one reviewer who felt uncomfortable delivering feedback commented that : not having received feedback on own performance throughout my career affects my personal confidence level in giving feedback - hard to empathise or know how it feels for recipient.(reviewer 1 ) not having received feedback on own performance throughout my career affects my personal confidence level in giving feedback - hard to empathise or know how it feels for recipient . conversely another felt that participation in this process had helped with the development of what is an increasingly important skill : this process is also a valuable educational tool for more senior staff in compiling and delivering effective and successful feedback.(reviewer 4 ) this process is also a valuable educational tool for more senior staff in compiling and delivering effective and successful feedback . documentation contributes to transparency and accountability in the process , but it also has the potential to compromise confidentiality . regardless of who controls the information , trust is required that it will be handled with professional integrity and used for staff development . the choice to give the recipient control of their information is a choice to empower and respect them as professionals who are committed to personal learning and professional development . the most rewarding aspect of this process for one reviewer was the ability to give ownership of the feedback and professional development to the individual rt . another noticed that giving staff control over their information led to : increased confidence of planners in their own ability and the extension of their skills as a result.(reviewer 4 ) increased confidence of planners in their own ability and the extension of their skills as a result . the most demanding aspect of the process was reported as managing case - specific forms and finalising the feedback . all respondents agreed that appropriate allocation of time , training , and managerial support for the process would increase its effectiveness . a performance review process is often seen as time consuming in the already busy schedules of rts and so the provision of protected time is important to achieve a successful feedback process.10 emphasis was also placed on the need to be empowered with opportunity to follow through on development strategies . one reviewer found that they were : not always in a position to follow through on development needs due to lack of knowledge of rostering for me and the recipient.(reviewer 3 ) not always in a position to follow through on development needs due to lack of knowledge of rostering for me and the recipient . identifying the need for managerial support raises a worthy discussion of the balance between managerial responsibility and personal responsibility in professional development . while the support of management is necessary to empower staff in achieving their development goals , shared responsibility must be emphasised to foster a culture where individuals are supported to take responsibility for their own development . achieving consistency of approach is a challenge in the implementation of any peer review process.10 guidelines were provided to support consistency in managing this process ; however it was acknowledged that reference to them was required in order for that to be effective . suggestions for improving the guidelines included ; defining individual responsibilities , clarifying feedback follow - up and access to mediation . these aspects of a process contribute to the perceptions of justice and impact on whether the feedback is accepted or not . fairness is achieved through full disclosure of the process , allowing input from the recipient before the feedback is finalised , and basing all judgements on evidence.12,14 one recipient supported the value of input by identifying the lack of it : there is no real pathway to discuss with senior rts what aids junior rts in learning and development this may help in developing the process further.(recipient 7 ) there is no real pathway to discuss with senior rts what aids junior rts in learning and development this may help in developing the process further . while both groups felt that transparency and objectivity had largely been achieved , it was suggested that the presence of a third party during discussions and documentation of conversations would improve this in some situations . training was also recommended for reviewers to maintain objectivity and thus improve the quality of the feedback . while adequate attention was given to both areas of strength and development needs , this was weakened by the limited ability to respond to strengths with pathways for progression , and to weaknesses with meaningful development plans . the need to train all staff in the goals of staff empowerment and skills development were also recurrent in comments made by both groups , and highlighted in the comment : it is often difficult to give constructive feedback on areas of weakness if rts are under the impression that this can be used against them in developing a career path . it needs to be reinforced that this is more a tool for development and empowerment and will be used to develop them further.(reviewer 5 ) it is often difficult to give constructive feedback on areas of weakness if rts are under the impression that this can be used against them in developing a career path . it needs to be reinforced that this is more a tool for development and empowerment and will be used to develop them further . one recipient expressed frustration in : being given the feedback and knowing what needed to be worked on , then not having the resources , time and support to set goals and improve in these areas.(recipient 1 ) being given the feedback and knowing what needed to be worked on , then not having the resources , time and support to set goals and improve in these areas . two recipients indicated the need for greater evidence on performance but did not indicate where evidence was lacking . evidence was provided through individual forms , and the management of these was cited as being the most onerous tasks in the process . documenting additional evidence may be ideal , but could add to the complexity of planning practice and the demand on the existing workload . stake12 discusses the inevitable presence of bias in any evaluation , where the desire to see the reviewed process supported or further developed may influence respondents in a particular direction . positive responses to the framework may indicate that those who participated were inclined to support the process and promote its development . researchers may have been at equal risk of such bias , however , as health professionals , rts are challenged to continually reflect on their actions in order to nurture a professional and ethical approach to all aspects of practice . this paradigm is particularly relevant to the concept of bracketing where reflection on all stages of the paf development , implementation and evaluation allowed an objective approach to be maintained . accordingly the professional and academic authors of the piece have drawn on these skills to engage in personal and group reflection on the different project stages . a reflective journal approach to bracketing was proposed by tufford and newman.15 although this research relates to social work , it can be seen that the prevalence of reflection in the radiation therapy profession can enable a more active and group approach to using reflection to support bracketing . this approach was taken in order to gain insight into individual experience of the framework . rts experienced the paf at different stages of development and implementation , which may have influenced both experience and memory of it . this potentially excluded rts who may have participated as peer reviewers to individual aspects of feedback but were not instrumental in finalising it . in hindsight , criteria for eligibility may not have been clear , limiting the respondents to only appointed senior rts . there was additional interest in identifying how many respondents contributed to feedback while acting at a higher level , to determine how this may have impacted on feedback satisfaction and confidence . this project was developed from a need to establish consistent practice and active development among rts in a dynamic work environment . clearly defined expectations for practice and a process for providing feedback on performance were necessary to achieve this . the rt - paf was developed to meet these needs and to promote a culture of performance appraisal that was consistent , objective , transparent , based on evidence and focussed on skills development . evaluation of this framework revealed its value in empowering staff to maintain an appropriate level of skill and reach their professional potential , in an environment of continually evolving technologies and practice change . this framework achieved its aims of defining expectations for practice and providing a fair and objective feedback process that focussed on skills development . obstacles to its success were related to the support and time resources required for rts to be able to develop meaningful development plans and implement strategies to meet these goals . the provision of protected time to conduct the review , provide feedback and complete development plans in a timely manner was highlighted as critical in ensuring the relevance of the feedback . ongoing management support of this framework was needed through rosters and training to meet development needs . training in the principles of the process and in feedback delivery skills was also recommended to ensure consistency in quality of the feedback delivered and how the process was conducted . the goal of empowering and developing staff required emphasis to reduce rt concerns of having their performance reviewed and the feedback documented . further development of the rt - paf requires commitment to ongoing development of the framework , and a commitment to empower staff to allow for realistic goal - setting and development of skills as they keep pace with changes to technology and practice . the benefits arising in terms of staff satisfaction and development , however , highlight the importance of this commitment to the modern radiation therapy workforce . additional supporting information may be found in the online version of this article : data s1 . radiation therapy performance appraisal framework ( rt - paf ) sample survey questions for reviewers and recipients .

introductionconstantly evolving technology and techniques within radiation therapy require practitioners to maintain a continuous approach to professional development and training . systems of performance appraisal and adoption of regular feedback mechanisms are vital to support this development yet frequently lack structure and rely on informal peer support.methodsa radiation therapy performance appraisal framework ( rt - paf ) for radiation therapists in planning and simulation was developed to define expectations of practice and promote a supportive and objective culture of performance and skills appraisal . evaluation of the framework was conducted via an anonymous online survey tool . nine peer reviewers and fourteen recipients provided feedback on its effectiveness and the challenges and limitations of the approach.resultsfindings from the evaluation were positive and suggested that both groups gained benefit from and expressed a strong interest in embedding the approach more routinely . respondents identified common challenges related to the limited ability to implement suggested development strategies ; this was strongly associated with time and rostering issues.conclusionsthis framework successfully defined expectations for practice and provided a fair and objective feedback process that focussed on skills development . it empowered staff to maintain their skills and reach their professional potential . management support , particularly in regard to provision of protected time was highlighted as critical to the framework 's ongoing success . the demonstrated benefits arising in terms of staff satisfaction and development highlight the importance of this commitment to the modern radiation therapy workforce .

Introduction Radiation Therapy Performance Appraisal Framework development RT-PAF evaluation Methods Data collection Participation Results Discussion RT-PAF effectiveness How it was experienced by RTs Challenges of implementation Suggestions for improvements Limitations of results Conclusion Conflict of Interest Supporting Information

vitrectomy surgery has emerged as a cornerstone for treatment of proliferative diabetic retinopathy and its complications . advances in surgical techniques and equipment continue to evolve and shape up the future of this modality of treatment . the use of anti- vascular endothelial growth factor ( anti - vegf ) agents as an adjuvant pharmacotherapy in the perioperative period has been proposed to provide better surgical outcomes . these agents reduce the vascularity of neovascular tissues and inhibit vascular proliferation,1 which will conceptually provide a better view for the surgeon and lower the chances of vitreous cavity hemorrhage following the procedure . this review will address the use and efficacy of anti - vegfs in the perioperative period in diabetic vitrectomy and will scrutinize the available data on this issue . we searched pubmed , medline , embase , central and lilacs for all trials that looked at the use of anti - vegfs in the perioperative period for diabetic vitrectomy . pars plana vitrectomy ( ppv ) is an important modality to treat proliferative diabetic retinopathy ( pdr ) . in one report , ppv was required in up to 10% of patients presenting with pdr within one year.2 the most common indications for surgery are nonclearing vitreous hemorrhage ( vh ) , tractional retinal detachments ( trds ) threatening the macula and combined tractional / rhegmatogenous detachments.3 a significant and rather common complication is vitreous cavity hemorrhage which may range from 10 to 80% of cases.4 this hemorrhage can either be early ( present within the first postoperative days ) or late , recurring within two to six months after a clear vitreous cavity postoperatively . causes of early or persistent hemorrhage include : remnants of new vessels or oozing dissected tissues during or after surgerysclerotomy site bleedingblood clot lysishemorrhage from residual anterior vitreous shake out bleeding . remnants of new vessels or oozing dissected tissues during or after surgery sclerotomy site bleeding hemorrhage from residual anterior vitreous late or recurrent hemorrhage can result from : new vessel growthrecurrent traction on remnant dissected tissueentry site neovascularization5 in which new anterior vessels grow at the inner sclerotomy sites accompanied with traction . this occurrence can be visualized by indirect ophthalmoscopy with deep scleral depression or documented with anterior segment high resolution ultrasonography.6 recurrent traction on remnant dissected tissue entry site neovascularization5 in which new anterior vessels grow at the inner sclerotomy sites accompanied with traction . this occurrence can be visualized by indirect ophthalmoscopy with deep scleral depression or documented with anterior segment high resolution ultrasonography.6 management of this complication ranges from observation7 to timely revision surgery . this depends on a number of factors including the status of the eye , the status of the fellow eye and the general condition of the patient . it is estimated that 30 to 50% of patients with postoperative vitreous hemorrhage would need revision surgery.8 a number of surgical strategies are considered the standard of clinical care and are aimed to prevent the occurrence of postoperative vitreous cavity hemorrhage . achieving adequate hemostasis , removal of posterior vitreo - retinal traction and aggressive panretinal photocoagulation treatment are widely adopted surgical principles . cryotherapy or laser to the sclerotomy site is thought to inhibit entry - site neovascularization.9 identifying the true vitreo - retinal plane and eliminating recurrent traction is crucial to prevent neovascularization . physical agents like air , gas and silicone oil play an important role as tamponade agents and therefore reduce the incidence of postoperative bleeding . pharmacological agents such as triamcinolone have been used intra - operatively to reduce inflammation and vascular proliferation.10 oral tranexamic acid has been given to patients after vitrectomy for pdr to inhibit clot dissolution and fibrinolysis.11 anti - vegf therapy has become the standard of care in many ophthalmic conditions , and is considered an important modality for treatment of diabetic macular edema . an electronic search was performed last on december 8 2011 to identify studies that addressed the use of anti - vegfs in the perioperative period to reduce bleeding in patients undergoing vitrectomy for pdr . a total of 96 records were screened after eliminating duplicates . after excluding irrelevant and retrospective reports , we will highlight the characteristics of these randomized studies and aim to draw conclusions based on each study design . the different methodology used in those studies summary of the six randomized prospective clinical trials with pertinent characteristics and results the only double - masked prospective randomized clinical trial comes from iran , where ahmadieh et al randomized 68 eyes of 68 patients undergoing ppv for pdr.13 thirty five eyes received 1.25 mg of intravitreal bevacizumab ( ivb ) one week prior to surgery while 33 received sham injections . the study clearly randomized and allocated patients into the treatment and control group and it accounts for participants who failed to complete the study protocol . the primary outcome measure in this study was the incidence of early postoperative vitreous hemorrhage ( defined as occurring before or at 4 weeks after surgery ) . secondary outcomes included mean change in best corrected visual acuity ( bcva ) and injection related adverse events . the authors reported the outcomes based on the intention to treat analysis and the per - protocol analysis . in the intention - to - treat analysis , the incidence of postvitrectomy hemorrhage 1 week and 1 month after surgery was significantly lower in the treatment group compared with the control group ( p = 0.023 and p = 0.001 , respectively ) . mean bcva improved from 1.88 logarithm of minimum angle of resolution ( logmar ) units in both study groups before surgery to 0.91 logmar units and 1.46 logmar units 1 month after vitrectomy in the treatment and control groups , respectively ( p= 0.001 ) . the per - protocol analysis included 16 eyes in the treatment group and 18 eyes in the control group . postvitrectomy hemorrhage occurred less frequently 1 week and 1 month after surgery in the ivb group compared with the control group ( p = 0.033 and p = 0.003 , respectively ) . mean improvement in bcva 1 month after vitrectomy was -1.05 logmar units in the ivb group and -0.42 logmar units in the control group ( p = 0.004 ) . the main reasons for not completing this study protocol were reabsorption of the vitreous hemorrhage before surgery ( 9 patients ) and the use of silicone oil tamponade ( 6 patients ) . modarres et al undertook a prospective surgeon - masked randomized clinical trial where 40 eyes of 40 diabetic patients were given either an ivb injection of 2.5 mg 3 to 5 days before operation or no injection at all.14 no details of the randomization methods used were presented by the authors . the study looked at the facilitation of surgery as a primary endpoint ( including the duration of surgery , number of endodiathermy applications and number of backflush needle applications ) . mean surgical time was 62+/-57.3 minutes in the injected group versus 95.5+/-36 minutes in the noninjected group ( p=0.03 ) . preoperative and 3-month postoperative visual acuities were the same for both groups . in the last follow - up examinations ( mean 7+/- 3.6 months ) , the injected group had better visual acuities than the noninjected group ( 1.1+/-0.4 and 1.4+/-0.3 logmar , respectively , p=0.006 ) . this study had a high rate of silicone oil tamponade ( 10/22 in the treatment group and 7/18 in the control group ) . di lauro et al conducted a prospective randomized clinical trial on 72 eyes of 68 participants who required vitrectomy for pdr.15 this study had three treatment groups : group a received sham injections ; group b received 1.25 mg of bevacizumab one week before vitrectomy ; and group c received 1.25 mg of bevacizumab 3 weeks before the surgery . the authors did not comment on the randomization or sequence of allocation of participants and there were no comments on the masking of participants or investigators . the primary outcome measures were the clearing of vitreous hemorrhage , the incidence of adverse effects and the need for other procedures during surgery . the average difference in the surgical time was statistically significant between group a and group b ( p = 0.025 ) , and between group a and group c ( p = 0.031 ) . again this study had a high rate of silicone oil tamponade ( 33.3% overall ) and those patients were not excluded in the statistical analysis . rizzo et al preformed an interventional , consecutive , randomized prospective study on 22 eyes of 22 participants.16 the treatment group received 1.25 mg of ivb 5 to 7 days prior to ppv and the control group received ppv alone . the authors used a table of random numbers to assign each participant to a study group . farahvash and collaborators randomized 35 eyes of 35 patients undergoing ppv for dense diabetic vitreous hemorrhage.17 despite the less number of patients , both groups were divided into 4 subgroups according to patient characteristics , mainly history of pan retinal photocoagulation ( prp ) and the presence of trd . eighteen eyes received 1.25 mg of ivb one week prior to surgery versus 17 in the control group who received nothing and were considered the control group . an intraoperative complexity score and pdr stage the primary outcome measures in this study were the facility of surgery ( severity of intraoperative bleeding and break formation , quantified by a surgeon based scoring system ) and the incidence of early and/ or late postoperative vitreous hemorrhage ( defined as occurring before or after 4 weeks of surgery , respectfully ) . follow up was 1 day , 1 week and 1 month and every 3 months after surgery . the mean scores of postoperative bleeding between the two groups were not significantly different ( p=0.35 ) , nor were the endodiathermy applications and break formations . anatomical outcome and visual acuity at three months and at the final . limitations of this study include the use of endotamponade in 9 patients and the lack of evaluation of induction or progression of trd by ultrasonography . the surgeries were done by three different surgeons and this might be of significance given the small sample size . the largest prospective randomized clinical trial to date comes from korea where ahn et al randomly assigned 107 eyes of 91 patients to three groups : group 1 received 1.25 mg ivb injection 1 to 14 days before ppv for pdr related complications . group 2 received the same injection at the end of surgery and group 3 got no injection.18 the primary outcome was the incidence of early ( less or equal to 4 weeks ) and late ( more than 4 weeks ) recurrent vh . secondary outcome measures were the initial time of vitreous clearing ( itvc ) and bcva at 6 months after surgery . the incidences of early recurrent vh were 22.2 , 10.8 and 32.4% in groups 1 , 2 and 3 respectively ( p=0.087 ) . a subgroup pairwise analysis showed significantly decreased early vh incidence in group 2 compared to that of group 3 ( p=0.026 ) . the itvc was significantly shorter in group 2 when compared with groups 1 and 3 ( p=0.45 and p=0.015 , respectfully ) . the period in which ivb was injected was variable ( 1 - 14 days ) nor were sham injections performed . all cases which needed endo - tamponade were excluded from the study , which introduces a possible bias towards including less complicated pdr patients . the researchers point out that the proportion of those patients where similar among the three groups and their inclusion did not alter the main outcomes of the study , but no data is provided . the authors therefore found no evidence to support the use of preoperative ivb to reduce the recurrence of vh in vitrectomy for pdr but maintained that in some cases , its intraoperative use may be of benefit . they recommend a multicenter prospective trial with still a larger number of patients to obtain more conclusive evidence . pars plana vitrectomy ( ppv ) is an important modality to treat proliferative diabetic retinopathy ( pdr ) . in one report , ppv was required in up to 10% of patients presenting with pdr within one year.2 the most common indications for surgery are nonclearing vitreous hemorrhage ( vh ) , tractional retinal detachments ( trds ) threatening the macula and combined tractional / rhegmatogenous detachments.3 a significant and rather common complication is vitreous cavity hemorrhage which may range from 10 to 80% of cases.4 this hemorrhage can either be early ( present within the first postoperative days ) or late , recurring within two to six months after a clear vitreous cavity postoperatively . causes of early or persistent hemorrhage include : remnants of new vessels or oozing dissected tissues during or after surgerysclerotomy site bleedingblood clot lysishemorrhage from residual anterior vitreous shake out bleeding . remnants of new vessels or oozing dissected tissues during or after surgery sclerotomy site bleeding hemorrhage from residual anterior vitreous late or recurrent hemorrhage can result from : new vessel growthrecurrent traction on remnant dissected tissueentry site neovascularization5 in which new anterior vessels grow at the inner sclerotomy sites accompanied with traction . this occurrence can be visualized by indirect ophthalmoscopy with deep scleral depression or documented with anterior segment high resolution ultrasonography.6 recurrent traction on remnant dissected tissue entry site neovascularization5 in which new anterior vessels grow at the inner sclerotomy sites accompanied with traction . this occurrence can be visualized by indirect ophthalmoscopy with deep scleral depression or documented with anterior segment high resolution ultrasonography.6 management of this complication ranges from observation7 to timely revision surgery . this depends on a number of factors including the status of the eye , the status of the fellow eye and the general condition of the patient . it is estimated that 30 to 50% of patients with postoperative vitreous hemorrhage would need revision surgery.8 a number of surgical strategies are considered the standard of clinical care and are aimed to prevent the occurrence of postoperative vitreous cavity hemorrhage . achieving adequate hemostasis , removal of posterior vitreo - retinal traction and aggressive panretinal photocoagulation treatment cryotherapy or laser to the sclerotomy site is thought to inhibit entry - site neovascularization.9 identifying the true vitreo - retinal plane and eliminating recurrent traction is crucial to prevent neovascularization . physical agents like air , gas and silicone oil play an important role as tamponade agents and therefore reduce the incidence of postoperative bleeding . pharmacological agents such as triamcinolone have been used intra - operatively to reduce inflammation and vascular proliferation.10 oral tranexamic acid has been given to patients after vitrectomy for pdr to inhibit clot dissolution and fibrinolysis.11 anti - vegf therapy has become the standard of care in many ophthalmic conditions , and is considered an important modality for treatment of diabetic macular edema . an electronic search was performed last on december 8 2011 to identify studies that addressed the use of anti - vegfs in the perioperative period to reduce bleeding in patients undergoing vitrectomy for pdr . a total of 96 records were screened after eliminating duplicates . after excluding irrelevant and retrospective reports , we will highlight the characteristics of these randomized studies and aim to draw conclusions based on each study design . the different methodology used in those studies summary of the six randomized prospective clinical trials with pertinent characteristics and results the only double - masked prospective randomized clinical trial comes from iran , where ahmadieh et al randomized 68 eyes of 68 patients undergoing ppv for pdr.13 thirty five eyes received 1.25 mg of intravitreal bevacizumab ( ivb ) one week prior to surgery while 33 received sham injections . the study clearly randomized and allocated patients into the treatment and control group and it accounts for participants who failed to complete the study protocol . the primary outcome measure in this study was the incidence of early postoperative vitreous hemorrhage ( defined as occurring before or at 4 weeks after surgery ) . secondary outcomes included mean change in best corrected visual acuity ( bcva ) and injection related adverse events . the authors reported the outcomes based on the intention to treat analysis and the per - protocol analysis . in the intention - to - treat analysis , the incidence of postvitrectomy hemorrhage 1 week and 1 month after surgery was significantly lower in the treatment group compared with the control group ( p = 0.023 and p = 0.001 , respectively ) . mean bcva improved from 1.88 logarithm of minimum angle of resolution ( logmar ) units in both study groups before surgery to 0.91 logmar units and 1.46 logmar units 1 month after vitrectomy in the treatment and control groups , respectively ( p= 0.001 ) . the per - protocol analysis included 16 eyes in the treatment group and 18 eyes in the control group . postvitrectomy hemorrhage occurred less frequently 1 week and 1 month after surgery in the ivb group compared with the control group ( p = 0.033 and p = 0.003 , respectively ) . mean improvement in bcva 1 month after vitrectomy was -1.05 logmar units in the ivb group and -0.42 logmar units in the control group ( p = 0.004 ) . the main reasons for not completing this study protocol were reabsorption of the vitreous hemorrhage before surgery ( 9 patients ) and the use of silicone oil tamponade ( 6 patients ) . modarres et al undertook a prospective surgeon - masked randomized clinical trial where 40 eyes of 40 diabetic patients were given either an ivb injection of 2.5 mg 3 to 5 days before operation or no injection at all.14 no details of the randomization methods used were presented by the authors . the study looked at the facilitation of surgery as a primary endpoint ( including the duration of surgery , number of endodiathermy applications and number of backflush needle applications ) . mean surgical time was 62+/-57.3 minutes in the injected group versus 95.5+/-36 minutes in the noninjected group ( p=0.03 ) . preoperative and 3-month postoperative visual acuities were the same for both groups . in the last follow - up examinations ( mean 7+/- 3.6 months ) , the injected group had better visual acuities than the noninjected group ( 1.1+/-0.4 and 1.4+/-0.3 logmar , respectively , p=0.006 ) . this study had a high rate of silicone oil tamponade ( 10/22 in the treatment group and 7/18 in the control group ) . di lauro et al conducted a prospective randomized clinical trial on 72 eyes of 68 participants who required vitrectomy for pdr.15 this study had three treatment groups : group a received sham injections ; group b received 1.25 mg of bevacizumab one week before vitrectomy ; and group c received 1.25 mg of bevacizumab 3 weeks before the surgery . the authors did not comment on the randomization or sequence of allocation of participants and there were no comments on the masking of participants or investigators . the primary outcome measures were the clearing of vitreous hemorrhage , the incidence of adverse effects and the need for other procedures during surgery . the average difference in the surgical time was statistically significant between group a and group b ( p = 0.025 ) , and between group a and group c ( p = 0.031 ) . again this study had a high rate of silicone oil tamponade ( 33.3% overall ) and those patients were not excluded in the statistical analysis . rizzo et al preformed an interventional , consecutive , randomized prospective study on 22 eyes of 22 participants.16 the treatment group received 1.25 mg of ivb 5 to 7 days prior to ppv and the control group received ppv alone . the authors used a table of random numbers to assign each participant to a study group . farahvash and collaborators randomized 35 eyes of 35 patients undergoing ppv for dense diabetic vitreous hemorrhage.17 despite the less number of patients , both groups were divided into 4 subgroups according to patient characteristics , mainly history of pan retinal photocoagulation ( prp ) and the presence of trd . eighteen eyes received 1.25 mg of ivb one week prior to surgery versus 17 in the control group who received nothing and were considered the control group . an intraoperative complexity score and pdr stage the primary outcome measures in this study were the facility of surgery ( severity of intraoperative bleeding and break formation , quantified by a surgeon based scoring system ) and the incidence of early and/ or late postoperative vitreous hemorrhage ( defined as occurring before or after 4 weeks of surgery , respectfully ) . follow up was 1 day , 1 week and 1 month and every 3 months after surgery . the mean scores of postoperative bleeding between the two groups were not significantly different ( p=0.35 ) , nor were the endodiathermy applications and break formations . anatomical outcome and visual acuity at three months and at the final . limitations of this study include the use of endotamponade in 9 patients and the lack of evaluation of induction or progression of trd by ultrasonography . the surgeries were done by three different surgeons and this might be of significance given the small sample size . the largest prospective randomized clinical trial to date comes from korea where ahn et al randomly assigned 107 eyes of 91 patients to three groups : group 1 received 1.25 mg ivb injection 1 to 14 days before ppv for pdr related complications . group 2 received the same injection at the end of surgery and group 3 got no injection.18 the primary outcome was the incidence of early ( less or equal to 4 weeks ) and late ( more than 4 weeks ) recurrent vh . secondary outcome measures were the initial time of vitreous clearing ( itvc ) and bcva at 6 months after surgery . the incidences of early recurrent vh were 22.2 , 10.8 and 32.4% in groups 1 , 2 and 3 respectively ( p=0.087 ) . a subgroup pairwise analysis showed significantly decreased early vh incidence in group 2 compared to that of group 3 ( p=0.026 ) . the itvc was significantly shorter in group 2 when compared with groups 1 and 3 ( p=0.45 and p=0.015 , respectfully ) . the period in which ivb was injected was variable ( 1 - 14 days ) nor were sham injections performed . all cases which needed endo - tamponade were excluded from the study , which introduces a possible bias towards including less complicated pdr patients . the researchers point out that the proportion of those patients where similar among the three groups and their inclusion did not alter the main outcomes of the study , but no data is provided . the authors therefore found no evidence to support the use of preoperative ivb to reduce the recurrence of vh in vitrectomy for pdr but maintained that in some cases , its intraoperative use may be of benefit . they recommend a multicenter prospective trial with still a larger number of patients to obtain more conclusive evidence . an electronic search was performed last on december 8 2011 to identify studies that addressed the use of anti - vegfs in the perioperative period to reduce bleeding in patients undergoing vitrectomy for pdr . a total of 96 records were screened after eliminating duplicates . after excluding irrelevant and retrospective reports , we will highlight the characteristics of these randomized studies and aim to draw conclusions based on each study design . the different methodology used in those studies summary of the six randomized prospective clinical trials with pertinent characteristics and results the only double - masked prospective randomized clinical trial comes from iran , where ahmadieh et al randomized 68 eyes of 68 patients undergoing ppv for pdr.13 thirty five eyes received 1.25 mg of intravitreal bevacizumab ( ivb ) one week prior to surgery while 33 received sham injections . the study clearly randomized and allocated patients into the treatment and control group and it accounts for participants who failed to complete the study protocol . the primary outcome measure in this study was the incidence of early postoperative vitreous hemorrhage ( defined as occurring before or at 4 weeks after surgery ) . secondary outcomes included mean change in best corrected visual acuity ( bcva ) and injection related adverse events . the authors reported the outcomes based on the intention to treat analysis and the per - protocol analysis . in the intention - to - treat analysis , the incidence of postvitrectomy hemorrhage 1 week and 1 month after surgery was significantly lower in the treatment group compared with the control group ( p = 0.023 and p = 0.001 , respectively ) . mean bcva improved from 1.88 logarithm of minimum angle of resolution ( logmar ) units in both study groups before surgery to 0.91 logmar units and 1.46 logmar units 1 month after vitrectomy in the treatment and control groups , respectively ( p= 0.001 ) . the per - protocol analysis included 16 eyes in the treatment group and 18 eyes in the control group . postvitrectomy hemorrhage occurred less frequently 1 week and 1 month after surgery in the ivb group compared with the control group ( p = 0.033 and p = 0.003 , respectively ) . mean improvement in bcva 1 month after vitrectomy was -1.05 logmar units in the ivb group and -0.42 logmar units in the control group ( p = 0.004 ) . the main reasons for not completing this study protocol were reabsorption of the vitreous hemorrhage before surgery ( 9 patients ) and the use of silicone oil tamponade ( 6 patients ) . modarres et al undertook a prospective surgeon - masked randomized clinical trial where 40 eyes of 40 diabetic patients were given either an ivb injection of 2.5 mg 3 to 5 days before operation or no injection at all.14 no details of the randomization methods used were presented by the authors . the study looked at the facilitation of surgery as a primary endpoint ( including the duration of surgery , number of endodiathermy applications and number of backflush needle applications ) . mean surgical time was 62+/-57.3 minutes in the injected group versus 95.5+/-36 minutes in the noninjected group ( p=0.03 ) . preoperative and 3-month postoperative visual acuities were the same for both groups . in the last follow - up examinations ( mean 7+/- 3.6 months ) , the injected group had better visual acuities than the noninjected group ( 1.1+/-0.4 and 1.4+/-0.3 logmar , respectively , p=0.006 ) . this study had a high rate of silicone oil tamponade ( 10/22 in the treatment group and 7/18 in the control group ) . di lauro et al conducted a prospective randomized clinical trial on 72 eyes of 68 participants who required vitrectomy for pdr.15 this study had three treatment groups : group a received sham injections ; group b received 1.25 mg of bevacizumab one week before vitrectomy ; and group c received 1.25 mg of bevacizumab 3 weeks before the surgery . the authors did not comment on the randomization or sequence of allocation of participants and there were no comments on the masking of participants or investigators . the primary outcome measures were the clearing of vitreous hemorrhage , the incidence of adverse effects and the need for other procedures during surgery . the average difference in the surgical time was statistically significant between group a and group b ( p = 0.025 ) , and between group a and group c ( p = 0.031 ) . again this study had a high rate of silicone oil tamponade ( 33.3% overall ) and those patients were not excluded in the statistical analysis . rizzo et al preformed an interventional , consecutive , randomized prospective study on 22 eyes of 22 participants.16 the treatment group received 1.25 mg of ivb 5 to 7 days prior to ppv and the control group received ppv alone . the authors used a table of random numbers to assign each participant to a study group . farahvash and collaborators randomized 35 eyes of 35 patients undergoing ppv for dense diabetic vitreous hemorrhage.17 despite the less number of patients , both groups were divided into 4 subgroups according to patient characteristics , mainly history of pan retinal photocoagulation ( prp ) and the presence of trd . eighteen eyes received 1.25 mg of ivb one week prior to surgery versus 17 in the control group who received nothing and were considered the control group . an intraoperative complexity score and pdr stage the primary outcome measures in this study were the facility of surgery ( severity of intraoperative bleeding and break formation , quantified by a surgeon based scoring system ) and the incidence of early and/ or late postoperative vitreous hemorrhage ( defined as occurring before or after 4 weeks of surgery , respectfully ) . follow up was 1 day , 1 week and 1 month and every 3 months after surgery . the mean scores of postoperative bleeding between the two groups were not significantly different ( p=0.35 ) , nor were the endodiathermy applications and break formations . anatomical outcome and visual acuity at three months and at the final . limitations of this study include the use of endotamponade in 9 patients and the lack of evaluation of induction or progression of trd by ultrasonography . the surgeries were done by three different surgeons and this might be of significance given the small sample size . the largest prospective randomized clinical trial to date comes from korea where ahn et al randomly assigned 107 eyes of 91 patients to three groups : group 1 received 1.25 mg ivb injection 1 to 14 days before ppv for pdr related complications . group 2 received the same injection at the end of surgery and group 3 got no injection.18 the primary outcome was the incidence of early ( less or equal to 4 weeks ) and late ( more than 4 weeks ) recurrent vh . secondary outcome measures were the initial time of vitreous clearing ( itvc ) and bcva at 6 months after surgery . the incidences of early recurrent vh were 22.2 , 10.8 and 32.4% in groups 1 , 2 and 3 respectively ( p=0.087 ) . a subgroup pairwise analysis showed significantly decreased early vh incidence in group 2 compared to that of group 3 ( p=0.026 ) . the itvc was significantly shorter in group 2 when compared with groups 1 and 3 ( p=0.45 and p=0.015 , respectfully ) . the period in which ivb was injected was variable ( 1 - 14 days ) nor were sham injections performed . all cases which needed endo - tamponade were excluded from the study , which introduces a possible bias towards including less complicated pdr patients . the researchers point out that the proportion of those patients where similar among the three groups and their inclusion did not alter the main outcomes of the study , but no data is provided . the authors therefore found no evidence to support the use of preoperative ivb to reduce the recurrence of vh in vitrectomy for pdr but maintained that in some cases , its intraoperative use may be of benefit . they recommend a multicenter prospective trial with still a larger number of patients to obtain more conclusive evidence . the use of preoperative ivb has been shown to reduce the incidence of postoperative vitreous hemorrhage in one randomized double blind prospective study ( ahmadieh et al),13 although the number of patients who completed the protocol was small . a prospective randomized ( but not double blind ) trial by ahn and collaborators18 maintained that preoperative ivb had no significant effect on postoperative bleeding but reported that intra - operative injections may be of added benefit . other randomized ( and nonrandomized ) trials that looked at different outcome measures produced inconclusive recommendations . using anti - vegfs in the perioperative period for diabetic vitrectomy may offer certain advantages but multicenter double masked , prospective studies should address the occurrence of early and late vitreous cavity bleeding as primary end points , with clear grading of the hemorrhage based on established criteria ( e.g. diabetic vitrectomy study criteria ) . the studies should also incorporate the effects of using endo - tamponade with gas or silicone oil on the statistical analysis of the data .

pars plana vitrectomy is an established surgical method for the treatment of proliferative diabetic retinopathy and its complications . anti - vascular endothelial growth factor agents suppress vascular proliferation and may be used as pharmacological adjuvants to reduce the incidence of postoperative hemorrhage in the vitreous cavity and to facilitate the surgical approach . we conducted an electronic search to identify prospective randomized controlled trials looking at the use of perioperative vascular endothelial growth factor suppression in diabetic patients undergoing vitrectomy . we found six prospective randomized trials with only one being double - masked . we present a summary of the findings . four studies suggest that the use of perioperative , anti - vascular endothelial growth factor agents facilitate vitrectomy surgery , but only one study supports their use to reduce the chances of early postoperative vitreous bleeding . two studies did not find a significant benefit for their use before surgery to reduce the recurrence of vitreous hemorrhage in proliferative diabetic retinopathy . more randomized double blinded studies with a larger number of patients are needed to establish a clear recommendation regarding the use of these agents . those studies should factor in the use of endo - tamponade with gas or silicone oil following vitrectomy .

INTRODUCTION Background Management of postoperative vitreous cavity bleeding and the proposed role of Anti-VEGFs Methods Studies considered for review CONCLUSION

v(d)j recombination is a mechanism of vertebrate genetic recombination that assembles gene segments into functional immunoglobulin ( ig ) and t - cell receptor ( tcr ) genes . this site - specific recombination reaction generates the enormous repertoire of tcr and ig molecules that are necessary for the recognition of diverse antigens from bacterial , viral and parasitic invaders . this reaction is directed by recombination signal sequences ( rsss ) , which flank each of the hundreds of potential donor gene segments . the v(d)j recombinase , comprised of the rag1 and rag2 proteins , introduces double - strand dna breaks at the junction between a rss and the flanking gene segment . dna repair activity then re - joins breaks at two distant cuts to generate a functional gene through chromosomal rearrangement . each rss is composed of seven conserved nucleotides ( a heptamer ) , residing next to the gene encoding sequence , followed by a spacer ( containing either 12 1 or 23 1 poorly conserved nucleotides ) and a conserved nonamer ( 9 bp ) . the rsss are present on the 3-side of a v region , on both sides of d segments , and on the 5-side of the j region . assembly of the correct composition of gene segments is directed by spacer length ; recombination only joins gene segments flanked by rsss with different spacer lengths . aberrant v(d)j recombination activity has been associated with oncogenic chromosomal translocations in lymphoid leukemia and lymphomas . the mechanisms of translocation remain unclear , but appear to include aberrant cutting of rss - like sequences ( cryptic rsss ) by the v(d)j recombinase at sites outside the ig and tcr loci . joining of these breaks to recombinase cuts within the ig or tcr loci potentially leads to high expression of an oncogene in lymphocytes and leukemic transformation ( 1,2 ) . considering the impact of this process in the context of translocation related to leukemia , a tool for predicting crsss on a genome - wide scale would be very useful to identify potential recombination sites . this task is complicated by the modular nature of rsss and the variability of nucleotide sequences within the heptamer , spacer and nonamer elements . using correlations between nucleotides at several positions within the 12 and 23 spacers and correlations between the identities of nucleotides at key positions , it is possible to predict the overall recombination efficiency of a rss ( 35 ) . it is now recognized that while rsss are defined by a strict requirement for highly conserved nucleotides in the heptamer and nonamer , the quality of rss function is determined in an analog manner by numerous complex interactions between the rag proteins and the less - well conserved nucleotides in the heptamer , the nonamer , and , importantly , the spacer . for the latter , the importance of consensus nucleotides in defined points is emerging as a determinant for the efficient recognition of rsss by rag proteins ( 5 ) . to accomplish the prediction of cryptic rss , we developed a software tool , dnagrab , which scans the whole genome to identify candidate crsss . this algorithm makes use of probability models , which can be recasted to bayesian networks , taking into account the correlations between groups of positions of a sequence , developed starting from specific reference sets of physiological rsss ( 3 ) . these bayesian models are created by searching statistical correlations for each position in rss patterns with all the other positions , placing no restrictions on the number of correlations or in the spatial positions relationship in the sequence . this approach determines , from all possible combinations of disjoint probability distributions , the set of distributions that most effectively distinguishes functional sites from non - functional sequences . although the family of models to consider is very large , the use of mutual information allows a fast model selection by maximizing the mean recombination information content ( ric ) for physiological rsss ( 3 ) . the ric score , defined as the natural logarithm of the joint probability function of mutually correlated positions , is finally used to predict the possible functional cryptic rsss . the prediction capability of this score with respects to the effective recombination efficiency of rsss has been long investigated and validated with in vitro testing ( 35 ) . however , some recent experiments for determining how well ric scores correlated with the levels of rag - mediated cleavage and v(d)j recombination activity demonstrate that the prediction capability of the ric score with respect to in vivo testing is quite weak ( 6 ) . this aspect is explained , at least partially , by experiments showing the effect of the chromatin structure on the rag cleavage efficiency , which confirms the role of chromatin in discriminating the rss functionality ( 7 ) . this information is actually not included in the ric score , which functional prediction capability must be considered , in the light of this limitation , only as a preliminary screening of genome - wide predictions . the implementation of dnagrab , written in c++ and based on modern computational optimization techniques , allows a time - efficient screening of genome - wide data . to perform a genome scale analysis , we started from the previously developed models for rsss with 12 and 23 spacer nucleotides ( 4 ) , which define the groups of spatial positions to be considered for correlation screening . while other models can be developed which take into account different nucleotide spacers , in a genome - wide perspective our choice was to focus on 12 and 23 spaced rss , since only these models are experimentally validated . concerning the mouse genome , we used a non - redundant version of the reference datasets employed in the original work ( 4 ) , for a total of 143 rss12 and 145 rss23 sequences . regarding the human genome , the reference sets of 168 rss12 and 178 rss23 were compiled in the context of this study , selecting sequences available from the imgt database ( 8) . the human and mouse rss reference datasets can be downloaded from the rsssite homepage . these models were used by dnagrab to scan all human and mouse chromosomes , searching for candidate crss sequences . to provide a reasonable dataset of crsss predicted to be possible functional substrates for the v(d)j recombinase , we scored the dnagrab - provided rsss using the ric score . in detail , the algorithm computes a score for each sequence starting with ca and compares it with a score that has been experimentally correlated with the rss function . in the current version of the system , pass / fail ric thresholds are set accordingly to the work of cowell and colleagues ( 3,4 ) : rss12 are scored as functional ( pass ) with ric 38.81 , while rss23 pass with ric 58.45 . using dnagrab , the rss reference models and the ric pass thresholds , a total of 3 089 308 and 1 833 319 potential rss12 sequences were identified in human and mouse genomes , respectively , while for rss23 3 218 664 and 2 091 561 rsss passing ric were identified and mapped . considering that 5% of crss locations are scored as both 12 and 23 rss in both genomes , these values indicate a global density of about 1 crss per 500 bp for human and 1 crss per 720 bp for mouse . these crss densities compare with the estimates from lewis and colleagues ( 9 ) of 1 crss per 600 bp , which were based only on inference from functional testing of plasmid dna . statistics of the genome - wide distribution of crsss , which are predicted to be functional according to this scoring scheme , are reported in tables 1 and 2 . there is no significant difference in the number of 12 and 23 crsss within the human and the mouse genome . on the other hand , the difference in the frequency of 12 and 23 crsss between these two eukaryotic genomes can be attributed to the different reference datasets . table 1.number and density of human putative crsss for intragenic and extragenic regionschr.rss12rss23rss countintrag . dens.extrag . dens.chr1248 570890.411002.74261 241872.17954.10chr2251 136858.59968.40258 916924.03939.30chr3214 721872.26922.23205 105939.34965.47chr4205 738875.10929.12183 8161001.061039.92chr5197 938864.89914.01186 426940.04970.44chr6183 662860.50931.68175 554951.26974.71chr7169 932858.72936.48174 965876.46909.55chr8155 781863.59939.55153 573930.73953.06chr9126 912874.461112.69137 272873.301028.71chr10144 072856.05940.74154 524868.69877.11chr11141 666874.92952.99148 507876.23909.09chr12139 185881.05961.68146 332876.15914.71chr1399 259865.341160.3096 400972.041194.71chr1489 376859.311201.1099 218873.301081.96chr1581 216882.691262.4596 756864.901059.69chr1678 857895.371145.81106 180727.97850.96chr1773 289922.981107.88108 155726.65750.73chr1875 549844.881033.4780 168890.12973.92chr1971 216915.51830.2888 168645.67670.64chr2061 670871.311021.9877 213781.58816.26chr2141 415837.221162.1439 510784.121218.17chr2240 042900.601281.2753 014664.33967.76chrx164 182833.67945.72159 189949.14975.38chry33 924774.60750.1928 450908.62986.94tot3 089 3083 218 664 table 2.number and density of mouse putative crsss for intragenic and extragenic regionschr.rss12rss23rss countintrag . dens.extrag . dens.chr1136 5091426.421444.56154 1441175.161279.29chr2127 4171407.531426.40148 6191148.641222.91chr3112 6231408.351417.12121 9631213.851308.59chr4108 4631456.201434.87124 7081168.661247.96chr5108 4711495.781406.25125 7471126.751213.05chr6105 3881433.101418.73120 4451155.721241.37chr7102 8741449.901482.63118 9381123.631282.39chr892 5611392.151423.27105 3241109.161250.80chr990 4971375.521371.05104 0611112.281192.34chr1095 5771380.141360.09102 6151187.031266.81chr1189 2311310.351365.49104 0881103.981170.59chr1287 2441323.811389.8796 9121180.011251.21chr1387 4631396.701375.2695 9331175.181253.84chr1479 4581403.241575.6198 5951141.771269.79chr1565 3471440.151583.7882 3241150.061257.17chr1662 6981417.321568.1477 0271174.761276.42chr1761 6591408.091545.1578 0121093.061221.26chr1858 0311405.721564.2071 9891166.831260.92chr1948 0791429.581275.8748 4471158.451266.18chrx111 8501472.871489.94109 8861414.721516.57chry19061448.931343.4217841596.391513.99tot1 833 3192 091 561 number and density of human putative crsss for intragenic and extragenic regions number and density of mouse putative crsss for intragenic and extragenic regions data about crss predictions performed on the human chromosome sequences ( hg18 ) and mouse chromosome sequences ( mm9 ) , as downloaded from the ucsc genome browser ( 10 ) , have been collected in a mysql database . a web interface has been developed using html and the perl scripting language . using this interface , it is possible to query the database about predicted human and mouse crsss , both with 12 and 23 spacers , and also to analyze user - provided human or murine sequences using the dnagrab algorithm directly . three different interfaces have been provided to query the database and retrieve predicted crss subsets : a genomic region using the first option , the crss search is performed in a specific cytoband of the selected chromosome , according to annotations reported in our local database . using the second option , the crss analysis is restricted to a specific region of a single chromosome , according to the ucsc genomic coordinates . gene search in all chromosomes is based on the string entered by the user in a text area , which is compared with all the gene symbols , refseq and protein accession identifiers stored in the local annotation database . all the crsss predicted within the user - specified gene will be displayed with the relative genomic coordinates and ric score . the query region extends from the transcription start site to the transcription end site of the gene and can be expanded upstream and downstream using the menu in the text area . the output results can be obtained in two different formats : tab - delimited text ( figure 1 ) or ucsc - uploadable format . if the ucsc - uploadable format option is selected , from the generated report it will be possible to display the rss predictions as user - generated tracks into the ucsc genome browser . the system creates a temporary text file in bed format and uploads it directly to the ucsc web site ( 10 ) , hence this option may be slow depending on the number of sequences selected . an example of a crss track displayed on the ucsc annotation background of the gnb211 mouse gene is shown in figure 2 figure 1.sample tabular output from a crss chromosomal search database query on a region of human chr8 ( nt . are the chromosome range , organism , type of crss requested and the putative signal sequences ( 7 ) with the sequence , the location , the ric score and a clickable link to ucsc . are the location of 12 ( red ) and 23 ( black ) crsss passing the ric score and mapping in a window encompassing the specified gene1000 bp .. sample tabular output from a crss chromosomal search database query on a region of human chr8 ( nt . are the chromosome range , organism , type of crss requested and the putative signal sequences ( 7 ) with the sequence , the location , the ric score and a clickable link to ucsc . sample graphical output from a crss gene search query for the mouse gene gnb2l1 . shown are the location of 12 ( red ) and 23 ( black ) crsss passing the ric score and mapping in a window encompassing the specified gene1000 bp . the analyse your own sequence section of the web interface enables users to identify the presence of putative crsss within any sequence . the user can choose to use the human or the murine model for searching both for 12 and 23 spaced rsss . for multiple analyses only sequences in fasta format are accepted while , for single sequence analysis , sequences with or without the fasta definition line are accepted . the dnagrab algorithm is used to predict crsss within user - provided sequences . while predicted crsss stored in the database are all considered functional according to the defined thresholds , this section provides a ric score , with respect to the selected model , for all the substrings starting with ca , providing information also for sequences that did not pass the ric filter . the output is also available in tabular form from the web page ( figure 3 ) . figure 3.sample tabular output of the analyse your own sequence section of the rsssite . the sequence has been analyzed using both the human and murine models , searching for 12 and 23 spaced crss . in the output , the start and end position the last column provides , according to the ric thresholds , an indication about the possibility of the sequence to be a dna breaking point . the sequence has been analyzed using both the human and murine models , searching for 12 and 23 spaced crss . in the output , the start and end position the last column provides , according to the ric thresholds , an indication about the possibility of the sequence to be a dna breaking point . data about crss predictions performed on the human chromosome sequences ( hg18 ) and mouse chromosome sequences ( mm9 ) , as downloaded from the ucsc genome browser ( 10 ) , have been collected in a mysql database . a web interface has been developed using html and the perl scripting language . using this interface , it is possible to query the database about predicted human and mouse crsss , both with 12 and 23 spacers , and also to analyze user - provided human or murine sequences using the dnagrab algorithm directly . three different interfaces have been provided to query the database and retrieve predicted crss subsets : a genomic region using the first option , the crss search is performed in a specific cytoband of the selected chromosome , according to annotations reported in our local database . using the second option , the crss analysis is restricted to a specific region of a single chromosome , according to the ucsc genomic coordinates . gene search in all chromosomes is based on the string entered by the user in a text area , which is compared with all the gene symbols , refseq and protein accession identifiers stored in the local annotation database . all the crsss predicted within the user - specified gene will be displayed with the relative genomic coordinates and ric score . the query region extends from the transcription start site to the transcription end site of the gene and can be expanded upstream and downstream using the menu in the text area . the output results can be obtained in two different formats : tab - delimited text ( figure 1 ) or ucsc - uploadable format . if the ucsc - uploadable format option is selected , from the generated report it will be possible to display the rss predictions as user - generated tracks into the ucsc genome browser . the system creates a temporary text file in bed format and uploads it directly to the ucsc web site ( 10 ) , hence this option may be slow depending on the number of sequences selected . an example of a crss track displayed on the ucsc annotation background of the gnb211 mouse gene is shown in figure 2 figure 1.sample tabular output from a crss chromosomal search database query on a region of human chr8 ( nt . are the chromosome range , organism , type of crss requested and the putative signal sequences ( 7 ) with the sequence , the location , the ric score and a clickable link to ucsc . are the location of 12 ( red ) and 23 ( black ) crsss passing the ric score and mapping in a window encompassing the specified gene1000 bp .. sample tabular output from a crss are the chromosome range , organism , type of crss requested and the putative signal sequences ( 7 ) with the sequence , the location , the ric score and a clickable link to ucsc . sample graphical output from a crss gene search query for the mouse gene gnb2l1 . shown are the location of 12 ( red ) and 23 ( black ) crsss passing the ric score and mapping in a window encompassing the specified gene1000 bp . the analyse your own sequence section of the web interface enables users to identify the presence of putative crsss within any sequence . the user can choose to use the human or the murine model for searching both for 12 and 23 spaced rsss . for multiple analyses only sequences in fasta format are accepted while , for single sequence analysis , sequences with or without the fasta definition line are accepted . the dnagrab algorithm is used to predict crsss within user - provided sequences . while predicted crsss stored in the database are all considered functional according to the defined thresholds , this section provides a ric score , with respect to the selected model , for all the substrings starting with ca , providing information also for sequences that did not pass the ric filter . the output is also available in tabular form from the web page ( figure 3 ) . figure 3.sample tabular output of the analyse your own sequence section of the rsssite . the sequence has been analyzed using both the human and murine models , searching for 12 and 23 spaced crss . in the output , the start and end position are reported , with the strand and the ric score . the last column provides , according to the ric thresholds , an indication about the possibility of the sequence to be a dna breaking point . the sequence has been analyzed using both the human and murine models , searching for 12 and 23 spaced crss . in the output , the start and end position the last column provides , according to the ric thresholds , an indication about the possibility of the sequence to be a dna breaking point . the rsssite web server provides a valuable tool to build preliminary in silico hypothesis on the sequence - based mechanisms regulating both physiological and aberrant v(d)j recombination . for example , an earlier version of rsssite was used in a study by dik and colleagues ( 11 ) focusing on the ( 11;14)(p13;q11 ) translocation , which is presumed to arise from an erroneous t - cell receptor delta tcrd v(d)j recombination and to result in lmo2 activation . using our algorithm , this group was able to determine ric scores for lmo2 crsss , which were used to drive functional experimentation . furthermore , they analyzed the lmo2 locus ( 10 kb to + 30 kb ) for the occurrence of 12- and 23-bp crsss predicted to be functional according to the ric score . in our laboratory experiments linked with this work , we selected 33 rsss from 11 chromosomal regions outside the immunoglobulin and tcr loci for functional testing . the dataset is fully described in table 3 . using ligation - mediated pcr ( lm - pcr ) , v(d)j recombinase - mediated dna breaks at rsss were analyzed in genomic dna prepared from mouse primary thymocytes , where rag1 and rag2 were expressed . we observed breaks of putative crsss in 12 out of the 33 sites tested ( 7 out of 15 for rss12 and 5 out of 18 for rss23 ) . concerning the distribution of these 12 rsss with respects to genes , four of them are intragenic , while eight are outside genes ( five upstream and three downstream ) . dna sequencing of lm - pcr products confirmed that breaks had occurred precisely at the 5 boundary of rsss , which is consistent with bona fide v(d)j recombinase - mediated cuts . v(d)j recombinase - mediated breaks were detected at 8 out of the 11 chromosomal sites tested , including previously uncharacterized rsss . no translocation products involving these break sites were detected in the assays performed so far . no selection was made for live cells , therefore many unrepaired breaks may have led to cell death . table 3.functionally tested sequences using putative crsss , according to the ric score , predicted by the dnagrab algorithm available at the rsssiterss12 sequences are in blue , rss23 sequences are in pink.cut * sequences are defined according to the visualization by lmpcr of a cleavage product at the 5-end of the rss . functionally tested sequences using putative crsss , according to the ric score , predicted by the dnagrab algorithm available at the rsssite rss12 sequences are in blue , rss23 sequences are in pink . cut * sequences are defined according to the visualization by lmpcr of a cleavage product at the 5-end of the rss . as discussed earlier in the algorithmic section of this work , while in vitro experiments have moderate correspondence with functional predictions achieved with the ric score , in vivo tests demonstrate a lower accordance with the proposed results , which can be partially attributed to the influence of the chromatin structure in recombination signal sequences recognition by rag proteins . therefore , the ric score provided by our algorithm should be considered only as a screening method for a preliminary identification of functional crsss in the context of genome - wide analyses , which takes into account the sequence patterns considered outside the chromosomal context . nonetheless , we believe that our results , as confirmed by the presented experimental validation , can be valuable for the identification of potential recombination sites , although these predictions must be considered taking into account the actual limitations of the developed algorithm that lacks , for example , of information about the chromatin structure . the identification and mapping of putative rsss in the human and mouse genomes has significant applicative potential , given the well - established observation of translocations at crss sites in lymphoid malignancies . we described here rsssite , a web - based database and search tool for the retrieval of predicted crsss from the human and mouse genomes , starting from a given chromosome region , a given gene identifier or user - supplied sequences . the surprisingly high frequency of observed v(d)j breaks suggests that many cryptic rsss may be cut by rag proteins during lymphocyte development . the control mechanisms that prevent more frequent involvement of aberrant v(d)j breaks in potentially oncogenic chromosomal translocations are currently unknown . we hope that the rsssite web server will provide a valuable tool to systematically test genomic and eventually epigenetic mechanisms regulating rss accessibility and usage at different chromosomal sites . italian fund for basic research ( firb - miur ) project grants italbionet and litbio. funding for open access charge : national research council .

recombination signal sequences ( rsss ) flanking v , d and j gene segments are recognized and cut by the vdj recombinase during development of b and t lymphocytes . all rsss are composed of seven conserved nucleotides , followed by a spacer ( containing either 12 1 or 23 1 poorly conserved nucleotides ) and a conserved nonamer . errors in v(d)j recombination , including cleavage of cryptic rss outside the immunoglobulin and t cell receptor loci , are associated with oncogenic translocations observed in some lymphoid malignancies . we present in this paper the rsssite web server , which is available from the address http://www.itb.cnr.it/rss . rsssite consists of a web - accessible database , rssdb , for the identification of pre - computed potential rsss , and of the related search tool , dnagrab , which allows the scoring of potential rsss in user - supplied sequences . this latter algorithm makes use of probability models , which can be recasted to bayesian network , taking into account correlations between groups of positions of a sequence , developed starting from specific reference sets of rsss . in validation laboratory experiments , we selected 33 predicted cryptic rsss ( crsss ) from 11 chromosomal regions outside the immunoglobulin and tcr loci for functional testing .

INTRODUCTION THE DNAGrab ALGORITHM: GENOME-WIDE SEARCH AND CLASSIFICATION OF RSSs Using the RSSsite web interface to identify and score cRSSs EXPERIMENTAL VALIDATION AND SUMMARY CONCLUSIONS FUNDING

floppy eyelid syndrome ( fes ) was first described by culbertson and ostler in 1981 . fes is characterized by very elastic upper lids that became easily distorted and evertible with minimal lateral traction and chronic papillary conjunctivitis of the upper palpebral conjunctiva and typically affects obese middle - aged men . the prevalence of fes within the general population varies within a range of 2.3% and 3.8% . since that first report , a growing number of publications have reported a high prevalence of other ophthalmic pathologies in patients with fes ; these include corneal abnormalities [ 3 , 4 ] , eyelid abnormalities [ 57 ] , and glaucoma . fes has also been associated with a variety of systemic diseases such as obesity , hypertension , ischemic heart disease , diabetes mellitus , skin pathologies , and most commonly obstructive sleep apnea syndrome ( osas ) [ 911 ] . osas is characterized by recurrent episodes of partial or complete upper airway obstruction causing cessation of breathing during sleep . it causes sleep disorder , waking the patient during night and resulting in excessive daytime somnolence . osas has high associated morbidity and mortality and is linked to hypertension , heart failure , stroke , and motor vehicle accidents [ 13 , 14 ] , and some cases can result in death . this proposed link was further reinforced by mcnab , in which 26 of 27 fes patients were found to have osas . up to now , the prevalence of fes in the osas population varies from 2% [ 17 , 18 ] to 32% . these studies are based on the findings that both fes and osas have similar pathologic changes in these two diseases , that is , a common defect in elastic tissue , and similar patient profiles , that is , middle - aged and obese men . were the first to find an increase in the elastolytic metalloproteinase enzymes , reporting a marked reduction in the amount of elastin fibers in the tarsal plates of patients with fes . . demonstrated a substantial loss of elastic fibers and ultrastructural abnormalities in residual fibers , together with an increased expression of elastin - degrading enzymes in the tarsus and skin of eyelid specimens from fes patients . other studies showed elastin fiber network disorganization and a loss of elastic fibers in the distal uvula tissue from patients with osas undergoing uvulopharyngoplasty [ 17 , 19 , 21 ] . these changes in elastic fibers provide evidence to those who have proposed a link between fes and osas and these changes may explain how osa and fes could be different manifestations of the same disease . in another way , both fes and osas symptoms seem to be associated with sleep posture . mcnab claimed that an association may exist between sleeping posture and pressure on the eye . the upper lid in fes patients has been observed to be everted during sleep and on waking . many patients have strictly unilateral disease , and the affected side is commonly more symptomatic on the side that the patient is used to sleeping on [ 16 , 22 ] . recently , fes was suggested as a useful way to identify individuals with a greater probability of having glaucoma in the osas population . all of these seem to suggest a strong link between disturbed sleep and their eyelid changes . others argued that both fes and osas are independently associated with obesity , male gender , and increasing age , which raises concern that these per se may be confounding factors . thus it is unclear whether osas and fes are causally associated , whether they merely share common risk factors , or whether they have a common pathophysiological cause . why patients with osas are at risk for fes is not known . previous results suggest that the risk is determined by an increased body mass index and the patients with fes are younger and more obese and have higher apneas or hypopneas index ( ahi ) than typical patients with osas [ 16 , 22 ] . however that relationship between the presence of fes and osas exists even when controlling for this variable . the contradictory reports suggesting this association are less conclusive due to the small number of patients [ 9 , 22 , 24 , 25 ] , inclusion of continuous positive airway pressure - treated patients [ 2 , 9 ] , or lack of systematic objective assessment of sleep apnea syndrome [ 2 , 24 ] . in view of the association between fes and osas may provide awareness for the otolaryngologist to facilitate further management . therefore , it is necessary to make clear whether fes is more prevalent in osas patients . thus , we performed this meta - analysis to evaluate the prevalence of fes in osas patients compared with that in non - osas subjects . our meta - analysis was conducted in strict accordance with the prisma ( preferred reporting items for systematic reviews and meta - analyses ) statement , which is reporting guideline for meta - analyses . the search was performed in the pubmed , embase , and cochrane databases up to december 2015 . the search terms were floppy eyelid or floppy eyelid syndrome and obstructive sleep apnea or obstructive sleep apnea syndrome or obstructive sleep apnea hypopnea syndrome or osa or the inclusion criteria for the current meta - analysis were the following : ( 1 ) only studies that were of an observational design and concerned the prevalence of fes in osas patients ; ( 2 ) only studies that obtained consent from the patients ; ( 3 ) only studies where polysomnography or oximetry was used and definition of hypopnea and apnea was presented for diagnosis of osas ; ( 4 ) only studies where fes and osas could be used as an outcome in the analysis ; and ( 5 ) only studies with an effective control group . two investigators ( dr . ping wang and dao - jiang yu ) independently screened all identified studies using the above - mentioned criteria . when any disagreement emerged , a third reviewer ( dr . meta - analyses were then conducted regarding the prevalence of fes in osas by calculating odd ratio ( or ) with 95% confidence interval ( ci ) . cochrane 's i index was calculated to assess heterogeneity , and if the data were not significant ( p > 0.05 , i < 40% ) , the ors were pooled according to the fixed - effect model . the meta - analyses were performed using the review manager ( revman , version 5.2 ) from the cochrane collaboration [ 27 , 28 ] . because there is no consensus as to the best standardized method for assessing the quality of observation studies , we designed a five - item scoring scale ( each item scoring 0 or 1 ; 1 being better ) [ 29 , 30 ] . the items on the integer scale were representativeness of the cases , whether the diagnosis criteria of fes were given , whether the assessment of osas was objective , and whether osas severity was assessed and controls for confounding factors . quality while 4 or 5 was considered to indicate high quality [ 29 , 30 ] . the quality of each study was independently assessed by two investigators ( dr . hui li and gang feng ) . the articles were initially identified by electronic and manual searching . after a review of the titles and abstracts , we excluded reviews , case reports , letters , and studies not about the relationship between fes and osas . the resultant eleven studies are related to the relationship between fes and osas . as the aim of this meta - analysis is to review whether fes is more prevalent in osas , two articles addressing the prevalence of osas in fes were excluded . among the nine studies about the prevalence of fes in osas , finally six studies that met the inclusion criteria were included in the meta - analysis . first , the diagnosis criteria for osas were somewhat different among all the included studies . most studies adopted ahi to diagnose osas [ 18 , 3133 ] , while others used oxygen desaturation index or respiratory disturbance index . second , the definitions for ahi and the grouping criteria for osas were slightly different in studies with ahi as diagnosis criteria . all these may affect the prevalence of fes in osas , especially that in different osas severity group . third , the diagnosis criteria for fes were subjective in all included studies [ 2 , 18 , 3133 ] except for one study without any diagnosis criteria . finally , the relationship between fes and osas is based on the hypothesis of the study . for example , the expression of fes and osas which could be different manifestations of the same disease was referred to in two studies and the prevalence of fes in osas patients was higher than non - osas population [ 31 , 32 ] . the meta - analysis data were derived from 6 studies of 609 cases in the osas group and 158 cases in the control group [ 2 , 9 , 18 , 3133 ] . as shown in figure 2 , the heterogeneity was not statistically significant ( i = 21% , p = 0.28 ) , and thus a fixed - effect model was used . the test for the overall effect revealed that fes was statistically prevalent in osas patients when compared with that in non - osas subjects ( z = 4.98 , p < 0.00001 ) . in the subgroup analysis by osas severity , the heterogeneity was not statistically significant ( i = 0% , p = 0.62 ; i = 21% , p = 0.28 ; and i = 13% , p = 0.32 for mild , moderate , and severe subgroup , resp . ) , and thus fixed - effect models were used in the following analysis . the test for the overall effect revealed fes was statistically prevalent in osas patients when compared to that in non - osas subjects , regardless of whether the severity is mild ( p value of z - test = 0.04 ) , moderate ( p value of z - test = 0.0004 ) , or severe ( p value of z - test < 0.0001 ) . the incidence of fes in osas increased with severity of osas as indicated with increased or values ( or = 2.56 , 4.62 , and 7.64 for mild , moderate , and severe subgroups , resp . ) . the 95% ci for mild , moderate , and severe subgroups were 1.056.28 , 1.9910.73 , and 3.4416.96 , respectively . the subgroups analysis results were summarized in figure 3 and table 2 . to evaluate the sensitivity of the meta - analysis , each study was sequentially excluded from the meta - analysis , and the corresponding heterogeneity results and results of the tests for overall effect are shown in table 3 . as shown in table 3 , the heterogeneities and overall effect of prevalence of fes in osas did not alter significantly when excluding any study from the meta - analysis , with the heterogeneity changing between 0% and 30% and all p values of overall effect remaining less than 0.0005 . the shape of the funnel plot was asymmetric ( figure 4 ) which suggested that the publication bias may exist . in this meta - analysis , we investigated the prevalence of fes in osas including 767 subjects . we found that individuals with osas showed an increased risk of fes in the overall population . the result from our meta - analysis suggested that osas patients had 4.12 times higher fes risk compared to those non - osas individuals . among the six included studies in this meta - analysis , four studies suggested that fes is more prevalent in osas patients and two studies did not report the close relationship between fes and osas [ 18 , 33 ] . the main result of meta - analysis is consistent with most of the included studies . actually the prevalence of fes in osas is greatly different among all the included studies , varying from 2.27% ( 1/44 ) to 64.57% ( 164/254 ) . this difference may arise from the different definitions for apnea or hypopnea and different criteria for osas severity grouping in these studies . as demonstrated in table 1 , three studies used the american academy of sleep medicine 2007 criteria in sleep staging and in determining the respiratory pathologies . the hypopnea was defined as a 30% reduction in airflow accompanied by a 4% oxygen desaturation or a 50% reduction in airflow accompanied by a 3% oxygen desaturation or arousal [ 9 , 31 , 32 ] , while in karger et al . 's study obstructive apnea was defined as cessation of airflow despite respiratory effort for at least 10 s and hypopnea was defined as at least a 30% drop in airflow for at least 10 s despite respiratory effort and at least a 4% drop in oxyhemoglobin saturation . in the study of muniesa royo et al . , obstructive apnea was defined as an absence of airflow for at least 10 s and hypopnea was defined as a clear ( 50% ) airflow reduction for at least 10 s , with a drop in oxygen saturation of at least 4% or an arousal . the differences are whether or not to include the time of airflow reduction and the amount of airflow reduction when determining a respiratory event . the definition of respiratory event is the basic for diagnosis of osas and thus may impact the grouping of osas and non - osas population and severity of osas . in addition , one study used only oximetry , which is a method not considered valid for osas diagnosis . all these detailed differences may impact the prevalence of fes in osas . in diagnosis of fes , this may be based on the experience of the examiner and thus impact the occurrence rate of fes in osas patients . in the stratified analysis by osas severity , the significant prevalence was observed in mild , moderate , and severe osas and the fes risk increased with the severity of osas with or as 2.56 , 4.62 , and 7.64 from mild to severe osas . this is consistent with previous report showing that a floppy eyelid rate was positively correlated with respiratory disturbance index values [ 9 , 32 ] . as ahi values increase , the clinical course of the osas becomes more severe , the tissue mechanic stress increases , and the eyelid floppiness progresses due to hypoxemia . however , only three studies reported fes prevalence in different severity of osas [ 9 , 32 , 33 ] and the sample number was small . thus , the results lacked sufficient reliability to confirm or refute an association in a definitive manner . in the future , more studies with fes in different severity of osas are still needed to address the role of osas on fes risk . the higher prevalence of fes in osas patients may indicate that the improvement of accompanying systemic and ocular symptoms may be expected after the appropriate treatment of osas . this was confirmed by a study in which the symptoms and signs of fes disappeared after continuous positive airways pressure during sleep . the author indicated the change of mechanical force to the eyelid during sleep , which varied with body position from prone to supine as the continuous positive airways pressure treatment progresses , may help alleviate the symptoms of fes . specifically , patients with osas may initially sleep prone ; the mechanical pressure to the eyelid is bigger . as continuous positive airways pressure treatment progresses , the symptoms of osas may be relieved . certainly , this hypothesis needs to be confirmed with large number of samples in the future . if this is the case , it may also provide another therapy choice for fes patients and ophthalmologists , although the reversal of fes with continuous positive airways pressure in osas patients was not common . given the strength of the association between fes and osas , the current findings raise the questions of whether fes screening should be performed routinely in osas patients . sleep physicians and ophthalmologists should be aware of this association and thus refer suspected patients for diagnosis and therapy . the limitation of this meta - analysis is that there is a referral bias in the population studied , as none of the studies selected their participants randomly . most were based on patients attending a clinic for evaluation of osas and without therapy except one study including osas patients after continuous positive airway pressure therapy . this may not be representative of the entire osas population , majority of whom remain undiagnosed ; thus selection bias may exist . the referred group of patients would include those with significant symptoms and identified as suspects by the general practitioner in the community . meanwhile , the control group in these included studies , which constitutes suspects of osas and is referred to sleep center , raises the question of whether the non - osas patients are representative of the general population . it is expected that this non - osas had higher ahi than general population because they all had symptoms of osas more or less . from this point of view , the occurrence rate difference between fes in osas and fes in the true sense of non - osas population should be more statistically significant . this indicated that the pooled results in this study represent unadjusted estimates , which are likely to be confounded by other risk factors ; these results should be confirmed in further studies . thus it is difficult to draw a definitive conclusion from this meta - analysis because of the different quality assessment of the included studies . in conclusion , this study confirmed that fes is very common in osas but that fes is not so common among the general non - osas population . large prospective randomized studies are required to better elucidate the exact prevalence of fes in patients with osas and the direct prognostic value .

controversial findings are reported about the relationship between floppy eyelid syndrome ( fes ) and obstructive sleep apnea syndrome ( osas ) . the main goal of this study was to evaluate whether fes is more prevalent in osas patients by performing a meta - analysis . a comprehensive literature search of pubmed , embase , and cochrane databases was performed . only studies related to the prevalence of fes in osas were included in the meta - analysis . we estimated a pooled odds ratio ( or ) for the prevalence of fes in osas . in total , 6 studies with 767 participants met the inclusion criteria . using a fixed - effects model , the pooled or was 4.12 . the test for the overall effect revealed that fes was statistically prevalent in osas patients when compared with that in non - osas subjects ( z = 4.98 , p < 0.00001 ) . in the subgroup analysis by osas severity , the incidence of fes in osas increased with severity of osas as indicated with increased or values ( or = 2.56 , 4.62 , and 7.64 for mild , moderate , and severe osas ) . in conclusion , the results indicate that fes is more prevalent in osas patients . however , this result was based only on unadjusted estimates . prospective cohort studies are needed to determine whether osas is an independent risk factor for fes .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

the amount of body fat may significantly change over the seasons , particularly in latitudes away from the equator , where seasonal changes in climate , temperature and duration of daylight are greater . these lead to changes in availability of certain foods and in individuals feeding habits and outdoor activity ( resulting e.g. in picnics being more common in summer ) ( reilly and peiser 2006 ) . plasqui and westerterp ( 2004 ) have shown that there is a significant seasonal variation in physical activity and total energy expenditure , with lower amounts in winter , in young dutch adults . the biological significance of such seasonal and daily environmental rhythms has long been appreciated ( reinberg 1972 ) and is best seen in seasonal animals and hibernators , which adjust their physiology both in preparation for , and in response to , changing demands of the environment ( ebling and barrett 2008 ) . however , in humans living in modern societies , the impact of seasonality has somewhat diminished following the introduction of artificial lighting and heating and air - conditioning systems . the use of these artificial aids reduces the exposure of individuals to fluctuations in ambient temperature and light , and this is more convenient for practising a modern lifestyle . however , these natural fluctuations contribute to the normal adjustment of the body clock to a 24-h period ; their extensive use ( artificial aids ) will lessen this synchronization and may increase the risk of developing mismatches between the natural environment and the body clock ( similar to the problems observed after a time - zone transition or during night work ) . it has been claimed that these misalignments may lead to alterations in metabolism and thermoregulation that promote obesity ( johnson et al . while seasonal rhythmicity in energy storage and expenditure is significantly influenced by changes in the external environment ( reilly and peiser 2006 ) , the nature of daily rhythms in metabolism is more complex . in this respect , humans possess internal timing mechanisms which can act independently of daily changes in the environment . all cells show a genetic potential for daily rhythmicity , but in practice , this rhythmicity is manifested in only some regions of the body . these regions include the liver ( which possesses a food - entrainable oscillator ) and the suprachiasmatic nucleus ( scn ) paired structures in the base of the hypothalamus . the scn normally coordinates rhythmic activity throughout the body ( acting via the autonomic nervous system , temperature regulation , hormone secretion , sleep , and feeding behaviour ) and is known as the body clock . evidence is accumulating to suggest that the disruption of these body clocks may contribute to metabolic disorders and predispose to obesity ( eckel - mahan and sassone - corsi 2013 ) . rhythmicity seen in many processes , including metabolism , reflects both personal habits ( e.g. sleep , activity and mealtimes ) and the impact of internal body clocks . humans , like other organisms , possess a timing system that consists of self - sustained oscillators that are reset by various synchronizers . in the absence of time cues , the dominant component of this system free runs with a period of 2425 h , giving rise to a so - called circadian rhythm ( from the latin : circa diem about a day ) . normally , this rhythm is entrained or synchronized to a 24-h cycle ( called daily in this review ) , predominantly by natural light dark cycles and , to lesser extent , by cycles of rest and activity or feeding and fasting . external stimuli that can synchronize the body clock to a 24-h cycle are called zeitgebers ( from german : time givers ) ( reilly and peiser 2006 ) . the molecular mechanisms that underlie the function of cellular clocks are the oscillating post - translational modifications of proteins ( e.g. phosphorylation ) and the autoregulatory feedback loops that control gene transcription and translation . the main loop comprises a positive and a negative limb that are interconnected ( albrecht 2012 ) . it consists of the transcriptional activators clock and bmal1 and their target genes per ( period ) and cry ( cryptochrome ) . products of these genes accumulate gradually and inhibit clock - bmal1 transcription . in turn , the feedback loop controlling bmal1 involves nuclear receptors rev - erb and ror/ that inhibit and activate bmal1 transcription , respectively . in addition , the activity of rev - erb links metabolism to the clock system ( liu et al . administration of rev - erb ligands in mice has been found to alter expression of both the clock genes in the hypothalamus and the metabolic genes in the liver , skeletal muscles and adipose tissue , resulting in increased energy expenditure ( solt et al . the reader is referred to recent excellent reviews ( albrecht 2012 ; bass 2012 ; bass and takahashi 2010 ; mohawk et al . the observation that all cells , tissues and organs contain the molecular potential to manifest a clock gave rise to the concept of peripheral and central ( master ) oscillators , the former normally being subservient to the latter . the central pacemaker in mammals is located in two hypothalamic clusters of neurons , the scn . these centres control behavioural , metabolic and physiological rhythms and can synchronize the peripheral oscillators ( welsh et al . an important peripheral oscillator is the food - entrainable oscillator ( feo ) , which controls food - anticipatory activity ( faa ) in rodents , the exact location of which is unknown ( mieda et al . faa in rodents is an increase in activity just before the food is regularly available . daily rhythms of locomotor activity , body temperature and corticosterone secretion can thus synchronize with the rhythm of food availability ( stephan 2002 ) , even when food is presented during the resting phase or when the central oscillator is destroyed ( mistlberger 2011 ) . these observations indicate that the feo ( and , possibly , peripheral clocks in general ) can act independently of the scn , at least in some animal . this independence of activity may become important when the master oscillator and lifestyle become misaligned . in normal circumstances , the master clock , the environment and peripheral clocks are synchronized to one another ( reilly and peiser 2006 ) . whereas periodicity of the master clock is controlled mainly through the light dark cycle ( acting as a zeitgeber ) , peripheral oscillators are affected either by behaviour ( cycles of rest and activity or feeding and fasting ) or by fluctuations in the levels of circulating hormones , such as catecholamines ( dibner et al . for example , peripheral oscillators in the pancreas and the liver ( marcheva et al . 2010 ) can be adjusted by regular food intake , even if this intake is timed unusually . the scn produces the endogenous component of the observed rhythm , and the exogenous component is superimposed upon it and corresponds to the environment and lifestyle ( e.g. inactivity and fasting when asleep ) . in practice , therefore , rhythms measured in the presence of an exogenous component may not give clear information regarding the activity of the internal oscillators , and the implications of this will be considered at the end of this review . however , normally , the endogenous and exogenous components are in phase but may become desynchronized ( e.g. by night work ) , because the scn is rather slow to adjust ( reilly and peiser 2006 ) . under such circumstances , the environment and lifestyle may also adjust the timing of the master oscillator ( e.g. by changing the light dark cycle ) and of the peripheral oscillators ( e.g. by changing feeding times , which will impact on feo ) . food intake in humans shows not only a daily rhythm ( daytime rather than nocturnal eating ) but also a rhythm related to the intake of individual meals with a period of about 45 h. this rhythm is ultradian , having a period of oscillation less than 20 h. such rhythms are probably seen in the activity of all hormones associated with food metabolism . while they are linked directly to food intake ( and , as such , can be considered exogenous ) , other rhythms are likely to be derived from internal oscillators . episodic release of hormones may exhibit yet another periodicity that lasts for minutes and reflects pulsatile release of a hormone followed by its removal and breakdown . there are also infradian rhythms ( with periodicity greater than 28 h ) including seasonal rhythms . since there is no clear evidence that endogenous circannual oscillators exists in humans , seasonal rhythms observed are attributed rather to exogenous factors . as indicated earlier , they include seasonal variations in food intake , physical activity , ambient temperature and the duration of daylight . this lack of information is partly due to the obvious fact that such studies demand a more elaborate protocol which covers the four seasons . moreover , if any interactions between daily rhythms and seasonal rhythms are sought , then the a full set of data covering the 24 h must be collected four times per year . daily food intake in humans usually consists of two to three main meals consumed at times that depend largely on lifestyle and social factors . it has been observed that food intake in the morning is more satiating than the same meal eaten in the evening ( de castro 2009 ) ; it has also been observed that the amount of food eaten shows seasonal variations , with increased meal size and total calorie intake occurring in the autumn ( de castro 1991 ) . the hunger and satiety centres in the hypothalamus contain receptors for mediators that affect feeding behaviour . these substances are either orexigenic ( stimulate feeding ) or anorexigenic ( inhibit feeding ) ( naslund and hellstrom 2007 ) . short - term regulation of food intake involves cholecystokinin ( little et al . 2005 ) , peptide yy , glucagon - like peptide and insulin ( suzuki et al . 2012 ) , all of which act anorexigenically , and ghrelin , which stimulates appetite ( cummings 2006 ) . these mediators display daily and ultradian rhythms in phase with food intake and some of them ( ghrelin , leptin , insulin ) are also involved in long - term regulation of body weight and energy homeostasis ( stutz et al . the mechanisms controlling seasonal food intake and energy balance in seasonal animals and hibernators differ across the species and reflect different strategies employed to survive in a harsh environment ( florant and healy 2012 ) . the seasonal changes are executed through fluctuations in humoral signals , including leptin and ghrelin ( adam and mercer 2004 ; florant and healy 2012 ) . interestingly , while leptin concentrations in humans do not exhibit consistent seasonal changes , it has been observed that the levels of leptin , cholesterol and triglycerides in obese males are significantly higher in winter ( kanikowska et al . 2013 ) . the digestive system shows rhythmicity in many functions , including basal gastric acid secretion , epithelial cell proliferation and gastrointestinal motility ( ekmekcioglu and touitou 2011 ) . it is attributed primarily to the timing and the size of meals and exhibits daily and ultradian components . however , some aspects of this rhythmicity may reflect the function of a peripheral clock . for example , it has been demonstrated in animals that rhythmic expression of clock genes within the neurons of the myenteric plexus modulates colonic motility by controlling the expression of neuronal nitric oxide synthetase ( nnos ) and vasoactive intestinal peptide ( vip ) ( hoogerwerf 2010 ) . indirect evidence for a role of biological clocks in gastrointestinal functions in humans comes from observations of night workers who have altered appetites and a higher prevalence of constipation , diarrhoea and abdominal discomfort ( nojkov et al . 2010 ) . metabolism of absorbed foodstuffs shows rhythmicity that reflects changes in the release of endocrine regulators . hormones associated with metabolism ( including glucagon , insulin , catecholamines , glucocorticoids and thyroid hormones ) show both circadian and ultradian oscillations . these rhythms are dominated by food intake ( and as such are exogenous and ultradian ) but may also be influenced by the scn that produces daily fluctuations in sympathetic nervous system outflow which may affect hormone secretion ( e.g. insulin ) . rhythmicity in clock gene expression and adipocytokine release is seen also in white and brown adipose tissues ( wat and bat , respectively ) ( gavrila et al . 2003 ) . body temperature in adult humans is about 1 c higher during the day , which is attributed both to the sleep - wake cycle ( exogenous component ) and to the scn - generated rhythmicity in metabolism and peripheral vasculature tone ( endogenous component ) . in babies , however , these rhythms of temperature regulation are not fully developed , and bat plays an important role in heat production . 2006 ) and shows a 24-h rhythm of glucose uptake ( van der veen et al . 2012 ) . moreover , bat activity is regulated via the sympathetic nervous system and by several hormones , all of which express daily variations ( kriegsfeld and silver 2006 ) . seasonal changes in bat have been observed in humans , with bat growth induced by exposure to cold and associated with the shorter hours of daylight in the autumn and winter ( au - yong et al . it is now clear that several aspects of metabolism show daily rhythmicity related to scn function ( bass 2012 ; bass and takahashi 2010 ; marcheva et al . however , the scn is also adjusted by feeding behaviour and metabolic products , which results in additional ultradian rhythmicity . moreover , many organs involved in food metabolism , such as the liver ( balsalobre et al . 2000 ) , pancreas ( sadacca et al . 2011 ) , intestine and stomach ( bostwick et al . 2010 ) and the adipose tissue ( johnston 2012 ) , have autonomous peripheral oscillators ( fig . 1 ) . therefore , it is not surprising that factors altering these interactions might adversely impact on metabolism and be associated with an increased risk of obesity . in this respect , it has recently been shown that the impairment of peripheral clocks may be associated with the development of diabetes ( pappa et al . 1interactions between the master clock ( scn ) , peripheral oscillators and the environment interactions between the master clock ( scn ) , peripheral oscillators and the environment however , other lifestyle - related factors that have been implicated in obesity ( such as sleep duration , eating habits , shift work ) have not always received enough attention ( chaput et al . . short sleep ( defined as 6 h of sleep per day ) and sleep disorders have been associated with lower concentrations of leptin and higher levels of ghrelin and with increased hunger and appetite ( taheri et al . in addition , it has been demonstrated that sleep deprivation may contribute to obesity through modulating plasma levels of leptin ( mullington et al . 2003 ) . also , the lack of orexin , a hypothalamic wakefulness - inducing neuropeptide ( sakurai et al . 1998 ) , affects sleep , feeding and metabolism ( adamantidis and de lecea 2008 ) and is associated with increased likelihood of developing obesity ( funato et al . narcolepsy , when patients suffer from extreme daytime sleepiness due to the loss of orexin - producing neurons ( tsujino and sakurai 2013 ) , has also been linked with increased body mass and obesity ( kotagal et al . patients with night eating syndrome , in whom the patterns of sleep and eating are disrupted , are often obese ( colles et al . 2007 ) and have altered rhythms of plasma leptin , insulin , cortisol , ghrelin , melatonin and glucose ( goel et al . it appears that most overweight and obese people sleep less than normal , and therefore , they have more time to eat ( chaput et al . 2011 ) and snack ( nedeltcheva et al . 2009b ) , and they are also at increased risk of insulin resistance and type 2 diabetes ( chao et al . interestingly , sleep architecture changes with seasons , with increased rapid eye movement ( rem ) sleep during the winter ( kohsaka et al . 2possible effects of altered sleep - wake cycles on metabolic hormones and body weight possible effects of altered sleep - wake cycles on metabolic hormones and body weight the exact mechanism linking sleep disturbances and it has been postulated that loss of sleep reduces glucose tolerance and increases insulin resistance ( nedeltcheva et al . on the other hand , sleep deprivation may increase food intake and appetite ( brondel et al . 2010 ) by decreasing leptin or by increasing ghrelin ( taheri et al . 2004 ) and orexin ( zeitzer 2013 ) . since sleep is believed to allow the brain to replenish energy stores , it has been speculated that altered sleeping habits might through the autonomic nervous system and hypothalamic - pituitary - adrenal axis impact on the release of metabolic hormones and the control of food intake ( spiegel et al . eating patterns when to eat , the amount of food eaten and the circumstances leading to finishing a meal affect energy intake ( blundell and cooling 2000 ) . an important study on the effect of meal frequency / pattern on body fat and metabolic functions in humans was by fbry and tepperman ( 1970 ) , who found that excessive weight , hypercholesterolemia , impaired glucose tolerance and ischemic heart disease were more common among persons who ate larger meals less frequently rather than smaller meals more often . grazers , who eat smaller meals throughout the daytime , may be metabolically advantaged compared to gorgers ( who eat fewer but larger meals ) , since larger meals may lead to increased fat synthesis and storage ( verboeket - van de venne and westerterp 1991 ) . it has been demonstrated that the same meal eaten at different times of the day may exert different metabolic effects ; thus , it appears that a morning meal is associated with better control of body mass than when the same meal is eaten later in the day ( keim et al . such an effect may be related to the amount of physical work performed during the daytime and endocrine responses to food intake ( with the insulin response to food intake being time - of - day dependent , for example ) . in this respect , it has recently been demonstrated in mice that a feeding regimen that restricted feeding time but not calorie intake showed improved nutrient utilization and energy expenditure ( hatori et al . 2012 ) . patterns of food intake can be determined by the social context , time availability and night work . binge eating involves food intake greatly in excess of metabolic requirements , often with the loss of control over the amount eaten . it has been observed that breakfast was the least , and dinner the most , common meal associated with this practice , and binge eating was often associated with evening snacking ( harvey et al . 2011 ; stunkard and allison 2003 ) . a positive relationship between appetite and food intake no longer exists for meals eaten during the working day ; in these circumstances , a proper meal is often replaced by fast food eaten at amounts reflecting time availability rather than appetite . by contrast , if there is plenty of time and one is with friends , food intake is often in excess of metabolic requirements , particularly if alcohol is drunk as part of the occasion s conviviality ( de castro 2009 ; waterhouse et al . the type of food eaten varies also over the day with greater intake of carbohydrates at breakfast and of fat at dinner ( westerterp - plantenga et al . short and irregular sleeps are associated with consuming more fats , fast foods and sweet beverages , and less vegetables ( baron et al . 2011 ) , as well as with more palatable foods high in sugar , fat and salt rather than rich in protein or roughage ( st - onge et al . these problems are particularly evident when snacking late at night or during a nocturnal waking episode . the mechanisms linking frequency of food intake and the type of food eaten remain unclear , but it has been suggested that lower leptin and higher ghrelin concentrations are involved ( taheri et al . for example , meals with a high fat - to - carbohydrate ratio decrease plasma concentrations of leptin ( havel et al . 1999 ) and ghrelin ( monteleone et al . 2003 ) concentrations . shift and night work may disturb 24-h rhythms , including endocrine rhythms ( morris et al . night work is associated with an increased risk of metabolic syndrome ( esquirol et al . 2009 ) , obesity ( pandalai et al . 2013 ) and sleep disturbances ( ohayon et al . 2003 ) and tend to snack ( on foods high in salt and carbohydrate ) rather than eat a full meal during the shift . in addition , attempts to eat with their families whenever possible often mean that total daily food intake increases . the combination of these factors increases the likelihood of developing indigestion , obesity and metabolic disorders ( karlsson et al . lack of synchrony between body clocks and lifestyle may change the secretory profiles of metabolic hormones , including ghrelin and leptin ( crispim et al . 2011 ) , which may contribute to increased appetite and higher energy intake ( spiegel et al . 2004 ) . gaining weight is more likely if night workers also experience social disturbances due to their abnormal lifestyle ( waterhouse et al . 2005 ) . thus , disturbed expression of clock genes in wat was detected in genetically obese mice of the kk and kk - a(y ) strains ( ando et al . these mice showed also abnormal leptin secretion , sleep disturbances , altered locomotor activity and changed rhythms of adiponectin and resistin release . also , mice with adipose tissue - targeted deletion of bmal1 displayed increased adiposity and body weight , impairment of feeding rhythms and alterations in the expression of hypothalamic neuropeptides that regulate appetite ( paschos et al . similarly , obese humans were found to differ in the expression patterns of several clock and metabolic genes in adipose tissue ( garaulet et al . 2011 ) , in the rhythms of plasma adipokines ( saad et al . 1998 ) and in daily rhythms of leptin and ghrelin secretion ( heptulla et al . 2001 ; yildiz et al . in addition to being a key component of the body clock , adipocyte bmal1 has also been implicated in adipose tissue differentiation and lipogenesis ( shimba et al . 2005 ) . moreover , the expression of uncoupling protein-1 ( ucp1 ) that is in involved in bat thermogenesis was found to associate with winter accumulation of visceral fat ( nakayama et al . the central and peripheral clocks act to coordinate behavioural and metabolic responses with the environment . light dark cycles entrain the central clock in the scn , which then synchronizes peripheral clocks and the rest of the body through autonomic innervation , body temperature , endocrine signalling and feeding - related cues . feeding can regulate peripheral clocks independent of the central clock through local metabolites and signalling pathways . increasing evidence suggests that this harmony may become disturbed either through behavioural misalignment ( such as shift work or jet lag ) or metabolic challenges ( e.g. high - fat feeding ) ( bass and takahashi 2010 ) . this may lead to further weakening of links between the clocks and result in abnormalities that promote weight gain , obesity and development of metabolic syndrome . future research will need to elucidate the exact molecular mechanisms linking biological clocks with metabolic homeostasis and nutrient state . ultimately , such studies may help to prevent metabolic derangement and obesity in individuals with sleeping disorders or working on shifts and to optimize weight loss regimens . also , whilst there is considerable evidence that altered daily rhythms are associated with obesity and allied problems , there is an interpretive problem associated with such results . as already mentioned , a measured daily rhythm reflects not only the activity of the central and peripheral oscillators controlling metabolism but also exogenous effects directly due to the pattern of food intake ( including the period of fasting during sleep ) . that is , a measured rhythm is not necessarily an accurate reflection of the activities of the internal mechanisms ( the oscillators ) ; it might be masked by the exogenous component . it is important to distinguish between these two causes of a rhythm if detailed information regarding the interactions between the internal oscillators and metabolic processes in obesity is sought . one way to separate the effects of these two causes is to minimize the exogenous component ; this could be achieved by giving identical meals at equal time intervals throughout the 24 h , also prohibiting sleep . the rhythms observed in these circumstances ( when the patterns of food intake and the sleep - wake cycle have been removed ) would then reflect those of the internal processes far more closely . such studies are of fundamental importance to a better understanding of obesity and need to be performed in future research .

while the significance of obesity as a serious health problem is well recognized , little is known about whether and how biometerological factors and biorhythms causally contribute to obesity . obesity is often associated with altered seasonal and daily rhythmicity in food intake , metabolism and adipose tissue function . environmental stimuli affect both seasonal and daily rhythms , and the latter are under additional control of internal molecular oscillators , or body clocks . modifications of clock genes in animals and changes to normal daily rhythms in humans ( as in shift work and sleep deprivation ) result in metabolic dysregulation that favours weight gain . here , we briefly review the potential links between biorhythms and obesity in humans .

Seasonal and daily influence on metabolism and body weight Basic principles of chronobiology Peripheral and central oscillators The rhythmic organism Rhythmicity of food intake, gut function and metabolism Factors associated with increased risk of obesity Conclusions and perspectives

labour pain is a universal experience among women but the perception differs between individuals and this is influenced by both physical and psychological factors . the difference in pain perception among parturients depends on factors such as pain threshold , culture , social status , parity , prenatal education , counseling , support during labour and medical intervention.12 the practice of obstetric analgesia may have well been established in developed countries , it is still in infancy in most developing countries . in fact in some countries , the majority of the pregnant women do not even know that labour pains can be relieved.3 the benefits of pain relief in labour , however , extend far beyond mere humanitarian ones and it should be clearly recognized that appropriate pain relief may improve the general course and success of labour.34 as a result of increased knowledge and sophistication in most developed countries , parturients are aware of pain relief in labour and may demand and experience practically pain - free labour.5 in our environment , due to the lack of resources and competing priorities , pain - free labour is not achieved in most cases.6 studies on patients perception of the quality of care in labour have identified that pain relief , information obtained about labour and presence of support person in labour as important aspects of quality care in labour.7 ekiti state university teaching hospital , ado - ekiti south - western nigeria , is an emerging teaching hospital established in 2008 ; hence , there is a need to study the practice of obstetric analgesia in her labour ward . the aim of this study would be to evaluate the perception of labour pains among the parturients , their knowledge and awareness of pain relief during labour , the types of obstetric analgesia available and the outcome of their labour . the outcome of this study would help to strengthen or modify the practice of obstetric analgesia in the labour ward of the hospital . the study was a cross - sectional design involving pregnant women who presented at the labour ward of the ekiti state university teaching hospital , ado - ekiti and the study was carried out between january 2009 and december 2010 . the ekiti state university teaching hospital , ado - ekiti serves as a referral centre for all the specialist and general hospitals in the state . the study involved the use of structured questionnaire administered to parturients within 2 hrs of delivery . the inclusion criteria were all women with singleton pregnancy who presented in labour , whose gestational age was between 37 and 42 weeks , the cervical dilatation was at least 4 cm but not more than 7 cm and gave a verbal consent to participate in the study . the exclusion criteria were all women whose gestational age was less 37 weeks , those with multiple pregnancy , those who were scheduled for elective caesarean section and those who did not agree to participate in the study . the data was collected using structured questionnaires that were administered to consecutive consenting parturients by members of the study team . the research instrument has three sections : the first section obtained information about socio - demographic characteristics of the parturients - age , parity , occupation of parturients and their husbands , tribe and religion ; the second section elicited information about labour history and its outcome - gestational age at presentation in labour , onset of labour , duration of labour , outcome of labour , baby 's outcome , apgar score and birth weight of baby while the third section inquired information about pain perception in labour - pain perception was assessed using a three - point verbal rating scale of mild , moderate and severe , use of analgesia in labour , type of analgesia given , degree of pain relief obtained assessed on a four - point verbal rating scale of no , minimal , moderate and maximum relief , back rubbing and companionship in labour . the first and third sections were obtained directly from the parturients while the second section was obtained from the labour record in their case files . the social class of each parturient was determined by adding the scores from the husband 's occupation and woman 's level of education as described by olusanya et al.8 the data collected was entered into and analyzed using the statistical package for social sciences ( spss ) version 15 . the results were presented using frequency tables and percentages and summarized using relevant descriptive statistics such as means . table 1 showed that the age of the respondents ranged between 17 and 40 years with a mean of 26.50 4.58 . majority of the respondents were yoruba , christians with tertiary education and belonged to the upper social class . sociodemographic characteristics of the respondents table 2 showed that 936 ( 92.5% ) of the parturients had spontaneous labour while 76 ( 7.5% ) of them had induction of labour . majority ( 75.2% ) of the women described their labour pains as severe in nature and 13 ( 1.3% ) of them described it as being mild . three hundred and fifty - seven ( 35.3% ) of the parturients were given analgesia in labour and pentazocine injection was the form of analgesic given to all of them . majority 159 ( 44.5% ) of those that were given analgesic in labour had mild relief of their labour pains while only 65 ( 18.3% ) obtained maximum relief of their pains . obstetric data and labour summary of respondents table 3 showed that 611 ( 93.3% ) of the respondents that were not given analgesia would have wanted the relief of the pains while 44 ( 6.7% ) of them would not want to receive analgesia in labour . the reasons given by those who would have wanted relief of their labour pains were that this would have helped to tolerate the labour experience better and also made them to co - operate with labour room staffs . out of 44 parturients who did want analgesia given in labour , 19 ( 43.2% ) of them want to experience the pains of labour believing that it is natural for a woman to have this , 13 ( 29.5% ) believed that the analgesia would slow down the progress of their labour while 6 ( 13.6% ) of them thought it may affect their baby . majority , 609 of the parturients had their back rubbed by an attendant or relative during labour while 403 of them did not have their back rubbed during labour . out of the 609 women whose back were rubbed in labour , 426 ( 69.9% ) of them reported that this decreased their labour pains while 183 ( 30.1% ) reported that this practice was not helpful in relieving the pains . four hundred and forty - two ( 43.6% ) of the respondents had one form of companion or the other during labour while 570 ( 56.4% ) of them did not have any form of companion during labour . for those who had companion , majority 81% of the companion were the labour ward staff while the rest were their relatives . majority , 79% , of those who had companions reported that this helped them to cope better the labour pains and the whole labour process while the rest 21% of them reported no benefit . majority 75.1% of the respondents were taught about pain relief in labour during their antenatal clinic visits while 24.9% of them were not taught . pain perception and factors relating to it among respondents table 4 showed that the degree of pain relief obtained from the analgesic given during labour statistically influenced the outcome of labour and that of the baby for each parturient with p value of 0.030 and 0.028 , respectively , but not statistically associated with the duration of labour with the p value of 0.809 . pain relief among respondents and the outcome of labour table 5 showed that the verbal pain score described by the respondents was statistically influenced by the antenatal education received by the respondents , higher parity , upper social status and higher educational attainment of the respondents with p value of 0.01 , 0.03 , 0.03 and 0.02 , respectively , but not by their tribe , religion and the type of labour with p value of 0.21 , 0.08 and 0.30 , respectively . the outcome of labour and of the baby were also not statistically influenced by the pain score of the respondents with p = 0.31and 0.11 , respectively . higher level of educational attainment was found out as significant predictor of pain perception among parturients because this exposes them to educational materials that change their orientation that labour pain is a myth and with improved knowledge about the availability of modern methods of pain relief in labour . this is similarly reported by olayemi et al.6 and onah et al.9 but differs from the report of kuti et al.10 that it is not influenced by parity and educational status . however , perception of labour pains did not differ significantly among parturients from the different ethnic groups in this study and similar perception of labour pains have been reported previously by studies from different ethnic regions in nigeria.91011 also interestingly , parturients expressed no difference in pain perception in both spontaneous and induced labour which is in contrast to reports by olayemi et al.6 and onah et al.9 that induced and augmented labour are more associated with a higher perceived pain than spontaneous labour . despite the high level of awareness of pain relief in labour among the parturients having been taught in the antenatal clinic , only 35.3% of them had analgesic given in labour and pentazocine injection was the only analgesic given . the analgesic used in this centre may be ineffective in most cases as only very few parturients had maximum relief of their pains , the outcome of labour in these parturients- majority had spontaneous vaginal deliveries- and their babies were still better . this reflects the low level of practice of obstetric analgesia in this centre despite the availability of more effective methods of pain relief but the well documented benefits of the obstetric analgesia to the mother and their babies have been demonstrated in this study.4 this is similarly reported by onah et al.9 many of the parturients who were not given analgesic desired to have pain relief in labour . this desire for analgesic in labour was significantly associated with the higher educational level , social status and the antenatal education received among the parturients . most educated women and those of upper social class live a less physically stressful life that would make them less accustomed to pains and suffering compared to the less educated women . they are also well informed about the documented advantages of pain relief in labour.6 this is contrast to the less educated women who do not desire pain relief because labour pain is considered as natural and a mark of womanhood and that drugs used may affect the progress of their labour and the outcome of their babies.912 parturients who had companions with them and also had their back rubbed during labour reported a decrease in their labour pains perception and that this helped them to cope better with the labour process . this was similarly reported in previous studies by hodnet et al.7 and abushaika et al.12 in places where doulas ( lay women who are trained in labour support ) are available , continuous labour support provided by them has been shown to consistently decrease the use of obstetric intervention.13 this practice should be encouraged since companionship in labour is one of the components of the active management of labour as opposed to what obtains in most centres in nigeria where relatives even their spouses are not allowed to stay with parturients in labour.10 this study has shown that many of the parturients described the labour process as being painful with only few having access to obstetric analgesia while majority would rather want a pain - free labour by accepting analgesics in labour . it is therefore essential that the practice of obstetric analgesia be stepped up with the introduction of parenteral opioids , epidural and inhalational analgesia into obstetric practice in our health care facilities in nigeria.39 in developed countries , obstetric anaesthesiology is a well - established subspecialty that takes care of analgesic need of parturients in labour to ensure a pain - free labour but in nigeria , the derth of anaesthesiologists has been a major challenge facing subspecialisation among the few available even in well - established tertiary health centres and teaching hospitals . government should encourage training of more doctors in the field of obstetric anaesthesiology to administer this in the labour wards by providing additional incentives for them . the support of health care personnel in labour , provision of infrastructure and maternal involvement in decision making are clearly central in achieving a positive child birth experience . it is , therefore , imperative that attention is paid to this not only in this centre but in nigeria so that our parturients would enjoy the benefits of a pain - free labour . this would give our women the dignity they deserve and feel honored when performing their god giving role and reduce adverse maternal and fetal outcomes . limitations to this study include the subjective assessment of labour pains and the relief obtained from analgesia by the parturients rather than by an objective method . the study was also limited by the fact that other methods of pain relief in labour are not readily available in this centre . my acknowledgment goes to all pregnant mothers that participated in this study and to the registrars , house officers and labour ward staff who helped in administering the questionnaires .

introduction : labour pain is a universal experience . relief of labour pains and companionship in labour are important aspects of quality of care in labour.objectives:to evaluate perception of labour pains among parturients , their knowledge and awareness of pain relief during labour , the types of obstetric analgesia available and the outcome of their labour at the ekiti state university teaching hospital , ado-ekiti.materials and methods : a cross - sectional study using questionnaire administered to pregnant women between 37 and 42 weeks gestational age in labour ward of the hospital.results:the study revealed that 75.2% of the parturients experienced severe labour pains and 35.3% of them received analgesia in labour with pentazocine injection being the only analgesic used . only 18.3% had maximum relief of their pains . parturients with increasing parity , higher social class and educational attainment and who had antenatal education on labour pains were associated with severe perception of labour pains with p values of 0.03 , 0.03 , 0.02 and 0.01 , respectively . parturients who were given pentazocine injection for pains and had relief in labour had more spontaneous vaginal deliveries , p = 0.030 and better outcome for their babies , p = 0.028 . majority of the women reported that the practice of companionship and back rubbing in labour helped them to cope better with the labour process.conclusion:most women desire relief of pains of labour but the practice is still suboptimal in this centre . efforts should be made towards developing the practice of obstetric analgesia and companionship in labour in this environment .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION

enantiomerically pure alcohols and amines constitute important synthetic building blocks and key targets in the manufacturing of a wide range of chemical products , such as agrochemicals , food additives , fragrances , and pharmaceuticals . consequently , significant efforts have been dedicated to the enantioselective synthesis of these compounds , including catalytic protocols for carbon - heteroatom bond formation , hydrogenations of ketones / imines , nucleophilic addition to carbonyl compounds , and kinetic resolution ( kr ) . of these methods , enzymatic kr of racemic mixtures is the most common way to access enantiomerically pure alcohols and amines on an industrial scale , owing to its high performance in terms of activity and selectivity . for the kr of these compounds , the process can be made either ( r)- or ( s)-selective depending on whether a lipase or a serine protease is chosen as the enzymatic resolving agent . in most of the reported kr protocols of alcohols and amines , the enzyme resolves the racemic substrate through selective acylation of one of its enantiomers , which allows for the isolation of the enantiopure alcohol or amine using conventional purification techniques . the acyl group being transferred to substrate by the enzyme comes from a so - called acyl donor , which is added to the reaction in at least equimolar amounts in regard to the substrate . since this transesterification process is fully reversible , highly activated esters or enol esters are commonly employed as acyl donors to push the reactions toward the formation of the acylated product . unfortunately , enzymatic kr , as all other resolution methods , suffers from the limitation that the maximum theoretical yield is only 50% . an efficient way to overcome this drawback and achieve a theoretical yield of 100% is to combine the resolution process with in situ racemization in a so - called dynamic kinetic resolution ( dkr ) ( scheme 1 ) . to date , a variety of protocols for the racemization of alcohols and amines have been developed , and these involve for example acid / base catalysts , transition - metal complexes , metal nanoparticles , or enzymes . however , the design of a successful dkr system is far from simple , given that the following requirements must be fulfilled : ( i ) the kr must display a sufficient enantioselectivity ( e value = kfast / kslow 20 ) ; ( ii ) the enzyme and the racemization catalyst must be compatible with one another ; ( iii ) the rate of racemization ( krac ) must be at least 10 times faster than the enzyme - catalyzed reaction of the slow reacting enantiomer ( kslow ) ; and ( iv ) the racemization catalyst must not react with the product formed from the resolution . among these requirements , the compatibility between the enzyme and racemization catalyst is generally the critical issue , since these catalysts often operate optimally under very different condition . it is also common that the racemization catalyst interferes with the enzymatic resolution or that the enzyme and its accompanying additives ( e.g. , surfactants and stabilizers ) have an inhibitory effect on the racemization catalyst . as a result of this compatibility issue , the identification of reaction conditions that enable both high enantioselectivity of the kr and efficient racemization has been a reoccurring challenge within the field of dkr . this perspective summarizes the key features of the extensive research that has been dedicated to the chemoenzymatic dkr of alcohols and amines during the past two decades . the aim is to cover both the biological and chemical aspects of the dkr , by discussing topics ranging from the design of enzyme - compatible racemization catalysts to enzyme engineering . further , we wish to point out the current state - of - the - art dkr protocols and their respective limitations , in an attempt to highlight novel avenues for future research . in 1996 , williams reported the dkr of an allylic acetate derivative by the combinative use of pseudomonas fluorescens lipase and pdcl2(mecn)2 to give the corresponding allylic alcohol in 81% yield and 96% ee after 19 days ( scheme 2a ) . in this reaction , the acetylated alcohol was deracemized by the coupling of a lipase - catalyzed ester hydrolysis to a racemization proceeding via palladium(ii)-mediated 1,3-acetate shift . although , the reaction proceeded at an impractical rate , this seminal study was an important first step that demonstrated the possibility of combining transition metal catalysis with enzyme catalysis for achieving dkr . in a subsequent study , williams and co - workers developed a method for the dkr of secondary alcohols lacking adjacent c = c double bonds by utilizing racemization catalysts operating through a reversible hydrogen - transfer mechanism . among the studied catalysts , rh2(oac)4 gave the best results and was combined with lipase - catalyzed transesterification , affording ( r)-1-phenylethanol with 60% conversion and 98% ee ( scheme 2b ) . unfortunately , this method suffers from several critical drawbacks , such as a low conversion of the overall dkr and the necessity of both o - phenanthroline and acetophenone as additives for efficient racemization . however , despite the disadvantages of this particular dkr system , this study was important since it demonstrated the potential of using metal catalysts operating via transfer hydrogenation mechanisms as a general method for racemizing alcohols . this work became a great source of inspiration for subsequent catalyst design , triggering the development of a great number of racemization catalysts based on different transition metals , which all functioned through different transfer hydrogenation mechanisms ( vide infra ) . unfortunately , it is beyond the scope and purpose of this perspective to provide an in - depth mechanistic discussion for these transfer hydrogenative racemization processes , and thus we kindly refer interested readers to some recent reviews that cover this topic . following the pioneering work of williams , the group of bckvall developed the first practical system for the dkr of secondary alcohols , which involved candida antarctica lipase b ( calb ) immobilized on acrylic resin ( also known under the trade name novozyme-435 ) and shvo s dimeric ruthenium complex 1 ( scheme 3 ) . this protocol was found to be compatible with a wide range of aliphatic and benzylic alcohols , providing the corresponding ( r)-acetates in high yields and ee s . a drawback of this dkr system is that the shvo complex 1 requires 70 c to efficiently split into the two monomeric species 1a and 1b , which mediates racemization through an outer sphere redox mechanism . because of this heat activation , the shvo complex 1 can only be combined with thermostable lipases , which limits the number of enzymes that can be used in the dkr . for example , sensitive serine proteases that exhibit ( s)-selectivity according to kazlauskas rule can not be used together with 1 . another issue of the shvo complex 1 was that it had to be used together with activated aryl esters such as p - chlorophenyl acetate , since simpler alkenyl acetates as acyl donors were found to interfere with the racemization and lead to substantial formation of ketone side products . despite these limitations , complex 1 has been successfully combined with several lipases for the dkr of -azido alcohols , benzoins , -halo alcohols , heteroaryl ethanols , hydroxylalkanephosphonates , -hydroxy amides , hydroxyl acid esters , hydroxyl aldehydes , -hydroxy alkyl sulfones , and -nitrile alcohols . in addition , complex 1 has been applied as racemization catalyst in the dkr of -substituted primary alcohols , where racemization occurs through enolization of the intermediate aldehydes . subsequent research aimed at discovering more active catalysts that could efficiently racemize alcohols under milder reaction conditions , enabling the use of a wider range of enzymes for dkr . the group of park reported on ( -indenyl)rucl(pph3)2 , 2 , as an efficient racemization catalyst for alcohols at room temperature , which unlike 1 only produced negligible amounts of ketone byproduct . however , a severe drawback of complex 2 is that it requires koh to display catalytic activity , which is detrimental for applications in dkr as the base can hydrolyze the product acetates and also cause enzyme deactivation . it was later found that complex 2 could instead be activated by o2 and et3n , but unfortunately a higher reaction temperature ( 60 c ) was needed for efficient racemization in this case . in the latter study , complex 2 was successfully combined with pseudomonas cepacia lipase ( ps - c ) for the dkr of a small scope of simple secondary alcohols ( scheme 4 ) . the initial success achieved by combining enzymes with ruthenium - based complexes strongly influenced the subsequent research within the field of dkr , which resulted in the development of a number of protocols utilizing different transfer hydrogenation - type ruthenium complexes as the racemization catalyst . however , the first main breakthrough came in 2002 when the group of park prepared the monomeric ruthenium aminocyclopentadienyl complex 3 and demonstrated that it could efficiently racemize secondary alcohols at room temperature . unlike complex 1 , this racemization catalyst did not require any heat activation but was activated by kotbu . in contrast to koh , kotbu shows a higher compatibility with most dkr systems commonly used . complex 3 was successfully paired with novozyme-435 for the synthesis of a variety of functionalized aliphatic and benzylic ( r)-acetates in high yields and ee s at room temperature ( scheme 5 ) . an advantage of this dkr protocol was that the cheap and readily available isopropenyl acetate could be used as the acyl donor instead of activated esters , such as p - chlorophenyl acetate . unfortunately , the dkr reactions were found to progress slowly , requiring reaction times of up to 7 days , which is in sharp contrast to the separate racemization and kr reactions , which were generally complete within a few hours . this significant difference in efficiency between the dkr and the separate reactions suggests that complex 3 and calb are not fully compatible with one another , leading to partial deactivation of both catalysts . because of its good racemization activity at room temperature , complex 3 could also be combined with the more sensitive protease subtilisin carlsberg , which opened up for ( s)-selective dkr protocols of secondary alcohols . in addition , the group of kim and park has in a recent study demonstrated that complex 3 can be used together with ionic surfactant - stabilized burkholderia cepacia lipase for the dkr of allylic secondary alcohols at room temperature in excellent yields and ee s . shortly after the development of 3 , the group of bckvall prepared a related monomeric ruthenium pentaarylcyclopentadiene complex 4 , which proved to be a highly efficient catalyst that managed to fully racemize enantiomerically pure 1-phenylethanol within 10 min , even at catalyst loadings as low as 0.5 mol % . this racemization catalyst displayed many similarities to complex 3 , both in terms of structure and activation method ; however , the absence of an amino - functionality in the cyclopentadienyl ligand resulted in an improved compatibility with enzymes . thus , combination of complex 4 with calb ( novozym-435 ) afforded a fast dkr of secondary alcohols , e.g. , 1-phenylethanol was transformed to its acetate in high yield and > 99% ee in 3 h. complex 4 paved the way for a new generation of dkr and dynamic asymmetric transformation ( dykat ) protocols involving several different enzymes , many of which had not been possible to incorporate previously . dkr protocols utilizing complex 4 have been applied to the deracemization of a wide range of functionalized secondary alcohols in excellent yields and ee s , including aliphatic alcohols , allylic alcohols , chlorohydrins , diols , homoallylic alcohols , and n - heterocyclic 1,2-amino alcohols ( figure 1 ) . in the case of the dkr of 1-phenylethanol , a large scale reaction with only 0.05 mol % of complex 4 was carried out on a 1 mol - scale to furnish 159 g ( 97% yield ) of the corresponding ( r)-acetate in 99.8% ee . dkr and dykat systems involving complex 4 have also been employed in the synthesis of several biologically relevant molecules and pharmaceuticals . as with complex 3 , the racemization activity of 4 at room temperature allowed it to be combined with subtilisin carlsberg for ( s)-selective dkr protocols . for some chlorohydrins and for alcohols containing a distant olefin group , such as homoallylic alcohols and 5-hexen-2-ol , racemization occurs significantly slower with ruthenium - based catalysts such as 4 , which calls for increased reaction temperatures in the dkrs . for these substrates , further research into more efficient racemization protocols one promising way to achieve a more efficient racemization of chlorohydrins could perhaps be to match the electronic properties of catalyst and substrate as recently reported by the group of bckvall . in this study , it was found that a highly electron - deficient analogue of complex 4 gave a 1030 times faster racemization of chlorohydrins than the standard catalyst . the authors ascribed the improved racemization rate to the higher efficiency of the electron - deficient catalyst in abstracting the hydride from this electron - deficient class of substrates . in recent years , several analogous enzyme - compatible racemization catalysts based on the cyclopentadienyl ruthenium core have been synthesized ( figure 2 ) . particularly , the group of kim and park has made several key contributions to the field of alcohol dkr by developing ruthenium - based racemization catalysts exhibiting improved stability and broader scope . one of these catalysts is the benzyloxy derivative 5 that has been used as a racemization catalyst in the dkr of a number of aliphatic and benzylic secondary alcohols under air atmosphere . the possibility to run the reactions open to air constitutes a significant practical improvement compared to the previous systems involving catalysts 3 and 4 , which both require the use of dry and inert conditions to prevent catalyst deactivation . another advantage of the benzyloxy motif was that it could be exploited as a handle for linking 5 onto polystyrene to create a heterogeneous version . interestingly , the dkrs involving the polymer - bound catalyst 6 gave comparable results in terms of yield and ee to those employing homogeneous 5 , demonstrating that heterogenization of the catalyst had a negligible effect on the racemization activity . moreover , catalyst 6 exhibited good recyclability that allowed it to be reused three times in the dkr of 1-phenylethanol , where the ( r)-acetate could be obtained in 95% yield and 99% ee over all cycles . in a subsequent study , catalyst 6 was employed in the key step of the synthesis of the enantiomerically pure pharmaceutical ( )-rivastigmine . kim , park , and co - workers have also developed another air - stable analogue of complex 4 by replacing one of its carbon monoxide ligands with pph3 . the resulting catalyst 7 could be activated at room temperature by ag2o and used together with novozyme-435 for the dkr of a small set of aliphatic and benzylic alcohols in excellent ee s . recently , the group of kim and park reported on an interesting ruthenium complex 8 containing an acyl substituted cyclopentadienyl ligand . this catalyst allowed for a significant extension of the scope of enantiomerically pure secondary alcohols that can be accessed through chemoenzymatic dkr . by combining catalyst 8 with ionic surfactant - coated burkholderia cepacia lipase , the dkr of a variety of secondary alcohols was accomplished at 2560 c , including -arylpropargyl alcohols , b(pin)-substituted benzylic alcohols , -chloro alcohols , and tms - propargyl alcohols . the groups of leino and kanerva reported on the preparation of the related pentabenzylcyclopentadienyl ruthenium complex 9 , which displayed comparable activity and scope of utility to that of complex 4 . however , an advantage of 9 is that its benzyl - substituted ligand can be conveniently synthesized on a large scale from the simple and cheap starting materials cyclopentadiene and benzyl alcohol . this can be compared to the syntheses of complexes 1 and 3 - 8 , which require the significantly more expensive precursor tetraphenylcyclopentadienone . another useful racemization catalyst , which does not require the use of strong alkoxide bases , was very recently reported by nolan and co - workers . the cationic ruthenium indenyl complex 10 is efficiently activated by the mild base , k2co3 , and was successfully combined with novozyme-435 for the dkr of a variety of secondary alcohols in high yields and ee s at room temperature . recently , the group of martn - matute showed that a ruthenium catalyst , formed in situ from the readily available complex [ ru(p - cymene)cl2]2 and the ligand 1,4-bis(diphenylphosphino)butane , could be employed in combination with lipase tl from pseudomonas stutzeri for the efficient dkr of -hydroxy ketones at room temperature . the dkr of these substrates provides straightforward access to a variety of functionalized molecules , such as enantiomerically pure amino alcohol and diol derivatives . so far this perspective has mainly described protocols employing ruthenium - based racemization catalysts , but it is important to also highlight the work on secondary alcohol dkr that involves other metals . for instance , feringa , de vries , and co - workers have developed a procedure for synthesizing enantiomerically pure epoxides in one step from the corresponding chlorohydrins , by utilizing the cationic iridacycle 11 together with a doubly mutated haloalcohol dehalogenase ( hhec ) in a biphasic system comprising toluene and 50 mm hepes buffer ( scheme 6 ) . as with the monomeric ruthenium - based racemization catalysts 3 - 9 , iridacycle 11 was activated by kotbu , enabling efficient dkr at room temperature . iridacycle 11 displayed an intriguing complementary reactivity to the ruthenium systems by exhibiting a significantly higher racemization activity and selectivity toward chlorohydrins compared to conventional benzylic secondary alcohols . another iridium - catalyzed protocol for the base - free dkr of nonfunctionalized aliphatic and benzylic secondary alcohols was disclosed by marr et al . this dkr utilized a series of piano - stool-type iridium nhc complexes together with calb . a general topic of concern regarding ruthenium- and iridium - based catalytic systems for racemization is the relatively high cost and low natural availability of these metals . therefore , efforts have been made to develop more cost - effective and readily accessible metal catalysts . an example addressing this requirement is the alme3/binol / calb system designed by berkessel et al . , which was used for the dkr of both aliphatic and benzylic alcohols at room temperature . in addition , a number of vanadium - based catalytic protocols for the dkr of alcohols have been developed during the past decade . akai and co - workers demonstrated that the oxyvanadium(v ) complex [ vo(osiph3)3 ) ] could racemize secondary allylic alcohols through 1,3-transposition of the hydroxyl group under mild reaction conditions . accordingly , this catalyst was found to be compatible with several lipases , such as burkholderia cepacia lipase , novozyme-435 and pseudomonas fluorescens lipase , which allowed for dkr of a wide range of linear and cyclic allylic secondary alcohols . moreover , the developed methodology could be used to transform a stereoisomeric mixture of dienols into a single dienyl acetate product in excellent yield and ee ( scheme 7 ) . the authors also prepared heterogeneous analogues of this oxyvanadium(v ) catalyst , which were immobilized on both a polymer and a mesoporous silica . the latter heterogeneous catalyst proved to be recyclable over six cycles without any loss in activity , and furthermore , it was capable of racemizing benzylic , heteroaromatic and propargylic alcohols . the ability of this catalyst to mediate the racemization of substrates lacking the allylic alcohol motif indicates that it can also operate through a more general dehydrative mechanism proceeding via a carbocation intermediate . another heterogeneous protocol for the dkr of secondary alcohols involving vanadium catalysis was reported by wuyts et al . in this system , voso4 was combined with novozyme-435 to achieve deracemization of several benzylic alcohols in octane at 80 c . there are also a number of reports on the use of heterogeneous acids and zeolites as racemization catalysts together with lipases for dkr of secondary alcohols . however , the major limitation of most of these protocols is that they can only racemize alcohols through a dehydration mechanism , which limits their scope to substrates that can form stable carbocations . furthermore , many of these systems suffer from reduced yields of the desired dkr products due to substantial formation of elimination side products . in sharp contrast to secondary alcohols , tertiary alcohols are a significantly more cumbersome class of substrates for which there exist no practical dkr protocols . although , there are a few enzymes that can resolve tertiary alcohols , it has proven difficult to couple these kr processes to in situ racemization . since the quaternary stereocenter of tertiary alcohols lacks a hydride substituent , it is not possible to utilize any of the transfer hydrogenation - type racemization catalysts . thus , the list of available racemization catalysts for tertiary alcohols is primarily limited to those operating through dehydrative mechanisms ( e.g. , lewis acids or vanadium catalysts ) proceeding via the formation of a tertiary carbocation . the latter carbocation intermediate is formed much more readily than the corresponding secondary one , which should facilitate the racemization of tertiary alcohols . the development of a general and practical dkr protocol for tertiary alcohols would be considered as an important milestone within the field of asymmetric synthesis , given the high prevalence of this structural motif in natural products and pharmaceuticals . as with alcohols , there are a variety of efficient methods for obtaining enantiomerically pure amines by the use of enzymatic kr . however , the available dkr protocols are drastically fewer in number due to the lack of efficient amine racemization catalysts . the main reason for the difficulty of racemizing amines is that they can act as strong coordinating ligands , which may lead to inhibition or even complete deactivation of the metal catalysts . thus , high temperatures are generally required to disrupt this undesired coordination and promote the racemization reaction . as previously discussed , the use of elevated reaction temperatures is undesirable from a dkr perspective , since it restricts the set of enzymes that can be employed . an additional challenge associated with the racemization of amines is that the generated imine intermediate is highly reactive and can thus take part in several side reactions , which reduces the yield of the desired dkr product . for example , the imine is prone to undergo hydrolysis into the corresponding ketone in the presence of water . the imine intermediate can also be subject to nucleophilic attack by another amine molecule to produce an aminal , which upon elimination of an ammonium ion forms a secondary imine that can be further reduced to a secondary amine byproduct . it has been found that both of these side reactions are usually favored by an elevated temperature , which further highlights the importance of efficient and mild amine racemization protocols . the first dkr of an amine was reported by reetz and schimossek in 1996 , where resolution of 1-phenylethylamine was accomplished by coupling calb - catalyzed amine acylation to pd / c - catalyzed racemization . unfortunately , the dkr reaction , which was performed in triethylamine at 5055 c using ethyl acetate as the acyl donor , was found to proceed slowly , and despite a reaction time of 8 days , it only gave a moderate conversion of 60% . following this work , the group of bckvall demonstrated that the shvo dimer 1 can be used as an efficient racemization catalyst for primary amines at 110 c . as a result of the high temperature , the enzymatic resolution was run separately at a lower temperature , and therefore the racemization and resolution had to be done stepwise . however , this problem was later circumvented by changing to the methoxy - substituted shvo analogue 12 , which enabled efficient racemization at 90 c . by using complex 12 together with novozyme-435 , the one - pot dkr of several aliphatic and benzylic primary amines was achieved in high yields and excellent ee s ( scheme 8) . this protocol enabled the dkr of 1-phenylethylamine to be performed on a multigram scale , with a low catalytic loading ( 1.25 mol % ) and with a substrate concentration of up to 0.9 m , affording the corresponding ( r)-amide in good isolated yield and 98% ee . a noteworthy feature of the dkr protocol involving 12 was that isopropyl acetate could be used as the acyl donor . although , this acyl donor may seem as the ideal choice given its low price and high availability , the use of carboxylic esters as acylating agents is generally undesired in amine dkr as they give an amide product that requires harsh reaction conditions to be reconverted back to the amine . commonly , strong acids and elevated temperatures are required to cleave the stable amide bond , which may be detrimental for substrates containing sensitive functional groups . to address this issue , bckvall and co - workers developed an improved procedure for the dkr of both aliphatic and benzylic primary amines involving complex 12 and calb that worked efficiently with dibenzyl carbonate as the acyl donor . in contrast to the amide functionality , the installed benzyloxy carbonyl group can be easily removed under mild reaction conditions through pd - catalyzed hydrogenolysis . together , the broad substrate scope involving both aliphatic and benzylic primary amines , the possibility of using carbonate - based acyl donors and the scalability make this catalytic protocol one of the most practical methods for the dkr of primary amines available to date . the group of bckvall has also demonstrated that complex 12 can be used in combination with the related enzyme candida antarctica lipase a ( cala ) immobilized on siliceous mesocellular foam ( mcf ) for the dkr of -amino esters . inspired by the seminal findings of reetz and schimossek , several research groups continued to study heterogeneous racemization protocols based on palladium for application in amine dkr . the first steps toward a practical dkr method for amines using this strategy were taken by jacobs and co - workers with their investigation on how alkaline earth supports affected the racemization activity of immobilized pd particles . among the tested catalysts , pd on baso4 was found to exhibit the highest activity and selectivity . the dkr with this racemization catalyst was performed at 70 c under 0.1 bar of h2 , using novozyme-435 as the resolving agent and either ethyl acetate or isopropyl acetate as the acyl donor . under these reaction conditions , a range of benzylic primary amines were converted into the corresponding ( r)-amides in high yields and ee s within 2472 h ( scheme 10 ) . andrade et al . later demonstrated that this protocol can also be applied for the dkr of selenium - containing benzylic primary amines with good results . however , a significant drawback of this dkr system is that it is limited mainly to benzylic amines , while aliphatic primary amines generally react too slowly . the only aliphatic amine that was tolerated by this system was 1-methyl-3-phenylpropylamine , which contained a distant aryl group that was most likely the reason for the success . despite , the presence of an aromatic moiety in the structure , this aliphatic amine was found to racemize significantly more slowly than the benzylic substrates and thus the corresponding dkr required both elevated temperatures and longer reaction times to give satisfactory results . in a subsequent study , the group of de vos demonstrated that the activity and selectivity of the pd / baso4 and pd / caco3 catalysts in the racemization of primary amines could be improved by using microwave irradiation as an alternative heating method . the reason for this phenomenon is that metal clusters are capable of efficiently absorbing microwave irradiation , which results in the generation of so - called hot - spots that can reach a temperature that exceeds that of the surrounding reaction media . the use of microwave irradiation was also found to lead to faster dkrs , although the ee s of these reactions were generally lower than those performed with conventional heating in an oil bath , because of a more facile background chemical acylation under the employed microwave conditions . a useful dkr method involving heterogeneous palladium has been reported by kim , park and co - workers ( scheme 9 ) . in this protocol , nanoparticulate pd immobilized on alo(oh ) is employed as the racemization catalyst together with novozyme-435 . this catalyst combination proved effective in the dkr of a range of benzylic primary amines , enabling the preparation of the corresponding ( r)-amide products in high yields and excellent ee s . however , in line with the protocol developed by jacobs and co - workers , this catalytic system required significantly harsher reaction conditions for the dkr of aliphatic substrates ( 12 mol % pd , 100 c and 1 atm h2 ) . interestingly , both the pd nanocatalyst and the enzyme could be recycled eight times in the dkr of 1-methyl-3-phenylpropylamine without any observable decrease in either conversion or ee . in a subsequent study , the group of kim and park extended the scope of this method to also include -amino amides . in addition , bckvall and co - workers have applied the pd / alo(oh ) catalyst in combination with cala - mcf for the dkr of -amino esters . xu et al . reported on the preparation of a heterogeneous racemization catalyst based on pd immobilized on a layered double - hydroxide - dodecyl sulfate anion support and demonstrated that it could be used with novozyme-435 for the dkr of benzylic primary amines at 55 c . unfortunately this protocol suffered from several drawbacks , such as high catalyst loadings , dilute substrate concentrations , and the need of the activated ester 4-chlorophenyl valerate as acyl donor . recently , the group of bckvall also developed a palladium - based heterogeneous racemization catalyst , consisting of 1.53.0 nm - sized pd nanoparticles immobilized on aminopropyl - functionalized mcf ( amp - mcf ) . this pd nanocatalyst ( pd - amp - mcf ) exhibited high activity in the racemization of 1-phenylethylamine , and moreover it displayed good enzyme - compatibility that allowed it to be used in dkr . the pd - amp - mcf catalyst was combined with novozyme-435 for the dkr of a range of primary benzylic amines at 70 c , producing the corresponding ( r)-amides in high yields and excellent ee s ( scheme 10 ) . furthermore , by increasing the catalytic amount of palladium from 1.25 to 5.0 mol % it was possible to maintain an efficient racemization even at 50 c , which allowed the catalyst to be used in a dkr of 1-phenylethylamine with the sensitive enzyme amano lipase ps - c1 ( burkholderia cepacia lipase immobilized on ceramic beads ) . remarkably , this is the first time that amano lipase ps - c1 has been successfully utilized in a dkr of an amine . it is also important to highlight the fact that the amount of pd nanocatalyst used in these dkr reactions with reasonably short reaction times is lower than previously reported for primary amines and that the reactions are run at a substrate concentration of 0.4 m , which is significantly higher than that used in previously reported systems . other practical advantages of the pd - amp - mcf were that it displayed high stability and low leaching , which allowed it to be recycled up to four times in the dkr of 1-phenylethylamine without any observable decrease in performance . as with other palladium - based systems , this dkr system does not work well for aliphatic amines . the group of li has also studied this system for the dkr of primary amines ; however , they used a slightly different version of the pd - amp - mcf catalyst that was impregnated with k2co3 and contained a lower palladium loading than the one used by bckvall and co - workers ( 2.0 versus 8.0 wt % pd ) . even though this alternative protocol allowed for an efficient and selective dkr of several amines , it is difficult to compare its performance to the system published by the group of bckvall , as it was studied under very different reaction conditions involving increased enzyme loadings and significantly lower substrate concentrations . the mcf material that was employed by the group of bckvall and li to support the pd nanoparticles has also been used to immobilize cala . with this versatility of the mcf in mind , the group of bckvall explored the possibility of co - immobilizing pd nanoparticles and an enzyme into the cavities of this support . this was done by first preparing the pd - amp - mcf catalyst with a moderate loading of palladium to leave a number of free aminopropyl groups for the enzyme , then functionalizing the free aminopropyl groups of the support with glutaraldehyde , and finally exploiting the aldehyde groups as linkers for the anchoring of calb . by this co - immobilization strategy , a metalloenzyme - resembling bifunctional catalyst was obtained that can perform both racemization and kr ( figure 3 ) . this hybrid catalyst was evaluated in the dkr of 1-phenylethylamine using ethyl methoxy acetate as the acyl donor under 1 atm . of h2 at 70 c . under these conditions the desired ( r)-amide product was obtained in 99% yield and 99% ee within 16 h. interestingly , the reaction involving the hybrid catalyst was found to proceed faster than that of separately supported pd(0)-amp - mcf and calb - mcf , highlighting that the close proximity of the two catalysts increases the rate of the dkr . the hybrid catalyst could be recycled , but unfortunately it was found to exhibit diminished activity from the third cycle as a result of partial enzyme denaturation caused by the hydrophilic silica support surface . dkr of an amine with a bifunctional biomimetic catalyst in which pd nanoparticles and a lipase ( calb ) are co - immobilized in mcf . it is not only palladium- and ruthenium - based racemization catalysts that have been employed in amine dkr systems . the group of de vos showed that both raney ni and raney co catalysts could be combined with novozyme-435 for the dkr of primary amines . unfortunately , these dkrs were found to proceed slowly , and even though they were performed at 7080 c for 25 days , the conversions and ee s were generally low . on the other hand , this catalytic system displayed an interesting preference for aliphatic primary amines , allowing for a faster dkr of these substrates . surprisingly , by following the ee of the starting material throughout the reaction , it was established that the long reaction time was due to a rate - limiting kr process . normally , the enzymatic kr of primary amines should proceed fast at these high temperatures . these results therefore suggest that the raney metal catalysts have an inhibitory effect on the enzyme . it was found that enzyme poisoning was caused by leaching of cobalt and nickel ions and that this problem could be circumvented by performing the kr and racemization in separate pots in a successive manner . however , this greatly diminished the practical utility of this dkr protocol . another interesting dkr system based on homogeneous [ ircp*i2]2 and candida rugosa lipase was reported by page and co - workers and was used for the deracemization of a secondary amine on a multigram scale . the dkr of secondary amines is significantly more challenging , because the extra substituent on the nitrogen brings additional steric bulk that prevents the substrate from being accepted by the enzyme . despite the fact that several secondary amines could be efficiently racemized by the iridium catalyst , the authors only managed to construct a working dkr for one isoquinoline derivative ( scheme 11 ) . in nature , the stereoinversion of some amino acids is carried out by a family of racemases that utilize pyridoxal phosphate as the catalytically active prosthetic group . these enzymes operate through a so - called schiff base - type mechanism , where the catalytically active pyridoxal phosphate group reacts with an amino acid to produce an imine intermediate , from which racemization occurs via a base - mediated enamine - imine interconversion . inspired by this class of natural racemases , felten et al . developed a synthetic active - site analogue by complexating zn(otf)2 to picolinaldehyde this racemization catalyst was successfully combined with the enzyme alacase ( another name for subtilisin carlsberg ) for the dkr of a small series of -branched amino acids in high enantioselectivity at room temperature . recently , several reports on metal - free methods for the dkr of amines have appeared . the group of gil and bertrand has shown that racemization of amines can be achieved by the use of in situ generated sulfanyl radicals and that it is possible to couple this process to enzymatic acylation . although this method has so far only been applied to the dkr of simple primary amines containing no or little functionality , it is promising since the racemization occurs under mild reaction conditions , which enables the use of sensitive proteases . another well - established method to racemize amino acid derivatives containing acidic -protons is to employ a base that is sufficiently strong to deprotonate these substrates . for example , tessaro and co - workers have successfully utilized the organic base 1,8-diazabicycloundec-7-ene ( dbu ) as a racemization catalyst in the mild dkr of several amino acid derivatives by combining it with subtilisin - catalyzed thioester hydrolysis . so far , this perspective has focused almost entirely on the design of efficient racemization catalysts as a means to broaden the scope of the dkr methodology . however , it is important not to overlook the considerable amount of work that has been done to improve enzymes as biocatalysts . the properties of an enzyme can be improved by immobilization , cross - linking , surfactant - stabilization , or enzyme engineering / directed evolution . the latter topic has been greatly propelled by advances in the field of molecular biology and genetic engineering , which has led to the development of new recombinant technologies that makes it possible to incorporate tailor - made dna fragments into organisms , such as escherichia coli and pichia pastoris , and use them as hosts for the expression of mutant enzymes with novel properties . unfortunately , it is beyond the scope of this perspective to summarize all key contributions to this vast research area , and therefore we kindly refer interested readers to a number of excellent recent reviews that cover this topic thoroughly . instead , our aim is to describe some selected techniques that we believe have the potential of making an impact on the field of alcohol and amine dkr . a classical way to improve the thermostability of enzymes and thus make them available for a wider range of dkr protocols is to immobilize them on a heterogeneous support . in fact , the majority of the commercially available enzymes that are used for dkr today are already supported on various types of carriers , including ceramic beads , diatomaceous earth , ionic liquids , resins , and silicas . in addition to improving the thermostability , the immobilization of enzymes also lead to several practical advantages , such as easier handling , simpler separation , and possibilities of recycling . another intriguing method to improve the general performance of enzymes is to polymerize them into so - called cross - linked enzyme aggregates ( cleas ) by the use of a bifunctional cross - linking agent . typically , glutaraldehyde is used for this purpose as it can react with free lysine residues on the surface of two neighboring enzyme molecules and covalently link them together through stable schiff - base - type bonds . this cross - linking leads to the formation and precipitation of large insoluble enzyme aggregates , which can be easily separated by either centrifugation or filtration . remarkably , these cleas often display comparable catalytic activity to that of the free enzyme , suggesting that the enzyme is locked in its active conformation in the aggregate and that diffusion of substrate into the enzyme s active site is not significantly hindered . furthermore , this aggregation strategy has been shown to lead to dramatic improvements of the stability of the enzyme toward elevated temperatures , hostile solvents , and autoproteolysis . these improvements are a direct consequence of the decrease in flexibility , which suppresses deactivation through denaturation . an additional advantage of the clea methodology is that it combines the processes of enzyme purification and immobilization into a single operation . consequently , it is possible to apply this method directly on crude extracts instead of pure enzyme solutions . today , a substantial number of cleas based on lipases and proteases have been developed and successfully used for the preparation of enantiomerically pure alcohols and amines . despite these achievements , there is to the best of our knowledge only one group , who has so far studied clea for applications in dkr . tessaro and co - workers studied the advantages of the clea methodology in dkrs of amino acid derivatives involving subtilisin carlsberg and dbu . here , subtilisin carlsberg was found to exhibit a higher tolerance to dbu when it was turned into a clea , which enabled a more efficient dkr . the promising results from this work suggest that the clea methodology could find applications in other dkr protocols as well . for example , in the case of amine dkr , where the racemization catalysts require elevated reaction temperatures , there is a need for thermostable enzymes and here the use of cleas could be advantageous . an alternative approach to prepare heterogeneous enzyme composites reminiscent of the clea methodology was recently reported by the group of zare . in this method , flower - shaped protein - inorganic hybrid nanostructures could be generated upon addition of cu(ii ) ions to enzymes , such as cala , carbonic anhydrase , laccase , and -lactalbumin . as in the case with the cleas , the nanoflowers were shown to exhibit a significantly higher thermostability than the free enzymes . interestingly , the nanoflowers also exhibited significantly enhanced catalytic activities , which were ascribed to the high surface area and the confinement of the enzymes in the nanoflowers . synthesized a similar protein - inorganic hybrid by mixing calb with pd(oac)2 in aqueous media . in this reaction , the calb acts as a reducing agent for the pd(ii ) ions , which leads to the formation of small pd nanoparticles within the emerging polymeric enzyme composite . the pd / calb composite was shown to display both acylation and racemization activity , which allowed it to be employed as a bifunctional catalyst in the dkr of 1-phenylethylamine in excellent yield and ee ( scheme 12 ) . for most dkr applications , it is crucial that the enzyme operates efficiently in organic solvents since most substrates and racemization catalysts are not soluble in aqueous media . one way to improve the activity and stability of a protease in organic solvents is to coat it with a lipid or surfactant before lyophilization . this treatment generates a reversed micelle around the enzyme with concomitant solubilization of small amounts of water , which provides the protease with a stable aqueous microenvironment that is maintained even when it is suspended in an organic solvent . several examples on the successful use of surfactants to stabilize enzymes and enable dkrs have already been presented in this perspective . alternative ways of obtaining enzyme mutants with improved properties involve rational enzyme design and directed evolution . in the rational design approach , amino acid residues that are anticipated to play a key role for the function of the enzymes are first identified by the means of for example x - ray crystallography , homology studies , or computational models . these amino acids are then selectively replaced by other amino acid residues that are expected to yield a mutant variant displaying the desired properties . unfortunately , the structural information on the enzyme that is needed to guide such efforts is in most cases limited , which imposes a severe restriction on when this methodology can be utilized . in fact , even with this knowledge in hand , it is often difficult to predict what structural modifications that should be incorporated in order to improve the performance of the enzyme . this is because our understanding of the chemical principles that govern the function and stability of enzymes is still very limited . despite this issue , rational design has been successfully used at multiple occasions for improving the performance of lipases . for example , hult and co - workers used a rational design approach to create a calb mutant that exhibited reverse enantioselectivity ( s ) as well as an improved substrate tolerance toward bulky secondary alcohols . interestingly , this dramatic alteration of the catalytic properties was achieved by exchanging a single amino acid residue in the so - called stereospecificity pocket of the enzyme . in the wild - type calb , the fast - reacting enantiomer places its medium - sized group in the stereoselectivity pocket and its large group toward the entrance of the active site . the access of the large group to the stereoselectivity pocket is effectively prevented by three sterically demanding amino acid residues : thr42 , ser47 and trp104 . the authors identified that a mutant with fundamentally different substrate preference could be generated by changing the sterically demanding trp104 to a smaller alanine residue . subsequently , bckvall and co - workers used this enzyme variant , denoted as calb w104a , together with complex 4 for the ( s)-selective dkr of a series of bulky 1-phenylalkanols in high yields and ee s ( scheme 13 ) . following this study , the group of bckvall and hult explored calb w104 as the resolving enzyme for diarylmethanols ; however , satisfactory e values were only obtained for substrates where the two aryl substituents differed significantly in size . as a result , the development of a dkr protocol for the latter substrate class was never pursued . the group of kim and park recently solved the dkr of this substrate class by using ruthenium complex 8 together with activated lipoprotein lipase . recently , ema , sakai , and co - workers redesigned burkholderia cepacia lipase by introducing two alterations , i287f and i290a , into the catalytically active site using a rational design approach . this double mutant removed a substantial part of the steric congestion in the active site , which enabled this enzyme variant to accept a wide range of extremely bulky secondary alcohols . furthermore , it was found that the phenylalanine residue introduced at position 287 could participate in an additional c h/-interaction with the substrate alcohol , which helped to stabilize the transition state of the acylation reaction and led to an improved ( r)-selectivity of the enzyme . although , the authors only evaluated this burkholderia cepacia lipase variant for kr purposes , it is reasonable to envision that a mild dkr protocol could be constructed by combining this enzyme with any of the available ruthenium - based racemization catalysts . in comparison to rational design , generation of large enzyme libraries with subsequent screening and selection ( e.g. , directed evolution ) is a more useful method for accessing enzyme mutants with improved properties . in this directed evolution approach , natural evolution is artificially mimicked under laboratory settings to create a darwinian - type selection process that will favor emergence of a desired mutant . in practice , this is done by performing iterative cycles of : ( i ) generation of gene libraries from the parent wild - type enzyme by the use of various mutagenesis techniques ; ( ii ) expression of the corresponding enzymes from the gene libraries ; ( iii ) screening of the enzyme mutants for a desired property using various high - throughput methods ; and ( iv ) selecting an improved mutant as a template for the next round of mutagenesis / expression / screening ( figure 4 ) . to date , the directed evolution methodology has been successfully used to modulate several properties of enzymes , including solvent tolerance , thermostability , and higher enantioselectivity for a broader scope of substrates . schematic representation of the directed evolution methodology , where an iterative number of mutagenesis , expression , screening and selection cycles is conducted until an enzyme mutant with desired properties has been obtained . the field of alcohol dkr has certainly advanced significantly during the past two decades and reached a high level of maturity . today , a wide range of functionalized primary and secondary alcohols can be efficiently resolved by the use of chemoenzymatic dkr . the key to this progress has been the successful design of several racemization catalysts , particularly those based on ruthenium , that can racemize alcohols under mild reaction conditions , thereby enabling the use of an increased number of enzymes . ultimately , it is the enzyme component of the dkr that determines what types of substrates that can be resolved and which enantiomer of the product that is favored . therefore , it is essential to have access to dkr protocols that involve a variety of enzymes . so far , lipases have been the enzymes of choice for dkrs , owing to their high activity and selectivity . moreover , these enzymes are associated with a number of practical advantages , including high commercial availability , high thermostability , and good tolerance toward organic reaction media . however , as mentioned previously most naturally occurring lipases preferentially give ( r)-selective resolution of secondary alcohols , and this imposes a limitation for the dkr , since the ( s)-product can not be accessed directly . it is therefore important to have access to ( s)-selective enzymes so that the ( s)-product can be prepared directly by dkr . examples of ( s)-selective enzymes in kr of alcohols are serine proteases , but they are unfortunately not very thermostable . for most secondary alcohols , racemization can be accomplished in reasonable times at room temperature thanks to the most recently developed ruthenium catalysts , which enable dkr systems involving serine proteases . however , for certain challenging substrate classes , such as chlorohydrins and alcohols containing distant olefin groups , the performance of the available ruthenium catalysts is not sufficient to allow for mild dkr s , and here there exists an opportunity for new catalyst design . another important topic of research within the field of alcohol dkr is to develop efficient racemization protocols for tertiary alcohols , which are compatible with the currently available enzymatic kr processes . in contrast to alcohols , the available dkr systems for amines are significantly fewer in number due to challenges associated with the racemization of these substrates . despite the considerable amount of research that has been dedicated to amine dkr , most of the reported protocols still involve racemization catalysts that require high reaction temperatures to function efficiently , which greatly restrict the set of enzymes that can be employed . moreover , the majority of these dkr protocols have only been successful with substrates that are readily racemized , such as -amino acid derivatives and benzylic amines . when it comes to aliphatic amines , the available dkr protocols are significantly fewer in number and generally involve harsh reaction conditions . here , the recently developed metal - free methods to racemize amines by the use of sulfanyl radicals show great promise and might hold the key to mild dkr of both aliphatic and benzylic amines . however , a major concern regarding the racemization by sulfanyl radicals is that it has so far only been combined with a limited number of enzymes , and it is still unclear how widely applicable this method is for dkr . research efforts dedicated toward improving the enzyme component will also play an important role in advancing the field of alcohol and amine dkr . with available molecular biological techniques , chemists now have access to methods for improving and expanding the portfolio of enzymes provided by nature . in particular , evolution of enzymes via generation of large libraries with subsequent screening and selection is a highly useful method for obtaining new enzyme variants with improved properties . until very recently , all screening studies on lipase libraries for increased enantioselectivity had dealt with the hydrolysis of esters in an aqueous medium . however , most dkrs of alcohols and amines are carried out as transacylations in an organic solvent . recently , a method was reported that enables evolution of a lipase for transacylation of secondary alcohols in organic solvent , and it was demonstrated that cala gave a double mutant ( calay93l / l367i ) with a significantly improved e value , 100 vs 3 , in the transacylation of 1-phenylethanol in isooctane . this method is promising and may provide new improved enzymes for the dkr of alcohols and amines . another promising technique to improve the thermostability of enzymes is the clea methodology , where enzymes are converted into heterogeneous aggregates through treatment with a bifunctional cross - linking agent . it is expected that the clea methodology will find future applications in dkr of alcohols and amines .

chemoenzymatic dynamic kinetic resolution ( dkr ) constitutes a convenient and efficient method to access enantiomerically pure alcohol and amine derivatives . this perspective highlights the work carried out within this field during the past two decades and pinpoints important avenues for future research . first , the perspective will summarize the more developed area of alcohol dkr , by delineating the way from the earliest proof - of - concept protocols to the current state - of - the - art systems that allows for the highly efficient and selective preparation of a wide range of enantiomerically pure alcohol derivatives . thereafter , the perspective will focus on the more challenging dkr of amines , by presenting the currently available homogeneous and heterogeneous methods and their respective limitations . in these two parts , significant attention will be dedicated to the design of efficient racemization methods as an important means of developing milder dkr protocols . in the final part of the perspective , a brief overview of the research that has been devoted toward improving enzymes as biocatalysts is presented .

Introduction Early Combinations of Metal Catalysts and Enzymes for the DKR of Secondary Alcohols Development of More Practical Protocols for the DKR of More Functionalized Alcohols The more Challenging DKR of Amines Methods for Improving Enzymes As Biocatalysts Summary and Outlook

the intensive care units ( icu ) admitted patients usually have wide range of physiological individualities . comparison of the patient 's outcomes without consideration of individual physiological differences does not seem to be sensible . it is obvious that , some measures to represent the physiological conditions of icu patients have to be considered in order to control and adjust mentioned differences . during the past decades , comparisons have been made on various models which have been applied for icu patients outcomes . the mentioned risk classification systems could also be applied to evaluate the performance of icus . in fact these systems are statistical models , by which the probability of an event such as fatality , could be measured from the characteristics and information of each individual patient . similar systems such as simplified acute physiology score ( saps ) , mortality probability model ( mpm ) , and acute physiology and chronic health evaluation ii ( apache - ii ) have been developed in various versions,[27 ] or estimation of the probability of mortality in the icus . the apache - ii is derived from 11 physiological variables , glasgow coma score ( gcs ) , patient 's age and the chronic health status scoring from zero in normal patients to 71 in worst scenario . the probability of mortality could be estimated using a logistic regression model , based on the model coefficients which are derived from categorization of icu admission causes . there have been many surveys for evaluation and comparison of the performances of apache - ii for different populations ; but no consistent pattern on accuracy of outcome predictions has been observed . the observed mortalities were more than model predictions in some cases , and less in some other cases , although some of the other studies have shown a good performance for the models . many surveys have been carried out to assess the performance of existing models in other populations . they mainly established that the intensive care risk prediction models , primarily developed in other countries , require validation and recalibration , prior to putting into actual exercise , within a new country setting.[1114 ] in this study the performance of apache - ii system was evaluated and customized on a number of icu patients in a tertiary referral center in order to develop a good model for evaluating the severity of illness for respiratory icu patients . patients admitted to nritld 's icu , in the period of time between jan 2009 to feb 2010 , were included in the study . data 's were collected by trained general physicians using forms designed for this purpose . to control the quality of data , first 10 sheets filled by each person were reviewed by the supervisor ; and finally the quality controlled data sets were entered to a data bank . the quantitative variables of different groups were presented in form of mean sd and the qualitative variables were presented by n ( % ) . student 's t test was applied to compare the quantitative variables between different groups , and the qualitative variables were compared using chi - square or fisher 's exact test . the total sample was divided into two subsamples : training set for refitting the variables into a modified model.test set for evaluating the model developed . the scores and probability of death were computed according to the original apache ii model . all variables of apache - ii were entered in a logistic regression model and new coefficients were estimated using training data set . the modified scoring system was developed based on significant regression coefficients ; non - significant variables , according to the wald test , were allocated zero score . the coefficients of the other significant variables were divided by the smallest coefficient and then rounded to obtain their scores . after preparing the modified scoring system , which we will call modified apache - ii hereafter , these scores were employed as independent variable in a logistic regression models with observed death as dependent variable . for assessment of the discriminative power of different models in both training and test data sets , the receiver operating characteristic ( sensitivity , specificity and accuracy rate were used to evaluate the calibration of different models . the kappa coefficient was also used for evaluating the agreement of observed and expected mortality according to the models . we used f - measure , f = ( 2 sensitivity ppv)/(ppv + sensitivity ) a harmonic mean of the sensitivity and positive predictive value ( ppv ) , as a measure of calibration . in addition , hosmer - lemeshow test , goodness of fit test was used to evaluate the calibration . for this purpose , the sample was sorted increasingly by the expected probability of death and then divided into 10 approximately equal size groups . then the observed and expected the hosmer - lemeshow test was not applicable here because the expected mortality in some groups was less than 5 ; so we just used groups made of this method . patients admitted to nritld 's icu , in the period of time between jan 2009 to feb 2010 , were included in the study . data 's were collected by trained general physicians using forms designed for this purpose . to control the quality of data , first 10 sheets filled by each person were reviewed by the supervisor ; and the quantitative variables of different groups were presented in form of mean sd and the qualitative variables were presented by n ( % ) . student 's t test was applied to compare the quantitative variables between different groups , and the qualitative variables were compared using chi - square or fisher 's exact test . the total sample was divided into two subsamples : training set for refitting the variables into a modified model.test set for evaluating the model developed . the scores and probability of death were computed according to the original apache ii model . all variables of apache - ii were entered in a logistic regression model and new coefficients were estimated using training data set . the modified scoring system was developed based on significant regression coefficients ; non - significant variables , according to the wald test , were allocated zero score . the coefficients of the other significant variables were divided by the smallest coefficient and then rounded to obtain their scores . after preparing the modified scoring system , which we will call modified apache - ii hereafter , these scores were employed as independent variable in a logistic regression models with observed death as dependent variable . for assessment of the discriminative power of different models in both training and test data sets , the receiver operating characteristic ( roc ) curve analysis and its area under curve ( auc ) was used . sensitivity , specificity and accuracy rate were used to evaluate the calibration of different models . the kappa coefficient was also used for evaluating the agreement of observed and expected mortality according to the models . we used f - measure , f = ( 2 sensitivity ppv)/(ppv + sensitivity ) a harmonic mean of the sensitivity and positive predictive value ( ppv ) , as a measure of calibration . in addition , hosmer - lemeshow test , goodness of fit test was used to evaluate the calibration . for this purpose , the sample was sorted increasingly by the expected probability of death and then divided into 10 approximately equal size groups . the hosmer - lemeshow test was not applicable here because the expected mortality in some groups was less than 5 ; so we just used groups made of this method . among 730 patients who were admitted during the study time in the icu , 143 ( 19.6% ) died in hospital . there were 423 ( 57.9% ) males and 307 ( 42.1% ) females in this sample . the mean age was 46.9 9.19 years ; that was significantly higher in death group compared to alive ( p < 0.001 ) . the mean of original apache - ii score in death group was significantly more than alive ( 16.84 7.2 vs. 7.374.6 ; p < 0.001 ) . the first diagnosis at the time of admission was categorized according to apache - ii classification [ table 2 ] . basic characteristics of patient 's , involved in the study first diagnosis , at the time of admission to icu the total sample was divided into two groups : 523 ( 71.6% ) in training dataset and 207 ( 28.4% ) in the test set . the comparison of basic characteristics like age , sex and mortality between training and test set shows no significant difference ( p = 0.081 , p = 0.905 and p = 0.823 , respectively ) . the variables of the original apache - ii system were entered in a logistic regression models and modified scoring system was developed [ table 3 ] . wald test showed that variables including temperature , mean arterial pressure , serum sodium , serum potassium and serum creatinine were not significant between two groups . accordingly , the modified apache - ii scores were computed for all patients ; and the following model was developed for estimating the probability of death : logit ( ) = -4.866 + 0.331 local apache - ii score nritld version of apache ii score ( modified ) in the training set , the area under the roc curve for original apache - ii was 0.860 ( 95% ci : 0.820 - 0.899 ) , and for modified apache - ii was 0.874 ( 95% ci : 0.832 - 0916 ) ; in the test set auc for original apache - ii was 0.908 ( 95% ci : 0.861 - 0.954 ) and for the modified apache - ii was 0.856 ( 95% ci : 0.789 - 0.923 ) [ figure 1 ] . roc curve analysis for comparing the discriminative power of original and modified apache ii systems ; ( a ) the roc curves for the test set ; ( b ) the roc curve for the training set the results of original apache - ii shows that on test set it had 84.1% accuracy , 16.3% sensitivity , 100% specificity , f - measure = 0.280 and kappa = 0.228 ; while the modified apache ii system had 87.9% accuracy , 56.4% sensitivity , 96.4% specificity , f - measure = .656 and kappa = 0.593 [ table 4 ] . comparison of original and modified apache ii systems to evaluate the calibration , observed probability was plotted against expected probability , in 10 groups of hosmer - lemeshow method [ figure 2 ] . in this plot , the solid line shows the reference line for the good calibration and the dashed line shows relationship between expected and observed probabilities . the deviation of the dashed line from the solid one shows the deviation of expected from the observed probabilities . accordingly , the modified apache - ii system shows better calibration in both training and test datasets . comparing the calibration of original and modified apache ii via 10 groups of hosmer - lemeshow method , ( a ) original apache ii on training set , ( b ) original apache ii on test set , ( c ) modified apache ii on training set , ( d ) modified apache ii on test set among 730 patients who were admitted during the study time in the icu , 143 ( 19.6% ) died in hospital . there were 423 ( 57.9% ) males and 307 ( 42.1% ) females in this sample . the mean age was 46.9 9.19 years ; that was significantly higher in death group compared to alive ( p < 0.001 ) . the mean of original apache - ii score in death group was significantly more than alive ( 16.84 7.2 vs. 7.374.6 ; p < 0.001 ) . the first diagnosis at the time of admission was categorized according to apache - ii classification [ table 2 ] . basic characteristics of patient 's , involved in the study first diagnosis , at the time of admission to icu the total sample was divided into two groups : 523 ( 71.6% ) in training dataset and 207 ( 28.4% ) in the test set . the comparison of basic characteristics like age , sex and mortality between training and test set shows no significant difference ( p = 0.081 , p = 0.905 and p = 0.823 , respectively ) . the variables of the original apache - ii system were entered in a logistic regression models and modified scoring system was developed [ table 3 ] . wald test showed that variables including temperature , mean arterial pressure , serum sodium , serum potassium and serum creatinine were not significant between two groups . accordingly , the modified apache - ii scores were computed for all patients ; and the following model was developed for estimating the probability of death : logit ( ) = -4.866 + 0.331 local apache - ii score nritld version of apache ii score ( modified ) in the training set , the area under the roc curve for original apache - ii was 0.860 ( 95% ci : 0.820 - 0.899 ) , and for modified apache - ii was 0.874 ( 95% ci : 0.832 - 0916 ) ; in the test set auc for original apache - ii was 0.908 ( 95% ci : 0.861 - 0.954 ) and for the modified apache - ii was 0.856 ( 95% ci : 0.789 - 0.923 ) [ figure 1 ] . roc curve analysis for comparing the discriminative power of original and modified apache ii systems ; ( a ) the roc curves for the test set ; ( b ) the roc curve for the training set the results of original apache - ii shows that on test set it had 84.1% accuracy , 16.3% sensitivity , 100% specificity , f - measure = 0.280 and kappa = 0.228 ; while the modified apache ii system had 87.9% accuracy , 56.4% sensitivity , 96.4% specificity , f - measure = .656 and kappa = 0.593 [ table 4 ] . comparison of original and modified apache ii systems to evaluate the calibration , observed probability was plotted against expected probability , in 10 groups of hosmer - lemeshow method [ figure 2 ] . in this plot , the solid line shows the reference line for the good calibration and the dashed line shows relationship between expected and observed probabilities . the deviation of the dashed line from the solid one shows the deviation of expected from the observed probabilities . accordingly , the modified apache - ii system shows better calibration in both training and test datasets . comparing the calibration of original and modified apache ii via 10 groups of hosmer - lemeshow method , ( a ) original apache ii on training set , ( b ) original apache ii on test set , ( c ) modified apache ii on training set , ( d ) modified apache ii on test set in this study we tried to customize one of the most commonly used models of icu risk - adjustment and embarked on developing a modified model with comparable performance based on our icu patients . the main reason of using apache - ii instead of apache - iv in this study was that the apache - iv system has some variables such as albumin and bilirubin that are not routinely registered for patients in our hospitals at first 24 hours of icu admission . accordingly using the performance of different predictive models has two main aspects ; discriminative power and calibration ( goodness - of - fit ) . the results of this survey showed that , the original apache - ii system had good discriminative power in our population , but poor calibration according to the goodness of fit measures . the modified apache - ii score showed a good discriminative power as well , although its area under roc curve in the test set was less than the original apache - ii . although the non - significant discriminative power and slight increase in accuracy ( 87.9% vs. 84.1% ) are among the outcomes of our study , the remarkable increase in sensitivity could be due to the sample selection ( all from a tertiary respiratory referral center ) . thus it is recommended that the external validity of our model would be considered in further studies . in this study , all calibration indices were in favor of the modified apache - ii except specificity and positive predictive values . this might be due to underestimation of the probability of death in our population by the original apache - ii score . there are several factors that have effect on the performance of models in different populations . some researchers believe that the different results of model calibrations could be the effect of various combinations of patients . most of the patients in the present study were suffering from respiratory and lung diseases making the combination of diseases different from which the original apache - ii has been developed . this might be one of the reasons for the difference in calibration of apache - ii score in our population . most of the previous studies have shown good discriminative power but different calibration , yet researchers try to improve the performance of these models . . showed that when a unit or patient population differs substantially from average condition , using the customized models is important . they showed that increasing frequency of patients with each disease characteristics above the original frequency may cause the discrimination and calibration to deteriorate . furthermore , apache - ii system is consisted on characteristics of patients at the first 24 hours of admission , and measures can not be considered independent from treatments and the quality of medical care . moreover , the starting point for this model is the time of admission which does not have standard definition and is often influenced by the condition of the icu such as number of beds , quality of pre - hospital care , etc . this all could explain why the modification of models for different populations or specific groups of patients such as respiratory patients is needed . our patient population , due to random factors , might have displayed a low incidence of renal failure , and so creatinine would not add much to the predictive model . omitting the mean arterial pressure ( map ) from the model , could be due to its strong co linearity with other variables such age , ph and wbc . since these variables were more powerful predictors than map ; and they were highly correlated with map , putting them together in a model could increase the variance of regression coefficients and consequently decrease the precision of model . definitely , it does nt mean that map is not a powerful factor in estimation of the severity of illness . although we discuss cogent points about customization , calibration and discrimination of apache models , the other aspect is that apache and mpm have primarily been developed on usa patients ; while saps has a more international component and the updated versions did not include usa patients , so we suggest future studies designed based on saps . definitely modified models would benefit the most if they are recalibrated in a larger icu patient population . it is anticipated that , a larger sample size that includes respiratory patients with different characteristics from different icus would lead to development of better models ; and could help to come across the deficiencies and improve this modified model . besides , the calibration and discriminative power of this customized model could be studied in other respiratory disease icus . the results of this survey show that the calibration of apache ii model on specific group of patients ( respiratory disease ) reduced the number of variables and enhanced its performance . it seems that apache - ii score has its own pros and cons and could be modified to increase its accuracy , performance , and adaptability in a local icu . obviously , with increasing developments in treatment methods and changing the mortality patterns in different populations , the scoring systems need frequently change and update . also , the results of this research emphasize that fitting the new models for specific groups of patients leads to reach more abstract models with fewer variables .

background : several models have been developed to measure the severity of illness in intensive care unit ( icu ) patients , it is suggested that the models should be customized depending on the characteristics of different population of patients . this study is aimed to assess and modify the performance of acute physiology and chronic health evaluation ii ( apache - ii ) model in a respiratory diseases referral center.materials and methods : a total of 730 patients , admitted to an intensive care unit during one year , were divided into two sets ( 71% training and 29% test ) . our modified apache - ii model was developed and calibrated on training set . then , the integrity of the customized model was checked and compared to the original apache - ii , on the test set . logistic regression was used to develop roc analysis , f - measure and kappa coefficient and were employed to calibrate the model.results:both original and our modified apache - ii scores performed acceptable discriminative power ( auc = 0.908 : 95%ci 0.861 - 0.854 ; and auc = 0.856 : 95%ci 0.789 - 0.923 , respectively ) ; the difference was not significant ( p = 0.132 ) . our modified apache - ii showed improved accuracy ( 87.9% vs. 84.1% ) and sensitivity ( 56.4% vs. 16.3% ) compared to the original model . f - measure and kappa also gave the impression of improvement for our modified apache - ii system.conclusion:the results demonstrated that a modified apache - ii system in a local icu of respiratory disease could have similar discrimination and comparable calibration to the original model .

INTRODUCTION MATERIALS AND METHODS Patient selection and data collection Statistical analysis Development of the modified APACHE-II Evaluation of the discriminative powers Calibration RESULTS Main characteristics of the patients Training and test sets Model building Comparing the discriminative powers Comparing the calibrations DISCUSSION CONCLUSION

vascular dementia sometimes can precede or accompany alzheimer 's disease , and in these cases the development of alzheimer 's disease becomes more dramatic . the dementia of ischaemic type and alzheimer 's disease is synergistic or additive in the earliest stages of alzheimer 's disease , although the interactive mechanisms are not known [ 1 , 2 ] . both types of dementia are neurodegenerative diseases and for both the dysfunction and degeneration of cholinergic projective systems in the cortex and the hippocampus from the forebrain nuclei are critical [ 38 ] . a number of studies on animal models demonstrated a possible trigger role of cholinergic projective neurons in brain ischaemia . the early activation of cholinergic projective neurons was found to occur simultaneously with glutamatergic activation in the cortex and the hippocampus [ 912 ] . correlations between the development of cholinergic dysfunctions and the destruction of pyramidal neurons in the hippocampus [ 6 , 13 , 14 ] , and also damage to the cognitive functions of animals [ 48 , 10 ] , led to the presumption that a dysfunction in cholinergic afferents plays a major role in the development of ischaemic pathologies [ 9 , 12 , 14 , 15 ] . modern electrophysiology accumulated numerous data that interneurons of the cortex and the hippocampus actively participate in the modulation of neuronal activity including the hippocampal pyramidal neurons [ 1618 ] . it was revealed that the cholinergic effects on the interneurons of the cortex and the hippocampus was substantially mediated through nicotinic receptors ( nachrs ) [ 1621 ] . on the other hand the role of the cholinergic interneurons in behavioural , and neurodegenerative mechanisms is still unknown . our investigations on the light and heavy synaptosomal fractions of the cortex and the hippocampus allowed the study of the major cholinergic projection systems of the cortex and the hippocampus and their minor intrinsic systems of cholinergic interneurons . according to immunochemical data , both the cortex and the hippocampus the first major sources are the neuronal projections from the forebrain nuclei basalis magnocellularis into the cortex ( precursor of the meynert nucleus in primates and humans ) and projections from the forebrain medial septal nuclei and vertical limb nuclei of the diagonal band of broca into the hippocampus . the synaptosomes are presynaptic parts of synapses with their junction complexes ; these shall be termed presynapses in the present study . we previously showed that for both the cortex and the hippocampus the cholinergic presynapses from different sources are isolated in different synaptosomal fractions during preparation in the sucrose density gradient . the presynapses of cholinergic projections from the forebrain nuclei are accumulated mainly in the light synaptosomal fractions whereas the presynapses of cholinergic interneurons are accumulated mainly in the heavy synaptosomal fractions [ 2931 ] . it is probable that the heavy synaptosomal fraction of the hippocampus may also accumulate a small part of the cholinergic projective presynapses ( lateral projection pathway into the hippocampus ) . our studies on the cortical synaptosomal fractions of cats allowed suggest the involvement of the cholinergic interneurons in cognitive functions . in the studies on the cortical and hippocampal synaptosomal fractions of rat we revealed that during the first three hours of chronic brain ischaemia the cholinergic projective neurons were reactive , as were the interneurons of the cortex and the hippocampus as well . at present , the molecular , genetic and neurochemical mechanisms of cognitive functions are widely investigated in different behavioural models and widely discussed as well . some among these data induce to revise generally conception that memory formation involves an irreversible passage via labile phases , such as working and short - term memory to the stable form of long - term memory . thus it was shown , that several drugs inhibited short - term memory without altering long - term memory and that working , short - term , and long - term memory were differentially regulated in the various brain regions by the various neurotransmitter systems , including cholinergic one [ 3437 ] . the authors concluded that different types of memory had the separate mechanisms , various neurotransmitter systems , and regions . the basis of our investigation is importance of the cholinergic systems in human and animal cognition , and also the existence of general mechanisms in the development of dementias of different aetiologies . in the present study , the cholinergic synaptic organization of different forms of learning and memory in rats with normal and chronic ischaemic brains a marker of cholinergic neurons , enzyme of acetylcholine synthesis choline acetyltransferase ( chat ; ec 2.3.1.6 ) was used for estimation of the cholinergic systems . chat activity and also protein contents ( total synaptic parameters ) were measured in subfractions of the synaptic membranes and the synaptoplasm isolated from light and heavy synaptosomal fractions of the cortex and the hippocampus . thus , the participation of projective and intrinsic cholinergic systems of the cortex and hippocampus in mechanisms of learning and memory under normal and ischaemic conditions was researched . in addition , the regulation of learning performance under prolonged action of selective agonist of 42 subtype of nachr metanicotine ( rjr ) and selective antagonist of non7 subtypes of nachr mecamlamine was studied . outbred white adult male rats ( 220270 g ) were supplied from the animal 's nursery light mountains ( russia ) and then kept in the vivarium of our institute of general pathology and pathophysiology . the rats were housed in a temperature - controlled room ( 2024c ) with free access to food and water and kept on a 12 h light / dark cycle according to the national institutes of health animal care and the principles of laboratory animal care guidelines and the study was approved by the ethical committees of the institutes . chronic rat ischaemia was induced by permanent occlusion of the common carotid arteries ( two - vessel occlusion , 2vo ) by ligation . the bilateral common carotid arteries were tied with silk threads whilst the rats were under an appropriate level of pentobarbital anaesthesia . the common carotid arteries were separated from the cervical sympathetic and vagal nerves through a ventral cervical incision . the sham - operated animals ( control groups ) underwent a similar surgery but vessel ligation was excluded . behaviour was studied in spatial contextual ( noncued ) or spatial cued models of learning and memory in the morris water maze following standard procedures . the experimental apparatus consisted of a circular water pool ( diameter , 120 cm ; height , 60 cm ) filled with milk - clouded water at 24c to a depth of 40 cm . a plexiglas hidden platform ( 10 10 cm ) was submerged 2 cm below the water surface and was placed at the midpoint of one quadrant . the rats were trained during three daily sessions in the contextual ( sham - operated/2vo rats ) or the cued learning models . in both learning models the rats were given four daily attempts to find the hidden platform in a 60 s time interval and the estimated swim time for platform achievement ( latency time ) was recorded . rats which failed to find the platform within 60 s were considered unable to solve the task and were softly guided there by the investigator with 60 s scored . the other rats remained on the platform for 30 s and were returned to their home cage during the intertrial interval ( 60 s ) . in the contextual model the location of the hidden platform remained the same throughout the training period . the pool was located in a test room containing no prominent visual marks . at the start of all trials , the rats were placed in the pool at one of four starting positions . in the cued model a prominent visual mark ( cue ) was placed on the maze wall over the hidden platform to help the animal locate the platform . in this model the rats had the same starting position but the hidden platform with its cue was moved to four different positions during the session . the following forms of cognitive functions were observed and investigated : the inherited abilities ( the first noncasual attempts at decision making in the task , that is , 1s1 trial in the cued model and 1s2 trial in the contextual model ) ; working memory in the first session ( 1s24 and 1s3 - 4 averaged out over the following trials , resp . ) ; learning in the second and the third sessions ( 2s24 and 3s24 averaged trials , resp . ) ; and long - term memory on the days after the first and the second sessions of training ( 2s1 and 3s1 trials , resp . ) . all behavioural experiments were carried out by investigators who had no knowledge of the experimental groups . metanicotine ( rjr 2304 , tocris ) , a selective agonist of 42 subtype of nachr and mecamilamine ( sigma ) , a selective antagonist of non-7 subtypes of nachr , were used . the preparations were subchronically administered ( i.p . ) three times daily in doses of 26 and 3.9 nmoles / kg , respectively . both the sham - operated and the ischaemic rats received the first injection of the preparations immediately after the end of narcosis ( 1.53 hours after surgical intervention ) . some of animals were decapitated for biochemical analysis 3 - 4 days after the third session of training . it means , the rats which trained from 6 day after the surgery were decapitated at 11 or 12 days after the surgery and so on . the biochemical group included the control/2vo animals trained in the contextual model ( contextual biochemical subgroup ) or the cued model ( cued biochemical subgroup ) . briefly , the brain , cortex and hippocampus were removed , separated and homogenized . from each sample the light and heavy synaptosomal fractions were isolated , with further separation of the subfractions of the synaptic membranes and the synaptoplasm , following preparative and the disruptive procedures and the discontinued gradients of sucrose density as described previously [ 30 , 39 ] . the fractions of the synaptosomes were obtained from the rough mitochondrial fraction by centrifugation using a bucket rotor ( 84,000 g 120 min , 24c ) in layers between 1.01.2 m sucrose densities ( the light synaptosomes ) and between 1.21.4 m sucrose densities ( the heavy synaptosomes ) . the synaptosomes were disrupted by combined shock procedures : the synaptosome pellets were suspended in hypo - osmotic solution containing 6 mm tris - ncl buffer , ph 8.1 ( 100 mg tissue / ml ) and they were then exposed by freeze - thawing . the synaptoplasm subfractions were obtained as supernatants by centrifugation from the disrupted synaptosomal fractions ( 14,000 g 30 min , 24c ) . the pellets were suspended in the hypo - osmotic solution and stratified on discontinued gradients again . the synaptic membrane subfractions were obtained by centrifugation using the bucket rotor ( 130,000 g 120 min , 24c ) in layers between 0.61.2 m sucrose densities . the clean synaptic membrane subfractions were free from glial , mitochondrial and synaptic vesicle contamination . the activity of chat in subfractions of synaptic membranes and synaptoplasm of the cortex and the hippocampus was determined by the radiometric method of fonnum and the protein contents were determined by the method of lowry et al . . accordingly , the membrane - bound mchat activity and m - protein contents were determined in the synaptic membrane subfractions , and the water - soluble cchat activity and c - protein contents were estimated in synaptoplasm subfractions . moreover , this is the reason why changes in mchat activity could be exposed and changes in cchat activity could be masked in the small presynapses , whereas changes in cchat activity , but not in mchat activity , could be exposed in the large presynapses . therefore , estimations of mchat and cchat activities ( as well as m- and c - protein contents ) could give additional information on the characteristics of changes caused by ischaemia . the reactive mixture contained a final concentration of 0.2 mm acetyl coasa ( fluka ) and [ 1-c]-acetyl coasa ( amersham pharmacia bioscience ) with spa 5 mci / mmol , 300 mm nacl , 3 mm mgcl2 , 0.2 mm physostigmine salicylate ( sigma ) , 10 mm choline chloride ( serva ) , 0.5% triton x-100 ( serva ) , 0.5 mg / ml albumin from bull serum ( koch - light ) , 10 mm sodium phosphate buffer/1 mm edta - na2 , ph 7.8 and the subfraction samples ( near 3.5 mg of protein ) at a common volume of 0.050.1 ml . the reactive mixture was incubated in a water shaker at 37c for 3060 min . the reaction was stopped by adding 2 ml of ice - cold stop solution ( 0.2 mm acetylcholine in 10 mm sodium phosphate buffer/1 mm edta - na2 , ph 7.8 ) and by placing the mixture in an ice bath . then , a 1 ml solution of sodium tetraphenylborate ( sigma ) in butyl acetate ( 15 mg / ml ) was added and quickly subjected to intensive mixing in a shaker ( 500 turns / min , 4 min , room temperature ) . the organic phase was separated from the inorganic phase by centrifugation ( 1000 g 15 min , 24c ) . the organic phase with acetylcholine ( 0.50.7 ml ) was placed into scintillation liquid for organic solutions and the radioactively synthesized acetylcholine ( dpm ) was quantified with a beta counter . reactive solution ( biuret reagent ) was prepared at the day of experiment by mixing 0.5 ml of 1% cupric sulfate with 0.5 ml of 2% sodium potassium tartrate , followed by the addition of 50 ml of 2% sodium carbonate in 0.1 n naoh . bovine serum albumin ( bsa ) powder was dissolved in distilled water and diluted to a concentration of 1000 g / ml . a series of dilutions of the basic bsa solution ( 50 , 100 , 200 , 400 and 500 g / ml ) was made by mixed thoroughly of the aliquots of basic bsa solution and water with repeated pipeting . samples were within the bsa standard range ( 120 g in assay volume ) . reaction was started by intensive mixed of 0.02/0.04 ml of bsa or subfractions samples with 1 ml of the reactive solution . the mixture was then allowed to incubate at room temperature for 1015 min prior to the addition of 0.1 ml per tube of 1.0 n folin & ciocalteu 's reagent . color was allowed to develop for 2 hours in dark at room temperature and the absorbance of the reduced folin reagent measured at 750 nm and blanked on the water only control . after then the reaction was found to be stable for up to an hour at room temperature and kept in refrigerator at 58c for up to 1 - 2 days . the behavioural results were expressed in terms of time taken to swim to the hidden platform ( s ) and the biochemical results were expressed in terms of chat activity ( nmoles acetylcholine / min ) or protein content ( mg ) in 1 g wet weight of cortex and hippocampus tissue , respectively . the data were calculated using the nonparametric fisher 's exact test and the r - criterion of the pearson 's correlative test in microsoft excel with a glance of adjusting formula for small number of observations . the period of 610 days of chronic brain ischaemia led to a strong decline in training efficiency in the morris water maze . learning and long - term memory were impaired in both the contextual and cued models ( figure 1 ) . learning in 2s24 and 3s24 were impaired in a similar manner in both of the behavioural models , whereas impairment of the long - term memory had the specificity in each model . in the contextual model ( figure 1 , top ) , impairment of long - term memory developed gradually and only 3s1 was significantly impaired . in the cued model ( figure 2 , bottom ) , long - memory 2s1 was impaired and 3s1 was the same as the control . it can be noted that although the investigated cognitive functions were impaired , they were still performed in ischaemic rats . from all of the investigated animals ( n = 27 ) , only two rats could not solve the tasks in our experimental conditions ( in the contextual model ) . as a rule , the prolongation of solving tasks and/or the delay in learning ( successful solving of the task only occurred in the third session ) was observed . the period of 1114 days of chronic ischaemia resulted in significant changes in chat activity and protein content in the investigated subfractions of the synaptosomes , both of the cortex and the hippocampus ( figure 2-all rats ) . in the cortical light synaptosomal fraction , mchat activity and m - protein content were increased , and these changes were positively correlated amongst themselves ( r = + 0.770 , n = 18 , total control and 2vo groups data , p < .001 ; in the control group r = + 0.788 , n = 9 , p < .02 ) . the cchat activity did not significantly vary or correlate with mchat activity but it was positively correlated with increasing c - protein content ( r = + 0.669 , n = 9 , p < .05 ; in the control group r = + 0.305 , n = 9 , this indicated a reorganization of the synaptic pool in more than one synaptic population of the cholinergic projective neurons in the cortex . but mchat activation was accompanied by a reinforcement in the positive correlation between its values and the m - protein content ( r = + 0.694 , n = 9 , p < .05 ; in the control group r = + 0.291 , n = 9 , p > .05 ) . also , a reinforcement of the positive correlation between the values of cchat activity and c - protein content was observed ( r = + 0.835 , n = 9 , p < .01 ; in the control group r = + 0.633 , n = 9 , p > .05 ) . at the same time , the correlation between the activity of mchat and the activity of cchat became weaker than in the control ( r = + 0.579 , n = 9 , p > .05 ; in the control group r = + 0.754 , n = 9 , p < .02 ) . this indicated a reorganization of the synaptic pool in more than one synaptic population of the cholinergic interneurons in the cortex . in the hippocampal light synaptosomal fraction , however , significant correlations between the values of mchat and cchat activity , and between the values of chat activity and protein content , were absent . this indicated a reorganization of the synaptic pool in more than one synaptic population in the hippocampus , in both the cholinergic systems and some noncholinergic systems . in the hippocampal heavy synaptosomal fraction , m - protein content decreased and c - protein content increased . changes in the m - protein content did not correlate with chat activity and thus reflected reorganization of noncholinergic presynapses in the hippocampus . the activities of mchat and cchat did not differ from the controls although at the same time a positive correlation arose between their values ( r = + 0.802 , n = 8 , p < .02 ; in the control group r = 0.300 , n = 9 , p > .05 ) . also , the positive correlations between values of c - protein content and mchat activity ( r = + 0.927 , n = 8 , p < .01 ; in the control group r = + 0.265 , n = 9 , p > .05 ) and cchat activity ( r = + 0.844 , n = 8 , p < .01 ; in the control group r = + 0.091 , n = 9 , this indicated a reorganization of the presynapses of the cholinergic interneurons / lateral pathway projective neurons in the hippocampus . it seems that the unchanged values of chat activity reflected parallel processes of activation and inactivation of chat in different synaptic populations of this fraction . also , the biochemical data of the contextual biochemical subgroup were compared with the cued biochemical subgroup . analysis of the biochemical parameters in the control biochemical subgroups did not reveal significant changes between the subgroups ( table 1 ) . only the values of mchat activity were lower in the cortical heavy and in the hippocampal light synaptosomal subfractions in the cued biochemical subgroup of rats as compared with the contextual one . analysis of the biochemical parameters in the 2vo biochemical subgroups confirmed our observations about the reorganization of the synaptic pool . in the cortical light synaptosomal fraction in the contextual biochemical subgroup ( figure 2-contextual ) , independent correlations were detected between the values of mchat activity and m - protein content ( r = + 0.765 , n = 10 , total control and 2vo contextual biochemical subgroups data , p < .01 ; in the control contextual biochemical subgroup r = + 0.774 , n = 5 , p > .05 ) and between the values of cchat activity and c - protein content ( r = + 0.987 , n = 5 , p < .01 ; in the control contextual biochemical subgroup r = + 0.441 , n = 5 , p > .05 ) . in the cued subgroup ( figure 2-cued ) , a correlation was only detected between mchat activity and m - protein content ( r = + 0.783 , n = 8 , total control and 2vo cued biochemical subgroups data , p < .05 ; in the control cued biochemical subgroup r = + 0.622 , n = 4 , p > .05 ) and a correlation was revealed between mchat and cchat activities ( r = + 0.995 , n = 4 , p < .01 ; in the control biochemical cued biochemical subgroups r = 0.404 , n = 4 , p > .05 ) . in the cortical heavy synaptosomal fraction in the contextual biochemical subgroup , independent correlations were detected between mchat activity and m - protein content ( r = + 0.981 , n = 5 , p < .01 ) and between the values of cchat activity and c - protein content ( r = + 0.966 , n = 5 , p < .01 ) , whereas there was no correlation between the activities of mchat and cchat among themselves ( r = + 0.652 , n = 5 , p > .05 ) . in the cued biochemical subgroup , an increase in mchat activity was detected ( a tendency ) whereas a decrease in m - protein content was revealed . the decrease in m - protein content allows to suppose the changes in noncholinergic presynapses . in the hippocampal light synaptosomal fraction in the contextual biochemical subgroup , an independent decrease in mchat activity and an increase in cchat activity only , and in the cued biochemical subgroup an increase in cchat activity only , and a decrease in m - protein content and an increase in c - protein content were detected . in the hippocampal heavy synaptosomal fraction in the contextual biochemical subgroup , positive correlations were detected between the increased values of c - protein content and mchat activity ( r = + 0.922 , n = 5 , p < .01 ) and cchat activitiy ( r = + 0.919 , n = 5 , p < .05 ) , and between mchat and cchat activities ( r = + 0.910 , n = 5 , p < .05 ; in the control contextual biochemical subgroup r = 0.266 , n = 5 , p > .05 ) . however , in the cued biochemical subgroup , a decrease in mchat activity and m - protein content was revealed and these changes did not correlate among themselves . this indicated a reorganization of the presynapses of the cholinergic interneurons / lateral pathway projective neurons and noncholinergic neurons in the hippocampus . differentiation of the rats into biochemical subgroups , tested in the contextual and cued models , permit to compare the behavioural performance and chat activity in these rats . under the normal conditions , each form had individual cholinergic composition ( table 2 : sham - contextual , figure 3-i : sham ) . the inherited ability 1s2 cholinergic composition included large presynapses of projective cortical neurons ( positive correlation with cchat activity ) and presynapses of hippocampal interneurons / lateral pathway projective neurons ( negative correlation with mchat and cchat activities ) . the same cholinergic structures associated with the long - term memory 3s1 , but with inverse symbols of r - criterions . the long - term memory 2s1 had composition other than 3s1 which involved small presynapses of the projective hippocampal neurons ( negative correlation with mchat activity ) and some populations of hippocampal interneurons ( positive correlations with mchat and cchat activities ) . the composition of the working memory 1s3 - 4 involved small presynapses ( positive correlation with mchat activity ) and large presynapses ( negative correlation with cchat activity ) of the projective hippocampal neurons . the composition of learning 2s24 and 3s24 was identical and involved small presynapses of the projective cortical neurons ( positive correlation with mchat activity in both forms of learning ) and the hippocampal interneurons / lateral pathway projective neurons ( positive correlations with mchat activity ) . the similar analysis in the cued biochemical subgroup revealed other individual cholinergic compositions of learning and memory ( table 2 : sham - cued , figure 3-ii : sham ) . according to our data , working memory 1s24 composition involved small presynapses of both cortical cholinergic systems ( negative correlation with mchat activity ) and of the hippocampal interneurons / lateral pathway projective neurons ( positive correlation with mchat activity ) . the composition of learning 2s24 and 3s24 was identical and comprised large presynapses of cortical interneurons and projective hippocampal neurons ( positive correlations with cchat activity in both cases ) . the long - term memory composition in 2s1 involved large presynapses of the cortical interneurons and small presynapses of projective hippocampal neurons ( in both cases , there were positive correlations with cchat or mchat activities ) . and the long - term memory composition in 3s1 involved large presynapses of the cortical projective neurons ( positive correlations with cchat activity ) . chronic brain ischaemia had considerable effects on the cholinergic organization of the investigated cognitive functions . in the contextual model ( table 2 : 2vo - contextual , figure 3-i : 2vo ) , inherited abilities 1s2 , learning 2s24 and long - term memory 3s1 completely lost correlations with the cholinergic populations and all forms of cognition lost correlations with the cortical cholinergic populations . the composition of working memory 1s3 - 4 only kept negative connections with the large presynapses of projective hippocampal neurons . the positive connections of learning 3s24 with the small presynapses of the hippocampal interneurons / lateral pathway projective neurons inversed to negative ones . the long - term memory composition 2s1 consisted of only new , positive connections with large presynapses of the projective hippocampal neurons . in the cued model ( table 2 : 2vo - cued , figure 3-ii : 2vo ) , long - term memory 2s1 and 3s1 completely lost correlations with the cholinergic populations and all forms of cognition lost correlations with the hippocampal cholinergic influences . the working memory 1s24 lost its negative connections with the small presynapses of cortical projective neurons and its negative connections with the small presynapses of cortical interneurons inversed to positive ones . the composition of learning 2s24 included a reversal to negative connections with the large presynapses of cortical interneurons , new negative connections with presynapses of cortical projective neurons and new negative connections with the small presynapses of cortical interneurons . the learning composition 3s24 kept its connections with the large presynapses of cortical interneurons and added new negative connections with presynapses of the cortical projective neurons . so , under 2vo conditions as in the contextual and in the cued biochemical subgroup quantity of the cholinergic connections with the cognitive functions significantly reduced and some new links arose . each form of cognition as resulting had 2vo cholinergic synaptic composition organized differently from normal ones . we attempted to analyze the dependence of impairment of the investigated cognitive functions in 2vo conditions from the reorganization of key cholinergic systems . it seems , in the contextual biochemical subgroup only preservation of the inherited abilities 1s2 from damage could be explaned by preservation of the key cholinergic populations , revealed in the normal conditions ( figure 4 , middle row ) . but long - term memory 3s1 had the same cholinergic composition with inverse symbols of r - criterions . in this case it would be expected that 3s1 would also be protected ; however , this did not take place . moreover , according to the cholinergic organization under normal conditions , working memory 1s3 - 4 would be considerably impaired , whereas long - term memory 2s1 and learning 2s24 and 3s24 would be considerably improved ; however , these did not occur either . on the other hand , the new cholinergic composition in 2vo conditions had accordance between reinforcement of the negative influence of the hippocampal interneurons / lateral pathway projective neurons on learning 3s24 and impairment of this function ( figure 4 , bottom row ) . but reinforcement of new negative and positive influences of projective hippocampal neurons was not reflected in the performance of either working memory 1s3 - 4 or long - term memory 2s1 . in the cued biochemical subgroup , only the preservation of long - term memory 3s1 could be explained by the resistance to ischaemia of the key synaptic population revealed in normal conditions ( figure 5 , middle row ) . at the same time , working memory 1s24 would be considerably impaired , learning 2s24 and 3s24 would be equally improved or otherwise unchanged and long - term memory 2s1 would be unchanged , but these were not observed . on the other hand , the absence of 1s24 impairment could also be explained by the new , weakly expressed positive cholinergic influence ( tendency ) of the large presynapses of cortical interneurons ( figure 4 , bottom row ) . then the distinctions in learning 2s24 and 3s24 performance in 2vo conditions would be explained by their new cholinergic compositions if we suppose more considerable influence of the large presynapses of the cortical interneurons on these functions in comparison with the influence of the other new key synaptic populations . the impairment of 2s1 also would be explained by the reduction of the link with this key synaptic population under the normal conditions . so , it seems that performance of the cognitive functions as in the contextual and in the cued model under 2vo conditions , as a rule , did not depend on their cholinergic organization , revealed in normal conditions . contrary , new cholinergic organization , revealed in 2vo conditions showed more significant correlations with changes in behavioral performance . whereas , the contextual and cued learning 2s24 and 3s24 revealed cholinergic synaptic compositions identical for normal conditions and different ones , revealed in 2vo conditions , it was investigated prolonged action on the learning performance of the selective agonist of 42 subtype of nachr rjr and the selective antagonist of non-7 subtypes of nachr mecamilamine . in the contextual model under normal conditions , both the agonist rjr and the antagonist mecamilamine did not influence on learning as 2s24 and 3s24 performance . it seems , this fact indicate that non-7 subtypes of nachr did not participate in the regulation of ones ( figure 6 , contextual learning ) . under the 2vo conditions , effects of rjr on both learning performance evidently , the 42 subtype did not participate in the regulation of the contextual learning , as before , while some non-7 and non-42 subtypes participated with negative influences on learning 3s24 . in the cued model under normal conditions , the agonist rjr impaired learning 2s24 and did not affect learning 3s24 ( figure 6 , cued learning ) . the negative effect of the agonist on learning in 2s24 was significantly greater than that of the antagonist ( p < .05 ) . the difference between the agonistic and antagonistic actions on learning in 3s24 at were also significant ( p < .05 ) . it follows that the 42 subtype ( negative influence ) and some non-7 and non-42 subtypes of nachr ( positive but weak influence ) participated in the regulation of learning in 2s24 . at the same time , the 42 subtype did not participate in the regulation of learning 3s24 , while some non-7 and non-42 subtypes participated with negative influences . under the 2vo conditions , rjr did not correct the impaired functions 2s24 and 3s24 , and mecamilamine did not correct learning 2s24 but resulted in normal learning 3s24 performance . it seems , non-7 subtypes of were removed from the receptor composition of learning 2s24 . at the same time , the importance of some non-7 and non-42 subtypes of nachr was reinforced or new connections arose in the receptor organization of learning 3s24 ( negative influence ) . so , in the contextual learning 2s24 and 3s24 , nachr were absent in normal and were acquired in 2vo receptor composition ( 3s24 ) . then , the cued learning 2s24 and 3s24 , with identical cholinergic synaptic compositions in the norm , had differences in receptor compositions . moreover , the cued learning 2s24 and 3s24 had also differences in 2vo receptor compositions via another means . this research showed that the chronic 2vo brain ischaemia model was an efficient model of neurodegenerative disorders , which was the first purpose of our investigation . the period of 610 days of 2vo ischaemia provoked typical attributes of vascular dementias such as impairment of learning and long - term memory in both spatial - contextual and spatial - cued models of behaviour in the morris water maze . the inherited abilities and working memory remained intact , and damage to the cued long - term memory was transient in this ischaemic period . a considerable reorganization of the synaptic pool of all investigated cholinergic systems in the cortex and the hippocampus was revealed in these same 2vo rats 1114 days after the surgery . a decrease in mchat or cchat activity in one synaptosomal fraction and an increase in another were obtained as result of the 2vo influence , but not of the training , whereas the biochemical parameters did not reveal similar changes between the control contextual and cued biochemical subgroups except one . it is possible that decrease in mchat activity in the hippocampal heavy synaptosomal fraction in the cued biochemical subgroup was result of the training ( see table 1 and figure 2-cued ) . the decrease in mchat and cchat activity reflected cholinergic hypofunction or a degeneration of the cholinergic presynapses . neurodegeneration was observed in different brain ischaemia models starting from the second day up to half a year of ischaemia [ 45 , 46 ] . dysfunction of chat in the projective fibres in the hippocampus ( representing 8090% of the total activity of this enzyme , and see table 1 ) was described at 714 days of ischaemia [ 6 , 13 , 14 , 47 , 48 ] . it was shown in vitro that the activity of mchat was selectively suppressed when the exchange of acetylcholine was damaged by inhibition of the vesicular acetylcholine transporter or the high affinity transport of choline . the function of the vesicular acetylcholine transporter depends on the proton gradient , which in turn is disturbed due to falling atp levels ( inhibition of the proton atpase ) or acidosis . we did not reveal a decrease in protein content correlated with chat activity in our research . but we did suppose that the correlation between chat activity and protein content could be masked because of the complex opposing processes that took place in some of the synaptosomal fractions . at the same time , according to data in the literature , sprouting and destruction with the swelling of neurons and their terminals predominates in late brain ischaemia or postischaemic reoxygenation ( in days and months ) [ 3 , 48 , 53 , 54 ] . in our research , activation of chat was also observed in the majority of the synaptic subfractions , and it could have reflected cholinergic hyperfunction or synaptogenesis ( sprouting ) . it is known that synaptic hyperfunction is accompanied with an enhanced structuring of proteins from the synaptoplasm . under these conditions , the m - protein content will increase and the c - protein content will decrease . therefore , the correlated increase between mchat activity and m - protein content will be reflected as cholinergic hyperfunction and synaptogenesis ( cortical light synaptosomes in the biochemical total group and both subgroups ) , whereas the correlated increase between cchat / mchat activity and the c - protein contect will only reflect synaptogenesis ( cortical light synaptosomes in the biochemical total group and the contextual subgroup , hippocampal heavy synaptosomes in the total group and the cued subgroup ) . selective activation of mchat in vitro was shown under conditions of impaired ionic balance such as an accumulation of [ ca]i and [ zn]i [ 55 , 56 ] . it was revealed that [ zn]i precedes [ ca]i accumulation and [ ca]i in turn results in the functional hyperactivation and swelling of synapses [ 53 , 57 , 58 ] . according to data in vitro activation of cchat reflect cholinergic hyperfunction under normal ionic and metabolic conditions [ 49 , 59 , 60 ] . therefore , we suppose that the activation of cchat reflected synaptogenesis , in agreement with other researchers . thus , chronic brain ischaemia for 1114 days resulted in a complex reorganization of the cortical and hippocampal synaptic pool which involved synaptogenesis or hyperfunctions in the unbalanced ionic conditions of one of the cholinergic synaptic populations and degeneration or dysfunction of the others . we supposed that all of the cholinergic processes revealed under 2vo conditions were present in both cholinergic subgroups of the rats but with different intensities ( see figure 2-all rats , contextual and cued ) . we also supposed that the variety of cholinergic reactions was revealed by the phenotypical variety of the outbred rats and it was useful for understanding some of the principles of the organization of different forms of cognition . the second purpose of our research was a comparative analysis of the behavioural and biochemical parameters for identification of the cholinergic composition of the investigated cognitive functions under normal and 2vo conditions . in the first place it is necessary to note that the results of this research confirmed and expanded the knowledge about cholinergic mechanisms of cognitive functions under normal brain conditions . our data showed the active involvement of cholinergic projective systems and also regional ones of the cortex and the hippocampus in cognitive processes . cholinergic synaptic connections with the investigated cognitive functions revealed under normal conditions indicate that each form of cognition has an individual cholinergic synaptic and probably receptor compositions . this conclusion is conformed to the results of investigations , obtained in the morris water maze and some other behavioural models [ 3437 ] . then , the data showed the participation of the cholinergic systems not only in mechanisms of learning and working memory , which was repeatedly observed in previous studies [ 35 , 45 , 61 ] , but also in mechanisms of the inherited abilities and long - term memory . our inherited abilities in the contextual task in the morris water maze was firstly detected by r g morris and u frey as a distinct type of memory and termed as it seems the problem of future discussions . our data , concerning individual cholinergic organisation of function support contextual inherited abilities as a distinct form of cognition . our data concerning the same cholinergic structures associated with inherited abilities 1s2 and long - term memory 3s1 also testify to possible tight interaction between these two forms of cognition . the involvement of cholinergic projective systems in mechanisms of the long - term memory is usually denied [ 6467 ] , and was only discussed in a few studies [ 36 , 68 , 69 ] . our data confirmed that synaptic populations of cholinergic projective neurons and of the interneurons of the cortex and the hippocampus can have positive and negative connections with cognitive functions . a negative dependence of cognitive functions on cholinergic cortical efficiency was also revealed earlier in cats using a similar methodology for researching the cholinergic synaptic organization of cognitive functions . therefore , our data demonstrate that the cholinergic mechanisms of learning and memory are more complex than currently perceived . it is evident that this can complicate the detection of cholinergic effects on some cognitive functions by means of nonselective influences on cholinergic efficiency . for example , according to our data in the contextual model , the nonselective pharmacological cholinergic means as well as use of different methods of degeneration of the cholinergic projective systems would certainly have revealed the participation of the cholinergic projective systems in learning 2s24 and 3s24 , but it would probably have concealed a cholinergic participation in the mechanisms of the inherited abilities , the long - term and the working memory . such results would correspond to the data in the literature [ 62 , 63 , 68 , 70 ] . from the numerous data in the literature , preservation of the cholinergic projective systems is critical for the success of cognitive processes , and it was thought that cholinergic dysfunction or degeneration results in the impairment of memory in neurodegenerative diseases of different aetiologies . therefore , we analyzed the connections between reorganization of the cholinergic synaptic pool and impairment of learning and memory under 2vo conditions . the comparative analysis showed that the connections between the functional and cholinergic parameters revealed under normal conditions were practically lost in the ischemic rats . in our research , only impairment of cued long - term memory 2s1 was really dependent on degeneration of the key synaptic population of the cholinergic cortical interneurons , and also , probably , the intact cued memory 3s1 by the unchanged key synaptic population of the cholinergic cortical projective neurons . at the same time , our data also showed different cholinergic compositions of the cognitive functions under 2vo and under normal conditions in as the spatial contextual and the spatial cued models . under 2vo conditions , most connections of the investigated functions with cholinergic synaptic populations revealed under normal conditions disappeared and new connections with other cholinergic synaptic populations arose . the quantity of cholinergic synaptic populations , involving in mechanisms of the investigated cognitive functions , was considerable reduced . furthermore , cholinergic connections in general disappeared from the mechanisms of the following forms of cognition : inherited abilities 1s2 , learning 2s24 and long - term memory 3s1 in the contextual model , and long - term memory 2s1 and 3s1 in the cued model . moreover , brain region specializations of both the contextual and the cued functions were changed . cortical cholinergic influences had been completely removed from the contextual functions and hippocampal ones from the cued functions . all considerable differences between cholinergic organisation of the cognitive functions in the normal and 2vo conditions stated above are clear demonstrated in figure 3 . it is important that a consistency between the performances of cognitive functions and their new key cholinergic synaptic populations was found in the majority of the remaining cholinergic - dependent functions under 2vo conditions ( from four to six functions ) . thus , according to our data , we suggest that the normal cholinergic synaptic connections in learning and memory were progressively reduced and changed during chronic ischaemia . it is known that anticholinesterase drugs are only effective in the early stages of alzheimer 's disease ( early and mild alzheimer 's disease ) . it can be noted that the new cholinergic connections with the cognitive functions were not necessarily a consequence of degeneration or dysfunctions in the key cholinergic synaptic populations ( it was evident for contextual learning in 3s24 and long - term memory in 2s1 , that is , these new links could arise by other , indirect reasons ) . the dependence on the inclusion of cholinergic links in the realization of cognitive functions from the functional background of neuronal environments was recently revealed . this corresponds with the theory by d. a. sakharov about the nonsynaptic transfer of chemical information [ 71 , 72 ] . according to this theory , any change in any functional system results in a change in all systems as a result of the change in neuroactive compounds of intercellular environments ( the matrix ) . the changes in the matrix determine the activation of one or another neuronal ensemble which finally determines the behavioural act . from all of these viewpoints , it seems that the main value for cognitive functions is its receptor composition and its change in neurodegenerative pathology . our data concerning the different consequences on the learning performance under normal and 2vo conditions by the action of rjr and mecamilamine on the same subtypes of nachr testify to this version . it seems that the reasons for changes in the cholinergic organization of cognitive functions in an ischaemic pathology can be any neurodegenerative or , on the contrary , reparative process ( sprouting ) of cholinergic and noncholinergic synaptic populations . in spite of the brain reparative potentials , the cholinergic and the whole neurochemical organization of cognitive functions under the chronic actions of pathological factors will be formed as optimally as possible under the new conditions . a new organization of cognitive functions will be constructed on neuronal elements which are stable against pathological influences . this new organization can provide an optimum realization of some cognitive functions but not of others . in any case , the study of new key neurochemical links in the organization of cognitive functions may be promising . the plasticity of neurochemical links in the individual organization of certain types of cognition can be used in the future for alternative corrections of vascular and other degenerative dementia .

the purpose of this research was a comparative analysis of cholinergic synaptic organization following learning and memory in normal and chronic cerebral ischaemic rats in the morris water maze model . choline acetyltransferase and protein content were determined in subpopulations of presynapses of light and heavy synaptosomal fractions of the cortex and the hippocampus , and the cholinergic projective and intrinsic systems of the brain structures were taken into consideration . we found a strong involvement of cholinergic systems , both projective and intrinsic , in all forms of cognition . each form of cognition had an individual cholinergic molecular profile and the cholinergic synaptic compositions in the ischaemic rat brains differed significantly from normal ones . our data demonstrated that under ischaemic conditions , instead of damaged connections new key synaptic relationships , which were stable against pathological influences and able to restore damaged cognitive functions , arose . the plasticity of neurochemical links in the individual organization of certain types of cognition gave a new input into brain pathology and can be used in the future for alternative corrections of vascular and other degenerative dementias .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

swallowing is an essential part of life ; disruptions in swallowing affect the quality of life , which is a major concern in an aging society . dysphagia or swallowing impairment refers to difficulty or discomfort during the progression of the alimentary bolus from the oral cavity to the stomach.1 it is strongly associated with aspiration pneumonia in older people and has been shown to result in extended hospital stay , worsening of other diseases and increased risk of death in the elderly . therefore , prevention , early detection and effective rehabilitation of dysphagia are important issues to improve health in the elderly.2 however , only a few epidemiological studies have reported the prevalence of dysphagia in community - dwelling populations . previously , serra - prat et al reported that the signs of dysphagia were prevalent in 27.2% of subjects aged 70 and older.3 additionally , previous studies have shown conflicting findings regarding the association between dysphagia and potential risk factors , such as age , malnutrition , oral conditions , lifestyle and medical history ( such as cognitive function).27 these studies included participants with various characteristics , such as pneumonia patients , residents in care homes , frail elderly people and the community - dwelling population . okamoto et al reported the relationship between dysphagia and tooth loss in community - dwelling independent elderly adults.4 another study showed that care dependency scale scores , malnutrition and disease clusters , such as dementia , nervous system disorders and cerebrovascular disease / hemiparesis , were associated with the presence of subjective dysphagia.5 lindgren and janzon reported complaints of swallowing difficulty with several clinical findings among 50- to 79-year - old males and females living in an urban area ; they described the consequences of these symptoms in terms of changing dietary habits and medical history but were unable to define potential risk factors of dysphagia.4,6 in previous studies , the number of examinations for participants was different . therefore , previous studies have not been able to clearly indicate potential risk factors for dysphagia . changes in physical performance , oral conditions and medical history generally begin at 50 years of age,6 and loss of skeletal muscle mass is prominent after this age in japanese adults.8 habitual exercise has been shown to be effective in maintaining physical performance in the elderly.9,10 the national dental survey conducted in japan revealed that the prevalence rates of periodontitis in those aged 4045 and 5559 years were 25.6% and 47.0% , respectively.11 several epidemiological studies have identified potential risk factors of dysphagia in community - dwelling older adults . epidemiological studies evaluating the prevention , early detection and effective rehabilitation of dysphagia should focus on patients above 50 years of age . thus , the present study aimed to examine the frequency of dysphagia and its risk factors in rural , community - dwelling people aged 5080 years . this study was a part of an ongoing community - based project entitled the iwaki health promotion project and comprised 651 volunteers ( 234 males and 417 females ; 50 years ) who participated in 2015 . these participants were rural residents of the iwaki district located in hirosaki in northern japan . all study subjects participated voluntarily in the iwaki project in response to a public announcement , and it was estimated that more than 1,000 males and females participated in this study . they participated cognitively and were physically capable of reaching the research venue by referring to a map . the exclusion criteria in the present study were medical histories of cancer , stroke , ischemic heart disease and parkinson s disease . additionally , patients with incomplete data were excluded from the study ; in total , 532 participants ( 185 males and 347 females ) were included . data collection for this study was approved by the ethics committee of the hirosaki university school of medicine , and all participants provided written informed consent . demographic data , such as age and sex ; lifestyle , such as smoking and drinking ; and medical history were obtained from self - reported questionnaires and interviews . to assess the exercise habits of the participants , they were also asked to choose from two items : none or 17 days / week . the height and weight of the participants were measured , and body mass index ( bmi ) was calculated using the following equation : bmi ( kg / m ) = weight ( kg)/(height [ m ] height [ m ] ) . malnutrition was assessed based on bmi < 20 kg / m . in this study , serum albumin ( alb ) concentration ( g / dl ) was considered as a confounding factor because it represents nourishment . non - fasting blood samples were obtained , and plasma concentrations of alb were measured using standard procedures in conjunction with a completely automated system ( srl ltd , tokyo , japan ) . mental state examination ( mmse ) was administered to all participants to measure their global cognitive status . this test assessed the orientation to place and time , short - term memory , episodic long - term memory , subtraction ability , ability to construct sentences and oral language ability . the maximum score was set as 30.12 to evaluate subjective dysphagia , we asked the question do you sometimes choke on drinks / food such as tea and soup ? . an objective assessment of dysphagia was carried out using the repetitive saliva swallowing test ( rsst ) , which detects patients who experience aspiration.13,14 the patients were asked to perform saliva swallows ( dry ) as many times as possible in 30 seconds . if the patient was unable to perform three consecutive swallows , he or she was likely to have dysphagia associated with aspiration . participants who responded positively to the question or presented with abnormal rsst findings were diagnosed with impaired swallowing . dental examinations were conducted by trained and experienced dentists under artificial lighting , with both the dentist and the patient in seated positions . differences between groups were determined using student s t - test and chi - square test . additionally , odds ratios ( ors ) with 95% confidence interval ( 95% ci ) were calculated using multiple logistic regression analysis . to explore the effect of potential risk factors of dysphagia on the prevalence of subjective dysphagia , a model was built by multivariate analysis using the forced entry method . variables presenting a p - value of < 0.25 in the univariate analysis were then included in the multivariate model as non - cases ( 0 ) and cases ( 1 ) of dysphagia . goodness of fit was performed based on the technique of hosmer and lemeshow . a p - value of < 0.05 was applied as the cutoff . the hosmer and lemeshow value was calculated to indicate the fit of the final model : the higher the p - value , the better the fit of the model . statistical package for the social sciences ( spss ) for windows ( version 24 ; ibm corp . this study was a part of an ongoing community - based project entitled the iwaki health promotion project and comprised 651 volunteers ( 234 males and 417 females ; 50 years ) who participated in 2015 . these participants were rural residents of the iwaki district located in hirosaki in northern japan . all study subjects participated voluntarily in the iwaki project in response to a public announcement , and it was estimated that more than 1,000 males and females participated in this study . they participated cognitively and were physically capable of reaching the research venue by referring to a map . the exclusion criteria in the present study were medical histories of cancer , stroke , ischemic heart disease and parkinson s disease . additionally , patients with incomplete data were excluded from the study ; in total , 532 participants ( 185 males and 347 females ) were included . data collection for this study was approved by the ethics committee of the hirosaki university school of medicine , and all participants provided written informed consent . demographic data , such as age and sex ; lifestyle , such as smoking and drinking ; and medical history were obtained from self - reported questionnaires and interviews . to assess the exercise habits of the participants , they were also asked to choose from two items : none or 17 days / week . the height and weight of the participants were measured , and body mass index ( bmi ) was calculated using the following equation : bmi ( kg / m ) = weight ( kg)/(height [ m ] height [ m ] ) . malnutrition was assessed based on bmi < 20 kg / m . in this study , serum albumin ( alb ) concentration ( g / dl ) was considered as a confounding factor because it represents nourishment . non - fasting blood samples were obtained , and plasma concentrations of alb were measured using standard procedures in conjunction with a completely automated system ( srl ltd , tokyo , japan ) . mental state examination ( mmse ) was administered to all participants to measure their global cognitive status . this test assessed the orientation to place and time , short - term memory , episodic long - term memory , subtraction ability , ability to construct sentences and oral language ability . to evaluate subjective dysphagia , we asked the question do you sometimes choke on drinks / food such as tea and soup ? . an objective assessment of dysphagia was carried out using the repetitive saliva swallowing test ( rsst ) , which detects patients who experience aspiration.13,14 the patients were asked to perform saliva swallows ( dry ) as many times as possible in 30 seconds . if the patient was unable to perform three consecutive swallows , he or she was likely to have dysphagia associated with aspiration . participants who responded positively to the question or presented with abnormal rsst findings were diagnosed with impaired swallowing . dental examinations were conducted by trained and experienced dentists under artificial lighting , with both the dentist and the patient in seated positions . differences between groups were determined using student s t - test and chi - square test . additionally , odds ratios ( ors ) with 95% confidence interval ( 95% ci ) were calculated using multiple logistic regression analysis . to explore the effect of potential risk factors of dysphagia on the prevalence of subjective dysphagia , a model was built by multivariate analysis using the forced entry method . variables presenting a p - value of < 0.25 in the univariate analysis were then included in the multivariate model as non - cases ( 0 ) and cases ( 1 ) of dysphagia . the hosmer and lemeshow value was calculated to indicate the fit of the final model : the higher the p - value , the better the fit of the model . statistical package for the social sciences ( spss ) for windows ( version 24 ; ibm corp . dysphagia was observed in 33 males ( 17.8% ) , 76 females ( 21.9% ) and a combination of 109 males and females ( 20.5% ) of the 532 participants , as shown in figures 1 and 2 , respectively . with respect to age , the highest prevalence was seen in 70- to 74-year - old males ( 40.0% ) and in 75- to 79-year - old females ( 41.4% ) , and the lowest prevalence was seen in both 55- to 59-year - old males and females ( 5.9% and 11.7% , respectively ) . in the univariate analysis , depending on the presence or absence of dysphagia ( tables 1 and 2 ) , significant differences in the number of teeth ( p=0.014 in males ) , oral dryness ( p=0.003 in males , p=0.007 in females ) and age ( p=0.014 in males , p=0.015 in females ) were noted . the average number of teeth in males without dysphagia was 23.87.3 and in males with dysphagia was 19.39.7 . the number of participants without dysphagia and oral dryness included 39 males ( 25.7% ) and 106 females ( 39.1% ) ; the number of participants with dysphagia and oral dryness was 17 males ( 51.5% ) and 43 females ( 56.6% ) . multiple logistic regression analysis was performed , where the dependent variable was the presence or absence of dysphagia and the independent variable includes the number of teeth , oral dryness , age , bmi < 20.0 kg / m , serum alb concentration , smoking habits , exercise habits , diabetes mellitus status and hypertension status . logistic regression analysis using the forced entry method showed statistical significance for oral dryness in males and females ( p=0.003 and 0.032 , respectively ; table 3 ) . in men , the ors for oral dryness and number of teeth were 3.683 and 0.946 ( 95% ci = 1.5708.644 and 0.8980.997 , respectively ) . this model was shown to be compatible with the hosmer lemeshow test ( p=0.109 ) , with a predictive accuracy of 83.8% . in females , this model was also shown to be compatible with the hosmer lemeshow test ( p=0.526 ) , with a predictive accuracy of 78.7% . dysphagia was observed in 33 males ( 17.8% ) , 76 females ( 21.9% ) and a combination of 109 males and females ( 20.5% ) of the 532 participants , as shown in figures 1 and 2 , respectively . with respect to age , the highest prevalence was seen in 70- to 74-year - old males ( 40.0% ) and in 75- to 79-year - old females ( 41.4% ) , and the lowest prevalence was seen in both 55- to 59-year - old males and females ( 5.9% and 11.7% , respectively ) . in the univariate analysis , depending on the presence or absence of dysphagia ( tables 1 and 2 ) , significant differences in the number of teeth ( p=0.014 in males ) , oral dryness ( p=0.003 in males , p=0.007 in females ) and age ( p=0.014 in males , p=0.015 in females ) were noted . the average number of teeth in males without dysphagia was 23.87.3 and in males with dysphagia was 19.39.7 . the number of participants without dysphagia and oral dryness included 39 males ( 25.7% ) and 106 females ( 39.1% ) ; the number of participants with dysphagia and oral dryness was 17 males ( 51.5% ) and 43 females ( 56.6% ) . multiple logistic regression analysis was performed , where the dependent variable was the presence or absence of dysphagia and the independent variable includes the number of teeth , oral dryness , age , bmi < 20.0 kg / m , serum alb concentration , smoking habits , exercise habits , diabetes mellitus status and hypertension status . logistic regression analysis using the forced entry method showed statistical significance for oral dryness in males and females ( p=0.003 and 0.032 , respectively ; table 3 ) . in men , the ors for oral dryness and number of teeth were 3.683 and 0.946 ( 95% ci = 1.5708.644 and 0.8980.997 , respectively ) . this model was shown to be compatible with the hosmer lemeshow test ( p=0.109 ) , with a predictive accuracy of 83.8% . in females , this model was also shown to be compatible with the hosmer lemeshow test ( p=0.526 ) , with a predictive accuracy of 78.7% . several studies have examined the methods used to evaluate dysphagia . the use of video fluorescence or video endoscopy of swallowing has been recommended for precise examination of dysphagia in the clinical setting.5 however , these methods are considered impractical for investigating a community - dwelling population due to their complex apparatus and the need of radiation exposure . one of the most common screening tests for dysphagia has shown a high correlation with video fluorography of swallowing , especially in the case of aspiration.14 the sensitivity and specificity of the rsst compared to the use of video fluorography were 98% and 66% , respectively . in order to increase the accuracy of detection , a simple questionnaire was also used to ensure that certain conditions such as inapparent dysphagia were included in the result.5 the prevalence rates of dysphagia in our study were 17.8% in males and 21.9% in females . as these results were similar to those reported by okamoto et al ( 15.1% ) , this study was considered appropriate for the evaluation of dysphagia.4 in this study , there were significant differences between dysphagia and oral dryness in both sexes.4 normally , crushed food is mixed with saliva to form bolus , thereby , enabling food to be swallowed easily . oral dryness is affected by the preparation phase ( chewing ) of the feeding / swallowing function . as dysphagia can result in reduced or altered oral intake of liquid , it increases the risk of oral dryness.3 in contrast to males , the number of teeth did not show any significant correlations with dysphagia in females . in some people with dysphagia , ingested food tends to remain in the mouth after a meal , as they are unable to facilitate the movement of the food from the oral cavity to the stomach by swallowing . as a result , it consists of occlusal support , mandibular stopping by occlusion , elevation of the hyoid bone , and movement of the larynx and tongue . the stability of the tongue apex has been shown to affect the swallowing process.15,16 when food is ingested , we use upper and lower teeth to crush the food . thus , decrease in the number of remaining teeth reduces the fixation and stability of the tongue tip , which , in turn , affects the squeezing movement seen during swallowing . age - related changes in the position of hyoid bone may be a component of decreased swallowing safety and aspiration in older adults.17 the distance between the hyoid bone and mandible increases with age , and the position of the hyoid bone is correlated with aspiration in older males . such difference was distinct between males and females , and it was further affected by the number of teeth and prevalence of dysphagia in the present study.18 considering the results obtained from this study , improvement in oral conditions might affect dysphagia.5,15,19 other studies have shown that oral hygiene is related to dysphagia and aspiration pneumonia . it is important for patients with dysphagia to maintain oral hygiene , as it prevents tooth loss and reduces the risk of aspiration pneumonia by reducing the number of pathogens ( such as bacteria implicated in periodontitis).20 moreover , stimulations caused during oral care may affect the swallowing reflex and may improve swallowing ability.21 furthermore , oral massage improves the flow rate of saliva in patients with xerostomia.22,23 thus , management of oral condition might prove beneficial because of its association with the teeth health and other conditions , such as oral dryness , dysphagia and pneumonia . although most of the studies investigated older or frail people , this study showed the relationship between oral conditions and dysphagia in middle - aged and elderly adults . the physiology of swallowing changes with aging.2,7 reductions in muscle mass and connective tissue and elasticity result in loss of strength and range of motion . van der maarel - wierink et al listed independent lifestyle , malnutrition , cognitive function , nervous system disease , cerebrovascular disease and hemiplegia as potential risk factors for swallowing disorders in elderly.5 some researchers have also pointed out that nutritional status and muscle strength are related to motor function during swallowing.5,6,15 furthermore , dementia patients are likely to be suffering with dysphagia due to impairment of the neurotransmission mechanism.2,7 in the current study , it was found that there was no significant difference in serum alb concentration , bmi or exercise habits among the population , and age was not significantly related to dysphagia . the results obtained from this study are not in accordance with those of previous studies . the reason for this may be due to the fact that unlike the previous studies , subjects who participated in this study were considered healthy . this is evident from the high mmse scores ( 28.82.1 in men and 29.01.8 in women ) obtained for current subjects . all study participants were cognitively and physically capable of arriving at the research venue by using a map . the results of this study are representative of dysphagia in the community - dwelling population or in the more healthy population compared to those of previous studies . using the volume - viscosity swallow test , they reported that the signs of dysphagia , impaired swallowing efficacy , impaired safety and aspiration were prevalent in 27.2% , 20.5% , 15.4% and 6.7% of the samples , respectively.3 however , they did not examine oral conditions . in this study , we did not clarify the type of dysphagia , and we examined oral conditions . we speculated that oral dryness and the number of teeth might have stronger associations with swallowing efficacy ( impaired labial seal , oral or pharyngeal residue and piecemeal deglutition ) rather than swallowing safety ( changes in voice quality , cough or decrease in oxygen saturation ) , especially because participants in this study were considered to be health conscious . therefore , for general population or healthy population , oral dryness and the number of teeth were more critical factors than other factors , such as nutritional status , lifestyle or previous medical history . as the study design was cross - sectional , it did not allow us to find out the detailed causal connections among the measured items . moreover , all subjects voluntarily participated in the iwaki project in response to public announcement , and it was estimated that more than 1,000 males and females participated in this study . therefore , the study participants were not randomly selected . certain impaired efficacies ( presence of oral , pharyngeal residue , piecemeal deglutition and impaired lip closure ) are important part of dysphagia , although swallowing efficacy was not evaluated ( only aspiration ) . the 10-item eating assessment tool ( eat-10 ) that is a self - reported questionnaire or the symptoms associated with dysphagia ( sydney swallowing questionnaire ) which assesses the severity of dysphagia . in this study however , the sensitivity and specificity of rsst compared to video fluorography were 98% and 66% , respectively . hence , we were unable to consider the effect of dentures ( denture position , form and suitability , etc . ) on dysphagia . future studies are warranted to dissect the impact of dentures , severity and type efficacy of dysphagia . in this study , the prevalence of dysphagia was approximately 20% in a japanese community - dwelling population aged 5079 years . this cross - sectional study revealed relationships between oral conditions , especially oral dryness , and dysphagia . thus , oral dryness and the number of teeth may be more important risk factors than aging , lifestyle and comorbidity when considering the dynamics of dysphagia in community - dwelling older adults . however , the interpretation of our results was hampered by lack of data , including insight about denture usage and longitudinal observations .

purposeprevention , early detection and effective rehabilitation of dysphagia are important issues to be considered in an aging society . previous studies have shown conflicting findings regarding the association between dysphagia and its potential risk factors , including age , malnutrition , oral conditions , lifestyle and medical history . herein , we assessed the prevalence and association of dysphagia with potential risk factors in 50- to 79-year - old adults dwelling in a community in japan.patients and methodsin this study , there were 532 participants ( 185 males and 347 females ) . participants who responded positively to the question do you sometimes choke on drinks / food such as tea and soup ? or those who presented with abnormal repetitive saliva swallowing test findings were diagnosed with dysphagia . the data collected from these participants included the following : number of teeth , occurrence of oral dryness , age , body mass index , serum albumin concentration , smoking , drinking and exercise habits , presence of diseases , such as diabetes mellitus and hypertension , and questions from the mini mental state examination.resultsdysphagia was observed in 33 males ( 17.8% ) and 76 females ( 21.9% ) . to explore the effect of the potential risk factors on the prevalence of dysphagia , a model was built by multivariate logistic regression analysis . using the forced entry method , oral dryness ( odds ratio [ or ] = 3.683 and p=0.003 in males ; or = 1.797 and p=0.032 in females ) and the number of teeth ( or = 0.946 and p=0.038 in males ) were found to be significantly related to dysphagia.conclusionthis cross - sectional study demonstrated associations between oral conditions and dysphagia . factors such as oral dryness and number of teeth may contribute to dysphagia more so than aging , lifestyle and comorbidity in community - dwelling adults over the age of 50 .

Introduction Patients and methods Participants Assessment of dysphagia Oral examination Statistical analysis Results Prevalence of dysphagia Univariate analysis Multiple logistic regression analysis Discussion Limitations Conclusion

there has been a substantial decrease in acute rejection rates , and in most centres , 1-year graft survival now exceeds 95% [ 13 ] . in contrast , recent years have seen only minimal gains in long - term transplant outcomes . death - censored graft survival has only marginally increased [ 13 ] , and life expectancy of kidney transplant recipients ( ktxrs ) remains markedly lower than that of the general population . in large part , this is due to complications associated with life - long immunosuppression . disease associated with bk virus ( bkv ) is one such complication . bkv is a small , ubiquitous , nonenveloped double - stranded dna virus of the polyomavirus family . its genome comprises early genes that code for the regulatory small and large t ( lt ) proteins and late genes that code for the viral capsid proteins ( vp1 , vp2 , and vp3 ) and agnoprotein . the genome also includes a noncoding control region ( nccr ) that contains the origin of viral replication and the transcription and promoter sequences that control viral gene expression . serological studies indicate that bkv infection has a worldwide adult seroprevalence rate of around 75% ( range of 4694% depending on the region studied ) [ 6 , 7 ] . primary infection is usually asymptomatic , but bkv establishes latency in urinary epithelium [ 68 ] . while reactivation and urinary shedding occurs in 10% to 60% of immunocompetent individuals [ 7 , 9 , 10 ] , markedly higher rates bkv appears to cause clinical disease only in individuals with changed or altered immune responses . this has been documented in pregnant individuals , those with human immunodeficiency virus-1 ( hiv-1 ) infection or receiving chemotherapy , and in bone marrow and solid organ transplant recipients . these include ureteric stenosis , haemorrhagic cystitis , transient renal dysfunction , and progressive renal impairment due to bkv - associated nephropathy ( bkvan ) . of these , bkvan is the most common and the most clinically significant because of its association with graft loss . bkvan was essentially a nonexistent entity in the 1980s and early 1990s , confirmed by a study that retrospectively reviewed biopsy slides of kidney transplant patients shedding decoy cells ( cells in the urine that contain viral inclusions ) between 1985 and 1996 . however , its incidence has steadily increased in the subsequent years , with reports from recent decades describing incidence rates as high as 10% . more importantly , bkvan has emerged as an important cause of graft loss , reported in 0% to 80% of cases depending on immunosuppressive regimen employed , cohort size , timing of detection , and management strategy instituted [ 6 , 13 , 16 ] . current knowledge regarding risk factors for bkvan in the posttransplant period is extremely limited and inconsistent . a number of clinical and demographic factors have been associated with increased risk ( table 1 ) [ 1735 ] , but most have been only variably implicated and have limited predictive value . more plausible is the notion that risk of bkvan is dependent on the interaction of multiple risk factors , with a primary contribution from immunosuppression , and additional contributions from such donor , recipient , and viral factors as those tabulated . immunosuppression is the most significant and the only widely accepted risk factor for posttransplant bkv replication . this is largely because bkv associated disease is seen only in immunosuppressed populations , and because multiple studies have shown reductions in bkv replication following immunosuppression minimisation . particularly , it remains unclear whether any particular agent can be specifically implicated , or whether overall potency of immunosuppression is responsible . surprisingly , in addition to its immunosuppressive properties , cyclosporine has been shown to possess antiviral activity in vitro against herpes simplex virus , vaccinia virus , hiv-1 [ 40 , 41 ] , and hepatitis c virus [ 4245 ] . similarly , some studies have shown a suppressive effect of mycophenolic acid ( mpa ; the active drug moiety of mycophenolate mofetil ( mmf ) and enteric - coated mycophenolate sodium ( ec - mps ) ) on the in vitro replication of various herpes viruses , hiv-1 [ 4749 ] , and hepatitis b virus [ 5052 ] . based on these data , acott et al . [ 53 , 54 ] investigated the impact of cyclosporine and mpa on bkv replication using vero e6 cells of green monkey origin infected with bkv ( vj isolate ) when 7090% confluence had been reached . clinically utilised concentrations of cyclosporine were shown to not only inhibit the primary bkv infection peak , but also to inhibit bkv reactivation and nccr gene rearrangements in a dose - independent manner . the magnitudes of these observed effects were similar at all cyclosporine doses tested ( 20012800 g was seen on transformed virus or in cells with high - level infection ( > 10 copies / ml ) . in a separate study , the same group showed a dose - dependent suppressive effect of clinically relevant concentrations of mpa ( 116 g / ml ) on early and persistent bkv infection . in contrast to cyclosporine , when added to culture at high doses ( > 8 g / ml ) , mpa was also able to inhibit high - level infection , and infection with nccr rearranged virus . a concern with both of the above - mentioned studies is that all results were obtained from a vero e6 monkey cell line . to prove relevance of these findings to humans , a recent study investigated the effect of cyclosporine on bkv-(archetype strain- ) infected human proximal renal tubular ( hk-2 ) cells . cyclosporine ( concentration range of 0.54 g / ml ) was shown to dose - dependently reduce the expression of bkv lt and vp1 gene transcripts and bkv lt protein and to prevent the lt protein - regulated increase in early promoter activity . interestingly , while actual data was not presented , this paper also reported that addition of tacrolimus ( 0.510 ng / ml ) to culture media had no impact on lt antigen expression , suggesting against a calcineurin inhibitor class effect . sirolimus has been shown to inhibit the expression of bkv lt antigen in both immortalized human renal cells and primary human renal tubular cells . the effect was dose - dependent , observed across the range of 30 to 150 ng / ml . as stated by the study 's authors ' , 30 ng / ml represents peak serum levels of sirolimus in transplant patients , while the higher concentrations may reflect tissue levels . in this study , use of sirolimus alone had no effect on bkv replication . however , its use in combination with leflunomide led to significant reductions in bkv dna . in vitro steroid treatment has been shown to confer enhanced host cell permissivity for bkv infection . . found that treatment of bkv - infected vero cells with dexamethasone increased the expression of viral transcripts ( particularly the capsid proteins ) up to 11-fold . dna - protein binding studies showed that this effect was mediated via a steroid hormone response unit in the nccr region of the bkv genome . one study has investigated the in vitro effects of immunosuppressive medications on bkv - specific t cell responses . exposed peripheral blood mononuclear cells ( pbmcs ) from nonimmunosuppressed bk seropositive healthy volunteers to increasing concentrations of tacrolimus , cyclosporine , mpa , and sirolimus . pbmcs were then stimulated with bkv large t - antigen , and t - cell responses were measured via an interferon gamma- ( ifn-- ) linked elispot assay . both tacrolimus and cyclosporine were shown to cause dose - dependent inhibition of ifn- expression . specifically , tacrolimus concentrations > 6 ng / ml were shown to cause > 50% inhibition of bkv - specific t cells , while concentrations of <3 ng / ml led to < 30% inhibition . inhibition by cyclosporine was > 50% at concentrations of 1920 ng / ml and < 30% at concentrations of < 960 ng / ml . in contrast , addition of clinically relevant concentrations of mpa ( up to 8 /ml ) and sirolimus ( up to 64 ng / ml ) had no effect on bkv - specific t cell ifn- production . of note , addition of increasing concentrations of sirolimus during t - cell expansion led to reductions in total cell count , consistent with the well - established anti - proliferative effect of this drug . this suggests that while sirolimus had no effect on t - cell activation , it was able to inhibit bkv - specific t - cell expansion . the above in vitro data suggest that cyclosporine , mpa , and sirolimus may be protective against bk replication , as compared to tacrolimus and corticosteroids . however , whether the postulated antiviral effects of these agents are sufficient to outweigh their immunosuppressive properties in vivo can not be elucidated from these studies . bkvan was essentially an unknown entity in the era of cyclosporine - based immunosuppression , with increasing identification of bkvan coinciding with inclusion of tacrolimus and mpa in immunosuppressive regimens . this has led to the suggestion that these agents may be specifically responsible . in support of this , multiple retrospective and observational studies have demonstrated substantial increases in the risk of bkv replication in the context of tacrolimus and mpa use [ 14 , 59 , 60 ] . table 2 details these studies . of importance , however , in the first four of the studies tabulated [ 14 , 20 , 59 , 60 ] , conversion to tacrolimus occurred in response to episodes of rejection . this makes it likely that the development of bkvan was preceded by overall intensification of immunosuppression . additionally , it is possible that the renal injury arising from rejection episodes may have had a predisposing effect . in the subsequent three studies tabulated [ 35 , 61 , 62 ] , no information was provided as to why recipients were administered a particular calcineurin inhibitor or antimetabolite over the other . given the general belief that tacrolimus and mpa have increased immunosuppressive potency compared to cyclosporine and azathioprine , use of these drugs may have been reserved for patients at higher immunological risk and were possibly dosed to achieve higher levels of immunosuppression . consequently , the potential for indication bias limits any conclusions that may be drawn regarding drug - specific effects on bkv replication . of note , while bkvan was not observed in previous decades in those receiving cyclosporine - based immunosuppression , a more recent study of 321 ktxrs treated with cyclosporine , azathioprine , and prednisolone showed an incidence of bkvan of 9.3% . given that no details regarding induction therapy or previous episodes of rejection were provided , it is difficult to evaluate in this study the influence of overall immunosuppressive burden on bkv replication . however , it does illustrate that bkvan can not be solely attributed to either tacrolimus or mycophenolate - based immunosuppression . a number of studies have attempted to examine the relationship between bkv replication and immunosuppressive burden , as quantified by tacrolimus trough levels and mmf and prednisolone drug doses [ 20 , 62 , 6467 ] ( see table 3 ) . in three of these studies [ 6466 ] , one of these studies reported that mmf and prednisolone doses were similar between groups , while another described an association between prednisolone but not mmf dose and bkvan . in contrast , three studies have shown no influence of tacrolimus trough concentrations on bk replication [ 20 , 62 , 67 ] . two reported no association between bkv and mmf dose [ 20 , 67 ] , while one reported no association between bkv and prednisolone dose . overall , these data perhaps suggest more prominent roles for tacrolimus and possibly prednisolone as predisposing agents for bk replication , compared to mpa . indeed , the authors of the second study tabulated concluded that reducing tacrolimus and prednisolone doses while maintaining mmf might be the most appropriate approach for treatment of bkvan . however , while it is generally accepted that tacrolimus trough concentrations are a reasonable surrogate measure of drug exposure , both mpa and prednisolone are drugs with high between - subject pharmacokinetic variability leading to poor relationships between drug doses and plasma concentrations [ 7072 ] . similar doses of these drugs in those with and without bk replication can not be equated with similar drug exposure . as a result , it is impossible to establish from these studies the individual contribution of mpa and prednisolone on bkv replication or to establish whether variable mpa and prednisolone exposure between groups may have confounded the data pertaining to tacrolimus concentrations . an open - label , prospective , single - centre randomized controlled trial ( rct ) of 200 de novo adult ktxrs provides the best evidence to date regarding the impact of the individual calcineurin inhibitors and antimetabolites on bk replication . this study randomly assigned recipients to tacrolimus or cyclosporine , administered in combination with azathioprine and tapering doses of prednisolone . mmf ( 1 g twice daily ) was substituted for azathioprine in those considered to be at high immunological risk or with a history of gout ( 49% in the tacrolimus group and 35% in the cyclosporine group ) . no difference was observed in the incidence of viruria in recipients assigned to cyclosporine compared to tacrolimus ( 36% versus 31% ; p = .61 ) or in recipients who received azathioprine compared to mmf ( 33% versus 38% ; p = .52 ) . similarly , there was no difference in viraemia with tacrolimus compared to cyclosporine ( 12% versus 11% ; p = 1.0 ) or with azathioprine versus mmf ( 13% versus 9% ; p = .46 ) . while viruria was highest with the combination of tacrolimus and mmf ( 46% ) and lowest with cyclosporine and mmf ( 13% ) ( p = .005 ) , viraemia tended to be highest with cyclosporine and azathioprine ( 15% ) and lowest with cyclosporine and mmf ( 4% ) ( p = .27 ) . these data fail to demonstrate a single role of any particular cni or antimetabolite in promoting bkv replication . the finding of the highest incidence of bk viruria with tacrolimus and mmf does suggest that this combination may be the most permissive for bkv replication . however , it should be noted that cyclosporine lowers mpa concentrations while tacrolimus does not , raising the possibility of confounding due to higher mpa exposure in the tacrolimus arm of the trial ( mpa concentrations not measured ) . further , those with both transient and sustained viruria were included in this analysis . given that transient viruria can be observed in a variety of individuals including healthy donors and nonkidney solid organ transplant recipients , this data may be misleading with regard to the influence of immunosuppression on clinically significant bkv replication . the relative effects of cyclosporine versus tacrolimus on bkvan are currently being assessed as a secondary end - point of the direct ( diabetes incidence after renal transplantation : neoral c-2 monitoring versus tacrolimus ) trial , a randomized , six - month , open label , international multicentre trial in which 682 patients who received kidney transplants were treated with either cyclosporine microemulsion formulation or tacrolimus to prevent organ rejection . use of pulse steroids as treatment for acute rejection has been independently associated with bkv replication and bkvan . however , as discussed above , it is difficult to differentiate whether bkvan following rejection results from augmented immunosuppression or from a drug - specific effect . there has been only limited study of the role of maintenance steroid therapy in promoting bkv replication . in a nonrandomized prospective study of 120 ktxrs of whom 71 were maintained on steroids and 49 were treated with early steroid withdrawal , dadhania et al . identified steroid maintenance therapy as an independent risk factor for bkv replication ( odds ratio 8.3 ( 95% ci 2.1 , 32.7 ) ; p = .003 ) ) . a retrospective single - centre study of 213 kidney and 14 pancreas - kidney transplant recipients found a lower incidence of bkvan in those that had steroids ceased early or withdrawn , as compared to those that continued on steroids ( 0% versus 3.5% ; p < .05 ) . a us optn database review of 48,292 ktxrs reported that the risk of treatment for bkv replication was increased in those discharged on maintenance steroids versus those that were not ( adjusted hazards ratio 1.16 ( 95% ci 1.02 , 1.31 ) ; p = .0237 ) ) . none of these studies reported on whether immunological risk status and immunosuppression target levels differed between groups . however , the study of dadhania et al . used multivariable logistic regression analysis to account for the effects of antihuman thymocyte globulin ( atg ) induction , tacrolimus trough levels , tacrolimus and mmf dose and acute rejection , while the database review fitted a cox proportional hazards model to account for a very large number of possible confounding variables . bkvan has been uncommonly observed in patients receiving calcineurin inhibitor - free or mtor - based regimens . a small case series reported the development of bkvan in 3 ktxrs maintained on sirolimus , prednisolone , and mmf . a retrospective study reported nine cases of bkvan in 344 kidney and 34 pancreas - kidney transplant recipients treated with sirolimus - based immunosuppression ( cyclosporine - sirolimus in 6 recipients , tacrolimus - sirolimus in 1 recipient , mmf - sirolimus in 1 recipient , and cyclosporine - mmf - sirolimus in 1 recipient ) . eight of nine patients had been previously exposed to atg , while 3 had experienced acute rejection . in the us optn database review , 5380 of 48,292 ktxrs were discharged on mtor - based immunosuppression , of whom 83 ( 1.74% ) received treatment for bkvan within 2 years of transplant . multivariable analysis showed a reduction in risk of treatment for bkv replication with use of an mtor at discharge , as compared to no mtor use ( adjusted hazards ratio 0.69 ( 95% ci 0.54 , 0.89 ) ; p = .0048 ) ) . the majority of studies have shown an increase in bkv replication following use of lymphocyte depleting agents for induction or treatment of rejection . this is not surprising given the immunosuppressive potency of these agents . in a study of 120 ktxrs , multivariable analysis showed an independent influence of atg induction on risk of bkv replication ( odds ratio 5.83 ( 95% ci 1.60 , 21.35 ) ; p = .008 ) . similarly , in the retrospective study of 344 kidney and 34 pancreas - kidney transplant recipients mentioned above , multivariable analysis correlated exposure to atg for either induction or rejection treatment with a higher incidence of bkvan ( 3.53% versus 1.44% ; p = .048 ) . in a retrospective analysis of renal biopsy and urine cytology samples from 880 ktxrs , use of atg for induction exerted an independent risk for developing both high level viruria and bkvan . in a prospective study of 78 ktxrs , those with viraemia were more likely to have received antirejection treatment with atg than those without evidence of bkv replication ( 60% versus 20% ; p = .008 ) . interestingly , in the us optn database review , induction therapy with thymoglobulin was associated with an increased risk of treatment for bkv ( adjusted hazards ratio 1.42 ( 95% ci 1.24 , 1.63 ) ; p < .0001 ) , but induction therapy with atg had no independent effect ( adjusted hazards ratio 01.19 ( 95% ci 0.73 , 1.95 ) ; p = .4792 ) . to date , there are no antiviral drugs available with specific activity against bkv . while various therapies have been tried , including cidofovir [ 78 , 79 ] , intravenous immunoglobulin , and leflunomide , success has been variable and none have been appropriately studied in a randomized controlled clinical trial . consequently , reduction of immunosuppression remains the mainstay of treatment , despite the increased risk of immunological allograft damage associated with this approach . multiple strategies are currently utilised , including reducing or ceasing antimetabolite therapy , lowering calcineurin inhibitor target concentrations , switching from tacrolimus to cyclosporine , and conversion from calcineurin inhibitor to sirolimus [ 13 , 82 ] . however , as outlined in a recent systematic review , published reports on these protocols have yielded mixed results , and no randomized controlled trials have compared one strategy with another . of note , in more recent times , many centers have instituted routine screening of urine or blood for bkv dna . such programs have reported significant improvements in graft outcomes , possibly due to early detection of viral presence and reduction of immunosuppression prior to the onset of graft damage [ 17 , 21 , 84 , 85 ] . this is evidenced by the increase in viral replication observed in immunosuppressed populations and the decrease in viral replication that follows immunosuppression reduction . to date , however , there is no conclusive evidence that any one drug has specific influence over another in regard to the development of posttransplant bkv infection . cyclosporine , mycophenolate , and sirolimus have all been shown to possess antiviral activity against bkv in vitro . alternatively , in vitro data suggest that tacrolimus has no inhibitory potential , while corticosteroids may in fact enhance bkv replication . however , whether these characteristics are sufficient to counteract or exacerbate the immunosuppressive properties of these drugs in vivo has not been confirmed in clinical trials . in ktxrs this illustrates that no one drug is either necessary or sufficient . while multiple studies have associated tacrolimus and mmf use with increased risk , the majority of these were retrospective or observational in nature and confounded by variable and uncontrolled use of additional immunosuppression . this showed no independent influence of either tacrolimus or cyclosporine , nor any independent effect of either azathioprine or mmf . however , again , results were likely cofounded by variable immunosuppressive cotherapy exposure in the two arms of this trial . a small number of studies have shown lower incidence of bkv replication under steroid minimisation , cni - free , or mtor - based regimens . however , given that such protocols are often reserved for lower immunological risk patients requiring lower total doses of immunosuppression , little can be interpreted from this data . there is general consensus that reduction of immunosuppression is appropriate for management of significant bkv replication . further , it appears that early detection and intervention are important in preventing irreversible graft damage . however , there has been limited comparison of the various approaches , with the result that there is no good evidence to support one strategy over another . for the future , prospective trials specifically designed to address the impact of the various immunosuppressive agents on risk of bkv replication are required . to prevent confounding of results due to variable cotherapy exposure , it is vital that these studies incorporate proper immunosuppression exposure measures ( e.g. , mpa drug concentration measurements ) . additionally , assays are becoming available that allow direct assessment of immune cell function . incorporation of these measures could prove invaluable in distinguishing postulated antiviral effects of the various drugs from the influence of drug - induced immunosuppression .

the incidence of bk virus infection in kidney transplant recipients has increased over recent decades , coincident with the use of more potent immunosuppression . more importantly , posttransplant bk virus replication has emerged as an important cause of graft damage and subsequent graft loss . immunosuppression has been accepted as a major risk for bk virus replication . however , the specific contribution of individual immunosuppressive medications to this risk has not been well established . the purpose of this paper is to provide an overview of the recent literature on the influence of the various immunosuppressant drugs and drug combinations on posttransplant bk virus replication . evidence supporting the various immunosuppression reduction strategies utilised in the management of bk virus will also be briefly discussed .

1. Introduction 2. Immunosuppression and BKV 3. Immunosuppression Reduction as a Treatment Strategy for BKV Replication 4. Conclusions

hypersensitivity to mosquito bites ( hmb ) is characterized by intense skin reactions at bite sites , which consist of not only erythema or bulla but also ulcer or scar , and is associated with general symptoms , such as high fever , general malaise , cramps and renal failure . while this disorder is very rare and reports are scarce in the western literature , some cases of hmb have been reported from japan and taiwan . more than 90% of adults have been exposed to epstein - barr virus ( ebv ) and the virus persists in b cells and epithelial cells in the oropharynx . recently , ebv has been considered to be associated with not only b - cell malignancy but also t - cell malignancy and natural killer ( nk ) cell granular lymphocyte proliferative disorder ( glpd ) . here we report the first case of hmb associated with nk cell - derived large granular lymphocyte ( nk - lgl ) lymphocytosis in korea . a 19-year - old male was referred to our hospital with well - demarcated pustules on the erythematous base on the face / right ear ( figure 1 ) and with fever for 4 days . he had suffered hypersensitive reactions to mosquito bites , such as skin lesions ( ex , nodules , pustules , ulcerations ) , edematous change on the whole body and the extremities and fever , from childhood . laboratory tests showed white blood cell count 7.010/l , hemoglobin 15.0 g / dl , platelet 27210/l , biochemical profile , including lactate dehydrogenase , was in the normal range . ebv anti - ea - dr igg , anti - ebna was positive and serum titer of igg against ebv vca increased . immunophenotypic analysis demonstrated that cd16+cd56 + cells increased ( 79% ) and cd3 + , cd4 + , and cd8 + cells decreased ( 15% , 8% , 7% ) . we found rearrangement of tcr-chain gene by pcr analysis in this case ( figure 3 ) , as in some cases of the glpd . in our case , pcr amplification with specific primers for tcr-chain gene demonstrated a single band of approximately 160190 bp for v-j products . ebv , a widespread human herpes virus , infects > 90% of the population by adulthood and persists in b cells and epithelial cells in the oropharynx where reactivation and viral replication may intermittently occur . the virus has also been linked to various b cell , non - b cell neoplasms , such as endemic burkitt s lymphoma and nasopharyngeal carcinoma . ebv can infect t cells and peripheral lymphoproliferation of cd3 + cells may occur under caebv and , also , ebv can infect nk cells and may induce nk cell lymphoproliferation in patients with caebv . if the activity of ebv is responsible for the lymphoproliferation , the anti - ebv antibody titers , especially anti - vca igg or anti - ea igg might be related with lymphoproliferation . however , if our patient 's data , such as ebv anti - vca igg and ebv anti - ea igg , could not indicate caebv directly , this patient may be considered as caebv because nk cell lymphocytosis associated with ebv infection is frequently detected . to confirm the caebv , further examination of ebv , serial check - up for serum levels of anti - vca igg , iga and igm , anti - ea igg , iga and igm , and anti - ebna are required . according to ishihara et al , 31% of cases of caebv were complicated by hmb and suggested that the pathogenesis of hmb might be related to clonal lymphoproliferation of ebv dna - positive nk cells . this immunohematological abnormality may induce the characteristic symptoms of hmb . on the other hand , ishihara et al suggested that hmb might be one of the factors that induce ebv - associated lymphoproliferative disease . in this study , while three patients who did not manifest lymphoproliferation did not exhibit this history of hmb , four of six patients who did manifest monoclonal or oligoclonal lymphoproliferation had hmb . tokura et al have reported that nk cell - dominant mononuclear cells are infiltrated in mosquito bite sites of a severe hmb patient . it could be speculated that accumulation of nk cells augments reactions on the skin and that these nk cells may directly or indirectly mediate the systemic symptoms by the mosquito bites . recurrent and prolonged activated state of nk cells may induce additional genetic damage , including chromosomal changes that lead to the genesis of leukemias or lymphomas . in conclusion , we report the first case of hmb associated with nk - lgl lymphocytosis in korea , and further investigation of hmb pathogenesis and the relationship between ebv - infected nk cells and subsequent oncogenesis of nk - lgl lymphoma , including chronic nk - lgl lymphocytosis , is needed .

hypersensitivity to mosquito bites ( hmb ) is characterized by intense skin reactions at bite sites . the pathogenesis of hmb might be related to clonal lymphoproliferation of epstein - barr virus dna - positive natural killer ( nk ) cells . we report the first case of hmb possibly associated with nk cell - derived large granular lymphocyte ( nk - lgl ) lymphocytosis in korea .

INTRODUCTION CASE DISCUSSION

this study provides class ii evidence that teriflunomide generally does not adversely impact the ability of patients with rms to mount immune responses to influenza vaccination . the teriflunomide and vaccination ( teriva ) study ( nct01403376 ) was a multicenter , multinational , parallel - group study involving 128 patients in 3 treatment groups . groups 1 and 2 included patients with rms treated with either teriflunomide 7 mg or 14 mg once daily for at least 6 months over the course of 2 long - term extension studies ( nct00228163 : open - label extension of a phase ii study , which started in 2001 ; nct00803049 : blinded extension of the phase iii temso study , which started in 2004 ) . group 3 included patients with rms who had received a stable dose of ifn--1 for at least 6 months , and represents a reference population of patients with rms who have previously been reported to mount normal immune responses to seasonal influenza vaccination . the study comprised a screening period of up to 21 days , followed by administration of seasonal influenza vaccine on day 1 and antibody assessments at day 28 ( 2 days ) postimmunization ( figure e-1 on the neurology web site at www.neurology.org ) . patients were immunized with a single i m or intradermal administration of the 2011/2012 inactivated seasonal influenza vaccines , vaxigrip or mutagrip ( both sanofi pasteur , lyon , france ) . both vaccines contained the following influenza strains : a / california/7/2009 ( h1n1 ) , a / perth/16/2009 ( h3n2 ) , and b / brisbane/60/2008 ( b ) . choice of vaccine was performed according to country standards ; mutagrip was administered only in germany to 20 patients ( 12 in the ifn--1 group , and 3 and 5 in the 7-mg and 14-mg teriflunomide groups , respectively ) . the teriva study protocol and participation consents were submitted to independent ethics committees or institutional review boards , and subsequently reviewed and approved . the study was performed at sites approved to participate in either of the 2 long - term teriflunomide extension studies . subjects participating in these extension studies were asked to participate voluntarily in the teriva study . male or female subjects aged between 18 and 60 years with rms treated for at least 6 months with once - daily teriflunomide ( 7 mg or 14 mg ) or ifn--1 , and who were expected to remain on treatment for the duration of the study and who signed a specific informed consent form , were eligible for inclusion in the teriva study . subjects with concomitant infectious pathology at the time of vaccination , ms relapse within 1 month of vaccination , or administered systemic corticosteroids within 1 month of vaccination were excluded from the study . additional exclusion criteria included contraindication to influenza vaccine or in receipt of any vaccination within the last 6 months ; prior use of investigational drugs or participation in a clinical trial within 1 year before screening ( for patients in the ifn--1 group only ) ; prior / concomitant use ( within 1 year of study entry ) of cladribine , mitoxantrone , or other immunosuppressant agents ; prior / concomitant use of glatiramer acetate ( within 1 year of study entry ) ; or iv immunoglobulins ( within 3 months of study entry ) . pregnant or breastfeeding women were also excluded from the study . the hemagglutination inhibition assay ( hia ) hias were performed using serial 2-fold dilutions of serum and results were presented as titers ( i.e. , the highest dilution of serum that achieved complete inhibition of hemagglutination ) . evaluation of vaccine effectiveness in this study was performed in accordance with european guidelines , which apply 3 criteria to evaluate the immune response to influenza vaccine . first , the seroprotection rate ( i.e. , the proportion of subjects achieving a postvaccination titer 40 with the hia ) should be achieved in 70% of vaccinees postvaccination for individuals aged 1860 years and in 60% for those over 60 years . second , the mean geometric increase ( i.e. , the quotient of postvaccination and prevaccination geometric mean titers [ gmts ] ) should be 2.5 in individuals aged 1860 years and 2.0 in individuals over 60 years . third , the seroresponse rate ( i.e. , the proportion of previously seronegative subjects exceeding a postvaccination titer of 40 , and the proportion of previously seropositive subjects with a 4-fold increase in prevaccination and postvaccination sera ) should be achieved in 40% of vaccinees aged 1860 years and in 30% in those over 60 years . for each virus strain and each age class , at least one of the 3 criteria should be met . in this study , the primary efficacy endpoint was the proportion of patients who achieved seroprotection to the influenza vaccine strains , defined as an influenza antibody titer 40 for each strain 28 days postvaccination . the following secondary endpoints were also assessed : the proportion of patients with seroconversion ( i.e. , a prevaccination antibody titer 10 and a postvaccination antibody titer 40 ) ; the proportion of patients with a prevaccination antibody titer < 40 achieving seroprotection ; the proportion of patients with either a 2- or 4-fold increase in strain - specific antibody titers compared with prevaccination titers at 28 days postvaccination ; gmts at baseline and day 28 for each strain , and the corresponding gmt ratio ( postvaccination / prevaccination ) . assuming a response rate of around 70% ( measured as the proportion of patients with antibody titers 40 ) , sample size was calculated as 37 evaluable subjects per group based on the required number of subjects needed to obtain a precision of 12.5% ( corresponding to confidence interval [ ci ] widths of 25% ) around the point estimate of the true response rate under a 90% ci for the true response rate . efficacy analyses were conducted in the per - protocol population , which comprised all patients consenting to enter the study who received influenza vaccination and had an antibody titer at day 28 . the safety population comprised all patients who consented and were enrolled into the study . for the primary efficacy analysis , the proportion of patients with influenza antibody titers 40 at 28 days differences between each teriflunomide treatment group and the ifn--1 reference group were estimated along with 90% cis for each vaccine strain . although there were 2 teriflunomide treatments and 3 viral strains , no adjustments for multiplicity were made . for secondary efficacy analyses , the evaluation of 2- or 4-fold increases in influenza antibody titers compared with prevaccination titers , proportions of patients , and corresponding 90% cis using normal approximation were calculated for each strain and each treatment group . gmts at baseline and day 28 and the respective ratios ( postvaccination / prevaccination ) were summarized by strain and treatment group . for analysis of safety , incidences of treatment - emergent adverse events ( teaes ) , treatment - emergent serious adverse events ( saes ) , and adverse events leading to permanent treatment discontinuation occurring from time of vaccination to day 28 were documented within each group . the teriflunomide and vaccination ( teriva ) study ( nct01403376 ) was a multicenter , multinational , parallel - group study involving 128 patients in 3 treatment groups . groups 1 and 2 included patients with rms treated with either teriflunomide 7 mg or 14 mg once daily for at least 6 months over the course of 2 long - term extension studies ( nct00228163 : open - label extension of a phase ii study , which started in 2001 ; nct00803049 : blinded extension of the phase iii temso study , which started in 2004 ) . group 3 included patients with rms who had received a stable dose of ifn--1 for at least 6 months , and represents a reference population of patients with rms who have previously been reported to mount normal immune responses to seasonal influenza vaccination . the study comprised a screening period of up to 21 days , followed by administration of seasonal influenza vaccine on day 1 and antibody assessments at day 28 ( 2 days ) postimmunization ( figure e-1 on the neurology web site at www.neurology.org ) . patients were immunized with a single i m or intradermal administration of the 2011/2012 inactivated seasonal influenza vaccines , vaxigrip or mutagrip ( both sanofi pasteur , lyon , france ) . both vaccines contained the following influenza strains : a / california/7/2009 ( h1n1 ) , a / perth/16/2009 ( h3n2 ) , and b / brisbane/60/2008 ( b ) . choice of vaccine was performed according to country standards ; mutagrip was administered only in germany to 20 patients ( 12 in the ifn--1 group , and 3 and 5 in the 7-mg and 14-mg teriflunomide groups , respectively ) . the teriva study protocol and participation consents were submitted to independent ethics committees or institutional review boards , and subsequently reviewed and approved . the study was performed at sites approved to participate in either of the 2 long - term teriflunomide extension studies . subjects participating in these extension studies were asked to participate voluntarily in the teriva study . male or female subjects aged between 18 and 60 years with rms treated for at least 6 months with once - daily teriflunomide ( 7 mg or 14 mg ) or ifn--1 , and who were expected to remain on treatment for the duration of the study and who signed a specific informed consent form , were eligible for inclusion in the teriva study . subjects with concomitant infectious pathology at the time of vaccination , ms relapse within 1 month of vaccination , or administered systemic corticosteroids within 1 month of vaccination were excluded from the study . additional exclusion criteria included contraindication to influenza vaccine or in receipt of any vaccination within the last 6 months ; prior use of investigational drugs or participation in a clinical trial within 1 year before screening ( for patients in the ifn--1 group only ) ; prior / concomitant use ( within 1 year of study entry ) of cladribine , mitoxantrone , or other immunosuppressant agents ; prior / concomitant use of glatiramer acetate ( within 1 year of study entry ) ; or iv immunoglobulins ( within 3 months of study entry ) . the hemagglutination inhibition assay ( hia ) was used to detect strain - specific anti - influenza antibodies 28 days postimmunization . hias were performed using serial 2-fold dilutions of serum and results were presented as titers ( i.e. , the highest dilution of serum that achieved complete inhibition of hemagglutination ) . evaluation of vaccine effectiveness in this study was performed in accordance with european guidelines , which apply 3 criteria to evaluate the immune response to influenza vaccine . first , the seroprotection rate ( i.e. , the proportion of subjects achieving a postvaccination titer 40 with the hia ) should be achieved in 70% of vaccinees postvaccination for individuals aged 1860 years and in 60% for those over 60 years . second , the mean geometric increase ( i.e. , the quotient of postvaccination and prevaccination geometric mean titers [ gmts ] ) should be 2.5 in individuals aged 1860 years and 2.0 in individuals over 60 years . third , the seroresponse rate ( i.e. , the proportion of previously seronegative subjects exceeding a postvaccination titer of 40 , and the proportion of previously seropositive subjects with a 4-fold increase in prevaccination and postvaccination sera ) should be achieved in 40% of vaccinees aged 1860 years and in 30% in those over 60 years . for each virus strain and each age class , the primary efficacy endpoint was the proportion of patients who achieved seroprotection to the influenza vaccine strains , defined as an influenza antibody titer 40 for each strain 28 days postvaccination . the following secondary endpoints were also assessed : the proportion of patients with seroconversion ( i.e. , a prevaccination antibody titer 10 and a postvaccination antibody titer 40 ) ; the proportion of patients with a prevaccination antibody titer < 40 achieving seroprotection ; the proportion of patients with either a 2- or 4-fold increase in strain - specific antibody titers compared with prevaccination titers at 28 days postvaccination ; gmts at baseline and day 28 for each strain , and the corresponding gmt ratio ( postvaccination / prevaccination ) . assuming a response rate of around 70% ( measured as the proportion of patients with antibody titers 40 ) , sample size was calculated as 37 evaluable subjects per group based on the required number of subjects needed to obtain a precision of 12.5% ( corresponding to confidence interval [ ci ] widths of 25% ) around the point estimate of the true response rate under a 90% ci for the true response rate . efficacy analyses were conducted in the per - protocol population , which comprised all patients consenting to enter the study who received influenza vaccination and had an antibody titer at day 28 . the safety population comprised all patients who consented and were enrolled into the study . for the primary efficacy analysis , the proportion of patients with influenza antibody titers 40 at 28 days differences between each teriflunomide treatment group and the ifn--1 reference group were estimated along with 90% cis for each vaccine strain . although there were 2 teriflunomide treatments and 3 viral strains , no adjustments for multiplicity were made . for secondary efficacy analyses , the evaluation of 2- or 4-fold increases in influenza antibody titers compared with prevaccination titers , proportions of patients , and corresponding 90% cis using normal approximation were calculated for each strain and each treatment group . gmts at baseline and day 28 and the respective ratios ( postvaccination / prevaccination ) were summarized by strain and treatment group . for analysis of safety , incidences of treatment - emergent adverse events ( teaes ) , treatment - emergent serious adverse events ( saes ) , and adverse events leading to permanent treatment discontinuation occurring from time of vaccination to day 28 were documented within each group . the teriva study ran from september 2011 to january 2012 and involved 14 different study sites in 5 countries ( austria , canada , germany , russian federation , and ukraine ) . a total of 128 patients were enrolled into the study : 41 , 41 , and 46 patients in the teriflunomide 7 mg , teriflunomide 14 mg , and ifn--1 groups , respectively . the majority of patients in the ifn--1 group were receiving avonex ( ifn--1a ; 30 g once - weekly i m ; 34.8% ) , rebif ( ifn--1a ; 44 g 3 times per week sc ; 28.3% ) , or betaseron ( ifn--1b ; 0.25 mg every other day sc ; 21.7% ) ; the remaining patients received either genfaxone ( ifn--1a ; 44 g 3 times per week i m [ russia only ] ; 8.7% ) or rebif ( ifn--1a ; 22 g , 3 times per week sc ; 6.5% ) . of the 128 patients enrolled , 122 were included in the per - protocol population ( 40 , 39 , and 43 in the 7-mg , 14-mg , and ifn--1 groups , respectively ) . five patients were excluded from the per - protocol population because of recent or concomitant exposure to a systemic or topical corticosteroid , and one patient in the ifn--1 group was excluded because of poor compliance with study treatment . patients had received teriflunomide treatment for a median of 5.7 years ( range 1.610.4 years ) and the reference population received ifn--1 for a median duration of 5.7 years ( range 1.017.9 years ; tables e-1 and e-2 ) . more than half of the per - protocol population ( 57% ) had received influenza vaccination previously , more so for patients in the ifn--1 group ( 69.6% ) than either of the teriflunomide groups ( 43.9% and 56.1% for 7 mg and 14 mg , respectively ) . baseline demographics and characteristics ( per - protocol population ) more than 90% of patients achieved postvaccination antibody titers 40 for h1n1 and b in all groups . the proportion of patients achieving seroprotection to h3n2 was lower in the teriflunomide 14-mg group ( 76.9% ) compared with the teriflunomide 7-mg ( 90.0% ) and ifn--1 ( 90.7% ) groups ( table 2 ) . rates of seroprotection in the teriflunomide groups were consistent regardless of the total duration of treatment exposure prior to vaccination or the nature of the extension study from which patients were enrolled ( tables e-3 and e-4 ) . thus , more than 70% of patients achieved an hi titer of 40 for all influenza strains across all treatment groups ( table 2 and figure 1 ) . primary and secondary efficacy endpoints european criteria for efficacy of influenza vaccination in an 18- to 60-year - old population require achievement of a hemagglutination inhibition titer 40 by 70% of patients , as indicated by the dashed line . a high proportion of patients across all treatment groups had high hi titers ( 40 ) for each vaccine strain at baseline ( table 1 ) . this reflects the fact that between 44% and 70% of the study population had received influenza vaccination previously and also because the influenza vaccines used in this study included the same strains as those used in the 2010/2011 vaccine . of the limited number of patients with a prevaccination titer 10 , approximately 50% or more achieved seroconversion ( day 28 titer 40 ) , with similar proportions across each of the treatment groups . the lowest seroconversion rates were observed for the h3n2 strain in all 3 groups ( table 2 ) . the proportion of patients with a prevaccination titer < 40 achieving seroprotection was 61% across the 3 treatment groups and 3 influenza strains . however , fewer patients in the teriflunomide 14-mg group than the teriflunomide 7-mg or ifn--1 groups exhibited seroprotection to the h3n2 strain ( table 2 ) . the proportion of patients with a 2- or 4-fold increase in influenza strain - specific antibody titers from baseline to day 28 was slightly higher in the ifn--1 group than either of the teriflunomide groups ( table 2 ) . across all influenza strains , the gmt ratio postvaccination / prevaccination ranged from 2.3 to 3.1 in the teriflunomide groups and from 3.4 to 4.7 in the ifn--1 group . the ratio exceeded 2.5 for all treatment groups and for all influenza strains , with the exception of h1n1 in the 14-mg teriflunomide group ( table 2 ) . the study demonstrated no new safety concerns with teriflunomide administration , and influenza vaccination was generally well tolerated by the entire study population ( table e-5 ) . the first was a case of thoracic herpes zoster in a female patient in the teriflunomide 14-mg group , which occurred 9 days postvaccination , persisted for 14 days , was mild in severity , and responded to antiviral treatment . the second was a case of infective cholecystitis , also in the teriflunomide 14-mg group , which was associated with an increase in white blood count and facial rash , which had recovered by the end of the study . these 2 cases were not considered as saes . indeed , no saes were reported in this study , and the overall incidence of teaes was higher in the ifn--1 group ( 45.7% ) than the 2 teriflunomide groups ( 26.8% and 36.6% for 7 mg and 14 mg , respectively ) . the teriva study ran from september 2011 to january 2012 and involved 14 different study sites in 5 countries ( austria , canada , germany , russian federation , and ukraine ) . a total of 128 patients were enrolled into the study : 41 , 41 , and 46 patients in the teriflunomide 7 mg , teriflunomide 14 mg , and ifn--1 groups , respectively . the majority of patients in the ifn--1 group were receiving avonex ( ifn--1a ; 30 g once - weekly i m ; 34.8% ) , rebif ( ifn--1a ; 44 g 3 times per week sc ; 28.3% ) , or betaseron ( ifn--1b ; 0.25 mg every other day sc ; 21.7% ) ; the remaining patients received either genfaxone ( ifn--1a ; 44 g 3 times per week i m [ russia only ] ; 8.7% ) or rebif ( ifn--1a ; 22 g , 3 times per week sc ; 6.5% ) . of the 128 patients enrolled , 122 were included in the per - protocol population ( 40 , 39 , and 43 in the 7-mg , 14-mg , and ifn--1 groups , respectively ) . five patients were excluded from the per - protocol population because of recent or concomitant exposure to a systemic or topical corticosteroid , and one patient in the ifn--1 group was excluded because of poor compliance with study treatment . patients had received teriflunomide treatment for a median of 5.7 years ( range 1.610.4 years ) and the reference population received ifn--1 for a median duration of 5.7 years ( range 1.017.9 years ; tables e-1 and e-2 ) . more than half of the per - protocol population ( 57% ) had received influenza vaccination previously , more so for patients in the ifn--1 group ( 69.6% ) than either of the teriflunomide groups ( 43.9% and 56.1% for 7 mg and 14 mg , respectively ) . more than 90% of patients achieved postvaccination antibody titers 40 for h1n1 and b in all groups . the proportion of patients achieving seroprotection to h3n2 was lower in the teriflunomide 14-mg group ( 76.9% ) compared with the teriflunomide 7-mg ( 90.0% ) and ifn--1 ( 90.7% ) groups ( table 2 ) . rates of seroprotection in the teriflunomide groups were consistent regardless of the total duration of treatment exposure prior to vaccination or the nature of the extension study from which patients were enrolled ( tables e-3 and e-4 ) . thus , more than 70% of patients achieved an hi titer of 40 for all influenza strains across all treatment groups ( table 2 and figure 1 ) . primary and secondary efficacy endpoints european criteria for efficacy of influenza vaccination in an 18- to 60-year - old population require achievement of a hemagglutination inhibition titer 40 by 70% of patients , as indicated by the dashed line . a high proportion of patients across all treatment groups had high hi titers ( 40 ) for each vaccine strain at baseline ( table 1 ) . this reflects the fact that between 44% and 70% of the study population had received influenza vaccination previously and also because the influenza vaccines used in this study included the same strains as those used in the 2010/2011 vaccine . of the limited number of patients with a prevaccination titer 10 , approximately 50% or more achieved seroconversion ( day 28 titer 40 ) , with similar proportions across each of the treatment groups . the lowest seroconversion rates were observed for the h3n2 strain in all 3 groups ( table 2 ) . the proportion of patients with a prevaccination titer < 40 achieving seroprotection was 61% across the 3 treatment groups and 3 influenza strains . however , fewer patients in the teriflunomide 14-mg group than the teriflunomide 7-mg or ifn--1 groups exhibited seroprotection to the h3n2 strain ( table 2 ) . the proportion of patients with a 2- or 4-fold increase in influenza strain - specific antibody titers from baseline to day 28 was slightly higher in the ifn--1 group than either of the teriflunomide groups ( table 2 ) . across all influenza strains , the gmt ratio postvaccination / prevaccination ranged from 2.3 to 3.1 in the teriflunomide groups and from 3.4 to 4.7 in the ifn--1 group . the ratio exceeded 2.5 for all treatment groups and for all influenza strains , with the exception of h1n1 in the 14-mg teriflunomide group ( table 2 ) . the study demonstrated no new safety concerns with teriflunomide administration , and influenza vaccination was generally well tolerated by the entire study population ( table e-5 ) . the first was a case of thoracic herpes zoster in a female patient in the teriflunomide 14-mg group , which occurred 9 days postvaccination , persisted for 14 days , was mild in severity , and responded to antiviral treatment . the second was a case of infective cholecystitis , also in the teriflunomide 14-mg group , which was associated with an increase in white blood count and facial rash , which had recovered by the end of the study . these 2 cases were not considered as saes . indeed , no saes were reported in this study , and the overall incidence of teaes was higher in the ifn--1 group ( 45.7% ) than the 2 teriflunomide groups ( 26.8% and 36.6% for 7 mg and 14 mg , respectively ) . the teriva study demonstrates that patients with rms treated with teriflunomide are generally able to mount effective immune responses to seasonal influenza vaccination . specifically , the proportion of patients with postvaccination antibody titers 40 to all influenza strains was greater than 70% in both teriflunomide treatment groups ( range 76.997.5% ) , thereby meeting the european criterion for the efficacy of influenza vaccine in adult subjects aged 1860 years , which was a priori identified as the primary criterion . neither the duration of teriflunomide treatment prior to vaccination nor the origin of the teriflunomide extension study in which patients were enrolled had a significant impact on overall seroprotection rates . thus , vaccine responses were preserved in patients treated with teriflunomide across a broad range of treatment exposure , ranging from 1.6 to 10.4 years . although the response to all vaccine strains in all treatment groups was sufficient for vaccination to be considered protective , it should be noted that there was a slightly diminished response in the teriflunomide 14-mg group with respect to some evaluations for the h3n2 and h1n1 strains . nevertheless , the development of adequate protective immune responses confirms the ability of teriflunomide to inhibit the activity of pathogenic autoreactive lymphocytes without causing global immunosuppression . no death , sae , or teae leading to treatment discontinuation was reported in the study , and no new or unexpected safety observations arose in either of the teriflunomide treatment groups . these safety data are in accordance with those from the temso trial , in which teriflunomide was generally well tolerated with a well - characterized safety profile . furthermore , the low incidence of serious infections and the absence of serious opportunistic infections seen in temso lends further support to the observations made in this study that immune surveillance is preserved under teriflunomide treatment . immune responses to influenza vaccine in patients with ms treated with ifn--1a were similar to untreated patients with respect to the proportion of patients achieving hi titers 40 . our study was consistent with the literature showing that effective immune responses were observed in a reference rms population treated with ifn--1 for at least 6 months , with good safety and tolerability . it should be noted that this study was not designed or powered to make direct comparisons between immune responses in the teriflunomide groups and the ifn--1 reference population . effective influenza vaccination remains important in patients with ms because influenza can cause serious complications and has been shown to be associated with a higher occurrence of exacerbations in patients with ms . the observations made in this study are therefore highly reassuring for patients administered teriflunomide treatment . we conclude that influenza vaccine was found to be safe and effective in patients with ms receiving teriflunomide . our findings , combined with efficacy and safety data from the temso study , support the view that while teriflunomide appears to effectively limit abnormal activation of pathogenic lymphocyte responses implicated in ms relapses , teriflunomide therapy does not significantly interfere with adaptive activation of immune responses and generally appears to spare the serologic response to influenza antigens . amit bar - or , franoise menguy - vacheron , deborah bauer , stefan jodl , philippe truffinet , and myriam benamor contributed to the development of the study design , data analysis , and interpretation . they were all involved in the development , reviewing , and revising of the manuscript . mark s. freedman , marcelo kremenchutzky , and paul w. oconnor all contributed to the development of the study design . deborah bauer was responsible for all statistical analyses of the data , as well as development and revising of the manuscript . scott chambers is a medical writer who provided editorial support in the preparation and editing of the manuscript text , figures , and tables , addressing author , reviewer , and editorial comments , and assisting with the submission process . a. bar - or : speaker , consulting fees , and/or research support : amplimmune , aventis , bayhill therapeutics , berlex / bayer , biogen idec , bioms , diogenix , eli - lilly , emd serono , genentech , genzyme - sanofi , gsk , guthy jackson greater good foundation , medimmune , mitsubishi pharma , novartis , ono pharmacia , receptos , roche , teva neuroscience , wyeth . freedman : research / educational grant support : genzyme ; consulting fees : actelion , bayer , biogen idec , celgene , genzyme , glycominds , teva , merck serono , novartis , opexa , and sanofi - aventis . m. kremenchutzky : consulting fees and/or research support from bayer , biogen idec , emd serono , genzyme , novartis , sanofi - aventis , and teva scott chambers : employee of fishawack communications ltd , contracted to provide editorial services to sanofi , funded by genzyme , a sanofi company . p.w . oconnor : consulting fees and/or research support : actelion , bayer , biogen idec , bioms , cognosci , daiichi sankyo , emd serono , genentech , genmab , novartis , roche , sanofi - aventis , teva , and warburg pincus .

objective : to investigate the effect of teriflunomide on the efficacy and safety of seasonal influenza vaccine.methods:the 2011/2012 seasonal influenza vaccine ( containing h1n1 , h3n2 , and b strains ) was administered to patients with relapsing forms of multiple sclerosis ( rms ) treated for 6 months with teriflunomide 7 mg ( n = 41 ) or 14 mg ( n = 41 ) , or interferon--1 ( ifn--1 ; n = 46 ) . the primary endpoint was the proportion of patients with influenza strain specific antibody titers 40 , 28 days postvaccination.results:more than 90% of patients achieved postvaccination antibody titers 40 for h1n1 and b in all groups . for h3n2 , titers 40 were achieved in 90% of patients in the 7 mg and ifn--1 groups , and in 77% of the 14-mg group , respectively . a high proportion of patients already had detectable antibodies for each influenza strain at baseline . geometric mean titer ratios ( post / prevaccination ) were 2.5 for all groups and strains , except for h1n1 in the 14-mg group ( 2.3 ) . the proportion of patients with a prevaccination titer < 40 achieving seroprotection was 61% across the 3 treatment groups and 3 influenza strains . however , fewer patients in the 14-mg than the 7-mg or ifn--1 groups exhibited seroprotection to h3n2 ( 61% vs 78% and 82% , respectively).conclusion : teriflunomide - treated patients generally mounted effective immune responses to seasonal influenza vaccination , consistent with preservation of protective immune responses.classification of evidence : this study provides class ii evidence that teriflunomide generally does not adversely impact the ability of patients with rms to mount immune responses to influenza vaccination .

Classification of evidence: METHODS Study design. Standard protocol approvals, registrations, and patient consents. Study population. Study endpoints. Statistical analysis. RESULTS Study disposition and baseline demographics. Efficacy. Safety and tolerability. DISCUSSION Supplementary Material AUTHOR CONTRIBUTIONS STUDY FUNDING DISCLOSURE

ayurveda as a medicinal system assesses deviation from health due to development of whole body system imbalance . these imbalances are among the basic concepts in ayurveda which are now well understood , and which include the doshas and subdoshas in terms of which the imbalances are expressed . it is now known that diet and lifestyle interventions derived from ayurveda can help restore the system to balanced functioning and for these reasons ayurveda interventions always have multiple components including diet and lifestyle components . ayurveda and other traditional medical systems often prescribe complex treatments consisting of a combination of drugs , diet , detoxification procedures , lifestyle changes , and yoga practices , customized to the needs of individual patients . the question therefore arises , can one report the outcome of such multi - component , individualized interventions with unique end points , in a manner that is not merely anecdotal , so that the report contributes positively to the corpus of ayurvedic clinical science ? various initiatives for methodological harmonization and quality improvement of clinical research have been published and are now widely accepted . the consolidated standards of reporting trials ( consort ) statement offers a minimum set of recommendations for reporting randomized clinical trials . recent work on the spirit initiative towards standards of designing protocols has been a welcome extension of reporting guidelines . in epidemiology , the strengthening the reporting of observational studies in epidemiology ( strobe ) statement similarly aims to strengthen reporting of observational studies , while the meta - analysis of observational studies in epidemiology ( moose ) group has prepared checklists for meta - analysis of observational studies . transparent reporting of evaluations with non - randomized designs ( trend ) statements concern methods of research design to eliminate biases in non - randomized studies . specifically related to ayurvedic intervention , narahari et.al have provided a standard framework under which to develop protocols and subsequent reports . in addition , the government of india has an official guidance on clinical trials of herbal remedies and medicinal plants , which is a part of the more general guidance on conduct of clinical trials in india . however , detailed development of guidelines for reporting of ayurvedic clinical trials has not been done to the fullest extent . this paper specifically considers the question of how to develop these standards of reporting of ayurvedic clinical trials designed in accordance with its holistic theory and practice , so that objective lessons can be learned from the experience . to address the question of standards of reporting clinical trials of ayurveda , one must ensure adherence to the three essential pre - requisites of good clinical science . firstly , reporting must be transparent , honest and accurate ; secondly , it must be scientifically unbiased ; and thirdly , all interventions used in the trial must be ethical . in the recent past , design of clinical trials for ayurvedic interventions has abandoned its multi - component , holistic approach . instead , trials have blindly emulated the simpler single drug - single disease intervention models used in western medicine . the limitations of this approach have been two - fold : ( 1 ) ayurveda 's complete interventions as used in normal clinical practice have not been put to the test ; rather , what has been tested has only been some part of the whole intervention normally prescribed ; ( 2 ) end - points used to signal positive outcome have not been defined in terms of ayurvedic clinical management principles ; nor have creative efforts been made to measure them . this mismatch between ayurvedic principles and scientific trial design has resulted in conclusions about treatment efficacy that are not in accord with the clinical experiences of practitioners of authentic ayurveda . these differences have in turn resulted in skepticism concerning ayurveda 's clinical claims in the world of professional medicine . in the context of developing standards for ayurvedic clinical trials , it must be firmly asserted that expertise in the use of ayurveda interventions lies solely with ayurvedic knowledge holders , and not with their collaborators , including statisticians . it is not wise to advise ayurvedic physicians on how to conduct ayurvedic clinical management of any particular health condition . however , fellow scientists and statisticians who are interested in appreciating the ayurveda knowledge system are fully justified in insisting on honest , accurate , scientifically unbiased , transparent and standardized reporting of all ayurvedic clinical trials . guidelines on reporting ( as opposed to conducting ) trials will play a major role in establishing credibility of ayurvedic clinical management . such guidelines , for example , will require transparency in reporting details for the following points : - reasons for conducting the clinical trial , its underlying rationale ? this is important in establishing the ethics , and in ensuring that all human experimentation is in line with the helsinki declaration on human experimentation.whether the goals of the clinical trial correspond to its stated rationale . internal validity of the trial.whether the trial 's design elements ensure that its goals can be achieved . this guideline should include discussion on how the patients / subjects were selected ( inclusion and exclusion criteria ) , how many ( to ensure lack of scientific bias ) , duration of the intervention , and the experience and qualifications of ayurvedic practitioners conducting the trial . if addressed transparently , the design elements lead to clear understanding of biases.whether the disease under study was appropriately defined i.e. based on ayurvedic knowledge ( listing all end - points associated with the disease with due weightages ) . clarity of disease definition helps minimize subjectivity in patient selection , as well as biases in assessing end - points related to the disease.were the various components prescribed for the intervention based on customized diagnosis ? were all details of its complexity included ? clear and full definitions of the interventions used , including placebo or comparator interventions allows others to assess reasons for the trial 's failure or success , and will help them replicate the trial should they be interested in doing so.definitions of the end - points indicating a positive outcome for the patient , and how to measure them - success or failure of an intervention depends on objective definitions of the presence or absence of pathology . along with the previous point on definition of the disease , this helps make reports clearly unbiased in assessing an intervention 's success.actual patient history during the trial , including details of patient selection procedures , patient drop - out and associated reasons , and all records of adverse reactions or events ? transparency in reporting individual patient flow during the trial helps in assessing the internal validity of the trial , which is a first step to establishing its external validity.discussions of net results obtained , keeping in mind the original rationale for conducting the trial . discussions should include points on ( 1 ) the internal validity of the trial including clear discussion on how closely conduct of the trial followed its written protocol ; ( 2 ) external validity of the trial including analyses of results vis - - vis the latest scientific indicators ( e.g. bio - markers , state of the art scientific breakthroughs ) and extrapolations of results to a wider population than that studied in the trial . reasons for conducting the clinical trial , its underlying rationale ? this is important in establishing the ethics , and in ensuring that all human experimentation is in line with the helsinki declaration on human experimentation . whether the goals of the clinical trial correspond to its stated rationale internal validity of the trial . whether the trial 's design elements ensure that its goals can be achieved . this guideline should include discussion on how the patients / subjects were selected ( inclusion and exclusion criteria ) , how many ( to ensure lack of scientific bias ) , duration of the intervention , and the experience and qualifications of ayurvedic practitioners conducting the trial . if addressed transparently , the design elements lead to clear understanding of biases . whether the disease under study was appropriately defined i.e. based on ayurvedic knowledge ( listing all end - points associated with the disease with due weightages ) . clarity of disease definition helps minimize subjectivity in patient selection , as well as biases in assessing end - points related to the disease . clear and full definitions of the interventions used , including placebo or comparator interventions allows others to assess reasons for the trial 's failure or success , and will help them replicate the trial should they be interested in doing so . definitions of the end - points indicating a positive outcome for the patient , and how to measure them - success or failure of an intervention depends on objective definitions of the presence or absence of pathology . along with the previous point on definition of the disease , this helps make reports clearly unbiased in assessing an intervention 's success . actual patient history during the trial , including details of patient selection procedures , patient drop - out and associated reasons , and all records of adverse reactions or events ? transparency in reporting individual patient flow during the trial helps in assessing the internal validity of the trial , which is a first step to establishing its external validity . discussions of net results obtained , keeping in mind the original rationale for conducting the trial . discussions should include points on ( 1 ) the internal validity of the trial including clear discussion on how closely conduct of the trial followed its written protocol ; ( 2 ) external validity of the trial including analyses of results vis - - vis the latest scientific indicators ( e.g. bio - markers , state of the art scientific breakthroughs ) and extrapolations of results to a wider population than that studied in the trial . as previously emphasized , demand for standardization in reporting should in no way restrict ayurvedic physicians from practicing their art faithfully . guidelines only go to promote the report 's objectivity , scientific validity and ethical considerations . in other words , standards of reporting are not about clinical management , but offer guidelines on how to report faithfully , comprehensively and accurately on what actually occurs during trials . such standards of reporting will allow all readers to enjoy objective conclusions , and obtain better pictures of detailed strategies and ethical norms used in trials . for example : a report may state , the inclusion criteria for subjects and choices of end - points are based on western definitions of the disease , only treatment is based on ayurvedic principles. such an admission will encourage the reader to ask how the western definition correlates with the corresponding ayurvedic definition , and the extent to which the ayurvedic end - points would correspond with western ones . ( one - to - one correspondences are unlikely ; generally , the relationship will be many - to - one.)an unclear rationale may raise questions about the ethics of conducting the trial . if the statistical reasoning used to determine the number of subjects is inadequate , it may prove impossible to draw conclusions from the resulting trial . using patients as subjects for such a trial could become an ethical issue , quite apart from being a scientific problem.any mismatch between the definitions of a disease and its cure , will cause problems in interpreting results . ensuring that both are clearly defined should eliminate them.state whether blinding was , or was not , carried out . blinding is conducted to eliminate performance or assessment bias . in ayurvedic trials , it is often inappropriate and not done . explicitly reporting this to be the case , and listing associated reasons , may stimulate readers to think of alternative strategies to minimize performance bias.similarly , randomization should be reported , as should the method of carrying it out , since randomization reduces biases arising in patient selection . any selection bias can be reduced by having a comparison group , and randomly assigning patients between intervention and comparison groups . the comparison group can be a placebo group or a standard treatment group , or , if neither is feasible , it could even be historical data ( clearly identified and defined in the protocol ) from previous studies.patient flow : all details of patients enrolled for the trial whether or not they completed it , inform the reader about deviations from the written protocol during implementation , so reporting them is important.all the points and examples emphasize the three basic principles under which reporting should be done - transparency , scientifically unbiased and ethically strong . a report may state , the inclusion criteria for subjects and choices of end - points are based on western definitions of the disease , only treatment is based on ayurvedic principles. such an admission will encourage the reader to ask how the western definition correlates with the corresponding ayurvedic definition , and the extent to which the ayurvedic end - points would correspond with western ones . ( one - to - one correspondences are unlikely ; generally , the relationship will be many - to - one . ) if the statistical reasoning used to determine the number of subjects is inadequate , it may prove impossible to draw conclusions from the resulting trial . using patients as subjects for such a trial could become an ethical issue , quite apart from being a scientific problem . any mismatch between the definitions of a disease and its cure , will cause problems in interpreting results . ensuring that both are clearly defined should eliminate them . in ayurvedic trials , it is often inappropriate and not done . explicitly reporting this to be the case , and listing associated reasons similarly , randomization should be reported , as should the method of carrying it out , since randomization reduces biases arising in patient selection . any selection bias can be reduced by having a comparison group , and randomly assigning patients between intervention and comparison groups . the comparison group can be a placebo group or a standard treatment group , or , if neither is feasible , it could even be historical data ( clearly identified and defined in the protocol ) from previous studies . patient flow : all details of patients enrolled for the trial whether or not they completed it , inform the reader about deviations from the written protocol during implementation , so reporting them is important . all the points and examples emphasize the three basic principles under which reporting should be done - transparency , scientifically unbiased and ethically strong . various initiatives for methodological harmonization and quality improvement of clinical research have been published and are now widely accepted . guidelines suggested by narahari and developed in consort for herbal medicines are good building blocks . narahari 's paper emphasizes the steps necessary for ensuring that good clinical science is practiced through a specific example . by building on his ideas and generalizing them the consort statement for herbal medicine is also a good buidling block and needs to be enhanced with ayurvedic specific definitions of interventions and disease cures . complexity arising due to these difficult issues needs to be added for reporting of ayurvedic clinical trials . this work clearly highlights the complexity involved in chinese interventions and definitions of disease state and cure . adaptations of conosrt priniciples , which allow for traditional chinese interventions offer guidance for ayurvedic scientists . an added complexity for an extensively practiced healthcare system like however , neither data collection nor reporting of any of these clinical data are standardized . currently generated reports include : data from single case studies , generally reported as anecdotes.data from observational studies reported without addressing their limitations , such as performance and selection biases and consequent shortcomings.data from clinical trials which are improperly designed or have interventions based on a reductionist approach . data from single case studies , generally reported as anecdotes . data from observational studies reported without addressing their limitations , such as performance and selection biases and consequent shortcomings . data from clinical trials which are improperly designed or have interventions based on a reductionist approach . in western bio - medicine case studies , observational studies , clinical trials and meta - analyses are used for specific purposes at successive stages of drug development . in contrast , in ayurveda all four kinds of study can be used to establish its evidence base . in particular , well - defined meta - analyses should be carried out to summarize clinical results across multiple trials . we need to develop guidelines for each one of them , namely , ayurvedic case reports , ayurvedic observation studies , and ayurvedic clinical trials . as has been seen in the western biomedicine case , standardizing reporting guidelines will ensure that good clinical science is practiced and reported . the time is now right to build on existing guidelines and , where none exist , develop them .

reported lack of efficacy of ayurvedic treatments in clinical trials is often not due to inefficacy of the treatment itself , but arises from inadequacies of trial design . this paper argues that trials of ayurvedic interventions should exclusively use its multi - component , individualized and inherently holistic approach , and that general guidelines for rigorously reporting such clinical trials should be developed . holistic ayurvedic clinical trials , rigorously conducted and with high standards of reporting should translate into good clinical science , and may be expected to generate higher credibility for clinical studies of the ayurvedic knowledge system .

INTRODUCTION STANDARDS OF REPORTING CLINICAL TRIALS OF AYURVEDA DISCUSSION

in 2012 , prostate cancer was the fifth most commonly diagnosed malignancy in korean men , with an estimated 9,258 new cases detected annually . nearly 50,000 korean men are living with prostate cancer , of whom 1,460 die from the disease each year . compared with western countries , prostate cancer is less common in korea ( and in asian countries in general ) , although its prevalence doubled between 2002 and 2007 , likely due to demographic aging , changes in diet , and increased prostate - specific antigen ( psa ) screening . for example , a retrospective analysis of prostate cancer patient data collected between 2006 and 2010 from a tertiary care center ( gangnam severance hospital , yonsei university college of medicine , seoul , korea ) revealed a steady increase in the prevalence of prostate cancer , with 21% of patients reporting advanced or castration - resistant prostate cancer ( crpc ) in 2010 . as prostate cancer progresses metastases develop , and most cases respond to androgen deprivation therapy ( adt ) , at least initially . unfortunately , resistance to adt always ensues , resulting in a transition to metastatic crpc ( mcrpc ) , a lethal clinical state defined by cancer progression despite effective lowering of serum testosterone to < 50 ng / dl or 1.7 nmol / l . use of docetaxel plus prednisone is the current standard of care for mcrpc in korea , but this regimen is associated with toxicity and , ultimately , drug resistance . recent evidence has highlighted the ongoing , central role of androgen receptor ( ar ) signaling in crpc , suggesting that further inhibition of this pathway with noncytotoxic therapies may confer a survival benefit . firstly , affected patients often have elevated serum psa levels and measurable intratumoral androgens despite castrate levels of testosterone . secondly , preclinical data showed that ar overexpression is sufficient to confer resistance to adt in prostate cancer cell lines . enzalutamide is an ar inhibitor that has shown an 8-fold greater affinity for the ar than bicalutamide in studies of lncap / ar cells ( half maximal inhibitory concentration , 21 nm vs. 160 nm ) . enzalutamide also has the capacity to reduce the efficiency of ar nuclear translocation and impair ar binding to dna . in chemotherapy - nave men with mcrpc , findings from the international , randomized , double - blind , phase 3 , prevail trial demonstrated that enzalutamide improved overall survival ( os ; 32.4 months vs. 30.2 months ; hazard ratio [ hr ] , 0.71 ; 95% confidence interval [ ci ] , 0.600.84 ; p<0.001 ) and radiographic progression - free survival ( rpfs ; not yet reached vs. 3.9 months ; hr , 0.19 ; 95% ci , 0.150.23 ; p<0.001 ) . prevail data showed that the benefit of enzalutamide extended to all secondary end points , including the time until the first skeletal - related event ( sre ) , soft - tissue response rate , time until psa progression , and psa response rate ( 50% decline ) , as well as patient - reported outcomes . enzalutamide was approved in korea in june 2013 for use in men with crpc postchemotherapy based on results of the affirm trial . in may 2015 the indication was expanded to include men with crpc regardless of prior chemotherapy exposure based on results of the prevail trial . in this post hoc analysis , we evaluated the treatment effects , safety , and pharmacokinetic exposure of enzalutamide versus placebo in prevail patients from korean study sites , as well as the consistency of these results with those in the overall population . the full methodology of prevail ( nct01212991 ) , including patient eligibility , end point definitions , and study conduct has been reported . prevail was approved by the independent review board at each participating site and was conducted according to provisions of the declaration of helsinki and good clinical practice guidelines of the international conference on harmonization of technical requirements for registration of pharmaceuticals for human use . briefly , consenting patients were chemotherapy - nave men aged at least 18 years with histologically or cytologically confirmed adenocarcinoma of the prostate that was castration resistant , with evidence of progression by imaging and/or testosterone level of 1.73 nmol / l ( 50 ng / dl ) or less . eligible patients had not received abiraterone acetate , had an eastern cooperative oncology group performance status ( ecog ps ) of 0 ( asymptomatic , fully active ) or 1 ( ambulatory but restricted in strenuous activities ) , and were asymptomatic or mildly symptomatic based on the brief pain inventory short form question 3 ( i.e. , pain score 03 ) . medical castration with a gonadotropin - releasing hormone analog was required in patients without orchiectomy . concomitant use of corticosteroids was permitted with doses equivalent to 10 mg / d of prednisone or prednisolone . patients with visceral disease or with new york heart association class i or ii heart failure were eligible . patients with conditions that could lower the seizure threshold ( e.g. , brain metastases , history of seizure , concurrent medications ) , prior use of chemotherapy , or new york heart association class iii or iv heart failure were excluded . patients were enrolled from september 2010 through september 2012 at 207 sites globally , seven of which were in korea . , san francisco , ca , usa and astellas pharma inc . , northbrook , il , usa ) or placebo once daily , with randomization stratified by study site . treatment was discontinued for occurrence of unacceptable side effects , confirmed radiographic progression , or confirmed sre and either initiation of cytotoxic therapy or an investigational agent for prostate cancer . radiographic progression - free survival was defined as the time from randomization to the first objective evidence of radiographic disease progression assessed by a blinded independent central review facility or death due to any cause within 168 days after treatment discontinuation , whichever occurred first . radiographic disease progression was evaluated using the prostate cancer clinical trials working group guidelines for bone disease and response evaluation criteria in solid tumors version 1.1 for soft - tissue disease and included confirmed new bone lesions and new soft - tissue lesions . imaging was performed at screening , at weeks 9 , 17 , and 25 , and every 12 weeks thereafter . secondary end points included rpfs by investigator review , time to initiation of chemotherapy , time to psa progression , psa response , overall soft - tissue response , and time to sre . time to initiation of chemotherapy was defined as the time from randomization to initiation of an antineoplastic agent for prostate cancer . time to psa progression was defined as the time from randomization to first confirmed psa progression . psa response was defined as a decline in psa of at least 50% from baseline as determined by the local laboratory ( confirmed by a second assessment conducted 3 weeks later ) . time to sre was defined as the time from randomization to first occurrence of radiation therapy or surgery to bone for prostate cancer , pathological bone fracture , spinal cord compression , or change of antineoplastic therapy to treat bone pain . safety was evaluated in all randomized patients who received at least one dose of study drug . adverse events ( aes ) were graded according to the common terminology criteria for adverse events version 4 . in all patients , blood was collected for the determination of predose minimum plasma concentrations ( cmin ) of enzalutamide and its active metabolite n - desmethyl enzalutamide at weeks 5 , 13 , and 25 . the samples were stored at 70 until required for analysis by covance ( princeton , nj , usa ) , as described elsewhere . briefly , both analytes were extracted from plasma by a liquid - liquid extraction procedure , separated by reversed phase high - performance liquid chromatography , and detected by tandem mass spectrometry , in accordance with u.s . food and drug administration guidance . the quantitation for both analytes was 0.02 to 50.0 g / ml . a post hoc analysis of patients enrolled in korean study sites at the time of study entry was performed for the coprimary end points , aes , and all secondary and exploratory end points . final results for the entire prevail study were based on 439 centrally assessed rpfs events ( cutoff date may 6 , 2012 ) and 540 deaths patients randomized after may 6 , 2012 , were not included in the final rpfs analysis . thus , an analysis of investigator - assessed rpfs at the final os data cutoff date ( september 16 , 2013 ) and an updated exploratory analysis of os at the prespecified final number of deaths ( 765 ; cutoff date june 1 , 2014 ) were also performed . in this post hoc analysis , baseline characteristics and treatment effects were evaluated in the intent - to - treat population ( all randomly assigned patients ) . the hr relative to placebo , with < 1.00 favoring enzalutamide , was determined using an unstratified cox regression model with treatment as the only covariate . the mean minimum concentration of the sum of enzalutamide plus n - desmethyl enzalutamide at weeks 5 , 13 , and 25 were adjusted for weight using log - linear regression . the relative systemic exposure to the sum of enzalutamide plus n - desmethyl enzalutamide in korean and non - korean patients was expressed as the ratio of their weight - adjusted cmin values , including 90% cis . the full methodology of prevail ( nct01212991 ) , including patient eligibility , end point definitions , and study conduct has been reported . prevail was approved by the independent review board at each participating site and was conducted according to provisions of the declaration of helsinki and good clinical practice guidelines of the international conference on harmonization of technical requirements for registration of pharmaceuticals for human use . briefly , consenting patients were chemotherapy - nave men aged at least 18 years with histologically or cytologically confirmed adenocarcinoma of the prostate that was castration resistant , with evidence of progression by imaging and/or testosterone level of 1.73 nmol / l ( 50 ng / dl ) or less . eligible patients had not received abiraterone acetate , had an eastern cooperative oncology group performance status ( ecog ps ) of 0 ( asymptomatic , fully active ) or 1 ( ambulatory but restricted in strenuous activities ) , and were asymptomatic or mildly symptomatic based on the brief pain inventory short form question 3 ( i.e. , pain score 03 ) . medical castration with a gonadotropin - releasing hormone analog was required in patients without orchiectomy . concomitant use of corticosteroids was permitted with doses equivalent to 10 mg / d of prednisone or prednisolone . patients with visceral disease or with new york heart association class i or ii heart failure were eligible . patients with conditions that could lower the seizure threshold ( e.g. , brain metastases , history of seizure , concurrent medications ) , prior use of chemotherapy , or new york heart association class iii or iv heart failure were excluded . patients were enrolled from september 2010 through september 2012 at 207 sites globally , seven of which were in korea . , san francisco , ca , usa and astellas pharma inc . , northbrook , il , usa ) or placebo once daily , with randomization stratified by study site . treatment was discontinued for occurrence of unacceptable side effects , confirmed radiographic progression , or confirmed sre and either initiation of cytotoxic therapy or an investigational agent for prostate cancer . radiographic progression - free survival was defined as the time from randomization to the first objective evidence of radiographic disease progression assessed by a blinded independent central review facility or death due to any cause within 168 days after treatment discontinuation , whichever occurred first . radiographic disease progression was evaluated using the prostate cancer clinical trials working group guidelines for bone disease and response evaluation criteria in solid tumors version 1.1 for soft - tissue disease and included confirmed new bone lesions and new soft - tissue lesions . imaging was performed at screening , at weeks 9 , 17 , and 25 , and every 12 weeks thereafter . secondary end points included rpfs by investigator review , time to initiation of chemotherapy , time to psa progression , psa response , overall soft - tissue response , and time to sre . time to initiation of chemotherapy was defined as the time from randomization to initiation of an antineoplastic agent for prostate cancer . time to psa progression was defined as the time from randomization to first confirmed psa progression . psa response was defined as a decline in psa of at least 50% from baseline as determined by the local laboratory ( confirmed by a second assessment conducted 3 weeks later ) . time to sre was defined as the time from randomization to first occurrence of radiation therapy or surgery to bone for prostate cancer , pathological bone fracture , spinal cord compression , or change of antineoplastic therapy to treat bone pain . safety was evaluated in all randomized patients who received at least one dose of study drug . adverse events ( aes ) were graded according to the common terminology criteria for adverse events version 4 . in all patients , blood was collected for the determination of predose minimum plasma concentrations ( cmin ) of enzalutamide and its active metabolite n - desmethyl enzalutamide at weeks 5 , 13 , and 25 . the samples were stored at 70 until required for analysis by covance ( princeton , nj , usa ) , as described elsewhere . briefly , both analytes were extracted from plasma by a liquid - liquid extraction procedure , separated by reversed phase high - performance liquid chromatography , and detected by tandem mass spectrometry , in accordance with u.s . food and drug administration guidance . a post hoc analysis of patients enrolled in korean study sites at the time of study entry was performed for the coprimary end points , aes , and all secondary and exploratory end points . final results for the entire prevail study were based on 439 centrally assessed rpfs events ( cutoff date may 6 , 2012 ) and 540 deaths patients randomized after may 6 , 2012 , were not included in the final rpfs analysis . thus , an analysis of investigator - assessed rpfs at the final os data cutoff date ( september 16 , 2013 ) and an updated exploratory analysis of os at the prespecified final number of deaths ( 765 ; cutoff date june 1 , 2014 ) were also performed . in this post hoc analysis , baseline characteristics and treatment effects were evaluated in the intent - to - treat population ( all randomly assigned patients ) . the hr relative to placebo , with < 1.00 favoring enzalutamide , was determined using an unstratified cox regression model with treatment as the only covariate . the mean minimum concentration of the sum of enzalutamide plus n - desmethyl enzalutamide at weeks 5 , 13 , and 25 were adjusted for weight using log - linear regression . the relative systemic exposure to the sum of enzalutamide plus n - desmethyl enzalutamide in korean and non - korean patients was expressed as the ratio of their weight - adjusted cmin values , including 90% cis . the may 6 , 2012 , cutoff date for the protocol - specified interim analysis of centrally assessed rpfs after 439 events in the overall prevail population occurred during the enrollment period in korea , thus 38 patients did not have rpfs evaluated by central review ( fig . 1 ) . the investigatorassessed rpfs and os results , as well as all other metrics , are reported using a data cutoff of september 16 , 2013 , determined by the occurrence of 540 protocol - specified deaths in the overall study population ; at this date all korean patients had enrolled . seventy - eight of 1,717 prevail patients were enrolled at korean study sites ( enzalutamide , n=40 ; placebo , n=38 ) . baseline demographic and disease characteristics were well balanced between the enzalutamide- and placebo - treated korean patients , except that the proportion of patients with an ecog ps of 0 was lower , and the proportion of patients with bone disease or with a prior radical prostatectomy was higher in the enzalutamide than placebo group ( table 1 ) . compared with the overall study population , korean patients had lower median body weight and body mass index , less baseline pain , lower median psa , less baseline use of corticosteroids , greater use of prior antiandrogens , and greater use of hormonal therapies , and more had a gleason score of at least 8 at initial diagnosis . among korean patients , median duration of treatment was more than twice as long in the enzalutamide as in the placebo arm ( 13.0 months vs. 5.1 months ) . more korean patients in the enzalutamide group than in the placebo group received at least 12 months of treatment ( 65% vs. 32% ) and continued to receive treatment as of the september 16 , 2013 , data cutoff date ( 53% vs. 18% ) . in the final prevail analysis of korean patients , enzalutamide was associated with a 77% reduction in the risk for centrally assessed radiographic progression or death ( hr , 0.23 ; 95% ci , 0.022.24 ; fig . for rpfs , the treatment effect associated with enzalutamide versus placebo in korean patients was similar to that observed in the overall study population . despite the limited number of patients available for centrally assessed rpfs , a similar relative risk was evident when rpfs was investigator assessed ( hr , 0.22 ; 95% ci , 0.110.46 ; fig . median investigator - assessed rpfs was not yet reached in the enzalutamide group ( 95% ci , 13.6not yet reached ) versus 8.0 months in the placebo group ( 95% ci , 1.98.4 ) . risk of death was reduced by 23% in korean patients treated with enzalutamide ( hr , 0.77 ; 95% ci , 0.282.15 ) relative to those who received placebo ( fig . 4 ) . in the updated analysis of os ( june 1 , 2014 ) that included an additional 9 months of follow - up , the risk of death in korean patients was reduced by 24% ( hr , 0.76 ; 95% ci , 0.341.68 ) with enzalutamide relative to placebo ( fig . 5 ) . with the exception of median time until first sre , enzalutamide was associated with improved outcomes versus placebo in korean patients for all secondary end points ( table 2 ) , as evidenced by longer median times to cytotoxic chemotherapy and psa progression and better psa response rates . among korean patients , subsequent therapies were used by 18% and 66% of enzalutamide- and placebo - treated patients , respectively ( table 3 ) . the most common subsequent therapies received by korean patients were docetaxel ( 13% and 58% , respectively ) and abiraterone acetate ( 2.5% and 10.5% , respectively ) . the incidence and nature of aes emergent in korean patients treated with either enzalutamide or placebo were comparable to those observed in patients treated with enzalutamide or placebo in the overall safety population ( table 4 ) . the median treatment durations for the enzalutamide and placebo groups were 13.0 and 5.1 months , respectively . among korean patients , the incidence of aes of grade 3 or greater was higher in the enzalutamide group than in the placebo group ( 33% vs. 11% , respectively ) , although none were considered to be treatment related . the most frequent aes ( with > 20% incidence ) in enzalutamidetreated korean patients were fatigue and upper respiratory tract infection , most of which were grade 2 or less . the mean cmin of the sum of enzalutamide and n - desmethyl enzalutamide in korean and non - korean patients at 5 , 13 , and 25 weeks are presented in table 5 . the cmin values of enzalutamide plus n - desmethyl enzalutamide at all of these time points were similar in korean patients and non - korean patients , with geometric mean ratios ( korean : non - korean ) of 0.97 , 1.04 , and 1.03 , respectively . the may 6 , 2012 , cutoff date for the protocol - specified interim analysis of centrally assessed rpfs after 439 events in the overall prevail population occurred during the enrollment period in korea , thus 38 patients did not have rpfs evaluated by central review ( fig . 1 ) . the investigatorassessed rpfs and os results , as well as all other metrics , are reported using a data cutoff of september 16 , 2013 , determined by the occurrence of 540 protocol - specified deaths in the overall study population ; at this date all korean patients had enrolled . seventy - eight of 1,717 prevail patients were enrolled at korean study sites ( enzalutamide , n=40 ; placebo , n=38 ) . baseline demographic and disease characteristics were well balanced between the enzalutamide- and placebo - treated korean patients , except that the proportion of patients with an ecog ps of 0 was lower , and the proportion of patients with bone disease or with a prior radical prostatectomy was higher in the enzalutamide than placebo group ( table 1 ) . compared with the overall study population , korean patients had lower median body weight and body mass index , less baseline pain , lower median psa , less baseline use of corticosteroids , greater use of prior antiandrogens , and greater use of hormonal therapies , and more had a gleason score of at least 8 at initial diagnosis . among korean patients , median duration of treatment was more than twice as long in the enzalutamide as in the placebo arm ( 13.0 months vs. 5.1 months ) . more korean patients in the enzalutamide group than in the placebo group received at least 12 months of treatment ( 65% vs. 32% ) and continued to receive treatment as of the september 16 , 2013 , data cutoff date ( 53% vs. 18% ) . in the final prevail analysis of korean patients , enzalutamide was associated with a 77% reduction in the risk for centrally assessed radiographic progression or death ( hr , 0.23 ; 95% ci , 0.022.24 ; fig . the treatment effect associated with enzalutamide versus placebo in korean patients was similar to that observed in the overall study population . despite the limited number of patients available for centrally assessed rpfs , a similar relative risk was evident when rpfs was investigator assessed ( hr , 0.22 ; 95% ci , 0.110.46 ; fig . median investigator - assessed rpfs was not yet reached in the enzalutamide group ( 95% ci , 13.6not yet reached ) versus 8.0 months in the placebo group ( 95% ci , 1.98.4 ) . risk of death was reduced by 23% in korean patients treated with enzalutamide ( hr , 0.77 ; 95% ci , 0.282.15 ) relative to those who received placebo ( fig . 4 ) . in the updated analysis of os ( june 1 , 2014 ) that included an additional 9 months of follow - up , the risk of death in korean patients was reduced by 24% ( hr , 0.76 ; 95% ci , 0.341.68 ) with enzalutamide relative to placebo ( fig . 5 ) . with the exception of median time until first sre , enzalutamide was associated with improved outcomes versus placebo in korean patients for all secondary end points ( table 2 ) , as evidenced by longer median times to cytotoxic chemotherapy and psa progression and better psa response rates . among korean patients , subsequent therapies were used by 18% and 66% of enzalutamide- and placebo - treated patients , respectively ( table 3 ) . the most common subsequent therapies received by korean patients were docetaxel ( 13% and 58% , respectively ) and abiraterone acetate ( 2.5% and 10.5% , respectively ) . the incidence and nature of aes emergent in korean patients treated with either enzalutamide or placebo were comparable to those observed in patients treated with enzalutamide or placebo in the overall safety population ( table 4 ) . the median treatment durations for the enzalutamide and placebo groups were 13.0 and 5.1 months , respectively . among korean patients , the incidence of aes of grade 3 or greater was higher in the enzalutamide group than in the placebo group ( 33% vs. 11% , respectively ) , although none were considered to be treatment related . the most frequent aes ( with > 20% incidence ) in enzalutamidetreated korean patients were fatigue and upper respiratory tract infection , most of which were grade 2 or less . the mean cmin of the sum of enzalutamide and n - desmethyl enzalutamide in korean and non - korean patients at 5 , 13 , and 25 weeks are presented in table 5 . the cmin values of enzalutamide plus n - desmethyl enzalutamide at all of these time points were similar in korean patients and non - korean patients , with geometric mean ratios ( korean : non - korean ) of 0.97 , 1.04 , and 1.03 , respectively . in this post hoc analysis of asymptomatic or mildly symptomatic korean men with chemotherapy - nave mcrpc who participated in the prevail study , treatment effects observed with enzalutamide versus placebo were consistent with those observed in the overall study population , including centrally assessed rpfs ( data cutoff may 6 , 2012 ) , investigator - assessed rpfs ( data cutoff september 16 , 2013 ) and a range of secondary end points . some differences in baseline disease characteristics were observed in the korean subgroup relative to the overall study population , which were likely related to difference in clinical practice . a higher percentage of korean patients had a gleason score of at least 8 suggesting a higher disease burden , yet they reported less pain and had a lower median psa level at baseline . compared with the overall population of prevail , a larger proportion of korean patients received prior antiandrogen therapy , which possibly contributed to the lower baseline median psa level . unlike the overall study population , no korean patients received corticosteroids at baseline . aside from differences in clinical practice , some differences between the baseline characteristics of korean patients and the overall prevail population may be related to ethnicity . in men without prostate cancer , it is uncertain if this difference in psa levels between ethnicities signifies that korean patients have a lower disease burden . regardless , the difference in the proportion of patients with confirmed psa responses ( i.e. , 50% reduction from baseline ) between the enzalutamide - treated and placebo - treated korean patients was large ( 70% vs. 11% ) , suggesting that regular psa monitoring may help physicians evaluate treatment response . as in the overall study population , korean patients receiving enzalutamide had a more than 2-fold longer duration of therapy than those receiving placebo , and incidences of aes were similar between korean patients and the overall study population . importantly , there were no drug - related aes of grade 3 or greater in korean patients and no instances of seizure in korean patients or the overall study population . notably , prevail was not designed to assess differences between enzalutamide and placebo in the korean subgroup given the small number of korean patients included in the study , limited follow - up duration , and low number of events . these limitations lead to median estimates of os and rpfs that could be considered tentative at the time of analysis and a restriction on the ability to detect differences between the aes of enzalutamide- and placebo - treated patients . acknowledging these limitations , we sought to examine the consistency of the results in korean patients with those in the overall study population , which was powered sufficiently to detect the treatment effects and safety of enzalutamide versus placebo . in conclusion , this post hoc analysis of multicenter , randomized , placebo - controlled prevail data confirms similar treatment effects and ae profile between korean patients and the overall study population .

purposethis post hoc analysis evaluated treatment effects , safety , and pharmacokinetics of enzalutamide in korean patients in the phase 3 , double - blind , placebo - controlled prevail trial.materials and methodsasymptomatic or mildly symptomatic chemotherapy - naive men with metastatic castration - resistant prostate cancer that progressed on androgen deprivation therapy received 160 mg / d oral enzalutamide or placebo ( 1:1 ) until death or discontinuation due to radiographic progression or skeletal - related event and initiation of subsequent therapy . coprimary end points were centrally assessed radiographic progression - free survival ( rpfs ) and overall survival ( os ) . secondary end points included investigator - assessed rpfs , time to initiation of chemotherapy , time to prostate - specific antigen ( psa ) progression , psa response ( 50% decline ) , and time to skeletal - related event.resultsof 1,717 total patients , 78 patients were enrolled in korea ( enzalutamide , n=40 ; placebo , n=38 ) . hazard ratios ( 95% confidence interval ) for enzalutamide versus placebo were 0.23 ( 0.022.24 ) for centrally assessed rpfs , 0.77 ( 0.282.15 ) for os , 0.21 ( 0.080.51 ) for time to chemotherapy , and 0.31 ( 0.170.56 ) for time to psa progression . a psa response was observed in 70.0% of enzalutamide - treated and 10.5% of placebo - treated korean patients . adverse events of grade 3 occurred in 33% of enzalutamide - treated and 11% of placebo - treated korean patients , with median treatment durations of 13.0 and 5.1 months , respectively . at 13 weeks , the plasma concentration of enzalutamide plus n - desmethyl enzalutamide was similar in korean and non - korean patients ( geometric mean ratio , 1.04 ; 90% confidence interval , 0.971.10).conclusionsin korean patients , treatment effects and safety of enzalutamide were consistent with those observed in the overall prevail study population ( clinicaltrials.gov identifier : nct01212991 ) .

INTRODUCTION MATERIALS AND METHODS 1. Study design and patients 2. Assessments 3. Statistical analysis RESULTS 1. Patients 2. Treatment effects 3. Subsequent antineoplastic and endocrine therapies 4. Safety 5. Pharmacokinetics DISCUSSION CONCLUSIONS

the maldi - tof ms spectrum of bacillus massiliogabonensis is available at http://www.mediterranee-infection.com/article.php?laref=256&titre=urms-database . the 16s rrna gene sequence of strain marseille p2639 was deposited in genbank database under accession number lt598571 . strain marseille p2639 was deposited in the collection de souches de lunit des rickettsies ( csur ) and registered under number p2639 .

the discovery of new bacteria from the human gut using a culturomics method is a novel field of increasing interest in microbiology . here the main characteristics of bacillus massiliogabonensis strain marseille p2639 , a new gram - negative bacterium isolated from the stool sample of a healthy 16-year - old gabonese boy , are reported .

MALDI-TOF MS spectrum accession number Nucleotide sequence accession number Deposit in culture collection

gypsies and travellers are recognized as one of the most disadvantaged minority groups in the united kingdom and globally . research into the health needs of this group is an emerging field , and gypsies and travellers could justly be described as an invisible minority1 in being rarely captured in health statistics , unassertive of their health needs and with few champions.2 , 3 in the united kingdom , gypsies and travellers are known to have poorer health status and a higher risk of mortality than socioeconomically matched comparison groups4 and to experience health inequalities which are greater than could be expected simply from socioeconomic disadvantage or from belonging to a minority ethnic group.5 gypsies and travellers access to health services is also known to be poor.6 , 7 in several european countries , roma people have been shown to have poorer health than other ethnic groups and poorer access to health services,8 , 9 , 10 and a recent literature review identified a higher prevalence of both communicable and noncommunicable disease in the roma community with significantly shorter life expectancies than national averages.11 gypsies and travellers share a history of persecution and rejection by mainstream society , which continues today.12 , 13 , 14 , 15 the umbrella term gypsies and travellers covers a diversity of people , including continental roma , english and welsh gypsies and irish and scottish travellers.3 in the 2001 , uk census gypsy or irish traveller was included as an ethnic category for the first time . gypsies have a shared common history , language and oral literature4 and share with travellers beliefs about the centrality of the family as a social structure , nomadism and ideals of purity which influence hygiene in daily life and behaviour towards the opposite sex.13 , 16 a component of the gypsy traveller identity is a strong awareness of the differences between gypsy culture and that of the majority population,17 , 18 which has been described as contributing to gypsies and travellers ability to maintain a unique identity as internaloutsiders within often hostile host populations.16 some community values such as taking a pride in resilience and resourcefulness , particularly in children , and setting a higher value on lived experience above learning from books diverge from those of the settled community.13 the common use of the word gorgio to describe nongypsies emphasizes the status of gypsy travellers as a race apart who share defined cultural characteristics which define and confirm their unique identity.18 given the poor health outcomes experienced by gypsies and travellers , it is important to explore their views on the services they receive from health providers and to establish their primary health needs . it has been noted that roma women are often overlooked in health research due to dual discrimination , both ethnic and gender , within and outside the community,19 and there is currently little qualitative research exploring women 's views on maternal and child health and the health services provided . the uk healthy child progamme20 stipulates that mothers should be visited antenatally to support infant feeding and that postnatal visits should promote breastfeeding , with introduction of solids foods at around 6 months , as recommended by the world health organization.21 encouraging engagement with children 's preventive health services has been recognized as the starting place for reducing health inequalities among gypsies and travellers , particularly in the area of nutrition.11 how a baby is fed in the first year of life has an impact upon health in the long and short term.21 , 22 , 23 breastfed babies are less likely to experience morbidity in the first year of life24 and to have a reduced risk of diabetes , hypertension and obesity in later life.25 health benefits for mothers who breastfeed include a reduced risk of breast and ovarian cancer and type 2 diabetes.24 in the united kingdom , infant feeding behaviour is strongly related to socioeconomic class and ethnicity , with highly educated professional women most likely to breastfeed among the white community , but mothers from nonwhite ethnic minority groups more likely to breastfeed than the general population.22 despite exclusive breastfeeding for 6 months being advised , only 34% of babies in the united kingdom are breastfed at all at 6 months of age and only 1% exclusively.22 a small quantitative study of gypsies and travellers feeding practices in one primary care trust in england , suggested very low breastfeeding rates , with only 3% estimated to have initiated breastfeeding and none continuing to 68 weeks ( data based on health visitors reports ) ; however , a survey of 20 gypsy traveller women suggested a rate of 15% who had ever breastfed.26 breastfeeding rates were found to be slightly above average among roma mothers in serbia27 , but no difference was identified between the duration of breastfeeding for czech and roma children ( a median duration of 3 months breastfeeding in both groups).28 no studies have looked at weaning practices among gypsies and travellers , although these are recognized as being important in relation to continuation of breastfeeding,22 and to later eating habits which have an impact upon risk of obesity and cardiac disease.29 , 30 the aim of this study was to explore the views of gypsy traveller mothers and grandmothers on infant feeding and health service provision . semistructured interviews were conducted with a purposive sample of mothers and grandmothers from english gypsy , irish traveller and romanian roma communities between november 2011 and february 2012 . grandmothers were included as they are considered to have an influence upon mothers infant feeding choices.31 the sample size was designed to facilitate in depth exploration of the subject and allowed the comparison of the views of roma mothers and grandmothers with their english gypsy and irish traveller counterparts . twentytwo mothers and grandmothers were recruited , half from the roma community , and half from english gypsy and irish traveller backgrounds ( see table 1 for demographic details ) . inclusion criteria were as follows : demographic details of participants nvq , national vocational qualification ( a uk occupational qualification ) . mothers of roma , english gypsy or irish traveller ethnicity with a child aged 3 years or undergrandmothers of roma , english gypsy or irish traveller ethnicity with one or more grandchildren mothers of roma , english gypsy or irish traveller ethnicity with a child aged 3 years or under grandmothers of roma , english gypsy or irish traveller ethnicity with one or more grandchildren recruitment was carried out by a researcher ( lc ) , following introductions by local authority and voluntary workers who were either members of , or wellknown to , the community . these workers initially identified potential participants , and the researcher checked inclusion criteria at interview , including the participant 's own selfdefinition of ethnicity . one interviewee was excluded at interview as she did not agree that her ethnicity was irish traveller , despite the link worker having identified her as such . participants were interviewed in their homes or in a community setting , including a church . travelling participants were interviewed in their homes on both privately owned and council caravan sites . the sample size was achieved with consideration to ritchie et al . 's view that further data collection would lead to diminishing returns in terms of new insights and ideas gained.32 ethical consent was granted by a university ethics committee ( application number hsc/11/09/89 ) . all were given directly to study participants by a link worker who could explain the content ; the researcher visited a few days later in the company of a link worker to offer an interview . prior to interview , the researcher read the information sheet with each participant and completed a consent form , thus ensuring that participants were able to opt out if they wished at this stage . all written materials were translated into romanian for roma participants and also read through at interview by an interpreter . consent was requested for audiotaping and use of anonymized quotations , in addition to participation . to preserve anonymity , questions were based upon a topic guide developed in collaboration with the steering committee , which included local experts in infant feeding and nutrition in addition to two members of gypsy traveller ethnicity . the topic guide was focused on personal experience of milk and solid feeding , as well as perceived family and community views of infant feeding . only one roma participant spoke english , so interviews with the roma were conducted in romanian , with professional interpreters providing concurrent translation . for all roma participants , romanian was a second language ; no romanyspeaking interpreters existed in bristol . liamputtong33 suggests that in crosscultural research , interpreters are best considered as joint researchers , as in facilitating communication through translation , they play a part in shaping the data collected . both interpreters who took part in the project were wellknown to the community and often acted as interpreters in health and educational settings . permission was requested to audiotape the interview . where participants agreed to audiotaping , interviews were subsequently transcribed . for the remainder , the interviewer ( lc ) documented responses contemporaneously by hand . no participants objected to notes being taken , but some interviewees felt that no traveller would allow their voice to be recorded . a sample of audiotaped interviews with roma participants was checked for validity of translation by an independent romanian interpreter ; all were confirmed to be accurate . data were coded using nvivo 9 as part of data analysis using a framework approach by which data are classified and organized according to key themes and concepts.34 members of the steering group contributed to the preliminary identification of codes from iterative reading of transcripts and contemporaneous notes and reached agreement on emergent categories . following coding of all transcripts , lc and ds identified dominant themes and developed a conceptual framework from an interpretation of the data set as a whole . the framework approach allowed movement from raw data to abstraction in the analytical process , without losing the voice of participants.35 semistructured interviews were conducted with a purposive sample of mothers and grandmothers from english gypsy , irish traveller and romanian roma communities between november 2011 and february 2012 . grandmothers were included as they are considered to have an influence upon mothers infant feeding choices.31 the sample size was designed to facilitate in depth exploration of the subject and allowed the comparison of the views of roma mothers and grandmothers with their english gypsy and irish traveller counterparts . twentytwo mothers and grandmothers were recruited , half from the roma community , and half from english gypsy and irish traveller backgrounds ( see table 1 for demographic details ) . inclusion criteria were as follows : demographic details of participants nvq , national vocational qualification ( a uk occupational qualification ) . mothers of roma , english gypsy or irish traveller ethnicity with a child aged 3 years or undergrandmothers of roma , english gypsy or irish traveller ethnicity with one or more grandchildren mothers of roma , english gypsy or irish traveller ethnicity with a child aged 3 years or under grandmothers of roma , english gypsy or irish traveller ethnicity with one or more grandchildren recruitment was carried out by a researcher ( lc ) , following introductions by local authority and voluntary workers who were either members of , or wellknown to , the community . these workers initially identified potential participants , and the researcher checked inclusion criteria at interview , including the participant 's own selfdefinition of ethnicity . one interviewee was excluded at interview as she did not agree that her ethnicity was irish traveller , despite the link worker having identified her as such . participants were interviewed in their homes or in a community setting , including a church . travelling participants were interviewed in their homes on both privately owned and council caravan sites . the sample size was achieved with consideration to ritchie et al . 's view that further data collection would lead to diminishing returns in terms of new insights and ideas gained.32 all were given directly to study participants by a link worker who could explain the content ; the researcher visited a few days later in the company of a link worker to offer an interview . prior to interview , the researcher read the information sheet with each participant and completed a consent form , thus ensuring that participants were able to opt out if they wished at this stage . all written materials were translated into romanian for roma participants and also read through at interview by an interpreter . consent was requested for audiotaping and use of anonymized quotations , in addition to participation . to preserve anonymity , questions were based upon a topic guide developed in collaboration with the steering committee , which included local experts in infant feeding and nutrition in addition to two members of gypsy traveller ethnicity . the topic guide was focused on personal experience of milk and solid feeding , as well as perceived family and community views of infant feeding . only one roma participant spoke english , so interviews with the roma were conducted in romanian , with professional interpreters providing concurrent translation . for all roma participants , romanian was a second language ; no romanyspeaking interpreters existed in bristol . liamputtong33 suggests that in crosscultural research , interpreters are best considered as joint researchers , as in facilitating communication through translation , they play a part in shaping the data collected . both interpreters who took part in the project were wellknown to the community and often acted as interpreters in health and educational settings . permission was requested to audiotape the interview . where participants agreed to audiotaping , interviews were subsequently transcribed . for the remainder , the interviewer ( lc ) documented responses contemporaneously by hand . no participants objected to notes being taken , but some interviewees felt that no traveller would allow their voice to be recorded . a sample of audiotaped interviews with roma participants was checked for validity of translation by an independent romanian interpreter ; all were confirmed to be accurate . data were coded using nvivo 9 as part of data analysis using a framework approach by which data are classified and organized according to key themes and concepts.34 members of the steering group contributed to the preliminary identification of codes from iterative reading of transcripts and contemporaneous notes and reached agreement on emergent categories . following coding of all transcripts , lc and ds identified dominant themes and developed a conceptual framework from an interpretation of the data set as a whole . the framework approach allowed movement from raw data to abstraction in the analytical process , without losing the voice of participants.35 three dominant themes were identified as influencing infant feeding behaviour , which were common to all participants and related to the culture of their community . these themes were as follows : the centrality of the family , beliefs and traditions related to culture , and a travelling lifestyle . a fourth crosscutting theme was identified as interaction with health professionals ; this was interwoven with the themes relating to gypsy and traveller culture throughout . it was apparent that at many points , interactions with health professionals led to some conflict with beliefs and attitudes prevalent in the community , and these conflicts are brought out in the account below . large families are commonplace among gypsies and travellers , and there is an expectation that older children help care for younger siblings . as a result , women considered that they had preexisting knowledge and skills when they became parents . few participants described themselves as requiring professional support in caring for children irrespective of age or number of children : the travelling community are reared up with children . i was married at 13 when you are small you are with your mum , and she shows you how to do things . roma 11 , mother i 'll tell you again , my mum had 10 kids and i helped look after them , i did n't need to learn from scratch , i already knew . some people do n't know , they have to learn from scratch and they need people telling them what to do and what not to do . i was married at 13 when you are small you are with your mum , and she shows you how to do things . i 'll tell you again , my mum had 10 kids and i helped look after them , i did n't need to learn from scratch , i already knew . some people do n't know , they have to learn from scratch and they need people telling them what to do and what not to do . english gypsy 4 , mother young motherhood increased participants view of themselves as being highly experienced in all aspects of child care . compared with the experiential knowledge that was easily available within the family , the advice of health professionals was often set at a low value by mothers and grandmothers.i do n't think they need any advice in feeding their children because it 's passed on from their own mums and grandmothers . irish traveller 1 , grandmother i 've no idea when health visitors say to introduce baby dinners . i think the health visitor advises 3 months but i would do what i thought , from my experience . lots of health visitors haven't had a baby ; they do n't know anything about it , just what they have read . irish traveller 4 , mother i do n't think they need any advice in feeding their children because it 's passed on from their own mums and grandmothers . irish traveller 1 , grandmother i 've no idea when health visitors say to introduce baby dinners . i think the health visitor advises 3 months but i would do what i thought , from my experience . lots of health visitors haven't had a baby ; they do n't know anything about it , just what they have read . irish traveller 4 , mother contact with health services was described as less common in the past ( early on they did n't want anything to do with travellers , now it 's more accepted. irish traveller 5 , grandmother ) , but english gypsies and irish travellers now expected contact with midwifery and health visiting services . roma women were sometimes unclear about difference in roles , referring to professionals generically as being from the doctor's. all described a minimal service with little routine contact beyond the immediate postnatal period . mothers took a pride in being capable mothers and did not see themselves as requiring extensive contact with health professionals.the health visitor came once or twice and saw i could get along , now she does n't come . translated roma 3 , mother the health visitor came once or twice and saw i could get along , now she does n't come . translated health professionals were described by all groups as relying on written materials to give health promotion information , despite low literacy levels . although none of the roma interviewees read english , most mentioned having been given leaflets . several nonroma participants also could not read health promotion materials : we received leaflets in english , about how to breast feed and what to expect when you 're a mum , but we do n't actually know how to read in english . translated roma 8 , mother she gave me some [ leaflets ] , but i ca n't read , not unless i ask someone to read it , then i find out what 's in it . i just looked at the pictures , but i did n't often look in the book . irish traveller 4 , mother we received leaflets in english , about how to breast feed and what to expect when you 're a mum , but we do n't actually know how to read in english . translated she gave me some [ leaflets ] , but i ca n't read , not unless i ask someone to read it , then i find out what 's in it i just looked at the pictures , but i did n't often look in the book . irish traveller 4 , mother these contacts often served to highlight the differences between travelling people and the settled community . many participants had little contact with people outside their community and were aware only of their own infant feeding norms . english gypsy and irish traveller participants frequently expressed pride in their culture , and where there was opposition between family traditions and health professional advice , a common response was to reaffirm the positives about the community way. one mother commented to the researcher : me meself , i like me own way of life , i 've been brought up as a romany gypsy and that 's the way i want me boys to be brought up to be truthful . i 'm not racialist against outsiders , but what we call like gaujes or house people like yourself you likes your way , we got our own way . english gypsy 5 , mother me meself , i like me own way of life , i 've been brought up as a romany gypsy and that 's the way i want me boys to be brought up to be truthful . i 'm not racialist against outsiders , but what we call like gaujes or house people like yourself english gypsy 5 , mother few demands were made on health services by gypsy and traveller mothers at the time of pregnancy and birth , and most health professionals were described as offering little beyond the minimum routine service . roma mothers described breastfeeding as the usual method of feeding , which babies much enjoyed . while the overriding reason given for preferring breastfeeding was its participants placed more emphasis on ease and the fact it was common practice : it is better to breastfeed because it is healthier . . we do n't give the bottle , you just put the breast in his mouth and that 's it . translated roma 6 , mother well it 's easier because you do n't have to spend the money on powder milk and bottles and all that stuff . it 's not just about the money ; it 's just that i find it better . translated roma 8 , mother we raise them with the breast . translated roma 10 , grandmother it is better to breastfeed because it is healthier . we do n't give the bottle , you just put the breast in his mouth and that 's it . translated well it 's easier because you do n't have to spend the money on powder milk and bottles and all that stuff . and it 's not just about the money ; it 's just that i find it better . . translated roma 10 , grandmother when a mother had a baby in the roma community , help with breastfeeding was available from within the extended family . mothers could learn by watching others breastfeed , and some described being explicitly taught how to breastfeed by their own mother.my mum showed him my breasts and she was showing this is how you do it then you take it out and you.pat him and when you feed him you put him like this . translated roma 4 , mother my mum showed him my breasts and she was showing this is how you do it then you take it out and you.pat him and when you feed him you put him like this . translated by contrast , english gypsies and irish travellers described a predominantly bottlefeeding culture . as with the roma , feeding behaviours prevalent within the community were passed on to the next generation , but in this case , community knowledge was of how to prepare and store bottles of formula milk , and which brand to choose . some harmful practices continued among english gypsies and irish travellers including adding foods such as rusks in the bottle at an early age which was justified as being a customary practice and as having been recommended by health professionals in the past . some of the younger mothers were aware that adding solid foods to bottles was no longer advised , citing the immaturity of the baby 's digestive system as a reason to delay the introduction of solid foods . both irish traveller and english gypsy mothers continued with pured foods beyond the recommended date ( even up to 18 months ) , due to a fear that the baby would choke if offered finger foods . to avoid this , two mothers offered food which they had prechewed , a practice which was described as being a traditional custom among gypsies . some irish traveller and english gypsy mothers wished to try breastfeeding , primarily because they were aware of the health benefits.they said it was better for the baby , and so because they said that , i thought , go on then , i 'll try it . english gypsy 2 , mother there are more natural things in your own breast milk than are ever in formula i saw some traveller girls who would dare to breastfeed and i wanted to try . irish traveller 2 , mother they said it was better for the baby , and so because they said that , i thought , go on then , i 'll try it . english gypsy 2 , mother there are more natural things in your own breast milk than are ever in formula i saw some traveller girls who would dare to breastfeed and i wanted to try . irish traveller 2 , mother although most interviewees knew of a relative or friend who had breastfed , choosing to initiate breastfeeding carried a sense of breaking a taboo . breastfeeding was frequently described as not being part of the gypsy or traveller tradition , instead being something that gauje women would do . breastfeeding conflicted with ideals of female behaviour , such as covering the body at all times , particularly in the presence of the opposite sex . many mothers considered that breastfeeding could only be carried out alone in a private place , not even in the presence of other women.breastfeeding is something that i would not do . i 'd never do it , no one in my family would you ca n't pull out a boob in front of a man , it would a bit embarrassing like some travelling women do it , in the trailer . i 'd never do it , no one in my family would you ca n't pull out a boob in front of a man , it would a bit embarrassing like some travelling women do it , in the trailer . irish traveller 6 , mother one mother stated that because men did not understand the health benefits of breast milk , they preferred women to bottlefeed.most traveller men are old fashioned in their ways and strict about how women behave . these men think that giving the bottle is exactly the same without a woman exposing herself . irish traveller 2 , mother most traveller men are old fashioned in their ways and strict about how women behave . these men think that giving the bottle is exactly the same without a woman exposing herself . irish traveller 2 , mother the one instance where irish traveller and english gypsy interviewees described themselves as actively seeking help from midwives and health visitors was in initiating and continuing breastfeeding . advice was then sought from professionals because no skilled help was available from within their communities . while messages about the health benefits of breastfeeding were successfully assimilated , postnatal support for breastfeeding was described as insufficient , with professionals failing to grasp the extent of cultural taboos which made breastfeeding as a dangerously immodest act . a mother , who had bottle fed in hospital due to fear of being observed by visitors , received only telephone advice ( just carry on trying ) from her midwife when she subsequently asked for help in attempting to breastfeed her baby at home . this parsimonious support offered by health professionals contrasted unfavourably with the support which was always available from within the family and community . a warm , trusting relationship which had developed over time appeared a prerequisite for successfully supporting a mother to breastfeed.i do n't know what decided me to breastfeed , perhaps it was the midwife . with the last one i had the best service from the midwife . i do n't know what decided me to breastfeed , perhaps it was the midwife . with the last one i had the best service from the midwife . irish traveller 5 , grandmother names of helpful and respected health professionals were known throughout the community even if they worked in another city . participants considered that being informed about gypsy traveller culture was an important characteristic of the respected health professional . grandmothers described a tradition in romania of breastfeeding until around 2 years , with solid foods , such as polenta , pork and nettle soup , being introduced at around 56 months . however , once in the united kingdom , some mothers started giving formula feeds , even though this had led to some problems with milk supply , babies refusing the breast and subsequent constipation . commercially manufactured baby foods were given in place of solid family foods , sometimes as early as 4 weeks of age . more civilized and thinking it is better to give the bottle ( roma 10 , grandmother ) . several mothers stated that in the united kingdom , they could afford to bottlefeed and give commercially made baby foods , and therefore did so . there was no evidence of awareness that a very high standard of infant nutrition was being replaced with a less healthy diet.in romania i did n't have the possibility to buy all the things i needed to bottle feed , so that 's why i breast fed . translated roma 7 , grandmother in romania i did not have enough money for baby food ; i had to feed the same food as everyone else . roma 11 , mother in romania i did n't have the possibility to buy all the things i needed to bottle feed , so that 's why i breast fed . translated roma 7 , grandmother in romania i did not have enough money for baby food ; i had to feed the same food as everyone else . when asked about any potential longterm health benefits of breastfeeding for babies or mothers , no participant was aware of any impact on lifelong health . two roma women responded to this question by quoting a gypsy proverb , how will a man live if he does not eat? which places emphasis upon the importance of food to avoid starvation rather than seeking additional health benefits . for roma women , economic opportunities played a part in dictating infant feeding patterns . some introduced formula feeds in order to be able to do housework , shop or work outside the home , as other family members could bottlefeed . others clung to the traditional maternal role of which breastfeeding was seen as a part , in order to have freedom from paid labour.some [ women ] bottle feed their babies and they have other occupations like selling newspapers or doing something to get money . i just stay at home and take care of the children and my husband goes and works translated roma 1 , mother some [ women ] bottle feed their babies and they have other occupations like selling newspapers or doing something to get money . i just stay at home and take care of the children and my husband goes and works i 'd rather stay at home , rather than going and selling newspapers . translated for english gypsy and irish traveller , interviewees travelling could lead to disrupted contacts with health professionals due to moving between sites . for mothers who wanted to breastfeed , breastfeeding while living in a caravan placed a responsibility on the mother to avoid the risk of offending others . the sociability of travelling life contributed to the difficulties of finding an acceptable place to breastfeed , and for some shortened the duration of breastfeeding.i did try with the last one and i did it for a couple of months . if was difficult because i had no privacy i did n't have a problem with breastfeeding but it was very hard living in a caravan with people in and out i did n't mind giving up.irish traveller 5 , grandmother i did try with the last one and i did it for a couple of months . if was difficult because i had no privacy i did n't have a problem with breastfeeding but it was very hard living in a caravan with people in and out large families are commonplace among gypsies and travellers , and there is an expectation that older children help care for younger siblings . as a result few participants described themselves as requiring professional support in caring for children irrespective of age or number of children : the travelling community are reared up with children . i was married at 13 when you are small you are with your mum , and she shows you how to do things . roma 11 , mother i 'll tell you again , my mum had 10 kids and i helped look after them , i did n't need to learn from scratch , i already knew . some people do n't know , they have to learn from scratch and they need people telling them what to do and what not to do . i was married at 13 when you are small you are with your mum , and she shows you how to do things . i 'll tell you again , my mum had 10 kids and i helped look after them , i did n't need to learn from scratch , i already knew . some people do n't know , they have to learn from scratch and they need people telling them what to do and what not to do . english gypsy 4 , mother young motherhood increased participants view of themselves as being highly experienced in all aspects of child care . compared with the experiential knowledge that was easily available within the family , the advice of health professionals was often set at a low value by mothers and grandmothers.i do n't think they need any advice in feeding their children because it 's passed on from their own mums and grandmothers . irish traveller 1 , grandmother i 've no idea when health visitors say to introduce baby dinners . i think the health visitor advises 3 months but i would do what i thought , from my experience . lots of health visitors haven't had a baby ; they do n't know anything about it , just what they have read . irish traveller 4 , mother i do n't think they need any advice in feeding their children because it 's passed on from their own mums and grandmothers . irish traveller 1 , grandmother i 've no idea when health visitors say to introduce baby dinners . i think the health visitor advises 3 months but i would do what i thought , from my experience . lots of health visitors haven't had a baby ; they do n't know anything about it , just what they have read . irish traveller 4 , mother contact with health services was described as less common in the past ( early on they did n't want anything to do with travellers , now it 's more accepted. irish traveller 5 , grandmother ) , but english gypsies and irish travellers now expected contact with midwifery and health visiting services . roma women were sometimes unclear about difference in roles , referring to professionals generically as being from the doctor's. all described a minimal service with little routine contact beyond the immediate postnatal period . mothers took a pride in being capable mothers and did not see themselves as requiring extensive contact with health professionals.the health visitor came once or twice and saw i could get along , now she does n't come . translated roma 3 , mother the health visitor came once or twice and saw i could get along , now she does n't come . translated health professionals were described by all groups as relying on written materials to give health promotion information , despite low literacy levels . although none of the roma interviewees read english , most mentioned having been given leaflets . several nonroma participants also could not read health promotion materials : we received leaflets in english , about how to breast feed and what to expect when you 're a mum , but we do n't actually know how to read in english . translated roma 8 , mother she gave me some [ leaflets ] , but i ca n't read , not unless i ask someone to read it , then i find out what 's in it . i just looked at the pictures , but i did n't often look in the book . irish traveller 4 , mother we received leaflets in english , about how to breast feed and what to expect when you 're a mum , but we do n't actually know how to read in english . translated she gave me some [ leaflets ] , but i ca n't read , not unless i ask someone to read it , then i find out what 's in it . i just looked at the pictures , but i did n't often look in the book . irish traveller 4 , mother these contacts often served to highlight the differences between travelling people and the settled community . many participants had little contact with people outside their community and were aware only of their own infant feeding norms . english gypsy and irish traveller participants frequently expressed pride in their culture , and where there was opposition between family traditions and health professional advice , a common response was to reaffirm the positives about the community way. one mother commented to the researcher : me meself , i like me own way of life , i 've been brought up as a romany gypsy and that 's the way i want me boys to be brought up to be truthful . gaujes or house people like yourself you likes your way , we got our own way . english gypsy 5 , mother me meself , i like me own way of life , i 've been brought up as a romany gypsy and that 's the way i want me boys to be brought up to be truthful . i 'm not racialist against outsiders , but what we call like gaujes or house people like yourself you likes your way , we got our own way . english gypsy 5 , mother few demands were made on health services by gypsy and traveller mothers at the time of pregnancy and birth , and most health professionals were described as offering little beyond the minimum routine service . roma mothers described breastfeeding as the usual method of feeding , which babies much enjoyed . while the overriding reason given for preferring breastfeeding was its participants placed more emphasis on ease and the fact it was common practice : it is better to breastfeed because it is healthier . we do n't give the bottle , you just put the breast in his mouth and that 's it . translated roma 6 , mother well it 's easier because you do n't have to spend the money on powder milk and bottles and all that stuff . it 's not just about the money ; it 's just that i find it better . translated roma 8 , mother we raise them with the breast . translated roma 10 , grandmother it is better to breastfeed because it is healthier . we do n't give the bottle , you just put the breast in his mouth and that 's it . translated well it 's easier because you do n't have to spend the money on powder milk and bottles and all that stuff it 's not just about the money ; it 's just that i find it better . translated we raise them with the breast . translated roma 10 , grandmother when a mother had a baby in the roma community , help with breastfeeding was available from within the extended family . mothers could learn by watching others breastfeed , and some described being explicitly taught how to breastfeed by their own mother.my mum showed him my breasts and she was showing this is how you do it then you take it out and you.pat him and when you feed him you put him like this . translated roma 4 , mother my mum showed him my breasts and she was showing this is how you do it then you take it out and you.pat him and when you feed him you put him like this . translated by contrast , english gypsies and irish travellers described a predominantly bottlefeeding culture . as with the roma , feeding behaviours prevalent within the community were passed on to the next generation , but in this case , community knowledge was of how to prepare and store bottles of formula milk , and which brand to choose . some harmful practices continued among english gypsies and irish travellers including adding foods such as rusks in the bottle at an early age which was justified as being a customary practice and as having been recommended by health professionals in the past . some of the younger mothers were aware that adding solid foods to bottles was no longer advised , citing the immaturity of the baby 's digestive system as a reason to delay the introduction of solid foods . both irish traveller and english gypsy mothers continued with pured foods beyond the recommended date ( even up to 18 months ) , due to a fear that the baby would choke if offered finger foods . to avoid this , two mothers offered food which they had prechewed , a practice which was described as being a traditional custom among gypsies . some irish traveller and english gypsy mothers wished to try breastfeeding , primarily because they were aware of the health benefits.they said it was better for the baby , and so because they said that , i thought , go on then , i 'll try it . english gypsy 2 , mother there are more natural things in your own breast milk than are ever in formula breast milk has more vitamins and stuff that they ca n't put in sma i saw some traveller girls who would dare to breastfeed and i wanted to try . irish traveller 2 , mother they said it was better for the baby , and so because they said that , i thought , go on then , i 'll try it . english gypsy 2 , mother there are more natural things in your own breast milk than are ever in formula i saw some traveller girls who would dare to breastfeed and i wanted to try . irish traveller 2 , mother although most interviewees knew of a relative or friend who had breastfed , choosing to initiate breastfeeding carried a sense of breaking a taboo . breastfeeding was frequently described as not being part of the gypsy or traveller tradition , instead being something that gauje women would do . breastfeeding conflicted with ideals of female behaviour , such as covering the body at all times , particularly in the presence of the opposite sex . many mothers considered that breastfeeding could only be carried out alone in a private place , not even in the presence of other women.breastfeeding is something that i would not do . i 'd never do it , no one in my family would you ca n't pull out a boob in front of a man , it would a bit embarrassing like some travelling women do it , in the trailer . i 'd never do it , no one in my family would you ca n't pull out a boob in front of a man , it would a bit embarrassing like some travelling women do it , in the trailer . irish traveller 6 , mother one mother stated that because men did not understand the health benefits of breast milk , they preferred women to bottlefeed.most traveller men are old fashioned in their ways and strict about how women behave . these men think that giving the bottle is exactly the same without a woman exposing herself . irish traveller 2 , mother most traveller men are old fashioned in their ways and strict about how women behave . these men think that giving the bottle is exactly the same without a woman exposing herself . irish traveller 2 , mother the one instance where irish traveller and english gypsy interviewees described themselves as actively seeking help from midwives and health visitors was in initiating and continuing breastfeeding . advice was then sought from professionals because no skilled help was available from within their communities . while messages about the health benefits of breastfeeding were successfully assimilated , postnatal support for breastfeeding was described as insufficient , with professionals failing to grasp the extent of cultural taboos which made breastfeeding as a dangerously immodest act . a mother , who had bottle fed in hospital due to fear of being observed by visitors , received only telephone advice ( just carry on trying ) from her midwife when she subsequently asked for help in attempting to breastfeed her baby at home . this parsimonious support offered by health professionals contrasted unfavourably with the support which was always available from within the family and community . a warm , trusting relationship which had developed over time appeared a prerequisite for successfully supporting a mother to breastfeed.i do n't know what decided me to breastfeed , perhaps it was the midwife . with the last one i had the best service from the midwife . i do n't know what decided me to breastfeed , perhaps it was the midwife . with the last one i had the best service from the midwife . irish traveller 5 , grandmother names of helpful and respected health professionals were known throughout the community even if they worked in another city . participants considered that being informed about gypsy traveller culture was an important characteristic of the respected health professional . grandmothers described a tradition in romania of breastfeeding until around 2 years , with solid foods , such as polenta , pork and nettle soup , being introduced at around 56 months . however , once in the united kingdom , some mothers started giving formula feeds , even though this had led to some problems with milk supply , babies refusing the breast and subsequent constipation . commercially manufactured baby foods were given in place of solid family foods , sometimes as early as 4 weeks of age . more civilized and thinking it is better to give the bottle ( roma 10 , grandmother ) . several mothers stated that in the united kingdom , they could afford to bottlefeed and give commercially made baby foods , and therefore did so . there was no evidence of awareness that a very high standard of infant nutrition was being replaced with a less healthy diet.in romania i did n't have the possibility to buy all the things i needed to bottle feed , so that 's why i breast fed . translated roma 7 , grandmother in romania i did not have enough money for baby food ; i had to feed the same food as everyone else . roma 11 , mother in romania i did n't have the possibility to buy all the things i needed to bottle feed , so that 's why i breast fed . translated roma 7 , grandmother in romania i did not have enough money for baby food ; i had to feed the same food as everyone else . when asked about any potential longterm health benefits of breastfeeding for babies or mothers , no participant was aware of any impact on lifelong health . two roma women responded to this question by quoting a gypsy proverb , how will a man live if he does not eat? which places emphasis upon the importance of food to avoid starvation rather than seeking additional health benefits . for roma women , some introduced formula feeds in order to be able to do housework , shop or work outside the home , as other family members could bottlefeed . others clung to the traditional maternal role of which breastfeeding was seen as a part , in order to have freedom from paid labour.some [ women ] bottle feed their babies and they have other occupations like selling newspapers or doing something to get money . i just stay at home and take care of the children and my husband goes and works i 'd rather stay at home , rather than going and selling newspapers . translated roma 1 , mother some [ women ] bottle feed their babies and they have other occupations like selling newspapers or doing something to get money . i just stay at home and take care of the children and my husband goes and works i 'd rather stay at home , rather than going and selling newspapers . translated for english gypsy and irish traveller , interviewees travelling could lead to disrupted contacts with health professionals due to moving between sites . for mothers who wanted to breastfeed , breastfeeding while living in a caravan placed a responsibility on the mother to avoid the risk of offending others . the sociability of travelling life contributed to the difficulties of finding an acceptable place to breastfeed , and for some shortened the duration of breastfeeding.i did try with the last one and i did it for a couple of months . if was difficult because i had no privacy i did n't have a problem with breastfeeding but it was very hard living in a caravan with people in and out i did n't mind giving up.irish traveller 5 , grandmother i did try with the last one and i did it for a couple of months . if was difficult because i had no privacy i did n't have a problem with breastfeeding but it was very hard living in a caravan with people in and out this qualitative study presents the views of gypsy and traveller mothers and grandmothers in an under explored area . its strength lies in giving voice to women from a marginalized group to comment on the health services provided for them and to indicate in which aspects of infant feeding culturally sensitive health promotion could be developed . there are indications from this study that cultural beliefs about infant feeding between uk gypsy travellers are not identical , and a wider range of views may have emerged from a larger sample . limitations of the study include the use of romanian , rather than roma interpreters , which could reduce the ability of participants to express themselves , and the use of interpreters who had ongoing contact with families in all aspects of their lives . however , this study adds to research which suggests that infant feeding in the united kingdom is influenced by ethnicity and culture36 , 37 , 38 and indicates that a common gypsy traveller identity influences infant feeding practices . shared aspects of gypsy life , such as observing defined gender roles , having large families and living within a close community13 , 16 , influence how mothers feed their babies . this study has highlighted that common aspects of the gypsy traveller identity affect feeding differently in different groups , a finding which has important implications for policy and practice in health promotion . in the roma community , studies of other ethnic groups have shown that migrant mothers who interact least with the host community are most likely to retain traditional feeding habits.38 , 39 romanian roma mothers exhibited a strong sense of community and could describe traditional feeding behaviours but took a pragmatic approach to changing these , particularly if infant feeding needed to fit in with the demands of paid and unpaid work . the economic necessity to return to work soon after delivery has been previously identified as a factor reducing migrant women 's ability to breastfeed.40 roma women described limited involvement with health professionals and seemed unaware of the potential cost to infant and maternal health in switching to formula feeding and early weaning . ceasing to practice traditional infant feeding behaviours , which are superior to the usual feeding practices of the uk settled population , poses an increased risk to child and maternal health . as roma children are known to have a higher prevalence of health risk factors,41 it is important to recognize where new risks are posed to child and maternal health . in this study , english gypsies and irish travellers exhibited a strong awareness of their political and cultural identity and commonly described their behaviour as characteristic of their community and unlike that of the gauje or nongypsies . shared infant feeding practices may play a part in maintaining family traditions and values , hence contributing to the community cohesion and resilience which can combat the effects of social disadvantage and racism.42 gypsies and travellers have been noted to define their identity as much by what is rejected as by what is chosen,18 and in this study , the identification of practices , such as breastfeeding , as something that a gypsy would not do demonstrates this . as demonstrated in other studies , developing a relationship with a trusted health professional appeared to be a key factor in facilitating breastfeeding , particularly for mothers who face exceptional barriers.43 , 44 in a community , where family support is ubiquitously present , it is vital that health professionals offer a responsive and accessible service . to this end , guidelines have previously been developed to assist health professionals in working effectively with gypsies and travellers.45 despite these , this study has highlighted again the futility of promoting the health benefits of breastfeeding in the antenatal period while failing to provide an adequate service postnatally.46 the disadvantage experienced by gypsies and travellers is exemplified by the demographic details of participants ( table 1 ) , which showed that participants had larger than average families , younger age of maternity and low educational achievement in comparison with the majority uk population ; all these factors increase disadvantage and reduce life chances.47 given the emphasis on providing health promotion and support to the most disadvantaged in the healthy child programme , it is remarkable that mothers in this study described receiving very little targeted support from health professionals . the frequency with which health professionals were reported as giving health education leaflets to women who did not read english suggests that cultural competency is undeveloped and not prioritized by practitioners.48 mcfadden et al . suggest that current provision of community breastfeeding support may be inappropriate and inaccessible to some women from minority ethnic backgrounds,49 and this study lends corroboration to this view . the effects of social disadvantage , exclusion and racism compound the difficulties faced by gypsy it is important to explore the views of gypsies and travellers in order to gain insight into their health needs and to address the extreme health inequalities they experience . this study has implications for policy and the practice of health professionals , both in indicating the customary feeding behaviours of some gypsy and traveller groups , and in suggesting how culturally sensitive support could facilitate optimal infant feeding practices . in a culture which prizes children , improving health outcomes by ensuring the best infant feeding is a potentially powerful health promotion message . this study was internally funded by the university of the west of england as part of the spur early career researcher funding stream .

abstractbackgroundgypsies and travellers are known to have poor health status and access to health services , even in comparison with other ethnic minority groups . people from this stigmatized ethnic group are rarely consulted about their health needs or health service provision . optimal infant feeding in the first year of life has the potential to improve lifelong health.objectivethe aim of this study was to explore mothers and grandmothers views on feeding in the first year of life , including the support provided by health professionals.methodssemistructured interviews were conducted with a purposively selected sample of 22 mothers and grandmothers of english gypsy , irish traveller and romanian roma ethnicity between november 2011 and february 2012 in a city in southwest england.resultsfew women perceived themselves as requiring help from health professionals in infant feeding , as acceptable and accessible support was available from within their own communities . roma mothers described a tradition of breastfeeding and appropriately timed weaning , while english gypsies and irish travellers customarily practised less healthy infant feeding . when mothers requested support , health service provision was often found inadequate.conclusionexploring the views of gypsies and travellers is important to gain insight into the provision of health services for this marginalized ethnic group . this study has implications for policy and the practice of health professionals , in indicating the customary feeding behaviours of some gypsy and travellers , and highlighting areas meriting culturally sensitive health promotion .

Introduction Methods Research design Participants Ethics Data collection Data analysis Results Centrality of the family Beliefs and traditions Travelling Discussion Conclusion Funding Conflict of interest

influenza a , b , and c viruses are members of the orthomyxoviridae , which is a family of enveloped viruses with segmented , single - stranded , negative - sense rna genomes ( palese , 2007 ) . they are classified according to antigenic differences in their nucleoprotein ( np ) and matrix protein ( m1 ) , which are major structural components of their virions . of the three members , influenza a viruses , which are further classified into 16 hemagglutinin ( ha ) subtypes and 9 neuraminidase ( na ) subtypes on the basis of their antigenic properties , cause annual epidemics in humans and recurring pandemics that spread globally with severe consequences for human health and the economy . the genome of influenza a virus consists of eight single - stranded , negative - sense rna segments that form ribonucleoprotein complexes ( rnps ) together with the viral rna ( vrna)-dependent rna polymerase complex [ basic polymerase 1 ( pb1 ) , basic polymerase 2 ( pb2 ) , and acidic polymerase ( pa ) ] and many nucleoprotein ( np ) molecules . the genome encodes at least 12 viral proteins , most of which are necessary for efficient virus replication in host cells and for virion formation . during virus infection , the virus attaches to receptors ( i.e. , sialic acid linked to galactose ) on the cell surface via ha virion surface proteins and enters the cell by clathrin - mediated endocytosis . the rnps within the virion are released into the cytoplasm by acidic ph - dependent membrane fusion , again via the ha proteins , and are transported to the nucleus of the infected cells . unlike most negative - sense rna viruses , transcription and replication of the influenza virus genome after the rnps are synthesized in the nucleus , they are exported to the cytoplasm with the help of two viral proteins , m1 ( bui et al . , 1996 , 2000 ) and nuclear export protein ( nep / ns2 ; oneill et al . , 1998 ; ye et al . 2001 ) , via the cellular chromosome region maintenance 1 ( crm1 ) protein - dependent pathway ( neumann et al . , 2000 ; elton et al . they are then transported to the budding site of the apical plasma membrane of polarized cells , through a rab 11-dependent vesicular transport pathway ( amorim et al . , 2011 ; eisfeld et al . , 2011 ; momose et al . , the transmembrane proteins ha , na , and an ion channel protein ( m2 ) are conveyed to the budding site by a standard exocytic pathway through the endoplasmic reticulum and golgi apparatus . m1 is presumed to interact with both the rnps and the cytoplasmic tails of ha , na , and m2 and to act as a bridge between them at the budding site ( zhang et al . , 2000 ; mccown and pekosz , 2005 ) . eventually , all of the viral components , including the viral transmembrane proteins , m1 , and rnps , are orderly assembled into progeny virions . the progeny virions are released from the apical plasma membrane into the extracellular environment , where na plays an important role in cleaving sialic acids bound to receptors on the cell surface and ha proteins on the progeny virions . although the morphology of influenza a virions , whether budding virions or purified virions , has been investigated extensively for many years by using various electron microscopic methods , knowledge of the virion structure remains limited , mainly due to pleomorphism , which complicates morphological and structural analyses and impedes investigators efforts to draw conclusions about the native structure of the virion . however , recent electron microscopic analyses have revealed several important morphological and structural features of influenza virions , which provide valuable information about the native morphology of these structures . this review summarizes the current progress in virion morphology studies and presents a model of the likely native morphology of influenza a virion . virions possess a lipid envelope that is derived from the host cellular membrane during the budding . they are generally spherical or elliptical in shape , ranging from approximately 80120 nm in diameter and are occasionally filamentous , reaching more than 20 m in length . sometimes , however , they take on an irregular shape . virions are covered with numerous membrane - spanning glycoproteins , ha and na , and small amounts of m2 . the peripheral membrane protein , m1 , which is one of the most abundant viral proteins in the virion , binds to the lipid envelope to maintain virion morphology . although small amounts of nep are present within the virion ( yasuda et al . , 1993 ) , its localization is unknown . most laboratory - adapted strains of influenza a virus are predominantly spherical or elliptical , whereas clinical isolates are predominantly filamentous and their adaptation in eggs results in the loss of this filamentous phenotype ( chu et al . , 1949 ; kilbourne and murphy , 1960 ) . the filamentous phenotype is thought to be a genetic trait , as shown by virus gene reassortment experiments ( smirnov et al . , 1991 ) and reverse genetics studies ( bourmakina and garcia - sastre , 2003 ; elleman and barclay , 2004 ) . such studies have demonstrated that the m1 and m2 proteins are important determinants of filamentous virion formation ( hughey et al . , 1995 ; roberts et al . , 1998 ; bourmakina and garcia - sastre , 2003 ) . in addition , the polarized cell phenotype and actin cytoskeleton network also play important roles in the formation of filamentous virions ( roberts and compans , 1998 ) , suggesting that several factors are involved in determining virion morphology . the significance of the filamentous morphology for virus replication and pathogenesis remains uncertain ; however , the fact that clinical isolates , independent of ha and na subtype , consistently form filamentous shapes strongly suggests that this filamentous phenotype is essential for virus survival in nature . the filamentous morphology may , for example , facilitate cell - to - cell transmission of progeny virions in the respiratory mucosa of infected individuals . in addition to the spherical , elliptical , and filamentous virions , irregular - shaped virions are sometimes observed when using negative - staining electron microscopy ( negative - staining em ) of purified virions ( figure 1 ; stevenson and biddle , 1966 ; almeida and waterson , 1967 ; wrigley , 1979 ) . interestingly , such virions have never been reported when ultrathin - section transmission electron microscopy ( ultrathin - section em ) or scanning electron microscopy ( sem ) is used to view budding virions . this discrepancy is thought to be partially due to artifacts caused during sample preparation for negative - staining , since artifacts can be introduced into fragile biological specimens on air drying ( nermut and frank , 1971 ; ruigrok et al . , 1992 ) . however , other artifacts may also be included , because such irregular - shaped virions are also reported when using cryoelectron microscopy ( cryoem ) of purified virions ( harris et al . , 2006 ; nayak et al . , 2009 ) , which does not involve air drying . earlier reports suggested that the pleomorphism is introduced during the storage of virions at 4c after they are harvested ( choppin et al . , 1961 ; more importantly , it was recently demonstrated that virion morphology is substantially affected by ultracentrifugation , which is often involved in the negative - staining process , resulting in the appearance of morphologically irregular virions ( sugita et al . , 2011 ) . when virions are chemically fixed with glutaraldehyde ( gla ) , which presumably creates cross - links among the membrane - bound m1 proteins that form the layer underneath the viral envelope as well as among m1 proteins and the cytoplasmic tails of the ha , na , and m2 transmembrane proteins , the virion morphology is relatively conserved even after ultracentrifugation . this finding is consistent with observations made when using ultrathin - section tem and sem of budding virions , where morphologically irregular virions have never been found ; sample processing of the ultrathin - section tem and sem usually begins with gla fixation , and does not involve ultracentrifugation . taken together , the pleomorphism of influenza virions observed when using negative - staining em and cryoem is an artifact that is mainly introduced during sample preparation procedures that involve ultracentrifugation and storage . ( a ) spherical virions [ a / wsn/33 ( h1n1 ) ] , ( b ) filamentous virions [ a / udorn/307/72 ( h3n2 ) ] , and ( c ) irregular - shaped virions [ a / puerto rico/8/34 ( h1n1 ) ] . the eight vrna segments , which contain conserved sequences at their 3 and 5 ends that are partially complimentary to each other and form the core promoter ( skehel and hay , 1978 ; robertson , 1979 ; desselberger et al . , 1980 ) , range in size from 890 to 2341 bases . each vrna segment is separately encapsidated by np to form the rnp , which has a flexible , twisted rod - like structure that is folded back and coiled on itself . the rod - like rnps are approximately 12 nm in diameter , but vary in length from approximately 30110 nm ( pons et al . , 1969 ; compans et al . , 1972 ) . the length of each rod - like rnp correlates with the length of each vrna segment ( compans et al . , 1972 ) . although the morphology of the purified rnps has been well characterized , the conformation of rnps inside virions had been largely unknown for a long time . when virions were examined by using negative - staining em or cryoem , a continuous strand of 78 nm in diameter , which was regularly packed in the form of a helix , was observed within disrupted virions ( hoyle et al . , 1961 ; apostolov and flewett , 1965 ; schulze , 1972 ; almeida and brand , 1975 ; murti et al . , 1980 , 1992 ; although the strands were observed only at a low frequency , they were the sole structure clearly visualized by using negative - staining em and cryoem . therefore , it was proposed that rnps exist as a single continuous helix , which would be fragmented into multiple rod - like rnps during the purification process of rnps ( almeida and brand , 1975 ; murti et al . , 1980 ) . later , however , it was demonstrated by use of negative - staining em ( ruigrok et al . , 1989 ) and cryoem ( calder et al . , 2010 ) , that the continuous helix found within disrupted virions was a layer of m1 proteins underneath the lipid envelope . how , then , are the rnp fragments arranged within the virion ? by using thin - section em , it is shown that progeny virions budding from the plasma membrane of infected cells contain a distinct arrangement of rnps ( oxford and hockley , 1987 ; noda et al . , 2006 ) . the rnps are always suspended from the top of the budding virions and are oriented perpendicular to the budding tip . many transversely sectioned budding virions contain eight rnps , in which a central rnp is consistently surrounded by seven others . serial transverse ultrathin - sections of whole budding virions have revealed that the eight rnps within a virion differ in length , suggesting that the budding virion contains different kinds of vrnas ( compans et al . , 1972 ) . interestingly , the eight rnps are confined to the top of the filamentous virion and the reminder of the filamentous virion is empty , indicating that such filamentous virions do not randomly incorporate multiple sets of eight rnps ( noda et al . , 2006 ) . recent cryoem analyses of purified virions confirmed that they contain eight rnps arranged in a distinct pattern ( harris et al . furthermore , the rnps are restricted to one end of the filamentous particles that are released from infected cells , which is consistent with the observations made by using ultrathin - section em of budding virions ( noda et al . , 2006 ) . thus , virions , regardless of their shape , incorporate an organized set of eight rnps . based on recent microscopic analyses , a schematic diagram of a native influenza virion is depicted in figure 2 . whether spherical , elliptical ( figure 2 left ) , or filamentous ( figure 2 right ) , the lipid - enveloped virions are regular in shape and covered with ha and na glycoproteins and m2 proteins . single na proteins can be observed anywhere on the virion surface , whereas a cluster of na proteins is also found at the end of the virion that is opposite to the end where the rnps are located ( calder et al . , 2010 ) . because the rnps bind to the inner leaflet of the envelope at the distal end of budding virion ( noda et al . , 2006 ; calder et al . , 2010 ) , the na cluster would play a role in release from the infected cell by destroying the ha binding to receptors on the cell surface . , 2010 ) , m2 proteins are likely located at the proximal end of the budding virion , on the same side as the na cluster . in addition , the m2 protein plays an important role in rnp incorporation ( mccown and pekosz , 2005 ; iwatsuki - horimoto et al . , 2006 ) , suggesting that it should also be present at the distal end of the budding virion , where the m2 protein and rnps could associate with each other . the m1 protein forms a matrix layer underneath the lipid envelope that is arranged as an ordered helix composed of multiple copies of m1 proteins that contribute to the virion morphology ( calder et al . , note that both virions contain an organized set of eight rnps at the end of the virion . the eight rod - like rnps , which differ in length , are arranged in a distinct pattern , in which seven peripheral rnps surround a central rnp ( oxford and hockley , 1987 ; noda et al . , 2006 ) . such an arrangement is observed not only in budding virions , but also in isolated virions ( harris et al . , 2006 ; 2010 ) , suggesting that there are interactions among the rnps to maintain this organization . the eight rnps are consistently associated with the inner envelope at the distal end of the budding virion and oriented almost perpendicular to the budding tip ( oxford and hockley , 1987 ; noda et al . , 2006 ) , indicating a polar organization of the virion . electron microscopic approaches are useful methods for investigating morphological and structural features of viruses . however , when fragile , lipid - enveloped viruses , including influenza viruses , are examined by using electron microscopy , artifactual effects such as ultracentrifugation , storage , and air drying should be considered . recent electron microscopic analyses of influenza virions clearly show that the native virion morphology is uniformly spherical , elliptical , or filamentous . irregularly shaped virus particles are mainly artifacts caused during sample processing , indicating that such virus particles unlikely reflect the true virion structure . accordingly , it is important to pay close attention to sample processing , when structurally examining influenza virions with electron microscopic techniques . despite the fact that various electron microscopic analyses have contributed to our knowledge of influenza virion morphology , much about the internal structure of the virion remains unknown . for instance , how do the rnps associate with the envelope at the tip of the virion ? how are the eight rnps arranged in their distinct pattern ? although solving these issues by using conventional two - dimensional electron microscopic methods , such as thin - section em and negative - staining em would be difficult , if not impossible , they can be addressed by using three - dimensional analysis of rnps within the virion by means of electron tomography , which provides higher resolution images . the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

influenza a virus is an enveloped virus with a segmented , single - strand , negative - sense rna genome . its virions show spherical or filamentous shapes of about 100 nm in diameter and occasionally irregular morphology , which exemplifies the pleomorphic nature of these virions . each viral rna segment forms a ribonucleoprotein complex ( rnp ) , along with an rna - dependent rna polymerase complex and multiple copies of nucleoproteins ; the rnps reside in the enveloped virions . here , we focus on electron microscopic analyses of influenza virions and rnps . based on the morphological and structural observations obtained by using electron microscopic techniques , we present a model of the native morphology of the influenza virion .

Introduction The Morphology of the Influenza a Virion RNPs within the Virion A Model of a Native Influenza a Virion Conclusion Conflict of Interest Statement

abelson murine leukemia viral oncogene large tumor suppressor runt - related transcription factor peroxisome proliferator - activator receptor gamma 2 transcriptional enhancer activator domain yes - associated protein based on a number of criteria , certain genes are categorized as oncogenes whereas the genes that oppose their function are regarded as tumor suppressors . this categorization is of enormous value in investigating the molecular basis of cell fate determination . by and large , these genes are components or targets of cellular signaling pathways that transmit bimodal on and off instructions . the extent to which this perception is correct forms the basis of our recent study . we investigated the crosstalk between the dna damage response pathway and the hippo pathway and revealed that a proto - oncoprotein switched to behave as a tumor suppressor . our finding therefore violates the on / off instruction rules by including a third option of opposing antipodal switching . the transcriptional coactivator yes - associated protein ( yap ) enters the nucleus by default to target specific genes . yap has been implicated as an oncogene in conjunction with the transcriptional enhancer activator domain ( tead ) family of transcription factors in human cancers , where it activates proproliferative and antiapoptotic target genes . the activity of yap is blunted by blocking its nuclear entry through the hippo pathway , which is important in regulating cell contact inhibition , organ size control , and cancer development . under conditions of cell cell contact , upstream elements of the hippo pathway transmit signals through the kinase large tumor suppressor ( lats ) , which phosphorylates yap on serine residues resulting in yap cytoplasmic sequestration , downregulation of its nuclear target genes , and compromised oncogenic potential . a twist was introduced into this coherent picture when it was recognized that yap is also a coactivator of proapoptotic genes . in response to dna damage , yap targets p73 , a member of the tumor suppressor p53 family , to induce proapoptotic gene expression and initiate a cellular death axis . the non - receptor tyrosine kinase abelson murine leukemia viral oncogene ( c - abl ) is activated under conditions of dna damage and phosphorylates tyrosine residues on both p73 and yap . tyrosine phosphorylated yap promotes the death axis at 2 levels : by enhancing the p73-mediated death axis and by supporting p73 accumulation . moreover , when coactivating the runt - related transcription factor ( runx ) , yap regulates the expression of itch , the p73 e3 ligase . upon tyrosine phosphorylation by c - abl , c - abl thus switches yap behavior from activating the e3 ligase that induces p73 degradation to coactivating the accumulated p73 . this regulatory module exemplifies the capacity of c - abl to switch yap behavior to promote the opposing function . a similar behavior was discovered at the level of targeting the tead transcription factor . the nuclear yap tead complex controls the survival axis by inducing transcription of a set of genes involved in 2 major activities : supporting cell proliferation on one hand and inhibiting apoptosis on the other hand . these two yap - regulated activities give rise to a net increase in the cell population . however , when c - abl is activated in response to dna damage , the tyrosine - phosphorylated yap still binds tead but is unable to induce the survival axis . thus , as modified yap also induces the death axis via p73 , activated c - abl switches yap from being an oncogene to becoming a tumor suppressor ( fig . these findings exemplify a newly emerging principle in signaling : not only turning on and off , but also dictating an opposing function . 1.yap is switched from oncogene to tumor suppressor function through phosphorylation by c - abl . ( a ) when the hippo pathway is inactive , as in proliferating cells , yes - associated protein ( yap ) coactivates the transcriptional enhancer activator domain ( tead ) transcription factor to express survival genes involved in the suppression of apoptosis . ( b ) under dna damage conditions , abelson murine leukemia viral oncogene ( c - abl ) is activated and phosphorylates p73 and yap . these modifications lead to yap - p73 induced transcription of proapoptotic genes and activation of a death axis . simultaneously , phosphorylation of yap by c - abl inhibits its potential to coactivate tead , resulting in blunting of the expression of antiapoptotic genes and promotion of apoptosis . yap is switched from oncogene to tumor suppressor function through phosphorylation by c - abl . ( a ) when the hippo pathway is inactive , as in proliferating cells , yes - associated protein ( yap ) coactivates the transcriptional enhancer activator domain ( tead ) transcription factor to express survival genes involved in the suppression of apoptosis . ( b ) under dna damage conditions , abelson murine leukemia viral oncogene ( c - abl ) is activated and phosphorylates p73 and yap . these modifications lead to yap - p73 induced transcription of proapoptotic genes and activation of a death axis . simultaneously , phosphorylation of yap by c - abl inhibits its potential to coactivate tead , resulting in blunting of the expression of antiapoptotic genes and promotion of apoptosis . at first it may appear odd that a cell would use the same protein as an oncogene and as a tumor suppressor . what could be the advantage of one effector controlling 2 opposing tasks ? recent studies revealed that il-2 exhibits paradoxical behavior in the determination of t - cell homeostasis . by applying a mathematical model , it was demonstrated that a single secreted molecule makes the homeostasis more robust against perturbations . in our system the hippo pathway turns yap effector activity on and off whereas the dna damage response switches yap activity to the opposing task . knows what to do when receiving 2 schizophrenic instructions . we hypothesized that one instruction must be dominant over the other . we found that when the hippo pathway is active , namely under cell cell contact conditions , the dna damage response is inactivated . this is accomplished by a double lock mechanism ; on one hand the hippo kinase lats neutralizes yap via its sequestration in the cytoplasm , while on the other hand it inhibits c - abl kinase activity . the next question is why the hippo pathway is dominant over the dna damage response . the hippo pathway is functional at the level of on / off in a reversible manner . yap enters and exits the nucleus based on received instructions and does not reach a fixed irreversible status . in contrast , c - abl is involved in irreversible nuclear processes , such as inducing the death axis activity of yap . recently , we found that c - abl promotes adipocyte differentiation , another irreversible process , by phosphorylation of the key adipogenic transcription factor peroxisome proliferator - activator receptor gamma 2 ( ppar2 ) and its subsequent stabilization and activation . it is therefore somewhat intuitive that the reversible processes would be dominant over the irreversible ones . in summary , we attribute signaling to not only the capacity to turn downstream effectors on and off , but also the ability to impose an opposing task on a given effector . the interplay between the hippo pathway and the dna damage response nicely exemplifies this principle . in addition , we think that this model is important to reach rapid homeostasis , provided that the involved pathways are hierarchically designed . 551/11 ) , and from the minerva foundation with funding from the federal german ministry for education and research .

cancer research has been significantly accelerated by viewing cancer as a functional collision between 2 dichotomous sets of genes : oncogenes and tumor suppressors . signaling pathways turn oncogenes and tumor suppressors on and off to dictate cell fate decisions . we contend that signaling also dictates opposing behaviors of a given effector .

Abbreviations None Disclosure of Potential Conflicts of Interest Funding

search for diabetes in youth ( search ) is a multicenter observational study of youth diagnosed with diabetes before age 20 years . participants were recruited from four geographically defined populations in ohio , washington , south carolina , and colorado , health plan enrollees in hawaii and california , and indian health service beneficiaries from four american indian populations . all registered patients were invited by mail , telephone , or at a clinic visit to complete a brief initial survey , and all survey completers whose diabetes was not secondary to other conditions were invited by telephone or at a clinic visit to complete a baseline research visit . participants whose diabetes was incident in 20022005 who completed the baseline study visit were invited for follow - up visits at 12 , 24 , and 60 months after their baseline study visit . during these visits , participants provided informed consent or , for those younger than 18 years old , assent and completed questionnaires . for metabolically stable participants ( i.e. , no episode of diabetic ketoacidosis during the previous month ) , fasting blood samples were drawn after a minimum 8-h overnight fast . these analyses include data from baseline visits of the 2001 ( prevalent ) cohort and from the baseline and 12-month follow - up visits of the 20022005 ( incident ) cohorts . baseline visits were completed by 45.7% of the eligible cohort ( 5,294 visits/11,592 subjects ) . after excluding 290 participants ( 5.5% ) without at least one pedsql module completed by youth or parent , the final baseline sample included 5,004 participants ( 2,462 prevalent and 2,542 incident subjects ) . these data were used to calculate mcids . of the 20022005 incident cohorts who completed the baseline and 12-month follow - up visits ( n = 1,449 , 57% of incident sample with baseline visits ) , 47 ( 3.2% ) without at least one pedsql module were also excluded , resulting in a final sample of 1,402 participants to identify predictors of qol change over time . youth and their parents reported on youths generic and health - related qol using the pedsql generic core and diabetes module ( 3 ) . all youth aged 5 years and older completed an age - appropriate self - report version of the two pedsql modules . items were read to the child or given to the child to read , depending on the child s reading skills . in addition , parents completed a parent - proxy version of the measures for all youth aged younger than 18 years at the time of their visit . thus , participants aged 18 years and older had one score for each module ( self - report ) , participants aged between 5 and 17 years received two scores for each module ( youth and parent report ) , and participants aged younger than 5 years had one score for each module ( parent report only ) . for all versions , items assess the degree of difficulty youth experience with different aspects of everyday living , including physical symptoms and emotional , social , and academic functioning ( generic core ) , as well as treatment adherence and barriers , diabetes - related worries , and communication with others about diabetes ( diabetes module ) . item scores were transformed , with subscale and total scale scores ranging from 0 to 100 . higher scores reflect better qol . the pedsql generic core and diabetes module are psychometrically sound ( 3 ) . demographic and medical information was collected by self - report or parent report and medical record review . those who self - reported being of hispanic ethnicity were categorized as hispanic , regardless of race , and the remainder were categorized by self - reported race . if no or multiple races were endorsed , participants were categorized as other / unknown race / ethnicity . health insurance was categorized as private , state / federally funded , other ( including indian health service , student health clinics , military , or other / unknown sources ) , or none based on self - report . clinical data , including physical measurement and fasting blood samples , were obtained at the baseline search study visit . glycemic control was assessed via glycated hemoglobin ( a1c ) using high - performance liquid chromatography ( tosoh bioscience , montgomeryville , pa ) . bmi was calculated from height and weight measured at the initial study visit , and z - scores ( zbmi ) were calculated using centers for disease control and prevention algorithms ( 14,15 ) . diabetes type was determined by physician diagnosis , and comorbid medical diagnoses were based on self - report or parent report . the mean ( sd ) and scale reliability ( cronbach ) values were calculated for each subscale and the total scores for the generic core and the diabetes module . mcids were calculated using a commonly used distribution - based method : one standard error of measurement ( sem ) ( 12,16 ) . the following equation is used to calculate the sem : sem = sd [ 1 ] , where is the total score or subscale reliability estimate . the sem estimates the variation in scores due to the measurement precision in the scale and assumes that a change in scores smaller than the value of the sem likely results from measurement error rather than a meaningful increase or decrease in the value of the construct being measured ( in this case , qol ) . a score change greater than or equal to the value of the sem represents meaningful variation in the measured construct that is likely not due to measurement error . mcids for various qol ( 17,18 ) measures , including the mcid for the pedsql across a range of pediatric chronic conditions ( 13 ) , have been calculated using this approach . this statistical methodology produces mcids that are expressed in the same units of measurement as the qol score ( 19 ) . the sem and resulting mcids were calculated for the sample of participants and parents for the total and subscale scores of the generic core and diabetes module and were also established separately for each diabetes type . the result is a single value that represents the magnitude of change in scores on qol measures that is detectable to the individual with the chronic condition . thus , the mcid represents a clinically meaningful or important difference in qol that can be referenced in clinical practice and research . to examine predictors of change in qol , two sets of regression analyses were conducted using data from the subset of participants diagnosed from 20022005 with a 12-month follow - up visit and complete data at both time points ( total n = 1,402 [ n range 1,0721,094 ] across the two modules and two reporters ) . general linear regression models were constructed to determine the demographic and clinical characteristics that predicted the amount and direction of improvement or worsening in pedsql change scores . pedsql change scores were calculated as total score at the follow - up visit minus total score at the baseline visit . predictors were demographic and clinical variables that have previously demonstrated associations with qol , including age , sex , insurance coverage , race / ethnicity , diabetes type , diabetes duration , z - bmi , and a1c , all of which were assessed at the baseline visit , and models controlled for pedsql scores at baseline . next , to determine the characteristics that predicted clinically meaningful improvement in qol , participants pedsql change scores for 1 year were categorized as positive ( score increase 1 mcid ) , negative ( score decrease 1 mcid ) , or no change . binary logistic regression models were constructed to predict positive qol change of at least 1 mcid compared with negative / no qol change , controlling for all measured demographic and medical variables noted above , time from baseline to follow - up , and baseline pedsql scores . separate analyses were conducted for parent and child report of the generic core and diabetes module total scores . the mean ( sd ) and scale reliability ( cronbach ) values were calculated for each subscale and the total scores for the generic core and the diabetes module . mcids were calculated using a commonly used distribution - based method : one standard error of measurement ( sem ) ( 12,16 ) . the following equation is used to calculate the sem : sem = sd [ 1 ] , where is the total score or subscale reliability estimate . the sem estimates the variation in scores due to the measurement precision in the scale and assumes that a change in scores smaller than the value of the sem likely results from measurement error rather than a meaningful increase or decrease in the value of the construct being measured ( in this case , qol ) . a score change greater than or equal to the value of the sem represents meaningful variation in the measured construct that is likely not due to measurement error . mcids for various qol ( 17,18 ) measures , including the mcid for the pedsql across a range of pediatric chronic conditions ( 13 ) , have been calculated using this approach . this statistical methodology produces mcids that are expressed in the same units of measurement as the qol score ( 19 ) . the sem and resulting mcids were calculated for the sample of participants and parents for the total and subscale scores of the generic core and diabetes module and were also established separately for each diabetes type . the result is a single value that represents the magnitude of change in scores on qol measures that is detectable to the individual with the chronic condition . thus , the mcid represents a clinically meaningful or important difference in qol that can be referenced in clinical practice and research . to examine predictors of change in qol , two sets of regression analyses were conducted using data from the subset of participants diagnosed from 20022005 with a 12-month follow - up visit and complete data at both time points ( total n = 1,402 [ n range 1,0721,094 ] across the two modules and two reporters ) . general linear regression models were constructed to determine the demographic and clinical characteristics that predicted the amount and direction of improvement or worsening in pedsql change scores . pedsql change scores were calculated as total score at the follow - up visit minus total score at the baseline visit . predictors were demographic and clinical variables that have previously demonstrated associations with qol , including age , sex , insurance coverage , race / ethnicity , diabetes type , diabetes duration , z - bmi , and a1c , all of which were assessed at the baseline visit , and models controlled for pedsql scores at baseline . next , to determine the characteristics that predicted clinically meaningful improvement in qol , participants pedsql change scores for 1 year were categorized as positive ( score increase 1 mcid ) , negative ( score decrease 1 mcid ) , or no change . binary logistic regression models were constructed to predict positive qol change of at least 1 mcid compared with negative / no qol change , controlling for all measured demographic and medical variables noted above , time from baseline to follow - up , and baseline pedsql scores . separate analyses were conducted for parent and child report of the generic core and diabetes module total scores . the study sample was composed of 5,004 children , adolescents , and young adults diagnosed with type 1 or type 2 diabetes in 2002 through 2005 ( n = 4,393 ) or whose diabetes was prevalent in 2001 ( youth were a mean age of 12.5 years ( sd , 4.7 ; range , 222 years ) , 51.3% female , 67.9% non - hispanic white , and 19.3% were publically insured ( medicaid / medicare ) . the demographic and clinical characteristics of participants in the study sample stratified by diabetes type are reported in table 1 . baseline demographic and clinical characteristics of 5,004 study participants , by diabetes type : search for diabetes in youth study , 20012005 the internal consistency of the pedsql total scores for the sample was excellent for the generic core ( youth report : = 0.87 , parent report : = 0.88 ) and diabetes module ( youth report : = 0.85 , parent report : = 0.88 ) . in most cases , the youth- and parent - reported total and subscale scores were consistently higher for type 1 diabetes than for type 2 diabetes , although the sds were quite large for both groups . mcids for the pedsql generic core and diabetes module at the baseline study visit , by reporter ( youth / parent ) and diabetes type : search for diabetes in youth study , 20012005 the pedsql was completed by 4,041 participant parent pairs for participants aged between 5 and 17 years , 551 participants aged 18 years or older , and 412 parents of participants aged younger than 5 years . the mcid for the generic core total score was 4.83 for youth - report and 5.16 for parent - report ( table 2 ) . for youth with type 1 diabetes , the generic core mcids were 4.72 for youth - report and 4.88 for parent - report . the generic core mcids were higher for those with type 2 diabetes compared the mcids for youth with type 1 diabetes ; mcids among youth with type 2 diabetes were 5.41 for youth - report and 6.27 for parent - report . the mcid for the diabetes module total score was 5.34 for youth - report and 4.64 for parent - report . the diabetes module mcids for participants with type 1 diabetes were 5.27 for youth - report and 4.54 for parent - report . as with the generic core mcids , the diabetes module mcids were higher among youth with type 2 diabetes : 5.96 for youth - report and 6.06 for parent - report . longitudinal analyses were conducted with data from 1,402 youth with at least one parent- or youth - reported score on the generic core or diabetes module at the 12-month follow - up visit ( n range 1,0721,984 across the two modules and two reporters ; table 3 ) . this subset comprises 55% of the 20022005 incident cohorts that were included in the baseline visit sample for this study . the 12-month follow - up visits occurred on average 14.6 months ( sd 3.1 [ range 6.028.1 ] ) after the initial visit . the youth in the longitudinal analysis were younger and had shorter disease duration ( due to the exclusion of the participants whose diabetes was prevalent in 2001 ) , and lower zbmi ( all p < 0.05 ) than youth in the larger cross - sectional sample used to calculate mcids at baseline . linear regression analysis of predictors of change in pedsql total scores * between the baseline and 12-month follow - up visit : search for diabetes in youth study , 20022005 incident cases in the linear regression models , improvements in youth - reported generic core and diabetes module pedsql change scores during 1 year were predicted by lower baseline qol ( = 0.50 and = 0.52 , respectively ; p < 0.0001 ) , male sex ( = 1.66 and = 1.93 , respectively ; p < 0.05 ) , and being privately insured ( = 2.25 and = 2.28 , respectively ; p < 0.01 ) . for parent - reported scores , lower baseline qol ( = 0.47 , p < 0.0001 ) , private insurance ( = 2.46 , p < 0.01 ) , diagnosis of type 1 diabetes ( = 4.36 , p < 0.01 ) , and lower baseline a1c ( = 0.98 , p < 0.001 ) predicted greater improvements in generic core change scores . lower baseline qol ( = 0.40 , p < 0.0001 ) , lower baseline a1c ( = 0.84 , p < 0.001 ) , and longer duration of diabetes ( = 0.12 , p < 0.01 ) predicted greater improvement in diabetes module change scores . across scales , reporters , and diabetes type , approximately one - third of the sample had pedsql change scores that decreased by 1 mcid , one - third had no significant qol change , and one - third had change scores that increased by 1 mcid . in the logistic regression models predicting a clinically meaningful improvement ( 1 mcid ) in pedsql change scores ( table 4 ) , baseline qol scores significantly predicted an improvement for both youth- and parent - reports on the generic core and diabetes module ( or range 0.920.94 , all p < 0.0001 ) . for youth - report , there were no additional predictors of clinically meaningful improvements on the generic core beyond baseline qol scores , whereas clinically meaningful improvements in diabetes module scores were also predicted by being privately insured ( or 0.68 [ 95% ci 0.480.98 ] , p < 0.05 ) . for parent - report , beyond baseline qol scores , clinically meaningful improvements on the generic core were also predicted by private insurance ( or 0.65 [ 0.450.94 ] , p < 0.05 ) , lower bmi ( 0.86 [ 0.741.00 ] , p < 0.05 ) , and lower a1c ( 0.85 [ 0.770.95 ] , p < 0.01 ) , whereas clinically meaningful improvements on the diabetes module were also predicted by lower a1c ( 0.87 [ 0.790.97 ] , p < 0.05 ) . logistic regression analysis to identify predictors of 1 mcid improvement on pedsql total score between the baseline and 12-month follow - up study visits : search for diabetes in youth study , 20022005 incident cases the study sample was composed of 5,004 children , adolescents , and young adults diagnosed with type 1 or type 2 diabetes in 2002 through 2005 ( n = 4,393 ) or whose diabetes was prevalent in 2001 ( youth were a mean age of 12.5 years ( sd , 4.7 ; range , 222 years ) , 51.3% female , 67.9% non - hispanic white , and 19.3% were publically insured ( medicaid / medicare ) . the demographic and clinical characteristics of participants in the study sample stratified by diabetes type are reported in table 1 . baseline demographic and clinical characteristics of 5,004 study participants , by diabetes type : search for diabetes in youth study , 20012005 the internal consistency of the pedsql total scores for the sample was excellent for the generic core ( youth report : = 0.87 , parent report : = 0.88 ) and diabetes module ( youth report : = 0.85 , parent report : = 0.88 ) . in most cases , the youth- and parent - reported total and subscale scores were consistently higher for type 1 diabetes than for type 2 diabetes , although the sds were quite large for both groups . mcids for the pedsql generic core and diabetes module at the baseline study visit , by reporter ( youth / parent ) and diabetes type : search for diabetes in youth study , 20012005 parent pairs for participants aged between 5 and 17 years , 551 participants aged 18 years or older , and 412 parents of participants aged younger than 5 years . the mcid for the generic core total score was 4.83 for youth - report and 5.16 for parent - report ( table 2 ) . for youth with type 1 diabetes , the generic core mcids were 4.72 for youth - report and 4.88 for parent - report . the generic core mcids were higher for those with type 2 diabetes compared the mcids for youth with type 1 diabetes ; mcids among youth with type 2 diabetes were 5.41 for youth - report and 6.27 for parent - report . the mcid for the diabetes module total score was 5.34 for youth - report and 4.64 for parent - report . the diabetes module mcids for participants with type 1 diabetes were 5.27 for youth - report and 4.54 for parent - report . as with the generic core mcids , the diabetes module mcids were higher among youth with type 2 diabetes : 5.96 for youth - report and 6.06 for parent - report . longitudinal analyses were conducted with data from 1,402 youth with at least one parent- or youth - reported score on the generic core or diabetes module at the 12-month follow - up visit ( n range 1,0721,984 across the two modules and two reporters ; table 3 ) . this subset comprises 55% of the 20022005 incident cohorts that were included in the baseline visit sample for this study . the 12-month follow - up visits occurred on average 14.6 months ( sd 3.1 [ range 6.028.1 ] ) after the initial visit . the youth in the longitudinal analysis were younger and had shorter disease duration ( due to the exclusion of the participants whose diabetes was prevalent in 2001 ) , and lower zbmi ( all p < 0.05 ) than youth in the larger cross - sectional sample used to calculate mcids at baseline . linear regression analysis of predictors of change in pedsql total scores * between the baseline and 12-month follow - up visit : search for diabetes in youth study , 20022005 incident cases in the linear regression models , improvements in youth - reported generic core and diabetes module pedsql change scores during 1 year were predicted by lower baseline qol ( = 0.50 and = 0.52 , respectively ; p < 0.0001 ) , male sex ( = 1.66 and = 1.93 , respectively ; p < 0.05 ) , and being privately insured ( = 2.25 and = 2.28 , respectively ; p < 0.01 ) . for parent - reported scores , lower baseline qol ( = 0.47 , p < 0.0001 ) , private insurance ( = 2.46 , p < 0.01 ) , diagnosis of type 1 diabetes ( = 4.36 , p < 0.01 ) , and lower baseline a1c ( = 0.98 , p < lower baseline qol ( = 0.40 , p < 0.0001 ) , lower baseline a1c ( = 0.84 , p < 0.001 ) , and longer duration of diabetes ( = 0.12 , p < 0.01 ) predicted greater improvement in diabetes module change scores . across scales , reporters , and diabetes type , approximately one - third of the sample had pedsql change scores that decreased by 1 mcid , one - third had no significant qol change , and one - third had change scores that increased by 1 mcid . in the logistic regression models predicting a clinically meaningful improvement ( 1 mcid ) in pedsql change scores ( table 4 ) , baseline qol scores significantly predicted an improvement for both youth- and parent - reports on the generic core and diabetes module ( or range 0.920.94 , all p < 0.0001 ) . for youth - report , there were no additional predictors of clinically meaningful improvements on the generic core beyond baseline qol scores , whereas clinically meaningful improvements in diabetes module scores were also predicted by being privately insured ( or 0.68 [ 95% ci 0.480.98 ] , p < 0.05 ) . for parent - report , beyond baseline qol scores , clinically meaningful improvements on the generic core were also predicted by private insurance ( or 0.65 [ 0.450.94 ] , p < 0.05 ) , lower bmi ( 0.86 [ 0.741.00 ] , p < 0.05 ) , and lower a1c ( 0.85 [ 0.770.95 ] , p < 0.01 ) , whereas clinically meaningful improvements on the diabetes module were also predicted by lower a1c ( 0.87 [ 0.790.97 ] , p < 0.05 ) . logistic regression analysis to identify predictors of 1 mcid improvement on pedsql total score between the baseline and 12-month follow - up study visits : the mcids for the pedsql generic core total score established in this study were consistent with published generic core total score mcids in other chronic conditions ( 4.36 for youth - report and 4.50 for parent - report ) ( 13 ) , ranging between 4 and 6 points across reporter and diabetes type . the values for most of the generic core subscales in the current sample of youth with type 1 and type 2 diabetes were higher than the pedsql generic core subscale mcids for youth with other chronic conditions , which are physical health , 6.66 , 6.92 ; emotional functioning , 8.94 , 7.79 ; social functioning , 8.36 , 8.98 ; and school functioning , 9.12 , 9.67 , for youth- and parent - report , respectively ( 13 ) . the only exception was the school functioning subscale , which was lower for both youth- and parent - reports . the higher mcids for most generic core subscales in this sample compared with other conditions suggests that across various domains of functioning , youth with diabetes seem to require more improvement or deterioration in qol to perceive a clinically meaningful difference in their subjective experience than do youth with other chronic conditions . given the complexity of the diabetes treatment regimen , a relatively large amount of change in qol ( 510 points ) may be needed for youth or their parents to perceive any meaningful differences in everyday functioning or activities . this pattern was especially pronounced in youth with type 2 diabetes , who had the highest mcid thresholds for clinically meaningful qol change . youth with type 2 diabetes report poorer qol and encounter multiple physical complaints and social and emotional difficulties ( 9,20 ) . in addition , given the higher zbmi scores among youth with type 2 diabetes compared with type 1 diabetes , there may be a cumulative effect of obesity and type 2 diabetes on some individuals qol ( 21 ) . as such , greater change in qol may be necessary for youth with type 2 diabetes to perceive an improvement or deterioration in their subjective well - being . for the diabetes module , the total score mcids were similar to the generic core total score mcids . although there is no direct point of comparison for the subscale mcids , it is notable that those of the diabetes module are substantially higher than the subscale mcids for the generic core . in particular , the treatment barriers , treatment adherence , and worries subscale mcids tended to be at or above 10 points . the higher mcids for these subscales reflect their lower cronbach s values . although these subscales have not been supported by factor analyses conducted on responses from youth with type 1 diabetes ( 7,10 ) , subscale mcids are reported here for comparison purposes and to illustrate mean score and similarities between parents of youth with type 1 and type 2 diabetes , which had not previously been reported by the search study . for clinicians and clinical trials researchers , fluctuations in qol that do not meet the mcid threshold may not be noticeable by patients or their families yet should be monitored to determine whether more substantial challenges arise and require intervention . the relatively higher mcids for youth with diabetes than for youth with other chronic conditions may translate to a need for interventions that explicitly target various components of general and diabetes - specific qol to achieve a change of sufficient magnitude for patient and parent perception of improvement . interventions to improve qol may benefit from a direct , targeted focus on barriers and facilitators of diabetes management and control . for example , given previous evidence of strong associations between lower qol and more depressive symptoms ( 10 ) , qol interventions that address diabetes - specific emotional concerns may be particularly beneficial . these data support published and ongoing intervention studies that demonstrate benefits of providing psychological support and skills training to youth with diabetes ( 12,23 ) . in addition , efforts to improve qol among youth with type 2 diabetes may achieve greater qol change by addressing diabetes and also weight - related domains of everyday functioning . associations between pedsql scores and a1c suggest that interventions that address and improve qol may ultimately translate to clinical improvements in glycemic control . one hypothesis is that patients who perceive better qol may encounter fewer barriers to diabetes management and may be more motivated to adhere to diabetes treatment recommendations such as self - monitoring . adaptation of established psychological interventions for youth with type 1 diabetes to meet the unique needs of youth with type 2 diabetes is needed ( 23 ) . longitudinal findings demonstrate that a number of demographic and clinical variables predicted increases in pedsql change scores over 1 year . the association between lower pedsql scores at baseline and larger change scores at 12 months likely reflects that participants with poorer initial qol had more opportunity for improvement . the associations of sex , insurance coverage , diabetes type , diabetes duration , and a1c at baseline with 12-month qol improvements are consistent with previous studies examining changes in qol after clinical or medical intervention ( 24,25 ) and help to identify patients who may experience qol fluctuations over time . given different predictors of parent- and youth - reported qol change , interventions will need to be tailored to individuals unique experiences to influence each of their perceptions of qol . because parent - reported change scores were predicted by bmi and a1c , efforts to improve physical well - being and glycemic control may be particularly influential on parental perceptions of their children s qol . however , the finding that children s qol change scores were predicted by sex and insurance status suggests that girls and those with public insurance may be most likely to benefit from additional qol monitoring . through this monitoring , it will be necessary to engage youth in discussion to identify and address the factors that are affecting their qol ratings . the identification of pedsql mcids also allowed us to determine the characteristics that predict who is most likely to experience clinically meaningful improvements in qol . private insurance coverage , lower bmi , and lower a1c at the baseline study visit predicted change that exceeded the threshold of perceived improvement in various qol domains . beyond the auto - correlational effects of baseline qol , these variables may represent protective factors , such as greater access to resources or better health status , that help individuals rebound from periods of lower qol and avoid long - lasting problems that may ultimately affect diabetes management and control . the interpretation of this study s findings should consider the response rates and limitations of the pedsql modules and their analyses . as previously reported , the baseline visit response rate was lower than expected ; older youth , african american youth , and those with type 2 diabetes were less likely to complete a visit ( 26 ) . the pedsql diabetes module was not developed or normed for youth with type 2 diabetes ( 3 ) , so the results of the pedsql diabetes module for youth with type 2 diabetes and their parents may have limited utility . concerns about psychometric properties of the diabetes module subscales among youth with type 1 diabetes have been reported ( 7,10 ) suggesting that use of total scores , and the associated mcids , is more appropriate than subscale scores . in addition , mcids were calculated for all pedsql subscales for consistency , but the lower values for some of these subscales resulted in larger mcids , which should be used with caution . finally , although we used the same approach to calculate mcids as used with the generic core for youth with other chronic conditions ( 13,18,19 ) and evidence demonstrates similarities between mcids established using statistical methods ( e.g. , 1 sem ) and those calculated in other ways ( 17 ) , alternative mcid calculations might have yielded different results . because concurrent measures of qol were not collected , alternative mcid calculations could not be conducted . nonetheless , the establishment of diabetes - specific mcids for the pedsql generic core and diabetes module for parents and youth provide an important reference point for clinicians and researchers interested in the effect of treatments on patient qol . clinical trials researchers may use these mcids to evaluate the degree to which an intervention improves participants qol in a clinically significant way . longitudinal research is needed to determine whether clinically meaningful changes in perceived qol are associated with clinically meaningful changes in indices of diabetes health status , such as glycemic control and risk for acute complications .

objectiveto establish minimal clinically important difference ( mcid ) scores representing the smallest detectable change in quality of life ( qol ) , using the pediatric quality of life inventory ( pedsql ) generic core and diabetes module among youth with diabetes and their parents , and to identify demographic and clinical correlates of qol change over 1 year.research design and methodsparticipants in the search for diabetes in youth study aged > 5 years and parents of youth aged < 18 years completed pedsql surveys at their initial and 12-month study visits . mcids for each pedsql module were calculated using one standard error of measurement . demographic and clinical characteristics associated with qol change were identified through multiple linear and logistic regression analyses.resultsthe sample comprised 5,004 youth ( mean age , 12.5 4.7 years ; mean diabetes duration , 3.4 3.7 years ) . of 100 possible points , pedsql total score mcids for youth with type 1 and type 2 diabetes , respectively , were generic core , 4.88 , 6.27 ( parent ) and 4.72 , 5.41 ( youth ) ; diabetes module , 4.54 , 6.06 ( parent ) and 5.27 , 5.96 ( youth ) . among 1,402 youth with a follow - up visit , lower baseline qol , male sex , private insurance , having type 1 diabetes , longer diabetes duration , and better glycemic control predicted improvements in youth- and parent - reported pedsql total scores over 1 year . clinically meaningful ( 1 mcid ) improvements in total score for at least one pedsql module were predicted by private insurance , lower bmi , and lower a1c at baseline.conclusionsthese diabetes - specific reference points to interpret clinically meaningful change in pedsql scores can be used in clinical care and research for youth with type 1 and type 2 diabetes .

RESEARCH DESIGN AND METHODS Data analysis RESULTS Characteristics of participants MCIDs Predictors of change in QOL scores during 1 year CONCLUSIONS

metachromatic leukodystrophy ( mld ) ( scholz 's disease ) is an autosomal recessive lysosomal storage disease caused by the lack of arylsulfatase a ( asa ) . this enzyme is essential for the normal metabolism of sulfatides which are vital elements of the myelin sheath . in mld , sulfatides accrue in many organs comprising brain , peripheral nerves , kidneys , liver , and gallbladder . sulfatide collects in white matter of the central nervous system and peripheral nerves and causes progressive demyelination and lethal neurological symptoms . mld is diagnosed biochemically by finding low levels of arylsulfatase in peripheral white blood cells and urine . mld is classified into three main clinical forms on the basis of the age of onset . the most common and lethal form is the late - infantile form , which begins before 4 years of age typically presenting between 12 and 18 months of age , and patients die by the end of the first decade . the juvenile form of mld comprises age onset between 4 and 16 years , whereas symptoms of adult mld begin after puberty . the patients usually present with signs and symptoms of peripheral neuropathy and alterations in intelligence , speech , and coordination . the disorder is progressive with gait disturbance , quadriplegia , decerebration , and mortality by the age of 6 months to 4 years . magnetic resonance ( mr ) imaging findings of the brain , especially t2-weighted imaging findings in the form of symmetric t2 hyperintense signal in the periventricular white matter , have been commonly reported in previous reports . however , diffusion - weighted imaging ( dwi ) findings have been sparsely reported and we present mr imaging , especially dwi findings in a 12-month - old patient of mld . a 12-month - old male presented with regression of milestones and progressive spasticity . mr imaging examination was performed and t2-weighted images revealed symmetrical hyperintensities , predominantly involving the deep white matter , corpus callosum with sparing of subcortical u - fibers [ figure 1 ] . the tigroid and leopard skin patterns of demyelination , which imply sparing of the perivascular white matter , were evident in the periventricular white matter and centrum semiovale [ figure 2 ] . the corpus callosum ( genu and splenium ) was also involved . using the transverse single - shot echo planar diffusion - weighted mr imaging , diffusion mr images were acquired . on b = 1000 mm / s images ( heavily dwis ) , hyperintensities were apparent in the deep white matter and corpus callosum with a signal intensity pattern the same as that of cytotoxic edema . on apparent diffusion coefficient ( adc ) maps , low levels of arylsulfatase in peripheral white blood cells and urine confirmed the diagnosis of mld . ( a and b ) bilateral symmetrical butterfly - shaped t2 hyperintensities of the periventricular and deep white matter with relative sparing of the subcortical u - fibers sagittal t2-weighted image showing tigroid pattern of due to hypointense linear bands against the background of hyperintense white matter the corresponding lesions showing restricted diffusion with high diffusion - weighted ( a and b ) and low apparent diffusion coefficient signal ( c and d ) symmetric confluent areas of t2 hyperintense signal in the periventricular white matter with sparing of the subcortical u - fibers and with no enhancement in postcontrast images are the most frequent reported mr imaging findings of mld . the tigroid and leopard skin patterns of demyelination are basically due to sparing of perivascular white matter and are visualized as dark spots or dark linear areas against a background of hyperintense white matter , giving the appearance of the skin of a leopard . the disease process also commonly affects the corpus callosum , internal capsule , and corticospinal tracts . the cerebellar white matter may also be affected and appears bright on t2-weighted images . in the advanced stage of mld , it has been reported that the diminished activity of asa leads to dysmyelination ( failure of myelin breakdown and reutilization ) . the corresponding distribution of the lesions on t2-weighted image and dwi in the present case strongly implied that the lesions on dwi were directly linked to the disease process . restricted diffusion ( cytotoxic edema ) was seen on the echo planar sequence as hyperintensities on heavily dwis ( b = 1000 s / mm ) and low adc values on adc maps [ figure 3 ] . sener reported a similar diffusion mr imaging pattern on echo planar diffusion images of a patient with mld with unchanged pattern on 6-month follow - up . phillips et al . also reported a similar pattern of restricted diffusion in patients of phenylketonuria which was hypothesized to be due to impaired myelination leading to reduced mobility of protons . hence , in the present case of mld , the restricted diffusion pattern perhaps was consistent with some breakdown of the white matter secondary to impaired myelination , which results in restricted mobility of protons leading to a diffusion mr imaging pattern similar to that of cytotoxic edema . to conclude , we report diffusion mr imaging of mld done using the echo planar sequence which has been sparsely reported . more diffusion mr imaging studies of dysmyelinating disorders might improve our interpretation of the imaging features of these diseases .

metachromatic leukodystrophy ( mld ) is caused by insufficiency of arylsulfatase a resulting in impaired myelination . diffusion magnetic resonance ( mr ) imaging features of this disease have been rarely reported . we report diffusion mr imaging of mld in a 12-month - old male who presented with regression of milestones and progressive spasticity .

Introduction Case Report Discussion Financial support and sponsorship Conflicts of interest

it is now firmly established that mitochondria are key players in different pathophysiological contexts ( duchen , 2004 ) . indeed , they are very efficient machines for decoding intracellular signals and in particular the calcium ( ca ) one ( clapham , 2007 ) . the mitochondrial respiratory chain , by pumping protons across the ion - impermeable inner membrane , establishes a gradient ( h ) , composed of an electrical potential ( ) and a concentration ratio ( ph ) , according to the nernst equation : h = zf + rt ln[h]i/[h]o . this has major implications for ca transport and distribution . in actively respiring mitochondria , considering that the buffering capacity is mostly provided by weak acids , the gradient is supposed to be maintained mostly as an electrical gradient across the inner membrane ( ~ 180 mv ) . this implies a strong thermodynamic force in favor of the accumulation of cations ( rizzuto et al . , 2000 ) . ca import across the outer mitochondrial membrane ( omm ) occurs through the voltage - dependent anion channels ( vdac ) ( simamura et al . , vdac is as a large voltage - gated channel , fully opened with high - conductance and weak anion - selectivity at low transmembrane potentials ( < 2030 mv ) , but switching to cation selectivity and lower conductance at higher potentials ( colombini , 2009 ; de pinto et al . , 2008 ; shoshan - barmatz et al . , ca mitochondrial traffic across the inner mitochondrial membrane ( imm ) takes place essentially through two pathways : i ) an electrophoretic uniporter that transports ca down the electrical gradient established by the respiratory chain , and ii ) a na / ca exchanger , mostly expressed in excitable cells ( muscle and brain ) , and a h / ca exchanger , that represents the prevailing route in most other tissues ( see fig . 1 ) . these electroneutral antiporters prevent the attainment of an electrochemical equilibrium ( rizzuto et al . , 2000 ) . despite intense investigations on the mitochondrial ca uniporter ( mcu ) for almost 50 years , the molecular identity of this gated channel is still a matter of debate . among the early candidates for the mcu were mitochondria - localized ryr1 ( beutner et al . , 2005 ) and the uncoupling proteins ( ucp ) 2 and 3 ( trenker et al . , 2007 ) , but such a role for these proteins is still controversial and further work will be needed to definitively elucidate the molecular identity of the channels or carriers involved . the identity of the mcu , as the product of the ccdc109a gene , has recently been reported by two recent publications ( baughman et al . , 2011 ; de stefani et al . , 2011 ) . in that respect , the report that the imm protein leucine - zipper - ef hand - containing transmembrane region ( letm1 ) , previously described as a k / h exchanger ( nowikovsky et al . , 2004 ) , could be a ca / h antiporter , catalyzing the uptake of ca into mitochondria was received with great interest ( jiang et al . , 2009 ) . these results remain a subject of intense discussion because they diverge in several issues from previous studies ; in particular , the effect of letm1 down - regulation / over - expression on mitochondrial ca transport could be a secondary effect of a decreased k / h exchange activity . finally , it is important to mention the recent description of an imm ca - binding protein named mitochondrial calcium uptake 1 ( micu1 ) , which appears essential for mitochondrial ca uptake ( perocchi et al . , 2010 ) . micu1 is a single - pass transmembrane protein and thus does not seem to participate in channel pore formation . it is possible that micu1 is part of a complex with the mitochondrial ca channel , or functions as ca buffer , or as a ca - dependent regulatory protein acting as a ca sensor ( through a pair of ca - binding ef - hand domains present in its sequence , the mutation of which eliminates the mitochondrial ca uptake ) . about the system for mitochondrial ca extrusion , the mitochondrial na / ca exchanger ( mncx ) and h / ca exchanger ( mhcx ) , have well known kinetic characteristics , are present in the imm ( bernardi , 1999b ) . recently , strong evidence has been provided that the na / ca exchanger isoform nclx is the long - sought protein responsible for the mitochondrial na - dependent ca efflux ( palty et al . , moreover , great attention has been drawn to the potential role of a large - conductance channel , commonly referred to as the permeability transition pore ( ptp ) . it is supposed to be a multiprotein complex activated in various pathophysiological conditions ( e.g. , mitochondrial ca overload ) ( see fig . 1 ) . its role in mitochondrial ca homeostasis , however , remains elusive ( bernardi and forte , 2007 ) . in a variety of cell systems ( ranging from epithelial cells to neurons ) , the cytosolic [ ca ] ( [ ca]c ) rises evoked by physiological stimulations are always paralleled by rapid mitochondrial [ ca ] ( [ ca]m ) increases , that reach values well above those of the bulk cytosol ( up to ~ 500 m in chromaffin cells ) ( giorgi et al . , 2009 ) . a ca signal originating in the cytoplasm that is propagated to mitochondria permits the transmission of an activatory signal to the energy powerhouse of the cell . here , three key metabolic enzymes ( pyruvate , -ketoglutarate , and isocitrate dehydrogenases ) are activated by ca . thus , in conjunction with the triggering of energy - consuming processes in the cytosol ( contraction , secretion , etc . ) , mitochondrial dehydrogenases are stimulated , adapting aerobic metabolism to the increased energy needs of an active cell ( rimessi et al . the alteration of the ca signals that reach mitochondria in association with different pathological conditions ( e.g. , oxidative stress ) can induce a profound alteration of organelle structure and function . as a consequence , the cell may be driven to its death ( giorgi et al . , 2008 ) . here , we review some human diseases associated with perturbations in mitochondrial homeostasis with particular emphasis on the role of ca signals . aging is not considered as an adaptative response , but rather as the price of other evolutionary selected functions , currently not completely specified ( kirkwood and austad , 2000 ) . this degeneration is characterized by a progressive loss of organ function , proposed to be at the basis of several degenerative disorders , promoted by a progressive occurrence of apoptosis in non - proliferating cells . it was recognized that mitochondrial atp synthesis is associated with reactive oxygen species ( ros ) production , including primarily superoxide ( o2 ) formation by the respiratory chain complexes i and iii ( balaban et al . , 2005 ; raha and robinson , 2000 ) . subsequently , superoxide leads to the formation of diverse oxidant products , such as peroxynitrite , hydrogen peroxide ( h2o2 ) and the highly reactive hydroxyl radical ( oh ) . mitochondria are considered the major source of ros and the extent of oxidative damage to macromolecules is generally thought to be the random consequence of physiological functions , as exemplified by lipid peroxidation , protein oxidation , and mitochondrial dna mutations ( cadenas and davies , 2000 ) . different works on the molecular routes of apoptosis have revealed the important role of mitochondria in decoding of oxidative insults , with the release of proteins such as cytochrome c ( cyt c ) or smac / diablo , which act as co - factors of effector caspases . ca overload , through a still controversial mechanism , can also lead to the opening of the ptp and swelling of the organelle in what appears to be a pivotal pathway ( duchen , 2000 ; ravagnan et al . , 2002 ) . one important implication of recent findings is that these phenomena are genetically determined at least in part and biochemically controlled by a stress - induced signal transduction pathway involving the 66-kda isoform of shc , p66shc . the relationship between mitochondria and p66shc emerged once the protein was effectively localized to the organelle and by the observation that , upon oxidative stress , part of the cytosolic pool of p66shc translocates to mitochondria ( orsini et al . the protein lacks a conventional mitochondrial targeting sequence , but its association with the mitochondrial tom / tim import complex and the mitochondrial heat shock protein mthsp70 has been reported ( orsini et al . , 2004 ) . within mitochondria , in particular in the mitochondrial intermembrane space , p66shc binds cyt c and acts as a redox - enzyme , generating h2o2 , which , in turn , induces the opening of the ptp and apoptosis . ros production by p66shc appears to be a specialized function whereby electrons are diverted from the mitochondrial electron transport chain to catalyze the partial reduction of molecular oxygen ( giorgio et al . , 2005 ) . furthermore , the ch2 domain of p66shc ( a specific n - terminus domain that distinguishes p66shc from the shorter shc isoforms , p46 and p52 ) seems to form a redox module responsible for apoptosis initiation , and can be activated through reversible tetramerization by forming two disulfide bonds ( donoghue et al . , the redox activity of p66shc is likely to be the explanation of the decrease in ros levels typical of p66shc knockout cells ( migliaccio et al . , 2006 ) , as well as an altered mitochondrial metabolism , characterized by lower oxygen consumption under basal conditions ( nemoto et al . , 2006 ) to exert its functions , p66shc has to be phosphorylated at serine 36 ( migliaccio et al . , 1999 ) : the responsible kinase was identified as pkc ( pinton et al . , 2007 ) . this phosphorylation of ser36 induces translocation of phosphorylated p66shc to mitochondria , and both h2o2 challenge and pkc activation promote binding of p66shc to pin1 , causing this translocation ( pinton et al . , 2007 ) . the enzymatic activity of pin1 is fundamental because the phosphorylated sites , once isomerized by pin1 , become targets for dephosphorylation by pp2a ( wulf et al . , 2005 ) . this solves the apparent paradox showing the importance of p66shc phosphorylation and the dephosphorylated state of p66shc within mitochondria . thus , the signaling pathway involving pkc , pin1 and pp2a controls the mitochondrial import of p66shc where the translocated protein can exert its oxidoreductase activity , generating h2o2 and inducing the opening of the ptp . all these events are finely linked to an altered mitochondrial ca homeostasis , revealing a primary mitochondrial perturbation both in structure and function . in fact , oxidative stress causes a drastic reduction of [ ca]m in mef cells , whereas knockouts for p66shc or pin1 are mostly insensitive to h2o2 treatment . moreover , the simple over - expression of pkc induces a strong decrease of mitochondrial ca uptake , underlining how mitochondrial ca alteration is one of the first events of oxidoreductase activity of p66shc . translocation of p66shc to mitochondria is a characteristic event of some kinds of tumors , such as prostate cancer . mef cells from p66shc knockout mice were more resistant to peitc ( an apoptosis - inhibitor ) and the same treatment increased both ser36 phosphorylation and p66shc - pin1 binding in pc-3 and lncap human cancer prostate cells ( xiao and singh , 2010 ) . moreover , growth stimulation of prostate cancer cells with 5-dihydrotestosterone ( dht ) is accompanied by increased p66shc levels , ros production , translocation of p66shc into mitochondria and an augmented interaction with cyt c ( veeramani et al . , 2008 ) . among other deleterious consequences described in this article , mitochondrial impairment has also been associated with the pathogenesis of some neurodegenerative and neuroinflammatory disorders . many lines of evidence suggest that both mitochondrial dna mutations and increased oxidative stress are major contributors in aging a process rendering the organism prone to neurodegeneration . particularly interesting , it has been demonstrated in many of the most frequently occurring diseases that mitochondria interact with proteins crucial to the development the pathology . in these interactions , ca homeostasis often plays a key role ( see fig . 2 ) . this brings hope for effective therapies that target mitochondrial processes and interaction sites of mitochondria and disease - related proteins . the most frequent age - related neurodegenerative disorder alzheimer 's disease ( ad ) is manifested by an impairment in cognitive abilities , particularly responsible for memory functions . at the molecular level , ad pathogenesis is associated with the accumulation of a number of proteins , including reelin and presenilins , which leads to the overproduction of -amyloid ( a ) . the -amyloid toxicity is further supported by the inability of neurons to properly regulate intracellular ca levels ( mattson , 2007 ; thibault et al . , 2007 ) . harmful consequences of a activity include increased ca influx into neurons , rendering them prone to excitotoxicity and apoptosis ( bezprozvanny and mattson , 2008 ) . also the interaction of a with other cations , such as fe and cu , leads to the generation of oh which causes membrane lipid peroxidation and , in turn , impairs the function of plasma membrane ca atpase ( pmca ) , a ca pump responsible for ca extrusion from the cell interior to the extracellular compartment . moreover , lipid peroxidation due to destabilization of membrane composition causes depolarization of plasma membrane and leads to the opening of nmdar and voltage - gated ca channels , additionally intensifying an influx of toxic amounts of ca into the cytoplasm ( berridge , 2010 ) . finally , mutants of presenilins lower the [ ca ] of intracellular store by increasing ca leakage and reducing ca uptake ( brunello et al . , 2009 ) . ca homeostasis can also be affected by ros due to the deterioration of membranes forming the intracellular ca stores ( e.g. , endoplasmic reticulum , er ) . it has also been demonstrated that a accumulates in mitochondria decreasing the activity of complexes iii and iv of the respiratory chain ( anandatheerthavarada and devi , 2007 ) . through such a decrease in mitochondrial function , a propels a vicious cycle in which an elevation in cytosolic ca levels , oxidative stress , and decreased atp synthesis , all induce a further ca overload and an even higher oxidative stress ( lin and beal , 2006 ) . taken together these findings indicate that suppression or blocking of ca influx could in effect promote the survival of degenerating neurons . several preliminary trials of l - type ca and nmdar channel blockers have been carried out with some success ; however , crucial obstacles still need to be overcome before the therapy can be considered successful ( mattson , 2007 ) . the most frequent movement disorder parkinson 's disease ( pd ) is caused by widespread neurodegeneration in the brain and a significant selective loss of nigrostriatal dopaminergic neurons ( thomas and beal , 2010 ) . processes underlying pd pathogenesis , especially in its non - familial occurrence , remain unclear . in both cases , the engagement of l - type ca channels is crucial in spontaneous pacemaking ( hausser et al . , 2004 ) , while at the same time it may contribute to elevated toxicity of mitochondrial toxins to nigrostriatal dopaminergic neurons . in this way , ca homeostasis is a regulator of their selective vulnerability ( chan et al . , 2009 ; surmeier et al . , 2010 ) . it has been proposed that the reduction in expression of the transient receptor potential cation ( trpc1 ) channel may be involved in dopaminergic neurodegeneration . trpc1 could also be neuroprotective against pd - inducing agents ( bollimuntha et al . , 2005 ) . huntington 's disease ( hd ) is characterized by severe motor , cognitive , and psychiatric symptoms . the genetic etiology of hd has been established to be a cag - triplet mutation within the huntingtin gene , inherited in an autosomal dominant fashion . possible links between mitochondrial dysfunctions and changes in ca homeostasis and hd pathology have been recently reviewed ( celsi et al . , 2009 ) . a number of authors report that this disease , prevalently initiated by neurological insults , is related to shifts in ca homeostasis . an increase in extracellular glutamate concentration sustains long - term distortions in ca signaling , a characteristic recognized as the epileptic phenotype ( delorenzo et al . a number of mitochondrial , as well as nuclear , dna mutations have been implicated with epileptic phenotypes . changes in electron transport chain activity increase metabolic demand in neurons and lead to distortions in ca homeostasis increasing its dendritic level . the number and distribution of mitochondria varies from one tissue to another , depending from the tissue 's dependence on oxidative phosphorylation for energy provision . for this reason , mitochondrial diseases ( mids ) predominantly manifest themselves in tissue / organs with high - energy requirements , and are aggravated by fever , infection , stress , toxic agents , or certain drugs ( finsterer , 2006 ) . mids are mainly due to mutations in mitochondrial or nuclear dna ( mtdna and ndna , respectively ) , resulting in the altered function of the respiratory chain or oxidative phosphorylation ( oxphos ) ( reinecke et al . , 2009 ) . the respiratory chain machinery consists of approximately 80 proteins , of which 13 are encoded by mtdna and all others are encoded by ndna ( dimauro , 2004 ; finsterer , 2010 ) . interplay between the mitochondrial and nuclear genomes is evident in disorders of nuclear- and mtdna - encoded subunits of the oxphos complexes ( smeitink et al . , deficiencies in oxphos result in immediate and downstream metabolic , structural , and functional effects . atp production , and consequently atp / adp homeostasis , is disturbed in oxphos deficiencies ( smeitink et al . , 2006 ) . the mitochondrial genome possesses a very high mutation rate , 10- to 17-fold higher than that observed in ndna . protective histones are also lacking , and although mtdna repair systems do exist ( de souza - pinto et al . , 2009 ) , they are not sufficient to counteract the oxidative damage sustained by the mitochondrial genome ( tuppen et al . , 2010 ) . at the same time , pathogenic ndna mutations are likely to be more numerous than pathogenic mtdna mutations ( finsterer , 2010 ) . point mutations have been correlated with numerous diseases , such as leigh syndrome , barth syndrome , neuropathies , cardiomyopathies , hepatopathy , nephropathy , multisystemic disease , and neurodegenerative diseases , including pd , ad and amyotrophic lateral sclerosis . ( dimauro , 2004 ; finsterer , 2008 ; guy et al . , 2002 ) . given the multi - systemic feature of mitochondrial cytopathies , different pharmacological approaches are required , but most of the proposed therapies are usually of scarce effectiveness ( zaffanello and zamboni , 2005 ) . non - specific drug therapy consists of different pharmacological agents , divided according to the type of action into antioxidants and lactate lowering agents ( which remove noxious metabolites ) , electron transfer mediators ( which bypass the defective site ) , alternative energy providers , cofactors , and other agents ( finsterer , 2010 ) , such as coenzyme q10 , ascorbic acid , vitamin e , lipoic acid , riboflavin , thiamin , niacin , and vitamin k ( phylloquinone and menadione ) . the general objective is to increase mitochondrial atp production and to slow ( or arrest ) the progression of the clinical symptoms ( marriage et al . , 2003 ) . in order to correct the energy imbalance in mids , it would be interesting to introduce drugs able to act on intracellular ca homeostasis since a strong relationship exists between ca and mitochondrial atp synthesis ( see fig . 2 ) . this ca content is actively maintained by the sarcoplasmic / endoplasmic ca atpase ( serca ) located on membranes of the er that constantly pump cytosolic ca into the lumen , at the expense of atp . this atp is mostly provided by mitochondria through the close contact sites between er and mitochondria ( giorgi et al . , 2009 ) . it has been reported , especially for defects on respiratory complex i , that cells from patients with mitochondrial diseases show reduced mitochondrial membrane potential together with reduced atp synthesis . this causes a reduced mitochondrial ca uptake under physiological stimulations ( visch et al . , 2006 ) . since ca uptake by mitochondria promotes krebs cycle activity and atp synthesis ( jouaville et al . , 1999 ) , it could be hypothesized that a negative feedback is created that maintains low intracellular ca levels and atp contents in cells derived from mids patients . in this scenario , it would be interesting to study the molecules capable of increasing er or mitochondrial ca levels . it has already been proposed that the pharmacological inhibition of the mitochondrial sodium ca exchanger ( that extrudes ca from mitochondria after physiological stimulation , recovering the basal [ ca ] ) is able to restore mitochondrial atp synthesis in cells harboring mitochondrial dna mutations ( brini et al . mitochondrial ca uptake is also a signaling event in the apoptotic cascade , especially in oxidative stress conditions . indeed , sustained mitochondrial ca elevations are able to induce the opening of the ptp , almost without stimulating the krebs cycle . since oxidative stress appears to be a common feature in many mids , therapeutical approaches based on ca handling should be considered together with antioxidant drugs or blockers of the ptp . the latter , especially cyclosporin a ( csa ) , are able to inhibit cytochrome c release induced by a number of apoptotic stimuli ( ow et al . , 2008 ; walter et al . , 1998 ) and cyclosporins are used as immunosuppressants ( probably independently from their mitochondrial activity ) and diverse studies support a protective role for csa in diseases with associated mitochondrial dysfunctions . myoblasts from patients affected by ullrich congenital muscular dystrophy display several mitochondrial alterations and a higher susceptibility to apoptosis due to the inappropriate opening of the ptp . in these conditions , it has been observed that csa inhibits apoptosis engagement ( merlini et al . , 2008 ) . a multitude of recent studies suggest that mitochondria play critical roles in both the life and death of terminally differentiated cardiac myocytes . mitochondria malfunction has been implicated in various cardiac pathologies , including ischemia reperfusion ( i / r ) injury , cardiomyopathy , and congestive heart failure ( gustafsson and gottlieb , 2008 ) . in healthy myocytes , mitochondria occupy a large portion of the cell and are located between the myofibrils and just below the sarcolemma ( andrienko et al . , 2003 ) . pathophysiological conditions that can open the ptp are characteristically present during myocardial i / r ( di lisa and bernardi , 2006 ) ( see fig . 2 ) . as such , an increasing number of pharmacological approaches are currently being developed to try to interfere with these parameters . cardioprotective effects of strategies that limit mitochondrial ca accumulation , have been demonstrated ( miyamae et al . , 1996 ) . for instance , treatment of hearts with ruthenium red ( rr ) or ru360 , inhibitors of the ca uniporter , reduced infarct size in hearts subjected to i / r ( garcia - rivas gde et al . , 2006 ; park et al . , 1990 ) . in addition , ru360 treatment dramatically decreased [ ca]m and inhibited ptp opening , which was associated with improved recovery of heart performance ( goldberg and bursey , 1992 ) . mitochondrial membrane potential is another critical regulator of ca accumulation : depolarization reduces the driving force for ca uptake by mitochondria and thereby prevents ca overload ; this prompted researchers to consider the decrease of the potential as a possible mechanism of cellular protection . this objective is attained with uncoupler agents such as 2 - 4-dinitrophenol ( dnp ) and carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone ( fccp ) ( brennan et al . , 2006 ) , and mitochondrial atp - sensitive k ( mitokatp ) channel openers , such as diazoxide ( garlid et al . , 1997 ; iwai et al . , 2000 ) or nicorandil , ( carreira et al . , 2008 ; iwai et al . , 2002 ) , which showed a cardioprotective effect in different models of i / r ( ishii et al . , 2005 ; kitakaze et al . pharmacological openers of the mitokatp channel have been developed for the treatment of angina pectoris ( nicorandil ) and hypertension ( cromakalim and pinacidil ) ( grover et al . cardiac mitokatp channels were also identified as important mediators of protection during ischemic preconditioning ( ipc ) , the most potent method for reducing reperfusion injury ( fryer et al . , 2000 ; murry et al . , 1986 ) , and thus represent a promising drug target . oxidative stress has also been associated with loss of cells in heart failure , i / r injury , and doxorubicin - induced cardiomyopathy , whereas reducing ros production is cardioprotective ( gustafsson and gottlieb , 2008 ) . moreover , there is an increased production of ros in failing hearts after myocardial infarction and consequently lipid peroxidation , decrease of mtdna copy number , and a reduced oxidative capacity due to lower activity of electron transfer enzymes ( ide et al . , 2001 ) . antioxidants , such as vitamin e ( sethi et al . , 2000 ) , ginkgo biloba extract ( seif - el - nasr and el - fattah , 1995 ) , trans - resveratrol ( goh et al . , 2007 ; ray et al . , 1999 ) , that remove radicals from cells , protect against i / r injury ( reeve et al . , 2005 ) . the use of novel mitochondria - targeted antioxidants has gained much interest ( victor and rocha , 2007 ) . mitoq , based on the endogenous mitochondrial ubiquinone coenzyme q , is such a compound ; a recent study demonstrated that feeding rats with mitoq significantly limited cardiac reperfusion injury ( adlam et al . , 2005 ) . moreover , inhibition of the ptp through direct targeting of its putative components ( i.e. , the adenine nucleotide translocator ( ant ) and cyclophilin d ( cyp - d ) ) may represent an effective therapeutic approach in protecting the heart against various cardiac pathologies ( reviewed in ( bernardi and forte , 2007 ; halestrap and pasdois , 2009 ) ) . csa has been demonstrated to be cardioprotective in isolated cardiomyocytes subjected to anoxia and reoxygenation ( griffiths et al . , 2000 ; nazareth et al . , 1991 ) , in langendorff - perfused hearts subjected to global i / r ( griffiths and halestrap , 1993 ) , as well as in animals subjected to coronary artery ligation and reperfusion ( hausenloy et al . , 2003 ) . one problem with the use of csa is that it can induce additional effects through inhibition of the ca - regulated protein phosphatase calcineurin , that has direct effects on heart function ( periasamy , 2002 ) and also undesirable immunosuppressive activity ( schreiber and crabtree , 1992 ) . to overcome this , other cyclosporine derivatives , such as the immunosuppressant sanglifehrin a ( sfa ) , as well as non - immunosuppressants [ n - methyl - ala]csa , [ n - methyl - val]csa , debio 025 , nim 811 and unil025 , were found to exhibit similar properties in terms of blocking pore opening , while being devoid of calcineurin inhibitory activity ( clarke et al . , 2002 ; gomez et al . in addition , studies on isolated mitochondria and cultured cardiomyocytes have confirmed a protective role of the ant inhibitor bongkrekic acid , via inhibition of pore opening ( akao et al . , recently , schaller et al . , ( 2010 ) have discovered a new mitochondrial - targeted cardioprotective drug , tro40303 ( 3,5-seco-4-nor - cholestan-5-one oxime-3-ol ) , that binds specifically to the mitochondrial translocator protein ( tspo , formerly known as peripheral benzodiazepine receptor ) and inhibits ptp opening triggered by oxidative stress . another interesting result was obtained with the free radical scavenger edavarone , which was shown to inhibit ptp opening and to prevent cardiac reperfusion injury ( tsujita et al . , 2006 ) . finally , the bcl-2 family proteins , which control mitochondrial membrane permeabilization and ca homeostasis , also play a central role in regulating apoptosis in the cardiovascular system ( gustafsson and gottlieb , 2007 ) . many of the pro - apoptotic bcl-2 proteins have been implicated in the pathogenesis of various cardiac diseases , including myocardial hypertrophy , infarction , and heart failure ( condorelli et al . , 1999 ; jung et al . , , anti - apoptotic interventions targeting the bcl-2 protein family provide opportunities for possible anti - ischemic therapies ( reed , 2001 ) , either through a gain of anti - apoptotic function or loss of pro - apoptotic function . the pharmacological strategy inhibiting mitochondrial outer membrane permeability , and thus apoptosis , by manipulation of the bcl-2 family to protect the myocardium against i / r injury is very recent but has already provided interesting results ( hetz et al . , 2005 ) , supporting the idea that clinical benefits may be obtained in the near future . from the above - described evidence , therapeutic interventions designed to prevent mitochondrial damage hold major promise as a novel strategy for reducing cardiac injury , the leading cause of death in western societies . effective therapies developed along these lines will thus represent a major advance in health care . diabetes mellitus is a disorder resulting from defects of both insulin secretion and action . to date , the cellular pathophysiology of diabetes - induced impairments , remain controversial . conceptually , the leading mechanism driving cellular pathology is believed to be associated with the accumulation of extracellular glucose , and generation of ros , thereby triggering pathological outcomes . there is abundant evidence suggesting a central role of mitochondria in diabetes , insulin resistance , insulin secretion and in complications derived from diabetes ( patti and corvera , 2010 ) . however , it is still difficult ( and controversial ) to establish whether the alterations in mitochondrial function are the cause or the effect of the disease . typically , the number and size of mitochondria is reduced in the muscle of diabetics , there is reduced mitochondrial biogenesis and switching of metabolic substrate use that is linked directly to mitochondrial enzymes ( chabi et al . , 2005 ) . mitochondria are an integral part of the insulin system found in the islet cells of the pancreas . ultrastructural examination of -cells has suggested that mitochondria are often in close proximity to the secretory insulin granules . this may facilitate metabolism secretion coupling , as atp is a major permissive factor for movement of insulin granules and for priming of exocytosis ( maechler and wollheim , 2001 ) . when blood glucose is high , the rate of glycolysis in -cells will increase and , as a consequence , the atp production is accelerated to provide the energy for insulin exocytosis . glucose - stimulated insulin release from -cells involves a complex series of signaling pathways . as in other exocytotic fusion events , ca is an essential trigger of insulin granule exocytosis ( rorsman and renstrom , 2003 ) . the ca signal resulting from mitochondrial activation completes the chain of events between glucose uptake into -cells and insulin exocytosis . a common motif in these cascades is the elevation of intracellular ca both in the cytosol and within the mitochondria . mitochondria take up ca through the mitochondrial ca uniporter in response to the glucose - induced rise in cytoplasmic ca , and release ca through the mncx ( bernardi , 1999a ) . [ ca]c rises may originate from ca influx at the plasma membrane or ca mobilization from the er . local ca influx or mobilization creates microdomains , local increases of ca exceeding the bulk [ ca ] in the cytosol ( giorgi et al . , 2009 ) . for both pathways , the amplitude and the duration of the [ ca]c rise may determine to what extent -cell mitochondria are activated . depending on the combination of secretagogues , the number and localization of such ca domains most certainly vary ( rizzuto and pozzan , 2006 ; rutter et al . , 2006 ) . the diabetic state is related to mitochondrial dysfunction and generally characterized by insufficient supply of energy or defects in the insulin signaling pathway . it has been shown that a reduction in mitochondrial ca accumulation is responsible for a reduction in insulin secretion ( wiederkehr and wollheim , 2008 ) , suggesting that modulation of mitochondrial ca may be a therapeutic target . restoration of the mitochondrial ca signal using an inhibitor of the mnce was shown to improve atp generation and insulin secretion ( lee et al . , 2003 ; visch et al . , 2004 ) , highlighting the exchanger as a potential new target for diabetes drug discovery . among the most convincing links between mitochondrial dysfunction and diabetes indeed , diabetes mellitus is very common in patients affected by mitochondrial diseases ( also referred to as mitochondrial encephalomyopathies , characterized by genetic disorders of mtdna , such as merrf and melas ) , while and a maternally - inherited form of diabetes is associated to mtdna mutations ( ballinger et al . , 1992 ) . accordingly , with the above suggested pivotal role of ca homeostasis in the cellular pathogenesis of diabetes , agonist - induced ca rises and atp generation were blunted and could be restored by applying an inhibitor of the mitochondrial ca efflux ( brini et al . , 1999 ) . finally , but not less important , an abnormal ca homeostasis and impaired mitochondrial function diabetic conditions affect er ca homeostasis in sensory neurons by lowering the er ca content due to reduced serca pump activity , inducing a mild condition of er stress that results in the decrease of nerve conductance velocity ( verkhratsky and fernyhough , 2008 ) . in conclusion , the signaling mechanisms that decode changes in nutrient supply are complex and not completely understood , but a control in intracellular [ ca ] , in particular at the mitochondrial level , appears to be of great significance for further research . mitochondria are the powerhouse of the cell being the place where oxidative metabolism takes place , rendering mitochondria crucial both in health and disease . many ( if not all ) of the mitochondrial activities are driven in a ca - dependent manner . the biochemical properties of the proteins involved in mitochondrial ca homeostasis have been known for over four decades , but only recently have we begun to unravel the dynamics of this cation at the molecular and mechanistic levels ( hajnoczky and csordas , 2010 ) . currently , specific drugs that act on mitochondrial ca homeostasis are being developed and these may open the way to new biochemically designed therapeutic approaches in the treatment of several disorders .

mitochondria are crucial in different intracellular pathways of signal transduction . mitochondria are capable of decoding a variety of extracellular stimuli into markedly different intracellular actions , ranging from energy production to cell death . the fine modulation of mitochondrial calcium ( ca2 + ) homeostasis plays a fundamental role in many of the processes involving this organelle . when mitochondrial ca2 + homeostasis is compromised , different pathological conditions can occur , depending on the cell type involved . recent data have shed light on the molecular identity of the main proteins involved in the handling of mitochondrial ca2 + traffic , opening fascinating and ambitious new avenues for mitochondria - based pharmacological strategies .

Introduction Mitochondria and aging Mitochondria and neurodegenerative diseases Mitochondrial diseases Mitochondria as drug targets in the treatment of cardiovascular diseases Mitochondria, Ca Conclusions

malaria is caused by a protozoa called plasmodium and spreads among humans by bites from anopheline mosquitoes . one species that has been identified as a malarial vector on lombok is anopheles aconitus . the species lives at locations ranging from sea level to 600800 m above sea level . aconitus can be found on rice fields ( planted and unplanted ) , various shallow pools ( rock , stream , and flood ) , and slow moving streams with grassy margins . this is commonly done in 3 ways : mosquito larvae control ( using larvicide ) , adult mosquito control ( using adulticide ) , and breeding habitat modification . the most effective approach is mosquito larvae control and this can be accomplished in several ways . the use of indigenous b. sphaericus is highly desirable as it would build a local capability to produce a biopesticide in developing countries . however , there is no information on prevalence of environmentally relevant b. sphaericus on lombok nor its potential to be used as a biopesticide . therefore , studies to reveal indigenous isolate of b. sphaericus are important to the island from both a public health and economic perspectives . in this study , isolates of b. sphaericus were taken from some areas close to beach area and villages known to be endemic to malaria . collection was done at 20 different locations close to beach area surrounding lombok island , west nusa tenggara , indonesia . five hundred grams of soil was collected from each chosen point compositely and stored in sterile screw - capped container . the chosen areas were close to village and/or river opening / estuaries presumably an appropriate location for anopheline breeding habitat . soil samples were homogenized with sterile physiological salt solution forming 10% w / v suspension . the suspension was heated to 80c for 30 minutes and then serially diluted with sterile physiological saline solution ( in 10 to 10 dilutions ) . diluted suspension was spread on nysm ( nutrient agar enriched with 0.5 g / l yeast extract , 0.2 g / l mgcl2 , 0.01 g / l mncl2 , and 0.1 g / l cacl2 ) plating medium supplemented with 100 g / ml streptomycin to avoid unwanted bacteria growth . colonies that showed gram positive rod with bulging endospore on the terminal end were purified for detailed characterization and toxicity testing . putative b. sphaericus isolates were further characterized using key biochemical / physiological tests such as catalase , oxidase , nitrate reduction , urease , sugar utilization , starch hydrolysis , and antibiotic sensitivity test . anopheline eggs came from mosquitoes reared intensively in rearing facility at ivrcrd ( institute for vector and reservoir control research and development ) , salatiga , central java , indonesia . larvae resulting from hatched eggs were reared for 6 days to reach 3rd instar stadium ( 3 - 4 mm in length ) . the b. sphaericus isolates were grown in nysm liquid medium at 30c for 72 hours with 170 rpm shaking . sixty anopheline larvae ( 60 larvae in 3 containers ) were put into 10% v / v isolated b. sphaericus grown on the nysm liquid medium . larva death on each test replication was observed and mean value of larva death was calculated . the test was also done with b. sphaericus 2362 for comparison . in order to obtain lc ( lethal concentration ) value , bioassay was done on isolates that showed more than 50% toxicity on initial toxicity testing . as mention by dulmage et al . , seven concentrations ( in 10-fold concentration differences with 3 replications ) of chosen isolated b. sphaericus grown in 3 24-hour nysm liquid medium were prepared ( totally there were 21 testing containers ) . aconitus larvae were distributed evenly in the container ( 20 larvae for each testing container ) . aconitus larvae in 3 containers ( 20 larvae per testing container ) were mixed with 10% v / v culture medium ( without bacteria ) as negative control and other 60 an . mortality rate of anopheline larvae was calculated using this formula:(1)mortality rate = number of dead larvaenumber of total larvae100%.if in the negative control group 520% dead larva are found , abbott 's correction formula is used to obtain corrected mortality rate:(2)corrected mortality rate = mortality rate of test groupmortality rate of control group100%mortality rate of control group100%.lethal concentration ( lc ) values in 24 and 48 hours were calculated using probit analysis applying software minitab v16 for windows . the use of b. sphaericus as a biocontrol agent to suppress anopheline larvae has been done intensively in some countries , such as united states , some european countries , and china . it is primarily used at standing waters , swamps / marshland , paddy fields , and lake shores that are known as primary mosquito breeding habitats [ 1113 ] . in this study 20 isolates of b. sphaericus ( and their toxicity attributes ) were found at 20 locations close to beach area around lombok island indonesia as presented in figure 1 . morphology characteristics of b. sphaericus isolated from lombok island are presented in figure 2 and their characteristics are presented on table 1 . the characteristics of putative b. sphaericus isolates were in agreement with standard characteristics mentioned in bergey 's manual of determinative bacteriology . we have found that all these isolates could be not collected from area exposed to sea water directly ( in form of rip - tide or salt dam / pool ) . b. sphaericus isolates were isolated from sand / soil covered / shaded with leaves and rich of organic matter ( grass , fallen leaves , branches , etc . ) . some locations formed small puddles , while other locations were dry and/or moist soil . from 20 locations explored , medium and highly toxic b. sphaericus was obtained only from 3 locations . compared to other locations , these 3 locations were areas that formed small puddles that were rich in organic matter , shaded from the sun , undisturbed by human activity , and inhabited by mosquito larvae , whereas other locations were dry or moist areas ( and no mosquito larvae was found ) . however , the latter were also rich in organic matter , shaded , and untouched . these locations were in accordance with the first discovery of b. thuringiensis in israel nagev dessert and discovery of b. sphaericus in the united states . those reports had similarities that those entomopathogenic bacteria were isolated from small puddle inhabited by mosquito larvae . soil is potential habitat for bacillus , as soil provides nutrients and growing factors for the bacteria . however , in this study richness in organic matter did not make a given area a suitable habitat for toxic b. sphaericus . it was seen that , from 20 locations explored , there were 10 locations that gave nontoxic b. sphaericus isolates , 7 locations that gave lowly toxic b. sphaericus isolates , and only 3 locations that gave very toxic b. sphaericus isolates . we suggest that richness in organic matter is not main factor for obtaining such toxic b. sphaericus isolates . contact with mosquito larvae should be taken into consideration and it was shown in some report in early discovery of entomopathogenic bacteria [ 14 , 15 ] . lc50 and lc90 values in 24 and 48 hours of three b. sphaericus isolates based on cell concentration ( cell / ml ) are shown in table 2 . isolates slg and tjl2 showed lc values that were close to those of b. sphaericus 2362 . lc ( lethal concentration ) value informs us how low the concentration or the dilution of certain microbe or ingredient able to kill targeted organism is . from these values it can be concluded that b. sphaericus isolate mnt was of lower toxicity than b. sphaericus 2362 , whereas b. sphaericus isolates slg and tjl2 had lc values that were almost similar to toxicity of b. sphaericus 2362 . these b. sphaericus isolates were the first toxic b. sphaericus isolated from lombok island , indonesia . other entomopathogenic bacterium that was isolated and tested was b. thuringiensis that came from some areas in indonesia . in contrast , b. sphaericus ' susceptible targets are culex and anopheles , whereas aedes is the least susceptible . the toxicity and lc value of this new isolated b. sphaericus suggest that it would be good candidate for local biocontrol agent on lombok island . there are 2 kinds of toxins : binary toxins / bin ( 51 and 42 kda ) are produced on sporulating stage and mosquitocidal toxins / btx ( 100 , 32 and 36 kda ) are produced on vegetative stage [ 16 , 17 ] . the binary toxins which are the most potent toxins can interact with receptor along larvae midgut specifically , whereas the mosquitocidal toxins are weaker toxins that will kill the larvae in longer period or will not kill at all ( just weaken the larvae ) . some strains may have both toxins ; others may have only one or none . that explains varied killing capability among strains of b. sphaericus worldwide . from its low toxicity and higher lc values ( compared to b. sphaericus 2362 ) , we predict that isolate mnt may only have binary toxins , while other 2 isolates may have binary toxin and mosquitocidal toxin altogether . compared to other biocontrol bacteria such as b. thuringiensis , b. sphaericus will last longer in environment ( some study reported 2030 days after application ) . even though many b. sphaericus strains from any places in the world have been collected , the existence of indigenous isolates is still important to study , as it will open opportunity to develop local - strain - based biopesticide production in developing countries such as indonesia . this capability will suppress cost used for importing commercial biopesticide from other countries and also promote local biopesticide industry as well . twenty local isolates of b. sphaericus were found from 20 locations close to beach area on lombok island with varied toxicity against anopheline larvae .

malaria is endemic to lombok island , indonesia . one approach to suppress malaria spread is to eliminate anopheline larvae in their habitat and the environmentally safe agent is bacteria , that is , bacillus sphaericus . however , there is no information regarding local isolate of b. sphaericus that is toxic to mosquito larvae from lombok . the aims of the study were to isolate b. sphaericus from soil in areas close to beach surrounding lombok island and to test their toxicity against 3rd instar anopheles aconitus larvae . soil samples were collected from 20 different sampling locations from lombok island and homogenized with sterile physiological salt solution . suspension was heat - shocked at 80c for 30 minutes and then spread onto antibiotic - supplemented nysm solid medium . colonies grown were characterized and subjected to initial toxicity test against anopheline larvae . isolates with more than 50% killing percentage were subjected to bioassay testing against anopheline larvae . from 20 locations , 1 isolate showed mild toxicity ( namely , isolate mnt ) and 2 isolates showed high toxicity ( namely , isolates slg and tjl2 ) against an . aconitus . those 3 isolates were potentially useful isolates , as they killed almost all larvae in 24 hours . the discovery of toxic indigenous isolates of b. sphaericus from lombok island opens opportunity to develop a biopesticide from local resources .

1. Introduction 2. Material and Methods 3. Results and Discussion 4. Conclusion

it is also a fundamental culprit in the pathogenesis of atherosclerosis , a chronic vascular disease that underlies the development of circulatory problems including coronary artery disease and cerebrovascular disease . in these scenarios , atherosclerosis runs a silent asymptomatic course , until stenosis or plaque rupture with superimposed thrombosis manifests clinically as stroke , myocardial infarction ( mi ) , or death . atherosclerosis involves a chronic and subclinical inflammatory process , initiated by endothelial damage and activation . subsequently , an elaborate cascade of steps involving various signaling molecules and leukocyte activation and migration follows , leading to the formation of atherosclerotic plaques . this complex process involves extensive interplay between various elements of the innate and adaptive immune systems . the innate immune system constitutes the first line of defense against pathogens and is highly conserved and universal from an evolutionary perspective . central to the function of innate immunity is the recognition of pathogen - associated molecular patterns ( pamps ) or disease - associated molecular patterns ( damps ) by immune - competent cells , such as tissue macrophages and endothelial cells . pamps are molecules of microbial origin , and damps are endogenous molecules that normally are sequestered and become released in response to cell injury or endogenous stress signals , such as heat shock proteins ( hsps ) . pamps and damps are recognized by a number of pattern recognition receptors ( prrs ) expressed on sentinel immune cells , which can trigger inflammatory and adaptive immune reactions . toll - like receptors ( tlrs ) constitute a major subgroup of pattern recognition receptors . subsequently , the human and murine homologs of toll were identified , and studies showed their essential role in mediating the immune response against bacterial lipopolysaccharide ( lps ) [ 6 , 7 ] . toll - like receptors are part of interleukin-1 receptor / toll - like receptor superfamily . they possess an extracellular n - terminal ligand - recognition domain with leucine - rich repeats and a cytoplasmic carboxyl terminal tail with toll / interleukin-1 receptor ( tir ) signaling domains . the extracellular leucine - rich domain binds to pamps and forms homo- or heterodimers with other tlrs . vertebrate tlrs are grouped into six subfamilies based on sequence homology , and their structure has been extensively described elsewhere [ 9 , 10 ] . at least thirteen mammalian tlrs are known , and these are expressed either on cell surfaces , where they bind microbial membrane components ( tlr1 , tlr2 , tlr4 , tlr5 , and tlr6 ) or endosomally ( tlr11 , tlr12 , and tlr13 ) where they recognize microbial nucleic acids ( tlr3 , tlr7 , tlr8 , and tlr9 ) . tlr4 is expressed in human coronary plaques , and that baseline tlr4 macrophage expression is upregulated by oxidized ldl ( oxldl ) . tlr2 and tlr4 are expressed by macrophages , neutrophils , and dendritic cells and have been implicated in the development of coronary artery disease ( cad ) through activation of nf-b pathways . furthermore , investigators have suggested that the phenomenon of molecular mimicry may be at play at activating innate immune pathways leading to atherosclerosis . tlr4 recognizes chlamydial antigens , and some studies have demonstrated a link between chlamydia pneumoniae infection and atherothrombosis [ 1315 ] . although other investigators have failed to replicate this association [ 16 , 17 ] , it nevertheless provides an intriguing insight into the mechanistic link between tlr pathways and atheroma formation . this review article aims to describe the immunological role of tlrs in promoting the development of atherosclerosis , with a primary focus on the function of tlr2 , tlr4 , and tlr9 . central to the development and progression of atheromas is the interaction between activated monocytes and oxidized ldl ( oxldl ) leading to formation of foam cells . the american college of cardiology / american heart association define hyperlipidemia associated with increased cardiovascular disease risk as ldl plasma levels above 70189 mg / dl ( 1.814.89 mmol / l ) . plasma ldl is transported by apolipoprotein - b lipoprotein complexes in the circulation ( apob - lps ) such as apob-100 . in vivo , ldl - c undergoes oxidative modification resulting in the formation of oxldl , which has strong proinflammatory and immunogenic properties . endothelial activation ensues following the lodging of oxldl in the subendothelial matrix of the tunica intima . here , oxldl triggers a response akin to that observed during chronic tissue injury , in that mononuclear leukocytes , primarily monocytes and lymphocytes , are recruited to the site of injury where oxldl lodges [ 23 , 24 ] . furthermore , as the plaque evolves , intermediate lesions are formed , characterised by smooth muscle cell ( smc ) proliferation and migration from the tunica media to the tunica intima . smcs also become lipid - laden . some authors postulate that angiogenesis is triggered by cytokines secreted from activated macrophages to provide monocytes that can differentiate into tissue macrophages , to phagocytose the apoptotic foam cells . other authors propose that angiogenesis in these lesions serves to perfuse the hypoxic environment created by the cellular debris . the consensus is that angiogenesis may set up a vicious cycle whereby there is a continual supply of monocytes which differentiate into tissue macrophages and become transformed into foam cells as a result of oxldl uptake . on the other hand , fibroatheromas consist of extracellular matrix secreted by smcs , and there is also evidence of calcified deposits . , fibroatheromas may further evolve into complicated unstable lesions with surface defects and multiple necrotic cores . complications from such lesions include erosion , rupture , fissure , and ulceration , resulting in various clinical manifestations , such as acute coronary syndromes [ 26 , 30 ] . cad remains a prevalent cause of global mortality , accounting for an approximate 1.8 million annual deaths in europe alone , which translates to 20% of total european mortality . low- and middle - income countries in eastern europe and central asia are now the most heavily affected by cad . this is primarily due to the fact that these regions constitute the majority of the world 's population . contributing to the shift in cad epidemiology is the increasing affluence in developing regions , including latin america , sub - saharan africa , middle east , and south east asia . the increasing urbanization , industrialization , and adoption of western - style lifestyles that follows from economic growth impact negatively on cardiovascular mortality and have extensive socioeconomic implications . furthermore , the shift in population demographics resulting in increased life expectancy further compounds the clinical and public health burden exerted by cad , particularly in western countries . tlrs are type 1 transmembrane glycoproteins comprised of extracellular , transmembrane , and intracellular signaling domains that are expressed either on the plasma membrane or on intracellular endolysosomal compartments . the cytoplasmic signaling domain is shared with that of the interleukin-1 ( il-1 ) receptor ( the toll / il-1r domain , tir ) , and as a consequence of this homology tlrs activate pathways shared with il-1r . upon ligand binding , the tlr signaling cascade is initiated by the tir domain via a number of cytoplasmic adapter molecules . these include myeloid differentiation primary response protein ( myd88 ) , tir - domain - containing adaptor protein ( tirap ) , and trif - related adaptor molecule ( tram ) . myd88 activates il-1r associated kinases ( iraks ) , irak-1 and irak-4 , and tnf - receptor associated factor-6 ( traf-6 ) [ 35 , 36 ] . consequently , recruitment of a number of proteins activates a complex containing tgf--activated kinase 1 ( tak1 ) , tak1-binding protein-1 ( tab1 ) , tab2 , and tab3 . the tak1/tab complex in turn leads to activation of both the mapk and nf-b signaling pathways . these steps result in activation of a number of genes coding for proinflammatory cytokines and chemokines , including tnf- , il-1 , and il-6 . this requires trif and tram adapter proteins that lead to phosphorylation of interferon regulatory factor 3 ( irf3 ) and nf-b transcription factors . the major outcome of trif - dependent tlr4 signaling is the production of type i interferons that have antiviral and antiproliferative activity [ 42 , 43 ] . the balance between myd88-dependent and trif - dependent tlr signaling is essential for proper immune function . wang et al . demonstrated that nrdp1 ( e3 ubiquitin ligase ) inhibits the production of proinflammatory cytokines but increases ifn- production in tlr - activated macrophages . conversely , liu et al . showed that intracellular mhc class ii molecules in antigen presenting cells can activate both myd88 and trif cascades , leading to production of proinflammatory cytokines and interferons . in addition to the activation of nf-b transcription factor in myd88-dependent and myd88-independent fashion , tlr2 has been implicated in the stimulation of proapoptotic pathways . subsequently they demonstrated that myd88 is the common mediator of tlr2-induced apoptosis and nf-b activation and that tlr2 induces apoptosis through the fadd - caspase pathway in a manner analogous to members of the tnfr family [ 47 , 48 ] . the in vivo relevance of tlr2 proapoptotic activity in either reinforcing or terminating the inflammatory response remains elusive , and its specific role in chronic inflammatory processes is poorly understood . primarily , they function as receptors for molecular domains borne on bacterial or viral pathogens . tlrs expressed on the plasma membrane recognize cell - wall components of bacteria and fungi , while those expressed on internal endolysosomal compartments bind viral pamps . specifically , tlr2 binds bacterial lipoproteins , and tlr4 is primarily activated by bacterial lipopolysaccharide and tlr9 by unmethylated cpg nucleotide sequences . in addition to exogenous ligands of microbial origin , tlrs are able to bind a wide range of endogenous ligands . these endogenous ligands are host - derived molecules that stimulate tlr signaling in the absence of infection , and they have been extensively reviewed elsewhere [ 49 , 50 ] . endogenous ligands include various extracellular matrix components , including fibronectin , fibrinogen , hyaluronic acid derivatives , heat shock proteins , and minimally oxidized low density lipoprotein . these ligands are either actively released by cells at sites of injuy or passively released by cells damaged from inflamed tissues . the evolving atherosclerotic plaque is a site of matrix turnover , tissue remodeling , and cell necrosis and hence contains a number of endogenous tlr ligands . of particular interest are heat shock proteins ( hsps ) , which have been identified as powerful activators of innate immune function . various investigators report that hsp60 induces a proinflammatory response in a tlr2 and tlr4 dependent fashion [ 5658 ] . investigations showed that contamination by lps in the hsp preparations is responsible for the observed tlr4 activation [ 59 , 60 ] . lps is a powerful inducer of tlr activation even in minute quantities , and lps contamination results from the production of recombinant hsp in escherichia coli . tlr signaling cascades can be activated by a broad range of host - derived molecules in the absence of exogenous infection , and this plays a central role in the development and progression of atherosclerosis . the subsequent sections of this review focus on the roles of tlr2 , tlr4 , and tlr9 in the pathogenesis of atherosclerosis . figure 1 outlines the key mechanisms from functional and animal studies implicating tlr2 and tlr4 in atherosclerosis . tlr2 is a cell surface receptor that binds a wide range of microbial components , such as gram - positive - derived lipoteichoic acid , bacterial lipoproteins , and zymosan . it is expressed in a number of immune cells , the endothelium , and epithelial cells . tlr2 has a unique ability to form functional heterodimers with either tlr1 or tlr6 resulting in relatively broad ligand specificity . tlr2 expression , along with that of tlr1 and tlr4 , is markedly increased in endothelial cells overlying atheromas . furthermore , endothelial tlr2 expression and activation may occur at areas of turbulent blood flow , such as the areas of lesion predilection within the aortic tree and heart . in vitro experiments using human coronary artery endothelial cells exposed to laminar blood flow showed lower levels of tlr2 expression when compared to endothelial cells exposed to static or turbulent flow . laminar flow at or above 0.5 n / m inhibits endothelial tlr2 expression via protein kinase ck2 , which phosphorylates a transcription factor known as specificity protein-1 ( sp1 ) . this prevents sp1 binding to the tlr2 promoter , thereby reducing tlr2 expression . in doing so showed that , in atherosclerosis - susceptible ldlr - deficient ( ldlr ) mice , complete deficiency of tlr2 leads to a reduction in atherosclerosis , whereas expression of tlr2 only on bone marrow derived cells has no impact on atherosclerosis . this experiment showed that tlr2 expression on non - bone marrow derived cells , such as vascular endothelium , at sites of nonlaminar flow contributes to the atherosclerosis . furthermore , the authors showed that on administration of pam3csk4 ( a synthetic tlr2/tlr1 agonist ) , atherosclerotic burden was dramatically increased in ldlr mice . the proatherogenic effect of pam3csk4 was not observed in ldlr mice with complete tlr2 deficiency or in ldlr mice with a deficiency of tlr2 only in bone marrow derived cells . this finding reinforces the role of tlr2 in promoting atherosclerosis through its action in cells of non - bone marrow origin . further investigations by mullick et al . using ldlr mice showed that aortic endothelial cell tlr2 expression was confined to areas of nonlaminar flow , specifically in the lesser curvature of the aorta , and that hyperlipidemia increases endothelial tlr2 expression . furthermore , the authors generated chimeric mice with green fluorescent protein ( gfp ) expression in bm derived cells ( bmgfp ) . bone marrow reconstitution of ldlr and ldlrtlr2 mice with bmgfp cells showed that hyperlipidemia increases lesser curvature bmgfp leukocyte accumulation , lipid accumulation , and foam cell generation , whereas hyperlipidemic double mutant bmgfpldlrtlr2 mice had reduced lesser curvature atherogenic activity . this study showed that endothelial tlr2 expression is linked to early atherosclerosis in murine models . using apoe atherosclerotic mice schoneveld et al . showed that exogenous tlr2 activation increases atherosclerotic plaque formation and plaque - media ratio . tlr2 is involved not only in the initial intimal lesion formation but also in development of occlusive disease . furthermore , tlr2 promotes vascular smooth muscle cell ( vsmc ) migration from tunica media to the intima in an il-6 dependent manner . genetic deficiency of tlr2 reduces diet - induced atherosclerosis in apoe mice , and tlr2 expression and activation regulates the inflammatory processes and ros production following vascular injury in mouse models . aside from the involvement of tlr2 in atheroma development , evidence shows that tlr2 contributes to coronary endothelial dysfunction after ischemia / reperfusion by activating neutrophils and free radical production . cd36 functions in the recognition of various endogenous ligands , including oxldl and the uptake of fatty acids . nonetheless , even though tlr2 participates in the immune response to oxldl , it is not a primary culprit in formation of foam cells unlike tlr4 and cd36 . other ligands implicated in tlr2 activation include high - mobility group box 1 protein ( hmgb1 ) . this is a nuclear transcription factor secreted by macrophages , monocytes , and dendritic cells that is expressed in atherosclerotic lesions . members of wnt family of glycoproteins such as wnt5a have been reported to be coexpressed with tlr2 and tlr4 in macrophage rich regions of more advanced staged atheromatous plaques . oxldl can induce mrna expression of wnt5a , which correlates with the severity of atherosclerotic lesions in human studies . recently , wnt5a pathways have shown playing a critical role in foam cell formation and oxldl uptake . the complex role of wnt5a pathways in atherosclerosis has been extensively reviewed by bhatt and malgor . taken together , these studies highlight the elaborate signaling pathways involving tlr2 and its important role in driving atherogenesis . tlr4 resides in the plasma membrane , where it recognizes a number of exogenous ligands and activates a series of inflammatory cascades in an nk-b - dependent fashion . several lines of clinical and experimental evidence support its role in the pathogenesis of atheromas . human studies showed that cd14 monocyte tlr4 expression is increased in unstable angina and acute mi compared to control and stable angina groups [ 82 , 83 ] . conversely , tapp et al . showed that mi is associated with increased numbers of tlr4 monocyte subsets , but not with higher tlr4 expression by individual monocytes . . showed that in murine models tlr4 is necessary for the oxldl - induced macrophage differentiation into foam cells . similarly , tlr4 is a critical mediator in oxldl - induced inflammatory cytokine expression in vascular smooth muscle cells . minimally modified ldl ( a subtype of oxldl that is essential for atherosclerosis ) induces ros production and macrophage cytoskeletal rearrangements in a tlr4 dependent and myd88-independent manner [ 55 , 88 ] . the spleen tyrosine kinase syk binds to the cytoplasmic domain of tlr4 and mediates macrophage activation , membrane ruffling , macropinocytosis , lipid accumulation , and their consequent transformation into lipid - laden foam cells [ 89 , 90 ] . further experimental evidence from loss - of - function animal models supports the role of tlr4 in atherosclerosis . atherosclerosis - prone apoe mice with deficiency of tlr4 or myd88 show attenuation in atherosclerosis development through decreased macrophage recruitment [ 91 , 92 ] . showed that tlr4 contributes to early - stage intimal foam cell accumulation at lesion - prone aortic sites in apoe tlr4 mice , with a 75% reduction in intimal lipid levels compared to apoe controls . this study also showed that tlr4 is a more powerful contributor to foam cell formation than tlr2 . demonstrated that mice lacking macrophage tlr4 expression have reduced atherosclerotic lesion size when fed low - fat diets , despite no observed difference in body composition and plasma lipids . this process compensates for loss of the vascular lumen due to plaque accumulation and involves collagen matrix degradation by matrix metalloprotease-9 ( mmp-9 ) , which is activated by tlr4 . tlr4 mice show no outward arterial remodeling in carotid artery ligation and femoral artery cutoff models . in addition to the established function of tlr4 in atheroma development , it also plays a critical role in the progression and eventual rupture of atherosclerotic plaques leading to the formation of occlusive thrombus . ishikawa et al . demonstrated an increased expression of tlr4 , but not tlr2 , in ruptured human coronary atherosclerotic plaques , with tlr4 immunostaining observed in the infiltrating macrophages . . showed that specific components of oxldl that accumulate in atheromas enhance the release of proinflammatory cytokines and upregulate mmp-9 in a tlr4/nf-b - dependent fashion . this investigation directly implicates the role of lipid derivatives as endogenous tlr4 ligands that contribute to matrix breakdown . the extracellular matrix ( ecm ) is a key player in the progression of atherosclerotic disease , and several studies have described the structural alterations that develop during atherosclerosis [ 83 , 99 ] . chronically inflamed tissues express extracellular matrix ( ecm ) proteins that regulate the migration of leukocytes and other immune cells to sites of injury . while healthy human endothelium rests on an ecm composed of collagen iv and laminin , the ecm of atherosclerotic vessels contains abundant fibronectin in both human and mouse models [ 100 , 101 ] . fibronectin occurs in two forms generated by alternative splicing , plasma fibronectin ( pfn ) and cellular fibronectin ( cfn ) . cfn is expressed in the ecm of various tissues and is a multimer that forms fibrils and contains extra domains a and b ( eda and edb ) . cfn is synthesised by vascular smooth muscle and endothelial cells . the extra domain a in fibronectin ( edafn ) deletion of the alternatively spliced eda exon reduces the number and size of atherosclerotic lesions in apoe null mice [ 103 , 104 ] . in vitro studies also suggested that the fn eda domain activates tlr4 . further investigations using apoe null mice with either constitutive expression or knockout of the fibronectin eda domain showed that edafn promotes progression of atherosclerosis through a mechanism that is partially dependent on tlr4 . showed that platelet tlr4 facilitates the prothrombotic effects of cellular edafn on platelet aggregation and arterial thrombosis . edafn mice lacking platelet tlr4 showed decreased thrombus formation and a slower thrombus growth rate compared with control mice expressing platelet tlr4 , further highlighting the proatherogenic effect of the fn - tlr4 interaction . while tlr2 and tlr4 are expressed on the cell surface , tlr9 colocalises to the endoplasmic reticulum in various cell types , including b - cells , macrophages , dendritic cells , and plasma cells . in the er , tlr9 is able to detect and bind unmethylated oligodeoxynucleotide cpg motifs in microbial dna sequences and trigger inflammatory responses . apart from its role in the recognition of bacterial dna , tlr9 has been closely linked with the development of atherosclerotic lesions , since it is activated by cpg motifs in nucleic acids that are released during vascular necrosis . activation of tlr9 stimulates the transformation of murine macrophages into foam cells in an nf-b- and irf7-dependent manner . this process is inhibited by the activation of liver x - receptors , which are transcriptional regulators of lipid and carbohydrate metabolism . in addition , activation of tlr9 via cpg - containing nucleotide sequences in plasmacytoid dendritic cells stimulates interferon- ( inf- ) secretion and increases the cytotoxic activity of cd4 t - cells towards vascular smooth muscle cells . however , some investigators have suggested that tlr9 is protective against atherosclerosis . in vitro activation of tlr9 stimulates interleukin-10 ( il-10 ) production , which in turn inhibits the expression of inf- secreted by plasma dendritic cells and inhibits cd4 cd25 t - cell proliferation [ 112 , 113 ] . loss - of - function animal models have been used to further elucidate the role of tlr9 in atheroma . used a double knockout mouse model lacking both tlr9 and apoe to compare aortic sinus atherosclerotic lesion development . the investigators showed a 33% increase in lipid deposition and atherosclerotic plaque size in apoe / tlr9 mice compared to apoe mice . furthermore , there was significant accumulation of macrophages , dendritic cells , and inf- in vasculature of apoe / tlr9 mice compared to control animals . loss of tlr9 function thus exacerbates atherosclerosis in apoe null mice exposed to a high fat diet . pharmacologic inactivation of tlr9 pathways in animal models results in a reduction in atherosclerotic lesion generation in apoe mice and reduced instability of vulnerable plaques . . showed that inactivation of tlr9 using immunoregulatory oligodeoxynucleotides such as irs869 reduces plaque burden and shunts the activities of proinflammatory macrophages ( m1 ) into anti - inflammatory macrophages ( m2 ) . investigated the effect of administering intravenous odn1826 ( type b oligodeoxynucleotide that activates tlr9 ) . they showed that stimulation of tlr9 impairs reendothelialization following acute vascular injury and increases plaque development in apoe mice . nonetheless , pharmacologic tlr9 activation did not alter the endothelium - dependent vasodilation , suggesting that tlr9 activation only affects the regenerative process but not the vasoactive function of endothelial cells . one possible explanation for the discrepancies between the investigations reported by krogmann et al . includes differences in the oligodeoxynucleotide dosage regime , as development of atherosclerosis requires chronic sustained inflammatory trigger . further investigations are needed to further define the role of tlr9 agonists in atherosclerotic disease . atherosclerosis is a highly complex process that involves multiple signaling cascades and a wide variety of cell types , including monocytes , macrophages , vsmcs , and various subtypes of t lymphocytes . th1 cells are proatherogenic while th2 and tregs ( t - regulatory cells ) have atheroprotective effects [ 117 , 118 ] . the function of different t - cell subtypes in atherosclerosis has been recently reviewed elsewhere [ 119 , 120 ] . ldlr mice were reconstituted with bone marrow cells where cd11c cells lacked the tlr adaptor myd88 . this decreases their ability to activate t - effector cells and was expected to produce a decrease in atherosclerosis . on the contrary , the study demonstrated an increase in aortic root atheroma size and monocyte infiltration , with no differences in lipoprotein levels . this effect was due to the loss of treg - mediated suppression of mcp-1 , and the data demonstrated the atheroprotective role of treg cells in murine models . it also provides interesting insight into the function of tlrs at the crossroads of innate and adaptive immunity in atherosclerosis , which might be useful in the development of novel therapeutic strategies . further evidence supporting the role of tlrs in atheroma development comes from genetic association studies . most studies have focused on two missense polymorphism types in tlr4 , asp299gly and thr399ile , and conflicting associations have been reported . some investigators reported a reduced risk of atherosclerosis in carriers of the asp299gly tlr4 polymorphism , which was not reproduced in other investigations . other case - control candidate genes have identified an association between the tlr4 polymorphism and acute myocardial infarction , with a reduced incidence of cardiovascular events in the 299gly allele [ 126 , 127 ] . however , a meta - analysis by koch et al . showed no association between tlr4 variants and myocardial infarction in a caucasian cohort . population - specific differences in risk allele frequency possibly account for the different associations reported in the literature [ 129 , 130 ] . tlrs form part of the innate arm of the immune system where they play an integral role in defense mechanisms against pathogens . they are expressed on a number of immune cells including macrophages , monocytes , and dendritic cells . tlrs distinguish between host molecules and pamps upon binding to the ligand and thereby trigger inflammatory response via nk-b pathways . myd88 deficiency in myeloid cells has been shown to inhibit macrophage recruitment and activation of the m1 system . in vivo and ex vivo studies have linked myd88-dependent growth factors produced by endothelial cells to initiating inflammation and development of atherosclerosis by priming the monocytes in arterial and adipose tissues to differentiate into m1-proinflammatory macrophages instead of m2 anti - inflammatory macrophages . tlr2 and tl4 are strongly implicated in atheroma development and progression , leading to coronary artery disease . this is evidenced by a number of in vitro and animal studies using atherosclerosis - prone ldlr or apoe deficient mice . moreover , the development of atherosclerotic plaques often requires more than just tlrs , and coreceptors such as cd36 have been linked to tlr2 activation and inducing atherosclerotic change . even though tlr2 and tlr4 are often labelled as atherogenic promoters , they are not considered as the primary culprit behind certain inflammatory changes such as formation of foam cells and generation of oxldl . furthermore , the presence of tlr4 in unstable plaques suggests that it is a major driver of cad progression . tlr4 upregulates matrix metallopeptidases such as mmp-9 which makes plaques prone to rupture . the precise function of tlr9 in atherosclerosis is yet to be defined , with conflicting studies reporting both proatherogenic and antiatherogenic effects . clearly , tlrs are promising therapeutic targets and possible biomarker candidates for a wide range of pathologies , but further studies are required in order to fully define their function .

toll - like receptors ( tlrs ) are key players in the pathogenesis of inflammatory conditions including coronary arterial disease ( cad ) . they are expressed by a variety of immune cells where they recognize pathogen - associated molecular patterns ( pamps ) . tlrs recruit adaptor molecules , including myeloid differentiation primary response protein ( myd88 ) and tirf - related adaptor protein ( tram ) , to mediate activation of mapks and nf - kappa b pathways . they are associated with the development of cad through various mechanisms . tlr4 is expressed in lipid - rich and atherosclerotic plaques . in tlr2/ and tlr4/ mice , atherosclerosis - associated inflammation was diminished . moreover , tlr2 and tlr4 may induce expression of wnt5a in advanced staged atheromatous plaque leading to activation of the inflammatory processes . tlr9 is activated by cpg motifs in nucleic acids and have been implicated in macrophage activation and the uptake of oxldl from the circulation . furthermore , tlr9 also stimulates interferon- ( inf- ) secretion and increases cytotoxic activity of cd4 + t - cells towards coronary artery tunica media smooth muscle cells . this review outlines the pathophysiological role of tlr2 , tlr4 , and tlr9 in atherosclerosis , focusing on evidence from animal models of the disease .

1. Introduction 2. TLR Signaling 3. Conclusion

rice is the main food resource worldwide and the staple food in korea . with the advent of a new hybridization technique in the late 1960s , the tongil variety was developed by setting up a cross between indica and japonica rice varieties ; up to the 1980s , this variety produced sufficient yield to meet the requirements of south korea . at present , improved japonica varieties are usually cultivated , and the tongil type varieties are only occasionally cultivated for processing and special purposes . since the 1980s , the demand for high - quality rice has increased due to the diverse requirements of consumers . therefore , improving the chemical composition and physical characteristics of rice is essential for developing rice varieties according to the preference of consumers and having health benefits as well as good quality . rice quality parameters such as the appearance , nutrition value , and taste and texture of cooked rice , color , and whole kernel morphology are important factors that affect consumer preferences . in addition , the major chemical components , that is , carbohydrates ( 7275% ) , proteins ( 710% ) , and lipids ( 1% ) , are important factors that reflect the nutritional aspect of rice ; the structure and compositions of the proteins and fatty acids are also important parameters . for cooked rice , texture and odor are important factors that determine the quality . in particular , texture is affected by the degree of solidity , adhesion , and cohesion ; the ratio of amylose to amylopectin can also affect the texture of cooked rice . although there are no methods to evaluate objectively the taste because it is a subjective parameter , taste is an important factor to evaluate the quality of rice . odor is also one of the important factors that affect the taste of cooked rice . the period for which rice is stored after harvest can affect the aroma of cooked rice irrespective of the varieties . rice that tastes good after cooking is known to have less than 7% protein and 15.516.5% water content . rice with high protein content is hard , less elastic , and less viscous after being cooked . in addition , the unsaturated fatty acid content of rice determines the appearance and quality of cooked rice grains [ 2 , 3 ] . the crude fatty acid contents of rice include palmitic acid , oleic acid , and linoleic acid . usually , rice with a high content of lipids is considered unhealthy ; however , if the fat content mostly comprises unsaturated fatty acids , the health risks are low . the qtls related to amylose , protein , and fat content were detected in previous researches . the qtls related to amylose and fat content , amy1 on chromosome 7 and amy2 on chromosome 8 with 20.4% of phenotypic variation and fat1 on chromosome 7 with 7.2% of variation , are detected from recombinant inbred lines ( rils ) population . six of qtls related to amylose content and five qtls for protein content were detected on chromosomes 2 and 12 . also rice protein content ( rpc ) and rice fat content ( rfc ) were identified from doubled haploid population including two rpc qtls ( qrpc-5 , qrpc-7 ) and two rfc qtls ( qrfc-2 , qrfc-5 ) . eight qtls , three qtls , and six qtls associated with protein , amylose , and fatty acid , respectively , were detected from rils population in three different locations which are wonju , daegu , and iksan of korea . five qtls for protein content ( pc ) were detected on chromosomes 3 , 4 , 5 , 6 , and 10 , respectively . a major qtl qpc-6 was located near the wx marker rm190 on the short arm of rice chromosome 6 , explaining 19.3% of the phenotypic variance and showing an additive effect of 0.471% . the other four qtls explained 3.910.5% of the phenotypic variance and had additive effects of 0.2130.343% . four qtls for fat content ( fc ) were detected on chromosomes 3 , 5 , 6 , and 8 , respectively . among these loci , qfc-5 on chromosome 5 was the qtl with the largest effect , explaining 12.9% of the phenotypic variance and showing an additive effect of 0.091% . qtl related to rice lipid content ( rlc ) with 2.37 of lod score was identified on chromosome 5 from the high lipid mutant line derived from dongjin by t - dna insertion . in this study , we investigated the growth characteristics and physicochemical properties of a cheongcheong nagdong double haploid ( cndh ) population and performed the analysis of qtl to attempt to determine quantitative trait loci ( qtls ) associated with the taste of cooked rice and breed a new variety that produces rice having good taste . a dh population consisting of 133 lines derived from a cross between cheongcheong and nagdong was cultivated by the institute of crop breeding , college of agriculture and biosciences , kyungpook national university , at an experimental field of the kyungpook national university , gunwi - gun , between 2010 and 2012 . the plant materials were sown on april 20 , 2012 , and transplanted by planting density of 30 15 cm with one plant per each line on may 20 , 2012 . all the cndh lines were planted with their parents with plants of only one line per planting row , and the planting density was 30 15 cm . the amount of fertilizer applied was n : p2o5 : k2o = 9.0 : 4.5 : 5.7 kg/10 a. phosphate and potassium were used as the main fertilizers , whereas nitric fertilizer was 70% of the main fertilizer used , and 30% of the topdressing fertilizer was used at the tillering stage . herbicides and insecticides for preventing diseases and pests were used as per the standard cultivation method of rice ( the rural development administration , 2000 ) . excluding the plants of the lines that showed remarkable sterility and separating lines , 70 cndh lines were used to determine the qtls associated with the quality of cooked rice . the growth characteristics of rice plants cultivated at the experimental field of kyungpook national university , gunwi , in 2012 , were investigated . the following growth characteristics of 5 plants of each line were measured : plant height , length of panicles , the number of spikelets per panicle , and percentage of fertile grains . the amylose , lipid , protein , and starch contents of rice were analyzed using near infrared spectroscopy ( nirs ) , and the crude fat with fatty acid content was determined by the bio - farming and functional materials for foods regional innovation center at the kyungpook national university , sangju campus . nirs ( foss nirsystems 6500 ; usa ) was performed three times after damaged and broken grains and green and red kernels were removed . qtls associated with rice quality were analyzed using 217 simple sequence repeat ( ssr ) markers obtained from the department of functional crop in rural development administration . the winqtl cartographer ( winqtlcart , version 2.5 ) was used for the qtl analysis . the composite interval mapping ( cim ) was used for whole genome scanning to detect qtls by using winqtlcart 2.5 at a threshold log of odds ( lod ) of 2.5 . the qtl analysis was performed after the cndh population was cultivated with their parents , cheongcheong and nagdong for 3 weeks in a green house . our growth characteristic analysis revealed that the plant height of the cndh population was lower than that of the parents . panicle length of the cndh population was lower than that of cheongcheong and higher than that of nagdong . the number of spikelets per panicle , 1,000 grain weight , and yield of the cndh population were lower than those of cheongcheong and nagdong ( see supplementary table 1 in supplementary material available online at http://dx.doi.org/10.1155/2014/781832 ) ; these parameters showed continuous variation in cndh , suggesting a normal distribution ( figure 1 ) . therefore , we analyzed the components of unpolished rice from the cndh population by using nirs . the amylose content of cheongcheong is lower than nagdong while the protein and lipid content of cheongcheong is higher than nagdong ( table 1 ) . thus , the correlation analysis revealed that the balance of the three components , amylose , protein , and lipid , is associated with the quality of rice ( table 2 ) . the major 3 factors associated with the taste of cooked rice are generally known as amylose , protein , and lipid . thus we performed the correlation analysis among these 3 factors in two consecutive years , 2012 and 2013 ( table 2 ) . as shown in table 2 , protein and lipid were significantly different from amylose suggesting that lipid was relatively higher impact factor for determining rice taste . however , in 2013 the factor of protein only seems to be significantly involved in the taste of cooked rice . this slight difference might be due to the differential environmental condition from two consecutive years . also , the frequency distribution of the three components in the cndh population showed a similar normal distribution except the frequency distribution in lipid content ( figure 2 ) . the qtls related to amylose content were qamy-7 , qamy-11 - 1 , qamy-11 - 2 , and qamy-11 - 3 and that related to protein content was qpro-2 . four of the qtls associated with amylose content were detected on chromosomes 7 and 11 . rm6776-rm21105 on chromosome 7 had a log of odds ( lod ) score of 6.25 . rm26771-rm26981 , rm26981-rm27242 , and rm25971-rm27123 on chromosome 11 had lod scores of 2.96 , 2.58 , and 3.59 , respectively . the one qtl associated with protein content was detected on chromosome 2 ; rm12532-rm555 had lod scores of 4.88 . further , one of the qtls associated with lipid content was detected on rm15064-rm15448 of chromosome 3 with an lod score of 3.55 ( table 3 , figure 3 ) . the index of coincidence for the qtls related to amylose content was 70% for rm21105 on chromosome 7 ( supplementary table 2 ) and 80 , 75 , and 70% for rm26771 , rm3482 , and rm26801 ( supplementary table 3 ) , respectively . the index of coincidence for the qtls related to protein content was 70% for rm12532 and rm555 on chromosome 2 and 75 , 75 , and 70% for rm506 , rm22198 , and rm22334 on chromosome 8 , respectively ( supplementary table 4 ) . the index of coincidence for the qtls related to lipid content was 70% each for rm15063 and rm15448 ( supplementary table 5 ) on chromosome 3 . we investigated the growth characteristics and analyzed the physicochemical properties of a doubled haploid population derived from a cross between cheongcheong and nagdong ( cndh ) to breed a rice variety that tastes good after cooking and to detect quantitative trait loci ( qtls ) associated with the taste of cooked rice . the results suggest that the plant height , panicle length , number of spikelets per panicle , 1,000 grain weight , and yield of the cndh population show a normal distribution . we further analyzed the contents of amylose , protein , and fatty acids in the rice grains . correlation analysis of the amylose , protein , and lipid contents indicated that each compound is related to the taste of cooked rice . in particular , the correlation coefficient of protein to amylose was significant at the level of 0.280 . the advent of molecular markers has helped to facilitate the understanding of the genetic behavior of complex quantitative traits . doubled haploid population derived from ir64 indica type and azucena japonica type is exploited for analysis of qtl related to rice quality . this study investigated the growth characteristics and analyzed the physicochemical properties of a doubled haploid population derived from a cross between cheongcheong and nagdong ( cndh ) to breed a rice variety that tastes good after cooking and detect quantitative trait loci ( qtls ) associated with the taste of cooked rice . because of these advantages , diverse methods such as the development of an automated system by using the nirs program were attempted to evaluate the taste of cooked rice in developed countries . despite remarkable development of measurement technology , several factors can affect the result of measurement . in the distribution histogram , the components are not perfect normal distribution because of damage or changing in the content of amylose , protein , and lipid by period and method of harvest , drying , heat , period of storage , and moisture . for determining amylose content and cohesion , alkali digestion value , and protein content , the basic physicochemical characteristics of rice measurement of the changes in amylogram characteristics yield more accurate results [ 13 , 14 ] . the lower the amylose content , the higher the viscosity of cooked rice . in contrast , the viscosity of cooked rice is low when the amylose content is high because amylose captures amylopectin and inhibits the swelling of starch particles . also , protein content decreases the taste of cooked rice because a layer of protein develops around the starch particles , reducing the viscosity and elasticity of cooked rice and directly affecting the gelatinization characteristics of starch . the amylose contents between two parental cultivars were shown to be significantly different ( table 1 ) suggesting differential gene expression of waxy gene , one of the key regulators determining amylose content . thus , as expected , intermediated extent of amylose content was detected in the hybrid of cheongcheong nagdong double haploid ( cndh ) population . although the smaller amounts of lipids are present in rice than the other components , they are a mediator of heat transfer and impart flavor . lipids have emulsification characteristics and combine with proteins , thereby affecting the properties of matter in tissues . the qtls related to amylose content were qamy-7 , qamy-11 - 1 , qamy-11 - 2 , and qamy-11 - 3 and those related to protein content were qpro-2 and qpro-8 . four of the qtls associated with amylose content were detected on chromosomes 7 and 11 . the index of coincidence for the qtls related to amylose content was 70% for rm21105 on chromosome 7 and 80 , 75 , and 70% for rm26771 , rm3482 , and rm26801 , respectively . the index of coincidence for the qtls related to protein content was 70% for rm12532 and rm555 on chromosome 2 and 75 , 75 , and 70% for rm506 , rm22198 , and rm22334 on chromosome 8 , respectively . the index of coincidence for the qtls related to lipid content was 70% each for rm15063 and rm15448 on chromosome 3 . the qtls for amylose content were detected on chromosomes 3 , 6 , and 7 commonly [ 4 , 7 , 11 ] . the qtls for protein content were detected commonly on chromosomes 3 , 6 , and 7 [ 68 ] . also the qtls for lipid content were commonly detected on chromosomes 5 , 6 , and 7 [ 69 ] . in spite of many results , several new qtls related to amylose and therefore , we can exploit several ssr markers on chromosomes 11 and 2 showing high coincidence ratio between genotype and phenotype . the markers can be useful to select desirable lines for rice breeding and facilitate further research .

we investigated the growth characteristics and analyzed the physicochemical properties of a doubled haploid population derived from a cross between cheongcheong and nagdong to breed a rice variety that tastes good after cooking and to detect quantitative trait loci ( qtls ) associated with the taste of cooked rice . the results showed that these compounds also represent a normal distribution . correlation analysis of the amylose , protein , and lipid contents indicated that each compound is related to the taste of cooked rice . the qtls related to amylose content were 4 qtls , protein content was 2 qtls , and lipid content was 2 qtls . four of the qtls associated with amylose content were detected on chromosomes 7 and 11 . the index of coincidence for the qtls related to amylose , protein , and lipid content was 70% , respectively . these markers showing high percentage of coincidence can be useful to select desirable lines for rice breeding .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

this study was approved by the western institutional review board ( olympia , wa , usa ) . morphologic data from 92 north american patients were derived from our prior study.14 ) the patient - reported ethnicities of this group included caucasian ( 90% ) , native american ( 4% ) , african american ( 4% ) , and hispanic ( 2% ) . these patients were from a single institution of the senior author , had a preoperative diagnosis of severe rotator cuff deficiency , and received computed tomography ( ct ) scans with 1.25-mm slice thickness ( ge lightspeed qz / i helical scanner ; ge healthcare , waukesha , wi , usa ) in the supine position . a convenience sample of 58 east asian patients who underwent open reduction internal fixation ( orif ) for a clavicle fracture with preoperative ct scans of the shoulder ( philips brilliance 64 ct scanner ; philips medical systems , cleveland , oh , usa ) was analyzed as a comparative demographic cohort . demographic data ( height , weight , and gender ) were acquired for all patients in the north american and east asian cohorts . study patients displayed little to no glenohumeral wear , osteophytes , or osseous defects as determined by a fellowship - trained orthopedic surgeon . the ct scans were imported into mimics 14.12 ( materialise , leuven , belgium ) and three - dimensional ( 3d ) renderings of the proximal humerus and scapula were generated . models were then imported into solidworks ( dassault systemes , vilzy - villacoublay , france ) as computerized assisted drawing entities . 1).14 ) a set of prescribed points denoting specific anatomic landmarks of the scapula and humerus were marked ( fig . 2 and table 1 ) and used to develop scapular- and humeral - based coordinate systems to assist with measurement of shoulder joint metrics . the scapular plane ( fig . 1a ) was defined by 3 intersecting points along the medial border , inferior angle of the scapula , and glenoid center ( fig . 1b and 2a ) , the humeral coronal plane was defined using the humeral shaft axis and a point on the greater tuberosity ( h ) . the shaft axis was created using the centers of 2 best - fit ellipses corresponding to 2 unique axial cross - sections of the humeral shaft in the 3d model . the greater tuberosity was identified as its most lateral point opposite to the humeral head 's surface center . points were marked along the cross - sectional coronal plane to denote the borders of the humeral head 's articular border and neck shaft . a best - fit circle was placed along the articular border to determine humeral head center ( g ) and radius of curvature ( g , l ) . the angle from the articular margins of the head from the head center was used to measure the articular angle ( ) . these articular margins were also used to define the line of the humeral neck and the neck shaft angle ( ) . in turn , the neck shaft line 's center was utilized to define humeral head thickness ( j , k ) . the humeral head center was used to supply the location for the orthogonal axial plane , and similarly the articular border of the humeral head was used to define a best - fit circle for the axial plane head radius of curvature ( n , o ) . the most lateral point of the acromion was marked ( i ) along with points corresponding to borders of the glenoid height ( p , q ) , and the superior ( r , s ) and inferior glenoid width ( t , u ) . humeral and scapular planes were used to reposition the humerus from its initial state to an ideal nonpathologic orientation ( fig . a soft tissue lateral offset of 4 mm between the glenoid and humeral head was used to standardize the thickness of articular cartilage in all patients ( f , l).15 ) both of these adjustments allowed statistical comparison to our prior work14 ) and use of our published glenoid height classification , which defines small , medium and large glenoid heights as < 33.4 mm , 33.438.0 mm , and > 38.0 mm , respectively.14 ) the coordinates for the adjusted humeral points were recorded and relative distances in each axes from the scapular landmarks were determined . these measurements quantified the acromial index ( ai ) , repurposing the methods used for plain radiographic measurements into our 3d model.16 ) for our purposes , the ratio of the distances of the lateral acromial ( f , i ) and greater tuberosity ( f , h ) were used relative to the scapular coordinate system . in addition , the critical shoulder angle metric utilized by moor et al.17 ) was also implemented in a modified fashion relative to the scapular coordinate system used in this study ( ) . the abduction lever arm of the middle deltoid ( g , v ) was determined according to a modified methodology of cabezas et al.14 ) and lemieux et al.18 ) measurements derived from both populations were evaluated for normality using shapiro - wilk test . normally distributed data were compared between populations using an independent t - test ( ibm spss ver . 20 ; ibm co. , armonk , ny , usa ) while a nonparametric mann - whitney u test was used to compare data that failed the normality test . statistical significance was set at p = 0.05 . 1).14 ) a set of prescribed points denoting specific anatomic landmarks of the scapula and humerus were marked ( fig . 2 and table 1 ) and used to develop scapular- and humeral - based coordinate systems to assist with measurement of shoulder joint metrics . the scapular plane ( fig . 1a ) was defined by 3 intersecting points along the medial border , inferior angle of the scapula , and glenoid center ( fig . 1b and 2a ) , the humeral coronal plane was defined using the humeral shaft axis and a point on the greater tuberosity ( h ) . the shaft axis was created using the centers of 2 best - fit ellipses corresponding to 2 unique axial cross - sections of the humeral shaft in the 3d model . the greater tuberosity was identified as its most lateral point opposite to the humeral head 's surface center . points were marked along the cross - sectional coronal plane to denote the borders of the humeral head 's articular border and neck shaft . a best - fit circle was placed along the articular border to determine humeral head center ( g ) and radius of curvature ( g , l ) . the angle from the articular margins of the head from the head center was used to measure the articular angle ( ) . these articular margins were also used to define the line of the humeral neck and the neck shaft angle ( ) . in turn , the neck shaft line 's center was utilized to define humeral head thickness ( j , k ) . the humeral head center was used to supply the location for the orthogonal axial plane , and similarly the articular border of the humeral head was used to define a best - fit circle for the axial plane head radius of curvature ( n , o ) . the most lateral point of the acromion was marked ( i ) along with points corresponding to borders of the glenoid height ( p , q ) , and the superior ( r , s ) and inferior glenoid width ( t , u ) . coordinates of all glenoid points were recorded to quantify the magnitude of each metric . humeral and scapular planes were used to reposition the humerus from its initial state to an ideal nonpathologic orientation ( fig . a soft tissue lateral offset of 4 mm between the glenoid and humeral head was used to standardize the thickness of articular cartilage in all patients ( f , l).15 ) both of these adjustments allowed statistical comparison to our prior work14 ) and use of our published glenoid height classification , which defines small , medium and large glenoid heights as < 33.4 mm , 33.438.0 mm , and > 38.0 mm , respectively.14 ) the coordinates for the adjusted humeral points were recorded and relative distances in each axes from the scapular landmarks were determined . these measurements quantified the acromial index ( ai ) , repurposing the methods used for plain radiographic measurements into our 3d model.16 ) for our purposes , the ratio of the distances of the lateral acromial ( f , i ) and greater tuberosity ( f , h ) were used relative to the scapular coordinate system . in addition , the critical shoulder angle metric utilized by moor et al.17 ) was also implemented in a modified fashion relative to the scapular coordinate system used in this study ( ) . the abduction lever arm of the middle deltoid ( g , v ) was determined according to a modified methodology of cabezas et al.14 ) and lemieux et al.18 ) normally distributed data were compared between populations using an independent t - test ( ibm spss ver . 20 ; ibm co. , armonk , ny , usa ) while a nonparametric mann - whitney u test was used to compare data that failed the normality test . statistical significance was set at p = 0.05 . the north american patient population was comprised of 48 females and 44 males ( average age , 71 years . ) while the east asian population was comprised of 35 males and 23 females ( average age , 69 years ) . the east asian cohort was shorter ( p = 0.038 ) and weighed less ( p < 0.001 ) . the smaller size of the east asian population was also indicative of smaller values for each shoulder - related metric when compared to the north american group , with a few exceptions . in the east asian group , the length of the acromion from the glenoid was greater compared to the north american group by an average of 3.6 mm ( p < 0.001 ) , which translated into a larger mean critical shoulder angle ( p < 0.001 ) . the abduction lever arm was not different between groups ( p = 0.408 ) , nor were the neck shaft and articular arc angles ( p = 0.305 and p = 0.185 , respectively ) . the ai was significantly larger in the east asian group ( p < 0.001 ) . applying our previously published classification schema for glenoid height to the east asian group , when analyzing gender differences between groups , the male subjects were found to exhibit all of the same statistical conclusions ( table 3 ) . as with the entire population , most of the metrics were smaller for the male east asian group compared to the male north americans , with the exception of the acromion length , which was again larger for the male east asian group ( average difference = 2.8 mm , p = 0.003 ) . consequently , the ai was also found to be larger for the male east asian group ( p < 0.001 ) . the angles were found to be no different between groups , as was the abduction lever arm measurement ( p = 0.061 ) . the female subjects were also found to have similar findings ( table 4 ) , with a difference in mean acromial length of 3.8 mm ( p = 0.001 ) . the ai was larger for the female east asian group compared to their female north american counterpart ( p < 0.001 ) . the majority of results in this study were unsurprising , given that the anthropomorphic metrics of height and weight were smaller in the east asian group . however , we did identify small morphologic differences between the two populations , which may impact ( 1 ) how we view and analyze shoulder anatomy and ( 2 ) how we understand the development of specific shoulder pathologies . it also provides insight regarding surgical procedures performed around the shoulder girdle , particularly relating to the area of shoulder arthroplasty . study results indicate significantly smaller metrics in nearly all measurement categories in east asians compared to north americans . on average , coronal and axial while the clinical meaning of this difference is unknown , it may be used as a design consideration by manufacturers of shoulder arthroplasty humeral head components intended for use in patients of east asian descent . currently , most arthroplasty prosthesis systems are based on average sizes for usa and european populations.567 ) in the setting of glenohumeral arthritis , placing a small humeral head component into a small east asian patient may lead to overstuffing of the joint , as the implanted head does not translate to a small component for this population . such a scenario may lead to stiffness , pain , component loosening , and a potentially inferior outcome . perhaps the most unexpected finding of this study was that the east asian group displayed a larger lateral acromial offset and ai while maintaining an equivalent abduction lever arm to their north american counterparts . several studies have explored the potential correlations between scapular morphology and different types of shoulder pathologies . nyffeler et al.16 ) were the first to examine the relationships between ai and the potential for developing rotator cuff tears ( rcts ) and glenohumeral osteoarthritis ( oa ) . they compared the ai in the following three age- and gender - matched groups : ( 1 ) a full - thickness rct group ; ( 2 ) a glenohumeral oa with intact rotator cuff group ; and ( 3 ) a volunteer control group with intact rotator cuff . the rct group demonstrated a mean ai of 0.73 , while the ai for the oa with intact rotator cuff group was 0.60 . their findings showed that a longer lateral extension of the acromion correlated with the development of rotator cuff pathology , whereas a shorter extension likely confers some protective effect . in a comparison morphometric study , yu et al.19 ) found a positive correlation between an increase in ai and subacromial impingement when compared to a normal control group . they also showed that patients with a lower ai demonstrated higher rates of traumatic instability . these findings were corroborated in another morphologic study by torrens et al,20 ) in which they compared the ai between a cohort of patients with rotator cuff tears and a control group without rotator cuff pathology . they again showed that the group with rotator cuff pathology had a significantly increased ai compared to the normal controls . the initial findings of nyffeler et al.16 ) have been substantiated in a recent follow - up case - control study by moor et al,17 ) wherein patients with rotator cuff tears demonstrated significantly higher ais . they also found that the critical shoulder angle , which is a measure of acromial length and glenoid inclination , was the best predictor for developing an rct . despite an apparent correlation between ai and rotator cuff pathology , miyazaki et al.21 ) have performed the only study , to our knowledge , to specifically analyze the association of ai and rotator cuff pathology between two distinctly different nationalities . they found a significant correlation between an increased ai and greater rates of rcts in a mixed brazilian cohort , but no association in a homogenous japanese population . similarly , in a prospective cross - sectional study by hamid et al,22 ) they found no association between ai and full - thickness rcts . there was a significant association , however , between the presence of an acromial spur along the insertion of the coracoacromial ligament and a full - thickness rct . it utilized the dataset generated from our prior work and , therefore , the results from the original population were not independently validated or re - measured . however , the interobserver measurement reliability was high ( intraclass correlation coefficient , 0.794 ) in our original investigation.14 ) given the smaller anthropometric and measured morphometric features of the east asian cohort reported herein , it may be reasonably assumed that reproducibility of the measurement technique in this population using identical methods as in our prior work would be high as well . secondly , the two data sets presented here represent patients treated by single surgeons in both patient cohorts . consequently , there may be a higher degree of variability in each population than reported in the current work . however , large - scale anthropomorphic studies indicate that our height / weight means for each group are well within a standard deviation of previously reported metrics.111213 ) thirdly , the number of east asian female subjects was smaller than the number of male subjects and this may have skewed the morphometric measures to be higher . however , to account for this discrepancy , we performed separate analyses for male and female subjects . finally , our measurements were made in a pathologic population with artificially positioned humeri , and as such , there may be other morphologic variables that were not considered in this study that would not be observed in normal , healthy controls . in conclusion , the east asian population exhibited smaller shoulder morphometrics than their north american cohort , with the exception of an extended acromial overhang . the morphologic data can provide some additional factors to consider when choosing an optimal shoulder implant for the east asian population , in addition to future designs that may better accommodate this population .

backgroundthe aim of this study was to determine if there were significant differences in glenohumeral joint morphology between north american and east asian populations that may influence sizing and selection of shoulder arthroplasty systems.methodscomputed tomography reconstructions of 92 north american and 58 east asian patients were used to perform 3-dimensional measurements . the proximal humeral position was normalized in all patients by aligning it with the scapular plane utilizing anatomic landmarks . measurements were performed on the humerus and scapula and included coronal and axial humeral head radius , humeral neck shaft and articular arc angles , glenoid height and width , and critical shoulder angle . glenohumeral relationships were also measured and included lateral distance to the greater tuberosity and acromion , abduction lever arm , and acromial index . parametric and nonparametric statistical analyses were used to compare population metrics.resultseast asian glenohumeral measurements were significantly smaller for all linear metrics ( p < 0.05 ) , with the exception of acromial length , which was greater than in the north american cohort ( p < 0.001 ) . the increase in acromial length affected all measurements involving the acromion including abduction lever arms . no difference was found between the neck shaft and articular angular measurements.conclusionsthe east asian population exhibited smaller shoulder morphometrics than their north american cohort , with the exception of an extended acromial overhang . the morphologic data can provide some additional factors to consider when choosing an optimal shoulder implant for the east asian population , in addition to creating future designs that may better accommodate this population .

METHODS Definition of Landmarks and Coordinate Systems Quantification of Glenohumeral Relationships Statistical Analysis RESULTS DISCUSSION

suspected coronary artery disease ( cad ) in patients with a concurrent malignancy greatly impacts prognosis and treatment decisions . faced with both diseases , appropriate prioritization of care is needed , as some cancer treatments may be cardiotoxic or lead to blood dyscrasias that could discourage the use of commonly prescribed cardiac medications , such as aspirin or heparin products . on the other hand , planning of cardiac invasive testing requires clinicians to take into account cancer status and possible delays in care . noninvasive coronary anatomical imaging could therefore help with accurate treatment planning prior to using invasive strategies . exclusion of significant obstructive cad in patients with left ventricular ( lv ) dysfunction and a prior history of exposure to cardiotoxic chemotherapy agents are both needed for the diagnosis of chemotherapy - induced cardiomyopathy ( cic ) . in addition , some cancer patients presenting with other findings suggestive of structural heart disease may need an accurate anatomical coronary evaluation . while invasive coronary angiography ( ica ) remains the procedure of choice in patients with high pretest probability of cad , it is not always justified in those with low to intermediate suspicion of cad . it also carries a risk of bleeding or infectious complications , which may be more likely in cancer patients because of blood dyscrasias or immunosuppression . there is therefore a need for an accurate noninvasive modality to define the coronary anatomy in some cancer patients . cardiac computed tomographic angiography ( ccta ) is such a modality , and has been shown to correlate well with ica [ 46 ] . specifically , its negative predictive value in excluding cad in the presence of a normal scan is very high . this retrospective data review study was approved by the institutional review board ( irb ) . the clinical presentation and baseline characteristics of patients who underwent ccta between january and december 2008 at the university of texas md anderson cancer center ( mdacc ) were reviewed . the indications for ccta were collected , as well as contraindications to invasive angiography , when explicitly documented . the downstream impact of ccta results on patient care decision - making was ascertained for both cardiac and cancer treatment by review of the medical records . specific diagnostic or therapeutic steps were considered to be based on cta results only when medical records explicitly referenced the cta results and directly linked those decisions with these findings . prior to ccta , patients with heart rates above 65 beats per minute on breath holding received beta - blockers ( metoprolol , up to 100 mg orally and/or up to 20 mg intravenously ) . patients needing heart rate control , but with contraindications to beta - blockers were managed with oral calcium - channel blockers . when blood pressure allowed , patients were also given sublingual nitroglycerin spray 0.4 mg immediately prior to undergoing scan . all studies were performed using a 64-detector scanner ( lightspeed vct , general electric healthcare ( gehc ) , milwaukee , wi ) . a calcium scoring scan was performed using 2.5 mm collimation and 25 cm display field of view with prospective cardiac triggering without intravenous contrast material . 150 ml of 320 mgi / ml contrast material ( iodixanol , visipaque 320 , gehc ) was administered at 5 ml / sec , followed by 50 ml of normal saline at 5 ml / sec . cardiac scanning was performed with 0.625 mm collimation using retrospective cardiac gating and electrocardiographic ( ecg ) dose modulation . in patients with significant beat to beat heart rate variability or frequent ectopic beats , data was reconstructed at 0.625 mm slice thickness and 0.4 mm interval at 70% , 75% , and 80% of the r - r interval for coronary analysis and a second series at 1.25 mm slice thickness and 1.25 mm interval with 10 cardiac phases from 5% to 95% of the r - r interval for functional analysis . additional coronary analysis phases were reconstructed as needed to address motion artifacts on the standard phases . examinations were interpreted by a thoracic radiologist ( gg ) with 5 years experience in cardiac ct in conjunction with a cardiologist ( i d ) . interpretation and postprocessing were performed using an advantage workstation ( gehc ) or an intuition workstation ( terarecon , inc . severe cad was defined as luminal diameter narrowing estimated to be greater than 70% in at least one vessel or more than 50% left main coronary stenosis . coronary stenosis that did not meet the defined criteria for severe was termed mild to moderate cad . a diagnosis of chemotherapy - induced cardiomyopathy was established by lv dysfunction with prior cardiotoxic chemotherapy exposure and absence of significant obstructive cad . thrombocytopenia was defined as platelet count less than 50,000 per l , and leucopenia as white blood cell count below 4,000 per l . medical management of cad was defined as treatment based on coronary calcification and/or the presence of plaque ( which defines atherosclerotic disease and therefore identifies more aggressive secondary prevention targets ) . cad risk factor modification was defined as primary prevention step in absence of demonstrated cad , which , for nondiabetic patients , has less stringent criteria . baseline demographics , cad risk factors and proportions of hematological malignancies are shown in table 1 . the mean age of the patient population was 58.8 12.8 years with 56% males . hypertension was found in 48% of the cases , hyperlipidemia in 36% of cases , diabetes mellitus in 13% and established cad in 9% of cases . about 45% of patients had hematological malignancy ( leukemia and lymphoma ) whereas the rest had a solid tumor . the most common indication for obtaining a ccta was lv dysfunction ( 41% of cases ) followed by chest pain and or dyspnea in 40% of the cases ( table 2 ) . about 20% ccta was obtained for atypical symptoms with abnormal stress test and 10% were for atypical symptoms with abnormal biomarkers . miscellaneous indications were present in 13 patients ( 16.3% ) , including suspected cardiac tumor and/or thrombus or pulmonary embolism . indications were not mutually exclusive and some patients had more than one at the time of ccta . overall , 72 pts ( 90% ) had at least one relative contraindication to ica ( table 3 ) . the majority of the patients ( 61% ) who underwent a ccta had a low to moderate pretest probability for cad . concomitant radiation or chemotherapy was present in 25% of cases and rest ( 19% ) had leucopenia and/or thrombocytopenia . normal coronaries were found in 26.3% of patients , while mild to moderate cad was found in 46% and severe cad in 28% . cardiac treatment decisions based on ccta findings are outlined in table 5 . due to concomitant advanced cancer or patient refusal , only 12 out of 22 patients with severe cad by ccta underwent ica , including 4 patients who eventually underwent coronary artery bypass grafting ( cabg ) . the majority of the patients were managed medically , with 38% of the patients treated medically for nonobstructive cad and 23% of patients treated for chemotherapy - induced cardiomyopathy ( figure 1 ) . in 25% of the cases a total of 41 pts ( 51.3% ) had their cancer management altered based on ccta findings ( table 6 ) . in 24% of cases , these findings directly influenced the decision to restarting chemotherapy ( figure 2 ) , while in 9% of cases , they led to a change in chemotherapy agents based on a diagnosis of chemotherapy - induced cardiomyopathy . in 2.5% of cases , chemotherapy was temporarily discontinued in order to proceed with invasive angiography ( figure 3 ) . in 10% of cases , exclusion of significant cad by ccta allowed for stem cell transplant evaluation to proceed . in 4% of cases , cancer surgery was performed without further cardiac testing , and 2.5% of cases subsequently started radiation therapy . the present study shows that ccta can have important consequences in evaluating cancer patients with suspected coronary disease , often leading to important decision in the care of both cancer and heart disease . for more than half the patients in our study coexistence of cad and malignancy often creates a therapeutic and diagnostic challenge : faced with two potentially life - threatening conditions , prioritization of care is paramount . this is because cancer , its treatment , and complications may render commonly used cardiac tests and therapies difficult , if not contraindicated . cancer patients receiving chemotherapy and experiencing bone marrow suppression are at higher risk of procedural complications should they undergo ica routinely . in other instances , postoperative status , ongoing infections , or radiation therapy may also preclude an invasive approach . in fact , a large majority of our patients had at least one relative contraindication to ica , and as such would have been less likely to be fully assessed without ccta . this often leads to the use of noninvasive stress imaging , traditionally echocardiography and nuclear imaging for a functional assessment of the coronaries . however , neither technique is able to show nonobstructive cad , nor are they free of false positives ( which could lead to unnecessary ica ) and false negatives ( which could fail to correctly identify cancer patients with significant cad ) . ccta also allowed the accurate diagnosis of cic , which has important implications in ongoing cancer care . current guidelines for the diagnosis of cic recommend a coronary angiogram to exclude the presence of significant cad . the acc / aha guidelines for the diagnosis and management of heart failure updated in 2009 recognize the use of noninvasive imaging for detection of ischemia in patients presenting with heart failure , if revascularization is contemplated , with the caveat that stress testing may not reliably distinguish between ischemic and nonischemic etiologies . this emphasizes the need for coronary anatomic assessment in these patients . in our practice in a tertiary cancer center , most patients with exposure to potentially cardiotoxic chemotherapy who experience a decrease in lv function are considered for angiography . prior to the use of ccta , however , many could not undergo ica , and a conclusive diagnosis was often lacking . as we have shown , however , significant cad in the setting of a cardiomyopathy could accurately be excluded , therefore confirming a suspicion of cic in a quarter of our patients ( figure 1 ) . noninvasive coronary artery imaging is therefore appealing in many instances in this patient population , particularly because of its high negative predictive value . indeed , ccta can conclusively demonstrate a normal coronary tree , therefore alleviating the need for ica . by accurately defining the coronary anatomy , ccta in fact allowed the prioritization of cancer and cardiac care in our patients , in some cases , by excluding significant cad suspected either based on cardiac biomarkers , symptoms , or previous testing ( figure 2 ) . the advantage of adequate identification of underlying severe cad in certain circumstances would allow stratification of patients who need modification of their chemotherapeutic regimen or who may benefit from concurrent treatment with cardioprotective therapeutic agents that promote positive remodeling , such as -adrenergic blockers , angiotensin converting enzyme inhibitors , or angiotensin receptor blockers . in others , by identifying patients who needed their cancer care to be put on hold until more pressing cardiac issues could be addressed , ccta also prevented undue delays in cancer care by seamlessly integrating with the flow of care . our results in a group of cancer patients with low to intermediate pretest probability for cad on the basis of risk factors alone suggest that it may be more prevalent than suspected . indeed , we found that nearly a third of pts had severe cad , and 5% of total population underwent cabg . this emphasizes the importance of coordinating cancer and cardiac care , as both can coexist with equal severity in the same patients . in fact , some cases will require the interruption of chemotherapy until the patient has undergone coronary revascularization ( figure 3 ) . the addition of noninvasive coronary imaging therefore adds an invaluable tool in the approach to these difficult cases . our study describes a single center experience , and included patients referred for ccta . in particular , there is no control group , and therefore no direct comparison to other noninvasive modalities ( stress testing ) can be drawn . whether ccta as a first step would compare favorably to other noninvasive modalities needs further study , particularly as a cost and radiation reduction measure . although we reported short - term outcomes in terms of cancer and cardiac care , long - term followup will be needed to show definitive improvement in overall outcomes . cardiac computed tomographic angiography is an attractive imaging modality in cancer patients with suspected structural heart disease when invasive coronary angiography may be too risky or impractical and allows improved coordination of cancer and cardiac care .

background . exclusion of underlying coronary artery disease ( cad ) is essential in the diagnosis of chemotherapy - induced cardiomyopathy . presence and severity of cad can also impact the choice of therapy in cancer patients . the value of cardiac computed tomographic angiography ( ccta ) in this setting has not been reported . methods . we collected data on the clinical presentation and indications for ccta performed from january to december 2008 at the university of texas md anderson cancer center ( mdacc ) . all examinations were performed using a 64-detector scanner . ccta results and subsequent treatment decisions were examined . results . a total of 80 patients underwent ccta during the study period for the following indications ( not mutually exclusive ) : cardiomyopathy of unknown etiology in 33 pts ( 41.3% ) , chest pain in 32 ( 40.0% ) , abnormal stress test in 16 ( 20.0% ) , abnormal cardiac markers in 8 ( 10.0% ) , suspected cardiac mass or thrombus in 7 ( 8.8% ) . chemotherapy - induced cardiomyopathy was diagnosed in 18 pts ( 22.5% ) . severe cad was detected in 22 pts ( 27.5% ) ; due to concomitant advanced cancer or patient refusal , only 12 underwent coronary angiogram . of these , 4 pts ( 5% of total ) underwent coronary artery bypass grafting . a total of 41 pts ( 51.3% ) had their cancer management altered based on ccta findings . conclusion . ccta is useful in evaluating cancer pts with structural heart disease and can have an impact on the management of cancer and cardiac disease .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions

the reported incidence of acute postoperative spinal infections ranges from less than 1% to 13% . . infection rate of lumbar open discectomies without fusion is known to be 1 - 2% , microdiscectomy 5% , instrumented posterior lumbar fusions 2 - 6% , respectively5,13 ) . c - reactive protein ( crp ) and erythrocyte sedimentation rate ( esr ) have been used to diagnose postoperative infections after spinal surgery . several studies have reported the usefulness of crp , esr , and wbc as a predictor of infection after spine surgery . however , it is difficult to detect infection before day 7 because incubation period where the immune system is activated to protect a living body from invading bacteria . the crp is a too sensitive marker to distinguish the case of early infection from postoperative inflammation . the purpose of this study was to assess the normal range and pattern of the change in plasma crp , esr , and wbc according to operative method and extents of spinal surgery . forty patients in two groups were prospectively enrolled from march of 2006 to june of 2006 . group a included 20 patients who had single level lumbar open discectomy ( lod ) and group b consisted of 20 patients who had posterior lumbar interbody fusion ( plif ) with stand - alone cage after decompression . all patients were operated by the same surgeon and all did not have postoperative infection . they did not have any constitutional symptoms of infection with normal surgical wound healing and normal crp three months after surgery . inclusion criteria included those 18 years - old older with surgical indication for elective surgery , either single level discectomy or plif and hospital length of stay of 7 days or more after the surgery . we excluded the patients who had previous spinal surgery , transfusion during the surgery , anemia , history of immunodeficiency , autoimmune disease such as rheumatoid arthritis , and any history of infection within 6 months of the surgery . all patients had blood draws for crp , esr , and wbc count 1 day before surgery and postoperative day ( pod ) 1 , pod3 , and pod7 . the normal crp value in the authors ' hospital was less than 0.3 mg / dl . esr is a manual test in which the level of blood collected in a western sedimentation rate tube is read by a technologist after a specific time period . statistical analysis was performed with using repeated measures anova , and changes in effect measures over time and between groups over time were assessed . the mean crp value of preoperative was 0.78 mg / dl ( 0.5 - 3 mg / dl ) , which increased to 1.11 mg / dl ( 0.2 - 3.8 mg / dl ) at pod1 . at pod3 and pod7 , it decreased to 0.54 mg / dl ( 0.1 - 2.0 mg / dl ) , 0.4 mg / dl ( 0.1 - 2.7 mg / dl ) respectively . change rates of crp value , comparing with preoperative crp were 42.3% increase at postoperative first day , 30.7% decrease at postoperative 3rd day and 40.8% decrease at postoperative 7th day . mm / hr ) , and postoperative mean esr value of first , third and seventh day were 2.14 mm / hr ( 2 - 27 mm / hr ) , 6.0 mm / hr ( 2 - 31 mm / hr ) , 6.4 mm / hr ( 2 - 43 mm / hr ) respectively . change rates of esr value , comparing with preoperative esr were 54.6% decrease at postoperative first day , 27.3% increase at postoperative 3rd day and 35.8% increase at postoperative 7th day . preoperative mean wbc values was 9.4410/mm ( 3.65 - 17.5810/mm ) , and postoperative mean wbc value of first , third and seventh day were 14.610/mm ( 7.44 - 26.7510/mm ) , 11.010/mm ( 5.44 - 23.1010/mm ) , 7.810/mm ( 4.33 - 16.6010/mm ) , respectively . change rates of wbc value , comparing with preoperative wbc were 54% increase at postoperative first day , 16.5% increase at postoperative 3rd day and 17.3% increase at postoperative 7th day . the mean crp value of preoperative was 0.67 mg / dl ( 0.5 - 1.6 mg / dl ) , which increased to 1.55 mg / dl ( 0.5 - 5.1 mg / dl ) , at postoperative first day . at postoperative third and seventh day , it decreased to 1.52 mg / dl ( 0.1 - 7.6 mg / dl ) , 1.03 mg / dl ( 0.1 - 4.2 mg / dl ) , respectively . change rates of crp value , comparing with preoperative crp , were 131.4% increase at postoperative first day , 126.8% increase at postoperative 3rd day and 53.7% increase at postoperative 7th day . mm / hr ) , and postoperative mean esr value of first , third and seventh day were 5.78 mm / hr ( 2 - 26 mm / hr ) , 15.2 mm / hr ( 2 - 22 mm / hr ) , 17.4 mm / hr ( 2 - 24 mm / hr ) , respectively . change rates of esr value , comparing with preoperative esr were 14.3% decrease at postoperative first day , 131.0% increase at postoperative 3rd day and 157.7% increase at postoperative 7th day . 8.0210/mm ( 4.86 - 11.4310/mm ) , and postoperative mean wbc value of first , third and seventh day were 14.6010/mm ( 8.21 - 20.5910/mm ) , 11.7010/mm ( 7.70 - 22.9010/mm ) , 9.9410/mm ( 5.39 - 16.5010/mm ) , respectively . change rates of wbc value , comparing with preoperative wbc were 82.0% increase at postoperative first day , 31.4% increase at postoperative 3rd day and 23.9% increase at postoperative 7th day . 1 shows the changes of mean crp , esr , and wbc on preoperative and postoperative 1 , 3 , and 7 days comparing between two groups according to the postoperative day . in open discectomy group ( group a ) , mean crp was normalized rapidly at postoperative 3rd day . while mean crp of group b showed steady decrease and mean crp did not normalized to preoperative state even in pod 7th day . mean esr level changes in both group a , b showed decrease on the 1st postoperative day , and then clearly elevated above its preoperative levels on the 3rd postoperative day , showing steady increase until 7th postoperative day . however , mean esr changes in group b showed steeper inclination than group a. mean esr value at postoperative third day increase more rapidly in group b than group a. both group a , b of mean wbc count elevated rapidly on 1st postoperative day , and then decreased to normalize after 3rd postoperative day . mean wbc changes of group a showed faster normalization than that of group b at pod 7th day . fig . 2 , 3 show the scattered pattern of crp , esr , and wbc . changes in crp , within treatment groups over time and between treatment groups over time were both statistically significant f(3,120)=5.05 , p=0.003 and f(1,39)=7.46 , p=0.01 , respectively . changes in esr , within treatment groups over time and between treatment groups over time were also found to be statistically significant , f(3,120)=6.67 , p=0.0003 and f(1,39)=3.99 , p=0.01 , respectively . these differences appear to be in part due to dramatic increases in esr in the plif group on pod3 and pod7 . changes in wbc values also were statistically significant within groups over time , f(3,120)=40.52 , p<0.001 , however , no significant difference was found in between groups wbc levels over time , f(1,39)=0.02 , p=0.89 . the mean crp value of preoperative was 0.78 mg / dl ( 0.5 - 3 mg / dl ) , which increased to 1.11 mg / dl ( 0.2 - 3.8 mg / dl ) at pod1 . at pod3 and pod7 , it decreased to 0.54 mg / dl ( 0.1 - 2.0 mg / dl ) , 0.4 mg / dl ( 0.1 - 2.7 mg / dl ) respectively . change rates of crp value , comparing with preoperative crp were 42.3% increase at postoperative first day , 30.7% decrease at postoperative 3rd day and 40.8% decrease at postoperative 7th day . mm / hr ) , and postoperative mean esr value of first , third and seventh day were 2.14 mm / hr ( 2 - 27 mm / hr ) , 6.0 mm / hr ( 2 - 31 mm / hr ) , 6.4 mm / hr ( 2 - 43 mm / hr ) respectively . change rates of esr value , comparing with preoperative esr were 54.6% decrease at postoperative first day , 27.3% increase at postoperative 3rd day and 35.8% increase at postoperative 7th day . preoperative mean wbc values was 9.4410/mm ( 3.65 - 17.5810/mm ) , and postoperative mean wbc value of first , third and seventh day were 14.610/mm ( 7.44 - 26.7510/mm ) , 11.010/mm ( 5.44 - 23.1010/mm ) , 7.810/mm ( 4.33 - 16.6010/mm ) , respectively . change rates of wbc value , comparing with preoperative wbc were 54% increase at postoperative first day , 16.5% increase at postoperative 3rd day and 17.3% increase at postoperative 7th day . the mean crp value of preoperative was 0.67 mg / dl ( 0.5 - 1.6 mg / dl ) , which increased to 1.55 mg / dl ( 0.5 - 5.1 mg / dl ) , at postoperative first day . at postoperative third and seventh day , it decreased to 1.52 mg / dl ( 0.1 - 7.6 mg / dl ) , 1.03 mg / dl ( 0.1 - 4.2 mg / dl ) , respectively . change rates of crp value , comparing with preoperative crp , were 131.4% increase at postoperative first day , 126.8% increase at postoperative 3rd day and 53.7% increase at postoperative 7th day . mm / hr ) , and postoperative mean esr value of first , third and seventh day were 5.78 mm / hr ( 2 - 26 mm / hr ) , 15.2 mm / hr ( 2 - 22 mm / hr ) , 17.4 mm / hr ( 2 - 24 mm / hr ) , respectively . change rates of esr value , comparing with preoperative esr were 14.3% decrease at postoperative first day , 131.0% increase at postoperative 3rd day and 157.7% increase at postoperative 7th day . preoperative mean wbc value was 8.0210/mm ( 4.86 - 11.4310/mm ) , and postoperative mean wbc value of first , third and seventh day were 14.6010/mm ( 8.21 - 20.5910/mm ) , 11.7010/mm ( 7.70 - 22.9010/mm ) , 9.9410/mm ( 5.39 - 16.5010/mm ) , respectively . change rates of wbc value , comparing with preoperative wbc were 82.0% increase at postoperative first day , 31.4% increase at postoperative 3rd day and 23.9% increase at postoperative 7th day . 1 shows the changes of mean crp , esr , and wbc on preoperative and postoperative 1 , 3 , and 7 days comparing between two groups according to the postoperative day . in open discectomy group ( group a ) , mean crp was normalized rapidly at postoperative 3rd day . while mean crp of group b showed steady decrease and mean crp did not normalized to preoperative state even in pod 7th day . mean esr level changes in both group a , b showed decrease on the 1st postoperative day , and then clearly elevated above its preoperative levels on the 3rd postoperative day , showing steady increase until 7th postoperative day . however , mean esr changes in group b showed steeper inclination than group a. mean esr value at postoperative third day increase more rapidly in group b than group a. both group a , b of mean wbc count elevated rapidly on 1st postoperative day , and then decreased to normalize after 3rd postoperative day . mean wbc changes of group a showed faster normalization than that of group b at pod 7th day . changes in crp , within treatment groups over time and between treatment groups over time were both statistically significant f(3,120)=5.05 , p=0.003 and f(1,39)=7.46 , p=0.01 , respectively . changes in esr , within treatment groups over time and between treatment groups over time were also found to be statistically significant , f(3,120)=6.67 , p=0.0003 and f(1,39)=3.99 , p=0.01 , respectively . these differences appear to be in part due to dramatic increases in esr in the plif group on pod3 and pod7 . changes in wbc values also were statistically significant within groups over time , f(3,120)=40.52 , p<0.001 , however , no significant difference was found in between groups wbc levels over time , f(1,39)=0.02 , p=0.89 . crp , esr , and wbc have been widely used for estimating the status of infection . several studies have reported the usefulness of this predictor and mentioned that the combination of normal crp , esr , and wbc levels reliably predicts the absence of infection after elective surgery5,10,12,18 ) . c - reactive protein was discovered and named for its reactivity to the c polysaccharide in the cell wall of s. pneumonia , which is produced by the liver in response to inflammation , infection , malignancy , and tissue damage with relatively high sensitivity , response speed , and range compared to other acute - phase reactants1 ) . normal crp concentration in healthy human serum is usually lower than 1 mg / dl , increasing slightly with aging . higher levels are found in late pregnancy , mild inflammation , and viral infections ( 1 - 4 mg / dl ) ; active inflammation and bacterial infection ( 4 - 20 mg / dl ) ; severe bacterial infections and burns ( > 20 mg / dl)3 ) . crp levels normally rise within 2 to 6 hours after surgery and then go down by the third day after surgery2 ) . the esr has been the measure of the quantity of rbc that precipitate in a tube in a defined time and is based upon serum protein concentrations and rbc interactions with these proteins . the esr increases in pregnancy or rheumatoid arthritis , and decreases in polycythemia , sickle cell anemia , hereditary spherocytosis , and congestive heart failure2 ) . multiple factors such as patient 's age , gender , rbc morphology , hemoglobin concentration , and serum levels of immunoglobulin influence the esr . the sample must be handled appropriately and processed within a few hours to assure test accuracy . while the esr is not a diagnostic test , it can be used to monitor disease activity and treatment response and signal that inflammatory or infectious stress is present . for example , in rheumatoid arthritis , the esr correlates well with disease activity ; however normalization of esr often lags behind successful treatment that causes resolution of the inflammatory state . immediately after surgery , lymphocytes decrease due to apoptosis but neutrophils increase causing overall rise in wbc count . therefore , the change in the peripheral wbc count , especially neutrophil count , over time serves as a useful marker of postoperative progress17 ) . there have been several studies that compared crp , esr and wbc in regards to early detection of infection . crp has been known to be more reliable than esr for monitoring the effectiveness of treatment for acute hematogenous osteomyelitis19 ) , septic arthritis in children15 ) , infected total joint arthroplasty6 ) , and spine infection7 ) . crp changes more quickly than esr and therefore crp may be a better reflection of current inflammation . khan et al.7 ) also mentioned that serial crp level measurement may be helpful in monitoring the treatment of postoperative wound infections after spinal surgery . however , esr does not appear to be as a sensitive marker of infection resolution . for the explanation of prompt response of crp , they assumed that crp , unlike the esr , is a fairly stable serum protein whose measurement is not time - sensitive and is not affected by other serum components . numerous studies have described the normal kinetics of crp as a rapid rise with peak value on pod2 or pod3 , followed by an initial sharp fall and then a gradual decrease with normalization by pod14 to pod216,7,8,15 ) . larsson et al.10 ) analyzed the prospective study of 89 patients who underwent uncomplicated spinal surgery showed that crp rises within the first two postoperative days and then sharply declines . esr shows more irregular peak at pod5 and then gradually returns to normal after several weeks5,7,10,18 ) . comparing with our analysis , in lod group , crp was normalized at postoperative 3rd day , however , in plif group , tendency of consistent high crp with steady declination was detected . however , there literatures did not design the study as limit to the single procedure . in current study , we compared the changes of crp , esr , and wbc according to the procedures ( lod and plif ) . it is hard for us to find the literatures asserting about changes of inflammatory markers according to the procedure . for that reason , we assert that our current study is valuable for that point . in general , the peak response is affected by the amount of iatrogenic tissue injury at surgery . our study shows that the peak crp level is low in lod group than plif 's one . there are a few literatures which measures and showed the normal ranges of crp , esr , and wbc in uninfected normal cases according to the procedures . understanding the determinants of peak value is important because values substantially higher than expected may indicate an abnormal inflammatory response due to a complication . deviations from normal kinetics of esr and crp may indicate an infectious complication . for spine surgery , kock - jensen et al.8 ) reported peak crp of 28.5 mg / l after lumbar disc surgery . larsson et al . presented peak values for microdiscectomy ( 46 mg / l ) , open discectomy ( 92 mg / l ) , anterior lumbar fusion ( 70 mg / l ) , and posterolateral interbody fusion ( 173 mg / l)18 ) . takahashi et al.16 ) found significantly higher peak values after posterior lumbar instrumented fusion ( 91.9 there have been several reports about value of crp , esr , and wbc according to infection . chung et al.2 ) reported that the postoperative esr in uncomplicated cases remained elevated 1 week after surgery , while the crp peaked at day 2 and normalized by day 7 after spine surgery . mun et al.13 ) reported that , if there is persistent elevation or second rise around a week after surgery , the wound infection should be considered . takahashi et al.16 ) found that the post - operative crp reached its peak on day 2 and remained abnormally elevated even after six weeks . and , mok et al.12 ) tried to derive the normal kinetics of crp , opening the way for recognition of deviations that may indicate a complication . the kinetics seems to be conserved regardless of operation , magnitude , or region . a second rise or failure to decrease as expected has a high sensitivity , indicating that an infection will cause a positive result . crp and esr reflect the degree of the inflammatory and surgical injuries as crp has shown to have a higher sensitivity and specificity and is more reliable than esr for detecting postoperative infection16 ) . in additions , some literatures asserted that crp is a predictable and responsive serum parameter in postoperative monitoring of inflammatory responses in patients undergoing spine surgery9,11 ) . knowledge of the postoperative peak value , either by routine measurement on pod2 or 3 or estimation of the peak value based on the analysis above , allows calculation of expected values at subsequent time points because the kinetics are defined . if a patient presents with findings concerning for infection days or even weeks after surgery , crp can be measured with a simple blood draw at that time and compared with the value expected if the patient adhered to normal kinetics12 ) . in current study we tried to minimize the influence of blood transfusion to crp , wbc , esr changes . enright et al.4 ) mentioned in his prospective study of crp concentrations pre- and post - transfusion , serum crp levels were analyzed before and after transfusion , using a fluorescence polarization immunoassay . he found that small rises in crp concentrations occurred following 55.6% of transfusions , however , there was no statistically significant difference between pre- and post - transfusion crp values , and these increases also failed to reach clinical significance . he added that crp post - transfusion of greater than 100 mg / l occurred on only one occasion , and was more likely to be due to underlying infection4 ) . however , esr and wbc may be affected by transfusion . in current case , we excluded the case of spinal surgery which needed transfusion , and that point made more reliable results than other literatures , reporting the crp , esr , and wbc with spinal surgery . we had very critical shortage that we used the average , median value and limitation of statistical analysis due to small number of prospective study cases . however , we tried to enumerate all the measured value of this study in the table . we presumed our prospective series might be the valuable report of comparison between surgical techniques without influence of transfusion . we assume that normalization of crp at postoperative 3rd day of lod may give us relief that surgery is not infected and checking and worrying about non - normalization of crp at postoperative 7th day of plif with stand - alone cage might be the unnecessary work . for esr , only the thing that we could find is both procedures showed steady increase until 7th postoperative day except steepness of inclination , which might mean that esr does not help for monitoring the infection after surgery . for wbc , we presumed that normalization of wbc at postoperative 3rd day of lod may provide us the non - infected state of surgery , and normalization of wbc at postoperative 7th day of plif with stand - alone cage probably may assure of non - infection . we tried to aware the typical pattern of crp , esr , and wbc in non - infected postsurgical patients and knowing the pattern may help to evaluate the early postoperative course . we found that in lod group of non - infection , dramatic decrease of crp was detected on pod3 and pod7 and dramatic increase of esr on pod3 and pod7 in plif group of non - infection . both group 's esr level showed typical kinetics , not decreased until pod7 and wbc level maintain nearly equal to pod7 . however , the crp level was sharply decreased at pod3 in lod group that of plif . so , we assumed that crp would be more effective and sensitive parameter than esr , wbc for early detection of infectious complications , especially in lod than plif .

objectivec - reactive protein ( crp ) and erythrocyte sedimentation rate ( esr ) are often utilized to evaluate for postoperative infection . abnormal values may be detected after surgery even in case of non - infection because of muscle injury , transfusion , which disturbed prompt perioperative management . the purpose of this study was to evaluate and compare the perioperative crp , esr , and white blood cell ( wbc ) counts after spine surgery , which was proved to be non-infection.methodstwenty patients of lumbar open discectomy ( lod ) and 20 patients of posterior lumbar interbody fusion ( plif ) were enrolled in this study . preoperative and postoperative prophylactic antibiotics were administered routinely for 7 days . blood samples were obtained one day before surgery and postoperative day ( pod ) 1 , pod3 , and pod7 . using repeated measures anova , changes in effect measures over time and between groups over time were assessed . all data analysis was conducted using sas v.9.1.resultschanges in crp , within treatment groups over time and between treatment groups over time were both statistically significant f(3,120)=5.05 , p=0.003 and f(1,39)=7.46 , p=0.01 , respectively . most dramatic changes were decreases in the lod group on pod3 and pod7 . changes in esr , within treatment groups over time and between treatment groups over time were also found to be statistically significant , f(3,120)=6.67 , p=0.0003 and f(1,39)=3.99 , p=0.01 , respectively . changes in wbc values also were be statistically significant within groups over time , f(3,120)=40.52 , p<0.001 , however , no significant difference was found in between groups wbc levels over time , f(1,39)=0.02 , p=0.89.conclusionwe found that , dramatic decrease of crp was detected on pod3 and pod7 in lod group of non - infection and dramatic increase of esr on pod3 and pod7 in plif group of non - infection . we also assumed that crp would be more effective and sensitive parameter especially in lod than plif for early detection of infectious complications . awareness of the typical pattern of crp , esr , and wbc may help to evaluate the early postoperative course .

INTRODUCTION MATERIALS AND METHODS RESULTS Changes of CRP, ESR, and WBC in lumbar open discectomy group (Group A) Changes of CRP, ESR, and WBC in decompression and posterior lumbar interbody fusion with stand-alone cage group (Group B) Overall differences CRP, ESR, and WBC between two groups Statistical analysis DISCUSSION CONCLUSION

this is the case of a patient with alcoholic liver cirrhosis ( lc ) who underwent emergency surgery for iliopsoas muscle hematoma and subsequently died in the hospital . iliopsoas muscle hematoma is well known as a major and well - documented complication of hemophilia and anticoagulant therapy [ 1 , 2 ] . it encompasses a wide spectrum of severity ( self - limiting to fatal ) depending of its size , etiology and the development of complications . the difficulties in the correct diagnosis frequently lead to delay in treatment or unneeded surgery . although lc is commonly associated with coagulopathy , including thrombocytopenia and hypoprothrombinemia , iliopsoas hematoma in lc patients has been rarely reported . however a high mortality rate has been reported in patients with intramuscular hematoma secondary to lc , especially alcoholic lc . it is important to take into account the characteristics of alcohol - related liver injury , such as the dysfunctional hemostasis due to fragile vascular walls , the inhibition of platelet adhesion to fibrinogen and the promotion of atherosclerosis . we herein present a case of bilateral iliopsoas hematoma mimicking rupture of iliac artery aneurysm in a patient with alcoholic lc and review the literature on iliopsoas muscle hematoma associated with lc . although rare , it is a condition with a poor prognosis , and a treatment plan should be carefully determined on a case - by - case basis . he had a history of alcoholic liver disease and emphysema since 45 years of age . a local orthopedic surgeon examined him and prescribed non - steroidal anti - inflammatory drugs with follow - up observation . he then visited a local physician as he developed numbness and sense of weakness of the left thigh , and he was subsequently hospitalized . rupture of bilateral common iliac artery aneurysms was suspected after computed tomography ( ct ) without contrast was conducted due to abnormal renal function , and the patient was transferred to our hospital while being administered catecholamines . on admission , he was awake and alert with a blood pressure of 106/76 mm hg and a heart rate of 133 bpm . the patient 's laboratory data upon admission to our hospital ( table 1 ) showed hemorrhagic anemia , hypoalbuminemia , hepatorenal dysfunction and coagulation abnormalities as follows : hemoglobin 5.3 g / dl , serum albumin 1.9 g / dl , aspartate aminotransferase 598 iu / l , alanine aminotransferase 148 iu / l , lactate dehydrogenase 873 iu / l , platelet count 6.6 10/l , prothrombin time 46% , activated partial thromboplastin time > 120 s and creatinine 2.4 mg / dl . the c - reactive protein level was increased ( 6.0 mg / dl ) and electrolyte disturbances were shown ( sodium 122 mmol / l and potassium 5.6 mmol / l ) . in the non - contrast ct performed by the previous physician , bilateral iliopsoas muscles were enlarged with huge hematoma with a maximal diameter of 7 cm accompanied by formation of fluid level ( fig . 1a ) , and the border between the bilateral iliac arteries and the hematoma was not clear ( fig . the patient was immediately admitted and an emergency operation was performed while administering red cell concentrates . it took 57 min from the start of patient transfer to that of operation , and 80 min from the start of the operation to aortic clamping . as for the operation findings , bilateral retroperitoneal hematomas were seen below the aortic bifurcation , and the bilateral external iliac artery and internal iliac artery were not identifiable within the hematomas . as for blood transfusion , 980 ml of packed blood cells and 960 ml of fresh frozen plasma were used . as for the postoperative course ( fig . 2 ) , the patient was extubated and feeding was started on postoperative day 4 ; however , he developed aspiration pneumonia . although postoperative progression of anemia was not observed using hematological drugs appropriately , follow - up ct on postoperative day 14 revealed that the sizes of bilateral iliopsoas muscle hematomas were almost the same as before the operation . tracheotomy was conducted and pneumonia treatment was continued ; however , the patient progressively deteriorated with liver dysfunction , accumulating ascites and intravascular volume depletion . although his renal failure became irreversible , dialysis treatment was declined by his family members , and the patient died on postoperative day 62 . lc is commonly associated with coagulopathies , including thrombocytopenia and hypoprothrombinemia , which often cause easy bruising and bleeding . sudden bleeding from gastrointestinal varices due to portal hypertension is also an established risk of lc . although muscle hematoma usually occurs either traumatically or spontaneously in patients with hemophilia and in patients receiving anticoagulant therapy , it is rare in cirrhosis . however , sugiyama et al . reported that the mortality rate of muscle hematoma in lc patients is extremely high in comparison to that in patients with hemophilia or under anticoagulant treatment and that these findings characterize muscle hematoma . actually the mortality rate of iliopsoas hematoma associated with hemophilia is extremely low after the induction of prophylaxis , and according to a review of iliopsoas hematomas in patients receiving intravenous heparin , only 1 of 54 patients ( 1.9% ) died . in these reports , it was generally accepted that early recognition and diagnosis of iliopsoas hematoma could reduce mortality . retrospective analysis of this case led to the consideration that bilateral iliopsoas muscle hematoma in this patient with alcoholic lc was caused either spontaneously or by trauma . we further studied cases of iliopsoas muscle hematoma accompanied by lc . during our search of japanese and english articles previous published in medical journals , we found 7 other cases ( table 2 ) [ 5 , 7 , 8 , 9 , 10 , 11 , 12 ] . the median age of these patients including the present case was 58 years ( range 4862 ) and all patients were male . a combination of hepatitis c- and alcohol - related lc and lc of unknown etiology were observed in 1 each out of 8 cases . this high rate of alcoholic lc was believed to be associated with hemorrhagic tendencies due to dysfunctional coagulation with lc as well as dysfunctional hemostasis due to fragile vascular walls caused by alcohol . huang et al . also reported that the overall incidence of spontaneous intracranial hemorrhage in lc was related to the etiology of lc , and the incidence was 6 times higher in alcohol - related lc than in virus - related lc . it seems likely that a common risk factor , namely alcohol , plays an important role in the promotion of non - variceal hemorrhage in lc patients . several actions of alcohol may promote hemorrhage , including the inhibition of platelet adhesion to fibrinogen , dose - related ethanol suppression of platelet aggregation induced by extravasation and the promotion of atherosclerosis . symptoms were frequently reported to include acute groin pain and femoral neuropathy . in our analysis of the previous literature , general fatigue and low back pain were often observed as a primary symptom ( 50 and 37.5% , respectively ) ( table 2 ) . as for the trigger of developing iliopsoas muscle hematoma in lc patients , 6 of 8 cases ( 75% ) revealed spontaneity and the others ( 25% ) contusion . however , case 1 was properly diagnosed with iliopsoas muscle hematoma only after getting the autopsy findings . treatment options include conservative therapy ( such as clotting factor replacement ) , transcatheter arterial embolization ( tae ) or hematoma removal . in this case , we had no choice but to perform the emergent operation without being able to obtain a ct image with contrast due to the patient 's renal dysfunction . even during the operation , iliac artery aneurysm could not be confirmed ; however , we proceeded with aortic bifemoral y - grafting as rupture of aneurysm could still not be excluded . as for the treatment strategy of iliopsoas muscle hematoma with lc , there is no standard therapy ; however , a treatment plan should be carefully determined on a case - by - case basis . because of the serious underlying condition , 5 of the 8 reported cases ( 62.5% ) led to in - hospital deaths . in this case , we could not reach a definitive diagnosis of iliopsoas muscle hematoma on admission . considering that this patient was a poor surgical risk case , conservative therapy such as tae . reported that 19 of 26 patients ( 73% ) recovered following tae in anticoagulant - related rectus abdominis and iliopsoas hematoma . however , an unsuccessful case of tae was also reported in an unsuited patient . urgent liver transplantation , which was reported by yamamoto et al . , might be a treatment choice in the future . taking into account its extremely high mortality rate , iliopsoas muscle hematoma should be recognized as a rare but clinically important bleeding complication of lc patients , especially in cases of alcoholic lc . in conclusion , iliopsoas muscle hematoma in patients with lc is rare but should not be overlooked , and an appropriate treatment plan should be determined on a case - by - case basis despite its poor prognosis .

iliopsoas muscle hematoma in a patient with alcoholic liver cirrhosis is rarely seen , however it has a high mortality . thus we should cautiously make a diagnosis and treatment . this is the case of a 60-year - old male . he had a 15-year history of alcoholic liver disease and emphysema . he presented with low back pain after a fall that had happened 2 months before . due to persistent back pain , he went to see a local physician who , after detailed examination , suspected rupture of bilateral common iliac artery aneurysms and transferred the patient to our hospital . the same presumptive diagnosis was made , and on this basis , an aortic bifemoral y - graft was implanted . he developed aspiration pneumonia and hepatic and renal dysfunction postoperatively , which led to multiple organ failure and subsequent in - hospital death on postoperative day 62 . this was believed to be a case of iliopsoas muscle hematoma developed in a patient with liver cirrhosis , and considering it was a case with poor surgical risk , a conservative treatment option such as transcatheter arterial embolization should also have been considered . although iliopsoas muscle hematoma with alcoholic liver cirrhosis is rare , an appropriate treatment plan should be determined on a case - by - case basis despite its poor prognosis .

Introduction Case Report Discussion Disclosure Statement

the maxillary sinus is the first of the paranasal sinuses to develop , and its growth ends with the eruption of the third molars at approximately 20 years of age.1 the inferior sinus wall is a curved structure formed by the lower third of the medial wall and the buccoalveolar wall,2 and the floor is formed by the alveolar process of the maxilla . the sinus floor extends between adjacent teeth or individual roots , creating elevations in the antral surface , commonly referred to as hillocks.4 the roots of the maxillary premolar , molar and occasionally canine teeth may project into the maxillary sinus.5 because of the implications this can have on surgical procedures , it is essential for clinicians to be aware of the exact relationship between the apical roots of the maxillary teeth and the maxillary sinus floor . wehrbein and diedrich6 described a positive correlation between the length of root projection into the maxillary sinus as observed on panoramic radiographs and the amount of pneumatization that occurs after extraction . sinus expansion following extraction can greatly decrease the amount of bone height available for implant placement.1 a periapical or periodontal infection of the upper premolars and molars may spread beyond the confines of the supporting dental tissue into the maxillary sinus , causing sinusitis.3,7,8 endodontic therapy or extraction of these teeth can result in penetration,9 oroantral fistulae or root displacement into the sinus cavity.10 the relationship between the dental roots and the inferior sinus wall is known to influence orthodontic tooth movement,11 and the intrusion or bodily movement of teeth across the sinus floor that occurs with orthodontic treatment has been shown to cause moderate apical root resorption and a high degree of tipping.6 the aim of this study was to assess the relationship between the maxillary sinus floor and the apices of the maxillary teeth roots using dental cone - beam ct . the study material comprised dental conebeam ct images ( imtec imaging , ardmore , ok , us ) taken from 92 patients obtained from the archives of a private dentomaxillofacial radiology center . of these , 50 ( 54.3% ) were female and 42 ( 45.7% ) were male , with a mean age of 38.815.3 years ( range : 1066 years ) . in total , 87 dentulous right maxillary sinus regions and 89 dentulous left maxillary sinus regions were studied . lines were drawn on the cross - sectional images between the deepest point of the maxillary sinus floor and the root tips of the maxillary first and second premolars and first , second and third molars , and the distances were measured using built - in measurement tools . images were grouped according to the relation between the root tips and the maxillary sinus floor , as follows : group 1 : root tips in contact with the sinus floor ( figure 1 ) ; root tips penetrating into the sinus ( figure 2 ) ; and group 3 : root tips below the sinus floor ( figure 3 ) . root tips in group 1 were numbered as zero , those in group 2 were given negative numbers and those in group 3 were given positive numbers . means , standard deviations and minimum and maximum values were calculated for all right and left premolars and molars . t - tests were used to compare measurements between left and right sides and between female and male patients . means , standard deviations and minimum and maximum values obtained from right and left premolars and molars are given in tables 1 and 2 . the distance between sinus floor and root tip was longest for the first premolar root tip and shortest for the second molar buccodistal root tip for both right and left sides . no statistically significant differences were found between the measurements for right and left sides ( p>.05 ) or between female and male patients ( p>.05 ) . totally , for the right side 60% of the root tips were included in group 3 , 30% in group 1 and 10% in group 2 whereas on the left side 68% were included in group 3 , 21% in group 1 and 11% in group 2 . our study documents the anatomical relationship between the root tips of the maxillary posterior teeth and the maxillary sinus floor . both left and clinicians conducting preprosthetic and preimplant surgical procedures in the posterior maxilla must be aware of the degree to which teeth roots protrude into the sinus because of the associated risk of post - extraction pneumatization,6 which reduces the amount of bone available at the implant or denture site . in most cases , only a panoramic radiograph is available to the clinician , and only a few clinicians order a cone - beam ct.12 in one study that aimed to compare the relationship of maxillary teeth roots to the bottom of the maxillary sinus , panoramic radiographs and cts of 30 patients were examined . the topographic relationships between the roots and sinus as measured using panoramic radiographs were significantly different from those measured from cts , which were taken an average of 2.5 months after the radiographs . in the panoramic radiographs , 64 out of 129 roots appeared to penetrate the maxillary sinus , as compared to 37 of 129 roots in the ct transversal slices.13 in order to avoid disadvantages such as superposition of anatomic structures , horizontal and vertical magnification and a lack of cross - sectional information that are associated with panoramic radiographs , the present study was conducted using cone - beam ct images only . in a study that measured mean distances between maxillary posterior teeth apices and the maxillary sinus floor and between the apices and the adjacent lateral bony surfaces using ct display data from 12 autopsy specimens and 38 human subjects , the apex of the maxillary second molar mesiobuccal root was found to be closest to the sinus floor ( mean : 1.97 mm ) and farthest from the buccal bony surface ( mean : 4.45 mm ) , whereas the apex of the maxillary first premolar buccal root was found to be closest to the adjacent lateral bony surface ( mean : 1.63 mm ) and farthest from the sinus floor ( mean : 7.05 mm).14 the present study also found the first premolar root tip to be farthest and the second molar buccodistal root tip to be closest to the sinus floor on both right and left sides . another study by kwak et al15 that used ct images and described 5 vertical relationships and 3 horizontal relationships found the most frequent vertical relationship to be one in which teeth roots had no contact with the sinus floor . similarly , in the present study the most frequent vertical relationship was found to be one in which roots had no contact with sinus floor for both right and left sides . because of the very close anatomical relationship that exists between the maxillary posterior teeth root tips and the sinus floor , endodontic surgery of premolars and molars can result in accidental oroantral communication1618 that can allow bacteria from infected periapical tissue , resected root tips , or bony drilling dust to be displaced into the sinus and cause acute or chronic sinusitis.18 surgical treatment of posterior teeth is also complicated by the restricted space of the oral vestibular region , which in turn makes it difficult to raise a flap.19 various authors have studied the relationship between the maxillary molar and premolar roots and the maxillary sinus.3,4,14,20 eberhardt et al14 found the mean distance between the maxillary posterior teeth and the maxillary sinus floor to be 1.97 mm . the roots of the maxillary first and second molars have been shown to be in an intimate relationship with the maxillary sinus floor in 40 percent of cases.20 the palatine roots have been shown to be closer to the antral floor than to the palate and in close proximity to the maxillary sinus in 20 percent of cases.4 this location complicates a surgical approach through the sinus , so that palatinal access is usually required.14 although the vestibular roots of the upper posterior teeth are also in close contact with the maxillary sinus floor , vestibular roots are much easier to access than palatinal roots , so that treatment can usually be carried out without perforating the sinus wall.3 in some cases , the root apices protrude into the sinus so that treatment requires raising the sinus membrane.21 as with other apicoectomies , complications encountered during periapical surgery of the maxillary molars and premolars can include damage to a neighboring tooth . with regard to the specific treatment of maxillary molars and premolars , careful aperture of the maxillary sinus wall or floor is necessary , and attention must be paid to avoid sinus membrane perforation and the introduction of foreign bodies into the maxillary sinus.18,20,22,23 ericson et al6 reported that out of 159 maxillary premolars and molars treated with periapical surgery , aperture of the wall or floor of the maxillary sinus occurred in 18 percent of cases . the authors also stated that the introduction of foreign bodies into the maxillary sinus during surgery could cause thickening of the sinus mucosa and symptoms of maxillary sinusitis . in order to avoid penetration by foreign bodies , jerome and hill24 friedman et al25 performed periapical surgery on 94 maxillary teeth roots , 12 of which were maxillary molar roots , and reported an 11.8 percent rate of aperture of the sinus wall or floor . according to selden,26 pathological exposure of the maxillary sinus floor during periapical surgery predisposes to orosinusal communications . regarding sinus membrane perforation , persson23 performed periapical surgery on 18 maxillary molars , with a perforation rate of 44 percent . despite this complication , the reported surgical success rate was 78 percent , and no relationship was observed between membrane perforation and surgical outcome . ioannides and borstlap17 performed surgery on 47 maxillary molars , with a perforation rate of 14.8 percent . according to these authors , perforation of the membrane did not affect the formation of periapical bone . knowledge of the anatomical relationship between the maxillary sinus floor and the maxillary posterior teeth root tips is important for the preoperative treatment planning of maxillary posterior teeth . in view of the proximity of the maxillary sinus floor and maxillary root tips , clinicians must be particularly cautious when performing dental procedures involving the maxillary posterior teeth .

objectives : the aim of the present study was to assess the relationship between the maxillary sinus floor and the maxillary posterior teeth root tips using dental cone - beam ct.methods:a total of 87 right and 89 left maxillary sinus regions from 92 patients were examined using dental cone - beam ct . images were analyzed by a specialist in oral and maxillofacial radiology . perpendicular lines were drawn on the cross - sectional images between the deepest point of the maxillary sinus floor and the root tips of the maxillary first and second premolars and first , second and third molars , and the distances were measured using built - in measurement tools . means , standard deviations and minimum and maximum values were calculated for all right and left premolars and molars . t - tests were used to compare measurements between left and right sides and between female and male patients.results:the distance between sinus floor and root tip was longest for the first premolar root tip and shortest for the second molar buccodistal root tip for both right and left sides . no statistically significant differences were found between the right and left side measurements or between female and male patients ( p>.05).conclusions : knowledge of the anatomical relationship between the maxillary sinus floor and the maxillary posterior teeth root tips is important for the preoperative treatment planning of maxillary posterior teeth .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

acute lung injury ( ali ) and its more severe form , acute respiratory distress syndrome ( ards ) , can arise after many types of injury to the lung , including sepsis , mechanical and chemical injury and bacterial and viral infections 1 . in ali , the mortality rate is in the range 2030% , with about 55% of the cases progressing to ards within a few days . ards causes significant morbidity and approximately 40% mortality , resulting in 75 000 deaths / year in the usa alone 2 . in the past two decades , five emerging viruses have been known to cause significant ards - related mortality , including influenza h1n1 2009 and , in particular , the highly pathogenic avian influenza h5n1 and h7n9 viruses and the sars and mers coronaviruses . in this review human coronavirus ( cov ) infections have traditionally caused a low percentage of annual upper and lower respiratory infections 3 , including severe disease outcomes in the elderly , immunocompromised patients and infants . hcov - oc43 ( oc43 ) and hcov-229e ( 229e ) were the first documented human covs but , more recently , hcov - nl63 ( nl63 ) 4 and hcov - hku1 ( hku1 ) 5 were identified as a consequence of increased viral surveillance efforts in the early twenty - first century ( table 1 ) . these four viruses usually cause acute infection of the upper respiratory tract and less frequently are associated with lower respiratory tract 6,7 diseases as well . severe disease is both rare and typically associated with co - morbidities and/or immunosenescence . the past 15 years have also seen the emergence of two new human coronaviruses that cause significant disease and mortality . sars - cov was identified in 2003 and caused an acute , atypical pneumonia and diffuse alveolar damage ( dad ) in roughly 8000 patients 8,9 . those over 65 years of age often developed ards , resulting in mortality rates that exceeded 50% . overall , sars - cov infection caused nearly 800 fatalities , representing a nearly 10% mortality rate . more recently , in 2012 , a new human coronavirus , designated mers - cov , was identified . mers - cov continues to circulate in camels and humans , with over 857 official cases and 334 deaths , representing an approximately 35% case fatality rate to date in humans 10,11 . mers - cov - induced disease is particularly severe in aged patients and those with pre - existing co - morbidities . human coronaviruses , their receptors , emergence , disease and infection data sars - cov and mers - cov have clear zoonotic origins , although their exact paths from animal reservoir to human infection are not yet clear . viruses with high nucleotide identity to sars - cov were found in key amplifying hosts such as palm civets and raccoon dogs in guangdong province china during the 20022003 sars epidemic 12 . later studies identified highly conserved viruses circulating in horseshoe bats , including some strains that are able to bind to , and infect , human cells 1316 . the existence of novel bat sars - like coronaviruses that also use bat , civet and human angiotensin 1 converting enzyme 2 ( ace2 ) receptors for entry , such as sars - cov , strongly suggests an opportunity for further zoonotic disease outbreaks in human and animal populations . sars causes an atypical pneumonia characterized by cough , fever and infiltrates with a ground - glass appearance on x - ray 17,18 . early - stage disease was characterized by acute dad , with oedema , fibrin and hyaline membranes in the alveolar spaces , typical of ali 19 . other patients predominantly showed an acute fibrinous and organizing pneumonia pattern or a mixture of the two patterns 20,21 . longer - term disease courses typically progressed to organizing phase dad and eventual deposition of fibrous tissue . autopsy of fatal sars - cov cases also revealed denuded airways , haemorrhage and increased macrophage populations in the lung 22,23 . during the sars epidemic , researchers noted that late - term disease progression was unrelated to viraemia but was more likely to be associated with immunopathological damage 24 . mers - cov has caused sporadic infections , along with several local outbreaks throughout the middle east since its discovery in 2012 25,26 . although much remains unknown , closely related viruses have been isolated from camels 27 and highly homologous mers - like bat covs have been identified in african neoromicia capensis bats 28 . local surveillance efforts have detected high levels of antibodies that recognize mers - cov in dromedary camels 29 ; furthermore , sampling of archived camel serum samples has revealed mers antibodies from as early as 1992 30 . these data suggest that bat to camel to human transmission routes may have seeded the 2012 outbreak in human populations , perhaps associated with the expanding camel trade that has emerged between equatorial africa and saudi arabia over the past 20 years . animal models of human disease should recapitulate many of the pathological and immune outcomes seen in human infections . numerous models have been established to better enable our understanding of the mechanics of sars - cov infection and pathogenesis , although few recapitulate the human disease phenotypes ( table 2 ) . initial studies utilized late epidemic strains in non - human primates 3133 , where mild to severe disease was observed , depending on the study location and animal age . to date , the differences in disease severity noted in primates have not been reconciled , but may reflect differences in virus strains or infection conditions . although still under development , mers - cov replication and disease have been reported in both rhesus macaques and common marmosets 34,35 . sars - cov replication resulted in limited disease in young models of immunocompetent mice 3638 ; however , mild clinical disease was noted in 1 year - old mice 39 . a mouse - adapted sars ( ma - sars ) strain was also developed that provides a model for moderate to lethal disease , depending on infectious dose , animal age and genetic background of the host 4042 ( table 2 ) . the ma - sars model faithfully replicates the age - dependent susceptibility observed in human patients , as well as key features of human lung pathology , including virus tropism to airway epithelial cells and type ii pneumocytes , pneumonia , hyaline membrane formation , development of dad and denudation of airway epithelial cells 40,43 . a limitation may be the rapid clearance of virus titres that is seen in younger and , to a much lesser extent , in aged animals . development of the ma - sars model has allowed for in - depth studies of viral pathogenesis and the host immune response , taking advantage of immunological tools and reagents for the mouse as well as the existence of knockout mouse strains . use of these tools has greatly added to our understanding of sars - cov pathogenesis , far beyond what could be learned in in vitro experiments or observational studies of human cases . because of receptor incompatibilities , mers - cov does not replicate in mice unless the animals are first transduced with adenovirus vectors encoding the receptor for entry , human dipeptidyl peptidase-4 ( dpp4 ) 44 . non - human primate and mouse models of sars - cov and mers - cov infection ; less common models include hamster 145 , ferret 146 and cat in this review we focus solely on hcov interactions within the context of the respiratory system and infection of relevant cell types . more specifically , we review some cov host interactions that alter cell - intrinsic antiviral defence programmes and other host pathways that contribute to pathological findings of ards , with its associated exudative and organizing phase diffuse alveolar damage and pulmonary fibrosis . nf-b signalling is an important component of numerous cellular responses , including stress , cytokine signalling , response to bacterial or viral infection and apoptosis 45,46 . the sars - cov envelope ( e ) protein stimulates nf-b signalling 47 , leading to lung cytokine signalling and inflammatory cell recruitment . the sars - cov papain - like protease ( plp ) has also been shown to antagonize nf-b signalling 48 in vitro . chemical inhibitors of nf-b signalling reduce lung pathology and inflammation following ma - sars infection , demonstrating the importance of this pathway 47 in pathogenesis . while the sars - cov e protein is not required for viral replication , it is important for inhibition of the host cellular stress response , apoptosis and unfolded protein response 49,50 . the e protein , along with the sars - cov orf3a and orf8a proteins , has ion channel activity 50 and may contribute to vascular permeability and fluid accumulation in the lung following sars - cov infection . sars - cov lacking e has been shown to be an effective vaccine 51,52 and a mers - cov clone lacking e has been generated 53 , although replication requires expression of e in trans . sars - cov , and to a greater extent mers - cov , are highly sensitive to interferon treatment in cell culture . interestingly , sars - cov pathogenesis does not significantly change in various type i interferon ( ifn ) knockout mouse models , except for a slight increase in overall virus titres 5456 . despite this , stat1- and myd88-deficient mice are significantly more vulnerable to lethal outcomes following infection 56,57 . like many viruses , covs encode a suite of genes that antagonize cell - intrinsic innate immune defence programmes in the infected host cell ( reviewed in 58 ) . numerous in vitro studies have demonstrated the ifn antagonist activity of both sars - cov and mers - cov proteins 5961 , and a detailed review of sars - cov evasion of the innate immune response was recently published by totura and baric 62 . analysis of ifn - stimulated gene ( isg ) expression in calu-3 human airway epithelial cells highlighted the ability of sars - cov and mers - cov to avoid detection by the host 63 . as compared with influenza a viruses , isg transcripts and proteins are not induced until late after sars - cov and mers - cov infection , when peak titres have already occurred in culture ( 1824 h ) . late in infection , isgs showed nearly universally increased expression following sars - cov infection , except for ace2 and serping1 . however , a much larger subset of isgs had significantly decreased expression following mers - cov infection . like mers - cov , h5n1 vn1203 infection also resulted in significant down - regulation of subsets of isgs . no consistent pattern in up - regulation or down - regulation of gene expression correlated with transcription factor usage , suggesting that a novel mechanism may be responsible for expression of the isg subsets . cells infected with mers - cov and h5n1 avian influenza were shown to have specifically altered open and closed chromatin structures , potentially limiting the ability of transcription factors to access and bind certain isg promoter regions . the mechanism by which mers - cov induces this chromatin structural alteration is as yet unknown . in contrast , the ns1 protein of h5n1 was responsible for the chromatin changes in influenza - infected cells . although speculative , it seems likely that many rna viruses may encode strategies to epigenetically alter host chromatin structure , influencing host gene expression . this mechanism may be partially mediated by the production of double - membrane vesicles , which could sequester rna replication intermediates away from the host - sensing machinery 64,65 . mda5 and ifit1 are important host antiviral sensor or antiviral defence isgs that detect viral rnas . ifit1 recognizes unmethylated 2-o rna 66 and alters efficient translation / stability of uncapped viral mrnas 67 . sars - cov and other coronavirus rnas are protected from ifit recognition because they encode a 2-o - methyltransferase ( 2-omt ) activity in the viral replicase protein , nsp16 68,69 . sars - cov is much more sensitive to interferon treatment in the absence of functional nsp16 methyltransferase activity , and mutant viral titres drop rapidly in both infected epithelial cells and mice . deletion or knockdown of either mda5 or ifit1 restored mutant sars - cov viral loads demonstrated the essential role of these host proteins in detecting pathogen - associated molecular patterns . ablation of the 2-omt activity may provide a universal strategy to rationally design live attenuated mutants of contemporary and newly emerging cov . both in vivo and in vitro studies have addressed the role of specific proteins in the innate immune system , often isgs , in sars - cov pathogenesis . transcriptional analysis on autopsy tissue from sars - cov - infected patients revealed increased expression of stat1 along with other ifn - induced cytokines 70 . the sars - cov accessory protein orf6 was identified as an interferon antagonist important for viral replication in low multiplicity of infection ( moi ) in vitro infections 71,72 . orf6 was subsequently found to sequester karyopherin 2 , a nuclear import factor , and block the nuclear translocation of stat1 after sars - cov infection . interestingly , stat1 translocation to the nucleus is not blocked in mers - cov - infected cells , so it remains uncertain whether antagonists of nuclear import are encoded in the viral genome 73 . transcriptional profiling of sars - cov - infected macaques revealed robust ifn signalling , including stat1 translocation to the nucleus , in the lung but not in the cells that stained positive for viral antigen 74 . these data highlight the importance of in vivo studies versus high moi in vitro studies . significantly , they also highlight the need to examine , or at least consider , expressing and signalling differences in specific cell types instead of global transcriptomic studies in those in vivo experiments . stat1 knockout mice have been studied extensively in the context of viral infection , typically showing a heightened susceptibility to disease , due to the lack of a type i ifn response 75 . these knockouts were first tested for sars - cov susceptibility using the tor2 strain in a sublethal model ; animals deficient in stat1 were unable to clear virus from the lung and developed a more severe and longer - lasting pneumonia than the control mice 76 . frieman et al showed that stat1 knockout mice are highly susceptible to infection with ma - sars in a novel , ifn - independent mechanism 56 . ma - sars - infection causes massive inflammatory cell influx in the lungs of stat1 knockout mice , including large numbers of macrophages , neutrophils and eosinophils . stat1 knockout mice have gross pathological changes in their lungs , including massive haemorrhage as well as increased lung size and stiffness . as seen in some humans , these mice develop severe pulmonary fibrosis and succumb to disease at late time points after infection . stained lung sections revealed the presence of collagen protein in alveolar exudates in stat1 knockouts , indicating development of early - stage pulmonary fibrosis . subsequent studies demonstrated that stat1 knockout animals developed a th2-skewed immune response and had significant numbers of alternatively activated or m2 macrophages in their lungs 77 . stat6 is required for the development of alternatively activated macrophages , and stat1/stat6 double knockout mice do not develop the severe lung disease and pro - fibrotic lesions observed in the stat1 single knockout 78 , thus demonstrating that these macrophages are essential for development of the pulmonary fibrosis phenotype . further elegant experiments showed that it is the stat1 deficiency in monocyte / macrophage cells , not the infected epithelial cells , that drives alternatively activated macrophage production and induction of fibrotic lung disease following sars - cov infection . alternatively activated macrophages are typically induced by the th2 cytokines il-4 and il-13 ; they have an anti - inflammatory role and play an important role in wound - healing processes 151 . ace2 is expressed on well - differentiated airway epithelial cells 79 and its expression increases following type i interferon treatment 63 . both ace2 protein levels and rna expression are down - regulated after either in vitro or in vivo sars - cov infection 63,80 and nl63 also down - regulates ace2 expression following in vitro infection 81 . it has previously been reported that ace2 and angiotensin-2 protect mice from sepsis- and acid aspiration - induced ali 82 . additionally , histopathological lung disease worsens when spike - fc is inoculated into mice with ali 80 . the normal function of ace2 is to inactivate angiotensin-2 , a negative regulator of the renin this system controls blood pressure and is involved in the development of pulmonary hypertension and pulmonary fibrosis . the renin multiple genome - wide association studies have investigated an association between genetic variation in ace and susceptibility to ards , with mixed results 86 . the role of dpp4 in mers - cov infection is discussed in detail by haagmans et al in a separate review in this issue 152 . furthermore , it has been suggested that unregulated ifn responses contributed to development of immunopathology and severe disease following sars - cov infection 88 . the data discussed above support this hypothesis and suggest that early control of isg signalling may be a means of preventing or controlling the development of severe lung disease . expression of the isg serping1 is also decreased following sars - cov infection of epithelial cells ; it functions by inhibiting the complement system as well as several proteases in the coagulation pathway . the role of the coagulation , fibrinolysis and wound healing in ards development are discussed below . the alveoli of the lung are where gas exchange occurs , providing oxygen to blood flowing through capillaries in the alveolar membrane . the alveolar walls are composed of type i and type ii pneumocytes , along with alveolar macrophages 89 . type i pneumocytes cover 95% of the alveolar surface area and allow for gas exchange with blood in the capillaries of the lung . type ii pneumocytes are the progenitors of type i pneumocytes and are also responsible for generating pulmonary surfactant 90 , a mixture of lipids and surfactant proteins that is crucial in reducing surface tension in the lung . sars - cov infection causes desquamation of pneumocytes in humans and mice , contributing to alveolar dysfunction , oedema and haemorrhage . alveolar macrophages play an essential role in surveillance of the local environment and inhibit an excessive immune response , although this inhibition can also block an effective response to sars - cov infection 91 . the functions of these cell types are critical in maintaining balance between inflammation , coagulation and wound repair , especially following lung injuries such as viral infection 92 . in ards patients , uncontrolled inflammation , fluid accumulation and developing fibrosis severely compromise gas exchange and lead to respiratory failure . sars - cov and influenza infect type i and type ii pneumocytes in the lung 93,94 . ards patients exhibit decreased surfactant levels 95 and ma - sars infection results in decreased surfactant transcript and protein levels 43 . decreased surfactant , and the consequent increase in surface tension , reduces the ability of the lung to expand and contract during normal respiration respiratory dysfunction occurs when the alveolar membranes are obstructed , or when the ability of the lung to expand and contract , circulating oxygenated air , is compromised . lethal sars - cov infection in the mouse and human is characterized by a breakdown of alveolar membrane integrity , resulting in accumulation of fluid exudates in the alveolar spaces . virus infection also results in an overwhelming cytokine response , severe lung tissue damage and respiratory failure 9699 . the progression from initial disease to diffuse alveolar damage and the exudative and organizing stage of dad is often independent of high - titre viral replication 24 , indicating that this severe disease outcome is primarily driven by an immunopathological response , including inflammatory cell recruitment and viral damage to type ii pneumocytes . this conclusion is further supported by non - human primate and mouse models of sars - cov infection , where lethal disease is more often associated with severe pulmonary lesions , alveolar exudates and respiratory dysfunction than with high viral load 43,100 . ma - sars infection results in peak viral titres at 12 days post - infection , along with airway denudation and resulting debris , which can occlude the small airways . severe lung disease , including inflammatory cell infiltrates , haemorrhage , alveolar oedema and hyaline membrane formation typical of the exudative stage of dad ( figure 1 ) , occurs at days 47 post - infection , when virus loads in the lung are dropping rapidly and/or are below the limit of detection . many isgs stimulated by sars - cov infection are involved in wound - healing responses and thus may contribute to sars - induced ali and ards . ma - sars lung immunopathology . ( a ) mock - infected lung stained with haematoxylin and eosin . ( b ) large airway of a c57bl/6 j ( b6 ) mouse , 7 days post - infection , with 10 plaque - forming units ( pfu ) ma - sars , shows denudation of the epithelial cells . ( c , d ) immunohistochemical staining of the sars - cov n protein at 2 days post - infection shows staining consistent with infection of airway epithelial cells and type ii pneumocytes , respectively . ( e ) msb staining highlights fibrin in the parenchyma of the lung ( red staining ) in b6 mice , 7 days post - infection with 10 pfu ma - sars . ( f ) perivascular cuffing in a b6 mouse , 4 days post - infection with 10 pfu ma - sars . ( g ) hyaline membranes in the parenchyma of the lung of a b6 mouse , 7 days post - infection with 10 pfu ma - sars . ( h ) inflammation in the lung of a b6 mouse , 7 days post - infection with 10 pfu ma - sars . ( i ) haemorrhage in the lung of a serpine1 mouse , 7 days post - infection with 10 pfu ma - sars . model of an infected alveolus in the lung . type i and type ii pneumocytes make up the alveolar walls and resident alveolar macrophages and pulmonary surfactant exist in the airspace ( a ) . in the acute phase of sars - cov infection ( b ) , type i and type ii pneumocytes are infected and secrete inflammatory cytokines , while surfactant levels decrease . during the late stage / tissue damage portion of viral infection , viral titres decrease , while airway debris , pulmonary oedema and hyaline membrane formation all impede respiration ( c ) . while sars - cov evades detection by the host immune system and causes minimal changes in transcript and protein levels for the first 24 h of infection 43 , it ultimately induces a massive signalling response in infected lungs . pro - inflammatory cytokines and chemokines , including il-6 , tnf , il-1 and ccl2 57 , recruit inflammatory cells to the site of infection . neutrophils and cytotoxic t cells , along with these cytokines , can induce tissue damage , including vascular leakage , and stimulate pulmonary fibrosis 101 . pro - fibrotic genes , including tgf1 , ctgf and pdgfa and numerous collagen transcripts , have increased expression following ma - sars infection . fluid exudates , haemorrhage and fibrin are all observed in the alveolar spaces of sars patients , as well as in animal models of disease 17,43,102 , increasing in severity as a function of age . in response , the coagulation cascade is activated , including increased factor 10 ( fx ) , f2 , f3 ( tissue factor ) , f11 , f12 and f7 transcript levels . activation of the coagulation cascade results in f10 cleavage of prothrombin into thrombin and subsequent thrombin cleavage of fibrinogen into fibrin 103 . fibrin clots in the alveoli are a prominent feature of sars - cov infection in humans and mice . the goal of this coagulation response is likely to protect the host by sealing the alveoli , preventing alveolar flooding and haemorrhage , which limit oxygen exchange and endanger patient survival . collagen accumulation , fibrin and fibrin clots all contribute to a developing fibrotic lung state , while at the same time stimulating the infected host to up - regulate fibrinolytic pathways 92 . profibrinolytic genes include members of the urokinase pathway , such as urokinase ( plau ) , tpa and plasmin ( plg ) 104 . the urokinase signalling pathway leads to cleavage and activation of plasmin into plasminogen ; this protease then cleaves fibrin clots . serpine1 and serpine2 are negative regulators of urokinase pathway and inhibit urokinase and tissue plasminogen activator ( tpa ) activity . urokinase signalling is highly active in the absence of serpine1 , and this imbalance often results in haemorrhage in knockout mice . serpine1 is highly expressed in sars patients , non - human primates and small animal models 43,74,105 . ards studies , independent of coronavirus infection , have attributed this serpine1 expression to alveolar macrophages and type ii pneumocytes 106,107 . ma - sars infection in a serpine1 knockout mouse model results in lethal disease with extreme lung pathology 43 . conversely , ma - sars - infected mice with a genetic deficiency in tpa have increased exudates in the lung . the dysregulation of these coagulation / anti - coagulation cascades can result in worsening end - stage lung disease conditions , resulting in death . profibrotic and profibrinolytic signalling are part of the wound - healing response , along with other extracellular matrix ( ecm ) remodelling pathways 101 . sars - cov infection causes massive tissue remodelling through urokinase and coagulation pathways activity , as discussed above . other important wound - healing pathways and ecm proteins with altered signalling following sars - cov infection include matrix metalloproteinases , egfr and collagens 43 . successful recovery from ali requires a delicate balance of pro - inflammatory , profibrotic and profibrinolytic responses . by altering isg expression , including ace2 , stat1 and serpine1 , sars - cov infection of alveolar epithelial cells sets the stage for the development of severe lung disease , including ards . sars and mers patients with severe lung disease exhibited lung consolidation , decreased blood oxygen saturation and often required intubation and ventilation 99,108,109 . small animal models of severe lung disease typically lack physiological readouts of respiratory function that can be directly correlated back to human signs of disease . whole - body plethysmography captures respiratory data in unrestrained animals , allowing for longitudinal measurement of pulmonary function ; these data can also be directly related to some human respiratory metrics 110,111 . sars - cov infection causes increased penh , a calculated measure of airway resistance and increased ef50 ( mid - breath exhalation force ) , indicating that respiratory function is compromised and animals must do more work to breathe 69 . unpublished data ( gralinski and menachery ) indicate that it is the exhalation portion of each breath that is impacted by sars - cov infection , likely due to extensive debris clogging the conducting airways . further experiments have shown that stat1 knockout mice have increased penh levels early after infection , and that at late timepoints they have reduced lung capacity , corresponding with the profibrotic histopathological changes observed in the lung ( gralinski , unpublished data ) . ards is a devastating end - stage lung disease with no cure . despite numerous clinical trials , improved several highly pathogenic emerging virus infections cause ards with high frequency , underscoring the critical public health importance of understanding the virological components and molecular mechanisms that drive this devastating end - stage lung disease . furthermore , a portion of ards cases may progress to pulmonary fibrosis , another clinically devastating end - stage lung disease with few treatment options . consequently , understanding the development of ards following viral infection remains a high - priority research topic that is germane to global health and pandemic disease control . the twenty - first century has demonstrated that zoonotic events will continue to introduce coronaviruses and other viruses into the human population , and that these viruses have the potential to spread rapidly , cause significant disease in communities and disrupt the global economy . an emerging theme is the connectivity between virus infection , complement and coagulation cascade activation , pro - inflammatory and profibrotic cytokine responses and disease severity . more studies are needed to unravel the complex interactions between these pathways that can interact to promote or dysregulate wound recovery after life - threatening respiratory virus infection . in particular there is a need for including well - articulated animal models that faithfully recapitulate disease processes across species . only through a better understanding of the interplay between a dysregulated host immune response and ali and ards

respiratory viruses can cause a wide spectrum of pulmonary diseases , ranging from mild , upper respiratory tract infections to severe and life - threatening lower respiratory tract infections , including the development of acute lung injury ( ali ) and acute respiratory distress syndrome ( ards ) . viral clearance and subsequent recovery from infection require activation of an effective host immune response ; however , many immune effector cells may also cause injury to host tissues . severe acute respiratory syndrome ( sars ) coronavirus and middle east respiratory syndrome ( mers ) coronavirus cause severe infection of the lower respiratory tract , with 10% and 35% overall mortality rates , respectively ; however , > 50% mortality rates are seen in the aged and immunosuppressed populations . while these viruses are susceptible to interferon treatment in vitro , they both encode numerous genes that allow for successful evasion of the host immune system until after high virus titres have been achieved . in this review , we discuss the importance of the innate immune response and the development of lung pathology following human coronavirus infection .

Introduction Innate immune response Acute respiratory distress syndrome (ARDS), coagulation, fibrinolysis and respiratory function Concluding thoughts Author contributions

health information organizations ( hios ) combine prescription information purchased from pharmacies with anonymized patient medical records purchased from health insurance companies to determine which drugs individual physicians prefer for specific diagnoses and patient populations . this information is used to tailor marketing strategies to individual physicians and to assess the effect of promotions on prescribing behavior . the american medical association ( ama ) created the prescription data restriction plan in an attempt to address both the privacy concerns of physicians and industry concerns that legislation could compromise the availability of prescribing data . however , the pdrp only prohibits sales representatives and their immediate supervisors from accessing the most detailed reports . less than 2% of us physicians have registered for the pdrp , and those who have signed up are not the physicians who are targeted for marketing . although it has been argued that prescription tracking benefits public health , data gathered by hios is designed for marketing drugs . these data are sequestered by industry and are not generally available for genuine public health purposes . the ama met with industry and surveyed physicians , finding that although two - thirds of physicians opposed the availability of prescribing data to drug representatives , 77% of those would have their concerns alleviated if doctors could choose to protect their individual prescribing information.3 in response to industry and physician concerns , the ama created the prescription data restriction plan ( pdrp ) . interested physicians visit a website ( http://www.ama-assn.org/go/prescribingdata ) and fill out a form requesting that their prescribing data be withheld from pharmaceutical sales representatives . to activate one s pdrp rights online , a physician must check off or provide a reason for opting out and accept the vaguely ominous statement that , some of the information provided may be shared with entities outside the ama in order to register and/or follow up on your concern . requests expire 3 years from when the physician signs up , and requests may take 6 months to take effect because drug companies are required to check the list quarterly and then have 90 days to restrict sales representatives access to these data.12 a pilot version of the pdrp was launched in california and several other states in may 2006 ; the program officially launched nationally in july 2006.7 few doctors know about the pdrp . soon after the program launched , a poll of 210 physicians found that only 18% were aware of the program ; more than half ( 53% ) were interested in signing up.13 the ama mailed no announcements to physicians because studies have shown that direct mail to physicians is very inefficient , according to the ama s media outreach coordinator,7 who also noted that office staff often intercept physicians mail . instead , the ama placed ads or announcements in ama publications and several medical journals , and e - mailed announcements to about 100,000 physicians , about 1,000 of whom subsequently signed up for the pdrp.7 by july 18 , 2006 , 2,900 of 800,000 practicing physicians had signed up for the pdrp.13 by may 2007 , a year after the program began , about 1% of active physicians ( 7,476 ) doctors had opted out.14 by january 2008 , almost 12,000 doctors ( 65% of whom are not ama members ) have signed up.15 such low participation in the pdrp is predictable because opt - out options are designed to maximize participation while preserving a patina of choice.16 default choices are perceived to be socially preferable , so opt - out options have resulted in increased default participation in situations as diverse as prenatal hiv testing , 401(k ) savings plans , and medical journal reviewing.16 in its current form , the pdrp withholds some information from some industry representatives some of the time . all other employees retain full access to all the individual - specific prescribing information . as an industry article puts it , the information available to the home office will not change , regardless of physician enrollment in pdrp . this program will not affect headquarter business systems.17 the most relevant information is still available to pharmaceutical representatives because the pdrp exempts information on ( a ) deciles at the market or therapeutic class level , ( b ) segmented data that are not likely to reveal the actual or estimated activity of an individual physician , or ( c ) data on products ordered by physicians from pharmaceutical companies.17 in other words , the pdrp does not restrict pharmaceutical companies from seeing , analyzing , and using prescribing data to promote specific drugs to the doctors who have opted out . x is in the top 10% of doctors in terms of prescribing volume for antidepressants ; in the top 20% for antibiotics ; and is an early adopter of drugs . pharmaceutical representatives enjoy full access to drug - specific details on chemotherapy or any other drugs dispensed or administered in physicians offices . an industry article notes that that although the pdrp limits reps and their supervisors from accessing data on some physicians , it nonetheless enable companies to continue to run essential business applications that rely on prescriber data.17 whereas a massive physician sign - up of high prescribers for the pdrp would inconvenience pharmaceutical companies , the opt - outs to date are generally lower - prescribing physicians , with 72% in the market - five decile or lower.3 doctors in the bottom half of prescribers are not targeted for marketing anyway . in 2006 , new hampshire passed a law banning commercial sale of prescribing data and was promptly sued by ims and verispan . on april 30 , 2007 , a federal judge ruled the new hampshire law unconstitutional on the grounds that it restricts commercial free speech.18 the state plans to appeal the decision . other states are considering similar bills , but the decision in new hampshire and the availability of the pdrp has had an effect . a preliminary injunction was granted to prevent enforcement of a maine law ( which is also being appealed ) , and vermont is now modifying a law that it passed.15 the availability of the pdrp was key to a 2006 decision not to reintroduce california assembly bill 262 ( narrowly defeated in 2004 ) , which would have severely restricted the sale and use of prescription data.5 an industry publication explains , pharmaceutical manufacturers need to make the pdrp work , since the alternative , government legislation , is not in anyone s best interest . without pdrp , long - term access to physician - level data within the healthcare system is called into question.5 the ama s pdrp brochure states that the program responsibly honors the opinions of all physicians and provides a more reasonable alternative to a one - size - fits - all legislative ban that ignores vital aspects of prescribing data use.19 the industry - friendly nature of the pdrp is revealed in an article coauthored by ama and ims employees , which states , the rules allow the industry to retain access to prescribing data for most purposes , but they require companies to police their own sales forces . if they succeed , legislators will turn their attention elsewhere , and the industry can hang onto one of its most valuable data sources.17 pharmaceutical interests argue that unrestricted access to prescribing data saves lives , money , and physicians time . the pharmaceutical research and manufacturers of america ( phrma ) has argued that prescriber data are needed to send out communications informing doctors of drug recalls and serious adverse effects.20 however , when the fda orders a recall or mandates a new black box warning on a drug , the manufacturer is usually required to send a access to prescriber data allows pharmaceutical companies to target necessary prescription information to physicians which helps avoid a saturation of less relevant information to a broader physician audience.20 an industry article states that , prescribing data allow pharmaceutical promotion to be relevant and specific , making the whole process more cost - effective ( and sparing physicians from being bombarded with extraneous promotional materials and sales calls) .17 certainly , these data enable the identification of physicians relevant and specific to drug sales . sales calls and promotional materials are preferentially showered on high - prescribing physicians and others who affect market share.1 the ama , in testimony opposing legislation to restrict industry access to prescriber data , states that , this information is critical to improving the quality , safety , and efficacy of providing patient care through evidence - based medical research.21 the ama is misguided on this point . researchers do not need hio data , which are too expensive for most individual researchers to purchase anyway.4 ( ims has occasionally provided limited data to some researchers without cost or at discount.20 anecdotally , free data are older data and do not contain dates of service or other information routinely provided to paying customers . ) although hios claim that the fda relies on their data , the use of hio data by the fda for public health purposes is trivial . according to ims 2006 annual report , sales to the pharmaceutical industry accounted for substantially all of our revenue in 2006 , 2005 , and 2004.22 it bears noting that hios are not the only source of prescription data . medicare , medicaid , the military , the veteran s administration , and large health maintenance organizations ( hmos ) all have databases of medical and prescription information that have been extensively used by health services researchers . the inclusion of race and ethnicity data makes medicaid ideal for researching health outcomes in diverse populations ( medicaid covers 1 in 5 americans , including 1 in 3 african americans and 1 in 4 latinos).23 government data may be the most reliable for research purposes . one recent analysis found prescription data available from the center for medicaid and medicare services ( cms ) superior to medicaid data available from a commercial vendor.24 the coverage of outpatient prescription drugs through medicare part d will create a trove of prescription data on elders for researchers . hios package physicians names with prescriptions and patient medical records to facilitate pharmaceutical marketing efforts to manipulate individual prescribing habits . there is no obligation for hios to share information with government regulators even when such information affects public health . for example , sluggish sales of a migraine drug in 2003 led an unnamed company to hire ims health to determine why.25 ims researchers hypothesized that lower - than - forecasted sales of the migraine drug could be because of decreased menopausal hormone use . millions of women stopped taking menopausal hormones in mid-2002 when the women s health initiative showed that risks outweighed benefits for the most popular estrogen progestin regimen . ims examined the medical records of 41,403 women with migraines and found that those who stopped hormone therapy halved their use of migraine drug prescriptions . women filled an average of 2.94 prescriptions for migraine medication during a 6-month period on hormone therapy compared with an average of 1.49 prescriptions during a 6-month period after they stopped hormones . more than half of the women ( 54% ) filled no migraine drug prescriptions in the 6 months after quitting hormones.25 this very large study , which definitively linked migraines and hormone therapy , was never shared with physicians nor published in the medical literature . it was mentioned , in a publication directed exclusively at pharmaceutical marketers , as an example of the services hios provide to pharmaceutical companies . pharmaceutical companies are required by law to report adverse events associated with their products to the fda . the commercial sale and use of prescriptions and medical records compromises the privacy of individual prescribers and patients , is not necessary for health services research , and may well have adverse effects on public health . the purpose of prescription tracking is to promote the use of the newest , most expensive drugs , a practice that threatens rational prescribing . assessment of the benefits and harms of medications should be a government function , implemented by researchers without commercial ties , and conducted solely in the public health interest . public monies should be allocated to integrate existing public sources of health data and expand accessibility to researchers who will use these data in the interests of patients rather than industry profits .

introductionmonitoring and modifying physicians prescribing behavior through prescription tracking is integral to pharmaceutical marketing . health information organizations ( hios ) combine prescription information purchased from pharmacies with anonymized patient medical records purchased from health insurance companies to determine which drugs individual physicians prefer for specific diagnoses and patient populations . this information is used to tailor marketing strategies to individual physicians and to assess the effect of promotions on prescribing behavior.discussionthe american medical association ( ama ) created the prescription data restriction plan in an attempt to address both the privacy concerns of physicians and industry concerns that legislation could compromise the availability of prescribing data . however , the pdrp only prohibits sales representatives and their immediate supervisors from accessing the most detailed reports . less than 2% of us physicians have registered for the pdrp , and those who have signed up are not the physicians who are targeted for marketing.conclusionalthough it has been argued that prescription tracking benefits public health , data gathered by hios is designed for marketing drugs . these data are sequestered by industry and are not generally available for genuine public health purposes .

INTRODUCTION DISCUSSION CONCLUSION THE PDRP AN ALTERNATIVE TO LAW RISKS AND BENEFITS OF RX TRACKING

a self - expandable metal stent ( sems ) is an efficient and established tool for solution of biliary obstruction due to both benign and malignant diseases . meanwhile , there have been some reports regarding adverse events by a sems : migration , dislocation , ulceration , perforation of the bowel and so on.1 we sometimes encounter the case with kinking of the common bile duct ( cbd ) by a sems which is caused by inappropriate length or strong axial force of a sems and necessary for appropriate coping , for example , exchange of a sems or addition of another sems . a double - pigtail plastic stent is less costly than a sems and frequently used for decompression of the bile duct of patients with poor prognosis or undergoing heavy particle radiotherapy or proton therapy . furthermore , recently , a double - pigtail plastic stent is also feasible for endoscopic ultrasonography - guided2,3 or transpapillary gallbladder drainage.2 we present a rare case with ampullary carcinoma treated by a sems and additional stenting of a double - pigtail plastic stent for correction of kinking of the cbd . a 72-year - old man with jaundice was referred to our hospital . a contrast - enhanced computed tomography ( ct ) scan showed a 15-mm , weakly enhancing mass at the ampulla of vater ( fig . 1 ) and dilatation of the bile duct . biopsy of the mucosa indicated adenocarcinoma . for biliary decompression , a transpapillary , fully covered , self - expandable metal stent ( fcsems ) ( 1050 mm bonastent ; standard sci - tech , seoul , korea ) was deployed . because stent obstruction or migration was suspected , endoscopic carbon dioxide cholangiography was performed , which revealed kinking of the cbd 1 cm above the proximal end of the fcsems ( fig . a 7-f double - pigtail plastic stent ( 100 mm ; olympus medical systems , tokyo , japan ) was therefore placed through the fcsems to correct the kink , straightening the cbd ( fig . this is the first report of a unique use of a double - pigtail plastic stent to correct cbd kinking . it is reported that placement of a metal stent across the main duodenal papilla can predispose to cholangitis4 which is mainly caused by food impaction in a stent or reflux of duodenal contents to the bile duct . in this case , however , acute cholangitis was cured after the correction of cbd kinking , which indicates that cholangitis was caused by cbd kinking , not by placing a stent across the ampulla of vater . cbd kinking can occur by inappropriate placement of a sems or a large - bore diameter plastic stent5 with strong axial force . nakai et al.6 recommends a new method of sems stenting to reduce early stent - related complications including kinking of the bile duct by longer stent placement with the center of the stent located in the center of the biliary stricture . however , the new method was impossible in this case , because the site of the biliary stricture was located at the ampulla . there has been one report by park et al.7 in which a double - pigtail plastic stent with a fcsems was used for anchoring . they focused on the pigtail shape and revealed that it could help to prevent fcsems migration . meanwhile , we expected the correction of cbd kinking from the stent shaft in addition to anchoring . a use of a double - pigtail plastic stent can correct cbd kinking without exchange or addition of another sems and can save cost .

a 72-year - old man with jaundice by ampullary adenocarcinoma was treated at our hospital . for biliary decompression , a transpapillary , fully covered , self - expandable metal stent ( fcsems ) was deployed . four days later , the patient developed acute cholangitis . endoscopic carbon dioxide cholangiography revealed kinking of the common bile duct above the proximal end of the fcsems . a 7-f double - pigtail plastic stent was therefore placed through the fcsems to correct the kink , straightening the common bile duct ( cbd ) and improving cholangitis . this is the first report of a unique use of a double - pigtail plastic stent to correct cbd kinking . the placement of a double - pigtail plastic stent can correct cbd kinking , without requiring replacement or addition of a fcsems , and can lead to cost savings .

INTRODUCTION CASE REPORT DISCUSSION

patients with ra have systemic inflammation , as well as increased morbidity and mortality from cardiovascular disease [ 1 , 2 ] . various risk factors have been identified that contribute to atherosclerosis in ra patients [ 3 , 4 ] . recent years have seen increased attention devoted to inflammation associated insulin resistance . increasing numbers of research studies toll - like receptors ( tlrs ) , as key molecular components of the innate immune system , are of a central interest in innate system associated insulin resistance . the predominant site of tlrs expression is on cells of the innate system , particularly on monocytes . the tlrs recognize numerous ligands ; for example , tlr2 and tlr4 were hypothesized to recognize components of the bacterial cell wall such as peptidoglycan and lipopolysaccharide , respectively , and to interact with lipid - containing molecules . tlrs participate in the pathogenesis of insulin resistance in animal models [ 12 , 13 ] and mediate vascular inflammation and insulin resistance in diet - induced obesity . additionally , tlrs can link innate immunity and fatty acid - induced insulin resistance . activation of tlrs in adipocyte has been implicated in the onset of insulin resistance in obesity and type-2 diabetes . high glucose can induce tlr2 and tlr4 expression , activity , and inflammation via nuclear factor ( nf)-b . dasu and jialal further reported that free fatty acids in the presence of high glucose may amplify monocyte inflammation through tlrs . saturated fatty acid may serve as a ligand for several members of the tlr family [ 19 , 20 ] . furthermore , tlr2 , following ligation with specific ligands , can activate signal transduction , promote interleukin ( il)-6 production , and mediate initial events related to fatty acid - induced insulin resistance in muscle . tumor necrosis factor - alpha ( tnf- ) stimulation was also suggested to disrupt insulin signal transduction and induce insulin resistance . additionally , the inflammatory kinase i - kappa - b kinase- ( ik ) contributes to insulin resistance by activating nf-b and induces production of various inflammatory cytokines , including tnf- and il-6 [ 23 , 24 ] . although numerous studies have demonstrated that tlr2- and tlr4-dependent signaling are involved in the development of insulin resistance , data on a similar mechanism in the pathogenesis of insulin resistance in ra patients are still lacking . tlrs activation has been described as being involved in the pathogenesis of ra , and both tlr2 and tlr4 are potentially important receptors in the initiation and perpetuation of the inflammatory cycle in arthritis . tlrs are present on tissue synoviocytes and blood monocytes which are recruited to the site of inflammation and involved in the pathogenesis of synovial inflammation [ 2629 ] . thus , tlrs - inducing inflammatory cascades potentially can contribute to the pathogenesis of insulin resistance in ra patients . however , the evidence of the relationship between tlrs and insulin resistance in ra patients remains rare . this investigation tests the hypothesis that expressions of tlr2 or tlr4 on monocytes and related inflammatory cytokines , such as tnf- and il-6 , might be associated with insulin resistance in patients with ra . tlr2 still plays a role in the development of insulin resistance in patients with ra even in the absence of hyperglycemia , which is a well - known risk factor for metabolic syndrome . the study population included 30 consecutive ra patients that fulfilled the american college of rheumatology ( acr ) 1987 classification criteria and 10 healthy volunteers . patients or normal controls with diabetes mellitus were excluded . the study was in agreement with the guidelines approved by the human research ethics committee at our hospital . demographic data , clinical characteristics , and current medications of the patients were recorded by two independent observers . overnight fasting blood samples were taken to determine blood glucose , serum insulin level , triglyceride ( tg ) , and cholesterol profiles including total cholesterol , ldl cholesterol , and hdl cholesterol . high sensitivity c - reactive protein and westergren erythrocyte sedimentation rates were determined at the e - da hospital clinical laboratory . ra disease activity was measured using the disease activity score in 28 joints . to measure the insulin resistance , the homoeostasis model assessment ( homa ) , as described by matthews et al . , was calculated using the formula : ( [ fasting plasma glucose ( mmol / l ) fasting plasma insulin ( u / ml)]/22.5 ) . monocyte staining for tlr2 and tlr4 expression was performed on the whole blood of ra patients and healthy controls before and stimulation with peptidoglycan from staphylococcus aureus ( 10 g / ml , sigma - aldrich , mo , usa ) or lipopolysaccharide from e. coli o26 : b6 ( 10 ng / ml , sigma - aldrich ) as ligands for tlr2 and tlr4 . whole blood specimens were incubated with antibodies against tlr2 ( ebioscience , ca , usa ) or tlr4 ( pe labeled , biolegend ) and anti - cd14 conjugated with fitc ( bd biosciences , ca , usa ) for 30 min at room temperature in the polystyrene round - bottom falcon tube ( bd biosciences ) . two ml working 1x bd facstm lysing solution was added to the reaction tube for 10 min at room temperature to lysis red blood cells , and the excess unbound antibody was then washed with pbs ( phosphate - buffer saline ) . cells were finally resuspended in pbs and analyzed with a flow cytometer ( becton - dickinson immunocytometry systems , san jose , ca , usa ) by counting 10,000 cells . expressions of tlrs were calculated as mean fluorescence intensity ( mfi ) and percentage of cd14 monocytes expressing tlr2 or tlr4 using winmdi98 software ( bd biosciences ) . whole blood samples were collected from both patients with ra and healthy volunteers via venipuncture into heparin containing tubes and diluted with 1x hbss buffer at a 1 : 1 ratio . the blood samples were incubated in the presence of 5% co2 at 37c in 24-well plates using 10 g / ml peptidoglycan or 1 ng / ml lipopolysaccharide or medium alone . the cells were then pelleted via centrifugation ( 400 g for 2 min ) , and the cell - free supernatants were stored at 70c for cytokine determination . concentrations of il-6 and tnf- of cell supernatants were determined using elisa kits ( bender medsystems , ca , usa ) . all analyses were performed using the spss statistical software ( version 15.0 ; spss inc . , chi - square test or fisher 's exact test , when necessary , was used for categorical values and wilcoxon 's rank sum tests for continuous variables . a total of 30 patients with ra ( 25 females and 5 males ) aged 36 to 82 years were enrolled and 10 healthy controls aged 51 to 64 years were also recruited . demographic characteristics , lipid profiles , cardiovascular risk factors , and the homa index for ra patients and normal controls are presented in table 1 . age , sex , systolic blood pressure , diastolic blood pressure , total cholesterol , low - density lipoprotein , high - density lipoprotein , triglycerides , bmi , and blood glucose between groups showed no significant differences . homa index was significantly higher in ra patients than in healthy subjects ( ra patients versus healthy subjects p = 0.017 , figure 1 ) . eighty - three percent of ra patients are current users of corticosteroid , but the cumulative dose of steroid showed no significant correlation with insulin resistance of these patients ( r = 0.081 , p = 0.675 ) . the datum suggests that perhaps better control of inflammation after receiving treatment with steroid may counterbalance the deleterious effect of corticosteroids on glucose metabolism . additionally , as would be expected , hscrp ( high sensitivity c - reactive protein ) and esr ( erythrocyte sedimentation rate ) revealed significantly higher concentrations in ra patients compared with normal controls ( p = 0.008 and p = 0.002 , resp . ) . homa index was significantly correlated with waist ( r = 0.381 , p = 0.038 ) , bmi ( r = 0.374 , p = 0.042 ) , tg ( r = 0.444 , p = 0.014 ) , and ratio of tg to hdl cholesterol ( r = 0.423 , p = 0.020 ) in nondiabetic ra patients ( figure 2 ) . however , homa index was not correlated with total cholesterol ( r = 0.076 , p = 0.689 ) and ldl cholesterol ( r = 0.093 , p = 0.626 ) in ra patients . these results imply that monitoring traditional risk factors might also be important in managing insulin resistance in ra patients , even in the absence of hyperglycemia . as shown in figure 3 , homa index was significantly correlated with tlr2 expression ( calculated as percentage of cd14 monocytes expressing tlr2 ) after stimulation with 10 g / ml of peptidoglycan ( r = 0.514 , p = 0.009 , figure 3(a ) ) , but not with tlr2 expressions before stimulation by peptidoglycan ( r = 0.387 , p = 0.056 ) . in contrast , homa index was not significantly correlated with tlr2 expression in monocytes after stimulation with 10 g / ml of peptidoglycan in normal controls ( r = 0.150 , p = 0.682 , figure 3(b ) ) . the experimental results revealed no significant correlation between homa index and tlr4 expression in monocytes before ( r = 0.288 , p = 0.162 ) or stimulation with 10 ng / ml lipopolysaccharide ( r = 0.230 , p = 0.268 , figure 3(c ) ) . as shown in figure 4 , homa index significantly correlated with fold increase of tlr2 expression ( calculated as mfi ) in monocytes of ra patients after stimulation with 10 g / ml peptidoglycan ( r = 0.441 , p = 0.027 , figure 4(a ) ) , while failing to reveal significant correlation in normal controls ( r = 0.249 , p = 0.492 , figure 4(b ) ) . additionally , there was no significant correlation between homa index and tlr4 expression ( calculated as mfi ) after stimulation with 10 ng / ml lipopolysaccharide ( r = 0.057 , p = 0.787 , figure 4(c ) ) . as shown in figure 5 , tlr2 expression ( calculated as percentage of cd14 monocytes expressing tlr2 ) significantly correlated with tnf- concentration following stimulation with 10 g / ml of peptidoglycan ( r = 0.484 , p = 0.014 ) , while failing to reveal significant correlation in normal controls ( r = 0.469 , p = 0.172 ) . in addition , homa index was significantly correlated with concentration of tnf- ( r = 0.433 , p = 0.019 ) in ra patients . as shown in figure 6 , tlr2 expression ( calculated as percentage of cd14 monocytes expressing tlr2 ) was significantly correlated with il-6 concentration following stimulation with 10 g / ml of peptidoglycan ( r = 0.611 , p = 0.001 ) in ra patients , but not significant in healthy volunteers ( r = 0.449 , p = 0.193 ) . in addition , homa index was significantly correlated with concentration of il-6 ( r = 0.468 , p = 0.009 ) in ra patients . the major finding of the present study is that tlr2 expression , after stimulation with peptidoglycan , showed significant correlation with insulin resistance in patients with ra in the absence of hyperglycemia . ra is one of the most prevalent autoimmune diseases and affects about 0.51% of the adult population . patients with ra have increased morbidity and mortality from cardiovascular disease such as atherosclerosis compared to patients without ra . both systemic inflammation and insulin resistance insulin resistance was considered to contribute to the increased cardiovascular risk in the general population [ 33 , 34 ] . inflammation has been identified as fundamental in insulin resistance in patients with ra [ 5 , 35 ] . the inflammatory pathways can be integrated to cause insulin resistance by activating membrane receptors such as tlrs [ 12 , 36 , 37 ] . tlrs are critical in the recognition of invading pathogens and activation of subsequent immune responses against them . upon stimulation , tlrs induce the activation of nf-b and mitogen - activated protein kinases ( mapk ) and the expression of inflammatory cytokines [ 36 , 38 ] . palmitate treatment of differentiated c2c12 myotubes , through tlr2 activation , led to a time - dependent inhibition of insulin - activated signal transduction . the inhibition of tlr2 expression can rescue cells from the activation of mapk8 and improve insulin resistance . tlr2 is crucial for diet - induced metabolic syndrome because mice lacking tlr2 are substantially protected from diet - induced adiposity and insulin resistance . additionally , polymorphisms in the tlr2 receptor gene have been linked to populations at high risk of developing type 2 diabetes [ 41 , 42 ] . overall , tlr2 could be a key modulator between inflammatory pathways and metabolic disorders such as insulin resistance . obesity can induce increased c - jun n - terminal kinase ( jnk ) activity [ 44 , 45 ] which is activated in response to inflammatory cytokines , free fatty acids , activated nf-b , and inflammatory mediators , including tnf- and il-6 , and may contribute to insulin resistance [ 23 , 24 ] . furthermore , raised free fatty acids and tg in obese individuals and animals can be important etiologies of insulin resistance . high glucose induces inflammatory cytokines , chemokines , p38 mapk , nf-b activity [ 4752 ] , and tlr2 expression . recently , dasu and jialal further indicated that free fatty acids in the presence of high glucose amplify monocyte inflammation via tlrs . collectively , tlrs inducing inflammatory pathway may contribute significantly to insulin resistance , particularly in the presence of obesity related conditions or high blood sugar . however , rare data exist describing the roles of tlrs in the development of insulin resistance in the absence of raised blood sugar in ra patients . the present study found that tlr2 expression in circulating cd14 monocytes , after stimulation with peptidoglycan , correlated significantly with homa index in nondiabetic ra patients . additionally , the expression of tlr2 , upon stimulation with peptidoglycan , was correlated with levels of tnf- and il-6 . homa index was also significantly correlated with concentration of il-6 and tnf- in ra patients . these data implied that tlr2 could contribute to the development of insulin resistance in patients with ra without concurrent hyperglycemia . additionally , the significant correlations of the homa index with tg and ratio of tg to hdl in ra patients are consistent with the previous literature . waist circumstance and bmi , as parameters measured while evaluating obesity , displayed a significant correlation with homa index in this study . control of obesity thus may also be critical in treating ra patients at a high risk of insulin resistance . further study is required to elucidate the detailed roles of peptidoglycan or other tlr2 ligands in the occurrence of insulin resistance . this study indicates that body mass index , waist , tg , and ratio of tg to hdl were significantly associated with homa index in nondiabetic ra patients . these findings are consistent with previous investigations showing that insulin resistance is associated with low hdl cholesterol and high tg in patients with inflammatory arthritis [ 53 , 54 ] . therefore , the so - called traditional risk factors also need to be closely monitored and carefully treated . the results demonstrating the close relationship between homa index and tlr2 expression in monocytes and inflammatory cytokines such as tnf- and il-6 could provide therapeutic interventions against insulin resistance in nondiabetic ra patients . treating traditional factors such as bmi , waist , tg , and ratio of tg to hdl cholesterol is crucial in minimizing the development of insulin resistance in ra patients , even in the absence of hyperglycemia . this study may lead to a better understanding of the relationship between tlr2 expression and insulin resistance in patients with ra .

the close relationship between increased tlr-2 expression in blood monocytes and insulin resistance in ra patients is shown in this study . traditional risk factors for metabolic disorders , including the waist circumstance , body mass index ( bmi ) , triglyceride ( tg ) , and ratio of tg to high density lipoprotein ( hdl ) cholesterol , were closely correlated with homa ( homoeostasis model assessment ) index in patients with nondiabetic ra . expressions of tlr2 in peripheral blood monocytes , following stimulation with peptidoglycan which is known as a tlr2 agonist , were closely correlated with the homa index , tnf- , and il-6 concentrations . accordingly , tlr-2 receptor and its related inflammatory cytokines could be potential therapeutic targets in managing insulin resistance in ra patients .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

clinical and epidemiological studies have identified several factors that increase the risk of coronary heart disease and heart attack . these factors include gender , ageing , heredity , smoking , hypercholesterolemia , hypertension , physical inactivity , obesity and overweight , and diabetes mellitus . risk factors often occur in clusters and may build on one another , such as obesity leading to diabetes and elevated blood pressure . when grouped together , as in the metabolic syndrome , these factors lead to an even greater risk of coronary artery disease . special attention has been given to the effect of psychological stress , oral contraceptives , excessive alcohol intake , sleep apnea , elevated c - reactive protein levels , fibrinogen , homocysteine and lipoprotein and , finally the presence of an oxidative stress . sies has defined oxidative stress as an imbalance between oxidants ( e.g. , free radical species derived from oxygen ) and antioxidants in favour of the oxidants , leading to a disruption of redox signalling and/or molecular damage . a large number of studies have indicated that oxidative damages to proteins , lipids or dna resulting from an increased production of reactive oxygen species ( ros ) from pathological processes or external origins are involved in the development of cardiovascular diseases and cancer . to regulate ros production and fight against their deleterious effect , the organism responds with a large and complex battery of antioxidants including enzymes , proteins , iron chelators , low molecular weight compounds , trace elements , and antioxidants arising from our diet ; among them , vitamins c and e , carotenoids and polyphenols are particularly important . many epidemiological and clinical studies have also shown that the lower the antioxidant status , the higher the risk of developing cardiovascular diseases and cancer [ 313 ] . a few prospective , large - scale , european studies have shown a negative correlation between vitamin c plasma levels and all - cause or cardiovascular mortality . plasma vitamin c levels below the normal range 48.8 mg / l ( or 2350 m ) were found to double the risk of developing cardiovascular disease and cancer [ 1418 ] . for -carotene , the upper reference limit was estimated to be 0.22 mg / l ( or 0.4 m ) . factors like male gender , age , race , body mass index ( bmi ) , smoking , alcohol consumption , triacylglycerol concentration , and inadequate dietary antioxidant intakes contribute to lower plasma levels of antioxidants [ 1928 ] . to our knowledge , so far no study did investigate how lifestyle behaviours such as smoking , physical activity , and diet alone or in association contribute to reach critical vitamin c ( < 6 mg / l or 34.2 m ) and -carotene ( < 0.22 mg / l or 0.4 m ) levels . currently , an increasing number of general practitioners strive to assess the antioxidant status of their patients for prevention purposes . in this respect this prompted us to undertake a wide epidemiological study based on a sample of the belgian population . elan ( etude ligeoise sur les antioxydants ) is a cross - sectional epidemiological study conducted from march through july 2006 as a joint project between the university of lige , the university hospital of lige , and the public health services of the province of lige ( belgium ) . the province of lige , one of the 10 belgian provinces , has 1.040.297 inhabitants for a geographic area of 3862 km . a stratified random sample of 55 general practitioners working in the province was selected as follows : 21 ( 38% ) in urban environment , 15 ( 27% ) in semiurban , and 19 ( 35% ) in rural living environment . each physician was asked to recruit in his / her practice 20 presumably healthy volunteers aged 4060 years . exclusion criteria included intake of antioxidant supplementation and previous history of cardiovascular diseases , diabetesm or cancer . a total of 897 eligible subjects were finally enrolled in the study : 349 ( 39% ) men and 548 ( 61% ) women . the day before the examination visit , subjects fasted for at least 12 h and were not allowed to drink fruit juice and to perform physical activity . the day of the visit , information including age , height , weight , consumption of fruits and vegetable by means of a home - made questionnaire , and intake of alcohol and oral contraceptive pills was collected . the body mass index ( bmi ) smoking ( yes / no ) and physical activity ( inactive or active 2 - 3 times a week ) were also recorded . social status was classified in 7 categories , as follows : professionals ( i ) , managerial and technical occupations ( ii ) , manual ( iiia ) and nonmanual skilled workers ( iiib ) , partly skilled workers ( iv ) , retired ( va ) , and unemployed ( vb ) . all contacted volunteers received written information about the goal of the study and signed an informed consent form prior to enrolment . they were immediately centrifuged on site and plasma was frozen as aliquots on ice packs coming from a 80c freezer and placed in a refrigerating box . for the assay of vitamin c , 0.5 ml plasma was immediately transferred to ice - cold tubes containing 0.5 ml of 10% metaphosphoric acid . analyses were performed on the day of blood collection by a spectrophotometric method using the reduction of 2,6-dichlorophenolindophenol ( perkin elmer lambda 40 norwalk , usa , sensitivity : 2 mg / l , inter- and intra - cv : 4 and 6% ) . mg / l , inter- and intra - cv : 5.35 and 10.73% ) was determined by hplc procedure ( alliance waters , usa ) coupled with a diode array detector ( pda 2996 , waters , usa ) . both vitamin c and -carotene were analyzed in a routine way . independently of gender , our reference values , as published earlier on a population of 128 healthy subjects , were 6.218.8 mg / l ( 35.3107.1 m ) for vitamin c and 0.050.62 mg / l ( 0.091.15 m ) for -carotene [ 31 , 32 ] . results were expressed as means standard deviation ( sd ) for quantitative variables , while frequencies and proportions ( % ) were used for categorical variables . mean values between groups were compared by one - way analysis of variance or the kruskal - wallis nonparametric method if normality assumptions could not be fulfilled . correlation coefficients ( classical or spearman ) were calculated for measuring the association between two quantitative variables . logistic regression analysis was used to predict vitamin c and -carotene deficiency from risk factors . the association between deficiency and risk factors was measured by the odds ratio ( or ) and its 95% confidence interval . calculations were always carried out on the maximum number of data available . missing data were not replaced . results were considered to be significant at the 5% critical level ( p < .05 ) . data analysis was carried out using sas ( version 9.1 for windows ) and s - plus ( version 9.0 ) statistical packages . the gender - specific demographic , biometric , and clinical characteristics of the study population are given in table 1 . in the elan population , respectively 27% of women and 25% of men were smokers . height , weight , bmi , and both systolic and diastolic blood pressures were significantly higher in men than in women . the dietary intake of vitamin c and -carotene ( mg / day ) derived from fruits and vegetables consumption generally was significantly lower in men than in women ( p < .0001 ) . table 2 displays vitamin c and -carotene mean levels with respect to subject 's characteristics given in table 1 . as expected , vitamin c and -carotene values were lower by , respectively , 15 and 32% in men when compared to women ( p < .0001 ) . smokers had significantly decreased vitamin c and -carotene levels by 19% ( p < .0001 ) and 39% by contrast , regular physical activity was associated with a mean increase of 9% for vitamin c ( p < .0001 ) and 25% for -carotene ( p a positive relationship was demonstrated between the amount of daily fruit intake and both vitamin c and -carotene levels . the latter were , respectively , reduced by 27% and 46% ( p < .0001 ) in non consumers when compared to people eating 2 - 3 fruits / day or more . the group apple - pear - grapes - banana , kiwi , citrus fruits , orange , and all kinds of red fruits and strawberry had a positive influence on vitamin c. in contrast , none of the vegetables except endives had a positive effect on vitamin c ( data not shown ) . with respect to -carotene , apricot , the group apple - pear - grapes - banana , kiwi , citrus , mango , and orange contributed to improve the plasma level of -carotene but only asparagus , carrot , and tomato for vegetables ( data not shown ) . women taking oral contraceptive had significantly lower -carotene levels but vitamin c levels were unchanged . no relevant effect of blood pressure , intestinal disorders , environment , and age ( < and > 50 years ) has been evidenced on the plasma level of both studied antioxidants . figure 1 depicts the distribution of plasma vitamin c and -carotene levels in men and women . only 2.1% of the subjects had higher values than the upper reference value ( 18.8 mg / l ) . if a large majority ( 81.5% ) of participants were within the recommended values in vitamin c ( 6.218.8 mg / l ) , it should be noted that 16.4% of them were , however , either in a subdeficiency ( 10.3% ) or deficiency ( 6.1% ) status with respect to the cutoff value of 6 mg / l ( 34.2 m ) . as far as the -carotene is concerned , a small portion of the study subjects ( 7.0% ) had higher concentration in -carotene than the upper normal value ( 0.68 mg / l or 1.23 m ) and more than 90% of the elan cohort had a plasma -carotene concentration within the conventional and usual values ( 0.050.68 mg it is worth noting that 46.7% of the studied population had plasma values below the critical point of 0.22 mg / l ( 0.4 m ) . by logistic regression analysis applied to vitamin c data ( table 3 ) , it was found that male gender ( or = 1.70 ; p = .011 ) , smoking ( or = 2.84 ; p < .0001 ) , lack of physical activity ( or = 2.05 ; p = .0015 ) , and intake of less than 2 fruits / day ( or = 2.96 ; p = .0003 ) were significant risk factors of vitamin c deficiency . overweight ( or = 1.18 ; p = .42 ) and oc use for women ( or = 1.24 ; p = .61 ) were not significant . when adding the social status , the risk of vitamin c was significantly increased ( or = 1.34 ; p = .0044 ) , when being unemployed compared to the other professional categories . for -carotene deficiency , logistic regression analysis showed that male gender ( or = 2.67 ; p < .0001 ) , smoking ( or = 3.03 ; p < .0001 ) , overweight ( or = 2.28 ; p < .0001 ) , lack of physical activity ( or = 1.51 ; p = .0091 ) , intake of < 2 fruits / day ( or = 1.41 ; p = .045 ) , and oc use for women ( or = 4.67 ; p < table 4 examines the probability of vitamin c and -carotene deficiency ( i.e. , lower than the critical values ) with respect to an increasing number of risk factors . healthy habits consisting in no smoking , practising a regular physical activity , and eating at least two fruits per day resulted in a weak probability ( less than 5% ) to have plasma vitamin c below 6 by contrast , poor healthy living habits ( smoking , no physical activity , nonconsumption of fruits , and overweight ) significantly increased this probability up to 46.2% for men against 33.5% for women . when accounting for unemployment , these probabilities raised to 66.3% for men and 44.3% for women , respectively . it should also be noted that men were always associated with higher probabilities of vitamin c levels below 6 mg / l than women ( except those of social class iv and va ) smoking , practising no physical activity , and eating none fruits regardless of the combination of lifestyle behaviours . for -carotene , healthy habits ( nonsmoking , practising sport , and eating two or one fruits per day ) resulted in 13.7% and 29.7% of chance to get critical level in -carotene for women and men , respectively . smoking by itself significantly increased this probability but in a higher extent in men ( 56.1% ) than in women ( 32.4% ) . the combination of smoking , absence of regular physical activity , and any intake of fruits contributed to a high probability in men ( 73.2% ) and to a less extent in women ( 50.5% ) . the addition of overweight resulted in an increase of about 20% in women ( 69.9% ) and of only 13% in men ( 86.1% ) . finally , the worst situation ( 91.6% ) was observed for women by adding the intake of oral contraceptive pills . it should be noted that for both genders , all probabilities were largely higher than those derived for vitamin c risk of deficiency . the reference values for vitamin c established by our group are comprised between 6.2 mg / l ( 35.3 m ) and 18.8 mg / l ( 107.1 m ) . this is in agreement with other studies performed in different european populations [ 20 , 35 ] . in the work of rutkowski and grzegorczyk , ten ranges of concentrations given by medical handbooks and textbooks have been compared in detail with 15 parallel ranges taken from scientific papers , paying attention to their significant discrepancies . based on source values and basic statistical calculations , a reliable mean range of vitamin c normal concentrations " in blood plasma has been obtained : 6.314 mg / l ( 36.179.4 m ) . according to le grusse and watier , the critical range of accepted marginal vitamin c deficiency was between 3.5 and 6.2 mg / l ( 20 m35 m ) . for -carotene , we found the following normal range 0.050.62 mg / l ( 0.091.15 m ) which is in good agreement with the study of olmedilla et al . performed in five western european populations . with respect to gender , our mean values in antioxidants were in perfect agreement with those found in the reference french suvimax study ( vitamin c : men : 8.8 4.0 mg / l ; women : 10.6 5.5 mg / l ; p < .0001 ; -carotene : men : 0.22 0.16 mg / l ; women : 0.31 0.20 mg / l ; p < .0001 ) [ 20 , 38 ] . fruits and to a less extent vegetables are the primary dietary sources of both antioxidants . as confirmed in table 1 , it is recognized that women have dietary intakes richer in vitamin c and -carotene than men due to a higher intake of fruit and vegetables . as expected , our data clearly indicate that smoking was associated with a significant decrease of the mean plasma vitamin c ( 19% ) and -carotene ( 39% ) after adjustment for gender and all demographic variables described in table 1 . our observations were in good agreement with other reports [ 26 , 27 ] and , more particularly , the suvimax study performed on 3128 french men and women aged 3560 years . cigarette smoke contains a large number of free radicals species able to induce an oxidative stress on both the respiratory and circulatory systems with as consequence greater antioxidant depletion . after adjustment for all covariates , we also evidenced that a regular physical activity contributes to improve the vitamin c and -carotene levels . a simple explanation could be given by the fact that active people eat more fruits than inactive ones . southeast showing a significant association between fruit and vegetable consumption and physical activity ( p < we also evidenced a positive relationship between the frequency of fruit and the mean plasma level of vitamin c and -carotene . when compared to high consumers , people eating any fruit were characterized by plasma concentrations of both antioxidants which are extremely closed to the normal inferior value . by contrast , we were unable to evidence an association between antioxidant biomarkers and the consumption of vegetables . moreover , we also evidenced that only the intake of carrot and tomato among vegetables may significantly influence to a higher extent the plasma level of -carotene whilst a larger range of fruits were able to do it . it could be also possible that -carotene is a better predictor than -carotene as suggested in the european prospective investigation into cancer and nutrition ( epic study ) performed on a stratified random subsample of 3089 men and women . ideal bmi is the range of 2025 kg / m while a bmi of over 25 kg / m and 30 kg / m is , respectively associated with overweight and obesity . as described earlier [ 41 , 42 ] , we confirmed that a bmi > 25 kg / m resulted in a significant decrease by 31% of the plasma -carotene level . the use of oral contraceptives had a deep negative impact on the plasma level of -carotene . it has been speculated that estrogens induce an activation of the retinol binding protein , hence possibly increasing the conversion of -carotene into retinol . we have also shown that women taking oral contraceptives had higher oxidative damages to lipids than the others . it could be assumed that part of the antioxidant defences were more solicited in women using oral contraception to limit deleterious damages . neither blood pressure nor intestinal disorders ( which could explain a decrease in antioxidants due to possible malabsorption ) or environmental parameters had an influence on the mean plasma level of both antioxidants . based on large - scale epidemiological studies , it is now accepted that an alteration of antioxidant defences was significantly implicated in the development of several pathologies . however , if some variations of the mean plasma values may be evidenced with respect to lifestyle behaviours in all studies , no indication was ever given about the biological interpretation of these variations . therefore , the establishment of reference or usual values for antioxidants markers such vitamin c and -carotene is needed to detect abnormalities . as explained above , determination of normal ranges for both antioxidants has been achieved on a separate population of 128 healthy persons as earlier published [ 3133 ] . based on our reference values , we were able to detect that 16.4% of the whole elan population had non optimal plasma concentration in vitamin c ( < 6 mg / l or 34.2 m ) against 46.7% for -carotene ( < 0.22 mg / l or 0.4 m ) . at the light of the following but not exhaustive studies , the evidence of such abnormalities could therefore be of primordial interest as a preventive tool for health [ 44 , 45 ] . recently , langlois et al . proposed that plasma vitamin c should be considered as a predictor of cardiovascular disease in addition to being a classical nutritional biomarker . based on a large number of studies including the famous monica study [ 14 , 18 ] performed on 14 european populations , plasma cutoff levels ( 4.47.0 mg / l or 2540 m ) in vitamin c have been proposed , above which the risk for apparent cardiovascular events should decrease . in haemodialysis patients , the cutoff value of 5.66 mg / l ( 32 m ) was predictive of the appearance of adverse cardiovascular outcomes . in patients with peripheral arterial disease , the cutoff value of 4.9 mg / l ( 28 m ) was associated with increased levels of inflammation parameters . recently , myint et al . described in the european prospective investigation into cancer ( epic)-norfolk population that the relative risks for risk of stroke diminished inversely to the quartile of plasma vitamin c concentration as follows : 1.0 ( < 41 m or 7.27 mg / l ) , 0.83 ( 4153 m or 7.279.32 mg / l ) , 0.63 ( 5465 m or 9.5 or 11.44 mg / l ) , and 0.57 ( > 66 m or 11.6 , it was interesting to highlight that there was a continuous relation with mortality through the whole distribution of ascorbic acid concentrations . when compared to 3.66 mg / l , each 3.52 mg / l ( 20 m ) rise in plasma ascorbic acid concentration was associated with about a 20% reduction in risk of all - cause mortality ( p < .0001 ) , regardless of age , systolic blood pressure , blood cholesterol , cigarette smoking habit , diabetes , and supplement use . for -carotene , gey established that a plasma value below 0.22 mg / l ( 0.4 m ) was associated with an increased risk of developing cardiovascular diseases and cancer . among the elan population , 16.4% of the subjects presented a plasma vitamin c below the cutoff value of 6 mg / l ( 34.2 m ) and 46.7% a concentration in -carotene below the critical point of 0.22 mg / l ( 0.4 m ) . the availability of a statistical model for predicting the probability of getting a plasma value below the cutoff values of 6 mg / l ( 34.2 m ) for vitamin c and 0.22 mg / l ( 0.4 m ) for -carotene could be of interest for health prevention . cumulative probabilities were therefore presented instead of odd ratios to stress on the cumulative effect of the risk factors on low levels in both antioxidants . after adjustment for all covariates between them , table 3 clearly indicates that using such values rather than mean plasma value afforded better indications about the relationship between antioxidant biomarkers and lifestyle behaviours . a good base of healthy life could be nonsmoking , regular physical activity and eating more than 3 fruits / day . this resulted in a small probability to have inadequate plasma level of vitamin c. smoking and nonphysical activity significantly but moderately contributed to increase this probability for vitamin c for both genders . in contrast , the nonconsumption of fruits added to the parameters above produced a dramatic increase which was more pronounced in men ( 42.1% ) than in women ( 30% only for those belonging to social classes i , ii , iiia , and iiib ) . this suggests that the frequncy of fruit intake appears to be one of the most important regulators of the plasma concentration of vitamin c [ 50 , 51 ] with a more pronounced effect in men ( 65.3% ) having partial or no working activity . economical difficulties to buy fruits linked to a precarious social status can explain this last observation . as observed with the mean plasma values , no effect of bmi , intestinal disorders , and environment could be evidenced on the probability to get low plasma level in vitamin c. with respect to -carotene , it was quite interesting to note that despite a healthy way of life there was a significant risk ( 29.7% in men and 13.7% in women ) to get a value below 0.22 mg / l ( 0.4 m ) . malabsorption of this antioxidant could be a rationale explanation although we did not find any influence of intestinal disorders on the plasma level of -carotene . when compared to basal level and according to the different steps described in table , smoking contributes to a mean increase of 27% for men and 18% for women of getting critical plasma value . this is not surprising since the negative impact of smoking on antioxidant is well known . lack of physical activity was associated with a further but moderate increase of around 9% . this was less pronounced than those observed for vitamin c confirming that the latter is a better biomarker than -carotene of fruits intake . kg / m and oral contraceptives in women , significantly contributed to finally reach a high probability ( 86.1% for men and 91.6% for women ) to detect nonoptimal plasma value in -carotene . although representative of the province of lige , the study sample may not be considered as a national probability sample of the belgian population . moreover , we only focused our attention on people in the age range of 4060 years since we considered that lifestyle behaviours were well anchored in this population . it is clear that ageing ( > 60 years ) could also contribute to decrease the plasma level in antioxidants so that our observations in the elan population could be modified if taking this parameter in account . we only distinguished nonsmokers from both past and current smokers , this last one category being , however , in a large majority in the elan population . further , no question has been addressed with respect to environmental tobacco smoker which could possibly have a negative influence on plasma antioxidants . during our study , we also met some difficulties to integrate data about alcohol consumption due to a large underestimation of intake made by participants . as this parameter has been shown to reduce the level of plasma -carotene , it could partially explain the relatively high probability of getting a value below 0.22 mg / l ( 0.4 m ) even when observing optimal lifestyle behaviours ( no smoking , physical activity and eating more than 2 days a day ) . except for oral contraceptive , the influence of other drugs intake was not taken into consideration . to the best of our knowledge , the present study is the first one to address the relationship between plasma antioxidants and lifestyle behaviours in a belgian population . we have demonstrated that smoking regular physical activity and eating fruits were directly associated with the modulation of the mean plasma concentration of both vitamin c and -carotene . however , such variations , if well described in the literature , always remained within the normal or usual range of concentration . by using cutoff values associated with increased risk of developing cardiovascular diseases and cancer ( vitamin c < 0.22 mg / l or 0.4 m ) , we described how lifestyle factors alone or associated contribute to lower plasma concentrations . however , as vitamin c and -carotene are unstable constituents when not protected against air and light , a rigorous preanalytical sample handling and treatment ( immediate centrifugation , plasma precipitation and keeping the sample at 80c until analysis ) is required to interpret the data correctly . the elan ( etude ligeoise sur les antioxydants ) study was conducted from march through july 2006 as a joint project between the university of lige , the university hospital of lige , and the local health services of the province of lige ( belgium ) . sc j pincemail ( credec and dept of cardiovascular surgery ) were the main coordinators of the elan study . professor c charlier and professor jp chapelle ( laboratories of clinical biology ) allowed the analysis of -carotene while vitamin c determination was performed by mr jp cheramy - bien ( credec ) . g collette ( dept of general medicine ) allowed the recruitment of all general practitioners around the province of lige , belgium . sc s vanbelle ( dpt of medical informatics and biostatistics ) were involved in the statistical analysis of all data . all investigators critically revised the manuscript for the intellectual content and gave their final approval of the version to be published .

several factors , including fruit and vegetables intakes , have been shown to significantly influence the plasma concentrations of the two antioxidants vitamin c and -carotene . deficiency levels of 6 mg / l ( 34.2 m ) for vitamin c and of 0.22 mg / l ( 0.4 m ) for -carotene have been suggested below which cardiovascular risk might be increased . the present study performed on 897 presumably healthy subjects aged 4060 years aimed to examine how modifiable lifestyle factors may be related to vitamin c and/or -carotene deficiency . gender , smoking , lack of regular physical activity and of daily fruit consumption ( 2/day ) , and social status ( in particular , unemployment ) were found to be significant risk factors for vitamin c deficiency . for -carotene deficiency , the same factors were identified except social status ; moreover , overweight and oc use in women were also found to have a deleterious effect . for non exposed subjects , the probability of developing vitamin c deficiency was 4% in men and 2.4% in women . this probability increased to 66.3% for men and to 44.3% for women ( and even to 50.4% under oc use ) , when all risk factors were present . for -carotene deficiency , the corresponding probabilities were equal to 29.7% in men and 13.7% in women ( no risk factor present ) , and to 86.1% for men and 69.9% ( 91.6% for oc use ) for women ( all factors present ) , respectively .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusions Authors' Contribution

with advances in high - resolution ultrasound and other diagnostic techniques , minimally invasive foetoscopic procedures , also called foetendo procedures , are being carried out more frequently . foetoscopic laser ablation ( fla ) involves minimal insult to uterus , while obtaining access to foetus through a single atraumatic trocar insertion . twin pregnancy with shared monochoronic placenta may be complicated with twin - to - twin transfusion syndrome ( ttts ) and twin reversed arterial perfusion syndrome ( trap ) . in ttts , arteriovenous vascular communications occur in a shared monochorionic placenta of the twins . there is an imbalance in the blood flow between the developing foetuses . donor twin. in trap a twin with absent or non - functioning heart , called this results in overloading the heart in pump twin , resulting in polycythaemia , hydrops foetalis and congestive cardiac failure . interruption of abnormal vascular communications of placental vessel by laser or radiofrequency coagulation , . is the treatment of choice for ttts and trap . the anaesthetic concerns for foetoscopic procedures are those related to anaesthetising any pregnant patient for surgery such as preventing supine hypotension syndrome and maintaining uteroplacental blood flow , preventing unnecessary exposure of developing foetus to anaesthetic drugs . they also decrease the possibility of causing premature labour , bleeding and premature rupture of membranes . foetal anaesthesia is not required for trap and ttts as there is no foetal surgery involved . we present a retrospective analysis of anaesthetic management of 41 foetoscopic procedures , for ttts and trap done in our institution . we carried out a retrospective analysis of patients who had undergone foetoscopic procedures in our hospital from 2010 to 2015 . , 37 patients underwent fla of placental vessels for severe ttts and four patients for trap . all patients were evaluated preoperatively for any associated comorbidities such as pregnancy - induced hypertension , gestational diabetes , hypothyroidism , heart disease , bronchial asthma and anaemia . pre - operative laboratory investigations included complete blood count , serum creatinine and blood glucose . all patients had received antacid prophylaxis with injection ranitidine 50 mg intravenously along with injection ondansetron 4 mg intravenously 1 h before surgery . all patients were explained about the surgical and anaesthetic procedure , and a written informed consent was obtained . in all patients , 5 mg nitroglycerine patch anaesthetic technique consisted of spinal anaesthesia in most patients with bupivacaine 0.5% , or ropivacaine 0.75% , 2 3 ml given intrathecally with 27-gauge whitacre spinal needle , in the left lateral position . in some patients with expected prolonged procedure , combined spinal - epidural ( cse ) procedure was performed . an infusion of 0.5% bupivacaine was started epidurally , at the rate of 6 - 8 ml / h when patients started complaining of slight pain due to wearing off of spinal effect . some patients under spinal or cse analgesia requested for sedation and were sedated with midazolam 2 mg and fentanyl 25 g . patients who refused spinal anaesthesia , and patients whose spinal anaesthesia effect wore off due to prolongation of procedure , were given general anaesthesia with propofol 2 mg / kg , fentanyl 2 g / kg and atracurium 0.5 mg / kg with endotracheal intubation . anaesthesia was maintained with oxygen - air with sevoflurane 2% through a circle system at low flow rate of 1l / min . patients were monitored with electrocardiogram , oxygen saturation and non - invasive blood pressure and end - tidal co2 . at the end of the procedure , the effect of muscle relaxant was reversed with neostigmine 2.5 mg and glycopyrrolate 0.4 mg . hypotension was treated with phenylephrine 100 g or ephedrine 6 mg bolus doses ; nitroglycerine patch was continued postoperatively and renewed every 24 h for tocolysis for next 2448 h. no other tocolytic agent was used . a total of 41 american society of anesthesiologists grade 1 and 2 patients underwent fla procedures . of these , thirty seven patients were for ttts and four patients were for trap procedure [ table 1 ] . all patients were comfortable and tolerated the procedure well . among 41 patients , 34 patients received spinal anaesthesia , three patients received cse block ; three patients received general anaesthesia and one patient received sedation with local anaesthetic infiltration of abdominal wall . two patients with subarachnoid block required conversion to general anaesthesia because of prolongation of procedure [ table 2 ] . the mean age of pregnant mothers was 28.95 4.54 years ( range 2136 years ) . of these , seven patients had gestational diabetes , three patients were hypothyroid , one patient had chronic hypertension , one patient was anaemic and one patient was known asthmatic . mean gestational age at which patients underwent foetoscopic procedure was 22.70 3.1 weeks ( range 1629 weeks of pregnancy ) . thirty - four patients were given bupivacaine 0.5% and the mean volume used was 2.43 0.32 ml ( range 23 ml ) . three patients received ropivacaine 0.75% and the mean volume given was 2.85 0.19 ml ( range 2.53 ml ) . mean duration of surgery was 117.07 28.1 min ( range 60180 min ) . out of three patients who received cse , two patients required activation of epidural with 68 ml of bupivacaine . the outcome of the surgical procedure was that 13 patients delivered live twins , 15 patients delivered single live baby with other twin baby had intrauterine death ( iud ) . ttts and trap are complications of monochorionic placental twins due to sharing of the blood supply through common chorionic blood vessels , and selective laser photocoagulation of abnormal vascular communications is the treatment of choice . fla procedures for ttts and trap involve manipulation of placenta and umbilical cord vessels , and do not involve foetal surgery , and hence no foetal anaesthesia is required . ten of our patients had nausea intraoperatively ; however , none of them had vomiting and all of them had received ondansetron intraoperatively . nitroglycerine , magnesium sulphate and nifedipine are commonly used as tocolytic agents . in this study , all patient received nitroglycerine 5 mg patch 1 h before fla and was continued postoperatively for 2448 h. preterm labour , bleeding and premature rupture of membranes are the problems associated with fla . robinson et al . have reported pulmonary oedema due to large amount of saline irrigation of amniotic cavity used during the foetoscopic procedure to facilitate surgical exposure . it was hypothesised that the pulmonary oedema resulted from irrigating fluid absorbed through myometrial venous channels accessed by the passage of the operating trocars . with relaxed uterus , tocolytics may further facilitate fluid absorption , especially when magnesium sulphate is used , which in addition can cause an increase in pulmonary capillary leak and may lead to pulmonary oedema . foetoscopy procedure may be done under local anaesthesia / monitored anaesthesia with sedation , central neuraxial block and/or general anaesthesia . local anaesthesia involves infiltration of anterior abdominal wall and the peritoneum at the site of trocar insertion . bilateral transverse abdominis plane ( tap ) block may also be administered . however , these patients require some degree of sedation . fentanyl or morphine with or without midazolam may be used to induce acceptable maternal analgesia and sedation . midazolam crosses placenta and may cause reduction of foetal tone and help in stabilising the foetus for the procedure . there has been one report of sedation with dexmedetomidine use in foetoscopic blood transfusion but requires further studies are required as dexmedetomidine may cause maternal hypotension and bradycardia and reduction in placental blood flow . regional anaesthesia for foetoscopy avoids need for rapid sequence induction and intubation and risk of difficult intubation of the mother . regional anaesthesia has further advantage of avoiding sedative and other anaesthetic agents which may cause respiratory depression . the level of block should be at least till t8 level , preferably up to t6 level to avoid any pain or discomfort during foetal manipulation . in our patients vasopressors like phenylephrine or ephedrine may be preferred over fluids to treat maternal hypotension , during foetoscopic procedures , as large volumes of fluid may precipitate pulmonary congestion or oedema , in the presence of tocolytics . in addition , correction of aortocaval compression by left lateral tilt helps in reducing the severity and incidence of maternal hypotension . we found phenylephrine to be advantageous in our study as it reduced the heart rate , high probably due to nitroglycerine absorption . in this study , this may be related to the level of block , and variation in the dose of drug used between different anaesthesiologists . however , subsequently , there was no further incidence of hypotension during rest of the foetoscopic procedure . in previous study in patients undergoing selective fla , the incidence of maternal hypotension under epidural anaesthesia was 53.4% in the second trimester . however , there was no statistically significant correlation between intraoperative maternal hypotension and foetal outcome in their study . general anaesthesia has the advantage of increased ability of the patient to tolerate extreme positions and the surgical manipulations of the uterus which may be required in technically difficult cases . general anaesthesia may be preferred in patients with anterior placenta , as these patients require extreme positions for trocar insertion . however , use of drugs like sedatives analgesics and muscle relaxants has implication on foetal well - being . the patient should be positioned with left lateral tilt for left uterine displacement to prevent aortocaval compression , which may decrease venous return , resulting in decreased maternal cardiac output and low placental perfusion . doses of anaesthetic agent should take into consideration the increased sensitivity of parturient for drugs . foetal monitoring of heart rate is important as a direct evidence of the adequacy of foetal circulation . the foetal heart rate can be monitored during the procedure with ultrasound by the surgeon . anaesthesia for foetal surgery involves coordination and communication between surgical and anaesthesia team and patient herself . the limitation of this retrospective analysis is that no uniform dose of drug was used for regional spinal anaesthesia as different anaesthesiologists used different dosages . in addition , nitroglycerine patch which was used to tocolysis also could have contributed to hypotension . however , we did not find any incidence of hypotension during rest of the procedure . our experience with general anaesthesia has been limited to few cases , and hence comparison between the two techniques is not possible . multiple factors can influence the choice of anaesthesia , for foetoscopic procedures , such as the surgical procedure planned , pre - operative maternal history , history of previous uterine activity and the experience of surgeon for successful and good outcome . anaesthesia for ttts and trap may be undertaken under regional anaesthesia , even though general anaesthesia may be required in some cases .

background and aims : twin pregnancy with monochorionic placenta may be associated with arteriovenous vascular anastomosis of the placental vessels resulting in twin - to - twin transfusion syndrome ( ttts ) and twin reversed arterial perfusion syndrome ( trap ) . foetoscopic laser ablation ( fla ) is the treatment of choice in reducing foetal mortality related to this.methods:a retrospective review of medical records of 41 fla procedures for ttts and trap were analysed for anaesthetic management . thirty - four patients received subarachnoid block , three combined spinal - epidural block , three general anaesthesia and one local anaesthesia with sedation . nitroglycerine 5 mg patch was used for tocolysis 1 h before the procedure and continued for 2448 h postoperatively.results:bupivacaine was used in 34 patients , and ropivacaine in three patients . mean dose of bupivacaine 0.5% was 2.43 0.32 ml and ropivacaine 0.75% was 2.85 0.19 ml . the mean duration of surgery was 117.07 28 min . mild hypotension occurred in all patients under spinal anaesthesia and was treated with vasopressors . the foetal outcome among all 41 patients were 13 delivered live twins , 15 had a single live baby with intrauterine death of other twin baby . in 12 patients , both babies were intrauterine death . one patient was lost for follow-up.conclusion:foetoscopic procedures can be done under central neuraxial block , however occasionally general anaesthesia may be required .

INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

scar endometriosis is a rare entity reported in the gynecological literature , and is present in women who have undergone an abdominal or pelvic operation . the diagnosis of endometriosis is usually established by a biopsy . because endometriotic lesions can be present as a mass lesion , it seems feasible to investigate them by the non - invasive method of fine needle aspiration cytology ( fnac ) . we present a case of abdominal wall scar endometriosis in a woman who presented as a tumor on the abdominal wall . it was a case of endometriosis suspected by fnac , but confirmation was performed by cell block preparation , and subsequently by histology . a 27-year - old woman presented with a nodule at the lower abdominal caesarean section scar . the tumor appeared over the caesarean section scar 4 years after surgery , which gradually increased in size . the patient described cyclical pain at the site of the mass , which coincided with her normal menstrual cycle . the patient had a firm mass with restricted mobility along the right upper lateral aspect of the caesarean section scar with a black appearance . the mass measured about 5 cm 5 cm in size and was clinically diagnosed as a tumor on the abdominal wall . fnac was performed and the aspirate was obtained using a disposable 10 ml syringe and 22 gauge needle . the material was collected on glass slides and was wet fixed and then stained by the papanicolaou method , while the remaining aspirate was allowed to clot . to facilitate clotting , few drops of blood from the finger prick of the patient smear preparation of the fnac sample showed extensive areas of hemorrhage and scattered small cells with compact nuclei and scanty cytoplasm , resembling stromal cells and columnar epithelial cells , which represented the endometrial glands . ( a ) fine needle aspiration cytology smears show glandular epithelium with hemosiderin pigment - laden macrophages ( pap 400 ) . ( b ) the cell block section shows endometrial lining epithelium and stroma ( h and e , 100 ) . ( c ) tissue section of growth showing lining dermal epithelium and sub - epithelial dilated endometrial glands ( h and e , 40 ) . ( d ) tissue section of growth shows endometrial glands and stroma ( h and e , 100 ) the cell block findings were confirmatory of endometriosis as they revealed endometrial lining columnar epithelium with subepithelial dense and compact endometrial stroma along with extensive areas of hemorrhage [ figure 1b ] . histopathological findings after complete excision of the nodule also confirmed endometriosis [ figure 1c and d ] . it rarely involves the bladder , kidney , omentum , bowel , lymph node , pleura , umbilicus , hernial sac and abdominal wall . , endometriosis remains a diagnostic and therapeutic enigma even today , largely due to its variable presentations . clinically , the features diagnostic of scar endometriosis are lump in the scar , pain , increasing size of lump , bleeding and skin discoloration . in the literature , the mean size of the masses has been 3.1 cm ( range 1.5 - 4.8 cm ) . in our case , the size of the mass was 5 cm 5 cm , which was relatively large and confused with development of tumor on scar endometriosis . patients may present from months to years ( mean 21 months ) after their last obstetric / gynecologic surgery . in our case , the skin coloration at the site of endometriosis was black , which may be due to recurrent hemorrhage . the symptoms are non - specific , typically involving abdominal wall pain at the time of menstruation . clinical examination and other investigations also give non - specific results . in our case , the patient described cyclical pain at the site of the mass , which coincided with her normal menstrual cycle . because endometriotic lesions can present as a mass lesion , it seems feasible to investigate them by the non - invasive method of fnac . gupta also stated that the remaining aspirate was spun and a cell block was made from the sediment and sections cut and stained with hematoxylin the cytology smear was prepared from half of the aspirate material while the remaining aspirate was allowed to clot . to facilitate clotting , few drops of blood from the finger prick of the patient was added on the aspirate and the clot was transferred to a formalin vial and cell block was made . smear from the abdominal mass shows varying cellularity comprising epithelial and spindle stromal cells , with a variable number of hemosiderin - laden macrophages and inflammatory cells . the presence of any two of three components ( endometrial glands , stomal cells and hemosiderin - laden macrophages ) has been used for the cytological diagnosis of endometriosis . in our case , the cytology smear showed extensive areas of hemorrhage and scattered small cells with compact nuclei and scanty cytoplasm , resembling stromal cells and columnar and hemosiderin pigment - laden macrophages [ figure 1a ] . gupta stated that the fnac report was further confirmed on examination of cell block , which showed histological features of endometriosis characterized by endometrial glands separated by endometrial stroma and rare siderophages . in our case , the cell block findings were confirmatory of endometriosis , as they revealed endometrial columnar epithelium , with subepithelial dense and compact stroma along with extensive areas of hemorrhage [ figure 1b ] . histopathological findings after complete excision of the nodule also confirmed endometriosis [ figure 1c and d ] . in our case , the abdominal wall mass was larger ( 5 cm 5 cm ) in size and clinically thought to be a metastatic deposit or skin appendage tumor . the history of caesarean section and fnac from nodule on caesarean scar was suggestive of endometriosis . therefore , although endometriosis is diagnosed by fnac , cell block preparation is a good method for the diagnosis of endometriosis before excision of the mass .

presence of endometrial glands and stroma in places other than the uterus is called endometriosis . it can be pelvic or extra - pelvic . abdominal scar endometriosis is an extra - pelvic endometriosis that can occur after surgery involving the uterus . post - caesarean section , scar endometriosis is a rare event . the diagnosis is frequently made only after excision of disease tissue . we present a case of post - caesarean section abdominal scar endometriosis presenting as a tumor on the abdominal wall , which was diagnosed by fine needle aspiration cytology and confirmed by cell block preparation .

Introduction Case Report Discussion Conclusion

it has a horse shoe shaped body which lodges the teeth and a pair of rami which project upwards from the posterior ends of the body and provide attachment to muscles . each half of the mandible ossifies from only one center which appears at about the 6 week of intrauterine life in the mesenchymal sheath of meckels cartilage near the future mental foramen . the mental foramen opens below the sockets for the two deciduous molar teeth near the lower border , this is because the bone is made up of only the alveolar part with sockets . the mental foramen opens mid way between the upper and lower borders because the alveolar and sub alveolar parts of the bone are equally developed . the alveolar border is resorbed , so that the height of the body is markedly reduced . the mental foramen is an important anatomical structure situated in antero lateral aspect of the body of the mandible which transmits mental nerve , artery and vein . mental nerve is a terminal branch of the inferior alveolar nerve which supplies sensory innervations to lower lip , buccal vestibule and gingiva mesial to the first mandibular molar . mental foramen serves as an important anatomical landmark , the orientation and position of which facilitate local anesthetic , surgical and other invasive procedures for oral and maxillofacial surgeries . its location and the possibility that an anterior loop of the mental nerve may be present mesial to the mental foramen needs to be considered before any surgery in the foramina area in order to avoid injuring of the neurovascular bundles passing through these foramina and notches . the foramen may be occasionally misdiagnosed with a radiolucent lesion in the apical area of the mandibular premolar teeth . so identification of accurate anatomical position of mental foramen is very important in periodontal surgery especially during flap surgery in lower teeth , apical curettage of mandibular premolars , retrograde amalgam fillings and surgical orthodontics . replacement of missing teeth by dental implant and the increasing frequency of orthognathic surgery have increased the possibility of surgical procedures near the mental foramen . local anesthesia of the terminal incisive branches of the inferior alveolar and mental nerves can be obtained effectively if the mental foramen is correctly identified . localization of mental foramen radiographically is difficult due to lack of consistent anatomical landmarks for reference and the foramen can not be clinically visualized or palpated . therefore the location of the mental foramen had been studied by means of direct measurement on dry mandibles or by using radiographs of dry mandibles in patients . as the bone density increases the mental foramen becomes more difficult to identify on radiographs . knowledge of the most common position of the mental foramen in the population may give additional information in the mental nerve blocks and related mandibular surgeries . so the present study had been undertaken to determine the morphological features and morphometrics of mental foramen with reference to surrounding anatomical landmarks in both dentate and edentulous mandibles in coastal andhra population of andhra pradesh state . a total of 219 dry dentate and edentulous mandibles are examined in this study , out of these 127 were dentulous and 92 were edentulous . various parameters were measured by using digital vernier caliper , metallic wire and metallic scale on both the right and left sides . before measuring , the mandibles were placed on a standard horizontal plane to which the lower border of the mandible get its most contact when vertical pressure is applied to the molar region . [ pr - mf ] distance between posterior border of ramus [ pr ] of mandible and most anterior margin of mental foramen [ mf ] [ figure 1][sm - mf ] distance between symphysis menti [ sm ] and most anterior margin of mental foramen [ figure 2][mf - ifb ] distance between inferior margin of mental foramen and inferior border of mandible . [ ifb ] [ figure 3][ac - mf ] distance between alveolar crest and inferior margin of mental foramen [ figure 4][a1 ] distance between alveolar crest to upper most point of mental foramen [ figure 5][a2 ] distance between alveolar margins to the apex of second premolar socket [ figure 6][a1-a2 ] distance between alveolar crest to upper most point of mental foramen - distance between alveolar margins to the apex of second premolar socket [ figure 7]incidence of mental foramen in relation to first premolar , second premolar and first molar on right side in dentulous mandibles [ figure 8]incidence of mental foramen in relation to first premolar , second premolar and first molar on left side in dentulous mandibles [ figure 9]edentulous mandible showing accessory mental foramen [ figure 10 ] . [ pr - mf ] distance between posterior border of ramus [ pr ] of mandible and most anterior margin of mental foramen [ mf ] [ figure 1 ] [ sm - mf ] distance between symphysis menti [ sm ] and most anterior margin of mental foramen [ figure 2 ] [ mf - ifb ] distance between inferior margin of mental foramen and inferior border of mandible . [ ifb ] [ figure 3 ] [ ac - mf ] distance between alveolar crest and inferior margin of mental foramen [ figure 4 ] [ a1 ] distance between alveolar crest to upper most point of mental foramen [ figure 5 ] [ a2 ] distance between alveolar margins to the apex of second premolar socket [ figure 6 ] [ a1-a2 ] distance between alveolar crest to upper most point of mental foramen - distance between alveolar margins to the apex of second premolar socket [ figure 7 ] incidence of mental foramen in relation to first premolar , second premolar and first molar on right side in dentulous mandibles [ figure 8 ] incidence of mental foramen in relation to first premolar , second premolar and first molar on left side in dentulous mandibles [ figure 9 ] edentulous mandible showing accessory mental foramen [ figure 10 ] . [ pr - mf ] distance between posterior border of ramus of mandible and most anterior margin of mental foramen [ sm - mf ] distance between symphysis menti and most anterior margin of mental foramen [ mf - ifb ] distance between inferior margin of mental foramen and inferior border of mandible [ ac - mf ] distance between alveolar crest and inferior margin of mental foramen [ a1 ] distance between alveolar crest to upper most point of mental foramen [ a2 ] distance between alveolar margins to the apex of second premolar socket [ a1-a2 ] distance between alveolar crest to upper most point of mental foramen - distance between alveolar margins to the apex of second premolar socket incidence of mental foramen in relation to first premolar , second premolar and first molar - right side of dentulous mandibles incidence of mental foramen in relation to first premolar , second premolar and first molar - left side of dentulous mandibles edentulous mandible showing accessory mental foramen the mean values are expressed as mean standard deviation with confidence interval of 95% . [ pr - mf ] distance between posterior border of ramus [ pr ] of mandible and most anterior margin of mental foramen [ mf ] [ figure 1][sm - mf ] distance between symphysis menti [ sm ] and most anterior margin of mental foramen [ figure 2][mf - ifb ] distance between inferior margin of mental foramen and inferior border of mandible . [ ifb ] [ figure 3][ac - mf ] distance between alveolar crest and inferior margin of mental foramen [ figure 4][a1 ] distance between alveolar crest to upper most point of mental foramen [ figure 5][a2 ] distance between alveolar margins to the apex of second premolar socket [ figure 6][a1-a2 ] distance between alveolar crest to upper most point of mental foramen - distance between alveolar margins to the apex of second premolar socket [ figure 7]incidence of mental foramen in relation to first premolar , second premolar and first molar on right side in dentulous mandibles [ figure 8]incidence of mental foramen in relation to first premolar , second premolar and first molar on left side in dentulous mandibles [ figure 9]edentulous mandible showing accessory mental foramen [ figure 10 ] . [ pr - mf ] distance between posterior border of ramus [ pr ] of mandible and most anterior margin of mental foramen [ mf ] [ figure 1 ] [ sm - mf ] distance between symphysis menti [ sm ] and most anterior margin of mental foramen [ figure 2 ] [ mf - ifb ] distance between inferior margin of mental foramen and inferior border of mandible . [ ifb ] [ figure 3 ] [ ac - mf ] distance between alveolar crest and inferior margin of mental foramen [ figure 4 ] [ a1 ] distance between alveolar crest to upper most point of mental foramen [ figure 5 ] [ a2 ] distance between alveolar margins to the apex of second premolar socket [ figure 6 ] [ a1-a2 ] distance between alveolar crest to upper most point of mental foramen - distance between alveolar margins to the apex of second premolar socket [ figure 7 ] incidence of mental foramen in relation to first premolar , second premolar and first molar on right side in dentulous mandibles [ figure 8 ] incidence of mental foramen in relation to first premolar , second premolar and first molar on left side in dentulous mandibles [ figure 9 ] edentulous mandible showing accessory mental foramen [ figure 10 ] . [ pr - mf ] distance between posterior border of ramus of mandible and most anterior margin of mental foramen [ sm - mf ] distance between symphysis menti and most anterior margin of mental foramen [ mf - ifb ] distance between inferior margin of mental foramen and inferior border of mandible [ ac - mf ] distance between alveolar crest and inferior margin of mental foramen [ a1 ] distance between alveolar crest to upper most point of mental foramen [ a2 ] distance between alveolar margins to the apex of second premolar socket [ a1-a2 ] distance between alveolar crest to upper most point of mental foramen - distance between alveolar margins to the apex of second premolar socket incidence of mental foramen in relation to first premolar , second premolar and first molar - right side of dentulous mandibles incidence of mental foramen in relation to first premolar , second premolar and first molar - left side of dentulous mandibles edentulous mandible showing accessory mental foramen the mean values are expressed as mean standard deviation with confidence interval of 95% . morphometric features of 219 mandibles revealed the following results [ table 1 ] . depicting the values of all parameters distance between anterior margin of mental foramen to the posterior border of ramus of mandible [ mf - pr ] in dentate mandibles is 69.61 6.02 mm on right side and 69.17 6 mm on left side , whereas for edentulous mandibles the values have slightly decreased and it is 68.39 6.4 mm on right side , 68.82 6.55 mm on left side . the distance between symphysis menti and most anterior margin of mental foramen [ mf - sm ] , appears to be the same for both dentate and edentulous mandibles . mf - sm in dentate mandibles on right side is 28.24 5.09 mm and on left side it is 27.45 3.7 mm whereas for edentulous mandibles it is 28.51 4.53 mm on right side and is 27.99 4.5 mm on left side . distance between alveolar crest and inferior margin of mental foramen [ ac - mf ] in dentate mandibles is 15.29 3.71 mm on right side and is 14.38 3.75 mm on left side whereas for edentulous mandibles it is 15.50 3.71 mm on right side and on left side it is 14.70 4.43 mm which also appears to be the same for dentate and edentulous mandibles . distance between inferior margin of mental foramen and inferior border of mandible [ mf - ifb ] in dentate mandibles on right side is 12 2.37 mm and on left side is 12.39 2.74 mm whereas for edentulous mandibles on right side it is 13.40 4 mm and on left side it is 13.56 4.04 mm which shows that the distance between these parameters is increased in edentulous mandibles compared to dentulous mandibles . distance between alveolar crest to upper most point of mental foramen [ a1 ] in dentate mandibles is 13.18 2.32 mm on right side and is 12.56 2.40 mm on left side , whereas for edentulous mandibles on right side it is 11.09 3.41 mm and on left side is 10.01 2.68 mm which shows the distance has significantly reduced in edentulous mandibles . distance between alveolar margins to the apex of second premolar socket [ a2 ] in dentate mandibles is 8.93 2.33 mm on right side and is 8.75 2.34 mm on left side whereas for edentulous mandibles it is 6.16 2.60 mm on right side and is 5.38 2.42 mm on left side which reflects a decrease of values for edentulous mandibles . distance between alveolar crest to upper most point of mental foramen - distance between alveolar margins to the apex of second premolar socket [ a1-a2 ] , in dentate mandibles on right side is 4.18 1.71 mm and on left side it is 3.85 1.85 mm and for edentulous mandibles on right side it is 4.80 2.10 mm and on left side is 5.18 1.92 mm . the shape of mental foramen was visually observed and analyzed in all the 219 mandibles and it was found that in 87 cases it is round and 40 cases it is oval in dentate mandibles and in 56 cases it is round and in 36 cases it is oval in edentulous mandibles on right side . on left side , it is round in 99 cases and oval in 28 cases in dentate mandibles and in edentulous it is round in 57 cases and oval in 35 cases . the position of mental foramen on right side and left side in both dentate and edentulous mandibles in maximum cases is in between the first and second premolars [ 40 - 50% ] . the mental foramen is usually a single opening on the antero lateral surface of the mandible , 13 - 15 mm superior to the inferior border of the body of mandible which is generally seen to be circular or oval in shape . through this foramen , the mental neurovascular bundles exits . it remains incomplete till the 12 gestational week until the mental nerve separates into several fasciculi at that site . identifying the precise location of mental nerve is critical while performing peri apical surgery , cyst enucleation , endosseous implant , periodontal surgery and surgical orthodontic treatments such as a mandibular body osteotomy , in order to prevent damage to the mental nerve . in addition , knowing the site of the mental foramen allows for the accurate delivery of local anesthesia during dental procedures . there can be neuro sensory disturbances encountered if this important landmark is ignored while doing any invasive treatment in this region . the interval between the two premolars is the most common site of the mental foramen and the apex of second premolar is the second most common site . the results of the present study conclude the same . in 42.5% mandibles on right side and 40.9% on left side of dentate mandibles the common site is the interval between the two premolars and in 18.1% of the mandibles it is at the apex of second premolar . santini and land et al . , reported that in chinese the mental foramen was in line with second premolar and in british between first and second premolars . in edentulous mandibles , 50% of the mandibles on right side and 40.9% of the mandibles on left side , the mental foramen was located between first and second premolars . the second most common site is apex of second premolar which is seen in 25.2% of the mandibles , which is in accordance with the study conducted by shankland , wang , green . the position of the mental foramen varies depending on various factors like symmetry of mental triangle , microscopic and macroscopic morphology and maturity of the human mandible , bone remodeling activity and anthropologic features of the facial skeleton in different populations . according to the present study the distance between the apex of lower second premolar socket to superior border of mental foramen ( a1-a2 ) is 3.8 mm in dentate mandibles and 4.5 mm in edentulous mandibles . thus , when endodontic treatment is planned for these teeth , over obturation of root canals can lead to impingement and irritation to the mental nerve . wang reported that the average distance between the apex of the lower second premolar socket and superior border of mental foramen is 2.5 mm . such a short distance can harm the mental nerve if the root canal is overfilled during root canal treatment procedures . in the present study , the distance between inferior margin of mental foramen to the inferior border of mandible ( mf - ifb ) is 12.39 mm in dentate mandibles whereas 13.5 mm in edentulous mandibles which is in correlation with chung et al . , they reported that the average distance from the inferior border of the mandible to the center of mental foramen was 15.5 mm for korean males and 14.0 mm for korean females . gershchenson et al . , reported that the location of the mental foramen in relation to the border of the mandible and teeth depended on age , tooth condition and the degree of resorption . in adults as age advances , mental foramen shifts toward the superior border of mandible . this can be attributed to tooth loss followed by bone resorption . with the loss of teeth , there is change in the relative position from midlevel toward the upper border of the mandible . in severe cases of alveolar resorption the bone loss is so much that the incisive and inferior alveolar nerves are exposed out of bone and lie just below the mucosa which may cause pain on denture wearing . accordingly , the results of the present study also showed that the values decreased in edentulous mandibles . soikkonen et al . , reported that the mental foramen moves toward the lower cortex of the mandible as a result of alveolar atrophy and was situated on average 3.8 mm lower in edentulous jaws than in dentate jaws . in our present study , the distance between alveolar crest and inferior margin of mental foramen [ ac - mf ] in dentate mandibles is around 3.71 mm and in edentulous mandibles it slightly increased to 4.43 mm . the distance between the posterior border of ramus of mandible and most anterior margin of mental foramen is very important , as nerve and vessels pass through it and care is taken to avoid complications during inferior alveolar nerve blocks , peri apical surgeries , apical curettage and implant placement and nerve repositioning . in the present study , in dentate mandible the distance between most anterior margin of mental foramen and posterior border of ramus of the mandible ( mf - pr ) on right side is 69.61 6.03 mm , left side is 69.17 6.0 mm and in edentulous mandibles ( mf - pr ) on right side is 68.39 6.4 mm , left side is 68.81 6.55 mm . the distance between symphysis menti and most anterior margin of mental foramen ( mf - sm ) in dentulous mandible on right side is 28.24 5.09 mm , and on left side is 27.45 3.7 mm and in edentulous mandible ( mf - sm ) on right side is 28.51 4.53 mm , and on left side are 27.99 4.50 mm . clinically , there may be instances where the mental foramen can not be localized without a fixed reference point . in such cases , it can be localized if the distance from the symphysis menti is known . even so , in order to avoid neurovascular complications , particular attention should be paid to the possible occurrence of one or more accessory mental foramen during surgical procedures . utmost care to the accessory mental foramen and nerve is essential during dental implant surgery and any surgical procedure involving the mandibular molar and premolar region . this care may reduce the rate of paralysis and hemorrhage in mental region , lower lip and gingiva from the mental foramen to the midline of the ipsilateral side . in the present study out of 127 dentulous mandibles 13.97% accessory mental foramens were found whereas out of 92 edentulous mandibles 2.76% of accessory mental foramen were found . out of these 219 mandibles none of them presented with more than two accessory mental foraminae . gershchenson et al . , reported 7.51% of double foramina in indian and israeli mandibles . and zografos and mutzuri studied the incidence of double foramina in 11.48% of north indians and 6.68% of greek population , respectively . in the present study , 68.5% of dentulous mandibles on right side and 78% on left side , the foramen is round in shape and oval in 31.5 and 22% , respectively . in edentulous mandibles , it is found that 60.9% is round on right side and 62% is round on left side , whereas , it is oval in 39.5 and 38% , respectively . these values are similar to study reported by mbajiorgu et al . , and junior et al . according to al - khateeb et al . , majority of foraminae were round in shape which is similar to the present study . the present analysis revealed variations in position , shape and size of the mental foramen . the knowledge of exact position and various distances of mental foramen are necessary for its anesthetic and surgical intervention . thus , it is important to know the anatomical variations of mental foramina in order to avoid nerve damage in connection with surgical procedures . since this study had been conducted in the local population , the ethnic and racial variations are not highlighted . further studies focusing on racial and ethnic variations in different population groups need to be conducted on a large scale to substantiate this study . the knowledge of the position of mental foramen is important for day - to - day clinical practice . this variability of mental foramen should alert the dental surgeons while performing periodontal or endodontic surgery . the knowledge of the relationship of the foramen , in surgical procedures , in relation to anatomical landmarks can be of assistance in the localization of important maxillofacial neurological structures .

aim : the aim of this study is to determine the morphological features and morphometrics of mental foramen with reference to surrounding anatomical landmarks in coastal andhra population of andhra pradesh state.materials and methods : two - hundred and nineteen dry dentate and edentulous mandibles are examined in this study . out of these 127 were dentate and 92 were edentulous . various morphological and morphometrical parameters were measured by using digital vernier caliper , metallic wire and metallic scale on both the right and left sides.results:in the present study , the distance between most anterior margin of mental foramen and posterior border of ramus of the mandible is [ mf - pr ] , mf - pr is 69.61 6.03 mm on the right side and is 69.17 6 . 0 mm on left side in dentate mandible . in edentulous type , mf - pr is 68.39 6.4 mm on right side and 68.81 6.55 mm on left side . in the present study , the distance between symphysis menti and most anterior margin of mental foramen [ mf - sm ] in dentate mandible is 28.24 5.09 mm on right side and is 27.45 3.7 mm on left side . in edentulous mandible ( mf - sm ) is 28.51 4.5 mm on right side and on left side is 27.99 4.50 mm.conclusion:acquiring the knowledge and importance of anatomy of mental foramen is helpful in avoiding neurovascular complications , during regional anesthesia , peri apical surgeries , nerve repositioning and dental implant placement .

INTRODUCTION MATERIALS AND METHODS The parameters assessed were Statistical analysis RESULTS DISCUSSION CONCLUSION

we tested the stability of ebov on 3 materials commonly found in an ebola treatment unit ( etu ) in west africa : 1 ) utility - grade ( 308 ) stainless steel washers ( mcmaster - carr , atlanta , ga , usa ) ; 2 ) plastic ( teflon [ polytetrafluoroethylene ] ; mcmaster - carr ) ; and 3 ) tyvek ( from the front of a coverall ) . for each time point , 3 disks ( 4-cm diameter ) of each material were placed individually into wells of a 6-well plate . five samples ( 10 l / sample ) containing a total dose of 10 50% tissue culture infectious doses ( tcid50s ) of ebov in cell - free medium were evenly distributed on the disks . the plates were divided into groups , and each group was placed into a plastic hepa - filtered box and placed at 21c , 40% rh , or 27c , 80% rh . the samples were dried naturally , and virus titers were determined over a 14-day period . in the surface and fluid stability experiments , all samples were stored at 80c until titration ( 1 freeze thaw cycle of ebov samples that did not change virus titer ) . the tcid50 per milliliter for each sample at each time point was calculated by using the spearman - karber method ( 11 ) . because viral decay rates often exhibit first - order kinetics ( 12 ) , we log10 transformed our tcid50 calculations to represent virus titer and used a linear regression analysis ( prism version 6.05 ; graphpad , san diego , ca , usa ) to determine the log10 reduction rate of ebov on each surface at both environmental conditions ( figure 1 ; table 1 ) . we also determined whether linear regression models were significantly different from each other at the p<0.05 level by using an analysis of covariance equivalent test in prism . linear regression model showing the effect of different environmental conditions and surfaces on survival of ebola virus ( ebov ) . virus was dried on 3 surfaces found in outbreak settings at a ) 27c , 80% relative humidity ( rh ) ( west african tropical conditions ) and b ) 21c , 40% rh ( climate - controlled hospital conditions ) . virus concentration was reduced at a significantly slower rate on all surfaces in hospital conditions than in tropical conditions ( p<0.0001 for all surfaces ) . an analysis of covariance equivalent test was used to compare linear regression models and determine differences in virus reduction rates . tcid50 , 50% tissue culture infectious dose . * na , not applicable . y , log10 50% tissue culture infectious dose / ml ; x , days . in hospital conditions , virus titer on steel was reduced significantly faster than on plastic ( p = 0.004 ) and on tyvek ( p<0.0001 ) , but there was no significant difference in reduction between tyvek and plastic ( p = 0.13 ) . in tropical conditions , there was no significant difference in virus titer reduction on steel and on plastic ( p = 0.78 ) . however , virus decayed more slowly on tyvek than on steel ( p<0.0001 ) and on plastic ( p<0.0001 ) . there was no significant difference in reduction rate in virus titer in drying human blood in hospital or tropical conditions ( p = 0.92 ) . virus was reduced significantly faster at 27c than in water at 21c ( p = 0.0001 ) . the stability of ebov in water was assessed by diluting 150 l virus stock in 2.85 ml of ambion diethylpyrocarbonate treated water ( thermo fisher scientific , pittsburgh , pa , usa ) and removing residual protein and medium with 1 initial and 2 rinse spins on amicon ultra centrifugal filters 100k mwco ( merck , darmstadt , germany ) . ebov was more stable in water at 21c and had an 1 log10 reduction / day in water at 27c ( table 1 ; figure 2 , panel a ) . linear regression model showing stability of ebola virus ( ebov ) in fluids under different environmental conditions . virus concentration was reduced at a significantly faster rate in 27c water than in 21c water ( p = 0.0001 ) . b ) stability in drying or liquid ebov - spiked human blood samples at 2 environmental conditions . virus concentration was reduced at a significantly faster rate by drying than in liquid blood at both conditions ( p<0.0001 for each condition ) . no significant difference between reduction rates in virus titer in drying human blood at both conditions was found ( p = 0.92 ) . error bars indicate mean sem virus titer . dashed line indicates the limit of detection for the assay . an analysis of covariance equivalent test was used to compare linear regression models and determine differences in virus reduction rates . the stability of ebov in human blood was assessed by spiking blood samples from a healthy human volunteer to achieve a 10 tcid50/ml virus titer . the spiked blood was distributed in 1-ml aliquots into closed screw - top vials to maintain a liquid state , spread in 50-l aliquots onto the bottom of a 24-well plate , and dried . one group of samples was stored at 21c , 40% rh , and the other group was stored at 27c , 80% rh . ebov stability in drying blood exhibited first - order kinetics and was viable for up to 6 days at tropical conditions ( table 1 ; figure 2 , panel b ) . to approximate the stability of ebov in naturally infected human blood blood was collected during necropsy from 3 macaques that were previously enrolled in an animal care and use committee approved ebov pathogenesis study and were euthanized because they exhibited signs of evd and viremia . blood samples were divided into 2 groups with 2 sets of 150-l aliquots for each time point ; each group was stored at the conditions described above with each set in the liquid or drying state , and virus viability was assessed over a 14-day period . because of variation in calculated virus titer from each of the individual nhps , the log10 reduction rate could not be approximated and only the initial titer and the duration of viability are shown ( table 2 ) . in general , ebov maintained viability for a longer duration in liquid than in drying blood regardless of initial titer or environmental condition . * tcid50 , 50% tissue culture infectious dose ; nhp , nonhuman primate ; rh , relative humidity . we found that ebov can persist on surfaces common in an etu , highlighting the need for adherence to thorough disinfection and doffing protocols when exiting the etus and careful handling of medical waste . in addition , ebov maintains viability for a longer duration in liquid than in dried blood . ebov in blood of experimentally infected nhps persists for a similar duration as ebov in spiked human blood . a recent study showed that blood in the body cavity of an nhp contained viable ebov for up to 7 days after death ( 13 ) . we detected viable ebov in drying blood for up to 5 days at both environmental conditions in human and nhp blood . therefore , dried and liquid blood from an infected person in their home or etu should be treated as potentially infectious . the finding that ebov remains viable in water for as long as 3 ( 27c ) or 6 ( 21c ) days at the experimental concentration warrants further investigation into the persistence of the virus in aqueous environments , such as in wastewater or sewage canals . viable ebov has been isolated from urine ( 14 ) but not from human stool ( 8) . first , because standard volumes for samples were used , different volumes or matrices could influence the stability of ebov under the tested conditions . second , blood samples from the nhps might have different immunologic or biochemical conditions , which can potentially influence virus stability . third , the experimental conditions in the laboratory are sterile , but in disease - endemic areas and etus , bacteria or chemicals could influence ebov viability . overall , we found that different environmental conditions , fluids , and surfaces influence the persistence of ebov . these findings demonstrate that such factors are crucial in understanding transmission and improving safety practices .

we evaluated the stability of ebola virus on surfaces and in fluids under simulated environmental conditions for the climate of west africa and for climate - controlled hospitals . this virus remains viable for a longer duration on surfaces in hospital conditions than in african conditions and in liquid than in dried blood .

The Study Conclusions