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Breast cancer is the second most common cancer in new zealand and the third leading cause of cancer death.1 in 2011, the most - recent full year for which statistics are available, 2,867 new cases of breast cancer and 636 deaths were reported.2 approximately 18% of breast cancers in new zealand are human epidermal growth factor receptor-2 (her2)-positive.3 trastuzumab is standard treatment for her2-positive early breast cancer (ebc) and metastatic breast cancer (mbc), being administered as an intravenous (iv) infusion, in part with chemotherapy, as an every-3-weeks regimen for 12 months in ebc (17 cycles), or until disease progression in mbc . Trastuzumab iv needs to be reconstituted into solution for infusion, with the dose calculated on the basis of patient bodyweight.4 iv trastuzumab treatment starts with a loading dose infused over 90 minutes, followed by subsequent maintenance doses infused over 30 minutes.4 given the requirement for up to 12 months of adjuvant or maintenance treatment, such a regimen consumes considerable health care resources, including drug preparation and administration, clinic and chair time, and physician time dedicated to patient interaction.5 furthermore, insertion of an indwelling venous catheter is common in patients requiring prolonged iv infusion therapy, adding the costs of theatre and anesthetist time; complications such as infection or thrombosis6,7 may also be associated with additional costs in some cases . A subcutaneous (sc) formulation of trastuzumab was developed to allow drug administration over a shorter time period, with the goal of improving convenience and compliance, particularly during long - term therapy . The ability to do this was facilitated by the fact that trastuzumab has a wide therapeutic window8 with no known dose - limiting toxicities.9 sc administration of volumes> 1 ml has been limited by the structure and physiology of the sc layer, which contains a matrix of hyaluronan and collagen fibers.8 to overcome this issue, the trastuzumab sc formulation includes recombinant human hyaluronidase as a novel excipient.10 hyaluronidase hydrolyses hyaluronan, decreases resistance of the interstitial matrix11,12 and degrades the extracellular matrix . This allows monoclonal antibodies to diffuse through the channels,12 thereby facilitating sc administration of larger volumes of fluid and improving delivery of sc drugs to the systemic circulation . The effects of recombinant human hyaluronidase are temporary and reversible within 24 hours.12 trastuzumab sc can be administered over approximately 5 minutes, a much shorter period of time than that required for the trastuzumab iv loading and maintenance doses.4,10 in addition, trastuzumab sc is given as a fixed dose that does not require adjustment based on bodyweight.10 pharmacokinetic modeling, based on data from an open - label phase i / ib study,13 determined that a 600 mg fixed dose of trastuzumab sc provided drug exposure that was comparable to the iv trastuzumab regimen, without the need for a loading dose . This dose of trastuzumab sc was subsequently tested in the phase iii hannah study, which showed that trastuzumab sc was noninferior to trastuzumab iv with respect to efficacy (pathological complete response rate) and had an equivalent tolerability profile.14 one of the key principles guiding the new zealand ministry of health medical oncology national implementation plan15 is to effectively, equitably, and sustainably meet the future demand for medical oncology services, given the significant workforce and resource constraints that exist . A second aim is to maintain high quality of care and improve the quality of life for people with cancer . It also offers the potential to move treatment delivery to the outpatient setting and away from infusion services that are facing challenges due to increased demand for complex treatments.15 the aim of this study was to determine medical resource utilization associated with administration of trastuzumab sc injection compared with trastuzumab iv infusion in patients with her2-positive breast cancer in new zealand . This noninterventional, descriptive study was conducted alongside the ongoing safeher clinical trial (nct01566721), a study investigating the safety and tolerability of trastuzumab sc via handheld syringe or single - injection device . The time and motion study was conducted at the outpatient oncology centers at auckland city hospital (trastuzumab iv) and tauranga hospital (trastuzumab sc and iv). Patients receiving trastuzumab sc via handheld syringe were recruited by the health care professionals (hcps) who were administering trastuzumab to participants in the safeher clinical trial at tauranga hospital . Patients with ebc were approached during a routine visit from march 2013 to may 2013 as part of their enrolment in safeher and given information about the time and motion study by a member of the clinical team . Prior to trastuzumab administration, patient consent was sought for an observer to be present during that visit . Patients receiving trastuzumab iv were being treated in the metastatic and adjuvant settings and were undergoing routine clinical care in the outpatient clinic of either tauranga or auckland hospital in april 2013 and may 2013 . At the start of the clinic visit they were given information about the time and motion study by the hcp who would be administering trastuzumab . Under the same protocol as the sc group, patient consent to allow an observer to be present during their clinic visit was sought prior to trastuzumab administration . This study was an audit that does not involve the use, collection, or storage of human tissue, and therefore the health and disability ethics committees advised that the study did not require their review . Active hcp time captured during the time and motion study related to tasks associated with administration of trastuzumab sc or iv, as detailed below . New zealand - specific standardized treatment observation forms were created, based on those used in a time and motion study conducted in the united kingdom.16 times for entering the bed / chair, starting trastuzumab treatment, stopping trastuzumab treatment, and exiting the bed / chair were recorded in the treatment observation form in hours and minutes (hh: mm) by a single trained observer . A stopwatch was used by the same observer to record times for active involvement in specific trastuzumab treatment - related tasks as follows: install venous catheter / line flushing; prepare and administer premedication (if required); prepare for trastuzumab administration in the care unit; initiate trastuzumab therapy; monitor patient during treatment; disconnect infusion / flush infusion line / dispose of materials (iv only); monitor patient after treatment . Other variables recorded on the same treatment observation form were type of iv access (iv only), the occurrence of any unexpected events that disrupted typical patient flow, use and type of premedication, and consumables used for each task (including the exact product and the number of units used). Trastuzumab solution for iv infusion was prepared in the hospital pharmacy, where the total time was calculated as the time from taking the prescription to leaving the prepared solution for collection . Specific timed tasks for trastuzumab iv preparation were: prepare and label; add trastuzumab and mix with diluent; stand vial and check for impurities; calculate and check dose; add to infusion and mix; finalize for release . Consumables used during the preparation and delivery of both formulations were noted on the treatment observation form . Costs for indwelling venous lines (eg, portacath, hickman, and groshong) were not included in cost calculations because patients who required or had these access devices in place had had them inserted for previous chemotherapy . Unit costs for hcp and personnel time were derived from pharmaceutical management agency (pharmac) published costs, and were $45 nzd (new zealand dollars) per hour for a registered nurse, $50 nzd per hour for a hospital pharmacist, and $65 nzd per hour outpatient bed per chair cost.17 a simple cost - minimization model was developed to estimate cost savings associated with delivery of trastuzumab sc compared with trastuzumab iv in the outpatient setting . All outpatient consumables and times directly related to both routes of administration were quantified from the standardized treatment observation forms . Average estimates for delivery of trastuzumab iv and trastuzumab sc were calculated for all hospital and pharmacy data . These data were used to estimate resource inputs, which were then applied to the pharmac unit cost data for hourly rates and to consumable costs to allow determination of the estimated difference in costs between the two treatment groups . Only one trastuzumab iv formulation is available in new zealand (herceptin, roche products ltd, auckland, new zealand). It was assumed that drug acquisition costs for trastuzumab iv and trastuzumab sc would generally be equivalent, and therefore these were not included in the analysis . This noninterventional, descriptive study was conducted alongside the ongoing safeher clinical trial (nct01566721), a study investigating the safety and tolerability of trastuzumab sc via handheld syringe or single - injection device . The time and motion study was conducted at the outpatient oncology centers at auckland city hospital (trastuzumab iv) and tauranga hospital (trastuzumab sc and iv). Patients receiving trastuzumab sc via handheld syringe were recruited by the health care professionals (hcps) who were administering trastuzumab to participants in the safeher clinical trial at tauranga hospital . Patients with ebc were approached during a routine visit from march 2013 to may 2013 as part of their enrolment in safeher and given information about the time and motion study by a member of the clinical team . Prior to trastuzumab administration, patient consent was sought for an observer to be present during that visit . Patients receiving trastuzumab iv were being treated in the metastatic and adjuvant settings and were undergoing routine clinical care in the outpatient clinic of either tauranga or auckland hospital in april 2013 and may 2013 . At the start of the clinic visit they were given information about the time and motion study by the hcp who would be administering trastuzumab . Under the same protocol as the sc group, patient consent to allow an observer to be present during their clinic visit was sought prior to trastuzumab administration . This study was an audit that does not involve the use, collection, or storage of human tissue, and therefore the health and disability ethics committees advised that the study did not require their review . Active hcp time captured during the time and motion study related to tasks associated with administration of trastuzumab sc or iv, as detailed below . New zealand - specific standardized treatment observation forms were created, based on those used in a time and motion study conducted in the united kingdom.16 times for entering the bed / chair, starting trastuzumab treatment, stopping trastuzumab treatment, and exiting the bed / chair were recorded in the treatment observation form in hours and minutes (hh: mm) by a single trained observer . A stopwatch was used by the same observer to record times for active involvement in specific trastuzumab treatment - related tasks as follows: install venous catheter / line flushing; prepare and administer premedication (if required); prepare for trastuzumab administration in the care unit; initiate trastuzumab therapy; monitor patient during treatment; disconnect infusion / flush infusion line / dispose of materials (iv only); monitor patient after treatment . Other variables recorded on the same treatment observation form were type of iv access (iv only), the occurrence of any unexpected events that disrupted typical patient flow, use and type of premedication, and consumables used for each task (including the exact product and the number of units used). Trastuzumab solution for iv infusion was prepared in the hospital pharmacy, where the total time was calculated as the time from taking the prescription to leaving the prepared solution for collection . Specific timed tasks for trastuzumab iv preparation were: prepare and label; add trastuzumab and mix with diluent; stand vial and check for impurities; calculate and check dose; add to infusion and mix; finalize for release . Consumables used during the preparation and delivery of both formulations were noted on the treatment observation form . Costs for indwelling venous lines (eg, portacath, hickman, and groshong) were not included in cost calculations because patients who required or had these access devices in place had had them inserted for previous chemotherapy . Unit costs for hcp and personnel time were derived from pharmaceutical management agency (pharmac) published costs, and were $45 nzd (new zealand dollars) per hour for a registered nurse, $50 nzd per hour for a hospital pharmacist, and $65 nzd per hour outpatient bed per chair cost.17 a simple cost - minimization model was developed to estimate cost savings associated with delivery of trastuzumab sc compared with trastuzumab iv in the outpatient setting . All outpatient consumables and times directly related to both routes of administration were quantified from the standardized treatment observation forms . Average estimates for delivery of trastuzumab iv and trastuzumab sc were calculated for all hospital and pharmacy data . These data were used to estimate resource inputs, which were then applied to the pharmac unit cost data for hourly rates and to consumable costs to allow determination of the estimated difference in costs between the two treatment groups . Only one trastuzumab iv formulation is available in new zealand (herceptin, roche products ltd, auckland, new zealand). It was assumed that drug acquisition costs for trastuzumab iv and trastuzumab sc would generally be equivalent, and therefore these were not included in the analysis . A total of six patients receiving trastuzumab sc as part of the ongoing safeher clinical trial, and 12 patients receiving routine clinical treatment with trastuzumab iv (six each in tauranga and auckland) consented to allow an observer to record data for the time and motion study during their treatment visit and were included in the final analysis . Administration of trastuzumab sc reduced nursing time by an average of 43% and chair time by an average of 75% compared with trastuzumab iv (table 1). Although the two trastuzumab formulations were prepared in different settings (iv by the pharmacy and sc by the clinic nurse), the hcp time dedicated to drug preparation was also substantially lower for trastuzumab sc (table 1). Usage of consumables was low in both the trastuzumab sc and iv groups, but some savings were associated with the use of trastuzumab sc (table 2). Overall time to prepare and deliver trastuzumab therapy was markedly lower for the sc vs iv formulation (table 1). Average hcp nurse time, chair cost, and pharmacist time savings associated with the use of trastuzumab sc injection compared with trastuzumab iv infusion were estimated to be $61.67 nzd per administration (table 1), and consumables cost savings were estimated as $15.27 nzd per administration, with the largest single item being the cost of the delivery line (note that costs for indwelling venous lines were not included) (table 2). Thus, estimated cost saving achieved by switching to the sc formulation for delivery of trastuzumab in the new zealand district health board outpatient oncology clinic setting was $76.94 nzd per patient per cycle . The results of this descriptive time and motion study suggest that switching to trastuzumab sc injection from trastuzumab iv infusion in a new zealand oncology outpatient treatment setting decreases trastuzumab treatment - related tasks in the care unit, drug preparation time, chair time, and the volume and cost of consumables . Although differences between health care systems limit the generalizability of results, these new zealand data are similar to those obtained in europe18 and the united kingdom16 showing reduced chair time and hcp resource requirement in patients treated with trastuzumab sc compared with trastuzumab iv . The european and uk data were obtained in patients being treated with the two different trastuzumab formulations as part of the prefher trial,19 a global, randomized, crossover preference study conducted in patients with ebc . In europe, using estimates of the typical number of ebc patients being treated in the participating centers, total estimated savings in chair time associated with switching to trastuzumab sc ranged from 4490 8-hour days annually . In addition, there was an associated 21%52% reduction in the time hcps spent actively involved in administering treatment.18 patients treated with trastuzumab sc in the united kingdom also spent considerably less time in the care unit (30.3 minutes vs 94.5 minutes) and infusion chair (19.8 minutes vs 75.0 minutes) than did those receiving trastuzumab iv.16 in addition, mean hcp time was markedly lower when the sc formulation was used (24.6 minutes vs 92.6 minutes for iv administration), and consumable costs were lower (2.94 vs 8.81, respectively).16 it should be noted that the latter study included total infusion duration in hcp time, as opposed to the european study, which considered only active patient monitoring during infusion . On the basis of the results of the uk study, it was concluded that switching from trastuzumab iv to the sc formulation could substantially reduce overall treatment costs.16 these findings are similar to those documented after a switch from iv to sc rituximab in non - hodgkin lymphoma patients in the united kingdom, which was also associated with reduced active hcp and chair time and lower costs.20 in a separate trastuzumab analysis, hcps from a subset of prefher centers were asked to rate a variety of factors on their ability to impact cancer center efficiency and time required for trastuzumab administration.21 the biggest perceived impact of switching to trastuzumab sc, according to care unit respondents, was a reduced waiting list for any iv treatment at the infusion unit, followed by the potential for a more - flexible treatment schedule and the ability to treat more patients in the infusion unit.19 respondents perceived that a reduction in dosing errors, reduced drug wastage, and increased staff availability for other tasks would be the main impacts of switching from trastuzumab iv to sc.15 factors such as reduced waiting times and the flexibility to deliver treatment outside the infusion clinic offer the possibility of developing new patient care pathways defining when, where, how, and by whom trastuzumab therapy is administered . A switch to trastuzumab sc fits well with the new medical oncology model of care described by the new zealand ministry of health, which includes identification of patient care pathway tasks and functions that can be performed by staff other than senior medical officers.15 in this study, trastuzumab sc was prepared by a registered nurse prior to administration to the patient . This was considered to be the most likely way that trastuzumab sc would be prepared outside of a clinical trial situation or on a ward . Should trastuzumab sc become available for general use within an oncology outpatient setting, preparation of the treatment dose may take place in the pharmacy or in the outpatient clinic depending on the preference or protocol of the individual hospitals . However, regardless of which hcp prepares trastuzumab sc prior to administration, it is reasonable to assume that the time savings achieved would be similar to those observed during this study . Although new zealand time and motion data from the current study were consistent with those obtained in other trials, it should be noted that these preliminary assessments of the impact of switching from trastuzumab iv to the sc formulation are likely to underestimate the potential time and cost savings . Firstly, the costs of indwelling iv catheter insertion and maintenance, and of managing complications, were not included in the analysis . Vascular access devices are one of the most common causes of health care - associated infections, including sepsis.22 the rate of sepsis after implantation of a venous access system for chemotherapy in a new zealand population was 11%.7 catheter - related sepsis indirectly affects outcomes of chemotherapy by compromising dose intensity,23 is associated with significant morbidity and increased health care costs,2427 and is fatal in approximately 15% of patients.22 as a result, cost savings associated with switching to trastuzumab sc could be greater than those reported here . Using our estimate based on costs associated with hcp time and consumables only, multiplying the cost saving per cycle by the total number of treatment cycles given (6,752) to all the patients with ebc and mbc treated with trastuzumab in new zealand in 2012 (the most - recent set of complete pharmac special authority data; n=460), annual cost savings to the new zealand health care system associated with a switch from trastuzumab iv infusion to trastuzumab sc injection could be more than half a million dollars ($519,499 nzd). The calculations made based on the results of the current study assume that the total cost of trastuzumab therapy to the new zealand healthcare system would be equivalent whether the sc or iv formulation was used . These calculations could be influenced by the introduction of a biosimilar iv trastuzumab, but this is not expected to occur in new zealand until at least the first half of 2019 (on the basis of clinical trial data timelines on clinicaltrials.gov and estimated regulatory review processes). On a patient - by - patient basis, the cost of trastuzumab therapy may vary, given that the dosage for trastuzumab iv is calculated based on patient bodyweight and trastuzumab sc is given as a single fixed dose . Nevertheless, on a population basis, overall drug costs are likely to be similar, although exact figures for the new zealand setting remain to be determined, on the basis of negotiations around access and funding . Another reason why the current data may underestimate the resource - use impact of switching from trastuzumab iv to the sc formulation is that only in - hospital direct medical costs were considered . This means that costs incurred by the patient either directly (eg, transport, parking) or indirectly (eg, time and loss of productivity for both patients and their caregivers) have not been quantified . Patient preference, well - being, and quality of life were also not assessed in this study, but the prefher trial found that patients overwhelmingly preferred trastuzumab sc over the iv formulation.19 by far the most common reason cited by patients for this preference was that receiving trastuzumab as a sc injection saved time . Other reasons for preferring trastuzumab sc were less pain or discomfort and fewer side effects, the avoidance of problems associated with iv treatment (ie, venous access), and convenience.19 these findings suggest that switching to trastuzumab sc not only offers financial and logistical advantages to the health care system but is also perceived as a more - attractive treatment option by patients . This study had a number of limitations, including the small sample size (particularly in the trastuzumab sc treatment group) and the administration of trastuzumab sc in only one of the two study centers . However, trastuzumab sc is not yet funded in new zealand and is therefore not administered in routine clinical practice . As a result, the only available data on administration of trastuzumab sc was from consenting patients who were receiving trastuzumab sc at the single new zealand center participating in the ongoing safeher trial . For both the sc and iv groups, it was not possible to report patient demographic data because the patient consent obtained applied only to observation and recording of tasks and times associated with trastuzumab administration and not access to patient medical files . For the iv group, it is likely that the patients treated were a relatively heterogeneous group because no selection was undertaken and any patient receiving trastuzumab was eligible (eg, ebc or mbc). Patients in the sc group had only ebc and therefore it is possible that disease severity was a confounding factor because patients who were more unwell might have needed more hcp involvement . Despite these issues, the range of values obtained was relatively narrow, and expected time savings were comparable to those from international studies with broadly similar study design and objectives . It is possible that the results of this study cannot be generalized to centers other than the two that participated in this study . However, there are only six regional oncology centers in new zealand, and 40% of new zealand s cancer patients are treated at auckland city hospital, which contributed patients to this study . It should also be noted that in new zealand there is no regional variation in drug costs because these are mandated nationally by pharmac, and unit costs for hcp and personnel time were derived from pharmac - specified national values.17 therefore, it is likely that the findings will be relevant across the new zealand health care setting . The new sc formulation of trastuzumab represents a clinical opportunity to improve the efficiency of her2-positive breast cancer treatment delivery by providing time and cost savings and the potential to modify pathways of patient care . This contributes to new zealand ministry of health goals for increasing oncology treatment capacity and allowing treatment to be administered away from regional oncology centers.15 it should be noted that the time and equipment savings associated with a switch to trastuzumab sc are not a true financial saving but instead represent the value of the best available alternative use of a resource (opportunity cost). Fewer health care staff and infusion chairs would be needed to treat the same number of patients with trastuzumab sc, and this resource is then available to be redeployed in the treatment of other patients . Thus, at least in the short term, the main result of switching from trastuzumab iv to trastuzumab sc would be to ease pressure at capacity - constrained treatment centers . Data from this new zealand time and motion study provide an indication that switching from iv to sc administration of trastuzumab has beneficial effects on resource use, including hcp time and consumables needed to administer treatment . These reductions contribute to health care cost efficiencies, could help to address current capacity issues at new zealand district health boards, and have the potential to influence how and where her2-positive breast cancer treatment is delivered in the future.
Painful uterine contractions cause maternal hyperventilation and increased catecholamine concentration resulting in maternal and foetal hypoxaemia . An effective analgesia takes away the disadvantages and results in better maternal and foetal outcome . Hence, the control of pain should form an integral part of labour management at any level . An ideal labour analgesic technique should provide adequate and satisfactory analgesia without any motor blockade or adverse maternal and foetal effects . Among the variety of labour analgesia techniques ranging from parenteral and inhalational agents, combined spinal epidural (cse) analgesia is increasingly used to provide pain relief during labour . It combines the advantage of rapid onset of spinal analgesia and the flexibility of the epidural catheter . Dose adjustments and frequency of administration of the drug according to parturients requirement is possible with the epidural route which can also be extended to provide anaesthesia for caesarean delivery if need arises . Addition of opioids to local anaesthetics reduces its requirement by synergistic effect of opioid receptors in the spinal cord . Although fentanyl and sufentanil are the most commonly used opioids for epidural labour analgesia, studies comparing cse technique employing sufentanil and fentanyl are very few and give conflicting results . At the assumed equipotent doses of sufentanil to fentanyl ratio of 6:1, there is some evidence that sufentanil is clinically superior to fentanyl as an adjunct to bupivacaine in labour epidurals . Hence, we decided to compare the efficacy, quality and duration of analgesia and maternal and foetal effects of adding the two highly lipid - soluble opioids, sufentanil and fentanyl to low - concentration bupivacaine for cse labour analgesia . A randomized, prospective, single - blinded study was carried out on 60 parturients over a period of 6 months after obtaining clearance from the institutional ethical committee . Parturients belonging to asa grade i and ii with singleton, term pregnancy in spontaneous labour with cervical dilatation of less than 4 cm, with normal foetal heart tracings requesting labour analgesia, were included in the study . Parturients with medical disorders, obstetric complications, malpresentation of foetus and those with contraindication for regional analgesia were excluded from the study . After obtaining informed written consent, parturients were randomly allocated by a computer - generated table of random numbers by a person blinded to the procedure to avoid selection bias into two groups of 30 each as group s (n=30) and group f (n=30). Before institution of cse, parturients were administered 1000 ml ringer lactate solution and monitors were connected and baseline readings recorded . Patients were placed in the left lateral position and, under aseptic precautions, cse analgesia was administered through a midline approach (l2-l3 or l3-l4 level). Lumbar epidural space was identified with an 18 g tuohy needle using loss of resistance to saline technique . After negative aspiration for blood and cerebrospinal fluid, the epidural catheter was threaded on through the tuohy needle and the needle was carefully withdrawn . Spinal analgesia was instituted using a 25 g whitacre needle one space below . On identification of subarachnoid space, 1 ml of drug containing 0.5 ml 0.5% bupivacaine heavy (2.5 mg) and 0.5 ml sufentanil (5 mcg) for group s or 0.5 ml fentanyl (25 mcg) for group f the parturient was turned supine immediately after subarachnoid block and the uterus was displaced to the left using a wedge . Pulse rate, blood pressure, respiratory rate and spo2 were monitored at 0, 5, 10, 15 and 30 min and thereafter every 30 min until the women delivered . Motor block was assessed by modified bromage scale and the level of sensory block was recorded . Pain relief was assessed using visual analogue scores (vas) (0 = no pain, 10 = worst possible pain experienced). When the parturient experienced pain equivalent to a vas score of 5, epidural top - up was administered . The time interval between initiation of intrathecal analgesia and patient developing pain equivalent to vas parturients of group s received 10 ml of 0.125% bupivacaine with 0.30 mcg / ml of sufentanil and group f received 10 ml of 0.125% bupivacaine with fentanyl 2.5 mcg / ml through an epidural catheter . Time of onset of epidural analgesia, total number of top - ups and duration between successive top - ups were recorded . The mode of delivery in the form of normal vaginal delivery, instrumental delivery or caesarean section was observed . Side - effects such as pruritis (rated as none, minimal, moderate and severe), hypotension (fall of> 20% from baseline systolic reading), motor blockade of limbs, shivering, sedation (categorized as none, mild, moderate and severe for awake, drowsy, sleepy and unarousable) and bradycardia (heart rate<60) were noted . Neonatal outcome in the form of apgar scores at 1 min and 5 min and need for intensive care were noted . All the data were recorded by a resident blinded to the drug administered during conduct of labour analgesia throughout the study period . The parturient was monitored for 2 h following delivery and the epidural catheter was removed . They were questioned after 24 h about their views on the procedure and the satisfaction . Enquiry about the symptoms related to post - dural puncture headache (pdph) was made . Sample size of 60 with 30 parturients in each group was determined with power of study of 80% . Sample size of 60 with 30 parturients in each group was determined with power of study of 80% . There were no significant differences between the groups with respect to maternal demographic characteristics, parity, cervical dilation at initiation of labour analgesia, duration of labour and delivery characteristics [table 1]. Maternal demographic characteristics the mode of delivery data suggests no significant difference between the groups regarding incidence of caesarean, instrumental or vaginal deliveries [table 2]. The mean duration of intrathecal analgesia was 109.7 (6.3) min for group s and 73.63 (15.76) min for group f. the mean time of onset of epidural analgesia was 2.48 (0.28) min and 2.55 (0.77) min for group s and group f, respectively . The mean duration between epidural top - ups was 90.51 (15.8) min for group s and 83.54 (24.64) min for group f [table 3]. The number of additional bolus supplementary analgesic top - ups were comparatively less in the sufentanil group . Eight women among group s and nine among group f experienced transient initial motor blockade (bromage score 1). None of the women had any motor blockade at the end of 1 h. the highest level of sensory block attained was t6 in 63.33%, t8 in 36.66% among group s and 66.66% and 33.33% among group f, respectively . Onset and duration of analgesia the pain scores during the first and second stages of labour were comparable without any significant difference between the groups using vas scores (vas: 0 = no pain, 10 = worst pain experienced); 93.3% and 90.0% women had vas scores less than 1, indicating excellent pain relief by the cse technique in both groups [table 4]. Mean visual analogue scale scores no significant alterations in intrapartum vital parameters like spo2, mean arterial pressure, pulse rate and fhr were noted between the groups . The incidence of side - effects did not differ between the groups [table 5]. Pruritis, shivering, nausea and vomiting, sedation, bradycardia and hypotension were reported and treated accordingly . The most common side - effect was pruritis, which was reported among 20% and 30% of the women belonging to groups s and f, respectively . Nine parturients (four in group s and five in group f; p = ns) experienced mild sedation during the study period . Neonatal outcome was assessed based on apgar scores at the 1 and the 5 minutes, which did not differ between the groups . Maternal satisfaction was assessed based on subjective assessment as excellent, good, satisfactory and poor . 93.33% of the women of group s and 90% of the women of group f opined labour analgesia as excellent . Highly lipid - soluble synthetic opioids such as sufentanil and fentanyl are being increasingly used along with very low concentrations of local anaesthetic agents such as bupivacaine (0.06250.125%) and ropivacaine (0.20.25%) to provide excellent relief of pain during labour . Unlike hydrophilic opiates (morphine) or intermediate lipid - soluble opioids (meperidine), lipophillic opioids do not spread rostrally in cerebrospinal fluid to any great extent, and they tend to have fairly segmental analgesic profiles . Quality of analgesia and overall satisfaction scores were found to be comparable in both groups in our study . In a dose response study, the analgesic potency of epidural sufentanil was reported to be approximately five - times that of fentanyl when it was administered as the sole analgesic after lidocaine anaesthesia for caesarean delivery . Addition of bupivacaine to intrathecal opioid prolongs duration of labour analgesia compared with either drug used alone . Evidence suggests that bupivacaine potentiates the binding of morphine to opioid receptors, especially the highly dense kappa receptors, as the result of an associated conformational change in opioid receptors . We observed that duration of analgesia provided by intrathecal sufentanil and bupivacaine was 109.706.37 min as compared with 73.6315.76 min by group f. this difference was statistically significant . These are similar to previously reported results using sufentanil: 11426 min and 99 min . This prolonged duration of analgesia with sufentanil could be attributed to the known superiority of sufentanil over fentanyl in terms of potency . Similarly, the number of supplementary bupivacaine top - ups was comparatively less in the sufentanil group (group s 1.220.75 and group f 2.060.081). Addition of bupivacaine 2.5 mg and sufentanil 10 g for labour analgesia showed that the combination, although effective, was associated with a higher incidence of hypotension and impairment of muscle power of the lower limbs, although mild in some parturients . Another investigation by halving the total amount of intrathecal sufentanil and bupivacaine reduced the incidence of side - effects . However, this reduced dose regimen was associated with slower onset and shorter duration of labour analgesia . As 10 mcg sufentanil is associated with severe hypotension and 2.5 mcg sufentanil produces analgesia of shorter duration and slower onset, in our study, we added 5 mcg sufentanil to bupivacaine 2.5 mg intrathecal . Cse has an added advantage of initiating labour analgesia even at the late stages of labour . We found that analgesia through intrathecal route resulted in immediate and predictable onset of analgesia enhancing the parturient's cooperation for the insertion of epidural catheter . The duration of first and second stage of labour and the mode of delivery between the groups did not differ significantly . The incidence of caesarean delivery was 20% and 16.66% between group s and f, respectively . Higher concentration of bupivacaine (0.25%) was used in the past, which resulted in a fairly higher incidence of motor block causing pelvic muscle relaxation, foetal malposition and maternal inability to push and a higher incidence of instrumental delivery . Incidence of motor blockade in our study groups was very minimal and was of bromage scale 1, which did not persist beyond the first hour . The pain relief during labour was assessed by vas scoring and the pain scores were comparable between the groups . It was graded as excellent in 93.33% parturients of group s and 90.0% parturients of group f, as in previous studies . The dose of opioids administered did not adversely affect the apgar scores or cause respiratory depression among newborns in our study . Pruritus was the most common side - effect between the two groups, which was not of a serious nature . The incidence of sedation and nausea was similar in the two groups, with no parturient in either group requiring any intervention . Continuous infusion of a dilute mixture of local anaesthetics and opioids offers the advantage of stable level of analgesia and increased maternal haemodynamic stability . However, automated regular bolus delivery of epidural analgesia when compared with continuous infusion is found to decrease the incidence of breakthrough pain and increased maternal satisfaction . Because of logistic constraints, we used intermittent epidural dosing on demand by the parturients, which, we found, could be comparable to continuous and automated delivery system . The limitation in our study was to combine primiparous and multiparous parturients, which could have altered the results of mode of delivery . We conclude that cse using sufentanil and fentanyl achieved high patient satisfaction and excellent labour analgesia without serious maternal or neonatal side - effects.
It stimulates the dopamine (d2) receptors and also inhibits the gamma amino butyric acid release in central nervous system . Common therapeutic doses of tramadol are 50 mg orally and 100 mg with parenteral and rectal route of administration up to 400 mg / day . The main adverse drug reactions of tramadol are nausea, dizziness, somnolence, drowsiness, increased sweating, vomiting, and dry mouth . Seizure and apnea are the most important life - threatening clinical presentations of tramadol in therapeutic and toxic doses . From this view, evaluation of laboratory findings including plasma electrolytes, kidney and liver function tests, and blood gas analysis have a critical role for patient monitoring . As there are few studies about laboratory findings in tramadol acute poisoning, we evaluated the clinical and laboratory findings in acute tramadol - intoxicated cases and their role in the prediction of seizure . This was a retrospective study on patients with acute tramadol poisoning who referred to loghman hakim hospital poison center, tehran, iran from january to april 2012 . The exclusion criteria were co - ingestion, intoxication with unknown dose of tramadol, uncertainty about time of tramadol ingestion, onset of a seizure before admission on hospital, the past medical history of epilepsy and history of drug / substance abuse . Data such as patients age, sex, time of ingestion, ingested dose, cause of intoxication, respiratory rate, pulse rate, systolic and diastolic blood pressure, temperature, coma grade scale on admission time, and therapeutic interventions and patients outcome were extracted from the medical records . Laboratory findings including blood sugar, blood urea nitrogen (bun), creatinin (cr), sodium (na), potassium (k), liver function tests, cell blood count, and blood gas on admission time were retrieved from patients medical records . The data were expressed as mean standard deviation / standard error (se) for continuous or discrete variables and as frequency and percentage for categorical variables . We used the student's t - test and mann whitney u - test for statistical analyses of continuous variables with and without normal distribution, respectively . Linear correlations between variables were assessed by spearman and expressed as the spearman correlation coefficient . The data were expressed as mean standard deviation / standard error (se) for continuous or discrete variables and as frequency and percentage for categorical variables . We used the student's t - test and mann whitney u - test for statistical analyses of continuous variables with and without normal distribution, respectively . Linear correlations between variables were assessed by spearman and expressed as the spearman correlation coefficient . We used spss software (version 13, spss inc ., chicago, il, usa). A total of 144 patients including 111 men (77%) and 33 (23%) women with the mean age of 23.7 6.9 (range = 15 - 57) years old included in this study . The average time between ingestion and admission on hospital (mean se) was 292.2 30 min (range = 30 - 3600 min). Mean duration of hospitalization was 17.9 10.6 h (range = 3.6 - 80 h). In all of the cases, the route of exposure was oral, and the most drug dosage form was tablet (n = 142) and then capsule (n = 2). The mean ingested dose (mean se) was 1971.2 233.4 mg (range = 100 - 20000 mg). In most of the cases (n = 99, 68.8%) the cause of intoxication was suicide and then abuse (n = 45, 31.2%) [table 1]. Demographic findings in tramadol - intoxicated patients major cases had stable vital signs on admission and the related data are summarized in table 2 . 128 (88.9%) of patients were conscious, and 16 of them had a decreased level of consciousness . Seizure (47.9%), nausea (29.9%), vomiting (22.2%), drowsiness (20.1%), dizziness (18.1%), lethargy (6.3%), apnea (5.6%), agitation (4.2%), headache (1.4%), blurred vision (1.4%), ataxia (0.7%), anxiety (0.7%), sweating (0.7%), and nystagmus (0.7%) were the most clinical findings in tramadol - intoxicated patients [table 2]. Laboratory findings on admission time in the intoxicated patients have been summarized in table 3 . Clinical findings in tramadol - intoxicated patients on admission time laboratory findings in tramadol - intoxicated patients on admission time we divided the cases with regard to occurrence of seizure during hospitalization . The results showed that there were significant differences between cases with seizure and cases without seizure according to time interval between tramadol ingestion and hospital admission (tibtiha) (mean se) (330.3 53.2 vs. 257.3 30.1 min, p = 0.01), ingested dose (mean se) (1395.7 218.3 vs. 2500.7 390.7 mg, p = 0.006), with odds ratio 2.7 (1.03 - 7.09, 95% confidence interval [ci]), dizziness (3 cases vs. 23 cases, p = <0.0001), with odds ratio 0.1 (0.29 - 0.36, 95% ci), pco2 (51.2 8.5 vs. 48.4 8.6 mmhg, p = 0.03), with odds ratio 0.58 (0.27 - 1.24, 95% ci), and total bilirubin (0.5 0.2 vs. 0.7 0.4 mg / dl, p = 0.002). There was a correlation between ingested dose (r = 0.2, p = 0.006), paco2 (r = 0.2, p = 0.03), tibtiha (r = 0.2, p = 0.01), total bilirubin (r = 0.3, p = 0.002), dizziness (r = 0.3, p = 0.000), and seizure . Tramadol abuse and overdose is one of the most frequent health problems in iran and worldwide . In this study, we report clinical and paraclinical findings in 144 cases with pure tramadol poisoning who referred to a referral - poisoning center in tehran, iran . Furthermore, the results indicated that oral route is the most common route of exposure, which is similar with the findings of our previous studies . The frequency of other clinical manifestations including lethargy, coma, nausea, vomiting, agitation, and respiratory depression in our study were different with previous studies . Tramadol is metabolized by cytochrome p450 (cyp450) enzymes (mainly 2d6 isoenzyme) to its active metabolites m1 (o - desmethyl tramadol), m2 (n - desmethyl tramadol), m3 (n, n - didesmethyl tramadol), m4 (o, n, n - tridesmethyl tramadol), and m5 (o, n - didesmethyl tramadol). M1 metabolite has more affinity (200 times) for the -opioid receptors and also it has more inhibitory effect on biogenic amine reuptake than that of parent drug molecule and may be responsible for tramadol induce analgesia or seizure in intoxicated patients . In this regard, genetic polymorphism in humans may affect the tramadol metabolism and its peak blood concentration resulting to a different frequency of tramadol adverse effects or clinical presentations during therapeutic doses or intoxication . Although the increasing of liver function tests, serum bun and cr due to liver and kidney damage have been demonstrated in chronic administration of tramadol in experimental model, but in our study due to acute onset of toxicity we did not observe any increase in liver function tests, bun and cr and this is in concordance of the previous study . Rhabdomyolysis and rise of creatine phosphokinase (cpk) have been reported as a rare and serious complication in tramadol intoxication in the previous studies, which was observed in our study too . Although in the previous studies, prolong immobilization and multiple seizures have been described as one of the reasons for the rise of cpk and rhabdomyolysis, but in our study there was no significant difference in level of cpk in the tramadol - intoxicated patients with seizure in comparison to cases without seizure . Le berre et al ., reported tramadol induced hyponatremia which described as a result of inappropriate antidiuretic hormone secretion . In this study, the level of na, and k was within the normal range . The pco2 level was above normal range which could be attributed to tramadol - induced respiratory depression, which has been reported previously . In this study, the mean of ingested dose in the seizure group was less than those cases without seizure which is in contrasts with the result of the previous study . One of the explanations is the difference between two groups with regard to tibtiha, which was significantly longer in seizure group . Furthermore, as mentioned previously, the other reason could be the genetic polymorphism in patients . The main limitation of this study is its retrospective design, which should be considered in the interpretation of the results . With this regard there were significant differences between seizure and nonseizure cases with regard to tibtiha, ingested dose, and pco2 . Furthermore, we showed poor correlation between tramadol ingested dose, tibtiha, pco2, and seizure in tramadol - intoxicated cases . Hrr involving in the data collection and data analysis . Ss and ksn involving in the study design, conducting of the study and data analysis.
Familial glucocorticoid deficiency (fgd) is a rare autosomal recessive disorder resulting from adrenal unresponsiveness to adrenocorticotropic hormone (acth). This disease is characterized by low serum cortisol concentrations in the presence of grossly elevated plasma acth levels . Affected individuals typically present with hyperpigmentation, hypoglycemic seizures, failure to thrive, failure to thrive and recurrent infections . Causal mutations of fgd have been identified in mc2r, mrap, mcm4, txnrd2, star and nnt . Mc2r accessory protein (mrap), a 19-kda single - transmembrane domain protein, is essential for trafficking of mc2r from the endoplasmic reticulum (er) to the cell surface and subsequent signaling in response to acth . Mutations in mrap are responsible for 1520% of fgd cases, named fgd type 2 . Until now, this mutation has not been reported in chinese han cases of fgd . In the current paper, we describe a chinese han child who presented with isolated hyperpigmentation at birth and a homozygous splice mutation (c.106 + 1delg) in mrap . A female infant was referred to our neonatal unit at the age of nine days for evaluation of hyperpigmentation of the skin . She was the product of an uncomplicated pregnancy and was born by caesarian section for concerning cardiotocographic changes during labor . Her sibling had died on the third day of life with hyperpigmentation of the skin . Birth size was normal (weight 2800 g, 0.99 sds; length 46 cm, 1.69 sds; head circumference 33 cm, 0.74 sds) and apgar scores was 8 and 9 at 1st and 5th minute, respectively . On physical examination, weight was 2740 g (1.31 sds), length 47 cm (2.47 sds), and head circumference was 34.5 cm (1.5 sds). Endocrine investigations revealed low baseline 8 am serum cortisol (<1.0 g / dl) with extremely elevated acth (1250 pg / ml). Testosterone, 17-oh progesterone, estradiol, progesterone, luteinizing hormone, folliculin, and prolactin, free t4, and tsh levels were normal . Based on these findings, a familial glucocorticoid deficiency was suspected and oral hydrocortisone treatment at dose of 20 mg / m per day was started . Hydrocortisone dose (varying from 10 to 15 mg / m per day) adjustment was based on acth and cortisol concentrations . After 4 weeks, acth level was 278.0 pg / ml and cortisol level was 42.4 g / dl, and hydrocortisone dose was reduced to 15 mg / m per day (given in divided doses twice daily). The acth level was suppressed to slightly above the normal limit and the cortisol level is normal during the follow - up period . On her most recent visit, at the age of 12 months, hydrocortisone dose was 11 mg / m per day (5 mg was give once daily in the morning), the acth level was 25.10 pg / ml and cortisol level was 33.4 g / dl, and her skin was slightly lightened (fig . Her weight was 10 kg (0.84 sds), length 70 cm (1.67 sds), and head circumference was 46.5 cm (1.12 sds). Genomic dna was extracted from peripheral blood leucocytes from the infant and her family after informed consent was obtained . For the molecular diagnosis, a custom panel - based next - generation sequencing approach has been used to sequence all known adrenal gland diseases - associated genes in this child . We found a homozygous deletion of one nucleotide at the canonical 5 donor splice site (c.106 + 1delg) in intron 3 of mrap gene . This would result in the skipping of exon 3 and a prematurely terminated translation product ((1)). Her parents and maternal grandmother were found to be heterozygous carrier for the same mutation (fig . We report a case of fgd with homozygous mutation of mrap in a chinese han neonate . This girl presented with isolated hyperpigmentation, have low cortisol and high acth with normal electrolytes . A homozygous splice mutation (c.106 + 1delg) of mrap confirmed the diagnosis of fgd type 2 . Her parents and maternal grandmother had the same mutation . Until now, this mutation has not been reported in chinese han cases of fgd . Fgd is a rare autosomal recessive disorder characterized in laboratory testing by glucocorticoid deficiency and markedly elevated acth levels . Patients with fgd usually present during neonatal period to late childhood with symptoms related to cortisol deficiency and acth excess . In the neonatal period, these symptoms may include hyperpigmentation, hypoglycemia, irritability, jitteriness, lethargy, respiratory distress, cyanosis, apnea, hypotonia, seizures, shock, or sudden death . Newborns can present with a positive family history of early - unexplained infant deaths or other affected family members supports a diagnosis . The severe pigmentation of the skin is due to the over - stimulation of mc1r (cutaneous msh receptors) by high circulating msh which is a byproduct of acth synthesis from proopiomelanocortin . This hyperpigmentation fades once proper treatment is initiated with glucocorticoids, which reduce acth concentrations . This would suggest that the fetal corticotrophs could produce excessive plasma acth in response to low fetal cortisol, which in turn acts on melanocytes to promote eumelanin synthesis before birth . In most of the fgd patients suppression of acth levels is difficult, and therefore is not used as a goal for therapy . In this case, replacement treatment with hydrocortisone suppressed acth level to slightly above the normal limit and partially resolved the hyperpigmentation . Acquired causes of adrenal insufficiency such as adrenal hemorrhage, trauma and infections were excluded by history and laboratory findings . Congenital adrenal hyperplasia was excluded by hormone analysis; congenital adrenal hypoplasia was also unlikely, because she had normal serum 17-oh progesterone and no mutation of the dax1 gene . Mrap, located at 21q22.1, is a small single - pass transmembrane domain protein, which is essential for the processing of the acth receptor (melanocortin 2 receptor, mc2r) and its trafficking from the er to the cell surface . This mutation will lead to skipping of exon 3 and early truncation of the protein and the absence of the mc2r interacting transmembrane domain . As a result, mc2r is retained within the er and fails to reach the cell surface which lead to acth resistance and adrenal insufficiency . Mutations in mrap causing fgd2 were first described in 2005 . So far over 9 different mutations of mrap in fgd type 2 patients have been documented all of which are splice site or nonsense mutations and are predicted to produce proteins lacking the transmembrane domain essential for interaction with mc2r, . In our patient, we found a newborn with fgd type 2, presenting with severe hyperpigmentation . She was found to have a splice - site mutation in the mrap gene, hence explaining the early presentation seen in the case . In their study, metherell et al . Identified the 1-bp deletion, c. 106 + 1delg, in 6 individuals from 5 families with glucocorticoid deficiency, making this the second frequent mutation causing fgd unrelated to defects in the mc2r gene . To our current knowledge, 1-bp deletion (c. 106 + 1delg) in intron 3 of mrap gene, identified in the dna of the patient, cases of the condition have been reported in white,, black, indian, and middle eastern populations . To our knowledge, our patient is the first reported chinese han patient with fgd type 2, with a known mrap mutation . The patient was the offspring of non - consanguineous parents and her sibling had died in the neonatal period, possibly due to glucocorticoid insufficiency . Prolonged acth excess or glucocorticoid deficiency increases linear growth, while early diagnosis and appropriate therapy in this case enabled the patient to achieve normal developmental milestones . Further studies describing new cases and mutations causing fgd will contribute to understanding the mechanism of this rare and potentially life - threatening disease.
Pulmonary arteriovenous malformation (pavm), first described in 1897 by churton, is characterized by abnormal communications between pulmonary veins and arteries, and is known to disturb the filtering action of pulmonary capillaries, causing thromboembolic event in systemic circulation . Most of the pavms have no symptoms, and are detected as abnormal shadow in the chest . Conclusive diagnosis is generally made by means of pulmonary arteriography or three - dimensional computed tomography (3d - ct) angiography . While hypervascular lesions can mimic pavm, we observed a rare case of a lung granuloma mimicking pavm, and performed video - assisted thoracic surgery (vats). A 76-year - old woman, who was otherwise symptom - free, was admitted to our hospital because an abnormal shadow was detected in the left lung field on her chest x - ray . Chest computed tomography (ct) revealed a 20 mm 14 mm nodule with well - defined margins and smooth contours in the left upper segment (fig . An enhanced solitary lung nodule, connected with linear structures suggestive of feeding artery and drainage vein, was revealed by contrast - enhanced 3d - ct (fig . Arterial blood gas analysis showed hypoxemia, with arterial oxygen pressure (pao2) 56 mmhg in room air . First, the patient was placed in the right lateral decubitus position, and the left lung was deflated . Intraoperatively, elastic hard nodule was palpable in the left upper segment and bruit was not convincing . The nodule was nontortuous in shape, covered with the visceral pleura, and neither the feeding artery nor the drainage vein was detected . Histopathological findings revealed multiple foci of coagulative necrosis surrounded by epithelioid cell granuloma containing langhans - type multinucleated giant cells, involving the medium - sized blood vessels in the pulmonary parenchyma . The lung granuloma was difficult to be preoperatively distinguished from pavm, because hypervascular lesion such, as inflammatory changes can present as strongly enhanced nodules after injection of contrast material . The patient had an uneventful postoperative course and was discharged 14 days after the operation . Causes of acquired or secondary pavm include chest trauma, thoracic surgery, hepatic cirrhosis, infections (actinomycosis, schistomiasis), metastatic carcinoma and systemic disease . Twenty eight percent of cases are considered to have no symptoms . It was reported that when a single isolated pulmonary arteriovenous malformation is 2 cm or smaller, no symptoms appear . Generally, when pavm is 2 cm or more, symptoms such as breathing difficulty, cyanosis, hypoxemia, finger clubbing and polycythemia occur, and the rate of occurrence of severe complications, such as rupture of the malformation, hemoptysis, cerebral infarction and cerebral abscess, is approximately 30% . The patient in the present case was asymptomatic and had no history of chest trauma, surgery, infection or systemic disease . Abnormal vascular structures, such as pavm are not convincing in histopathological findings . Coincidentally, the inflammatory granuloma of the lung looked almost identical to pavm because of the nontortuous shape with linear structures suggestive of feeding artery and drainage vein . The resection of lung granuloma is highly successful for an isolated malformation, and mortality rate is low in this surgery . Embolization is an appropriate treatment modality in multiple pavm for which surgery is not suitable . Complications of embolization include pleuritic chest pain, pulmonary infection, air embolism, migration of coils and paradoxical embolism . Although pulmonary angiography or contrast - enhanced 3d - ct has been the standard diagnostic tool for pavm, it has limitations in detecting pavms presenting as small nodules and enhanced nodules . In our case, since the enhanced lung nodule size was 22 mm in diameter, it mimicked pavm . In this patient, it was difficult to distinguish the lung granuloma from pavm, preoperatively . Though there is a report which shows lung cancer mimicking pavm, to our knowledge, this is the first report of a lung granuloma mimicking pavm . In conclusion, clinicians should pay particular attention to enhanced nodules to rule out a variety of disorders, including neoplasm, infection, inflammatory and vascular abnormality when pavm was suspected . For the purpose of not only diagnosis but also for safety in treatment of pavm, the surgical approach seems appropriate . 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 on request . Yoshinobu ichiki: study design, data collections, data analysis, writing; junji kawasaki: data collections; takayuki hamatsu: data collections; taketoshi suehiro: data collections; makiko koike: data collections; fumihiro tanaka: study design, data collections, data analysis; keizo sugimachi: data collections.
Although chronic hepatitis c (chc) in children is usually asymptomatic, it is often persistent and is a possible cause of morbidity in later life . Moreover, progressive liver disease, including cirrhosis, has been reported during childhood . Although new direct - acting antivirals (daas) are now the cornerstones in the treatment of hcv infection in adults, combination therapy with pegylated interferon (peg - ifn) alpha and ribavirin (rbv) (peg / rbv therapy) is still the current standard therapy in children [2, 3]. Although this regimen was found to be highly effective in children with genotypes 2 and 3, it has limited efficacy for those with genotype 4 [5, 6], the prevalent genotype in egypt . Therefore, the ability to predict response to this costly with many side effects antiviral regimen remains an important research goal . In spite of the extensive researches in this field, knowledge is still defective . On defining predictors of treatment response, many studies concentrated on virus genotype, gender, age, race, and fibrosis stage, among others . Nevertheless, all of these are nonmodifiable factors but only give a prediction about the likelihood of sustained virologic response (svr). Moreover, some of them are expensive and invasive . On investigating the role of interleukin-28b (il28b) polymorphism in pediatric patients with chc, one study suggested that il28b genotype was not a strong predictor of svr, while other studies have shown usefulness of il28b genotype as a predictor of treatment response [1012]. Virological responses during therapy, such as rapid virologic response (rvr) and early virologic response (evr), are widely used for predicting end of treatment response (etr) and svr . Nonetheless, it is obvious that predictions made before administration of therapy are more desirable than those done during treatment course . We aimed to investigate some inexpensive, easy to perform, and noninvasive modifiable (as adiponectin and vitamin d) and nonmodifiable (as alpha - fetoprotein afp) factors which may have a relation to treatment response in children with chc . If we found a significant relation to successful treatment, we can use them not just to predict the likelihood of treatment response but also to improve the svr rates by modulating the modifiable ones . This knowledge can also constitute a base for later on researches when daa became approved in pediatric age . This prospective cohort study included fifty children with chc recruited from pediatric hepatology department, national liver institute (a tertiary level institute), menofiya university, egypt, between june 2012 and june 2014 . Their mean age was 11.46 3.48 years and thirty three (66%) of them were males . Another 21 healthy children with comparable age (mean; 9.14 3.31 years) and sex (males; 13 61.9%) to the disease group were enrolled as controls . Chc was diagnosed on the basis of positive anti - hcv antibodies and positive hcv - rna for more than 6 months duration, together with the pathological picture of chc . Any case with associated liver disease (either identified by laboratory or histological examination) or out of the inclusion criteria for peg / rbv therapy, as defined by el naghi et al . A signed informed consent was obtained from parents of all recruited children before enrollment in the study . The study was approved by the research ethics committee of the national liver institute, menofiya university, and conforms to the 1975 declaration of helsinki and its later amendments . Alanine transaminase (alt), aspartate transaminase (ast), prothrombin time (pt), complete blood count (cbc), thyroid stimulating hormone (tsh), and serum autoantibodies (anti - nuclear antibodies, anti - smooth muscle antibodies, and liver - kidney microsomal antibodies) were performed for every patient . Viral markers (anti - hcv, hepatitis b virus (hbv) surface antigen, hbv core immunoglobulin (ig)m, and igg antibodies) were performed for all patients using chemiluminescence immunoassay (roche diagnostic inc ., real - time pcr for hcv - rna was performed using abbott m2000 real - time system (abbott molecular inc ., des plaines, illinois, usa) for every patient before starting therapy and at week 12 of therapy . For those who continued therapy, abdominal ultrasound was performed using 25 mhz curved linear and 48 mhz linear transducers (xario xg; toshiba, tokyo, japan). An ultrasonographic - guided liver biopsy was performed for all patients using a true - cut needle, size 16 g. biopsy specimens were fixed in formalin, embedded in paraffin, and finally the slides obtained were stained by hematoxylin and eosin (h&e), perls, and orcein for routine histopathological evaluation . Grades of necroinflammatory activity 13 were ascribed as minimal, grades 47 as mild, and grades 812 as moderate, whereas grades> 13 were ascribed as severe chronic hepatitis . Stages of fibrosis of 0 - 1 indicated absent / minimal fibrosis, stages 2 - 3 indicated significant fibrosis, and stages 46 indicated advanced fibrosis . All children with chc received peg - ifn-2b (pegintron; schering - plough, new jersey, usa) at a dosage of 60 g / m / week subcutaneously and rbv orally at a dosage of 15 mg / kg / day on two divided doses . Thirty - six (72%) children attained etr and svr with no relapses, while 14 (28%) children were nonresponders . Serum samples were collected from every patient before starting peg / rbv therapy and from healthy controls then stored at 80c until used . Serum adiponectin level was determined by the human adiponectin (acrp30) enzyme - linked immunosorbent assay (elisa) kit (orgenium laboratories, vantaa finland). Serum vitamin d was measured using 25-oh vitamin d enzyme - immunoassay (eia) kit (immundiagnostik, bensheim and biomedica, wien austria) intended for the quantitative determination of the 25-oh vitamin d in plasma or serum . Lastly, serum afp was measured by quantikine, a human afp eia kit (r&d systems inc ., minneapolis, usa). Serum levels of the three test parameters were expressed as nanograms per milliliter (ng / ml). Values were expressed as mean standard deviation (range) or number (percentage) of individuals with a condition . For quantitative data, statistical significance was tested by either independent samples t - test or nonparametric mann - whitney u test according to the nature of the data . For qualitative data, a multivariate analysis was performed using a binary logistic regression analysis for factors that significantly associated with treatment response on univariate analysis . The cutoff values for optimal clinical performance of adiponectin, vitamin d, afp, and level of hcv - rna for differentiation between responders and nonresponders were determined from the receiver - operating characteristic (roc) curves . The diagnostic performance was presented as sensitivity, specificity, negative predictive value (npv), positive predictive value (ppv), and accuracy . Body mass index of children with chc was within average with a mean of 18.56 2.79 kg / m . Level of hepatitis c viremia ranged from 8.04 10 to 6.23 10 with a mean of 6.28 10 1.23 10 iu / ml . Forty - five (90%) patients with chc showed mild stage of fibrosis and 5 (10%) cases showed moderate fibrosis . Most of (86%) chc children had mild activity, while 7 (14%) cases had minimal activity . Serum adiponectin was significantly higher in the chc group than healthy controls (8.92 2.85 and 6.049 1.04 ng / ml, resp . ; p <0.0001). Also, in spite of being insignificant, serum vitamin d and afp were higher in the chc group (71.6 49.1 ng / ml and 3.6 2.96 ng / ml, resp .) Than healthy controls (46.4 21.8 ng / ml and 3.0 0.39 ng / ml, resp .) Pretreatment factors that could be associated with response to peg / rbv therapy were compared between treatment responders (svr) and treatment nonresponders (figure 2). It was found that adiponectin was significantly higher in those with svr (9.79 2.7 versus 6.69 1.77 ng / ml; p <0.0001). On the other hand it was found that both afp and viremia were significantly lower in the treatment responders (2.84 0.51 ng / ml and 4.18 10 1.03 10 iu / ml, resp .) Than in nonresponders (5.69 5.1 ng / ml and 1.16 10 1.57 10 iu / ml, resp .) With p <0.0001 and p = 0.0003, respectively . Lastly, vitamin d was found to be higher in the treatment responders (77.2 46.6 ng / ml) than nonresponders (57.2 53.9 ng / ml), with borderline significance, p = 0.076 . Other studied pretreatment parameters showed no difference between responders and nonresponders (table 1), while in multivariate analysis adiponectin was shown to be the only significant independent predictor of treatment response (p = 0.044) (table 3). Cutoff points for variables showing significant associations with treatment response were analyzed by the roc curves (figure 3). For adiponectin it was found that at a cutoff value of> 8.04 ng / ml, it can predict treatment response by 77.8% sensitivity, 92.9% specificity, 96.6% ppv, 61.9% npv, and 82.3% accuracy, while afp and hcv - rna at cutoff values <3.265 ng / ml and <235,384 iu / ml, respectively, can predict treatment response with a sensitivity of 83.3% and 83.3%, specificity of 85.7% and 78.6%, ppv of 93.75% and 90.9%, npv of 66.7% and 64.7%, and accuracy of 82.36% and 79.38%, respectively . Adiponectin was found to be significantly negatively correlated with both afp (r = 0.29 and p = 0.043) and level of viremia (r = 0.39 and p = 0.005), with no significant correlation with other studied parameters . Also, there was no significant correlation between afp, vitamin d, and level of viremia and all other studied parameters (table 2). This study is, to our knowledge, the first to show the predictive value of baseline serum levels of adiponectin, vitamin d, and afp for the treatment response of chc in children . It is a protein hormone that modulates a number of metabolic processes including glucose and fatty acid catabolism . The anti - inflammatory effects of adiponectin could protect the liver from the development of inflammation and cell injury . In the present work, the significantly higher adiponectin in the chc group than the control group may be due to an anti - inflammatory role of adiponectin in those with chc . In previous studies, adiponectin was found to directly affect the inflammatory response by regulating both production and activity of cytokines . In addition, hypoadiponectinemia has been reported to enhance hepatic steatosis, inflammation, fibrosis, and hepatocarcinogenesis in animal models of liver diseases [1719]. Moreover, nonalcoholic steatohepatitis patients show lower levels of adiponectin with higher grades of inflammation . In this work, the pretreatment serum level of adiponectin was significantly higher in the treatment responders (svr) than nonresponders, and at a cutoff value of> 8.04 ng / ml it can predict the treatment response by a sensitivity of 77.8% and a specificity of 92.9% . . Found that lower adiponectin was an independent predictor of no virological response at the end of treatment (p <0.001). This may indicate the benefit of the anti - inflammatory role of adiponectin in those with chc . Adiponectin administration, in both alcoholic and nonalcoholic fatty liver in mice, was found to suppress hepatic production and the circulating levels of tumor necrosis factor- and ameliorates hepatic steatosis . Moreover, the significant negative correlation found in this work between serum adiponectin and viremia may indicate an antiviral role of adiponectin . On the other hand this later concept was suggested by the study of zografos et al . Who found a significant increase of adiponectin at the end of hcv treatment for those with etr . Abdel latif et al . Found that serum adiponectin levels were lower in hcv - infected patients with steatosis than in those without steatosis and these levels tend to decrease with the increase in the grade of steatosis, the advance in the grade of histological activity, and the stage of fibrosis . In our work, there was no significant correlation between adiponectin and stage of fibrosis or grade of necroinflammatory activity . This difference may relate to the difference in age range and the relatively mild histological affection of our chc group . According to the previous results, not only can adiponectin be used as a reliable pretreatment predictor of treatment response in combination with other defined parameters but also it can be tried as an adjuvant therapy with peg / rbv especially for those with pretreatment lower serum levels . To prove this, it needs a well controlled clinical trial . Beside its action in calcium homeostasis, vitamin d has a significant immunomodulatory action and is an important mediator of innate and adaptive immune systems . In spite of many researches, generally, in relation to vitamin d synthesis in the liver, mild to moderate liver dysfunction causes malabsorption of vitamin d. moreover, liver dysfunction of 90% or more results in inability to make sufficient 25-oh vitamin d . Some researchers showed that adults with chc have higher incidence of severe 25-oh vitamin d deficiency compared to the normal control . On the contrary, in the present study we found that vitamin d was higher in children with chc than normal controls with borderline significance (p = 0.071). Study may be due to younger age and milder liver affection in our children compared to their adult population . Hypothetically, this reported vitamin d increase in children with chc reflects a possible antiviral role of vitamin d. this is supported by matsumura et al . Who demonstrated in in vitro study that 25-oh vitamin d is an anti - hcv agent that targets viral particle assembly step . . Found that low vitamin d serum level is related to severe fibrosis in adults with chc . However, we did not find significant correlation between vitamin d and any of the stage of fibrosis or grade of activity or any other studied pretreatment parameter . However, no previous data are available for the pediatric age group . In the present work, a higher vitamin d level was found in hcv treatment responders than nonresponders but with borderline significance ., bitetto et al ., and nimer and mouch detected an association between lower vitamin d serum levels and failure to achieve svr in adults with chc . On the other hand, lange et al . Found that pretreatment serum level of vitamin d is not an optimal predictor of treatment response in hcv genotype 1 . Hypothetically, in the presence of vitamin d deficiency, it might be preferable to correct the deficiency before starting antiviral therapy . Nevertheless, to date there are few published reports on the role of vitamin d supplementation in patients with chc . . Found that vitamin d supplementation, in adults with recurrent hepatitis c postliver transplant, improves the probability of achieving a svr . Also, nimer and mouch found that adding vitamin d to conventional peg / rbv therapy for patients with hcv genotypes 2 - 3 significantly improved viral response the reported higher vitamin d among treatment responders in the present study together with the known immunomodulatory action of vitamin d and previous clinical trials of vitamin d supplementation in adults [29, 30] suggest that adding vitamin d to peg / rbv therapy in children may increase svr rates without serious adverse events . However, to prove these findings, well designed and large prospective studies are needed . Following birth, afp levels decrease rapidly to less than 20 ng / ml and increase significantly in certain pathologic conditions ., significant elevations of afp are commonly seen in nonhepatic malignancies and benign conditions, such as acute and chronic viral hepatitis . Previous studies reported that the prevalence of increased serum afp varies from 10% to 43% in adult patients with chc and suggested an association between an increased serum afp and advanced fibrosis or cirrhosis [3336]. In agreement with the above reports, we found that afp was higher in children with chc than healthy controls with statistically insignificant difference . The absence of a significant correlation between afp and stage of fibrosis in the present work may be due to the milder liver affection with low stages of fibrosis in reported cases when compared to the adult series . Some studies found significant correlation of pretreatment low afp serum level and treatment response in adults with chc [3739]. In accordance with the previous studies, we found that lower serum afp was significantly related to svr in children with chc but with borderline significance in multivariate analysis . The svr was 93.75% among children with afp below 3.265 ng / ml and 33.3% for children with afp 3.265 increased production of afp in hepatitis and cirrhosis was first thought to reflect the process of surviving hepatocytes, but this hypothesis has been refuted by other reports [33, 40]. More recent hypothesis ascribes the increased serum afp to the hepatic damage per se with selective transcriptional activation of the afp gene . In the present study, the reported negative correlation between adiponectin with its known hepatoprotective role and afp (p = 0.043) is supporting this hypothesis and may give an explanation to the link between the baseline serum afp and the treatment response . In the present study viral load in univariate analysis was found to be a significant pretreatment predictor of treatment response as it was shown in previous reports . However, in multivariate analysis, it was not an independent predictor of treatment response . The emergence of the new daas for the treatment of chc [41, 42] may be considered a limitation in the current study . However, peg / rbv is the still approved standard therapy for children with chc [5, 43]. Moreover, peg - ifn and rbv are still included with daas in the treatment regimens of those with genotype 4, the prevalent genotype in egypt . A major limitation of this study is the low number of cases and controls enrolled . Also, absence of testing for hcv genotype and il28b genotype could be considered another limitation of this work . In conclusion, serum adiponectin can be added to the list of the pretreatment determinants of svr in children with chc, with the advantage of being easy to perform, noninvasive, and modifiable . In spite of its significant prediction for the likelihood of response, we cannot state to screen and select the patients to be treated or not based on their pretreatment levels of this predictor . Instead, it can be used to prioritize patients to treatment when resources are limited, thus avoiding toxicities and cost for those unlikely to respond to treatment . Also as it is a modifiable factor, supplementation for deficient cases
Radical prostatectomy (rp) is considered to be a standard curative procedure for patients with organ - confined prostate cancer . Recently, the advent of pioneering nerve - sparing robot - assisted laparoscopic radical prostatectomy (ns ralp) has significantly increased the potency rate after rp [1 - 3]. However, even if ns ralp is performed, a large number of patients still experience erectile dysfunction (ed) after surgery . Therefore, the need for better penile rehabilitation is anticipated . This increase in need for penile rehabilitation stems from two pillars . First, the introduction of prostate - specific antigen (psa) testing in the 1990s made early detection of prostate cancer possible . The early screening using psa testing led to a significant drop in patient age, and younger prostate cancer patients have great interest in and need for erectile function (ef) after surgery . Second, recent advances in surgical techniques have motivated more prostate cancer patients to consider penile rehabilitation . Ns ralp by an experienced surgeon can minimize nerve injury, and early penile rehabilitation with oral administration of phosphodiesterase type 5 inhibitors (pde5-is) can maximize penile recovery in ed patients after rp . After the advent of the orally administered pde5-is in 1998, there was a huge shift in the ed treatment algorithm . Recent literature shows that pde5-is are safe and effective in treating ed patients after rp . Once daily administration of pde5-is has been experimentally proven to have a protective role in ed, and early administration of pde5-is helps to prevent cavernosal hypoxia, which leads to smooth muscle apoptosis and penile fibrosis [8 - 11]. Although pde5-is are one of the standard treatments for ed after rp, consensus has not been reached on their use, such as when to start, which drug is best, which dosage to use, how long to use, and when to stop . In the case of tadalafil, doses of 10 and 20 mg have been administered on demand to ed patients, and once - a - day dosing of 5 mg has been used since the korea food and drug administration approval in 2008 . Moreover, in a study that compared the efficacy, safety, and tolerability of on - demand tadalafil and daily dosed tadalafil for the treatment of ed, 72% of the patients preferred daily dosed tadalafil . However, clinical studies on the daily administration of low - dose pde5-is and the effect of such treatment on ed patients after rp are sorely lacking . The present study was therefore conducted to evaluate the efficacy and safety of once daily use of tadalafil 5 mg in the treatment of ed patients after ralp . We evaluated the records of 116 patients who, between september 2007 and december 2011, had undergone ralp by an experienced surgeon for localized prostate cancer at our institution . Ralp was first performed in 2004 as a joint effort between our institution and singapore general hospital, which was the site of the initial korean experience . Standard ralp techniques including bladder neck preservation and posterior urethral reconstruction were used because these techniques during ralp can have favorable impacts on the early postoperative recovery of continence . The 116 patients who developed ed after ns ralp, who had normal preoperative ef, and whose local clinical stage was t2 or lower with a gleason score lower than 8 and a serum psa of less than 20 ng before surgery were included in the study . The additional exclusion of 24 patients who underwent hormonal or radiation therapy preoperatively or postoperatively, who received any kind of preoperative ed treatment, or to whom pde5-is could not be prescribed resulted in 92 patients . The 92 patients were retrospectively evaluated in accordance with the ethical standards of the institutional review board at dong - a university hospital . In a study of the 92 patients, 47 patients were prescribed tadalafil at a dose of 5 mg per day . In the tadalafil group, all patients took 5 mg tadalafil per day 1 hour before bedtime . For all 47 patients, oral intake of tadalafil 5 mg was initiated early after foley catheter removal, between 2 and 3 weeks after the surgery . The tadalafil group was then stratified depending on the ns procedure they had undergone: bilateral ns (n=30) and unilateral ns (n=17). Postoperative ef was assessed through the patients' responses to the korean version of the five - item international index of erectile function (iief-5) questionnaire, which is also referred to as the sexual health inventory of men . The iief-5 questionnaire is the abridged version of the iief-15 and consists of 5 questions related to ed diagnosis and ef improvement . The iief-5 questionnaire is a self - administered questionnaire that can sensitively detect subtle changes in ef recovery . Each of the 5 questions is scored from 0 to 5 or from 1 to 5, where 0, the absence of sexual activity; 1, very low / almost never or never / extremely difficult; 2, low / a few times / very difficult; 3, moderate / sometimes / difficult; 4, high / most times / slightly difficult; and 5, very high / almost always or always / not difficult . The total iief-5 score is calculated by summing each score from the five questions and varies from 1 to 25, where a score of 1 to 7, severe ed; 8 to 11, moderate ed; 12 to 16, mild - moderate ed; 17 to 21, mild ed; and 22 to 25, no ed . In the present study, the iief-5 questionnaire was conducted 3 times: before surgery, at 6 months after surgery, and at 1 year after surgery . All patients were also counseled on ed, tadalafil intake, and its side effects at each visit . In the present study, positive responders were defined as those patients whose combined score for q2, " when you had erections with sexual stimulation, how often were your erections hard enough for penetration? " And q3, " during sexual intercourse, how often were you able to maintain your erection after you had penetrated your partner? " On the iief-5 questionnaire was 8 or more . Outcomes from the tadalafil group and the non - tadalafil group were compared and analyzed according to ns status on the basis of the patients' iief-5 score . Data were summarized by using descriptive statistics: frequency and percentage for categorical variables and meanstandard deviation and median (range) for continuous variables . Differences in the patients' demographic and clinical characteristics were compared across subgroups with fisher exact test for categorical variables or mann - whitney u - test for continuous variables . Wilcoxon's rank sum test was performed to test differences in iief-5 domains between time points . The cumulative incidence of recovery of erection firm enough for sexual intercourse was estimated by the kaplan - meier product limit method . The log - rank test was also employed to investigate the difference in recovery of erection between groups . A p - value lower than 0.05 was considered statistically significant . By its nature, this study was explorative and, therefore, no adjustment for multiple testing was applied . All statistical analyses were carried out by using ibm spss ver . 19.0 (ibm co., armonk, ny, usa) and medcalc 11.6.1.0 (medcalc, ostend, belgium). Demographic and clinical variables at baseline were well balanced between the tadalafil group and the non - tadalafil group (table 1). The 92 patients who were screened for eligibility according to the inclusion and exclusion criteria were retrospectively assessed in the present study . The patients in the tadalafil group (n=47) and those in the non - tadalafil group (n=45) were also stratified according to ns status . In the tadalafil group, 63.8% of the patients (n=30) underwent a bilateral ns procedure and 36.2% of the patients (n=17) underwent a unilateral ns procedure . In the non - tadalafil group, bilateral ns was performed for 60% of the patients (n=27) and unilateral ns was performed for 40% of the patients (n=18). Compared with the non - tadalafil group, the tadalafil group regardless of ns procedure reported a statistically significant improvement in ef (p=0.0049, log - rank test). At 6 months after the surgery, 11% of the patients in the tadalafil group (n=5) responded positively to tadalafil, whereas there were no positive responders in the non - tadalafil group . At 12 months after the surgery, 32% of the patients in the tadalafil group (n=15) were able to perform intercourse, and 9% of the patients in the non - tadalafil group (n=4) had erection sufficient for sexual intercourse . Once - daily use of tadalafil 5 mg resulted in ef improvement during the course of 1 year after ns ralp (fig . The mean iief-5 score at 1 year also differed between the tadalafil group and the non - tadalafil group as shown in table 2 (p<0.05, wilcoxon's rank sum test) and in fig . Most of the patients who had undergone ns ralp experienced ed after the surgery . At 6 months, the total iief-5 scores of the tadalafil group and the non - tadalafil group were 10.03.4 and 7.04.0, respectively (p<0.0001, mann - whitney u test). At 1 year after the surgery, the total iief-5 score in the tadalafil group was significantly greater than that in the non - tadalafil group (13.25.6 vs 7.74.8, p<0.0001; mann - whitney u test). Moreover, there was a significant increase in all 5 domains of the iief-5 in the tadalafil group, whereas in the non - tadalafil group no significant increases were observed in any of the domains at 1 year (q4, p<0.0001; q1, p<0.0001; q3, p<0.0001; q2, p=0.006; q5, p<0.0001). In the tadalafil group, the iief-5 score of the bilateral ns group was greater on all 5 domains of the iief-5 questionnaire than the score in the unilateral ns group at 1 year after surgery (table 3). 1, the patients' ns status largely influenced the response rate to tadalafil (p=0.0098, log - rank test). Among the patients who were prescribed tadalafil 5 mg per day, four patients (13%) in the bilateral ns group and one patient (6%) in the unilateral ns group positively responded to tadalafil at 6 months after the surgery . At 1 year, 12 patients (40%) in the bilateral ns group and 3 patients (18%) in the unilateral ns group responded positively to tadalafil and were able to perform intercourse . In the non - tadalafil group, only three patients (11%) for whom bilateral ns was performed and one patient (6%) for whom unilateral ns was performed had erections sufficient for vaginal intercourse . In the tadalafil group, two patients did not attempt sexual intercourse even though they had sufficient erections after the surgery . According to the results of the study, the once - daily dosage of tadalafil 5 mg was most effective when bilateral ns was performed . Daily use of tadalafil was well tolerated in the present study . At 6 months, flushing and dizziness were the most commonly reported adverse events . In the tadalafil group, four patients (8.5%) experienced flushing and one patient (2.1%) experienced dizziness at 6 months . At 1 year, two patients (4.3%) had headache, one patient (2.1%) experienced dizziness, and three patients (6.4%) were reported to experience flushing after taking the drug . In our retrospective review, we learned that the dropout rate was 8% (4 of 51) over a period of 1 year; two of the four patients had discontinued treatment owing to the high cost of the medication and the other two patients owing to a lack of efficacy . The results of the current study of daily administration of tadalafil 5 mg for 1 year demonstrate the efficacy and safety of once - a - day dosing of tadalafil 5 mg in ed patients after ralp . After the surgery, patients in the tadalafil group showed greater improvement in ef than did those in the non - tadalafil group . The potency rate in the tadalafil groups who underwent either unilateral or bilateral ns continued to increase after the surgery, whereas the magnitude of improvement in the bilateral ns group was far greater than in the unilateral ns group . According to the iief-5 scores at 6 months and 1 year in the tadalafil group, the scores of the bilateral ns group (10.73.3 and 15.05.3, respectively) were significantly higher than the scores in the unilateral ns group (8.73.2 and 10.14.7, respectively). Erection sufficient enough for sexual intercourse with the administration of tadalafil was observed in 40% of the patients in the bilateral ns group and 18% of the patients in the unilateral ns group . The efficacy of daily administration of tadalafil was highly dependent on the degree of neurovascular bundle preservation . Reported that the efficacy of sildenafil citrate for ed patients who underwent bilateral, unilateral, and non - ns retropubic rp was 71.7%, 50%, and 15.4%, respectively . In another study that evaluated 91 ed patients after rp, 52% of the patients (n=48) responded positively to sildenafil citrate and were able to have successful vaginal intercourse 1 year after the surgery . In a randomized, double - blind, placebo - controlled, parallel study, montorsi et al . Reported that tadalafil once - a - day significantly improved ef compared with the placebo group . After 12 weeks of drug administration, iief - ef domain scores 26 were obtained for 67 patients (45.9%) in the tadalafil group and 21 patients (31.3%) in the placebo group . However, in a randomized, double - blind, placebo - controlled study on the effects of nightly sildenafil citrate for the prevention of ed after bilateral ns rp, erection good enough for satisfactory sexual activity occurred only in 27% of the sildenafil group (n=14 of 51) and in 4% of the placebo group (n=1 of 25). Moreover, montorsi et al . Conducted a prospective, randomized, double - blind, double - dummy, multinational, multicenter, parallel group study and evaluated 628 patients after bilateral ns rp . They compared the efficacy of on - demand versus nightly dosages of vardenafil and reported that iief scores 22 were obtained in 24.8%, 32.0%, and 48.2% of the placebo, vardenafil nightly, and vardenafil on - demand groups, respectively . Response rates to sildenafil after ns rp varied from 10% to 76%, whereas the response rates for the non - ns group varied from 0% to 15% . The potency rate of 32% in the tadalafil group in the current study is slightly lower than the rates in previously published studies . The slightly lower response rate (32%) in the tadalafil group may stem from the rather stringent criteria applied in this study . For instance, several other studies did not provide a clear definition or criteria for a positive response, and in those studies a positive response did not necessarily mean the ability to achieve sexual intercourse . In the current study, a summed score of 8 or more on q2 (" when you had erections with sexual stimulation, how often were your erections hard enough for penetration? ") And q3 (" during sexual intercourse, how often were you able to maintain your erection after you had penetrated your partner? ") Of the iief-5 questionnaire was required for patients to be classified as positive responders . The relatively older age of the patients in the present study (67.96.8 years) might have had a negative impact on the potency rate . Furthermore, 2 patients in the tadalafil group did not attempt sexual intercourse even though they had sufficient erections after the surgery . Lastly, the biopsy gleason score, which is known to be related to psa failure, was slightly higher in the tadalafil group than in the non - tadalafil group even though a high gleason score of 8 or more was observed in only three of the patients (one in the non - tadalafil group and two in the tadalafil group). Diverse factors including preoperative ef, frequency of sexual intercourse before surgery, type of ns procedure, degree of postoperative potency, and the interval between surgery and the administration of pde5-is might have affected the potency results . The discrepancy among published results suggests that further confirmatory studies are needed on the effects of pde5-is and the optimal regimen . The mechanism of action of pde5-is is related to the etiology of ed after rp . Even though the ns technique with the advent of ralp significantly improved the potency rate, neurapraxia and nerve injury still persist even 1 year after surgery . The patients' iief-5 score at 1 year was significantly lower than the patients' iief-5 score at preoperative baseline, and most patients (n=116) experienced ed after the surgery even though ns ralp was performed . When neurologic injury occurs, penile hypoxia and fibrosis lead to the absence of spontaneous nocturnal erections, which decreases the release of nitric oxide . This decrease in nitric oxide production leads to a drop in the amount of available cyclic guanosine monophosphate (cgmp). Pde5-is inhibit pde5, which metabolizes cgmp, and this results in an increase in cgmp levels . This increase in the amount of cgmp coupled with nitric oxide induces corporal smooth muscle relaxation, and this leads to subsequent erection by allowing blood flow to the penis [21 - 24]. The whole process requires nerve preservation, because if there is no cgmp, there is no substrate for pde5 to metabolize . This mechanism of action explains why the effect of tadalafil was much greater in the bilateral ns group than in the unilateral ns group in the current study . Persistent use of pde5-is inhibits hypoxia - associated fibrosis by amplifying the depressed nitric oxide signaling pathway, and this leads to erectile recovery after rp . In a recent study that compared the effects of different pde5-is, tadalafil was the most effective agent, followed by vardenafil, although there was no significant difference in the safety profile of the drugs . In long - term studies, once daily administration of tadalafil 5 mg was effective and safe and thus became a viable alternative to the widely used on - demand administration of tadalafil for ed patients . Moreover, the mean half - life of tadalafil (17.5 hours) is longer than that of sildenafil and vardenafil (about 4 hours). The longer duration of the drug may allow patients with ed to enjoy sexual activity free from the burden of planning drug intake before intercourse, which can improve the patients' sexual quality of life [27 - 29]. Previously published studies maintain that pde5-is can be administered as early as at the time of removal of the foley catheter or within 1 month after rp . Early administration of pde5-is helps to prevent cavernosal hypoxia, which leads to smooth muscle apoptosis and penile fibrosis . In our study, all patients in the tadalafil group initiated oral intake of tadalafil 5 mg right after foley catheter removal, between 2 to 3 weeks after the surgery, and tadalafil was administered for a period of 1 year . The potency rate of the bilateral ns group who took tadalafil (40%) was significantly higher than that of the non - tadalafil group (11%) at 1 year after the surgery . The results of our study suggest that ed after ralp can be well treated with once daily dosage of tadalafil 5 mg if ns ralp is performed . Therefore, tadalafil should be considered as a first - line treatment for a well - selected subgroup of ed patients after rp . The current study was neither randomized nor placebo - controlled because it was carried out in a retrospective manner . Although the results of the study should be applied with caution, the findings suggest that once daily dosage of tadalafil 5 mg was effective and well - tolerated by ed patients after ralp at 1 year after the surgery . A second limitation of the current study is related to the relatively small number of patients . However, the current study can be a pilot for further studies that have larger randomized samples of patients . Despite these limitations, this study is the first long - term study that evaluated the efficacy and safety of tadalafil 5 mg once daily use in ed patients after ns ralp . More confirmatory studies that are prospective, crossover, randomized, placebo - controlled, and double - blind are clearly required to determine the effects and the optimal regimen of tadalafil in ed patients after ralp . This study is the first long - term study that evaluated the efficacy and safety of tadalafil 5 mg once daily use in ed patients after ns ralp . The results of the study indicate that once - a - day dosing of tadalafil 5 mg is effective and safe in properly selected ed patients after ns ralp . At 1 year after the surgery, a recovery rate of 40% was attained in the bilateral ns group who took tadalafil 5 mg once per day before bedtime . Once daily administration of tadalafil 5 mg was effective and safe for ed patients who had undergone ns ralp, and the patients showed gradual improvement in their ef over a period of 1 year.
Many older adults have substantial vision problems such as cataracts, glaucoma, and macular degeneration, which are the leading causes of reduced visual acuity1 . Poor vision is one of the most important risk factors associated with falls among older people2 . Vision plays a key role in maintaining balance by providing information about the environment and constant information to the central nervous system about body orientation in space and relative to other body parts3 . Achieving effectively maintained balance during functional dynamic balance tasks requires energy efficiency to minimize fatigue, which is associated with postural control deficits, and stability to prevent falling or injury4 . Among the clinical balance tests, the single - limb stand (sls) is a commonly used clinical tool for assessing postural stability in the elderly and individuals with various balance disorders . The y - balance test (ybt), which was developed by clinical applications of the star excursion balance test, involves maintaining a sls while reaching as far as possible with the other leg . As a dynamic sls balance test, the ybt has been used to prospectively identify individuals who have chronic ankle instability and greater risk of lower extremity injury in sports and as a post - rehabilitation test5 . Recently, the test has been used as a screening tool for elderly participation6 . However, no studies have investigated energy expenditure during the dynamic single - limb balance test in older adults with different visual acuities . Therefore, this study compared energy expenditure in the ybt, the functional dynamic single - limb balance test, in elderly people with good and poor binocular visual acuity (bva). A total of 21 elderly females from community housing were enrolled in the present study . Visual acuity was measured by use of a, jin s vision test chart (jv institute, seoul, south korea). Each participant s bva was evaluated with and without their own spectacles for classification into two groups: poor bva (corrected bva 0.4 logarithm of the minimum angle of resolution [logmar]) and good bva (corrected bva 0.3 logmar)7 . In the poor bva group, the participants average age (meansd), height, and weight were 78.555.24 years, 148.862.77 cm, and 49.466.05 kg, respectively; they had a left side va and right side va of 0.530.15 logmar and 0.540.16 logmar, respectively . In the good bva group, the participants average age, height, and weight were 76.406.13 years, 147.004.00 cm, and 48.994.91 kg, respectively; they had a left side va and right side va of 0.230.07 logmar and 0.230.09 logmar, respectively . All participants could walk independently without an assistive device and scored greater than 24 points on the korean version of the mini - mental state examination . They had no past or present neurological disorder, no musculoskeletal disease that could interfere with daily activities, and no significant auditory impairments, and they were not taking drugs that could have influenced the results of this study . Ethical approval was obtained from the inje university faculty of health science human ethics committee, and all subjects signed an informed consent form prior to their participation . A triaxial accelerometer (fit . Life, suwon, south korea) was used to measure energy expenditure during the ybt . We measured the raw data using the x, y, and z variables of acceleration reformed as acceleration due to activity by removing gravitational acceleration . Moreover, we calibrated the single vector magnitude (svm) by summing the acceleration of the three axes8:svm = in the present study, a range of 2 g was selected . Data were collected at a sampling rate of 32 hz . Before the task, the participant s dominant leg was determined by kicking a soccer ball . For normalization, the lower limb lengths of all participants were measured in supine position from the anterior superior iliac spine to ipsilateral medial malleolus using a cloth tape measure . Reach distance was normalized to limb length, and the maximum reach distance was expressed as a percentage of limb length (% maxd). The normalized value was calculated as reach distance divided by limb length and then multiplied by 100 . Composite reach distance was the sum of the three reach directions divided by 3 times the limb length, which was then multiplied by 100 . The accelerometer was fixed with double - sided adhesive tape over the l3 spinous process9 . For the test, the participants stood with one foot on the center footplate from the ybt kit (move2perform, evansville, in, usa) at the starting line and their hands on their pelvis . They were asked to push the reach - indicator block with the free limb in the anterior (a), posterior medial (pm), and posterior lateral (pl) directions in relation to the stance foot on the central footplate . The testing order was trial standing on the right foot reaching in the a direction (right a reach) followed by trial standing on the left foot reaching in the a direction . After the test trials were completed, each participant was given a 2-minute rest period and then conducted three test trials in each direction11 . A trial was classified as invalid if the participant did not return to the starting position, placed the reach foot on the ground on either side of the line or tube, raised or moved the stance foot during the test, or kicked the plate with the reach foot to gain more distance . If an invalid trial occurred, the data were discarded, and the participant repeated the trial . Statistical analysis was performed using the spss software (version 18.0 for windows, spss inc ., a paired t - test was used to examine differences between right and left limb reach distances . As no differences were found, for the data analysis, we used the dominant limb distance in each direction (right side). The differences in reach distance and energy expenditure were analyzed using the independent t - test for comparisons between groups . Effect sizes (cohen s d) for test differences were calculated by determining the difference between the poor bva and good bva group mean values and dividing by the pooled standard deviation12 . The normalized reach distance (% maxd) in the good bva group (a, 60.84.3; pm, 94.36.1; pl, 88.97.8) during the ybt in the three directions and composite reach (81.34.8) were significantly longer (p <0.05) compared with the values in the poor bva group (a, 55.65.9; pm, 84.29.9; pl, 81.75.4; composite, 73.86.2). The energy expenditure (cm / s) in the good bva group during the ybt in the three directions (a, 413.8145.2; pm, 490.0189.4; pl, 585.8209.7) was significantly reduced (p <0.05) compared with the values in the poor bva group (a, 697.4231.6; pm, 698.8227.3; pl, 791.6182.7). The effect size value of the reach distance and energy expenditure showed that the results were large for the a (1.064 and 1.525), pm (1.268 and 1.044), pl directions (1.187 and 1.104) and composite reach (1.412) in terms of the differences between the good and poor bva groups . This study was designed to compare energy expenditure and reach distance during the ybt in elderly women with good bva and poor bva . It showed that normalized reach distances in the a, pm, and pl directions and the composite reach of the good bva group were significantly greater compared with the values in the poor bva group . The reach distance during the ybt reflected the degree of dynamic balance control . A longer reach thus, our findings suggest that visual acuity affects the functional dynamic sls in the a, pm, and pl directions . Moreover, older adults with poor bva are more balance challenged compared with those with good bva . In the a reach direction, participants received visual feedback during the ybt . Wang et al.13 showed that reduction of the stability boundary of the multi - joint coordination patterns in maintaining the sls in the absence of vision could lead to increased rotation of segments around the center of mass . Hallemans et al.14 reported that no vision conditions limited movements of the hip and ankle in the sagittal plane compared with a full vision condition . Although, visual awareness is reduced in the pm and pl directions compared with the a direction, participants with good bva have a longer reach compared with those with poor bva . When one sense diminishes, it is common to find a greater dependence on other sensory cues to preserve balance15 . However, hazime et al.16 reported that vision had a key role in the sls, requiring greater balance ability, whereas perturbation in proprioception showed effects only in the double- limb stance . The stance platform of the ybt kit is elevated 1 inch, and its width is 5.2 inches; thus, maintaining balance in the dynamic sls on the balance platform of the ybt kit is greater challenge than doing so on the ground in elderly subjects . The results show that elderly people with good and poor bva rely more on visual input than other sensory inputs for postural control . In addition, in this study, the energy expenditure during the ybt in three directions in the poor bva group was significantly increased compared with the values in the good bva group . An accelerometer was used as an objective tool for assessing physical activity level related to energy expenditure . Accelerometry has been shown to have intra - rater reliabilities (0.9250.994) and correlation with scores on the commonly used physical activity questionnaire (r = 0.830)17 . Our results imply that the velocity of trunk segment motion frequently changes and fluctuates, and that ineffective biomechanical cost strategies in terms of energy efficiency are performed . Individuals with poor bva may habituated to discomforts visual acuity and rely more on ankle proprioception to control their posture during quiet standing . When balance challenged with conditions such as the sls, vision play a major role compared with in a normal standing position . Thus we surmise that these individuals compensate with trunk rotation during ybt in order to maintain postural stability16, 18 . Hence, participants with good bva have a longer reach in the ybt compared with those with poor bva . First, it only measured reaching distances and energy expenditure . Measurements of muscle activation and range of motion in the trunk and lower extremities were not taken . Thus, we could not explain the connection between energy expenditure and fatigue or trunk lean during the ybt . Second, the valid of the triaxial accelerometry in measurement of energy expenditure during a short period of activity has not been verified . In addition, the sample size was small, so caution should be used when generalizing the results . Further studies should investigate kinematic and kinetic data, as well as the correlation of the ybt with gait parameters in elderly subjects with poor bva.
C57bl/6j male mice (25 months of age) were housed individually on a 12 hr/12 hr light / dark schedule with lights on at 7 a.m. (zt0) and handled for 6 days . Mice were sleep - deprived (sd) in their home cages for 5 hours by gentle handling beginning at zt5 or left undisturbed (non - sleep - deprived mice, nsd). For contextual fear conditioning experiments, animals were placed in a novel chamber for 3 minutes, and received a 2-second, 1.5 ma footshock after 2.5 minutes . Mice received intra - peritoneal injections of rolipram (rol; 1 mg / kg) or vehicle (2% dmso in 0.9% saline) immediately and 2.5 hours post - training . Testing of contextual memory was performed 24 hours after training in the trained context and 48 hours after training in a novel chamber . 1-train ltp was induced by a single 100 hz, 1-second duration train of stimuli . 4-train ltp consisted of 4 trains applied with a 5-minute inter - train interval; for massed 4-train ltp a 5-second inter - train interval was used . Theta - burst stimulation (tbs) consisted of 40-ms duration, 100 hz bursts delivered at 5 hz for 3 seconds (15 bursts of 4 pulses per burst, for a total of 60 pulses). Chemical ltp was induced by treatment of slices for 15 minutes with 5 m forskolin (fsk) in 0.1% ethanol, or a combination of 50 m forskolin and 30 m 3-isobutyl-1-methylxanthine (ibmx, in water). Rolipram (0.1 m in 0.1% dmso) was applied for 60 minutes, beginning 30 minutes before tetanization . Camp assays on ca1 regions of hippocampal slices 10 minutes after treatment for 15 minutes with forskolin (50 m), forskolin + ibmx (30 m), or vehicle (0.1% etoh) were performed by radioimmunoassay according to kit instructions . Camp - specific pde activity assays29 and western blots for pde4a530 were performed as previously described . Full methods and any associated references are available in the online version of the paper at www.nature.com/nature.
Cell metabolism governing the growth and functioning of each cell and a whole organism refers to chemical transformations and enzyme - catalyzed energy producing and energy utilizing reactions of carbohydrates, proteins, and lipids . Amongst the most metabolically active organs are liver, brain, gut, kidneys, and heart [13]. Although the rate of metabolic reactions is lower in skeletal muscles, they account for around 20% of the total energy expenditure due to a 5060% contribution to a total body mass . Several micrornas were reported to control processes related to metabolism such as insulin secretion (mir-9, mir-375), adipocyte differentiation (mir-143), fatty acid metabolism (mir-122), and myogenesis (mir-1, mir-133a, mir-133b, and mir-206) (reviewed in). Of potential meaning is also mir-378a, located in the gene encoding master metabolic regulator, peroxisome proliferator - activated receptor gamma, coactivator 1 beta (pgc-1). Mir-378a was found to affect lipid and xenobiotic metabolism, lipid storage, mitochondrial function, and shift towards a glycolytic pathway (warburg effect) [5, 6]. Because nutrients supply for metabolic processes is a matter of circulation, metabolically active tissues require high vascular density . Recently, mir-378a was reported to regulate tumor angiogenesis mainly via inhibition of tumor suppressors sufu and fus-1 [8, 9]. Thus, a growing body of evidence suggests a role of mir-378a as a mediator controlling reciprocally dependent processes such as metabolism, muscle differentiation / regeneration, and angiogenesis . Micrornas (mirnas; mirs) are small noncoding rna molecules with an average length of 21 - 22 nucleotides which can regulate gene expression posttranscriptionally by targeting mostly the 3untranslated region (3utr) of mrnas . However, mirna target sites were also found on the 5utr regions of human mrna . Since their discovery in c. elegans in 1993, mirnas currently can be recognized as potent players in wide spectrum of biological processes like development, differentiation, cellular defense mechanisms, and others . Conservative estimates state that over 30% of mrna expression is regulated by mirnas [12, 13]. However, others suggest that even up to 60% of the mrna expression is targeted by mirnas . Mirnas are often located in the introns of coding genes or noncoding sequences but can also be located in exons . Intronic mirnas can be expressed together with their host gene mrna being derived from a common rna transcript [15, 16]. Other mirnas can also have their own promoters, which enable independent expression, or can be organized in clusters sharing a common transcriptional regulation [17, 18]. Mirnas transcription is rna polymerase ii - dependent . In the case of mirnas that are encoded in their own genes, the primary mirna transcript (pri - mirna) is several kilobases long, while mirnas encoded in intronic regions of other genes (mirtrons) have shorter transcripts . The mirna stem loop is excised from pri - mirna by endoribonuclease drosha / dgcr8 (microprocessor complex) and a hairpin called pre - mirna is exported from the nucleus by exportin-5 in a ran - gtp dependent manner . An endoribonuclease dicer removes the hairpin loop sequence from pre - mirna, creating a double stranded mirna duplex . Depending on the relative stability of the mirna duplex, one or, more rarely, both strands can be incorporated in a multiprotein rna - induced silencing complex (risc). When there is perfect pairing between the mirna sequence and its target site, mrna is cleaved by a protein part of the risc called argonaute (ago). If the pairing is partial, deadenylation of the mrna via recruitment of the ccr4-not complex by the gw182 proteins inside the risc takes place and the poly - a tail is lost, leaving the mrna vulnerable to rnase activity, ubiquitination, and mrna degradation . Alternatively, mirna - induced risc can also cause repression of translation by mechanisms such as, for example, the promotion of ribosome drop - off from the mrna transcript or destabilization of the mrna binding cap protein (figure 1) (reviewed in [20, 21]). The pre - mir gives rise to a leading strand (mir-378a-3p, previous ids for murine sequence: mmu - mir-422b, mmu - mir-378, and mmu - mir-378 - 3p; for human: hsa - mir-422b and hsa - mir-378) and a passenger strand (mir-378a-5p, previous ids for murine sequence: mmu - mir-378, mmu - mir-378, and mmu - mir-378 - 5p; for human: hsa - mir-378 and hsa - mir-378). Mirna-378a-3p mature strand was first identified in 2004 in humans (originally named mir-422b). Recently, other mirs with similar sequences but other localizations in the genome have been discovered and named: mmu - mir-378b, c, d in mouse and hsa - mir-378-b, c, d1,d2,e, f, g, h, i, j in human [2327] (table 1). In humans, mir-378a is by far the most expressed of the mir-378 sequences, with 7030 reads per million, in 78 experiments during deep sequencing, compared with 1013220 reads per million, in 4272 experiments for the other forms, respectively . In mice, mir-378a and mir-378b have similar expression levels, at 11700 and 11000 reads per million (mirbase, version 21, september 2015). The sequence of mir-378a mature strands is highly conserved between species, with the mir-378a-5p strand being identical in both human and mice and the mir-378a-3p strand only differing in one nucleotide (table 2) [6, 27]. Pgc-1 may regulate several facets of energy metabolism such as mitochondrial biogenesis, thermogenesis, and glucose and fatty acid metabolism . Both strands of mir-378a are coexpressed with pgc-1 as shown, for example, in the liver and during adipocyte differentiation [6, 29]. The coexpression of mir-378a with its host gene implies they may share the same transcriptional activators, and mir-378a might be involved in similar processes as pgc-1. Accordingly, high levels of (porcine) mir-378 - 1 (table 2) expression are found in developing muscle, postnatal muscle, and myocardium and in brown adipose tissue [29, 30]. To date, only a limited number of mir-378a targets, which can be predicted based on in silico analysis, have been experimentally validated . The latter, however, imply a role of mir-378a in mitochondrial energy homeostasis, glycolysis, and skeletal muscle development and in tumor angiogenesis and other processes (table 3). A major source of energy production comprises oxidation of glucose in glycolysis followed by oxidation of pyruvate in well - oxygenated cells (or followed by lactic acid fermentation in cancer, the warburg effect) and from -oxidation of lipids, which yields even more atp per gram then carbohydrates metabolism . A location of mir-378a in the gene encoding pgc-1 implies an involvement of mir-378a in metabolic pathways . Unlike its homologue, pgc-1, the expression of pgc-1 is not elevated in response to cold exposure but occurs in response to hypoxia, exercise, caloric restriction, or aging (reviewed in). Pgc-1 is preferentially expressed in tissues with relatively high mitochondrial content, such as heart, skeletal muscle, and brown adipose tissue . In 2002, pgc-1 was first cloned and shown to be upregulated in the liver during fasting . Pgc-1 strongly activates hepatic nuclear factor 4 (hnf4) and ppar, both of these nuclear receptors being important for the adaptation of hepatocytes to the effects of fasting . These findings could hint to a possible role of pgc-1 in the regulation of gluconeogenesis and fatty acid oxidation in the liver . Pgc-1 is also involved in the regulation of energy expenditure or in the pathway of estrogen receptor - related receptors (errs) [3337]. Since mirnas originating in the introns of host genes may modulate the protein encoded by their parental genes and may be involved in the same mechanisms [3840], mir-378a is proposed to be involved in the metabolic pathways affected by pgc-1 . It was reported that mice lacking the first intron of the ppargc1b gene (and thus mir-378a) have a significantly higher oxygen capacity and mitochondrial function . They identified a mediator complex subunit 13 (med13), involved in nuclear receptor signaling, and carnitine acetyltransferase (crat), a mitochondrial enzyme involved in fatty acid metabolism, as targets of mir-378a-5p and mir-378a-3p, respectively . Regulated also cytochrome p450 2e1 (cyp2e1) being involved in the metabolism of, for example, drugs and toxins . In addition, it has been discovered that transcription factor nuclear respiratory factor-1 (nrf-1), a critical regulator of the expression of some important metabolic genes in mitochondria regulating cellular growth, is inhibited by mir-378a-3p . Thus, mir-378a can be considered as a regulator of mitochondrial function in cells overexpressing mir-378a . Moreover, mir-378a-5p inhibits the mrnas of err and ga - binding protein- in breast cancer, which both interact with pgc-1 and together control oxidative metabolism . This leads to a reduction of tricarboxylic acid gene expression and oxygen consumption and an increase in lactate production, which shifts cells from an oxidative towards a glycolytic pathway . In this way, mir-378a-5p is believed to be a switch regulating the warburg effect in breast cancer . Moreover, in situ hybridization experiments in this study showed that mir-378a-5p expression correlates with progression of breast cancer . The proposed regulating role of mir-378a-5p on the warburg effect is in parallel with the effects of pgc-1, which mediates gluconeogenesis and fatty acid metabolism after periods of fasting or intense exercise . Coactivation by pgc-1 of err and ppar makes muscle fibers in pgc-1 transgenic mice more rich in mitochondria and highly oxidative . Accordingly increased glycolytic rates and increased cell proliferation can be related to lactate production by lactate dehydrogenase (ldh). Ldha was found to be a direct target of mir-378a in the study of mallat et al . . Hsa - mir-378a-3p represses cell growth and increases cell death by targeting ldha . Of note, hsa - mir-378a-3p and hsa - mir-378a-5p had opposite effects on ldha expression . In addition, mir-378a is also considered as an important factor in adipogenesis and lipid storage . There is a complex family of factors regulating those processes such as insulin, insulin - like growth factors (igfs), glucagon, and thyroid hormones t3 and t4 (reviewed in [4649]). As mentioned before, it was demonstrated that mir-378a - knockout mice do not get fat after 8 weeks of high fat diet . Such animals show an enhanced mitochondrial fatty acid metabolism and have elevated oxidative capacity of tissues targeted by insulin (e.g., liver, muscles, and adipose tissues). In accordance with that, it was shown that mature strands of bta - mir-378 - 1 (table 1) are expressed at higher level in cows with high (versus low) amount of back fat . Similarly, an inhibition of both mmu - mir-378a-3p and its host gene, pgc-1, by the flavonoid fisetin lowered the accumulation of fat in the liver . Interestingly, mmu - mir-378a-5p was downregulated in mice that were fed a high fat diet for five months . Overexpression of mir-378a-3p/-5p during adipogenesis increased the transcriptional activity of ccaat / enhancer - binding proteins (cebp) alpha and beta, which can stimulate the expression of leptin, a hormone produced mainly by adipocytes which controls the homeostasis of body weight (reviewed in [52, 53]). On the other hand, tnf-, il-6, and leptin are reported to increase the expression of mir-378a-3p in mature human adipocytes in vitro . These cytokines are mainly secreted in the adipose tissue and are suggested to be involved in development of insulin resistance [55, 56]. In addition, mir-378a-3p was shown to target insulin growth factor 1 receptor (igf1r) and reduce the akt signaling cascade in cardiomyocytes during cardiac development . Moreover, in tissues where igf1 levels were high (e.g., fibroblasts and fetal hearts), mir-378 - 3p levels were very low, showing an inverse relation and suggesting a negative feedback loop between mir-378a-3p, igf1r, and igf1 . The latter functions as a master regulator of adipogenesis and is involved in the formation of peroxisomes and the catabolism of very long chain fatty acids [58, 59]. Accordingly, the amount of adipose tissue does not increase in mice lacking ppar when they are fed a high fat diet . It was also reported that in cultured adipocytes mmu - mir-378a and pgc-1 expression is ppar, or rosiglitazone (a ppar ligand), dependent, finding two peroxisome proliferator response elements in the mir-378a loci . On the other hand, overexpression of mir-378a elevated the expression of ppar isoform 2, suggesting positive feedback loop and confirming the involvement of mir-378a in the storage of fat . There are several activators known to induce expression of ppar such as the members of the e2f transcription factor family and prostaglandin j-2 (pgj-2) [6365]. The latter may act through rar - related orphan receptor alpha (rora), which is frequently found in myocardium . In addition to ppar, rora regulates also myod, a major transcription factor involved in skeletal muscle differentiation [67, 68]. Interestingly, rora is a possible (but not yet validated) target for mir-378a-3p . A proteomics - based study revealed several other proteins that are potentially targeted by rat mir-378a-3p or mir-378a-5p . Mir-378a-3p was shown to regulate mannose-1-phosphate guanylyltransferase (gdp), dimethylarginine dimethylaminohydrolase 1 (ddah1), and lactate dehydrogenase a (ldha); all those proteins are participating in metabolic processes . On the other hand, tropomyosin beta chain, which is involved in the regulation of atpase activity, high levels of murine and rat mir-378a-3p, mir-378a-5p, and porcine mir-378 - 1 are reported in both developing and adult skeletal muscles [7, 30, 44]. Myod and myog play a role in the processes of myogenesis and muscle regeneration, in which dormant satellite cells are activated upon muscle damage and start proliferating and differentiating into muscle fibers (reviewed in [70, 71]). It has been shown that mir-378a-3p targets the myogenic repressor myor during myoblast differentiation, which directly inhibits myod . On the other hand, myod is upregulated in response to mir-378a-3p overexpression and, conversely, the level of mir-378a-3p may be enhanced by myod . Thus, there is evidence for a feedback loop in which mir-378a-3p regulates muscle differentiation via inhibiting myor, leading to an increase of myod, which in turn enhances mir-378a-3p . That mir-378a may also control the development of skeletal muscle mass after training . In this study, mir-378a (strand not specified) was significantly downregulated in men who obtained low training - induced muscle mass gain compared to men who obtained high training - induced muscle mass gain . Mir-378a-3p is expressed mostly by cardiomyocytes, but not by nonmuscle cells, whereas the level of mir-378a-5p was reported to be very low in the heart . Fang et al . Showed that mir-378 - 3p is significantly downregulated both in vitro in cardiomyocytes cell cultures exposed to hypoxia and in vivo during myocardial injury in rats . Overexpression of mir-378a-3p enhanced cell viability and inhibited apoptosis via caspase-3 inhibition . In contrast to this finding, another study found that mir-378a-3p downregulation enhanced the survival of cardiac stem cells via focal adhesion kinase activation and releasing connective tissue growth factor (ctgf), the latter being a target of mir-378a-3p . Overexpression of mir-378a-3p in the study of knezevic et al . Increased apoptosis of cardiomyocytes via the direct targeting of igf1r leading to a decrease of akt signaling . This is in opposition to the previously mentioned study of fang et al . Which showed apoptosis the converse findings of the studies could be explained by different models used by knezevic et al . And because of those discrepancies, the role of mir-378a in apoptosis of cardiomyocytes requires further investigation . The finding that mir-378a-3p affects both igf1r and the akt pathway was confirmed in a study which found that overexpression of mir-378a-3p in rhabdomyosarcoma suppressed igf1r expression and affected phosphorylation of the akt protein . Mir-378a-5p was shown to target heat shock protein 70.3 (hsp70.3) in mouse hearts in normoxic conditions, but in hypoxic conditions a transcript variant of hsp70.3 without mir-378a-5p target site in its 3-utr is not repressed and can exert its cytoprotective properties . Mir-378a-3p prevented cardiac hypertrophy by targeting either ras signaling or the mitogen - activated protein kinase (mapk) pathway [77, 78]. More studies on the effect of mir-378a expression in muscle disorders would also be desirable . In both golden retriever muscular dystrophy dogs and duchenne muscular dystrophy patients, all in all, these findings suggest mir-378a-3p can be considered as an important player in cardiac development, remodeling, and hypertrophy . Angiogenesis comprises development of new blood vessels from existing ones, regulated by cytokines and growth factors such as, for example, vascular endothelial growth factor (vegf), platelet - derived growth factor (pdgf), and angiopoietin-1 (ang-1). Their expression can be posttranscriptionally controlled by micrornas such as mir-126, mir-296, mir-210, mir-21, and the mir-17~92 cluster (reviewed in). Skeletal muscles and heart muscle are tissues which, due to their oxygen and energy consumption, need to be sufficiently vascularized . One of the major regulators of angiogenesis is the hypoxia - inducible factor-1 (hif-1), which controls over 100 genes involved mainly in the glycolytic pathway and blood vessel formation, including vegf - a or interleukin-8 [8385]. Vegf is generally induced by hypoxia, while il-8 in at least some cancers and endothelial cells can be diminished by hif-1 via inhibition of c - myc and sp-1 transcription factors [86, 87]. C - myc, known as a regulator of cell cycle progression, apoptosis, and cellular transformation, is also a potent activator of pgc-1 and, in turn, mir-378a-3p, upregulating their expression . In addition, mir-378a has been shown to affect vegf - a in two ways . Human hsa - mir-378a-5p (by the study of hua et al . Named as mir-378) can directly affect vegf - a by competing with hsa - mir-125a for the same seed - region in the vegf - a 3utr causing upregulation of vegf - a . Mir-378a-5p can also indirectly regulate vegf - a affecting sonic hedgehog (shh) signaling via sufu inhibition, which is an inhibitory component of this signaling pathway . The shh pathway in turn can upregulate vegf - a and also other regulators of blood vessels formation, ang-1 and ang-2 expression [9092]. Increased expression of vegf - a, as well as pdgf and tgf1, was also seen in mesenchymal stromal cells (mscs) transfected with rno - mir-378a-5p . In skeletal muscles, vegf - induced angiogenesis appears not to be regulated by the well - known hif pathway but by pgc-1, which coactivates estrogen - related receptor alpha (err-) on binding sites in the promoter and the first intron of the vegf gene, inducing its expression . This angiogenic pathway shows new roles for pgc-1 and err-, which are important regulators of mitochondrial activity in response to stimuli like exercise . If there might be a role for pgc-1 in this pathway, it is yet to be examined . It is noteworthy, however, that mir-378a-5p is known to affect the estrogen receptors by inhibiting err, another estrogen - related receptor . A role for mir-378a in cell cycle regulation and stimulation of cell growth is also proposed . In human mammary epithelial and breast cancer cell lines, mir-378a-3p can target the antiproliferative protein tob2, which is a suppressor of cyclin d1, which in turn is required for cell cycle g1-phase to s - phase progression . Whether mir-378 affects endothelial cell proliferation by regulation of cell cycle remains to be established . The role of mir-378a in the formation of blood vessels nourishing tumor and enabling tumor growth was revealed . Mir-378a was found to be differentially regulated in different types of cancers being downregulated in gastric cancer [96, 97], oral, and colon carcinoma, while being upregulated in renal and lung cancer [9, 101]. Since it is also changed in serum or plasma of patients with prostate cancer, renal cancer [100, 103], and gastric cancer and frequently found to be overexpressed in cryopreserved bone marrow mononuclear cells from acute myeloid leukemia patients, mir-378a might be considered as a biomarker . The role of mir-378a in tumorigenesis, tumor growth, and tumor vascularization was revealed for the first time by lee and coworkers in glioblastoma . They showed that mir-378a-5p enhances cell survival, reduces caspase-3 activity, and promotes tumor growth and angiogenesis, through repression of two tumor suppressors, sufu and fus-1 . Strikingly, nude mice injected with mir-378a-5p transfected cancer cells formed tumors of bigger volume and with larger blood vessels compared to gfp - transfected cells . On the other hand, high expression of mir-378a-5p in nsclc correlated with brain metastases due to higher cell migration, invasion, and tumor angiogenesis . Another study confirmed the downregulation of fus-1 by mir-378a-5p and showed that in the hepg2 liver cancer cells mir-378a-5p overexpression enhanced proliferation, migration, and, when injected in mice, invasion . Also in rhabdomyosarcoma, enhanced expression of mir-378a, vegf, and mmp9 correlated with increased vascularization and metastasis . Taken together, these studies suggest that mir-378a may serve as a prognostic marker in cancer due to its effects on angiogenesis . Our recent data confirmed the proangiogenic effect of mir-378a (both strands) in non - small cell lung carcinoma (nsclc) and pointed at its correlation with heme - degrading enzyme, heme oxygenase-1 (ho-1). An involvement of ho-1 in angiogenesis and vegf - a as well as il-8 signaling was shown by us previously; however, its action in tumors seems to be complex . In nci - h292 cell line overexpressing ho-1, mir-378a (both strands) also overexpression of the mir-378a precursor sequence diminished ho-1 expression . Conditioned medium from nci - h292 cells overexpressing mir-378a enhanced angiogenic potential of hmec-1 endothelial cell line . Tumors formed by such cells in subcutaneous xenografts showed enhanced growth, vascularization, oxygenation, and distal metastasis in vivo . These interactions between mir-378a and ho-1 were confirmed in our studies on the role of the nrf-2 transcription factor / ho-1 axis in nsclc cell lines [110, 111]. On the other hand, enhanced expression of mmu - mir-378a-5p in 4t1 murine breast cancer cells decreased the proliferation, migration, and invasiveness of these cancer cells in vitro and in vivo by targeting fibronectin, resulting in inhibition of tumor growth . Recent study showed that mir-378a may act as a biomarker for response to antiangiogenic treatment in ovarian cancer . Low expression of mir-378a was associated with longer progressive - free survival in patients with recurrent ovarian cancer treated with the antiangiogenic drug bevacizumab . Overexpression of the mir-378a precursor in ovarian cancer cells altered expression of genes associated with angiogenesis (alcam, ehd1, elk3, and tln1), apoptosis (rpn2, hipk3), and cell cycle regulation (swap-70, lsm14a, and rdx). High mir-378a (strand not specified) expression in renal carcinoma correlated with higher levels of endothelial surface marker cd34 in these tumors . Notably, a recent study suggested clinical relevance for mir-378a in metastatic colorectal cancer, in which enhanced mir-378a expression significantly improved the sensitivity to cetuximab treatment in these patients . Mir-378a-5p transfection of mscs has been shown to enhance their survival and angiogenic potential under hypoxic conditions in vitro . In coculture with human umbilical vein endothelial cells (huvecs), mir-378a-5p - transfected mscs formed a larger number of vascular branches on matrigel . In the mscs transfected with mir-378a-5p, the expression of bcl-2-associated x protein (bax), which is an important proapoptotic regulator, was decreased, leading to a better survival . It still has to be determined if the proangiogenic effect of mir-378a in vivo is confined to tumor angiogenesis, or if this effect is also present in physiological angiogenesis and regenerative neovascularization . Recently, it was reported that anti - mir-378a-5p enhances wound healing process by upregulating integrin beta-3 and vimentin . It was reported that pgc-1 induces angiogenesis in skeletal muscle, enhancing the expression of vegf both in vitro and in vivo after (transgenic) overexpression . Accordingly, it was also found that vegfa is upregulated in c2c12 myoblast cell line with pgc-1 overexpression . However, after a pcr - based gene array of 84 known angiogenic factors and further rt - pcr of individual genes, they concluded that pgc-1 triggered an antiangiogenic program . After inducing hind limb ischemia in pgc-1 overexpressing mice, an impaired reperfusion was noticed when compared to wild type littermates . The role of inflammation in angiogenesis is studied the most in the context of cancer (e.g., reviewed in [119, 120]) but is certainly not limited to this pathology . Both lymphoid (reviewed in [121, 122]) and myeloid (reviewed in) derived inflammatory cells affect angiogenesis in a stimulating or inhibitory manner . The role of mir-378a in inflammatory cells was reported and its anti - inflammatory effect could be suggested . Nk cells exert potent cytotoxic effects when activated by type i ifn from the host once infected . Mir-378a was found to be downregulated in activated nk cells and further proved to target granzyme b. thus, ifn- activation decreases mir-378a expression and in turn augments nk cell cytotoxicity . Accordingly, suppression of mir-378 targeting granzyme b in nk cells resulted in inhibition of dengue virus replication in vivo . Macrophages are known to play either inhibitory or stimulatory roles in angiogenesis (reviewed by). Mirnas have been proposed to regulate activation and polarization of macrophages (reviewed by [127, 128]). In a study of rckerl mir-378a-3p was identified as a part of the il-4-driven activation program of anti - inflammatory macrophages (m2). Mir-378a-3p was highly upregulated after stimulation with il-4 of peritoneal exudate cells of mice injected with the parasite brugia malayi compared to controls and infected il-4-knockout mice . The study identified several targets for mir-378a-3p within the pi3 k / akt signaling pathway, which are important for proliferation but only partially responsible for m2 phenotype . Another study found mir-378a (strand not specified) expression upregulated after stimulation with cytokines like, for example, tnf- and il-6 . In line with its potential role in macrophages, mmu - mir-378a (strand not specified) has been found to be upregulated during osteoclastogenesis in vitro . Furthermore, serum levels of mir-378a-3p have been shown to correlate with bone metastasis burden in mice injected with mouse mammary tumor cell lines 4t1 and 4t1.2 . A growing body of evidence suggests a role for mir-378a as a mediator controlling reciprocally dependent processes in metabolism, muscle differentiation / regeneration, and angiogenesis . As mir-378a was found to be differentially regulated in different types of cancers and its level is changed in serum of prostate, renal, and gastric cancer patients, it can be considered as a biomarker for those diseases . The correlation between mir-378a expression and disease progression in lung cancer, liver cancer, and rhabdomyosarcoma suggests a further role of this microrna as a prognostic marker . If more research will be done to the mechanisms of action, possibilities for therapeutic use of mir-378a could be sought in the field of metabolic disorders, obesity, or tumors . More studies on the effect of mir-378a expression in muscle disorders would also be desirable . The proangiogenic effect of mir-378a was observed in tumors; however, no studies have been performed on the angiogenic effects of mir-378a in physiological settings or diseases where angiogenesis plays important roles, such as diabetes and cardiovascular diseases . More study has to be done to assess the mechanisms of mir-378a function in blood vessel formation . Of note, in contrast with proangiogenic role of mir-378a, inhibition of mir-378a-5p enhanced wound healing process . This might suggest a role for mir-378a-5p in diseases such as diabetes or in decubitus ulcers, in which wound healing is impaired . Of note is the confusion that has arises because of a disarray in nomenclature with studies describing the same molecule, mir-378a, as mir-422b, mir-378, or mir-378 . In addition, it is not always clear which of the two mature strands of mir-378a is studied . This could lead to misunderstandings and errors in interpreting the data published so far.
A large number of patients (70 - 80%) with head and neck cancers are diagnosed having locally advanced primary disease along with high lymph nodal metastases . In recent years, intensity modulated radiotherapy (imrt) has shown benefits in lowering the toxicities as well as improvements in loco regional control in these patients. [35] a crucial step in radiotherapy treatment planning process is to determine the tumor location and its extent . Modern imaging modalities, such as computed tomography (ct), magnetic resonance imaging (mri), ultrasound, single - photon emission computer tomography, and positron emission tomography (pet) assist the radiation oncologists in localization of the target volume . Ct scan is the principle source of imaging data used for defining gross tumor volume (gtv) for planning conformal therapy for most sites . Recurrence pattern following imrt shows that most of the recurrences occur only in high - dose region . So apart from anatomical delineation, functional imaging may improve outcome with imrt . With the advent of pet scan, it is possible to demonstrate abnormal glucose metabolism in tumor cells using [18f]-2fluoro, 2deoxy d - glucose([18f]-fdg). Recently, [18f]-fdg - pet - ct scan has been applied to tumor volume delineation for many cancer sites in addition to diagnostic purpose . In this study, we compared quantitatively gtv delineated on [18f]-fdg - pet - ct scan to those delineated on contrast - enhanced ct scan (cect scan) in primary and nodal areas of head and neck cancers . Twenty - six patients with head and neck cancers underwent [18f]-fdg - pet and cect scans in a dedicated pet - ct scanner in single session from august 2008 to march 2010 . This study was approved by the institutional review board and the patients were enrolled for the study after an informed consent had been obtained . Characteristics of 26 patients underwent [18f]-2fluoro, 2deoxy d - glucose - positron emission tomography / computed tomography pet / ct was performed using the siemens biograph 40 true point pet / ct scanner (luetium oxyorthosilicate based 40 slice scanner). Following administration of 375 mbq of 18f - fdg, the patients were asked to wait in a quiet room for 1 h. we performed diagnostic pet / ct and radiotherapy planning pet / ct in subsequent sessions on the same day . The field of view for diagnostic pet / ct was from the skull to the upper thigh . After the completion of attenuation correction ct scan (120 kv, 80 ma), pet acquisition was performed . After completion of diagnostic pet / ct, the table top was changed to flat one for imrt planning pet / ct . The patients were immobilized with face mask and were properly aligned in radiotherapy treatment position with the help of lasers . The pet acquisition was performed with the field of view from frontal sinus to d4 vertebral level and acquisition time was 120 - 180 s / bed position . Immediately following the acquisition of the pet scan cect scan was performed using the same slice position with a slice thickness of 5 mm . All data were sent through digital imaging and communications in medicine to a workstation treatment planning system . Then cect scans were fused with pet images acquired in the treatment planning position using inbuilt software in the siemens coherence - oncologist treatment planning system [figure 1]. While evaluating cect images, pet images were blinded . Target volumes and organs at risk were contoured and transferred to oncentra treatment planning system for volume analysis . Positron emission tomography / computed tomography of 64 years female with nasal cavity cancer showing clear demarcation of tumor with thickened mucosa the primary tumor and abnormal lymph nodes in the neck were delineated by an experienced radiation oncologist . Gtv - ct scan (gtv ct) was delineated on the imrt planning cect scans according to standard protocols using all available clinical information from physical palpation, available imaging including ct and mri and direct laryngoscope findings [figure 2]. Lymph nodes with> 10 mm in shortest dimension, perinodal extension and necrosis in center were included in gtv ct . Gtv cts were contoured by a radiation oncologist after consulting with an experienced radiologist . While contouring gtv ct, fdg - pet images were completely blinded . But ct does not show clear demarcation of tumor with thickened mucosa gtv-[18f]-fdg - pet / ct (gtv pet) was delineated on fused images using the visual interpretation method along with the opinion of a nuclear medicine physician . Abnormal fdg uptake areas in imrt planning pet / ct scans above normal background uptake were included in gtv pet . The gtvs obtained from the pet / ct scans were compared with the ct - based gtv using student's t - test with the help of spss 16.0 software . The comparative analysis of two volumes is displayed as a schematic diagram [figure 3]. The mismatch between two volumes was analyzed by the following method: mismatch a to b = (a - intersection volume)/b 100 . The coverage between two volumes was analyzed by the following method: coverage a to b = intersection volume / b 100 . Gross tumor volume (gtv) positron emission tomography (pet) (black), gtv ct (red), pet out of ct (purple), ct out of pet (green) and intersection volume (turquoise) were analyzed pet / ct was performed using the siemens biograph 40 true point pet / ct scanner (luetium oxyorthosilicate based 40 slice scanner). Following administration of 375 mbq of 18f - fdg, the patients were asked to wait in a quiet room for 1 h. we performed diagnostic pet / ct and radiotherapy planning pet / ct in subsequent sessions on the same day . The field of view for diagnostic pet / ct was from the skull to the upper thigh . After the completion of attenuation correction ct scan (120 kv, 80 ma), pet acquisition was performed . After completion of diagnostic pet / ct, the table top was changed to flat one for imrt planning pet / ct . The patients were immobilized with face mask and were properly aligned in radiotherapy treatment position with the help of lasers . The pet acquisition was performed with the field of view from frontal sinus to d4 vertebral level and acquisition time was 120 - 180 s / bed position . Immediately following the acquisition of the pet scan cect scan was performed using the same slice position with a slice thickness of 5 mm . All data were sent through digital imaging and communications in medicine to a workstation treatment planning system . Then cect scans were fused with pet images acquired in the treatment planning position using inbuilt software in the siemens coherence - oncologist treatment planning system [figure 1]. While evaluating cect images, pet images were blinded . Target volumes and organs at risk were contoured and transferred to oncentra treatment planning system for volume analysis . Positron emission tomography / computed tomography of 64 years female with nasal cavity cancer showing clear demarcation of tumor with thickened mucosa the primary tumor and abnormal lymph nodes in the neck were delineated by an experienced radiation oncologist . Gtv - ct scan (gtv ct) was delineated on the imrt planning cect scans according to standard protocols using all available clinical information from physical palpation, available imaging including ct and mri and direct laryngoscope findings [figure 2]. Lymph nodes with> 10 mm in shortest dimension, perinodal extension and necrosis in center were included in gtv ct . Gtv cts were contoured by a radiation oncologist after consulting with an experienced radiologist . While contouring gtv ct, fdg - pet images were completely blinded . But ct does not show clear demarcation of tumor with thickened mucosa gtv-[18f]-fdg - pet / ct (gtv pet) was delineated on fused images using the visual interpretation method along with the opinion of a nuclear medicine physician . Abnormal fdg uptake areas in imrt planning pet / ct scans above normal background uptake were included in gtv pet . The gtvs obtained from the pet / ct scans were compared with the ct - based gtv using student's t - test with the help of spss 16.0 software . The comparative analysis of two volumes is displayed as a schematic diagram [figure 3]. The mismatch between two volumes was analyzed by the following method: mismatch a to b = (a - intersection volume)/b 100 . The coverage between two volumes was analyzed by the following method: coverage a to b = intersection volume / b 100 . Gross tumor volume (gtv) positron emission tomography (pet) (black), gtv ct (red), pet out of ct (purple), ct out of pet (green) and intersection volume (turquoise) were analyzed the distribution of all staging patterns for t, n, and m categories is shown in table 2 . Pet scan changed the ct scan - based staging in 8 cases (30.76%). Seven cases (26.92%) were up - staged by pet / ct scan, and only one (0.38%) was down - staged by pet / ct scan . In one patient (patient 12) with the unknown primary with secondaries in neck, pet / ct identified primary in soft palate . Pet / ct identified one second primary tumor in left pyriform sinus in the case of right pyriform sinus (patient no . 3). Impact of positron emission tomography / computed tomography on staging and comparison of gross tumor volume the impact of pet / ct on gtvs is shown in table 3 . The median value of gtv ct was 32.00 cc (range: 0.00 - 309.10 cc) and the mean was 54.78 cc 64.47 cc . The median value of gtv pet was 35.00 cc (range: 3.20 - 269.50 cc) and the mean was 48.43 cc 53.21 cc . The gtv ct was larger than the gtv pet in nine cases (37.5%) and was smaller in 15 cases (62.5%). Impact of positron emission tomography / computed tomography in primary and nodal gross tumor volume the median value of percentage of primary gtv pet covered by ct was 70.07% (range: 32.42 - 95.52%) and the mean was 65.18% 18.84% . The median value of percentage of primary gtv ct covered by gtv pet was 68.39% (range: 34.58 - 88.94%) and the mean was 69.12% 14.64% . The median value percentage of mismatch of primary gtv pet to ct was 25.01% (range: 5.00 - 141.41%) and the mean was 33.63% 28.96% . The median value of percentage of mismatch of primary gtv ct to pet was 31.97% (range: 2.60 - 142.67%) and the mean percentage was 46.05% 36.92% . Pet scan changed the gtv ct with a median value of -12.52% (range: -131.11 - 53.74%) and with a mean value of 15.18 41.49% . In most of the cases, pet scan identified gtvs outside the volume delineated by ct scan . The median value of primary gtv pet outside ct scan volume was 12.10 cc (range: 1.50 - 29.80 cc) and the mean value was 12.17 cc 7.81 cc . The median value of gtv pet was 7.00 cc (range: 0.40 - 71.70 cc) and the mean was 12.72 cc 15.46 cc . The median value of gtv ct was 6.50 cc (range: 0.20 - 67.00 cc) and the mean was 11.04 cc 14.87 cc . The gtv ct was larger than the gtv pet in nine cases (37.5%) and was smaller in 15 cases (62.5%). The median value percentage of nodal gtv pet covered by ct was 83.73% (range: 31.18 - 100.00%) and the mean was 81.46 19.48% . The median value of percentage of nodal gtv ct covered by gtv pet was 61.30% (range: 25.81 - 100.00%) and the mean was 61.02 18.00% . The median value of percentage of mismatch of nodal gtv pet to ct was 61.88% (range: 0.00 - 168.42%) and the mean was 63.90 47.53% . The median value percentage of mismatch of nodal gtv ct to pet was 10.86% (range: 0.00 - 81.82%) and the mean percentage was 18.66 24.65% . Mismatch pet to ct was significantly smaller than ct to pet (p = 0.04). The median value of primary gtv pet outside ct scan volume was 3.20 cc (range: 0.00 - 18.20 cc) and the mean value was 4.44 4.52 cc . The median value of gtv ct was 32.00 cc (range: 0.00 - 309.10 cc) and the mean was 54.78 cc 64.47 cc . The median value of gtv pet was 35.00 cc (range: 3.20 - 269.50 cc) and the mean was 48.43 cc 53.21 cc . The gtv ct was larger than the gtv pet in nine cases (37.5%) and was smaller in 15 cases (62.5%). Impact of positron emission tomography / computed tomography in primary and nodal gross tumor volume the median value of percentage of primary gtv pet covered by ct was 70.07% (range: 32.42 - 95.52%) and the mean was 65.18% 18.84% . The median value of percentage of primary gtv ct covered by gtv pet was 68.39% (range: 34.58 - 88.94%) and the mean was 69.12% 14.64% . The median value percentage of mismatch of primary gtv pet to ct was 25.01% (range: 5.00 - 141.41%) and the mean was 33.63% 28.96% . The median value of percentage of mismatch of primary gtv ct to pet was 31.97% (range: 2.60 - 142.67%) and the mean percentage was 46.05% 36.92% . Pet scan changed the gtv ct with a median value of -12.52% (range: -131.11 - 53.74%) and with a mean value of 15.18 41.49% . In most of the cases, pet scan identified gtvs outside the volume delineated by ct scan . The median value of primary gtv pet outside ct scan volume was 12.10 cc (range: 1.50 - 29.80 cc) and the mean value was 12.17 cc 7.81 cc . The median value of gtv pet was 7.00 cc (range: 0.40 - 71.70 cc) and the mean was 12.72 cc 15.46 cc . The median value of gtv ct was 6.50 cc (range: 0.20 - 67.00 cc) and the mean was 11.04 cc 14.87 cc . The gtv ct was larger than the gtv pet in nine cases (37.5%) and was smaller in 15 cases (62.5%). The median value percentage of nodal gtv pet covered by ct was 83.73% (range: 31.18 - 100.00%) and the mean was 81.46 19.48% . The median value of percentage of nodal gtv ct covered by gtv pet was 61.30% (range: 25.81 - 100.00%) and the mean was 61.02 18.00% . The median value of percentage of mismatch of nodal gtv pet to ct was 61.88% (range: 0.00 - 168.42%) and the mean was 63.90 47.53% . The median value percentage of mismatch of nodal gtv ct to pet was 10.86% (range: 0.00 - 81.82%) and the mean percentage was 18.66 24.65% . Mismatch pet to ct was significantly smaller than ct to pet (p = 0.04). The median value of primary gtv pet outside ct scan volume was 3.20 cc (range: 0.00 - 18.20 cc) and the mean value was 4.44 4.52 cc . In our study, all patients underwent imrt planning cect scans along with pet / ct . The use of non - ionic contrast material did not interfere in interpretation of data . Fused cect scan studies with pet scans for better anatomical delineation for various types of solid cancers . Incorporation of pet / ct in the management of head and neck cancers has resulted in significant changes in clinical staging. [1014] wang, et al ., evaluated the impact of fdg - pet fused with planning ct scans on tumor localization . Pet / ct changed ct - based staging in 16 of the 28 (57%) patients . In 12 cases, the ct - based t - stage was upgraded by the pet / ct . In six cases, the ct - based nodal information was up - staged by the pet / ct ., analyzed the use of [18f]-pet / ct images for staging and target volume delineation of patients with head and neck carcinoma . Pet / ct imaging changed the tnm categories in 5 of the 22 (22%) cases when compared with ct alone . T - stage changed in 3 of 22 (14%) and n - stage in 2 of 22 cases (10%). Similarly, in this study, pet / ct resulted significant discrepancy with ct - based staging in 7 of the 26 patients (up - staged in 6 and down - staged in 1). Various methods were proposed for accurate contouring of the gtv in pet / ct fusion - guided radiotherapy . Visual interpretation method,[1517] a fixed 50% threshold of the background subtracted tumor maximum uptake (thr), an appropriate thr level for an individual patient, software - based automated segmentation, were most commonly used for pet / ct - based contouring in clinical studies . The most appropriate method for pet / ct - based gtv delineation various studies compared the gtvs contoured in pet / ct scans with ct scans and demonstrated the role of pet / ct fusion for radiotherapy planning. [111516182226] scarfone, et al ., prospectively studied the impact of hybrid pet - ct simulation in tumor and normal tissue delineation for rt planning in patients with head and neck cancer . They contoured abnormal pet uptake areas using the visual interpretation method . We also adopted visual interpretation method to define gtvs in primary and nodal areas for our study . Heron, et al ., prospectively studied the impact of hybrid pet - ct simulation in tumor and normal tissue delineation for rt planning in patients with head and neck cancers . For primary disease, pet - based target volumes (mean = 42.7 cm) were significantly (p = 0.002) smaller compared with ct - based target volumes (mean = 65 cm) for all patients ., compared ct, mri, and [18f]-fdg - pet for delineation of gtv in pharyngolaryngeal squamous cell carcinoma . For oropharyngeal tumors and for laryngeal or hypopharyngeal tumors, average gtvs delineated at ct were 32.0 and 21.4 cm, respectively, whereas average gtvs at pet were smaller, 20.3 [p = 0.10] and 16.4 cm [p = 0.01], respectively . In various studies, pet - based gtvs were smaller than ct - based gtvs . In this study, gtv pet was significantly smaller than gtv ct (p <0.001) and consistent with above studies . In contrast, nishioka, et al . Showed that gtv volumes were not changed by image fusion between 18f - fdg - pet and mri / ct in majority of cases and ashish, et al . Variations in image acquisition procedures, registration methods, and target volume delineation methods are potential factors for this variation . Mismatch analysis is a potential tool to study the disagreement between ct and pet / ct in contouring of gtv . Many studies analyzed this issue and found that there is a significant mismatch existing between gtv pet and gtv ct . In a study by daisne, et al ., the average mismatch ct to fdg - pet was 73% and fdg - pet to ct was 14% for oropharyngeal tumors . For laryngeal tumors, average mismatch ct to fdg - pet was 48% and fdg - pet to ct was 17% ., found the mean value for mismatch of gtv pet / gtv ct as 28.9% s32.9% and the mismatch of gtv ct / gtv pet as 70.9% 50.9% . In this study, the mismatch gtv pet / gtv ct was significantly smaller than mismatch gtv ct / gtv pet (p = 0.03). In this study, the coverage of gtv pet to gtv ct and gtv ct to gtv pet was analyzed to evaluate the difference between two gtv volumes . This was comparable with results of studies done by ei - bassiouni, et al ., and schinagl et al ., in a study by ei - bassiouni, et al ., the median percentage of the gtv pet covered by gtv ct was of 99.5% (range: 41.6 - 100%) and a mean of 92.4% 16.4%, whereas the median percentage of gtvct covered by ptvpet was 95.1% (range: 44.3 - 100%) and a mean was 88.2 - 16.2% (p = 0.2). The major clinical implication of our study is delineation of the metabolically active volumes outside the ct scan . These volumes of diseases could have been missed during radiotherapy treatment planning and could result in in - field recurrences and marginal recurrences . The median value of primary gtv pet outside ct scan volume was 12.10 cc (range: 1.50 - 29.80 cc) and the mean value was 12.17 7.81 cc . Pet / ct has the advantage to identify metabolically active volumes that are used in simultaneous integrated boost sib - imrt and these volumes are also utilized for dose escalation of gtv . Performed studies in phantoms filled with radioactive material and proposed threshold - based contouring for pet / ct scans in head and neck cancers . However, we adopted the visual interpretation method for target volume delineation mainly because of limitations in our planning systems . Twenty - six patients were included in this study . In spite of these limitations, though this study demonstrated that pet / ct could improve target volume delineation for radiation treatment planning, we feel that more studies are needed to substantiate the importance of pet / ct . Pet / ct significantly alters the staging of tumor (t staging) and lymph node metastases (n staging) in head and neck cancers . Pet / ct also has the potential to identify gtv outside ct - based gtv that increases the accuracy of radiation planning . We believe that in the era of imrt / image - guided radiotherapy (igrt), pet / ct will increasingly be used for the radiotherapy planning.
There has been considerable interest in the possibility that the eradication of persistent viral reservoirs in hiv-1-infected patients could be achieved through specific upregulation of viral expression from quiescently infected reservoir cells [16]. These silent viral reservoirs, largely comprised of hiv-1-infected, resting cd4 + t cells, are long - lived despite continuous and lengthy administration of haart or antiretroviral therapy [3, 4, 7]. Eradication of these persistent reservoirs may be possible if a sufficient level of viral expression could be induced from the latent proviruses in order to trigger immune clearance or apoptosis of infected reservoir cells [36]. A number of diverse agents upregulate viral transcription from latent hiv-1 proviral templates in vitro and in vivo . These compounds include small chemical cellular activators such as prostratin, a member within the phorbol ester family, the macrolide lactone bryostatin-1, chromatin - remodeling agents, and select cytokines (primarily interleukins and interferons) [1, 5, 812]. Upregulation of latent hiv-1 expression via the phorbol ester family of compounds or bryostatin-1 occurs as a consequence of activating or modulating the protein kinase c pathway [2, 8, 9, 11, 12]. The ability of such compounds to upregulate hiv-1 transcription has been well documented in several cellular systems including the latently infected u1 or ach 2 cell - lines, primary t lymphocytes from haart patients as well as the scid - hu mouse model for hiv-1 infection [2, 8]. The concentration ranges of the pkc activating agents investigated in this study effectively upregulate latent hiv-1 expression in primary cells of infected patients . However, developmental and toxicological effects associated with administration of these agents in an intact whole animal model have not been thoroughly evaluated . One purpose of this study was to rapidly assess the gross effects of pkc activation or modulation on zebrafish embryos and larvae, particularly morbidity or lethality, as major indicators of whether nontumor promoting phorbols, including prostratin, or the lactone bryostatin-1, could be regarded as serious candidates for use in humans at reasonable and effective doses . The tumor - promoting property of some phorbol esters essentially excludes these agents from consideration for clinical administration . The observation that zebrafish are able to uptake pkc activating agents from the media also provides impetus to observe the effects of modulating protein kinase c pathway activity on zebrafish tissue formation and function . Modulation of the pkc signal cascade can have widely diverse effects upon cells and whole tissue systems . Notable effects, apart from the potential of hiv-1 reservoir eradication in humans, include the apparent extension of memory which may bear relevance toward the treatment of alzheimer's disease as well as the amelioration of certain malignancies [1416]. Wild - type ab strain and fli-1 transgenic strain zebrafish were maintained in separate tank systems for embryo and larvae harvesting . Fli-1 transgenic zebrafish bear an enhanced green fluorescent (egfp) open reading frame linked to the fli-1 promoter which drives zebrafish expression of the egfp transgene in blood vessels . Embryos a few hours after fertilization were distributed into 60 15 mm sterile culture dishes containing equal volumes of embryo media comprised 5 mm nacl, 0.17 mm kcl, 0.33 mm cacl22h2o, and 0.33 mm mgso47h2o . Typically, each dish contained 4050 embryos per dish then treated once daily by replenishing the media with the concentrations of agents as stated for three consecutive days or as stated otherwise . Samples included a negative vehicle control, 1% dimethyl sulfoxide (dmso) and varying concentrations of prostratin or a phorbol-12 myristate-13 acetate (pma) positive control at 10 um, each suspended in dmso . The treated embryos were then observed for developmental abnormalities or mortality using fluorescent or light microscopy . Fluorescent images under blue light were captured using an olympus ck40 fluorescent microscope equipped with a digital camera . Visible light photos were obtained using a fujifilm finepix a610 digital camera with the lens placed onto one eyepiece of dissecting microscope bearing an illuminated specimen base . Hatched larvae were evenly distributed into 60 15 mm sterile culture dishes at a minimum of 20 larvae / dish containing embryo media . The larvae were treated once with the concentrations of agents as indicated and harvested 6075 minutes after treatment then stored at 70 degrees c for subsequent page / western analysis . Typically, fourteen larvae from each treated population were harvested by transfer into individual 1.5 ml microfuge tubes on ice using 1 ml plastic transfer pipets . The remaining specimens remained in the media and were photographed either immediately or 24 hours after treatment using 0.4% tricaine as an anesthetic for surviving specimens . Zebrafish specimens harvested and frozen at 70c were thawed and suspended in 50 ul of laemmli protein loading buffer (biorad, hercules, ca, usa). Equivalent quantities of protein were loaded to each lane, quantified by using the lowry protein assay (biorad, hercules, ca, usa). Prior to gel loading, the samples were heated to 90c for 5 minutes and centrifuged at 12,000 g for 3 minutes . Samples were electrophoresed on 4%15% gradient gels (biorad, hercules, ca, usa) and proteins transferred to pvdf membranes using trans - blot semi dry transfer cell (biorad, hercules, ca, usa) at 15 v for 1 - 2 hours . Protein blots were incubated in the presence of anti - gfp antisera (bio - rad, hercules, ca, usa) or anti - mapk-8 zebrafish - specific antisera obtained from anaspec, freemont, ca, usa . Pvdf membranes were developed using the westernbreeze chromogenic western blot immunodetection kit as per instructions using anti - rabbit igg conjugated with alkaline phosphatase . Images of blots were captured by scanning with an hp officejet pro combination printer / scanner . As shown in figure 1, no abnormalities or increased mortality were observed one day after treatment of day 1 postfertilization embryos with varying concentrations of prostratin and 10 um pma compared to embryos treated with an equivalent concentration of the control vehicle, dmso . The development of the embryonic vascular systems appeared similar in all treated samples as assessed by egfp fluorescence of vascular endothelial cells which are visibly highlighted via expression of the fli-1 promoter / egfp transgene . The egfp fluorescence notably highlights the vascular endothelium in the anterior head portion and extending down the developing vertebral column of the embryos in these images . As shown in figure 2, observations by light microscopy of treated, unhatched embryos on day 3 postfertilization looked much the same with regard to development as those noted for the previous day treatments . These later - stage embryos, exposed to low (0.1 m) and medium (1 m) concentrations of prostratin, were not observably different from those treated with dmso . As well, exposure to 10 m prostratin elicited no observable degree of developmental abnormality . This was very much in contrast to specimens treated with 10 m pma treatment . As shown in the bottom right panel of figure 2, this representative pma - treated embryo was deformed and failed to survive . As shown in figure 3, overall development of the 4-day postfertilization embryos and newly hatched larvae continued to proceed without observable gross defects with increasing concentrations of prostratin . Differences began to emerge shortly after day 3 after fertilization and generally into day 4 when hatching is generally initiated . The most common effects observed were death and/or trapping in the egg sac with exposure to 10 m pma . The concentration - dependent effects of phorbol treatment were next compared on hatched, nontransgenic seven - day - old ab larvae as shown in figure 4 . Pma treatment at 10 m resulted in rapid and complete lethality as is clearly apparent for the decaying embryo specimen shown to the right side of figure 4 which was photographed 24 hours after treatment . However, very limited gross morphological deformities were apparent in larvae treated with 10 m prostratin at least from the time of treatment to visual inspection 24 hours later . Indeed, the 10 m prostratin - treated larvae appeared morphologically similar to those treated with the dmso vehicle control (middle and left panels of figure 4) with high survival rates 24 hours after treatment . Protein gel electrophoresis (page) of total proteins from treated larvae was employed to assess any major differences in gene expression as a consequence of phorbol ester exposure . Page comparing treated and vehicle control showed no remarkable difference in overall expression among treated or control embryos (data not shown) or larvae as shown in figure 5(a). Abundant structural proteins in larvae were similar in presence and abundance among all the phorbol - treated larvae samples versus the dmso control . Pma, known to be a potent apoptotic mitogen for most cells and tissue systems [15, 16], exhibited the highest degree of inducing developmental defects and lethality relative to prostratin - treated specimens at the same concentration ., western analysis was performed on hatched ab larvae treated with dmso, prostratin and pma using mapk-8 antisera in the expectation that phorbol exposure may alter the expression level of this specific signaling protein . As shown in figure 5(b), mapk-8 exhibited modest levels of upregulation following treatment with prostratin or pma relative to dmso - control - treated larvae in this representative blot . Western analysis was also employed to assess any changes in the expression of the egfp protein which remained at similar levels among fli-1 larvae treated with prostratin or pma versus dmso control (data not shown). The lethal effects of an expanded panel of pkc modulators were next compared in a concentration - dependent fashion using larvae eight days after fertilization . As shown in figure 6, treatment with different pkc modulators exhibited defined threshold doses for lethality . The phorbol ester, 12-deoxyphorbol-13-phenylacetate (dpp), had a concentration - dependent lethal effect at 10 m, similar to pma . Prostratin only elicited rapid death at 100 m which occurred in the same time frame as dosage with 10 m pma and 10 m dpp, usually within 6075 minutes after treatment . Of interest is the ability of larvae to tolerate concentrations of bryostatin-1 up to 10 m . Although 10 m bryostatin-1 treatment did not induce lethality in the short term, the fish appeared very lethargic 1 hour after treatment and no survivors were observed 24 hours after treatment . The treated fish were unaffected by concentrations of bryostatin-1 at or below 1 m . The survival profiles for each individual compound shown in figure 6 represent an average of four data sets for treated larvae eight days after fertilization with less than 2% standard deviation from the mean for each set . The lethal effects at the concentrations noted for this larval set also occurred reproducibly for either ab or fli-1 larvae treated even at earlier postfertilization times . In addition, these survival frequencies were similar for unhatched 4-day postfertilization ab or fli-1 embryos . Survival frequency for embryos and larvae, regardless of strain was always reproducibly ~94%99% at nonlethal agent doses, and lethality was consistently very near or at 100% with higher doses of the compounds at all times of treatment beyond three days after fertilization . The nontumor - promoting phorbol ester prostratin and the lactone bryostatin-1 modulate pkc cascade activity . These agents have been forwarded as potential candidates for the eradication of haart - persistent cellular reservoirs bearing silent hiv-1 proviral dna [24, 8, 9, 11]. These pkc modulators, including prostratin, upregulate latent viral expression in a number of cell - based systems [2, 8, 9, 11] but have not had widespread clinical use in patients except for bryostatin-1 . The primary objective of this study was to determine whether treatment with pkc modulators or activators, particularly the phorbol ester prostratin, might elicit a severe cytotoxic profile or induce development effects in tissue systems within a tractable vertebrate model . Specificity was further noted by modest upregulation in the expression of the signaling factor, mapk-8, within phorbol treated larvae . The map kinases (mapks) including mapk-8 (p38) are known to respond to mitogen stimulation, proinflammatory cytokines and environmental stress although this response is primarily mediated through a series of phosphorylation events of preexisting proteins [17, 18]. Our data suggest that some upregulation of mapk-8 via de novo synthesis can occur by exogeneous phorbol treatment of whole zebrafish larvae . This contrasts the expression of the egfp whose levels remained unchanged in fli-1 larvae treated similarly with the equivalent concentrations of prostratin and pma . We have some preliminary evidence that pkc modulating compounds induce apoptosis at high doses likely contributing to their obvious lethal effect . This is consistent with the action of phorbol esters and mapk-8 which can participate in a mitogen - activated cascade to initiate an apoptotic effect [16, 20]. Assessing the effects of pkc modulators using the zebrafish model are of interest given the ongoing concerns regarding the use of pkc activators or modulators as clinical candidates for administration to humans . This caution may be warranted, since this diverse class of compounds can broadly activate multiple cell - types and can rapidly advance cell - type specific differentiation, maturation, or apoptosis [2, 8, 19]. For instance, prostratin rapidly advances monocyte differentiation and bryostatin-1 induces accelerated maturation of human cord - blood derived dendritic cells . Interestingly, the broad effects of such properties induced by the phorbol ester family are unknown in a whole developing animal model . In contrast, bryostatin-1 has been evaluated clinically at low doses for the treatment of certain human cancers [2123]. Bryostatin-1 is also regarded as a potential candidate for the treatment of alzheimer's disease, as it appears that exposure to the compound can extend memory and rescue retrograde or anterograde long - term memory following cerebral ischemia / hypoxia [24, 25]. The data in this paper might be regarded as encouraging in that low concentrations of pkc modulators, including the phorbol esters, which upregulate latent hiv-1 expression in human cells within a range of 1 to 10 m [2, 9], showed no obvious effect upon zebrafish embryo or larvae development . In contrast, higher concentrations of pkc activators, elicited almost complete lethality in both zebrafish embryos and early - term larvae . Zebrafish embryos and larvae were particularly useful to study the effects of pkc activators or modulators in a whole animal model, since the compounds can be absorbed directly from the media and have clear effects on this organism's development and survival . Interestingly, effects on embryos were not observable until after day 3 into day 4 of agent treatment and were most notable with pma exposure . It is not clear why this very potent tumor - promoting phorbol did not elicit lethal effects on early - stage embryos as quickly as it did for hatched larvae . Zebrafish are recognized as a useful model to rapidly establish paradigms for the investigation and treatment of disabling human diseases . The absorption of exogenous phorbol esters by zebrafish at varying developmental stages appears to be a feasible way to begin to modulate pkc signaling in vivo to assess effects on specific tissue systems . Importantly, such processes affected by pkc modulators include, but are not limited to, memory extension and tumorigenesis as noted in other vertebrate systems . [22, 24, 25]. These studies also demonstrate that the nontumor promoting phorbol ester prostratin had no obvious deleterious effects on zebrafish development at concentrations below 10 m, which is sufficient to upregulate latent hiv-1 expression in human cellular systems [2, 8, 9]. This compound or related agents may deserve further consideration in clinical protocols toward the eradication of hiv-1 latent reservoirs.
Why have we not been able to use neuroimaging in the clinical management of psychoses? After 30 years of using this approach, we have established that structural brain alterations are present in individuals with psychoses . While this information has advanced academic knowledge on the pathophysiology of these disorders, it has had limited utility in clinical practice . The main reason can be found in the nature of brain alterations in psychoses: they are subtle and spatially distributed . Still, the time may now have come when quick and noninvasive structural neuroimaging abandons the academic solitude in which it has been operating, and brings its novel methods and findings into the clinical arena . This paper aims to appraise this evidence, and discuss it in the context of potential future applications of magnetic resonance imaging (mri) in the clinical management of psychoses . The first part of this review will present evidence on the presence of neuroanatomical alterations in psychoses . It will then discuss studies that have examined the relationship between brain alterations and various medium- to long - term clinical and functional outcomes . In the third section, it will discuss more recent evidence on the relationship between brain alterations and early outcomes, with a particular focus on the relationship between symptomatic responses and initial treatment with antipsychotic drugs . The paper will then present a discussion of more novel approaches for the analysis of mri data, such as support vector machine . Findings from these approaches have started to suggest that mri has the potential to provide clinically meaningful information . Finally, it will discuss the implications of having strong and valid neuroimaging markers of psychosis outcomes in developing targeted, as well as novel, treatment interventions for these disorders . Moving from the first evidence that individuals with established schizophrenia have subtle structural brain alterations, most notably an enlargement of the lateral ventricles, we have been studying individuals earlier and earlier along the disease course . More than 10 years ago, the seminal work by pantelis and colleagues showed that individuals at high risk of developing psychosis, but not ill yet, already show smaller volumes of the same brain areas that have been reported as smaller in individuals with an established psychosis, namely frontal and temporal cortices . Even more novel, however, was their report that this is particularly the case for individuals who then actually develop the illness, and that some of these alterations could be specific to the type of psychosis that these subjects developed . For example, they found that subjects who develop an affective type of psychosis showed reductions in the subgenual anterior cingulate cortex, an area involved in the generation of affective symptoms . Complementary to these gray - matter changes, ultra - high - risk individuals also have white matter alterations similar to, albeit less extensive than, first - episode patients . These include poorer integrity of the major associative fibers that connect fronto - parieto - temporal (superior longitudinal fasciculus) and fronto - parieto - occipital (inferior fronto - occipital fasciculus) regions, commissural fibers (corpus callosum), and cortico - subcortical pathways (corona radiata, corticospinal tract, and corticopontine tract). These findings, integrated with evidence from many reviews and meta - analyses, suggest that brain alterations become more extensive at the time of a first full psychotic episode, and then possibly even more marked over the years, when the illness becomes established . These are indeed two main factors that have limited our ability to use this information about brain alterations in the clinical management of the individual patient . Therefore, many studies have tried to establish whether these distributed brain changes are more likely to occur in individuals with a more severe illness course or a worse clinical and functional outcome . For example, a poorer clinical outcome has been variously defined as response to early treatment, number of subsequent episodes, severity of symptoms, hospitalizations, or duration of remissions; while functional outcome has been defined as the ability to live independently, maintain employment, or be in a relationship . Nevertheless, many studies have tried to establish if structural brain alterations are related to illness characteristics by comparing brain structure in patients and healthy controls and then evaluating the relationship with outcomes at a single time point; or conducting repeated evaluations of brain structure, and then investigating the relationship between longitudinal brain changes and outcomes . Most of these studies have evaluated the relationship with medium to long - term outcomes, and only more recently has the attention shifted to the evaluation of the relationship between brain alterations and early outcomes such as response to the first treatment . Studies that have evaluated brain structure at the time of illness onset and again following treatment, have often reported smaller volumes of gray matter in association with indicators of subsequent poorer clinical outcomes (eg, higher number of admissions or worse symptom severity), and with worse social or occupational outcomes . For example, smaller volumes of prefrontal areas have been found to predict poorer functioning (defined as a combination of social, occupational, and psychological functioning) 1 year later in a small sample of first - episode psychosis patients . Similarly, in a sample of patients with schizophrenia, drawn from an unselected general population sample, jskelinen and colleagues found that individuals showing higher density (an indirect measure of volume) of frontal and limbic areas had an overall better clinical (defined as a lower number of hospitalizations) and functional (defined as not being on a disability pension) outcome in the subsequent 16 years . Wassink and colleagues reported an association between smaller volumes of another brain area, the cerebellum, and longer negative and psychotic symptom duration, as well as psychosocial impairment (eg, quality of relationships, sexual activity, enjoyment of recreation, and work performance) in individuals with schizophrenia spectrum disorders after a 7-year follow - up . Still, others have not found an association between brain volumes at onset and subsequent outcomes . For example, the prospective study by van ilaren and colleagues found no relationship between brain volume measurements and symptom severity, level of functioning (defined as the ability to maintain a variety of social roles), need for care, and illness course after 2 years . Mitelman and colleagues examined white matter integrity using diffusion tensor imaging (dti), in a group of 104 patients with an average 4-year schizophrenia duration . They classified them as having a poor or good outcome, based on both symptom severity and level of functioning at this time point . They found that although both patient groups showed reduced overall white matter integrity of the prefrontal and temporal areas (as a measure of fractional anisotropy [fa]), these alterations were more extensive in patients with a poor outcome than in those with a good outcome . Longitudinal studies that have evaluated brain structure at multiple time points after illness onset can help clarify if brain alterations, evident at illness onset, progress differently in individuals who then developed a poorer outcome . For example, a larger decrease in the volume of the lingual gyrus, insula, and cerebellum has been reported in patients with a worse functional outcome (a combination of social, occupational, and psychological functioning) 4 years after the first psychotic episode . With a similar duration of follow - up, van ilaren and colleagues also reported a greater gray matter volume reduction, and a greater thinning of temporal and frontal cortices in individuals who had poorer outcomes (including a higher number of hospitalizations and a lower level of social and personal functioning) 5 years after illness onset . Similarly, in a sample of children and adolescents with early onset first - episode schizophrenia, arango and colleagues showed that more weeks of hospitalization and higher severity of negative symptoms were correlated with greater left frontal gray matter volume loss and greater cerebral spinal fluid increase, respectively . Dynamic changes in relation to outcomes were also observed by our group in a 6-year follow - up study of hippocampal volume in first - episode psychosis patients . In this sample, we specifically found that individuals who had an increase in hippocampal volume over the first 6 years of illness were the ones who showed a less severe illness course and lower symptom severity, as well as a better functional outcome at follow - up, thus suggesting an important role for brain neuroplasticity . Taken together, these findings suggest that brain alterations are present at illness onset and may be associated with worse medium- to long - term illness outcomes and ability to function . Still, two important considerations need to be made in the interpretation of these findings . First, it is not possible to establish what the casual link is between brain structure and illness course . While brain alterations could be the substrate of a more severe illness, the more severe illness itself could affect brain plasticity by increasing exposure to substance abuse, worse living conditions, or a less stimulating environment . It is, therefore, possible that patients with a more severe illness are exposed to longer periods of treatment or higher doses of antipsychotics, which may themselves affect brain volumes . From a clinical perspective, it may be more informative to study the relationship between brain alterations at illness onset and outcome measures that occur earlier in the course of illness, which is the topic of the next section . If brain alterations could be used to predict, as early as possible, which patients are destined to have a poorer response to the first few weeks or months of treatment with antipsychotic medications (the main stake for the treatment of psychosis), we could implement interventional strategies targeted at this specific subgroup . Currently, we treat the first psychotic episode on a trialand - error basis, and we simply have no way of saying who will respond to the first antipsychotic drug and who might benefit from longer or different interventions . In fact, we know that only -55% of patients respond to antipsychotics in the first 12 months of treatment . Yet, this early response is thought to be one of the strongest predictors of subsequent functional and clinical outcomes in psychosis . A pooled analysis, published in 1992, examined the relationship between response to antipsychotics and brain measures, such as ventricular - brain ratio, sulcal prominence, third ventricles, and other brain measures . The authors found that the composite effect sizes were very small and did not reach statistical significance . However, recent studies have lent renewed support to the notion that alterations in both gray and white matter may be associated with a poorer response to antipsychotics, possibly because they used more homogeneous populations, or in some cases, better imaging acquisition approaches and more sophisticated methods of analysis . For example, zipursky and colleagues examined global tissue volumes in firstepisode psychosis patients, and found that subjects with a poorer response (defined as having a lower reduction in symptom severity from baseline) to the antipsychotic haloperidol in the first month of treatment had smaller cortical gray matter volume than those who showed symptomatic improvement . Relatively consistent data have been reported on the relationship between response to clozapine and brain structure . Cortical measures, such as sulcal enlargement, particularly in the prefrontal cortex, have been frequently related to poor response to clozapine in patients with chronic schizophrenia . In a double - blind, 10-week trial of clozapine and haloperidol, arango and colleagues found that a larger prefrontal gray matter volume was associated with better treatment response in clozapine - treated patients, but with a poor response in haloperidol- treated patients . They also found no relationship between response to these drugs and hippocampal and caudate nucleus volumes . The authors suggested that the presence of larger brain volumes might characterize patients who are more likely to benefit from clozapine . This finding is also consistent with evidence from a study in patients with treatment - resistant schizophrenia who were treated for with clozapine 6 months . Patients with a larger baseline volume of the dorsolateral - prefrontal cortex were more likely to show improvement in their negative symptoms . Interestingly, amelioration of positive symptoms was related to a larger temporal cortex volume, and amelioration of disorganization symptoms was inversely related to hippocampal volume . Another temporal area has been reported as being related to early outcome the parahippocampal gyrus . Using voxel - based morphometry, bodnar and colleagues found that the parahippocampal gyrus was significantly smaller in first - episode psychosis patients who, after 6 months of antipsychotic treatment, had not remitted, compared with those who had achieved remission . Furthermore, by building a classification model using parahippocampal gray matter concentration, they were able to correctly classify remission status in 79% of the cases . In another study of patients with established schizophrenia treated with olanzapine or risperidone, 3-week response to these antipsychotics was associated with smaller volumes of other areas, such as the insula and rectal gyrus, and larger volumes of the basal ganglia . White matter has also been associated with treatment response, but the evidence is considerably more scant . Response to antipsychotics was evaluated by garver and colleagues in a small sample of patients with schizophrenia after 28 days of treatment . Here, somewhat counterintuitively, the 8 patients who responded to antipsychotics showed worse white matter microstructural integrity than the 5 patients who did not respond to medication . Of note, individuals, in this study, had an established illness, and therefore, in contrast, studies in antipsychotic naive or minimally treated patients have the advantage that any structural brain alterations would be less likely to reflect the effect of antipsychotics on brain structure, and more likely to be a marker of illness . Luck and colleagues analyzed a sample of first - episode psychosis patients using dti, and then reassessed them at 6 months, which is when they classified them as having either a poor or good treatment outcome . They specifically examined three white matter tracts connecting frontal and temporal regions: the cingulum, the superior longitudinal fasciculus, and the uncinate fasciculus . Patients with a poor outcome showed greater alterations in white matter integrity in the superior longitudinal fasciculus and uncinate than patients with a good outcome, suggesting that abnormal fronto - temporal connectivity may represent an early marker of short - term clinical outcomes . Our group has also recently focused on the evaluation of the relationship between neuroanatomical markers in white and gray matter at the time of presentation, and response to treatment at 12 weeks . This is a time when all patients would have received at least one full therapeutic course of antipsychotic medication . We examined white matter integrity, using dti and tractbased spatial statistics, to assess fa in a large sample of patients at their first episode of any psychosis . We operationalized response to treatment as having achieved symptomatic remission according to the criteria of the schizophrenia working group . We found that already at illness onset, patients who subsequently did not respond (n=40) had lower fa than healthy controls in the uncinate, cingulum, and corpus callosum . Furthermore, they also had lower fa in these regions than patients who responded to treatment (n=40; figure 1, tables i and ii). One of the most striking findings of this work was that the responders were indistinguishable from the healthy control subjects, suggesting that the original clinical sample was biologically rather heterogeneous . We rescanned these subjects at the 12-week clinical evaluation, and found that the same regional differences seen at baseline between the two patient groups were also present at 12 weeks, although the differences appeared less widespread . Furthermore, although there was some increase in fa over time, this was in the same direction in the two patient groups, with no significant time vs group interaction . We interpreted these findings to indicate that white matter integrity and brain connectivity are important moderators of response to antipsychotics . It is intriguing to speculate whether these alterations are established early on, as a neurodevelopmental deviation . The presence of a neurodevelopmental alteration would find further support in our gray matter findings from this sample . In these individuals, we performed the first 3d evaluation of cortical gyrification, a marker potentially indicative of early neurodevelopmental disturbances, in relation to subsequent treatment response . Gyrification defects have been associated with exposure to obstetric complications and with psychotic symptoms resistant to treatment . Interestingly, the earlier neuroimaging studies in treatment - resistant patients that were mentioned in the previous section, found that sulcal enlargement (which could lead to hypogyria) was associated with a poorer response to clozapine . In our samples, we found that, already at illness onset, patients who subsequently did not respond to treatment had significant cortical folding defects (hypogyria) of several frontotemporal regions and the insula when compared with the responders . They also had widespread deficits in gyrification extending to the precuneus, angular gyrus, and lingual gyrus when compared with healthy controls (figure 2, table iii). In contrast, and similarly to what we saw for white matter, patients who subsequently responded were virtually indistinguishable from the healthy controls . These findings are very consistent with the evidence described earlier that only the nonresponders have reduced integrity in the white matter tracts that connect these cortical regions . It is also interesting that the alterations in gyrification and white matter were evident in nonresponders with both affective (bipolar disorder or major depression with psychotic symptoms) and nonaffective (schizophrenia, schizophreniform disorder, schizoaffective disorder and psychosis not otherwise specified) psychosis, suggesting that as a group, nonresponders are likely to have a more homogeneous pathophysiological process underlying psychoses than the responders . Taken together, these findings suggest that individuals who are less likely to respond to treatment represent either a more severely affected group, or a group that has experienced a pathophysiological insult at an early neurodevelopmental stage than those who respond to treatment, possibly with a differential exposure to factors that determine axonal integrity (genetic or molecular), which, in turn, influences brain connectivity and gyrification . Although crucial to our understanding of psychosis, these group findings need to be extended if they are to inform clinical outcomes at the level of a single individual . Structural imaging has strong potential for clinical applicability as it is: (i) noninvasive; (ii) quick to acquire; (iii) widely available; and finally, (iv) cheap compared with other imaging methods such as positron emission tomography . Therefore, what progress have we made toward its clinical applicability? Over the last few years, the application of novel approaches to the analysis of structural imaging data has made us feel closer to their potential use in clinical management . To this end, machine - learning methods, such as support vector machine, have shown promise in differentiating patients or ultra - high - risk individuals from healthy controls, based on structural mri data . Obviously, an even more interesting translational question would be: can we use structural mri data at illness onset to predict which individuals will develop worse outcomes or poorer responses to treatment? About 2 years ago, our group published the first paper showing that structural brain scans at the first episode could be used to predict, with significant accuracy, outcomes at 6 years . In this study, we found that the mri scan obtained when patients first presented to services could be used to predict which patients developed a continuous nonremitting illness course; distinguishing them from both healthy controls (sensitivity, 71; specificity, 61) and from patients who had an episodic, more benign illness (sensitivity, 71; specificity, 68). Consistent with our recent data, discussed in the previous section, we were not able to distinguish the patients who went on to have an episodic course from the healthy controls . Other papers have since confirmed, in different clinical populations, the potential of machine - learning approaches to discriminate individual patients in predicting the onset and subsequent severity of psychosis in ultra - high - risk individuals . While promising, these approaches need replication in larger samples of patients at the same illness stages and treated with the same pharmacological interventions, and also need validation for scans obtained using different scanners . These are just some of the aims of an ongoing, large, multicenter study funded by the fp7 programme of the european commission, optimise (optimization of treatment and management of schizophrenia in europe, www.optimisetrial.eu). Across various centers in europe, we are acquiring mri scans from a very large sample of patients with first - episode schizophrenia, who are then all treated with the same antipsychotic, amisulpride, for 4 weeks, when their symptomatic response is evaluated . This study will use both univariate and multivariate image analysis approaches, ie, support vector machine, to establish whether it is possible to predict the 4-week response based on the mri scan obtained at the first episode, alone or in combination with, other biological markers, including magnetic resonance spectroscopy . We know that the outcome and response to available treatments for schizophrenia, and for psychosis in general, is heterogeneous . Still, we do not have strong biological markers that could help us disentangle this heterogeneity and be useful in clinical practice . There are, however, structural brain alterations that, if refined, have potential for use early in the stratification of patients with psychosis . For this to be achieved for example, validation with very large datasets could help establish a reference group (a databank), which clinical imaging centers could access for an automated classification of their patients' mris in order to obtain an estimate of the likelihood of a specific outcome . For this to be achieved, it would be essential to have standardized definitions of outcomes within the clinical academic community that are relevant to clinicians, patients, and their careers . Such a system could then be used to assign a patient to targeted assertive case management at first presentation to services, in the optimization of pharmacological treatment, or in cognitive and family interventions . These have all been shown to improve treatment adherence and reduce relapse rates, eventually improving outcome . At the same time, they could help identify those patients most likely to have a good remitting illness after their first episode, who could then avoid long - term exposure to antipsychotic medication . Furthermore, the identification of gray matter and connectivity markers that characterize poor response poses the question of whether these markers could be used in patient stratification in clinical trials of new treatment strategies, or even to inform drug development of agents that can enhance and restore brain connectivity, which may elicit a better response in those who currently do not respond to available antipsychotics.
The treatment of patients with multiple trauma requires a different approach to that of patients with regular trauma because they are threatened not only by the injuries themselves, but also by the metabolic disruptions that follow.1 delay in surgery, blunt trauma, extensive soft tissue damage, and combined orthopedic and vascular injuries have been associated with an increased risk of amputation, while associated nerve and bone injuries with extensive soft tissue damage are risk factors for a poor outcome.2 acute renal failure is the main cause of death in patients with war wounds and trauma of the extremities . It would be helpful to minimize mortality in these patients by managing shock in a timely manner and taking the decision to amputate appropriately and promptly.3 severe head injury is known to be a major determinant of mortality in patients with multiple injuries, but other injuries also contribute to the clinical outcome.4,5 different mechanisms of injury, such as motor vehicle crashes, falls, or pedestrians being struck by a motor vehicle, impart varying degrees of force and energy transfer that may impact outcomes; this was found to predict mortality and functional impairment independently at hospital discharge.6 acute lower extremity compartment syndrome is a devastating complication that often presents silently in critically injured patients.7 patients who underwent delayed fasciotomy had twice the rate of major amputation and a three - fold higher mortality rate.8 the aim of this study was to evaluate if injuries of the extremities are associated with a higher one - month mortality rate than other types of associated trauma . This prospective, observational, cohort study was carried out in the regional emergency center of hospital de base after prior approval by the ethics research committee of the so jos do rio preto medical school . The emergency department follows a systematic pathway to provide initial assistance to accident victims using the atls (advanced trauma life support) protocol . All live accident victims treated in the emergency department from july 2004 to june 2005 were included . Patients who were dead on arrival and not submitted to any type of inhospital resuscitation procedure were not included in the study . Accident victims were allocated to two groups, ie, those with severe injuries to the extremities or pelvis (abbreviated injury scale [ais] 34) and those without injuries or with minor injuries to the extremities (ais 02). The fisher s exact test and relative risk were used for statistical analysis, and an alpha error of 5% (p 0.05) was considered statistically significant . A total of 3489 patients were evaluated in this study; 3244 (92.98%) did not have severe trauma of the extremities (ais 02), 34 (1.05%) of whom died . Severe injuries of the extremities (ais 34) occurred in 245 (7.02%) of the patients, with 13 (5.31%) dying (fisher s exact test: p = 0.001, relative risk 5.063, 95% confidence interval [ci]: 2.7079.467, table 1). Table 2 shows the age, type of injury sustained, and time until death after trauma for the patients who died . Of the 245 patients with ais 34, 71 (28.98%) were women and 174 (71.02%) were men, with the mean age of the men being 40.1 20.5 years and of the women 60.2 23.6 years . This study assessed whether severe injuries of the extremities affect the overall mortality rate in accident victims . The death rate was found to be higher for patients with ais 34 than in those without injuries or with minor injuries to the limbs . Thus, these data serve as a warning in respect to increased risk of death in orthopedic patients compared with general trauma patients . When the severity of specific injuries in accident victims is reported in the literature, head trauma is cited as one of the main causes of death.5,6 however, there are few data in the literature about the association between death and injuries to the extremities . One study reported that the mortality rate in accident victims with extremity injuries was higher in pedestrians struck by motor vehicles (20%), and for those with head injuries, it was higher for motor cycle crash victims (16%).6 the first phase of management for these patients aims to control bleeding, by surgical intervention if necessary, and to prevent further wound contamination . The second phase consists of resuscitation in the intensive care unit, and the third phase aims at definitive repair of the injuries sustained.9 pelvic injuries represent a thorny and stubborn therapeutic challenge . Rapid diagnosis and effective treatment (damage control) of these injuries play a key role in the patient s survival, inasmuch as the mortality of multiply injured patients with pelvic ring disruption remains high (20%35%).10 the preclinical management of patients with multiple trauma influences the prognosis regarding mortality and morbidity . Diagnostic overview, protection of vital functions in the special circumstance of shock, immobilization of the spine, and treatment of isolated injuries are an essential part of preclinical management.11 the type of trauma is known to influence the mortality rate.12 in this study, all the patients received specialized pre - hospital assistance, and were treated in a regional trauma reference center . In spite of all the care given at the scene of the accident through to discharge from hospital, trauma of the extremities was a significant cause of death . Accident victims with injuries of the extremities are at higher risk for death than those with other types of trauma.
Voltage - gated ca channels are the signature feature of excitable cells, transducing electrical activity into increased intracellular [ca] that mediates specific cellular effects such as muscle contraction, hormone secretion, and release of neurotransmitters . Thus, many regulatory mechanisms have evolved to fine tune ca channel activity and the resultant ca influx, mostly by protein protein interactions with, or posttranslational modifications of, the pore - forming 1 subunit . Some are rapid, such as ca - dependent inactivation of l - type (cav1.2) channels (budde et al ., 2002); others occur after the activation of signaling pathways, such as pka potentiation of cav1.2 channels or g protein inhibition of n - type (cav2.2) channels (catterall, 2000). In contrast, mechanisms that result in finely graded responses to changes in the cellular environment developing over longer time scales have not been well described . Rgk gtpases (rad, rem, rem2, gem / kir), the most recently characterized group within the ras family of gtp - binding proteins (reynet and kahn, 1993; maguire et al ., 1994; finlin and andres, 1997; finlin et al ., 2000), have received special attention because they are potent inhibitors of ca channels and candidates for ca channel regulators under transcriptional control that can therefore integrate the influence of multiple extracellular signals . Experiments in a variety of cell types have shown a drastic reduction of peak current amplitude for multiple ca channels after expression of gem / kir (beguin et al ., 2001, 2005b; murata et al ., 2004; ward et al ., 2004), rem, rad (finlin et al ., 2003; crump et al ., 2006), and rem2 (chen et al ., 2005; finlin et al ., 2005). Among ras family members, rgks differ by having extended variable n - terminal regions and conserved c - terminal extensions lacking the caax motif for fatty acylation, and containing binding motifs for calmodulin and 14 - 3 - 3 proteins (kelly, 2005). Individual rgks have nonoverlapping patterns of expression, and are transcriptionally induced and repressed by different factors . For example, gem and rem2 transcription has been reported to be stimulated by glucose in insulin - secreting pancreatic cells but follow a different time course (ohsugi et al ., 2004; finlin et al ., 2005); rad is overexpressed in muscle of type ii diabetics (reynet and kahn, 1993), and rem transcription is repressed by lipopolysaccharide exposure (finlin and andres, 1997). Gem inhibits the rho / rhoa kinase pathway (ward et al ., 2002) and induces neuroblastoma morphological and ganglionic differentiation (leone et al ., 2001). Expression of both gem and rem2 has been shown to decrease glucose - stimulated insulin secretion (beguin et al ., 2001; finlin et al ., 2005). A two - hybrid experiment identified ca channel subunits as a gem - interacting protein in the insulin - secreting min6 cell line (beguin et al ., 2001). Since subunits have been implicated in trafficking 1 subunits to the plasma membrane, this led to the hypothesis that rgks prevent subunits from interacting with 1 subunits, thereby preventing membrane targeting and resulting in reduced channels at the cell surface (beguin et al . A number of recent studies suggest instead that rgks inhibit channels already resident at the cell surface (chen et al ., 2005; finlin et al ., 2005). Moreover, though it is their potency that has earned them interest, it is a more subtle and tunable response that likely has physiological ramifications . It has already been established that changes in ca channel currents less severe than the near complete reduction observed when rgks are expressed in heterologous systems lead to drastic pathophysiological consequences (splawski et al ., 2004). It is difficult to understand how rgk expression could result in a finely graded response . In this study, we provide new insights into how gem and rem2 regulate ca channels . Exploiting the xenopus oocyte system to control levels of expression (canti et al ., 2001), we found that gem and rem2 drive a dose - dependent inhibition of ca currents . Rem2, but not gem, also modulated both the kinetics of channel activation and inactivation in a manner that was dependent on subunit interaction with the 1 interaction domain (aid). Together, these results suggest that specific rgks contribute to the fine tuning of ca influx by different mechanisms . Constructs for 1c (pcardhe), 2, and the 1c c - terminal deletion (amino acids 16702171), and the gst i - ii loop have been previously reported (zhlke et al ., 2000; nm_000725) was cloned into the pgem - he oocyte expression vector using standard molecular biology techniques . Bc018219) was obtained as an est and cloned into the pcs2 + oocyte expression vector (gift from d. mckinnon, state university of new york, stony brook, ny). Rem2 full - length (ay916790), a gift from d. andres (university of kentucky, lexington, ky), was digested out of the original pcdna3.1 vector and ligated into compatible sites in pcs2 + . The 1c n - terminal deletion (amino acids 2139) the 1c3 concatemer included amino acids 12134 from 1c and the entire 3 with a valine linker between them . The mutant 1c and corresponding concatemer included mutations y467s and w470a created by quikchange (stratagene). The kchip2b clone was a gift from p. pfaffinger (baylor college of medicine, houston, tx). In vitro crna transcription and microinjection into xenopus oocytes the following amounts of crna were injected: 1c (1 ng), 2 (1 ng), 3 (0.22 ng). Two - electrode voltage clamp recordings were performed as previously described (kim et al ., 2004). During recordings, oocytes were constantly superfused with a solution containing 40 mm ba(oh)2 (or 40 mm ca(oh)2 in experiments recording ca currents), 50 mm naoh, 1 mm koh, and 10 mm hepes (adjusted to ph 7.4 with methanesulfonic acid). Recordings were performed with a standard two - electrode voltage clamp configuration using an oocyte clamp oc-725c amplifier (warner instrument corp .) Connected through a digidata 1322a a / d interface (axon instruments, inc .) To a personal computer . Ionic currents were filtered at 1 khz by an integral 4 pole bessel filter and sampled 10 khz and analyzed with clampfit 9.2 . Steady - state inactivation was analyzed with a two - pulse protocol in which a 5-s conditioning pulse (p1) from 60 mv to + 50 mv was followed by a 100-ms test pulse (p2) at + 10 mv . Normalized p2 values were fitted with a boltzmann equation (i / ipeak = (1 io)/[1 + exp((v v1/2)/k)] + io). Activation time constants were estimated by fitting the activating component of the current trace to the following equation: i = io + afastexp(t/fast) + aslowexp(t/slow). Bursts of pancreatic cell action potentials were simulated by a 5-s depolarization to 40 mv from 70 mv followed by a series of 26 100-ms voltage - clamp depolarizations between 40 and 0 mv at 5 hz (kanno et al ., 2002). All values are given as means sem, with statistical comparisons performed with a student's t test . Protein expression / gst pull - down assays were performed as previously described (maltez et al ., 2005). Oocytes were injected with either 1,000 pg of gem crna, 1,000 pg gem crna with 1,000 pg crna cam, or 4,000 pg of gem crna . Oocytes were incubated at 17c for 24 h, lysed in ice - cold oocyte extraction buffer (20 mm hepes, 5 mm kcl, 1.5 mm mgcl2, 1 mm edta, 10% glycerol, 0.1% np-40, and roche protease inhibitor tablets), and then solubilized in sds . Constructs for 1c (pcardhe), 2, and the 1c c - terminal deletion (amino acids 16702171), and the gst i - ii loop have been previously reported (zhlke et al ., 2000; nm_000725) was cloned into the pgem - he oocyte expression vector using standard molecular biology techniques . Bc018219) was obtained as an est and cloned into the pcs2 + oocyte expression vector (gift from d. mckinnon, state university of new york, stony brook, ny). Rem2 full - length (ay916790), a gift from d. andres (university of kentucky, lexington, ky), was digested out of the original pcdna3.1 vector and ligated into compatible sites in pcs2 + . The 1c n - terminal deletion (amino acids 2139) the 1c3 concatemer included amino acids 12134 from 1c and the entire 3 with a valine linker between them . The mutant 1c and corresponding concatemer included mutations y467s and w470a created by quikchange (stratagene). The kchip2b clone was a gift from p. pfaffinger (baylor college of medicine, houston, tx). In vitro crna transcription and microinjection into xenopus oocytes has been previously reported (kim et al ., 2004). The following amounts of crna were injected: 1c (1 ng), 2 (1 ng), 3 (0.22 ng). Two - electrode voltage clamp recordings were performed as previously described (kim et al ., 2004). During recordings, oocytes were constantly superfused with a solution containing 40 mm ba(oh)2 (or 40 mm ca(oh)2 in experiments recording ca currents), 50 mm naoh, 1 mm koh, and 10 mm hepes (adjusted to ph 7.4 with methanesulfonic acid). Recordings were performed with a standard two - electrode voltage clamp configuration using an oocyte clamp oc-725c amplifier (warner instrument corp .) Connected through a digidata 1322a a / d interface (axon instruments, inc .) To a personal computer . Ionic currents were filtered at 1 khz by an integral 4 pole bessel filter and sampled 10 khz and analyzed with clampfit 9.2 . Steady - state inactivation was analyzed with a two - pulse protocol in which a 5-s conditioning pulse (p1) from 60 mv to + 50 mv was followed by a 100-ms test pulse (p2) at + 10 mv . Normalized p2 values were fitted with a boltzmann equation (i / ipeak = (1 io)/[1 + exp((v v1/2)/k)] + io). Activation time constants were estimated by fitting the activating component of the current trace to the following equation: i = io + afastexp(t/fast) + aslowexp(t/slow). Bursts of pancreatic cell action potentials were simulated by a 5-s depolarization to 40 mv from 70 mv followed by a series of 26 100-ms voltage - clamp depolarizations between 40 and 0 mv at 5 hz (kanno et al ., 2002). All values are given as means sem, with statistical comparisons performed with a student's t test . Protein expression / gst pull - down assays were performed as previously described (maltez et al ., 2005). Oocytes were injected with either 1,000 pg of gem crna, 1,000 pg gem crna with 1,000 pg crna cam, or 4,000 pg of gem crna . Oocytes were incubated at 17c for 24 h, lysed in ice - cold oocyte extraction buffer (20 mm hepes, 5 mm kcl, 1.5 mm mgcl2, 1 mm edta, 10% glycerol, 0.1% np-40, and roche protease inhibitor tablets), and then solubilized in sds . To explore the mechanisms by which rgks inhibit ca channel currents, we expressed cav1.2 channels (1c, 2, and 3) with gem, rem, or rem2 in xenopus oocytes and recorded the resulting currents by two - electrode voltage clamp . As observed previously, expression of any of these rgks drastically reduced the iba peak current amplitude (fig . 1 d). To distinguish among the possible models by which rgks inhibit ca currents and to explore the physiological implications, we took advantage of the xenopus oocyte system, in which it has been shown that expression levels of proteins can be accurately titrated in a monotonic fashion (canti et al ., 2001), in order to test whether inhibition were dose dependent . B demonstrates a monotonic increase in gem protein with increasing amounts of gem crna injected over the range that we studied . Moreover, coinjection of crna for another unrelated protein (calmodulin) did not affect gem protein levels, suggesting that crna amounts in this range did not exceed the protein synthesis capacity in oocytes . With this confirmation, 1 c shows that gem, rem, and rem2 inhibit iba peak current amplitude at 10 mv in a dose - dependent manner . This was not a nonspecific effect of increasing the amounts of crna injection; coexpression of crna for kchip2b, a protein that modulates k currents and is of similar mass to the rgks (an et al ., 2000), did not alter cav1.2 current amplitude . Examination of individual current traces also revealed differences in the mechanisms by which gem and rem2 affect cav1.2 currents . While coexpression of all three rgks decreased current amplitude, coexpression of rem2 also appeared to affect kinetics of activation and inactivation (fig . Effects of gem and rem appeared similar, so we focused on gem in further studies . (a) normalized i - v relationships of iba from oocytes injected with 1c/3/2 without or with gem (130 pg) or rem2 (936 pg). (b) immunoblots with anti - gem antibody . On left is shown gst and gst - gem, demonstrating specificity of the antibody . The right panel shows an increase in the amount of gem protein detected with an increasing amount (as indicated) of crna injected per oocyte . Gem is indicated by an arrow; a nonspecific band seen even without injection of gem crna is shown by an asterisk . (c) dose response of gem-, rem-, or rem2-mediated inhibition of iba . (d) exemplar traces of cav1.2 channels expressed without a rgk, with gem (20 pg), with rem (100 pg), or with rem2 (468 pg). . Coexpression of gem appeared to inhibit cav1.2 currents by a direct scaling effect, while rem2 altered the kinetics of activation and inactivation (fig . These different effects upon channel activation and inactivation are better appreciated by examining scaled traces from cav1.2 channels compared with traces from cav1.2 channels coexpressed with gem (26 pg) or rem2 (936 pg). Quantitative analysis of the kinetics of inactivation for cav1.2 channels coexpressed with rem2 was complicated because the decay phases of currents were contaminated by overlapping slow activation . Thus, we analyzed steady - state inactivation with a two - pulse protocol in which the normalized residual peak current during a + 10-mv test pulse (p2) was plotted against the voltage of a 5-s inactivating prepulse (p1) and found that both gem (26 pg) and rem2 (936 pg) affected steady - state inactivation (fig . Rem2 mainly affected the pedestal from 0.23 0.02 to 0.12 0.02, (n = 1112, p <0.0001) but gem reduced the slope to 14.1 0.6 from 9.0 0.8 (a) top panels show exemplar current traces during a 2-s test pulse at + 10 mv for gem and rem2 at the indicated doses . Bars, 1 a . The bottom panels show scaled exemplar traces of 1c/3/2 (gray) coinjected with gem (65 pg) or rem2 (936 pg) (black) during a 2-s test pulse to + 10 mv . (b) steady - state inactivation for 1c/3/2 without or with gem (26 pg) or rem2 (936 pg). N = 1012 (c e) dose response of rem2-mediated effects upon kinetics of activation (fast, fraction aslow, and slow, respectively, at the indicated test potentials see legend in c and with the amounts of crna injected as indicated on the x - axis), n = 714 . (f) scaled exemplar traces of activation phases at + 10 mv for 1c/3/2 coexpressed with the indicated rem2 doses . (g) ca currents recorded during a simulated burst of action potentials in a pancreatic cell (see materials and methods) with no rgk or rem2 (468 pg). Bars: 200 na, 1 s. rem2 also affected cav1.2 channel activation in a dose - dependent and voltage - dependent manner (fig . 2 c, 0 pg), the activating phase for currents elicited with test potentials from 0 to + 30 mv was best fitted with two exponentials (i = afaste + aslowe + c) and the dominant component was fast (fraction afast was 8090% at all test potentials; fig . Instead, the slower activation of cav1.2 channels induced by higher doses of coexpressed rem2 could be explained by two effects upon slow: slow became longer with increasing doses of rem2 and the fraction of aslow increased . These effects were most prominent at test potentials near the peak of the i - v curve (fig . The overall consequences of these dose - dependent effects upon activation induced by rem2 are illustrated by the overlaid traces in fig . 2 f. since these effects upon channel kinetics suggested that rem2 could alter the temporal nature of channel responsiveness during action potentials, we tested whether rem2 altered simulated ca - dependent action potentials that underlie the rhythmic bursting of electrical activity essential for insulin secretion in pancreatic islet cells (mears, 2004). 2 g shows the resultant ca currents from cav1.2 channels (1c, 3, and 2) expressed in xenopus oocytes during 26 successive 100-ms depolarizations from 40 to 0 mv (at 5 hz), a protocol that simulates the bursting activity during insulin secretion and has been used in isolated cells (kanno et al ., 2002). In the absence of rgks, the peak ca current amplitude decreased sequentially during the 26 successive depolarizations so that the current amplitude during the last depolarization was 46 4% (n = 8) of the peak current during the first depolarization . Not only were the current amplitudes smaller with coexpression of rem2 (consistent with the effects of rem2 presented above), but rem2 accelerated the decrease in amplitude during the successive depolarizations so that the current amplitude during the last depolarization was 12 7% (n = 7; p = 0.001 compared with no rgk) of the peak current during the first depolarization . This accelerated decrement is likely due to the increased rate of inactivation observed in the presence of rem2 (fig . Coexpression of gem led to decreased current (compared with no rgk), but did not affect the rate of decrement of the current amplitude (unpublished data), consistent with the lack of effect upon channel kinetics shown above . Thus, the presence of rem2 would decrease the integrated ca influx and alter its kinetics during a burst of action potentials such as those that drive insulin secretion in pancreatic islet cells . We next tested whether the 1c n or c termini were necessary for these effects by testing whether deletion constructs (2 - 139 in the n - terminus or 1669 - 2171 in the c terminus) were modulated by gem or rem2 . These experiments were prompted in part by a recent report suggesting that rem, another rgk member, required the 1c c terminus, particularly the pka phosphorylation site at ser, for inhibition of ca channel currents (crump et al . In contrast, we found that gem (250 pg) and rem2 (936 pg) consistently reduced peak current amplitude for channels containing intact or truncated 1c subunits (fig . 3, a and b). Rem2 also maintained its effects upon kinetics of activation and inactivation in the truncated channels, as shown in the scaled exemplar current traces (fig . The ca channel subunit, but not the 1c n and c termini, are required for gem- or rem2-mediated effects . (a and b) normalized iba for the indicated combinations of 1c (wt), the n - terminal truncation (n), or the c - terminal truncation (c), with or with gem or rem2, as indicated . All combinations were coexpressed with 3 and 2. scaled exemplar current for each pair with (black) or without (gray) gem or rem2 are shown on right . (c and d) normalized iba for the indicated combinations of c and 2 with or without 3 and gem or rem2, as indicated . For the pair without 3, scaled exemplar currents with (black) or without (gray) gem or rem2 are shown on right . We also used the 1c c - terminal deletion to analyze whether subunits were necessary for gem or rem2 modulation . Truncation of the 1c c terminus produces increased current amplitude in the absence of subunits (wei et al ., 1994; klckner et al ., 1995; gerhardstein et al ., 2000; ivanina et al ., 2000), thereby providing a larger baseline current from which to assess rgk inhibition . 3 (c and d) shows that, in the absence of a coexpressed subunit, neither gem nor rem2 inhibited channel currents . Further, rem2 modulated neither activation nor inactivation in the absence of a coexpressed subunit . These results show that rgk modulation is independent of the 1c n or c terminus, but requires subunits . Recent models have suggested that rgks inhibit ca channels by direct competition with subunits for this high affinity interaction site on the 1 subunit (sasaki et al ., 2005). Although subunit interaction with the aid is not required for all aspects of subunit modulation of ca channel function (maltez et al ., 2005), aid mutations that block subunit binding render channel currents too small to accurately assess an inhibitory effect of rgks (singer et al ., 1991). Building upon a previous hypothesis that the aid interaction serves mainly to secure subunits to 1 so as to allow other, lower affinity regulatory interactions, we covalently tethered subunits directly to the 1c c terminus after amino acid 2134 (1c3). Currents from this concatemer (expressed with 2) were similar to currents from untethered channels (1c + 3 with 2), except that the peak of the i - v curve shifted to more depolarized potentials (fig . 4 a) as previously reported (dalton et al ., 2005). To prevent 3 interaction with aid we made an 1c with two mutations in aid, y467s and w470a (1c), either of which has been shown to singly disrupt subunit interaction and block subunit modulation (van petegem et al . Confirmation of abolished subunit binding to the mutant 1c i - ii loop is shown in a gst pull - down assay (fig . 4 b). Current amplitudes from an 1c subunit with the same mutations coexpressed with 3 (1c + 3) were very small (fig . 4, a, c, and d) and indistinguishable from currents from an 1c expressed without 3 (not depicted), which is consistent with an absence of 3 interaction . When 3 was tethered to the aid mutant (1c3) however, the resulting current amplitude was significantly larger than from 1c coexpressed with untethered 3 (fig . 4, a, c, and d) since the requirement for -aid interaction could be at least partially circumvented by tethering the subunit to 1, this supported the hypothesis that other interactions between 1 and are important for -dependent modulation . Having generated an 1c subunit that was modulated by a subunit independent of its aid interaction, we therefore could test whether -aid was required for rgk inhibition . Currents from channels containing 1c3 coexpressed with either gem (250 pg) or rem2 (628 pg) crna showed that they both produced a similar reduction of current as for 1c + 3 (compare fig . 4, d and e). Although coexpression of gem or rem2 also reduced currents from channels containing 1c3, the reduction was much more modest (fig . 4, e and f). Moreover, although the effects of rem2 on activation and inactivation were preserved when coexpressed with 1c3, rem2 did not affect activation or inactivation when coexpressed with 1c3 (fig . 4 e). These results show that rem2-mediated effects upon activation and inactivation require the -aid interaction while gem- or rem2-induced inhibition of current amplitude does not . Interaction between the ca channel subunits and the aid influence gem- and rem2-mediated effects . (a) normalized i - v relationships of iba from oocytes injected with the indicated constructs or combinations, all coinjected with 2. n = 3134 . (b) a hisx6 immunoblot of a pull - down experiment for purified 2 sh3-gk core (maltez et al ., 2005) using a gst-1c i - ii or i - ii mutant loop . (d f) normalized iba for the indicated concatemers or combinations (all expressed with 2), with or without gem or rem2 . Scaled exemplar traces are shown on right . Bars: 1 s (d and f) and 50 ms (e). Heterologous overexpression of several rgks in a variety of systems produces almost complete inhibition of coexpressed or endogenous ca channel current (beguin et al ., 2001, 2005b; finlin et al ., 2003; murata et al ., 2004; ward et al ., 2004; chen et al ., 2005; crump et al ., 2006) and rgk inhibition of ca influx has been proposed as a mechanism for physiologic control of ca channel activity for responses such as regulation of insulin secretion from pancreatic islet cells (beguin et al ., 2001; finlin et al ., 2005) or control of cardiac rhythm (murata et al ., 2004). Lacking a detailed molecular understanding of how rgks could fine tune ca influx eclipses how this mode of regulation could shape a specific physiological response; for example, up - regulation of an rgk (ohsugi et al ., 2004; finlin et al ., 2005) and subsequent channel inhibition (beguin et al ., 2001; finlin et al ., 2005) after glucose stimulation might protect islet cells from excessive ca influx during chronic hyperglycemia, but how would cells retain their ability to secrete insulin with the almost complete loss of ca currents observed in previous overexpression experiments? In this study we describe two unexpected means by which rgks regulate ca channels, providing a framework for understanding how a wide array of ca signaling events can be precisely regulated . Exploiting the xenopus oocyte system to control protein expression levels, we found that rgk inhibition of ca channel current was dose dependent . The mechanism(s) by which rgks lead to current amplitude reduction, previously a source of controversy, is not revealed by these experiments; direct effects upon channels resident at the cell surface (chen et al ., 2005; finlin et al ., 2005) or effects upon channel trafficking / assembly (beguin et al ., 2001) cannot be easily distinguished by the experiments presented here . The significant finding, however, is that the suppression of current depends upon the level of rgk expression, thus promising a predictable and titratable attenuation of ca current by specific rgks . Thus, our results suggest that the high glucose - stimulated induction of a specific rgk and the resultant down - regulation of the ca current in pancreatic islet cells contribute to a protective mechanism against the detrimental effects of an enhanced ca signal resulting from chronic glucose exposure (juntti - berggren et al ., 1993); conversely, a reduction of rgk protein in response to elevated glucose would serve to compensate hyperglycemia acutely by increasing ca channel activity and consequent insulin secretion . Furthermore we found that rem2, but not gem or rem, also altered cav1.2 gating kinetics, slowing activation and enhancing inactivation . These effects upon kinetics suggest that rem2 must act at least in part upon channels resident at the cell surface . Not only could rem2 decrease peak ca influx, it could also impart profound influence on the ca - dependent action potentials that underlie the rhythmic bursting of electrical activity essential for insulin secretion in pancreatic islet cells (mears, 2004), by shaping the temporal nature of channel responsiveness as suggested by our experiments in fig . 2 g. although the molecular basis for the effects of rem2 upon channel kinetics is not clear, we speculate that its extended n terminus and c terminus that flank the ras core may be responsible; neither extension is found in rem or gem (fig . The kinetic actions of rem2 could result from an additional contact between rem2 and the channel within these nonconserved regions . Clustal w (1.83) multiple sequence alignment (thompson et al ., 1994) the gray - boxed areas highlight the extended n and c termini in rem2 that flank the conserved ras - like core . These different modes of regulation by gem and rem2, in conjunction with their differential temporal patterns of expression, may yield an integrated response to oppose effects of hyperglycemia . Gem, up - regulated in min6 cells within 45 min after exposure to glucose (ohsugi et al ., 2004), would diminish ca influx during acute hyperglycemia; rem2, induced after 16 h of high glucose (finlin et al ., 2005), would serve to shape ca responsiveness during chronic hyperglycemia . Although our experiments were not designed to address the relative potency of gem vs. rem2since their comparative levels in pancreatic cells have not yet been determined it is intriguing that rem2 appears to have a broader dose response range, which supports the proposed role in fine tuning ca responsiveness over time . Our study provides several new insights that help clarify the molecular mechanisms by which rgks inhibit ca channels . First, we demonstrated that subunits are necessary for rgk inhibition, corroborating previous reports of subunit dependence (beguin et al ., 2001). By using 1c subunits with deletions in either the n or c terminus in order to increase basal current amplitude, we avoided the difficulties of accurately measuring the inhibition of an already small signal, which may explain the contrasting result obtained with rem (crump et al ., 2006). Second, our experiments with the truncated 1c subunits demonstrated that neither the 1c n terminus nor c terminus were required for gem- or rem2-mediated inhibition or alteration of channel gating, also in contrast to a recent report (crump et al ., 2006). While these differences may be attributed to rem- vs. rem2-specific effects, failure of the c - terminal deletion (1733) in that report to augment ca currents compared with those from intact 1c subunits, as has been reported previously (wei et al . Our results also help clarify a conflict between two recent biochemical studies concerning whether the aid competes with cav for gem binding (sasaki et al ., 2005) or is present as a complex with cav and rem (finlin et al ., 2006). Our studies support the latter, where the rgks function only when the 1 subunit is in association with a subunit through its high affinity interaction site aid . In this context, our findings offer additional insights into mechanisms by which subunits modulate ca channel currents . Coexpression of subunits with 1 subunits increases current amplitude and affects kinetics of activation and inactivation (dolphin, 2003). Within 1 subunits, the major interaction site for subunits is the aid (pragnell et al ., 1994). The aid, however, is not absolutely required for all aspects of subunit modulation as 2a still modulated channel activation and inactivation for an 1a (cav2.1) subunit in which the aid had been deleted (maltez et al ., 2005). Because the wa mutation in the aid completely blocks subunit interaction (leroy et al ., 2005), our experiments showing that the subunit dependent augmentation of current amplitude is partially preserved with the 1c3 concatemers demonstrate clearly that subunits can still modulate ca channels through interactions exclusive of the aid (walker et al ., 1998; leroy et al ., 2005; utilization of these concatemers also elucidated the mechanism of rgk modulation of ca channels: the partial preservation of the gem- or rem2-mediated decrease in current amplitude with the 1c3 concatemers rules out models in which rgks compete with subunits for aid interaction (beguin et al . In contrast, the complete loss of rem2-mediated effects upon activation and inactivation suggest that the aid interaction is necessary only for rem2-mediated effects upon channel gating.
The incidence of endometrial carcinoma in korea has increased rapidly in the last ten years possibly due to an increasingly westernized life style, decreased number of pregnancy, and increase of postmenopausal hormone therapy (1). Surgery is the treatment of choice and total hysterectomy with bilateral salpingo - oophorectomy is the cornerstone of both the staging and treatment of endometrial carcinoma . However, although the international federation of gynecology and obstetrics (figo) adopted surgical staging in 1988, and the national comprehensive cancer network (2) also recommended standard surgical procedures, there is a significant variety in the actual surgical procedures employed in the treatment of patients with endometrial cancer . The management of patients with cervical involvement remains controversial and treatment options range from extrafascial hysterectomy (eh) to radical hysterectomy (rh), with or without radiation (3, 4). However, the literature regarding the treatment of these patients is limited by its low frequency . Cervical extension of endometrial carcinoma represents about 10% to 15% of cases at the time of diagnosis (5). Such cervical invasion is considered to increase the risk of nodal metastasis; and those patients are reported to have a worse prognosis than those without it (6 - 9). The rationale for performing rh in patients with cervical involvement is to eradicate all possible parametrial involvement (pmi). However, the preoperative detection of cervical involvement or parametrial disease is not entirely reliable despite the progress in imaging techniques such as magnetic resonance imaging (10, 11). Thus, although the only definitive method of ensuring the status and extent of disease in the parametria is through radical excision and a histological study of the operative specimen, the unreliability of preoperative prediction, and the relative rarity of pmi may make it unwise to resort to rh as a matter of routine in patients with cervical invasion (12). We reviewed the medical records of endometrial cancer patients with cervical involvement to determine the relation of parametrial spread with other histopathological variables and to evaluate whether the type of primary surgery using eh or rh alters the patients' outcome from a histological perspective . The cancer database from five different institutes were reviewed; patients surgically diagnosed with stage ii and a part of stage iii or iva endometrial cancer with cervical involvement during the period of 1993 - 2005 were identified . The recorded data were then extracted by a medically qualified gynecology trainee in each institute . The original pathology reports and operation reports of 133 patients were reviewed for the following information: age at diagnosis, pre - operative data, operative procedure, adjuvant treatment, follow - up data including date of recurrence, the patient status at last visit . A pathologic data such as figo stage, histologic type, tumor grade, depth of myometrial invasion, and the status of parametrium, and retroperitoneal lymph node were also reviewed . Patients with mixed mllerian tumor, including carcinosarcoma or endometrial stromal sarcoma, were excluded from the review . When the tumor grade varied between pre - operative biopsy and final pathologic reports, the higher grade was recorded . Confirmation of cervical invasion and staging was made by reviewing pathology reports, although pathology slides review was not performed . The term' radical hysterectomy' included modified hysterectomy (mrh) that could not be exactly differentiated in operating records . Patients with surgically staged iia cancer who had undergone eh were selectively treated with adjuvant radiation according to the degree of depth of myometrial invasion . Otherwise, most patients with stage iib disease or above underwent post - operative pelvic irradiation or brachytherapy . If sufficient follow - up information regarding survival and recurrence was unavailable through medical records, we referred to death certificates to obtain the required information . One hundred thirty - three patients with endometrial cancer stage iia to iva were found to have cervical invasion . Ninety - three percent (124/133) of the patients underwent complete surgical staging with or without paraaortic lymph node dissection . We found no significant difference in age, body mass index (bmi), distribution of figo stage, histology, and tumor grade between the eh and rh groups . Preoperative evaluations for disease extent including cervical involvement were mostly done by magnetic resonance imaging (mri) and partly by computed tomography (ct) or endocervical curettage . Pre - operative mri was performed in 63.9% (85/133) of the patients and sensitivity for cervical invasion was 44.7% (38/85). When the cases with stromal invasion were analyzed, the sensitivity was 53.3% (24/45). Table 2 presents the data for the choice of the extent of hysterectomy according to the results of preoperative evaluation . Eh was performed in 12/62 (19.4%) patients who were preoperatively suspected of cervical involvement, and more than half (37/71, 52.1%) of the patients who underwent rh had no preoperative evidence of cervical invasion . The results show that many surgeons did not strictly follow the results of preoperative evaluation in choosing the type of hysterectomy and the decision regarding eh or rh was made at their own discretion . Fig . 1 shows the relationship between the depth of myometrial invasion and that of cervical stromal invasion . A total of 121 cases had available information about both about the depth of myometrial and presence of cervical stromal invasion . In patients with disease limited to the endometrium, no case showed cervical stromal invasion . On the other hand, a majority of patients with disease involving cervical stroma had deep myometrial invasion (p=0.001). Table 3 showed the relationship between prognostic factors and parametrial involvement in 71 patients undergoing rh . Eight of ten patients (80.0%) had pelvic node metastasis, 1 patient had grossly positive adnexal involvement, and 3 had tumor spread outside the pelvis (2 of 3 had coexisting pelvic lymph node metastasis). Depth of myometrial invasion and pelvic or paraaortic lymph node positivity were significantly correlated with paramatrial involvement . Of the 19 patients with pelvic lymph node metastasis, 8 patients (42.1%) had concomitant pmi . Conversely, of the 10 patients with pmi, 8 (80.0%) had lymph node metastasis . In 74 patients with surgical stage ii, i.e., disease showing no demonstrable evidence of tumor spread in the pelvic cavity, rh was performed in 41 cases and no patients showed parametrial involvement . Sixty six of 74 (89%) patients underwent adjuvant radiation therapy (35 whole pelvic irradiation, 7 vaginal brachytherapy, 23 both, 1 unknown) and there were 3 patients who developed recurrent disease in the rh and none in the eh group (mean follow - up: 51 months, table 4). Our results show that a large number of korean gynecologic surgeons have a tendency to choose the surgical extent of hysterectomy through their own disposition and do not strictly adhere the results of pre - operative evaluation . Actually, the type of hysterectomy procedure selected for endometrial cancer varies in each nation and in each institution . Watanabe et al . (13) reported the results of a survey of a japanese group showing that more than 70% of institutes never perform rh without regarding the preoperative status of cervical involvement . Moreover, according to a survey in north america, nearly 30% of centers never performed class ii or iii extended hysterectomy for the treatment of endometrial cancer (14). As shown in table 2, surgeons who overuse rh without regarding the pre - operative results may believe that the current pre - operative evaluation method is not sensitive enough to detect cervical invasion and thus, worry about possible pmi or post - operative stump recurrence . On the other hand, those who perform only eh despite the positive findings from pre - operative evaluation may believe that cervical stromal invasion should be followed by adjuvant radiotherapy and thus, the prognosis would not be changed by performing a' high morbidity producing surgery' considering the low incidence of pmi . This inconsistent treatment policy, in fact, necessitates the need for an evidenced - based reformed guideline to which most clinicians can conform, although the definition of rh or eh is not standardized among surgeons and thus, the actual extent of surgery investigated as rh or eh by medical records might be different for each patient and this is one of the limitations of this study . Our results also showed that the pmi rate in patients with cervical invasion is about 14% in the rh group . This is within the range reported by other investigators, and is not significantly lower than reported values (15 - 17). However, parametrial spread was not found in any of the patients with figo stage ii, i.e., patients showing no intraoperative tumor spread, but was identified in 8 patients with positive pelvic lymph nodes, and two showed grossly frank extrauterine spread around the adnexa and another site in the pelvic cavity . These findings indicate that a risk of leaving an occult metastasis in parametrial tissue when performing only eh in patients without evidence of extrauterine tumor spread seems to be very low, even though all lymph node metastases may not be completely screened by intraoperative frozen section . Although they reported a relatively high rate of parametrial involvement, the points we focused on is not the crude value of the pmi itself, but the necessity of parametrial resection . Undoubtedly, cervical stromal invasion is known to increase the risk of pmi . However, the pattern of spread in the invaded cervix of endometrial cancer is quite different from that of primary cervical carcinoma . As shown in fig . 1, the pattern of cervical spread of endometrial cancer seems to follow myometrial invasion and originate from the endocervical glandular region, and as it advances, it invades the stroma . Therefore, we can speculate that before the tumor of the endometrium reaches the deep myometrium, a chance to detect direct pmi in the patients with suspicious cervical invasion seems to be low . However, pmi in endometrial cancer cannot be completely excluded only by the absence of cervical stromal invasion because pmi is reported to consist histologically of 2 patterns of spread: direct invasion of cancer cells to the parametrial connective tissues like that seen in cervical cancer, and parametrial lymph - vascular space invasion frequently seen in patients with deep myometrial involvement without cervical involvement (17 - 19). Current guidelines for endometrial cancer show that cervical stromal invasion is regarded as one of the major indications of adjuvant radiotherapy . We might guess that adjuvant radiation is required due to the implication that cervical stromal invasion raises the risk of recurrence not just by direct pmi from the cervix, but also by increasing the risk of occult parametrial involvement from the preceding deep myometrial invasion or myometrial lymph - vascular space invasion . Suffice it to say, this suggest that performing rh only in patients with cervical involvement cannot guarantee the complete eradication of possible parametrial involvement . In addition, it is very likely that we may have' cut through' the invaded parametrium in stage ii patients who underwent eh even though it was not revealed by pathology because there was no direct connection between the invaded cervical stroma and parametrium itself . Even acknowledging that occult parametrial spread had existed, our data showed that no recurrences occurred in patients who only underwent eh followed by adjuvant radiation and thus, it suggests that occult parametrial metastasis in patients with no gross extrauterine spread can be successfully controlled by radiation therapy . We could not evaluate the value of rh in patients with stage iii or over because their survival outcome is much likely to be influenced by variables other than parametrial involvement, i.e., lymph node status, extent of tumor spread, different treatment strategy to the recurred patients, etc . We did not overcome the limitations of retrospective analysis, i.e., lack of verifying the actually performed surgical extent, lack of centralization of pathologic review, and limited number of cases . Nonetheless, we cautiously suggest that routine rh in patients with cervical invasion and having no grossly positive extrauterine spread should be reappraised and that these patients are worthy of consideration for less radical surgery performed in conjunction with pelvic and paraaortic lymphadenectomy considering the low rate of parametrial involvement, inaccurate preoperative prediction, high post operative morbidity, and successful control through adjuvant radiation . Since this is an area of continued debate and a principal shift from our current practice and attitudes, a randomized study would be required to define the low - risk population that would have a modification of the extent of parametrial resection performed . In this regards, we can conclude that the real value of rh in endometrial cancer patients with suspicious cervical involvement can be evaluated by performing a large scaled randomized controlled study comparing the survival and quality of life between the group who underwent rh only or followed by tailored adjuvant radiation and the group with eh and routine adjuvant radiation therapy.
Pyrazolines are nitrogen - containing heterocyclic compounds, well known for their pronounced biological activity . It was demonstrated that the combination of pyrazole with azole ring, linked to each by one sigma bond, led to more biologically active targets; for example, pyrazolylthiazoles showed excellent antimicrobial activities . Continuing our work in this research field [69] and in an attempt to identify new and potent antimicrobial agents, we tried here to generate new benzofuryl 2-pyrazolin-1-ylthiazoles as antimicrobial agents using simple methods . The starting pyrazoline-1-carbothioamide 5 was prepared by treatment of 2-acetylbenzofuran 1 with equivalent of 4-flurobenzaldehyde 2 in the presence of 10% alcoholic naoh in 90% ethanol with stirring at room temperature to give chaconne 3 . Reaction of chalcone 3 with equivalent amount of thiosemicarbazide was performed in ethanol in the presence of 2.5 equivalent of sodium hydroxide to the target precursor 5 . The resulting pyrazoline-1-carbothioamide 5 was cyclized to the corresponding 2-(3-(benzofuran-2-yl)-5-(4-fluorophenyl)-4,5-dihydro-1h - pyrazol-1-yl)-4-methyl-5-(p - subs.phenyldiazenyl)thiazole derivatives 8a d by reaction with hydrazonoyl halides 6a d in anhydrous ethanol and in the presence of an equivalent of triethylamine (scheme 1). The reaction product structures were elucidated by means of nmr, ms spectroscopy, and elemental analyses (table 1). H nmr spectra of 8a d contained two doublet - doublet and one triplet signals due to the presence of ch2 adjacent asymmetric carbon . The mass spectra of 8a d showed the molecular ion peaks at m / z 481, 499, 515 and 561, respectively in agreement with the calculated masses . The reaction between pyrazoline-1-carbothioamide 5 and the equivalent amount of -haloketones, for example, phenacyl bromides derivatives, 2-bromoacetylbenzofuran, and 3-bromoacetylcoumarin, was performed in refluxing ethanol to yield pyrazol-1-ylthiazoles 1315 in good yields via the intermediates a (scheme 2). The h nmr of compounds 1316 indicate the disappearance of nh2 signal due to blocking of nh2 with thiazole nucleus . The mass spectra of 13a, b16 showed the molecular ion peaks at m / z 473, 518, 479, 507, and 465, respectively in agreement with the calculated masses . All the new synthesized compounds were screened for their antibacterial and antifungal activities at 100 g / ml concentration against four gram - positive bacteria (staphylococcus aureus atcc 29213; b. subtilis atcc6633; b. megaterium atcc 9885; sarcina lutea), three gram - negative bacteria (klebsiella pneumoniae atcc13883; pseudomonas aeruginosa atcc27953; e. coli atcc 25922), and two yeast (saccharomyces cerevisiae and candida albicans nrrl y-477). Most of the newly synthesized compounds showed good antimicrobial activities with respect to the control drugs . Compound 8c exhibited the highest potency against all tested organisms with respect to reference drugs . Compounds 8d and 13b inhibited the growth of staphylococcus aureus atcc 29213 with inhibition zones 23, 22 mm, respectively, while compound 5 showed excellent activities against klebsiella pneumoniae atcc13883; pseudomonas aeruginosa atcc27953; and e. coli atcc 25922 with inhibition zone about 24 mm . Also, compound 8c showed the highest activity against staphylococcus aureus atcc 29213, saccharomyces cerevisiae, and candida albicans nrrl y-477 with inhibition zone about 23 mm . The minimum inhibitory concentration (mic) of the synthesized compounds against highly inhibited organisms is reported in table 3 . Compounds 5 revealed low mic (200 g / ml) against staphylococcus aureus atcc 29213, b. megaterium atcc 9885, and candida albicans nrrl, compound 8a exhibited high mic (16 g / ml) against b. subtilis atcc6633 (table 3). Elemental analytical data were carried from the microanalytical unit, cairo university, giza, egypt . The ir spectra were recorded in potassium bromide disks on a jasco ft / ir-6100 . H nmr spectra were run on joel - eca 500 mhz in deuterateddimethyl sulphoxide (dmso - d6). Chemical shifts values () are given in parts per million (ppm). The mass spectra were performed using mass varian mat ch-5 spectrometer at 70 ev . (e)-1-(benzofuran-2-yl)-3-(4-fluorophenyl)prop-2-en-1-one 3; hydrazonoyl halides; 1-(benzofuran-2-yl)-2-bromoethanone 9; 3-(2-bromoacetyl)-2h - chromen-2-one 10; and 2,3-dichloroquinoxaline were prepared according to the literature . To a suspension of chalcone 3 (10 mmol, 2.66 g) and sodium hydroxide (25 mmol, 1.0 g) in ethanol (50 ml), thiosemicarbazide (12 mmol, 1.1 g) was added . The mixture was refluxed for 12 h, then left to cool; the solid product was filtered off, washed with ethanol, and dried . Yield 52%; m.p .) Max / cm 3460, 3335 (nh2); h nmr (dmso - d6) 3.09, 3.14 (dd, 1h, ch, j = 3.05 hz, j = 3.05 hz), 3.94, 4.06 (dd, 1h, ch, j = 11.45 hz, j = 11.5 hz), 5.88 (t, 1h, ch, j = 3.05 hz, j = 7.65 hz), 7.137.43 (m, 9h, ar - h), 9.44 (s, 2h, nh2, d2o - exchangeable); ms m / z (%): 339 (m, 75), 60 (100). To a suspension of compound 5 (1 mmol, 0.34 g) in ethanol (20 ml), the 1 mmol of appropriate reagent {(appropriate hydrozonoyl chlorides 6 + et3n); (appropriate phenacyl bromides 9); (2-bromoacetylbenzofuran 10); (3-bromoacetylcoumarin 11); or (3,4-dichloroquinoxaline 12)} was added and heated under reflux for 4 h. after cooling, the precipitate was collected by suction filtration . Yield 58%; m.p.180 - 2c; h nmr (dmso - d6) 2.54 (s, 3h, ch3), 4.09, 4.11 (2dd, 2h, ch, j = 10.7 hz, j = 9.95 hz), 5.87 (t, 1h, ch, j = 10.7 hz, j = 9.95 hz), 7.187.73 (m, 14h, ar - h); ms m / z (%): 481 (m, 75), 95 (100). Yield 66%; m.p . 201 - 3c; h nmr (dmso - d6) 2.54 (s, 3h, ch3), 4.07, 4.12 (2dd, 2h, ch, j = 10.7 hz, j = 9.95 hz), 5.89 (t, 1h, ch, j = 10.7 hz, j = 9.95 hz), 7.197.73 (m, 13h, ar - h); ms m / z (%): 499 (m, 80), 95 (100). Yield 72%; m.p . 206 - 8c; h nmr (dmso - d6) 2.53 (s, 3h, ch3), 4.10, 4.34 (2dd, 2h, ch, j = 10.7 hz, j = 9.95 hz), 5.89 (t, 1h, ch, j = 10.7 hz, j = 9.95 hz), 7.197.73 (m, 13h, ar - h); ms m / z (%): 515 (m, 70), 95 (100). 128 - 30c; h nmr (dmso - d6) 2.55 (s, 3h, ch3), 4.10, 4.34 (2dd, 2h, ch, j = 10.7 hz, j = 9.95 hz), 5.87 (t, 1h, ch, j = 10.7 hz, j = 9.95 hz), 7.197.72 (m, 13h, ar - h); ms m / z (%): 561 (m, 62), 95 (100). Yield 49%; m.p . 4.014.05 (dd, 2h, ch2, j = 11.45 hz, j = 10.33 hz), 5.73 (t, 1h, ch, j = 5.35 hz, j = 6.1 hz), 7.177.92 (m, 14h, ar - h); ms m / z (%): 473 (m, 80), 91 (100). Yield 58%; m.p . 208 - 10c; h nmr (dmso - d6) 4.014.08 (dd, 2h, ch2, j = 11.45 hz, j = 10.33 hz), 5.71 (t, 1h, ch, j = 5.35 hz, j = 6.1 hz), 7.177.92 (m, 14h, ar - h); ms m / z (%): 518 (m, 49), 91 (100). Yield 63%; m.p . 252 - 4c; h nmr (dmso - d6) 4.044.07 (dd, 2h, ch2, j = 11.45 hz, j = 10.33 hz), 5.73 (t, 1h, ch, j = 5.35 hz, j = 6.1 hz), 6.88 (s, 2h, benzofuryl - h), 7.197.70 (m, 13h, ar - h); ms m / z (%): 479 (m, 100). 233 - 4c; h nmr (dmso - d6) 4.05, 4.08 (dd, 2h, ch2, j = 11.45 hz, j = 10.33 hz), 5.72 (t, 1h, ch, j = 5.35 hz, j = 6.1 hz), 7.227.72 (m, 14h, ar - h), 8.23 (s, 1h, coumarinyl - h); ms m / z (%): 507 (m, 100). ; h nmr (dmso - d6) 4.02, 4.06 (dd, 2h, ch2, j = 11.45 hz, j = 10.33 hz), 5.70 (t, 1h, ch, j = 5.35 hz, j = 6.1 hz), 7.227.72 (m, 13h, ar - h); ms m / z (%): 465 (m, 100). Chemical compounds were individually tested against a panel of gram - positive and gram - negative bacterial pathogens and yeast . Antimicrobial tests were carried out by the agar well diffusion method using 100 l of suspension containing 1 10 cfu / ml of pathological tested bacteria and 1 10 cfu / ml of yeast spread on nutrient agar (na) and sabourund dextrose agar (sda), respectively . After the media had cooled and solidified, wells (10 mm in diameter) were made in the solidified agar and loaded with 100 l of tested compound solution prepared by dissolving 100 mg of the chemical compound in one ml of dimethyl sulfoxide (dmso). The inculcated plates were then incubated for 24 h at 37c for bacteria and at 28c for yeast . Ciprofloxacin (50 g / ml) and ketoconazole (50 g / ml) were used as standard for antibacterial and antifungal activities respectively . After incubation time, antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms and compared with that of the standard . The experiment was carried out in triplicate, and the average zone of inhibition was calculated . The bacteriostatic activity of the active compounds (having inhibition zones (iz) 18 mm) was then evaluated using the twofold serial dilution technique . Twofold serial dilutions of the tested compounds solutions were prepared using the proper nutrient broth . The final concentration of the solutions was 200, 100, 50, and 25 g / ml . The tubes were then inoculated with the test organisms, grown in their suitable broth at 37c for 24 hours for bacteria (about 1 10 cfu / ml and 1 10 cfu / ml of yeast), and each 5 ml received 0.1 ml of the above inoculum and incubated at 37c for 24 hours . The lowest concentration showing no growth was taken as the minimum inhibitory concentration (mic). This precursor reacted with thiosemicarbazide in strong basic medium to afford the n - pyrazoline thioamide 5 . The new compounds were tested for their antimicrobial activities and significant activities due to presence of three nucleuses: benzofuran, pyrazole, and thiazole . Also, some substituent increases the antimicrobial activities such as chloro substituent in compounds 8c and 13a.
There is increasing evidence linking endoplasmic reticulum (er) stress coping responses with the development of diseases . This is not surprising, given that the er is a multifunctional essential organelle in eukaryotic cells; it functions as the critical entrance to the secretory pathways, and is also a regulator of ca homeostasis, as well as protein and lipid synthesis, all of which are highly sensitive to a variety of environmental, physiological, and pathophysiological conditions . The er is also closely associated with all cellular organelles, and therefore, capable of driving sophisticated mechanisms of intracellular signaling, including activation of transcriptional processes, ca mobilization, and influencing energy metabolism . In this review, we briefly summarize the er stress coping responses, particularly the unfolded protein response (upr), and discuss the potential role of upr as a physiologically relevant pathway in supporting and controlling proper embryonic development . It impairs er functions such as protein folding and the heat shock response, and leads to altered ca levels and nutrient starvation . Activation of er stress can result in the buildup of misfolded proteins in the er and activation of the upr as a coping strategy (figure 1). The upr involves distinct components designed to reestablish the protein synthesis machinery (figure 1). These include (1) translational attenuation to arrest the entry of new proteins into the er; (2) transcriptional activation of genes encoding proteins involved in protein folding to assist the maturation of proteins; (3) transcriptional activation of genes for components of the er - associated protein degradation (erad) system to decrease the number of misfolded proteins; and if these are not successful, (4) activation of apoptotic pathways to eliminate defective cells . Three er transmembrane proteins: dsrna - activated protein kinase - like er kinase (perk), activating transcription factor 6 (atf6), and inositol - requiring enzyme 1 (ire1), in combination with the er molecular chaperone immunoglobulin binding protein (bip, also known as glucose - regulated protein [grp] 78), compose the upr, an er stress coping response . It is a monomeric, globular protein that functionally sorts and releases terminally misfolded substrates to the erad pathway . Under non - stressed conditions grp78/bip interacts with ire, perk, and atf6, but upon an increase in misfolded proteins, grp78/bip is sequestered away from these inducers, leading to the activation of the upr . Release of grp78/bip from perk triggers its dimerization and autophosphorylation, followed by phosphorylation of eif2, at ser, which inhibits protein synthesis by sequestering the trnamet responsible for initiating the translation of nascent protein (figure 1). During this period, atf4 transcriptional activity induces both pro - survival (early) and pro - apoptotic (late) transcriptional programs . Atf6 is an er transmembrane protein cleaved in the golgi . Under non - stress conditions when bip is sequestered away from atf6, the atf6 is translocated to the golgi, where it undergoes cleavage by site-1 and site-2 proteases (s1p and s2p) (figure 1). Cleavage of atf6 produces a soluble basic leucine zipper (bzip) transcription factor (cleaved atf6) that binds to er stress response elements (erse - i and ii) to induce transcriptional activation of er stress response genes . Atf6 can induce the expression upr - induced genes including grp78/bip, proteins involved in erad, and xbp1 mrna . Interestingly, er stress triggered by ca depletion induces the formation of a nascent, partially glycosylated form of atf6 with reduced interaction with calreticulin, and a faster rate of traversing the golgi, resulting in higher transactivation of atf6 gene targets . Upon release of grp78/bip under stress conditions, ire1 homodimerizes, causing a conformational change that is transmitted across the membrane, leading to activation of its cytoplasmic kinase activity, autophosphorylation, and activation of its endoribonuclease activity (figure 1). Ire1 endoribonuclease activity cleaves 28s rrna and specific micrornas, inhibits protein synthesis, and also splices xbp1 the protein product from the sxbp1 variant binds to the specific promoter elements, erse, and upre, triggering transactivation upr responsive genes such as grp78/bip, calreticulin, and others, which are part of the protein folding quality control and degradation machinery . One part of the cellular recovery system that is turned on by the upr is er - associated degradation (erad). There is tight regulation of the three arms of the upr with respect to the timing and response amplitude . The activation of each arm of the upr is specific to the source of er stress and governs cell fate, supporting either an adaptive response (cell survival) or a maladaptive response (cell death). Under prolonged and irreversible er stress,, upr activates autophagy, a controlled self - degradation process that can promote cell survival by eliminating damaged cellular components . The intensity, timing, and duration of the upr appear to be important in determining cell fate . These observations suggest that er stress coping responses may also play an important role in the maintenance of physiological events associated with organ function and/or development . Although there is emerging evidence that activation of er stress coping responses, including upr, may play an important role in diverse human diseases, it is conceivable that these coping responses may also be critical under normal physiological conditions and during development . Interestingly, there exists a group of small molecules that may stabilize protein folding, prevent protein aggregation, and consequently affect the severity of upr activation . The most studied of these molecules are synthetic - fatty acid derivative 4-phenylbutyric acid and the natural bile acid tauroursodeoxycholic acid (tudca). Although the precise mechanisms of their action are not clear at present, these compounds have a profound impact on many pathologies including obesity pathways, brain ischemic - reperfusion, photoreceptor pathology, and steatosis, to name a few . These findings suggest that targeting er physiology and er stress signaling and stress - induced coping responses has therapeutic potential for treating diseases related to er stress . The development of preimplantation embryos is under the influence of various hormones and growth factors that originate from maternal tissues and/or the embryo proper . Preimplantation embryos synthesize and secrete a wide range of hormones and growth factors that promote embryonic survival; in the blastocyst stage, they increase in cell number via activation of transcription as well as protein synthesis . During this synthetic activity, the embryos may experience an inherent level of er stress and consequently activate specific coping responses to recover homeostasis and support development or death . For example, in the mouse, the upr contributes to preimplantation embryo death in the ddk syndrome, suggesting that the er stress coping mechanism counters the stresses intrinsic to embryos . Mammalian early embryos placed in vitro are vulnerable to a variety of physicochemical stresses such as shearing, temperature changes, altered ph, and higher oxygen pressure, all of which are known inducers of er stress coping responses . These stresses alter gene expression, epigenetic mechanisms, and metabolism, reducing the embryonic development and viability . Although the influence of er stress coping responses on cellular physiology in adult tissues has been widely studied, their effects on the oocytes and early embryos of mammals remains unclear . In a porcine model, the functional abundance of sxbp1 is low in mature oocytes and at the 1-, 2-, and 8-cell stages of embryos, but it is highly abundant at the germinal vesicle, 4-cell, morula, and blastocyst stages . In mouse cumulus - oocyte complexes, fatty acid - induced er stress impairs protein secretion, mitochondrial membrane potential, and embryo development . Er stress is a key mechanism mediating the developmental potential of oocytes . In mouse oocytes, the cancer drug doxorubicin affects the mitochondria, perk, and er - associated caspase-12, while it inactivates parp, resulting in the activation of apoptotic signals . This suggests that activation of er stress coping responses may contribute, at least in part, to ovarian failure in women treated for cancer . In mouse preimplantation embryos, er stress - induced coping response molecules such as perk, ask1, bip, chop, gadd34, ire1, atf4, atf6, and xbp1 are expressed . Taking into account that perk, ire1, and atf6 are the three major transducers of the upr, the expression of the major transducers of the upr suggests that preimplantation embryos use the upr coping response as one mechanism to deal with preimplantation stress . In mice lacking upr - associated genes such as grp78 and ppp1r15a / ppp1r15b, the embryos do not survive beyond preimplantation development, suggesting the indispensable role of these er chaperones and er stress - induced coping responses in preimplantation embryos . Xbp1 is an important transcription factor and regulator of a subset of genes activated during er stress - induced upr . In drosophila, xenopus, and mice, xbp1 is essential for early embryonic development . In mice, functional sxbp1 is abundant in the germinal vesicle of immature oocytes and on spindles in mii oocytes but is solely detected in the cytoplasm at the one - cell stage when sxbp1 mrna is scarce . This indicates that sxbp1 acts as an important regulator of er stress - induced coping responses during specific developmental stages from oocyte to blastocyst . In mouse embryos, upr inducers such as tunicamycin and sorbitol increased nuclear sxbp1 at the one- and two - cell stages and activate xbp1 mrna splicing at the 8-cell, morula, and blastocyst stages, indicating that the ire arm of the upr is activated as an important coping response and adaptation to er stress in preimplantation embryos . In early mouse embryos, grp78/bip (hspa5), a central regulator for the er stress - induced upr coping response, is essential for cell proliferation and protecting the inner cell mass from apoptosis . Considering the importance of stress - activated protein kinase mitogen - activated protein kinase kinase (sapk - map3k) activation during cellular stress and the early stage of er stress, as well as the indispensable role of stress - activated proteins and map3k for preimplantation embryonic development, er stress manifested by sapk - map3k is a normal event critical during preimplantation development . The coupling of autophagy and upr has been implicated in a variety of biological processes . Induction of autophagy promotes preattachment development of in vitro - produced bovine embryos by reducing er stress, suggesting that autophagy / er stress balance is important for the developmental competence of early embryos . The inhibition of er stress - induced upr responses by tudca was found to enhance the maturation and developmental potential of porcine oocytes by preventing er stress - mediated apoptosis in vitro . In mouse cumulus - oocyte complexes, fatty acid - induced er stress coping responses are normalized by the upr inhibitor salubrinal, demonstrating that er stress is a key mechanism affecting oocyte developmental potential . Preimplantation embryos, under in vitro conditions, experience an increase in reactive oxygen species (ros), oxidative stress, heat - shock stress, and/or the culture stress due to insufficiency of maternally - derived embryotropic factors . Growth factors and ros scavengers have been successfully used as a medium supplement in assisted reproductive technology (art) programs to prevent poor outcomes for embryo survival under in vitro conditions . Tudca attenuates xbp1 mrna splicing in embryos at the two - cell stage and abolishes dna fragmentation induced by tunicamycin or sorbitol in blastocysts and recovered nuclear localization of the sxbp1 protein . In addition, tudca inhibits hyperosmolar - induced er stress coping responses as well as the er stress - induced apoptotic coping response during preimplantation embryo development . Er stress - induced coping responses may also play a critical role for embryo development after implantation . Indeed, increased er stress in decidual tissue in pregnancy has been shown to be implicated in fetal growth restriction with and without pre - eclampsia . Sustained er stress acts as a cofactor of oxidative stress in decidual cells from patients with early pregnancy loss . In decidual cells, excessive oxidative stress influences upr pathways to activate erad by decreasing valosin - containing protein, which is a type ii er - associated protein and a member of the aaaatpase family that facilitates delivery of misfolded proteins to the proteasome, which results in cell damage, inhibition of cell growth, and activation of apoptosis . Among upr constituents, p - eif2, grp94, and c / ebp homologous protein (chop) are increased in the placenta from intrauterine growth restriction and pre - eclampsia, indicating that er stress responses are also inducible under diseased conditions . Inositol - requiring enzyme 1 (ire1) or xbp1 null mice are unable to produce functional placentas, which is lethal to the embryo, indicating that the ire1 arm of the upr coping response is essential for placental development and embryonic viability . In mice, cadmium - induced teratogenicity is associated with ros - mediated induction of er stress coping responses in the placenta . Maternal exposure to cadmium resulted in activation of perk, phosphorylation of placental eif2, and an increase in chop, indicating that upr signaling is activated in the placenta due to cadmium - induced toxicity . This further supports the notion that er stress - induced coping responses are important mechanisms in embryos and extraembryonic tissues following exposure to environmental toxins . Furthermore, er stress - induced coping responses in the maternal decidua and placenta may counteract developmental problems during implantation and postimplantation development . Stress coping mechanisms are critical to minimizing or overcoming damage caused by ever changing environmental conditions . The upr pathway is mobilized in response to the accumulation of unfolded proteins and to ultimately regain er homeostasis . Various arms of the upr, an er stress coping response, are expressed in oocytes and preimplantation embryos, suggesting that er stress as a normal physiological adaptive / coping mechanism plays a pivotal role in the development of preimplantation embryos . As such, upr - associated molecules and pathways such as sxbp1 mrna may become useful markers for the potential diagnosis of stress conditions for preimplantation embryos . Most importantly, upr inhibitors, such as tudca, were found to effectively support the in vitro development of preimplantation embryos under stress conditions . Further identification of er stress - induced coping responses in preimplantation embryos would be helpful for a comprehensive understanding of development from preimplantation through early postimplantation.
Osmotic demyelination syndrome (ods) including central pontine myelinolysis (cpm) and extrapontine myelinolysis (epm) following overly rapid correction of hyponatremia has been well recognized515). Although ods occurring after transsphenoidal resection of pituitary adenoma has been reported occasionally111618), to our knowledge, few preoperative epm cases with pituitary adenoma have been reported14). Unfortunately, the outcomes of patients with ods are often disappointing with high rate of mortality and irreversible neurological deficits5). Here, we report a case of preoperative epm with nonfunctional pituitary macroadenoma who made a great clinical recovery after supportive therapy and transsphenoidal adenoma resection . A 38-year - old man was admitted to our hospital in december 2012 because of nausea, malaise, altered mental status, behavioral disturbances, dysarthria and dysphagia . The patient had been well until 2 months before admission, when nausea and malaise developed . His wife also reported that the patient had two episodes of transient loss of consciousness . He was sent to the emergency department of an outside hospital . At presentation, the patient had no sign of dehydration like poor skin turgor, orthostasis or decreased urine output, nor sign of volume overload including peripheral edema, ascites or elevated jugular vein pressure . Serum sodium of 102 mmol / l, potassium of 3.9 mmol / l and phosphate of 1.08 mmol / l were reported . The complete blood count and the remainder of metabolic panel including intravenous hypertonic saline was given with an average correction rate of 8 mmol/24 h. despite slow rate of correction, neurologic symptoms including restlessness and irritability developed . In the following days, the patient developed apathy, confusion and obtundation, followed by dysarthria, dysphagia, paraparesis, behavioral disturbances and disorientation . Endocrine studies revealed a serum cortisol concentration (8: 00 a.m.) of 62.4 nmol / l (normal, 138635 nmol / l), plasma adrenocorticotrophin (acth) of 1.03 pmol / l (normal, 2.217.6 pmol / l). Miu / l); serum free thyroxine (ft4) was 6.91 pmol / l (normal, 11.522.7 pmol / l), and serum total thyroxine (t4) was 43.0 nmol / l (normal, 65155 cranial magnetic resonance imaging (mri) was performed and a pituitary macroadenoma (fig . One week before admission, mri was repeated and epm was detected at bilateral basal ganglion and thalamus (fig . The patient was referred to our hospital because of persistent neurologic deficits . On physical exam, he was unresponsive to verbal or pain stimuli . Hormone studies showed normal atch, cortisol, tsh, t4, and ft4, decreased growth hormone and insulin - like growth factor 1, normal prolactin, decreased gonadotrophin (follicle - stimulating hormone and luteinizing hormone), estrogen and testosterone . The patient had several episodes of restlessness and agitation with shouting and fighting lasting 10 minutes every day . The decision of surgery was made then based on the improved general state of the patient and the intent to remove the underlying cause of hypopituitarism and hyponatremia . Pathologically, the mass was confirmed to be nonfunctional adenoma by hematoxylin and eosin (h&e) staining and immunohistochemical examination . Pseudorosette patterns were found on h&e staining and nuclear division was less than one per 10 high power field . The tumor cells were scattered positive for p53 and ki-67 . And ki-67 index was <1% . The immunostainings for acth, fsh, lh, prl, tsh and gh were negative . Recent hormone studies showed normal level of acth, cortisol, tsh, ft4 and t4 . The patient was able to resume his work as a bank accountant three months after the surgery . The latest follow - up mri was obtained 7 months postoperatively, and these images showed demyelination at bilateral basal ganglions and thalamus resolved and pituitary adenoma resected (fig . Among various etiologies of hyponatremia, endocrine disorders, including adrenal insufficiency and hypothyroidism, are uncommon causes which may be overlooked1712). While hyponatremia can be caused by hypopituitarism, severe hyponatremia occurring as the presenting manifestation of pituitary adenoma is rare8). At the onset of hyponatremia, given the endocrine abnormality and histology - confirmed pituitary adenoma, we propose that secondary adrenal insufficiency and hypothyroidism resulting from hypopituitarism was the underlying cause of his hyponatremia . However, demyelination can occur even with this correction rate (as in our case), especially in those with other ods risk factors5). Except for the rate of correction, serum sodium concentration at presentation and the duration of the hyponatremia are also important risk factors . On presentation, our patient had been symptomatic for 2 months and the serum sodium level was as low as 102 therefore, patients with more than one risk factors for ods may still be at risk for development ods at " normal " rates of correction and may require slower rates . If ods does develop despite the steps taken to avoid overly rapid correction, effective management is rare . Although reintroduction of hyponatremia, plasmapheresis and corticosteroids has been used, most evidence comes from animal models and data in humans are limited to case reports2513). A more recent review of 32 german patients with ods showed a much better outcome with only 30% having irreversible and incapacitating neurological deficits9). This review suggests that patients with ods can survive if the nonspecific secondary complications of transient illnesses such as aspiration pneumonia, deep venous thrombosis, and pulmonary embolism can be avoided . Some patients with ods can recover function after prolonged periods of severe neurologic impairment59). As in our case, the patient had developed progressive neurologic symptoms for two months on admission to our hospital . The patient was able to resume the job as a banking account after surgery, indicating complete recovery from neurologic and cognitive deficits . Therefore, supportive therapy should be continued for at least six to eight weeks before concluding that the deficits are irreversible . Surgery is the ultimate effective treatment for nonfunctioning adenoma, which not only removes the underlying cause of hypopituitarism, but also reverses or prevents vision impairment . According to a systemic review and meta - analysis evaluating the outcome of surgical treatment for nonfunctioning pituitary adenomas, pituitary hypofunction improved in as many as 30 percent after surgery10). While for patients with epm, appropriate surgical timing of adenoma resection is of great importance . Neurologic deficits of preoperative epm such as decreased level of consciousness, paraparesis, dysarthria and dysphagia, posed great challenge to the transsphenoidal operation of pituitary adenoma . Risk of complications such as aspiration, respiratory failure, and deep vein thrombosis is much higher than patients without neurologic deficits49). To minimize the risk of surgery and to remove the underlying cause of hyponatremia, we selectively arrange the operation after dysphagia and dysarthria resolved, and the patient became ambulant and able to obey simple commands . We report a case of preoperative epm with nonfunctional pituitary macroadenoma who made a great clinical recovery after supportive therapy and transsphenoidal adenoma resection . More cases are required to clarify the prognosis of ods and identify the factors associated with better outcome.
Mood disorders (md) such as depression and bipolar disorders are one of the most disabling types of diseases . In 2004, depression was the leading cause of disability as measured by years lost due to disability (yld) and the 3rd leading contributor to the global burden of disease assessed using the disability - adjusted life year (daly), a time - based measure that combines years of life lost due to premature mortality and years of life lost due to time lived in states of less than full health . Bipolar disorder was one of the top 10 leading global causes of yld in 2004 . Presence of risk factors (e.g., excessive nicotine use and alcohol and other drug use), cooccurring anxiety disorders, and eating disorders can lead to the early development of severe medical conditions . Also personality disorders (pd) are a class of disorders that can significantly worsen a patient's quality of life . In fact, psychosocial impairment is one of the diagnostic criteria for personality disorders according to the dsm iv and there is empirical evidence that the most severe personality disorders (e.g., schizotypal personality disorder and borderline personality disorder) are a major cause of psychosocial disability compared to unipolar depression without personality disorders . Both clinical practice and empirical studies show that there is often interdependence between md and pd [69]. Data from national epidemiologic survey on alcohol and related conditions showed cooccurrence rates of lifetime prevalence of three pd (borderline, narcissistic, and schizotypal) with any md ranging from 17.2% to 10.3% [1012] one of the reasons for the interest in this issue is to contribute to improve treatments . Empirical evidence reports a tendency of poor outcome in case of cooccurrence of depression and pd compared to patients with a diagnosis of depression only . This has been attributed either to worse compliance of pharmacological therapy or to the difficulty of maintaining an active and efficient social support, which could protect against relapses [15, 16]. Moreover, there is some evidence that the presence of pd, especially of the avoidant type, interferes with treatment response at interpersonal psychotherapy of depression . Several hypotheses have been suggested to understand md - dp relationship . Among others, lewinsohn and colleagues proposed that low levels of mood could have a scar effect on individuals: pd could develop probably by one's modification of coping and appraisal styles . From another perspective, it is argued that some maladaptive personality traits could be seen as risk factors of developing both an md and also a true pd . Recently it has been suggested that the age of onset of md could be a more efficacious criterion of classification than polarity . Unfortunately, the studies that have explored the age of onset of md are not many and, with the exception cited above, have investigated bipolar or unipolar samples separately, with an implicit assumption of the polarity criterion . Moreover, the common denominator of these studies was a priori determined thresholds that could explain the clinical difference in terms of severity or comorbidity found between patients who have developed the disorder at different ages . Fava and colleagues found a higher prevalence of pd in patients who had earlier onset of major depressive disorder (below 18 years) compared to patients with later onset . This result was not replicated by skodol and colleagues who instead found that severity and recurrence of major depressive disorder were predictors of borderline personality disorder . Perlis and colleagues have compared bipolar disorder patients with very early onset with patients whose onset was after the age of 18 finding a poor outcome in the first group in terms of fewer days of euthymia and greater impairment in functioning and quality of life . In a recent study, bukh and colleagues found higher comorbidity of personality disorder between patients with relative early onset of md . The main goal of the present study is to evaluate the association between the age of onset of md and the complexity of the personality traits of the patient . Secondly, we will be interested in identifying an age threshold able to maximize the differences between early and later onsets in terms of the complexity of the personality traits . The patients for the study were recruited in three psychiatric wards in piedmont (italy). Patients consecutively admitted for major depressive episodes or manic / hypomanic episodes between april 2006 and april 2007 were considered . Inclusion criteria were age over 18 and an agreement to participate in the study with informed consent, whereas patients diagnosed with schizophrenia and other psychotic disorders, chronic substance abuse, severe medical illnesses, or cognitive disorders were excluded from the study . The study was approved by the ethic committees of the hospitals involved in the study, and written informed consent was obtained from all participants . Axis i diagnoses were evaluated with the structured clinical interview (scid-1) for dsm - iv . Results of a recent study showed that scid - i validity was high and that interrater reliability ranged from .60 to .83 . Age at onset of depression or mania / hypomania, number of episodes of each type, average duration of each phase of illness, number of admissions for mood disorders, and family history of psychiatric illness were assessed by an anamnestic interview controlled whenever possible with corroborating family reports and medical records . The severity of the mood disorder was analyzed in terms of age of onset, duration of episodes, and frequency of episodes . All the participants included in the study were administered the millon clinical multiaxial inventory - iii [27, 28]. The mcmi - iii is a 175-item true / false self - report instrument that assesses axis i and ii psychopathology . The mcmi - iii raw scores are transformed and reported as weighted base rate (br) scores . Good internal consistency (= .66.90) and stability (test - retest r = .84.96) have generally been found for the mcmi - iii scales . A recent study (zennaro, in press), carried out on the italian version of mcmi - iii, shows that the inventory falls short in assigning pd categorical attributions to patients . The reason of such results can be found in br cutoffs used to determine the presence of traits versus the presence of pd disorders . Otherwise the cited study shows how mcmi - iii can correctly and reliably distinguish between pathological and not - pathological individuals . Even for this reason, mcmi - iii was used with the most elevated anchor point with the purpose of exploring personality traits rather than assigning a diagnosis . The severity of the personality traits was indeed analyzed in terms of complexity of the pd, with reference to the number of dimensions of the mcmi - iii with a br score of 85 or above . According to mcmi - iii scoring guidelines, patients with br scores of 85 or above on any of the mcmi - iii personality scales (i.e., schizoid, avoidant, depressive, dependent, histrionic, narcissistic, antisocial, aggressive, compulsive, passive aggressive, self - defeating, schizotypal, borderline, paranoid) are to be considered personality disorder elevated . The sample was thus divided in three groups: participants with none, one (i.e., simple pd), or with more than one (i.e., complex pd) elevations on the mcmi - iii . Mcm - iii was administered just before discharge and after the patients had recovered from the affective episode . To have 90% power to detect an effect size of 0.30 in the comparison of complexity of pd with six hypothetical nodes produced by recursive partitioning with two - sided significance level alpha of 0.05, we required about 200 patients . The shape of the distribution of the continuous variables was evaluated, and comparisons amongst the three groups defined by the mcmi - iii scores were done . After excluding patients with a single major depressive episode, who could not be analysed in terms of duration and number of episodes, a recursive partitioning analysis was used to find the most characterizing variables for the different complexity distributions of the pd . The recursive partitioning analysis was realized with the party procedure ((for a methodological description of procedure),) of the system for statistical computation and graphicsr . To study the individual contribution of each variable to the prediction of the complexity of the personality disorder, a factorial analysis of variance after a graphical inspection, a logarithmic transformation was applied to the continuous variables which had a nonnormal distribution (i.e., the age of onset of the md and the average number of episodes per year). In the model, the independent variables were gender, duration of episodes (divided into less than 1 month, 1 to 3 months and over 3 months), the age of onset of the md (after logarithmic transformation), the average number of episodes per year (after logarithmic transformation), and the age of testing . Finally, an analysis of the profiles of the mcmi - iii scales was performed with a repeated measures anova, encompassing the cluster found by the recursive partitioning as a grouping variable and the elevations of the mcmi - iii scales as repeating measures . All the other statistical analyses were performed with the spss for windows, release version 17.0, (spss, inc . The final sample encompassed 209 patients (66 males and 143 females; mean age 55.48 sd 13.04). Sample characteristics and clinical data relative to the md are shown in table 1 and also allow comparison with the complexity of the pd . Regarding axis i diagnoses, 10.5% of the patients had unipolar depression - single episode, 52.2% unipolar depression recurrent, 22.5% bipolar type ii disorder, and 14.8% bipolar type i disorder . Regarding personality disorders, 17.2% of patients had no elevated mcmi - iii scores, 45.9% had one peak, and 36.9% had two or more elevated values . The prevalence of elevated scores (i.e., at least a scale with a score of 85 or above) on the mcmi - iii is 83% in the group of patients with mood disorders . This value drops down to 77% if one considers only patients with depressive disorder . From the recursive partitioning analysis, the age of onset of the md was the most explicative variable with a threshold of 29 years . Later on, the analysis found a further significant classification: the group of participants over 29 years of age was divided according to the duration of the episodes (under 3 months versus over 3 months). Consequently the type of md was not significant in the explanation of the different patterns of the personality complexity (figure 1). The three patterns differed from each other on all of the three levels of complexity of pd . In particular, node 2 is characterized by more than half the patients with high complexity of pd (absolute majority of the node). The other two nodes show for patients with later onset of the md a cluster (node 5) with high presence of pd (only 8% of patients do not have a pd) represented by patients with longer episodes (over 3 months) and a cluster of patients (node 4) with lower presence of pd and, more importantly, less complexity (only 16% have a complex personality disorder). The analysis was applied to the participants with at least one mcmi - iii elevated value once more excluding patients diagnosed with a single major depressive episode . In table 2, the results of the comparison between the two groups are presented . The only statistically significant predictor emerging from multivariate analysis was the age of onset of the md (f = 8.945; df = 1; p = .003; = .058). The significance of the age of testing disappears: its connection to the complexity of the pd was probably influenced by the age of onset of the disorder itself . The duration of the episodes between simple and complex disorders was not statistically significant, and this difference did not improve its significance in the multivariate analysis (f = 1.230; df = 2; p = .295; = .017). This led us to think it is not a mere type ii error but more likely a variable with heterogeneous distributions in both personality disorder conditions . The profile comparisons (figure 2) show statistically significant differences between the three clusters based on pd complexity (f = 9.346; (df = 2); p <.01; = .092) and so did the interaction between cluster and mcmi - iii profile (correction of huynh - feldt (f = 3.640; (df = 14.357); p <.001; = .038)). From the multiple comparisons with bonferroni corrections, the cluster of patients with early onset of md significantly differed from the cluster with later onset and longer episodes, but it did not differ from the one with shorter episodes . On the other hand, the cluster with later md onset and longer episodes significantly differed from the other clusters . In particular, there was a clear difference between the cluster with early onset and the other two . The difference is seen in the elevation of the three last scales representing, according to millon, severe personality disorders with more impaired functioning . The clusters with later onset differed in the three scales: avoidant, depressive, and dependent, partially differed from each other in the paranoid scale, and remained very close to each other in all the other scales . The aim of the present study was to explore the relationship between the age of onset of md and the complexity of the personality traits of the patients . Moreover, we were interested in identifying an age threshold able to maximize the difference of personality traits between early and later onsets and investigating the association between mood disorder severity and personality traits . To start with, the prevalence of the personality disorders (pd) evaluated with the mcmi - iii in patients admitted for a depressive / hypomaniac episode through a dimensional evaluation was higher than that in previous studies, regarding both outpatients with a depression diagnosis and dimensional evaluation and patients with unipolar and bipolar disorders [6, 16]. These results can be in part linked to the fact that all the patients recruited for the present study had at least one hospital admission, a strong indicator of worse severity of the disorder . In the recursive partitioning analysis, the age of onset was the most significant predicting factor . Interestingly, the type of mood disorder was not a significant predicting factor, as previously shown . The threshold of onset was higher compared to previous studies [21, 23, 33] while it overlaps with what was recently highlighted about depressive disorders . It is interesting to underline the relationship identified between the severity of md in terms of duration of episodes and of pd, particularly in the cluster with later onset . The presence of maladaptive personality makes more likely both the onset of an md and the creation of a more complex personality disorder . This interpretation could explain the independence between the complexity of the pd (with the presence of the elevations on the scales more often associated with severe cases) and the severity of the md . Conversely, in the later onsets, the development of the particularly severe md can increase the personality symptomatology and elevate the mcmi - iii scale scores . This is what can be found in the three scales: avoidant, depressive, and dependent . In fact, between the patients with later md onset, the ones with longer episodes have more elevations on these three scales, while, on the other scales, except partially for the paranoid scale, they are practically identical . In particular, avoidant and dependent are two scales whose elevations are expected in the presence of depressive disorder . To date, it is not possible to highlight a clear direction of causality for the examined relationship and state whether the presence of a more complex personality disorder condition is caused by the onset of a more severe md or influences the severity of the md itself (e.g., by reducing the presence of social support around the patient). Thus, further studies are necessary to clarify this relevant issue, as the presence of an md as an inclusion criterion and the difficulty to accurately establish an age of onset for an md do not allow any definitive conclusion . Firstly, the cross - sectional design did not allow us to detect precisely how the pd and md developed . Secondly, as the assessment was conducted at the end of admission period, we could have found more pd than in the euthymic phase . Finally, the use of a self - administered tool for the detection of the pd may have led to an overestimation of the presence and complexity of the personality traits, particularly in keeping with the psychometric characteristics of the mcmi - iii, as described in a recent study on the topic (zennaro, in press) even if a conservative cutoff criterion was adopted . However, mcmi - iii (as other self - report inventories, e.g., minnesota multiphasic personality inventory-2) has the advantage of including validity scales that can detect possible patterns of response sets, biases, and distortions that might compromise the validity of the clinical assessment assuring specific quality of data at the study . This study has introduced the issue of disorder severity aspects in the investigation of the relationship between md and pd . Our findings suggest that it is important to design prospective studies able to evaluate the level of comorbidity also on later md onset patients and to include variables about the severity of the disorders . From a clinical point of view, the results suggest the need to assess maladaptive personality traits not only during adolescence but also in young adults too in order to prevent and treat potentially severe md.
Cognitive decline in older adulthood remains an area of great concern as the population ages . Some changes in cognitive function, such as decreased processing speed, are considered normative aspects of the aging process . However, the impact of even mild cognitive impairment on functional capacity highlights the importance of maintaining cognitive function for as long as possible . Sometimes summarized by the adage use it or lose it, current evidence suggests that leading an active lifestyle using it may buffer the effects of age - related cognitive decline losing it [35]. The mechanisms by which an active and engaged lifestyle may be related to better or preserved cognitive function in older adulthood remain to be fully elucidated . However, the cognitive reserve hypothesis predicts that some individuals are better able to withstand the physiological insults to the brain without measurable cognitive deficits because they had greater capacity to begin with . Individuals may be able to actively increase their reserve through engaging in cognitively stimulating activities . Social activities are considered part of what constitutes an active and engaged lifestyle, alongside cognitive and physical activities [3, 4, 79]. However, the evidence for a relationship between social activity participation and cognitive function is mixed . Some studies have found a relationship between social activities and cognitive function, while others have failed to do so . In an intervention study, older adults living in residential care with normal cognitive function, after participation in daily short duration social and physical activity sessions, performed better cognitively than they had during their baseline assessment . Conversely, aartsen et al . Found no relationship between social activities and cognitive function six years later using a cross - lagged regression approach that attempted to elucidate the strongest causal pathway . Similarly, green et al . Did not find support for the hypothesis that social contact is protective against later cognitive decline . The inconclusiveness of results has been acknowledged previously and suggested to relate to differences in statistical techniques [4, 8]. However, among studies that have used similar analytical methods, such as logistic regression, results are still mixed (e.g., [1417]). Research examining whether social engagement can predict cognitive function and change in cognitive function over time, using growth modeling techniques, has also lacked a consistent finding . James and colleagues found that social activity at baseline was associated with a higher baseline level of global cognitive function and lower rate of decline . Ertel et al . Found that baseline social integration was associated with a slower rate of memory decline, but not baseline memory performance . In a study of danish twins, mcgue and christensen found that social activity at baseline was related to level of cognitive function but not to change in cognitive function over time . They also found that, within monozygotic same - sex twin pairs, there was no evidence that the more socially active twin was less likely to experience cognitive decline than the less active cotwin . Overall, these studies demonstrate that many questions remain about the relationship between social activity and cognitive function [4, 7]. It is not clear whether it is a lifetime of social engagement (the effects of social activity accrued over time) that is protective, or if changes in social activity are related to cognitive function . Small and colleagues examined the relationship between changes in social activity and changes in cognitive function and found stronger evidence for changes in cognitive function predicting changes in social activity than the reverse . Changes in social activity at any point in time may correlate with changes in cognitive functioning after controlling for its overall trend . The current paper builds on previous work by exploring whether including social activity as a variable that changes over time can clarify the relationship between social activity and cognitive function in older adulthood . Using multilevel growth modeling with social activity as a time - varying covariate allows an examination of the relations between time - specific changes in cognition and social activity . This allows for a detailed test of whether the impact of social activity on cognitive function is accrued over time or whether changing social activity levels might relatively quickly impact cognitive function . Examining the temporal relationship between social engagement and cognitive function is needed to inform theories of possible mechanisms . The current analysis examines the relationship between social activity, change in social activity, and four domains of cognition including: reasoning, memory, fluency, and semantic knowledge in four different populations . The same models are tested with data from four different longitudinal studies: the long beach longitudinal study (lbls), the seattle longitudinal study (sls), the victoria longitudinal study (vls), and the origins of variance in the oldest - old: octogenarian twins study (octo - twin). This addresses the possibility that differing analytical methods may produce differing results and provides the opportunity for immediate replication and direct comparison of results . The diversity of the samples, two american, one canadian, and one swedish, increases the generalizability of the results and decreases the possibility that findings might be due to the particular features of one country or community . This research, initiated as a partnership between the advanced psychometric methods workshop series (mungas et al ., nia conference grant) and the integrative analysis of longitudinal studies on aging (ialsa) network, brought workshop participants together with researchers from four ialsa member studies . These studies were specifically selected based on their collection of cognitive, physical, and social activity data along with a range of cognitive functioning measures over multiple occasions held in common across the four studies . While the activity and cognitive functioning variables are not always identical, the subsets of variables in each study were chosen based on the rationale that they tapped similar domains at the construct level (e.g., fluid reasoning (gf), crystallized knowledge (gc), short - term memory (gsm), and long - term storage and retrieval (glr; e.g., category fluency)). In some cases the measures are the same, but more often they differ, providing opportunities for both strict and conceptual replication . An exception to this is the octo - twin dataset, for which a fluency measure was not available . The octo - twin study is based on the oldest cohort of the swedish twin registry and includes 702 participants aged 80 years and older at the time of the first assessment . Beginning in 19911993, the longitudinal all individuals with a dementia diagnosis at baseline were excluded from the analyses (n = 98). The total sample includes 604 individuals, of whom 524 had the social activity measure and at least one of the cognitive measures . Approximately 20% of the sample was lost to followup at each wave (10% per year), but most of this attrition was due to death . The ratios for gender, education, socioeconomic status, marital status, and housing of the octo - twin sample correspond to population statistics for this age range of the swedish population . Octo - twin cognitive measures reasoning was assessed using koh's block design test . In this task, participants are presented with red and white blocks and several patterns on cards and asked to construct the design on the card with the blocks . Memory was assessed using the prose recall test in which participants are asked for immediate free recall of a brief (100 words) story that has a humorous point . Responses are coded for the amount of information recalled in a manner similar to the wechsler memory scale . Semantic knowledge was assessed using the swedish version of the information task, which includes questions of general knowledge . Reasoning was assessed using koh's block design test . In this task, participants are presented with red and white blocks and several patterns on cards and asked to construct the design on the card with the blocks . Memory was assessed using the prose recall test in which participants are asked for immediate free recall of a brief (100 words) story that has a humorous point . Responses are coded for the amount of information recalled in a manner similar to the wechsler memory scale . Semantic knowledge was assessed using the swedish version of the information task, which includes questions of general knowledge . Octo - twin social activityparticipants were asked at each wave: how many people do you see? The possible response was: none (0); 1 - 2 (1); 35 (2); 610 (3), or 11 or more (4). Participants were asked at each wave: how many people do you see? The possible response was: none (0); 1 - 2 (1); 35 (2); 610 (3), or 11 or more (4). The lbls was initiated in 1978 when participants were recruited from the family health plan health maintenance organization (hmo), including mainly residents of long beach and orange county . The ethnic composition of the older group (98% caucasian) was similar to the 65 + population for the area based on the 1970 census . Panel 2, initiated in 1992, included 633 individuals contacted from the same hmo (64 were excluded due to frank dementia or serious sensory or neurological problems). In order to include the same measures as those in the seattle longitudinal study, lbls panel 1 (n = 106) and panel 2 (n = 631) data from 1994 to 2003 were used in the current analysis, excluding individuals younger than age 55 in 1994 (baseline n = 565). During this period, additional information on the lbls design, measures, and participants can be found elsewhere [29, 30]. Lbls cognitive measures reasoning was assessed using a composite score of the schaie - thurstone adult mental abilities test (stamat; schaie,) letter and number series tests . In letter series, participants view a series of letters (e.g., a b c c b a d e f f) and are asked to discover the rule that governs the series by identifying the letter that should come next in the series . Participants were to complete as many of the 30 items as possible within six minutes . Word series was a parallel test to letter series but the letters were replaced with months (e.g., january) and days of the week (e.g., monday). Memory involved immediate written recall of a list of 20 concrete high - frequency nouns studied for 3.5 minutes . Fluency was assessed by a word fluency task where participants were instructed to write down as many words as possible in five minutes that begin with the letter s. participants were instructed that they could not use proper nouns or create words by changing endings of other listed words (e.g., if the letter was w and you already said want, you should not also say wants, wanting, or wanted). Semantic knowledge consisted of the stamat recognition vocabulary test . Participants were given a word and asked to circle a synonym of that word from four possible alternatives . Reasoning was assessed using a composite score of the schaie - thurstone adult mental abilities test (stamat; schaie,) letter and number series tests . In letter series, participants view a series of letters (e.g., a b c c b a d e f f) and are asked to discover the rule that governs the series by identifying the letter that should come next in the series . Participants were to complete as many of the 30 items as possible within six minutes . Word series was a parallel test to letter series but the letters were replaced with months (e.g., january) and days of the week (e.g., monday). Memory involved immediate written recall of a list of 20 concrete high - frequency nouns studied for 3.5 minutes . Fluency was assessed by a word fluency task where participants were instructed to write down as many words as possible in five minutes that begin with the letter s. participants were instructed that they could not use proper nouns or create words by changing endings of other listed words (e.g., if the letter was w and you already said want, you should not also say wants, wanting, or wanted). Semantic knowledge consisted of the stamat recognition vocabulary test . Participants were given a word and asked to circle a synonym of that word from four possible alternatives . Lbls social activity measurea measure of social activity was derived from a modified version of the life complexity scale that was originally developed for the seattle longitudinal study . Participants were asked to record the number of hours per week on average they spent doing various activities (e.g., going to parties). The lbls version of the scale included 34 specific activities, 7 of which were considered social . In the current study, these activity measures were dichotomized in order to distinguish those who reported no activity (coded as 0) from those who reported one or more hours of activity per week (coded as 1). This was done because the range of scores varied greatly within and between measures, and because some scores were highly deviant (skewed) from expected values (e.g., reporting more than 100 hours of reading per week). The social activity variable was created by selecting questions from the life complexity scale that fit the social activity construct . That is, a composite score was formed by summing up the number of social activity items that were endorsed as having one or more hours of activity per week . The measure consisted of seven questions including: phone conversations, voluntary activities, going to parties, going to dances, playing cards, visiting others, and attending church . A social activity change variable was computed by subtracting the social activity measure in 1994 from social activity in 1994, 1997, 2000, and 2003 . A measure of social activity was derived from a modified version of the life complexity scale that was originally developed for the seattle longitudinal study . Participants were asked to record the number of hours per week on average they spent doing various activities (e.g., going to parties). The lbls version of the scale included 34 specific activities, 7 of which were considered social . In the current study, these activity measures were dichotomized in order to distinguish those who reported no activity (coded as 0) from those who reported one or more hours of activity per week (coded as 1). This was done because the range of scores varied greatly within and between measures, and because some scores were highly deviant (skewed) from expected values (e.g., reporting more than 100 hours of reading per week). The social activity variable was created by selecting questions from the life complexity scale that fit the social activity construct . That is, a composite score was formed by summing up the number of social activity items that were endorsed as having one or more hours of activity per week . The measure consisted of seven questions including: phone conversations, voluntary activities, going to parties, going to dances, playing cards, visiting others, and attending church . A social activity change variable was computed by subtracting the social activity measure in 1994 from social activity in 1994, 1997, 2000, and 2003 . The sls is a very long - running longitudinal study initiated by schaie, who first recruited members of a local health maintenance organization in 1956 . Current analyses used up to four waves of sls data from 1984 to 2005, which include an expanded set of measures that also overlap with the lbls . Only participants of 55 years and older at baseline were included in the analysis . Baseline was defined as each participant's first study visit, and time was measured in all analyses as years in study (coded as 0, 7, 14, and 21). See table 3 for sls participant characteristics over the four waves of data analyzed here . Sls cognitive measuresin order to model roughly equivalent cognitive outcomes across the four studies included in this coordinated effort, our analysis included measures of reasoning, fluency, memory, and semantic knowledge from a larger battery of tests . Reasoning was assessed with the word series test from the schaie - thurstone adult mental abilities test (stamat), in which participants were asked to determine a rule that governs a series of words (months or days of the week) by identifying what word should come next in a given series . Participants were provided with a printed word series and instructed to choose the next word in the series in multiple - choice format . The test consists of 30 items and total score is based on number of correct responses completed in 6 minutes . As in lbls, fluency was assessed with the word fluency test from the primary mental abilities test . Memory was assessed with a task in which participants were asked to study a list of 20 printed words for 3.5 minutes and provide immediate written recall of the items . Semantic knowledge was assessed with the educational testing service (ets) test of advanced vocabulary, in which participants were asked to identify synonyms for printed words from 5 choices . Total score was based on number of correctly identified synonyms out of 36 test items completed within 4 minutes . In order to model roughly equivalent cognitive outcomes across the four studies included in this coordinated effort, our analysis included measures of reasoning, fluency, memory, and semantic knowledge from a larger battery of tests . Reasoning was assessed with the word series test from the schaie - thurstone adult mental abilities test (stamat), in which participants were asked to determine a rule that governs a series of words (months or days of the week) by identifying what word should come next in a given series . Participants were provided with a printed word series and instructed to choose the next word in the series in multiple - choice format . The test consists of 30 items and total score is based on number of correct responses completed in 6 minutes . As in lbls, fluency was assessed with the word fluency test from the primary mental abilities test . Memory was assessed with a task in which participants were asked to study a list of 20 printed words for 3.5 minutes and provide immediate written recall of the items . Semantic knowledge was assessed with the educational testing service (ets) test of advanced vocabulary, in which participants were asked to identify synonyms for printed words from 5 choices . Total score was based on number of correctly identified synonyms out of 36 test items completed within 4 minutes . Sls social activity measurewe followed the methodology described in the lbls method portion of this paper in order to generate a roughly equivalent index of social activity (see lbls section for a detailed description). Following this methodology, we created a composite activity measure by summing dichotomized responses from a modified version of the life complexity scale creating a seven - item social activity composite (volunteering, playing cards, phone conversations, visiting others, attending church, dancing, and partying). Social activity change was computed by subtracting baseline activity from each follow - up activity measure . We followed the methodology described in the lbls method portion of this paper in order to generate a roughly equivalent index of social activity (see lbls section for a detailed description). Following this methodology, we created a composite activity measure by summing dichotomized responses from a modified version of the life complexity scale creating a seven - item social activity composite (volunteering, playing cards, phone conversations, visiting others, attending church, dancing, and partying). Social activity change was computed by subtracting baseline activity from each follow - up activity measure . The victoria longitudinal study began in 1986 - 1987 with a sample of 484 community residing volunteers and three - year retest intervals . Using a longitudinal sequential design, second and third independent samples began in 1992 - 1993 (n = 530) and 2001 - 2002 (n = 550). Each sample is tested at three - year intervals . To date, sample 1 has been tested on seven occasions (over 18 years), sample 2 on five (over 12 years), and sample 3 on two occasions (over 6 years). Participants in all three samples were recruited between the ages of 55 and 85 years . Data from seven waves of sample 1 and five waves of sample 2 were included in the current investigation . Approximately 25% of the sample was lost to followup at each wave, or 8% per year . Further detail on the vls design, measures, and participants can be found elsewhere . Vls cognitive ability measures fluency was measured by performance on a similarities task . In this timed task, participants were presented with target words and asked to write as many words as possible with the same or nearly the same meaning within six minutes . Memory was indexed using a 30 item, noun list learning task comprised of five semantic categories . Participants studied the word list for two minutes followed by a five minute free recall task . Reasoning was indexed by letter series in which participants were presented with a series of letters and asked to identify the next letter in the sequence that was consistent with the sequence rule . Fluency was measured by performance on a similarities task . In this timed task, participants were presented with target words and asked to write as many words as possible with the same or nearly the same meaning within six minutes . Memory was indexed using a 30 item, noun list learning task comprised of five semantic categories . Participants studied the word list for two minutes followed by a five minute free recall task . Reasoning was indexed by letter series in which participants were presented with a series of letters and asked to identify the next letter in the sequence that was consistent with the sequence rule . Vls social activity / lifestyle measurethe social activity / lifestyle measure used in the presented investigation included a subset of items from the vls activity lifestyle questionnaire . Individual item distributions were reviewed and a small number of poorly distributed items were eliminated . Seven items were selected due to their social nature (eat at restaurants, visit friend / relative, give dinner party, attend church, meetings of service organizations, meetings of clubs, and do volunteer work). For each item, participants indicated the frequency of engagement in that activity over the past two years on a scale from 0 to 9 (i.e., never, less than once a year, about once a year, 2 or 3 times a year, about once a month, 2 or 3 times a month, about once a week, 2 or 3 times a week, daily). The social activity / lifestyle measure used in the presented investigation included a subset of items from the vls activity lifestyle questionnaire . Individual item distributions were reviewed and a small number of poorly distributed items were eliminated . Seven items were selected due to their social nature (eat at restaurants, visit friend / relative, give dinner party, attend church, meetings of service organizations, meetings of clubs, and do volunteer work). For each item, participants indicated the frequency of engagement in that activity over the past two years on a scale from 0 to 9 (i.e., never, less than once a year, about once a year, 2 or 3 times a year, about once a month, 2 or 3 times a month, about once a week, 2 or 3 times a week, daily). The current analysis was conducted as part of a larger effort to examine the effects of lifestyle activities on cognitive function using the same analytic approach across studies from the integrative analysis of longitudinal studies on aging (ialsa) network . Thus, final models were selected in part to maintain consistency across lifestyle activities . In order to improve ease of interpretation of our results, age, the means for baseline age, education, and social activity were subtracted from their baseline values for each individual . This centered the covariates so that the intercept and linear slope terms would be interpreted as the expected value for an individual at the mean age and with the mean level of education for the study . The reference category for sex was male . In order to examine the effects of social activity on cognition, a series of multilevel models was fit with social activity as a baseline and a time varying covariate, with time specified as time since baseline, using multilevel mixed - effects regression in statacorp, the restricted maximum likelihood estimator (reml), and an unstructured covariance matrix . In the octo - twin study, participants were nested within their twin pair . In the vls, we controlled for enrolment cohort . Separate models were fit for each of the four cognitive measures (reasoning, fluency, memory, and semantic knowledge) resulting in four reported models for each study . While the familywise alpha rate within each individual study may be somewhat liberal, given our use of p <.05 as significance criterion, our focus on repetition of findings across studies imposes a strict limit to any reliance on chance findings . Formal meta - analytic methods, which would require identical measures and a larger number of studies, were not used . An initial 19-term model included the following terms: (1) baseline age, (2) sex, (3) education, (4) baseline social activity, (5) baseline social activity age, (6) baseline social activity sex, (7) baseline social activity education, (8) individually defined time since baseline, (9) time baseline age, (10) time sex, (11) time education, (12) time baseline social activity, (13) time baseline social activity baseline age, (14) time baseline social activity sex, (15) time baseline social activity education, (16) change in social activity from baseline (activity change), (17) social activity change baseline age, (18) social activity change sex, and (19) social activity change education . However, several terms were not significant for most of the studies and outcomes and so were trimmed to facilitate model interpretation . This process eliminated 7 of the 19 terms, first the 3-way interactions were eliminated, then the interactions with change in social activity . This resulted in a 12-term final model, presented in table 5 for separate cognitive constructs of reasoning, memory, semantic knowledge, and fluency . Significant between person age differences were seen at the first occasion of measurement for all memory, reasoning, and fluency tests, with older adults performing less well than their younger counterparts (all p <.01). Semantic knowledge results were less consistent, with sls showing no age differences, b = 0.02, p = .35, lbls and octo - twin suggesting the older individuals score worse, b = 0.28, p <.01, and b = 0.55, p <.01, respectively, and vls finding that older individuals performed slightly better, b = 0.08, p = .02 . At baseline, individuals with more years of education had significantly higher cognitive performance on all tasks, across all studies (all p <.01). Women at the mean baseline age had higher memory scores than did same aged men across all studies (all p <.01). Lbls and sls women scored higher than men on all measures (reasoning: p = .02 and p <.01; fluency: both p <.01), except for lbls semantic knowledge (p = .13, sls p = .02). Octo - twin women had significantly lower information scores, considered semantic knowledge, at baseline than octo - twin men (b = 4.20, p <.01). Significant within person declines were seen over time in study in all cognitive abilities and all studies (all ps <.01). A significant timeage interaction indicated that within each sample, those who were older at baseline declined significantly faster than the younger participants on all vls, sls, and lbls measures except lbls immediate memory . Except for the information test, evidence for differential decline in older individuals was not seen in octo - twin, which has a much narrower age range . Women showed less decline than men in the sls data only, and only for the fluency and semantic knowledge measures . Evidence for differential decline related to education was seen only for the lbls reasoning measure . Higher social activity levels at baseline, for individuals of average age and education, were associated with higher scores on reasoning within the vls and octo - twin studies, b = 0.05, p = .03, and b = 1.01, p <.01, respectively . The most consistent finding was that individuals with higher social activity levels at baseline also had higher memory scores . This was true for the sls, vls, and octo - twin studies, b = 0.14, p = .01, b = 0.06, p <.01, b = 0.34, p <.01, respectively . Baseline social activity was also positively related to fluency in the lbls (though the same measure was not significant in sls sample). Social activity at baseline and semantic knowledge was positively related in the octo - twin sample (operationalized as information; b = 0.87, p = .03), but negatively related in the sls sample (operationalized as vocabulary; b = 0.22, p = .02). The octo - twin sample was the only one where all cognitive measures considered had a positive association with baseline social activity levels . The baseline agesocial activity interaction terms were significant and positive for lbls reasoning (b = 0.08, p <.01) and semantic knowledge (b = 0.08, p <.01), indicating a stronger effect of social activity for older participants than for younger participants . However, the interaction of age and social activity was not significant for any other studies or cognitive measures (ps>.05). The interaction between education and social activity at baseline was not significantly associated with any cognitive measure within any of the samples . In terms of within person age changes, social activity at baseline was significantly related to the slope of semantic knowledge for sls participants such that higher social activity at baseline was associated with less semantic knowledge decline (p = .01). There were no other significant relationships between social activity at baseline and rate of change for any cognitive measure within any of the samples (all p>.05). In examining associations with change in social activity as a time - varying covariate in each of the models, was positively associated with performance on the fluency measures in all studies except sls (lbls p <.01, vls p <.01, sls p = .21), such that individuals who increased their level of social activity from their own baseline level exhibited higher occasion - specific fluency performance relative to their expected linear trajectory over time . In vls and sls, a significant relationship was also found between change in social activity and the reasoning (vls p = .02, sls p = .04) and memory (vls p <.01, sls p = .03) measure, indicating that participants who increased their social activity level also scored higher relative to their expected trajectory on the reasoning or memory measure . Changes in social activity were related to all cognitive measures in the octo - twin study (all p <.05), such that octo - twin participants who increased their social activity level from baseline had higher occasion - specific cognitive scores relative to their own linear trajectory in all domains tested . Comparing results of the same statistical model across four longitudinal studies of aging, we observed relatively few consistent associations between social activity and cognitive functioning . Looking at within person change in social activity and the four cognitive measures, the most consistent finding was an association with fluency in two of the three studies measuring it, after adjusting for linear effects of time and other covariates . Change in social activity was associated with memory performance in only half of the four studies (sls and vls). In only one study, octo - twin, change in social activity was significantly related to performance in all three of the cognitive domains considered (fluency measure not available). The octo - twin sample was the oldest and had the narrowest range of ages . Social activity in the octo - twin study was defined by the number of people with whom participants had contact, whereas the other studies included a range of activities with a social component . Given that within person decreases in performance were seen across all cognitive measures in all studies, the positive relationships generally represent attenuated decline, rather than improvement in cognitive performance . The use of within person change in social activity as a time - varying predictor of cognitive function is somewhat unique . However, in another study examining the temporal relations of social activity change and cognitive function, small and colleagues found significant coupling of social activity change and three cognitive domains: semantic decision speed (similar to our fluency measure), episodic memory, and semantic memory . They, however, found greater support for models where cognitive measures predicted changes in social activity levels, than the reverse . There was little evidence that initial levels of social activity were related to within person rate of decline in cognitive function . Although higher initial levels of social activity were associated with less decline on the sls semantic knowledge measure, this was not evident in any other samples . Sls has the widest interval between measurements and so correspondingly greater attrition between waves (though a similar yearly rate), but this does not suggest an obvious reason for the difference . The findings from the other studies are consistent with mcgue and christensen, who found that social activity at the first assessment was related to initial level of cognitive function but not change in cognitive function . However, other groups have found that social activity levels are associated with both baseline cognitive function and a reduced rate of decline over time . . Found that social integration was related to a slower rate of memory decline . We did not include a composite measure of general cognitive function, but our results, and the results of others who have examined specific facets of cognition, suggest that it is important to examine cognitive domains separately . Specifically, individuals with higher initial levels of social activity performed better on the memory measures in three of the four samples . Interestingly, the sample that did not show the effect was the lbls, which used the same measure as the sls study . This suggests the discrepancy is not a function of different memory measures being used, or how social activity was characterized in the study . It is perhaps related to differences in samples, although both were similar in average years of education and were american, although from different regions . This, and the accessibility of the activities, may influence the association with cognitive function the mean age of lbls participants was about six years older and the first wave of lbls assessment included in the present study was conducted ten years later than the first assessment of the sls . The age differences are not an obvious explanation, because the mean age of lbls participants is only slightly older than sls and vls participants and is nearly ten years younger than octo - twin participants . The lbls is the only study for which the majority of participants are not female, though this difference is slim (49% female in the lbls versus 52% in sls at baseline). The lbls does have the highest yearly rate of attrition (17% versus 10%, 7%, and 8%), although the loss at each measurement wave is similar to that of sls . It is unclear how this would affect the association between baseline social activity and memory performance; however, particularly considering that the mean social activity and memory measure scores for the lbls and sls were similar . It would be interesting in future work to consider general health and its age gradient in each of the studies . The lack of obvious reasons for discrepant findings, however, supports the importance of considering the reproducibility of results in coordinated, rather than pooled, analyses . Social integration has been posited to influence general health through multiple pathways that may overlap with those influencing cognition . Unfortunately, our findings do not strongly suggest that increased engagement in social activities confers immediate benefit in terms of cognitive function . Nor do they suggest that social participation reduces risk of cognitive decline in any domain apart from fluency . The cognitive reserve hypothesis suggests that, over time, reserve can be built up through stimulating activities and that individuals with high reserve can withstand more physiological deterioration before cognitive decline is observable . That social participation does not reduce the risk of cognitive decline in a broad range of measures suggests that it is not conferring reserve . One possible mechanism through which social engagement and cognitive function are related may be the cognitively stimulating nature of social activities . The cognitive training literature has found that training in specific tasks (e.g., memory tasks) does not necessarily transfer to other cognitive domains . This may be similarly true of social activity participation, whereby a relationship is only seen in cognitive domains that are being challenged by the social activity . Social interactions do typically involve verbal communication, and thus fluency, likely specifically verbal fluency, may be the cognitive domain most similar to our participants' social activities . Discrepancies in which activities are considered social may contribute to the lack of consistent association between social activity and cognitive function . For example, some activities (e.g., card games) may generally be more cognitively demanding than others (e.g., visits from family members), or primarily tax different cognitive skills, but this is likely to differ across individuals and situations . Although plausible, further research would need to confirm the validity of such hypothesis . In the context of physical health, others have suggested that different social factors (e.g., social influence, social engagement, and social support) may act primarily through different behavioral, psychological, and physiological pathways . Similarly, cognitive function may be differentially influenced depending on the particular combination of social factors and pathways . In this way, how social activity is conceptualized and measured may be related to the mechanistic pathway and thus differentially related to various domains of cognitive function . These effects may have contributed to the discrepancies between octo - twin and the other three studies in the current analysis, as octo - twin focused on frequency of contact with people as the social activity measure, whereas the other three encompassed a variety of activities . However, the lack of consistency between the two studies employing the same measures suggests that conceptualization of social activity does not fully explain the findings . A clue to the source of inconsistency across lbls and sls may be the very small average change in the lbls social activities measure, though its variance is comparable to those of sls and octo - twin . The association between cognition and social activity in lbls in particular also seems to vary by age more than in the other studies . A considerable strength of our analysis is the replication of the models across four longitudinal studies from geographically separate regions . This limits the possibility of spurious findings taking on undue importance and provides an opportunity to examine consistencies and inconsistencies between sample groups . In terms of potential methodological limitations, (for slope covariances; for variance components) suggest that the power to detect correlated change is extremely low . However, analysis of longitudinal studies on aging, including those used in the current paper, has consistently reported statistically significant variances and covariances in rates of change in cognitive outcomes ([4354], for summary see). In addition, the tests used by hertzog et al . Are based on significance tests for variance components whereas change was evaluated in our paper by examining fixed effects which will generally have greater power . Lending support to the argument that analyses in the current paper were adequately powered are the findings of significant associations between the identical set of cognitive outcomes in these same studies and physical and cognitive activities [57, 58]. Another limitation to our analysis is that while the study samples were restricted to initially healthy older adults, efforts were not made to exclude individuals who developed dementia over the course of the study periods (dementia diagnosis was available only in the octo - twin study). We cannot assume that any protective effects of social activity on cognitive function would be equivalent across healthy and dementing older adults . Including individuals whose cognitive decline may have been driven by the dementia process may impact the associations between social activity and cognitive function over time in nondementing individuals, and this may also contribute to inconsistencies in the literature . Finally, it is difficult to rule out the possibility that individuals decrease their social activities because their declining cognitive abilities make it more difficult for them to maintain social ties . The difficulty of determining the directionality of the relationship has been well acknowledged in the literature and it is similarly difficult to determine causal pathways in the current analysis [4, 5, 10]. Some attempts have been made to determine the most likely direction of the relationship, but the evidence is limited . The longitudinal nature of the studies considered here, and allowing social activity to vary across time, have narrowed the temporal distance between the social activity and cognitive performance, at least partially disentangling whether the relationship is primarily based on historical activity levels or whether the two tracks over time . Across four studies in three countries, baseline social activity levels failed to predict rate of decline in most cognitive abilities, and changes in social activity were not consistently associated with within person fluctuations in cognitive functioning . Our findings leave little support for the hypothesis that changes in social activity correspond to immediate benefits in cognitive functioning, except perhaps for fluency.
The pt mice, tcr- mice, and tcr- mice have been described (6, 17, 18). Tcr- pt mice were bred in the animal colony of the basel institute for immunology . Breeding of tcr- pt mice was done in the animal facilities at the hpital necker (paris, france). The tcr- transgenic mice, with a transgenic tcr specific for the male antigen (h - y) in the context of h-2d mhc molecules, have been described previously and were crossed on the c57bl/6 (b6) background (19). Tcr- transgenic pt mice were bred in the animal colony of the basel institute for immunology . The following mabs were used for staining: anti - cd4 (h129.19, pe - conjugated; gibco brl, gaithersburg, md; or h129.19, fitc - conjugated; gibco brl), anti - cd8 (ly-2, fitc - conjugated; pharmingen, san diego, ca; or 53 - 6.7, biotinylated; gibco brl; or 53 - 6.7, red613conjugated; gibco brl), anti - cd25 (3c7, pe - conjugated; pharmingen), anti - cd44 (biotinylated km81; american type culture collection, rockville, md), anti - pantcr- (h57 - 597, fitc - conjugated), anti - tcr- (gl3, fitc - conjugated; pharmingen), t3.70 (specific for the tcr- chain of the hyreactive tcr, fitc - conjugated), and f23.1 (specific for the tcr- chain of hy - reactive tcr, fluos - conjugated). Two- and three - color stainings were performed with fitc-, pe-, and biotin - labeled antibodies at optimal concentrations . Biotin - conjugated antibodies were revealed by either streptavidin - pe (southern biotechnology, birmingham, al) or streptavidin - tricolor (caltag laboratories, san francisco, ca). All stainings were done in 96-well plates (0.5 10 cells per well) in 20 l of mab in pbs plus 2% fcs plus 0.1% sodium azide for 20 min on ice . Between first and second step reagents cells were washed in pbs plus 2% fcs plus 0.1% sodium azide as was done after the last step . Data were analyzed on a facscan (beckton dickinson, mountain view, ca), using lysis ii software (beckton dickinson). For intracellular / extracellular double staining of thymocytes cells were first incubated with culture supernatant of mab 2.4g2 to block fcrii / iii . Cells were then stained for surface markers as described above . After washing in pbs, cells were fixed in pbs plus 1% paraformaldehyde for 15 min at room temperature, followed by two washing steps in pbs . Cells were then permeabilized in 0.5% saponin (sigma, heidelberg, germany) for 10 min at room temperature and washed in pbs . Intracellular staining with fitc - conjugated antibodies diluted in pbs plus 0.5% saponin was performed for 20 min at room temperature, followed by two washing steps in pbs and 2 15 min on a rocking platform in pbs plus 2% fcs plus 0.5% saponin on ice . Finally, cells were washed in pbs plus 2% fcs and analyzed on a facscan, using lysis ii software . The pt mice, tcr- mice, and tcr- mice have been described (6, 17, 18). Tcr- pt mice were bred in the animal colony of the basel institute for immunology . Breeding of tcr- pt mice was done in the animal facilities at the hpital necker (paris, france). The tcr- transgenic mice, with a transgenic tcr specific for the male antigen (h - y) in the context of h-2d mhc molecules, have been described previously and were crossed on the c57bl/6 (b6) background (19). Tcr- transgenic pt mice were bred in the animal colony of the basel institute for immunology . The following mabs were used for staining: anti - cd4 (h129.19, pe - conjugated; gibco brl, gaithersburg, md; or h129.19, fitc - conjugated; gibco brl), anti - cd8 (ly-2, fitc - conjugated; pharmingen, san diego, ca; or 53 - 6.7, biotinylated; gibco brl; or 53 - 6.7, red613conjugated; gibco brl), anti - cd25 (3c7, pe - conjugated; pharmingen), anti - cd44 (biotinylated km81; american type culture collection, rockville, md), anti - pantcr- (h57 - 597, fitc - conjugated), anti - tcr- (gl3, fitc - conjugated; pharmingen), t3.70 (specific for the tcr- chain of the hyreactive tcr, fitc - conjugated), and f23.1 (specific for the tcr- chain of hy - reactive tcr, fluos - conjugated). Two- and three - color stainings were performed with fitc-, pe-, and biotin - labeled antibodies at optimal concentrations . Biotin - conjugated antibodies were revealed by either streptavidin - pe (southern biotechnology, birmingham, al) or streptavidin - tricolor (caltag laboratories, san francisco, ca). Stainings were done in 96-well plates (0.5 10 cells per well) in 20 l of mab in pbs plus 2% fcs plus 0.1% sodium azide for 20 min on ice . Between first and second step reagents cells were washed in pbs plus 2% fcs plus 0.1% sodium azide as was done after the last step . Data were analyzed on a facscan (beckton dickinson, mountain view, ca), using lysis ii software (beckton dickinson). For intracellular / extracellular double staining of thymocytes cells were first incubated with culture supernatant of mab 2.4g2 to block fcrii / iii . Cells were then stained for surface markers as described above . After washing in pbs, cells were fixed in pbs plus 1% paraformaldehyde for 15 min at room temperature, followed by two washing steps in pbs . Cells were then permeabilized in 0.5% saponin (sigma, heidelberg, germany) for 10 min at room temperature and washed in pbs . Intracellular staining with fitc - conjugated antibodies diluted in pbs plus 0.5% saponin was performed for 20 min at room temperature, followed by two washing steps in pbs and 2 15 min on a rocking platform in pbs plus 2% fcs plus 0.5% saponin on ice . Finally, cells were washed in pbs plus 2% fcs and analyzed on a facscan, using lysis ii software . In initial experiments, it was determined whether either the tcr- or the tcr- could be responsible for the production of cd48 t cells in pt mice by analyzing the cellular composition of thymuses from either pt tcr- or pt tcr- double mutant mice that can only produce the and the tcr-, respectively . As shown in table 1 both types of mutant mice contained cd48 t cells that were further analyzed by cytoplasmic staining with antibodies specific for tcr- and tcr- chains . For this purpose cells were double stained for surface expression of cd4 and cd8 molecules as well as either for cytoplasmic tcr- or tcr- chains by double fluorescence using cd4 and cd8 antibodies in one color (green) and tcr- or tcr- antibodies in another color (red). In this analysis single positive cd48 and cd48 cells show an intermediate fluorescence between that of cd48 and cd48 thymocytes and cells were gated accordingly into double negative, double positive (dp), and single positive cells (fig . 1 shows that 64% of cd48 cells in wild - type mice expressed tcr- chains, and that due to tcr- selection by the pre - tcr (22) the vast majority of cd48 cells contained tcr- chains in their cytoplasm . On the other hand, the picture was different in pt/ mice where, due to the diminution of rapidly cycling tcr-selected cd484425 cells (6), only 21% of the cd48 cells were tcr- positive . In addition, only 39% of the cd48 cells contained tcr- chains in their cytoplasm indicating that in the pt/ mice the majority of the cd48 cells were generated by a mechanism that did not involve tcr- selection . The fact that not all single positive cells in these mice were tcr- is due to the fact that these cells are in part immature tcr- single positive cells, on their way from cd48 to cd48 cells . The tcr- single positive cells had a mature cd48 phenotype as confirmed by independent three - color stainings indicating also that these cells expressed tcr- receptors on the cell surface . These cells were present in a higher number in pt mice consistent with the notion that the pre - tcr may have a role in regulating rearrangement and/or expression (23 and unpublished observations). In pt tcr- mice the proportion of tcr- cd48 and tcr- single positive cells was even further reduced . When looking at the absolute numbers of various cell subsets (table 1 and fig . 1) it is clear that there was a very marked reduction in cell numbers of cd48 thymocytes and more mature cells in pt and pt tcr- mice, whereas the numbers of cd48 cells were within the same range . Pt tcr- mice also had reduced numbers of dp cells but here the picture differed from that in pt and pt tcr- mice in that all of the cd48 cells were tcr- positive, i.e., were exclusively generated through a mechanism that involved tcr- selection . The single positive tcr- cells in pt tcr- mice were exported from the thymi and cd48 as well as cd48 cells could be detected in lymphnodes of these mice (not shown). This excludes the possibility that these cells belong exclusively to the nk1.1cd4 subset that exhibits an unusual phenotype (24). The above results were reproducible in the different mice with marginal deviations in either the percentage of cells or absolute cell numbers and are schematically presented in fig . The main message from this analysis is that the tcr- can generate cd48 cells through tcr- selection, i.e., by intracellular or cell - autonomous signaling only . In contrast, the tcr- can generate cd48 cells that are either tcr- or tcr- but all tcr- through a mechanism that may involve intercellular communication of unknown nature . If the tcr- would generate a significant number of dp cells by cell - autonomous signaling one might expect to find some tcr- expression in these cells . However, the fact that the cd48 cells are tcr- negative suggests that these cells are not selected by cell - autonomous signaling by the tcr- even though it cannot be entirely excluded that tcr- expression is abruptly switched off in cd48 cells . The notion of intercellular communication is in line with experiments that involved transfer of t cells into thymuses of rearrangement - deficient mice that resulted in generation of cd48 cells of host origin (25) and also with earlier data by shores et al . Our experiments suggest that in the latter experiments but not t cells promoted the development of cd48 thymocytes and make the additional point that the generation of dp cells was not due to an artefact caused by adoptive transfer of cells . The fact that in the absence of the pre - tcr the generation of cd48 cells by the tcr- is rather inefficient, i.e., 240 10 versus 2,880 10 in pt tcr- versus wild - type mice, could depend on the fact that the tcr- is inefficiently formed in cd48 cells due to the late tcr- rearrangement and/or the fact that tcr- can only inefficiently replace the pre - tcr . To analyze this question in some more detail we studied mice that express a transgenic tcr- early in development on cd48 cells, i.e., tcr- transgenic pt mice . The transgenic tcr- could indeed overcome the cellular deficiency in the cd48 compartment as tcr- transgenic pt mice contained approximately one - half the number of thymocytes found in tcr- transgenic pt mice and many more than the number found in nontransgenic pt mice (fig . However, there was a subtle difference between tcr- transgenic pt and tcr- transgenic pt mice in that the latter, but not the former, contained a discrete subset of cd25 cells, indicating that in spite of the presence of the transgenic tcr-, the pretcr had its role in the exit from this compartment . This could be due to the lack of expression of the transgenic tcr- in a fraction of cells in the cd25 compartment of the tcr- transgenic, pt mice . This was in fact confirmed by cytoplasmic staining: while only nine percent of cd25 cells in tcr- transgenic pt mice expressed the transgenic tcr- chain the majority of these cells expressed the transgenic tcr- chain suggesting that expression of the two transgenes is differentially regulated (fig . In tcr- transgenic pt mice it is the combined action of the pre - tcr and the tcr- (mice that have only a tcr- transgene still exhibit a significantly larger cd25 compartment than tcr- transgenic mice, not shown) that reduce the number of cd25 cells while in tcr- transgenic pt mice this compartment is bigger in size because of the absence of the pretcr . From these data it would appear that the tcr- can at least partially mimic the function of the pre - tcr and that in normal mice the contribution of the tcr- to the generation of the cd48 compartment is limited due to relatively late expression of most tcr- chains (1, 2). Thus, all of the three known tcrs can have a role in promoting the development of pre t cells: the tcr- most likely by intercellular communication that furthers the development of cd48 cells irrespective of whether or not they have succeeded in tcr- rearrangement, the tcr- that depends strictly on intracellular, cell - autonomous signals generated by the tcr- chains and the pre - tcr that operates by a similar mechanism as the tcr- but is much more efficient because of the early and abundant expression of the pt gene during the phase of tcr- rearrangement . Therefore, only mice that cannot produce any of these receptors will exhibit complete arrest at the cd48 stage of development as evident in rag mice or mice that are deficient in both tcr- and tcr- chains and therefore, can make neither tcr-, pre - tcr, nor tcr- (14). In normal mice, the contribution of the tcr- in development of cells of the lineage appears to be limited based on the fact that the vast majority of cd48 cells are tcr- and thus are tcr- selected . Likewise, in normal mice, the contribution of the tcr- to the transition of dn to dp cells may be limited because of the small number of dp cells in pt tcr- mice . However, in the absence of pt these receptors avoid a severe immunodeficiency by enabling the formation of a significant number of mature t cells . It would appear that both the pretcr and the tcr- do not only mediate maturation but also proliferation since in wild - type mice and pt tcr- mice the proportion of large cd48 blasts that are derived from dividing cd48 precursors (15) is very similar (table 2). There are only slightly fewer blasts in pt tcr- mice indicating that also the tcr- generates dividing cd48 cells . With regard to the role of the src kinases in early development, our data is consistent with the notion that signaling through the pre - tcr involves both lck and fyn kinases but is equally consistent with the idea that the fyn kinase is involved only in signaling through the tcr- or -, and thereby responsible for the incomplete developmental arrest observed in lck mice . The fact that the tcr- promotes development much in the same way as the pretcr, i.e., by cell - autonomous signaling and thereby tcr- selection, suggests that t cell development may have proceeded in this way before the advent of the pre tcr- chain in evolution and that the pre - tcr had simply the advantage of making the pairing of a single tcr- chain with different tcr- chains more effective . Cd48 thymocyte subsets of wild - type and mutant mice mean values were obtained of four (two for pt/ tcr-/) different mice of each genotype from 68-wk - old litter . Intracytoplasmic staining for tcr- (tcr-ic) and tcr- (tcr-ic) within thymocyte subsets from c57bl/6 (wt), pt mice (a), and pt tcr-, pt tcr- mice (b). Total thymocytes were surface stained with pe - conjugated cd4 antibodies, biotinylated cd8 antibodies followed by pe - streptavidin; cytoplasmic staining was performed with anti - pantcr- or anti tcr- antibodies . The percentages of cells and absolute numbers (in brackets) are indicated . A schematic overview of various gene - deficient mice and the corresponding defects in t cell development . Percentages indicate the proportion of cells with cytoplasmic tcr-. The thickness of the bars is meant to correlate with the numbers of cells within the various subsets . Comparision of surface phenotype of thymocytes from tcr- pt vs. tcr- transgenic mice . (top) total thymocytes were double stained for cd4 (fitc - conjugated anti - cd4) and cd8 (red613-conjugated anti - cd8) surface antigens as described . Tcr- pt transgenic mice contained approximately one - half of the number of thymocytes found in tcr- transgenic mice (2,870 10 vs. 4,720 10 cells). (bottom) cells were stained with fitc - conjugated cd4 and cd8 antibodies in combination with biotinylated cd44 and peconjugated anti - cd25 antibodies . The expression of cd25 and cd44 was analyzed by three - color flow cytometry, using electronic gating to exclude fitc - positive cells . Assessment of transgenic tcr- and tcr- expression by intracytoplasmic staining . For intracellular / extracellular double staining, thymocytes isolated from transgenic tcr- mice and transgenic tcr- pt mice were stained with pe - conjugated cd25 antibodies and then with fitc - conjugated t3.70 antibodies specific for the transgenic tcr- chain of the hy - reactive tcr or fluos - conjugated f23.1 antibodies, specific for the transgenic tcr- chain of the hy - reactive tcr . Proportion of cd48 lymphoblasts in wild type and mutant mice percentages of cd48 blasts were determined by facscan using forward scatter as an index of size . The various mice were analyzed on the same day in the same experiment.
In recent years, coupling of theoretical and experimental approaches in the study of protein folding has resulted in providing fruitful clues . Experimental and computational protein design provides vital clues to understand the protein folding process, and it is of considerable interest in the area of protein science to engineer proteins with novel folds and desired functions . The field of protein design has a unique history where researchers from diverse discipline come together to explore novel catalytic, pharmaceutical, structural, and sensing properties of amino acids in proteins . Interestingly, a designed eleven amino acid sequence folded as a helix in one position and as a sheet in another position in the protein sequence . This work has enabled to explore the role of nonlocal interactions in the formation of secondary structure . Subsequently, helices were transmuted into sheets to understand the conformation change phenomenon and illustrate that not all the amino acids play an equal role in specifying a fold . Utilizing the knowledge offered by several protein design groups, kuhlman et al . In 2003 have computationally designed a 93-residue / protein called top7 and found that the protein could be experimentally folded and extremely stable . This pioneering work has enabled further research to understand the contribution of each amino acid residue in a protein to adopt a certain fold . Hence, emphasizing that protein design could be a powerful experiment to understand the processes that underlie conformational plasticity in proteins . Explored how two proteins with almost similar amino acid sequences change their fold and function . Following the contribution of various theoretical and experimental protein science research groups, several such engineered proteins with selective nevertheless, the design of such a pair of proteins with high sequence identity with completely different topologies can be viewed as a challenge to the well - accepted paradigm that similar sequences always tend to fold into similar three - dimensional structures . An analysis of the literature reveals that the design of two highly identical proteins with different folds and functions is challenging and time bound as shown in [table 1]. Streptococcus protein g contains two types of domains (ga and gb) that bind to serum proteins in blood . The natural versions of ga and gb domains share no significant sequence homology and have different folds, 3 and 4 +, respectively . From the above two parent proteins, high - identity versions of ga and gb were synthesized . Interestingly, small and critical differences in the sequences of the two proteins determine the topology of the protein early on the folding pathway . In addition, two proteins named ga88 (pdb i d: 2jws) and gb88 (pdb i d: 2jwu) by mutation experiments from the streptococcus protein g with 88% sequence identity adopt different structures and functions and these proteins are valuable tools to understand the contribution of residues to adopt a particular fold . These two proteins vary only at seven positions out of 56 amino acids, which are shown in [figure 1]. This design has made a breakthrough in the field of protein science and contradicts the general statement that following this, we have carried out computational sequence and structural analysis on these two designed proteins . We have performed secondary structure prediction of these two proteins and observed that the methods such as multivariate linear regression combiner can predict some regions as extended structures for the helical protein sequence ga, which gave us a clue that there may be structural plasticity at the region of first 15 residues, which are identical in the both proteins . We also discovered some patterns in the nonidentical positions of two proteins with a rare combination of residues that are not present in any publicly available sequence databases . By analyzing the structures of the two designed proteins, we predicted nucleation sites at various positions in the sequence, which may start or terminate secondary structural elements (helix, sheet, and coil). We also observed drastic difference in the surrounding environment of nonidentical residues (7 out of 56) and difference in interaction energy . By observing the structural plasticity at the amino and carboxyl terminal of the sequences of two designed proteins and the influence of surrounding environment of each residue, we concluded that early on during the process of folding, both proteins may choose different energetically favorable pathways to attain the different folds . Literature review of design of two proteins with high sequence identity adopting different folds sequence, dictionary of secondary structure of proteins assigned secondary structures and tertiary structures of pair of homologous heteromorphs (the seven residues that vary in both sequences are indicated in rectangular boxes) other researchers have characterized the folding of these two proteins using biophysical and computational experiments . They also indicated that the final native structures of these proteins were dictated very early along the folding pathway by performing equilibrium unfolding of ga88 and gb88, folding and unfolding kinetics and molecular dynamics simulations experiments . Concurrently, energy calculations were performed on the two designed proteins in a vacuum, which indicated that current computer modeling / simulations experiments cannot explain why two highly similar sequences fold into different structures . However, it was suggested that improved modeling / simulations tools should be developed to predict the pair of sequences with different structures, which differ, by only few residues . In a recent study, folding and unfolding kinetics experiments performed on these two designed proteins indicated a detectable residual structure in the denatured state of gb88 whereas the denatured state of ga88 is unstructured . Interestingly, they explored these two proteins by value analysis based on 132 site directed mutants and concluded that the protein's topology is committed very early along the folding pathway . Based on the above studies, we suggest that, along with the suitable protein design experiments, better theoretical models including folding simulations coupled with structure prediction and sequence search in databases can shed light on the phenomenon of protein folding and conformation switching which may ultimately lead us to understand the contribution of each amino acid in these proteins to adopt a specific fold.
In this case report, we describe a case of asherman's syndrome treated with adult autologous stem cells for endometrial regeneration that resulted in conception after in vitro fertilization - embryo transfer (ivf - et). The basis of this case is that endometrium is a dynamic, cyclically regenerating tissue, a unique model of physiological angiogenesis in adults . Angiogenesis results either from sprouting of new vessels through recruitment of local endothelial cells from neighbouring blood vessels and/or by endothelial progenitor cells circulating in the peripheral blood after release from the bone marrow. [14] bone marrow stem cells also contribute to regeneration of the endometrium . On the basis of these facts, her past treatment included a dilatation and curettage (d and c) in february 2005 . She presented to us with infertility and scanty menstruation since her d and c. her first transvaginal ultrasound scan on day 3 of the menstrual cycle revealed normal size retroverted uterus with homogenous myometrium and thin single line endometrium, but intact endometriomyometrial junction . The left ovary measured 2.52 2.51 3.04 cm, had two antral follicles and a hemorrhagic cyst, and was adherent to uterus posteriorly . Right ovary measured 2.55 1.22 1.84 cm and had only one antral follicle . Doppler studies showed very poorly vascularised ovaries, even with minimum wall filter, pulse repetition frequency of 0.3, and gains -0.8 . A repeat scan on day 14 revealed that hemorrhagic cyst in left ovary had regressed partially . Right ovary showed a follicle of 20 mm which on color doppler showed vascularity covering more than three - fourth of the follicular circumference with resistance index (ri) of 0.47 and peak systolic velocity (psv) of 11.23 cm / s, but the endometrium was only 3.2 mm with branches of spiral vessels seen only up to endometrio - myometrial junction . Follow - up scan after three days still showed the same endometrial picture though follicle had ruptured . Midluteal ultrasound scan (ninth day post ovulation) showed that endometrium had failed to grow even during secretory phase, despite corpus luteum with vascular ring covering more than half of the corpus luteal circumference with ri of 0.43 and psv of 10.43 cm / s on right side . Severe endometrial adhesions were seen, which were cut [figures 1 and 2]. Hysteroscopic picture - endometrial adhesions postadhesiolysis hysteroscopic picture iucd - cu t was placed to maintain surgically established patency of the endometrial cavity . She was treated with cyclical estrogen and progesterones with ethinyloestradiol 0.05 mg from fifth to 25 day of the cycle and with medroxy progesterone acetate 10 mg from 20 to 25 day for 6 months to obtain a functional endometrium . During this period, she had withdrawal bleeding, which was scanty . Ultrasound assessment of the endometrium in the following cycle showed no growth of the endometrium in the periovulatory and secretory phase of the menstrual cycle despite normal follicular development, rupture, and corpus luteum formation . Thin endometrium after removal of iucd in preovulatory period due to poor endometrial development, she was advised surrogacy with ivf . Her fsh on day 3 of cycle was 19.70 iu / ml with an antral follicle count of 2 . In view of poor ovarian reserve, she was given oral oestradiol valerate tablets in increasing doses from 4 mg daily for 3 days, followed by 6 mg daily for another 3 days, and then 8 mg daily for a total of 25 days along with aspirin 75 mg daily for endometrial preparation . Ultrasound scans were done intermittently to assess the endometrium, but it never reached a thickness more than 3.6 mm . This hormone replacement therapy cycle was repeated for 6 months without improvement of the endometrium . Based on reports of adult autologous stem cells applications for regeneration of injured cartilage and cardiomyocytes in cardiac infarction, it was thought that use of stem cells for regeneration of endometrium was worth trying, especially because endometrium naturally has a regenerating capacity . If the basal layer of the endometrium is repaired and further stimulated, it should increase in thickness . On june 15, 2009, her bone marrow aspiration was done from the iliac crest under local anesthesia maintaining strict asepsis . Aspiration was done using bone marrow biopsy needle and 10 ml syringe prewashed with heparin . Collection was done in cpda (citrate - phosphate dextrose anticoagulant) medium using 1 ml of medium for 7 ml of bone marrow . It was sent to a dedicated stem cell laboratory working as per clinical gmp / gtp standard and having a working area with laminar airflow to class 100 . Bone marrow was centrifuged using histopaque density gradient at 1000 rpm for 10 mins and 102 million mononuclear cells were separated . These cells were further treated by column separation technique and customized cocktail of cd9, cd90, and cd133 antibodies was used for immunomagnetic isolation of endometrial angiogenic stem cells . Gene expression study for cd9, cd44, and cd90 using rt - pcr technique was done for differentiated cells . Total 39 million marker - positive endometrial angiogenic cells were supplied in 0.7 ml of pbs (phosphate buffer saline) with 2% autologous (patient's own) heat - inactivated serum on the next day for transplant. [616] patient was called with partially filled bladder on june 16, 2009, second day of her menstrual cycle . Curettage was done under anesthesia . With patient in lithotomy position, sim's speculum in place, anterior retractor was used to retract the anterior vaginal wall, and volsellum was used to hold the anterior lip of cervix . France) attached with 1-ml syringe, filled with 0.7-ml stem cell suspension, was advanced through cervix upto the fundal end of the endometrium under transabdominal ultrasound guidance . When the tip of the catheter was 0.5 cm below the fundus, piston was slowly advanced to allow slow steady flow of cell suspension in the uterine cavity . After instilling 0.3 ml of stem cell suspension at the fundus, injection was continued when cannula was gradually withdrawn out, till the tip reached mid cavity of the uterus . It was further very gently and slowly withdrawn out of the internal os and then external os, maintaining continuous pressure on the piston to prevent any back flow . Speculum and volsellum were removed, and patient was shifted when she recovered from anesthesia . She was given oestradiol valerate 6 mg daily, starting on the same day for 25 days . Ultrasound was done on 14 day and 19 day of the cycle, which showed endometrial thickness of 5.0 and 5.2 mm, respectively, and color doppler showed spiral vessels reaching subendometrial zone . Ultrasound scans done at midcycle showed improvement in endometrial thickness, morphology, and vascularity [figure 4]. Well - developed endometrium with low - resistance vascularity reaching zone 4 on november 2, 2009, endometrial thickness at ultrasound was 6.9 mm, with vascularity reaching intraendometrial region . There were dominant follicles in either ovary, which excludes the possibility of any endogenous luteinizing hormone surge . On november 6, 2009, three 4 - 6 cell grade i donor oocyte ivf embryos were transferred . At this time, her endometrium was multilayered, with thickness of 7.1 mm and intraendometrial vascularity . She was given progesterone vaginal gel (crinone 8%, manufactured by fleet laboratories, uk, marketed by merck serono) twice a day after et, as luteal support and ethinyl oestradiol was continued in a dose of 6 mg daily along with aspirin 75 mg daily . Once -hcg was positive, the dose of oestradiol valerate was increased to 8 mg daily . Progesterone vaginal gel 8% was continued twice a day along with aspirin 75 mg daily . A single gestational sac of 12 mm, yolk sac of 2.9 mm, embryonic pole of 2.4 mm, with m - mode showing embryonic heart rate of 112/mt were seen at ultrasound on november 30, 2009 . Follow - up scan was done at 8 weeks of pregnancy which showed a healthy fetus [figures 57]. Gestational sac, yolk sac, and embryonic pole after embryo transfer and positive -hcg test m - mode of cardiac activity of embryo 3d picture of 8 weeks scan this was a case of infertility with severe asherman's syndrome and bilateral cornual tubal block with low ovarian reserve . Anti - mullerian hormone, which is now considered to be the most reliable marker for ovarian reserve, was not routinely used at that time (2007) and was not easily available locally, and therefore was not done . Low ovarian reserve with blocked fallopian tubes had left her with an option of oocyte donation . Placement of iucd after surgery and cyclical hormonal therapy in association with low - dose aspirin or nitroglycerine is an established protocol for development of functional endometrium . Placing iucd after curettage, followed by cyclical oestrogen progesterone therapy with aspirin, could not improve her endometrium . Stem cells derived from tissues such as bone marrow, cord blood, adipose tissue, or the amniotic fluid have demonstrated regenerative potential in a variety of diseases and degenerative disorders . Therefore, we tried to regenerate the endometrium with adult autologous stem cells after curettage, which was further supported with cyclical estrogen, progesterone, and aspirin for improving vascularization . Bone marrow aspiration is fairly safe procedure with the risk of bleeding or infection, which are extremely rare when done meticulously in the hands of the expert . Currettage was done to evoke injury - induced inflammatory reaction and hyperemia as homing induction, which in turn would enhance the response of endometrium to cyclical hormones . We would like to qoute a study that has shown regeneration of injured endometrium using adult bone marrow cells . Donor - derived bone marrow cells have been identified in human uterine endometrium. Recent evidence has implicated bone marrow - derived cells as possible endometrial progenitors . It is unknown whether these cells originate from bone marrow mesenchymal stem cells or, alternatively, are circulating endometrial cells originally derived from the endometrium and harbored in bone marrow . These cells, regardless of their origin, may serve as a source of reparative cells for the reproductive tract . These data show the potential for stem cells to have a role in the regeneration or repair of this tissue after injury . Significant engraftment of endometrium by bone marrow is likely to occur after endometrial injury or inflammatory insult . Additionally, the proliferation and development of endometrium are entirely regulated by hormonal stimuli . Ovarian estrogen and progesterone drive endometrial growth and apoptosis . In the case described in this study, we tried to regenerate the endometrium by endometrial angiogenic stem cells isolated from autologous adult stem cells and transplanting them in the endometrium immediately after curettage . This was based on a study that describes that mononuclear cells collected from the menstrual blood contains a subpopulation of adherent cells and retains expression of the markers cd9, cd29, cd41a, cd44, cd59, cd73, cd90, and cd105; the markers used were cd9, cd44, and cd90 to isolate the desired cells . There is a minimal risk of developed cells presenting certain other markers during the process, but immunomagnetic isolation with specific marker antibodies reduces this possibility . This experimental therapy may be tried on larger scale when the basal layer of endometrium is also damaged by surgical insult . The risk for malignancies after the use of adult autologous stem cells may be a concern in view of several reports, but we would like to draw attention to the fact that most of these reports discuss the cases where adult autologous stem cells were used to treat malignancies . The total cost of the therapy in an indian set up comes to approximately rs . 50 000 (rupees fifty thousand), excluding the expense of ivf - et . Moreover, it has an emotional and social advantage that the patient can bear her own child as against surrogacy . To the best of our knowledge, no case of asherman's syndrome conceived after endometrial regeneration with adult autologous stem cells after failure of all other conventional modes of treatment has been reported in literature . This therapy can be used as an alternative to surrogacy in females with severe asherman's syndrome, though larger trials may be needed to establish this as proved line of treatment.
A total of 1380 eyes were treated with prp for the indication of pdr from march 2001 to september 2006.the modified air lie house classification was followed . Cases included pdr of varying severity such as flat new vessels, with raised new vessels, subhyaloid hemorrhage, vitreous hemorrhage, trd and macular edema with and without vitreous traction . Thus in some of these cases the clinical evidence of vitreous contraction27 was already present before laser treatment was applied . In order to study the course of changes in the vitreous tissue over long term, we selected 100 eyes photocoagulated for pure pdr without clinically evident vitreous traction (as defined below). Out of these 100 eyes 74 eyes showing complete regression of new blood vessels following prp were studied over a follow - up period of one to four years . Criteria for pure pdr (without clinically evident vitreous traction): selection (1 to 5) and follow - up (6 to 7). Proliferative diabetic retinopathy with flat new vessels on the disc (nvd) and /or flat new vessels elsewhere (nve). 27,28neither hemorrhage in the vitreous nor subhyaloid hemorrhageno tractional edema of maculano previous ocular surgeryabsence of systemic hypertension and renal diseasefundus fluorescein angiography (ffa) proven regression of new vessels aft er prp and no recurrence of new vessels during a follow - up of at least one year.presence of other signs of involution in addition to regression of new vessels such as decrease in venous dilatation, disc pallor, disappearance of retinal hemorrhages . 29 proliferative diabetic retinopathy with flat new vessels on the disc (nvd) and /or flat new vessels elsewhere (nve). 27,28 neither hemorrhage in the vitreous nor subhyaloid hemorrhage no tractional edema of macula no previous ocular surgery absence of systemic hypertension and renal disease fundus fluorescein angiography (ffa) proven regression of new vessels aft er prp and no recurrence of new vessels during a follow - up of at least one year . Presence of other signs of involution in addition to regression of new vessels such as decrease in venous dilatation, disc pallor, disappearance of retinal hemorrhages . 29 careful history was obtained regarding duration of diabetes, type of diabetes and any other systemic illness (hypertension, renal failure) contributing to retinopathy, concurrent ocular disease and any previous ocular surgery . Cases with such concomitant pathology were not included in the study [table 1]. Detailed ocular examination including indirect ophthalmoscopy, slit - lamp biomicroscopy, macular examination with three mirror contact lens / 90 d lens and ffa was done . Optical coherence tomography (oct) was done in 32 eyes with csme out of 57 eyes (2004 onwards) to rule out vitreous traction . Eyes with any evidence of vitreous traction before prp were not included in the study . Pan retinal laser photocoagulation with or without focal /grid macular photocoagulation was done in all the 100 eyes . Green 532 and red 810 diode laser delivered through slit - lamp, were used . Diode laser was preferentially used for macular photocoagulation in the presence of lenticular opacities.30 a mild grey whitening of retina was the end point of treatment with spot size 200 to 300 and exposure duration of 0.1 to 0.25 secs, with power level adjusted to produce the desired reaction . The prp was completed in three to four sittings at intervals of four to seven days each; mainster 165 panfundoscopic lens was used . Follow - up was available in terms of visual acuity and ocular examination at the first week in all cases . The ffa was done at six weeks (18 cases), eight weeks (64 cases) and 11 weeks (18 cases). Seventy - four out of 100 eyes responded by way of complete regression of new vessels with laser photocoagulation [table 2]. Twenty - six eyes required further prp or other adjunctive treatment . Out of 57 eyes with macular edema visual improvement of two lines or more was observed in 34 eyes, stabilization of visual acuity in 11 eyes and drop of visual acuity by one line or more in 12 eyes . Sixteen out of these 74 eyes required to undergo vitreo retinal surgery within a period of one to four years after complete regression of new vessels following laser photocoagulation [table 3]. Other signs of regression of pdr such as disc pallor, ghost vessels and reduced venous dilatation were present in only 39 out of 74 cases . Incidence of pdr was significantly high in type i dm (p 0.001) and was observed to increase with duration of dm (p 0.001) [table 1]. Incidence of pdr with and without csme was significantly high in the age group 41 to 55 years (p 0.05) and also regression of pdr with prp (p 0.001) [table 2]. Indications for vitreo retinal surgery were significantly higher in type i dm (p 0.01), irrespective of age and duration of dm [table 3]. Diabetes induces pathology throughout the body and also in the vitreous via non - enzymatic glycation of proteins.7,10 advanced glycosylation end products (ages) have been found to be elevated in the vitreous of diabetics along with aggregation of collagen fibers and alterations in the cortex and hyalocytes.8,31 the vitreous in diabetics shows glycated collagen and increased amount of other proteins . 7 - 9,32 degenerative vitreous changes occurring in diabetics at a much younger age produce anomalous pvd, which has been said to help formation of new retinal vessels.9,33 structural changes at the vitreoretinal interface promote migration and proliferation of vasogenic cells in the vitreous, consequent contraction can produce vitreous hemorrhage and macular edema.12,34 advanced glycation end products correlate with glycemic control and these reactive compounds form on dna, lipids and proteins where they represent pathophysiological modifications that precipitate dysfunction at a cellular and molecular level in diabetics.10,34 though the term diabetic vitreopathy exists in the literature it has not been used to address and identify the pathological complex of diabetic retinopathy in any of the existing classifications of diabetic retinopathy . 19 - 22 we suggest that changes in the vitreous primarily due to diabetes mellitus and occurring independent of diabetic retinopathy can be called primary diabetic vitreopathy. The methods currently available for examination of the vitreous in vivo including oct give good information about the vitreo retinal interface but not the vitreous body, therefore such changes in the vitreous may not be easily detected clinically . We may therefore also call primary diabetic vitreopathy as subclinical diabetic vitreopathy.33 the hallmark of pdr is development of new vessels . The growing vascular endothelium combines with the collagen of the vitreous and gives it a contractile property.2,13,14 simultaneously fibrous tissue developing along the new vessels lines up on the posterior hyaloid, this also imparts contractile property to the vitreous.11 contraction of the vitreous can pull new retinal vessels to produce a bleeding in the subhyloid space to begin with [fig . 1]. If the bleeding is forceful or the vitreous largely liquefied this blood can break into the vitreous tissue concurrently or later . It should be noted here that bleeding outside the tissue confines of retina (internal limiting membrane) is produced by vitreous contraction and not proliferative retinopathy per se and thus any hemorrhage outside the retinal tissue whether subhyloid or in the vitreous should be considered a sign of vitreopathy and not retinopathy . Bleeding further augments the contractile property of the vitreous by inclusion of vasogenic and fibrogenic elements and such recurrent bleeding may result in the formation of fibrovascular tissue in the vitreous cavity . 2], secondary rhegmatogenous retinal detachment, persistent macular edema, premacular hemorrhage in addition to recurrent vitreous hemorrhage.35,36 we propose to call the changes induced in the vitreous tissue by proliferative retinopathy secondary diabetic vitreopathy or clinical diabetic vitreopathy. These are mainly in the form of an increase in the contractility and detachment of the vitreous . These changes are different from the changes of primary diabetic vitreopathy. If total vitreous detachment was present before development of pdr no bleeding or tractional retinal detachment may occur . Similarly, if there was no separation of vitreous subsequent to development of retinal new vessels the above pathological events may not occur . There have been several attempts in the past for pharmacological vitreolysis so as to abort any complications of retinal neovascularization by pull of vitreous and there is a continuous suggestion in the literature for early vitrectomy in pdr cases with good vision so as to forestall the complications produced by vitreopathy . 37 - 39 however, the changes in the vitreous have never been included in any of the classifications of diabetic retinopathy . Laser ablation of the retina induces regression of new vessels in about two - thirds of cases . 1 - 3 this results in the disappearance of a ready source of bleeding i.e. New vessels . With laser treatment the incidence of non - resolving massive vitreous hemorrhage has drastically reduced . But the other indications for vitreo retinal surgery such as trd, rhegmatogenous retinal detachment [fig . 3], tractional macular edema, premacular fibrosis, small recurrent vitreous hemorrhages, retinal wrinkling, macular heterotropia and dense premacular hemorrhage have persisted . 4 - 6, 15 - 18 these are all produced by vitreopathy, vitreous traction and not by pdr alone . In our series, out of 74 eyes showing complete regression of new vessels with prp, 16 eyes (23%) required to undergo vitreous surgery for the indications of recurrent vitreous hemorrhage (eight), tractional retinal detachment (four) secondary rhegmatogenous retinal detachment(one) and tractional macular edema (three cases) [figs.4 a, b, c]. In other words 23% of cases showed a continued contraction of vitreous strong enough to produce the indications for surgery despite successful regression of new vessels . We invited data from leading retina centers in the country on incidence of vitreous surgery in cases of pdr fully regressed with prp, over a period of one to four years . It is logical to say that in these cases the changes of primary /secondary diabetic vitreopathy were relatively independent of the effect of laser treatment and the regressed status of pdr . In the remaining 77% cases there are several factors which explain the absence of complications produced by diabetic vitreopathy . Regression of new vessels is withdrawal of a ready source of bleeding, multiple chorioretinal adhesions produced by laser protect against trd and rhegmatogenous retinal detachment . Laser treatment in cases with csme helps resolution of edema, it is effective in tractional macular edema also if the vitreous traction is not very strong . In addition to the above, prp induces posterior vitreous detachment in 50% cases and in the large majority this is eventless . A small percentage of these cases may show vitreous bleeding or even trd during completion of prp if it is too aggressive and spaced closely.40 we therefore understand that in the large majority of cases (77% in our series) a balance is established between the beneficial effect of laser on retina and the damaging effect of diabetic vitreopathy . In the remaining cases (23% in our series) this balance might not be established and ongoing vitreous contraction might have produced indications for vitreo retinal surgery . Another aspect of the effect of laser photocoagulation is that though the new vessels disappear the fibrous tissue does not and a slow, late cicatrization of fibrous tissue can produce indications like retinal wrinkling, macular heterotropia41 and shallow trd; we had one such case in our series with retinal wrinkling and trd occurring in regressed retinopathy after a quiet period of three years [fig . We suggest diabetic vitreopathy as a separate subdivision in the existing classification of diabetic retinopathy, identifying the changes induced by diabetes mellitus as primary diabetic vitreopathy and the changes induced by pdr as secondary diabetic vitreopathy. It is the changes of secondary diabetic vitreopathy which can be clinically observed and which produce the indications for vitreo retinal surgery, these can be called diabetic vitreopathy as such or clinical diabetic vitreopathy. We further observe that certain cases of clinical diabetic vitreopathy might not be operable because of extensive neovascularization both in the anterior and posterior segment or many complex fibrovascular membranes in the vitreous . In other words we can have cases which are operable and may benefit by vitreous surgery i.e. Surgical vitreopathy and cases which are inoperable or may not benefit by vitreous surgery i.e. Non surgical vitreopathy. Recently, intravitreal drugs have been used to alter the course of pdr . First is purified ovine hyaluronidase (vitrase, ista pharmaceuticals) which produces vitreous liquefaction . The second group are anti - vegf drugs, which reduce neovascularization, contraction of fibrovascular proliferations when prp is applied and bleeding during surgery . Avastin (genetech pharmaceuticals), the commonest drug, has been used as a preoperative adjunct for pdr, trd with severe pdr, for iris rubeosis, florid disc neovascularization and for treatment of pdr complicated by vitreous hemorrhage . 23 - 26 this would mean that anti - vegf drugs can be used in cases which are either inoperable because of extensive neovascularization, massive vitreous hemorrhage or are risky to operate due to the possibility of intraoperative bleeding and other complications . Use of these drugs may render such cases suitable for surgery . In order to identify such cases in a logical manner we suggest a separate class in between surgical and non - surgical vitreopathy as intermediate vitreopathy. The cases put in this class can shift to the surgical vitreopathy group if response to drug is adequate and to the non - surgical vitreopathy group if the drug treatment does not make the case operable . The term diabetic retinopathy may be replaced by diabetic retino - vitreopathy and may be classified as below . Non - proliferative diabetic retinopathy (mild / moderate/ severe / very severe)proliferative diabetic retinopathydiabetic maculopathy (focal, diff use, ischemic, mixed)clinical diabetic vitreopathyi) surgical vitreopathyii) intermediate vitreopathyiii) non - surgical vitreopathy non - proliferative diabetic retinopathy (mild / moderate/ severe / very severe) proliferative diabetic retinopathy diabetic maculopathy (focal, diff use, ischemic, mixed) clinical diabetic vitreopathy i) surgical vitreopathy ii) intermediate vitreopathy iii) non - surgical vitreopathy posterior pole trd 5superior half trd 16tractional macular edema without macular ischemia 15premacular fibrosis 28recurrent vitreous hemorrhages in laser regressed pdr 4, 17secondary rhegmatogenous retinal detachement 27 - 28optic disc traction 27, 28macular heterotropia 41retinal wrinkling 41dense premacular hemorrhage 35 tractional macular edema without macular ischemia 15 premacular fibrosis 28 recurrent vitreous hemorrhages in laser regressed pdr 4, 17 secondary rhegmatogenous retinal detachement 27 - 28 optic disc traction 27, 28 macular heterotropia 41 dense premacular hemorrhage 35 florid neovascularization with /without any of the above indications 26anterior segment neovascularization 24neovascularization non - responsive to laser with/ without any of the above indications 24 - 26large non - resolving vitreous bleeding in laser - treated or untreated pdr 26 florid neovascularization with /without any of the above indications 26 anterior segment neovascularization 24 neovascularization non - responsive to laser with/ without any of the above indications 24 - 26 large non - resolving vitreous bleeding in laser - treated or untreated pdr 26 inferior peripheral trd 16recurrent vitreous hemorrhages in active pdr 28, 36tractional macular edema with ischemia 15, 27 inferior peripheral trd 16 recurrent vitreous hemorrhages in active pdr 28, 36 tractional macular edema with ischemia 15, 27 this classification represents all stages and manifestations of diabetic retinopathy including changes in the vitreous . The classification identifies surgical indications and places different manifestations in accordance with the pathology and indicated treatment . For example, tractional diabetic macular edema is classified as surgical vitreopathy as it is the vitreous traction which is the cause and surgery is the treatment . The classification incorporates the indications for using recent intravitreal anti - vegf drugs also . The study sets the direction for further research and investigations on vitreous changes using modern tools such as ultrasound and oct in diabetics before and after development of diabetic retinopathy and also long - term prospective observations on the retina and vitreous on a larger sample of pdr cases after prp . Non - proliferative diabetic retinopathy (mild / moderate/ severe / very severe)proliferative diabetic retinopathydiabetic maculopathy (focal, diff use, ischemic, mixed)clinical diabetic vitreopathyi) surgical vitreopathyii) intermediate vitreopathyiii) non - surgical vitreopathy non - proliferative diabetic retinopathy (mild / moderate/ severe / very severe) proliferative diabetic retinopathy diabetic maculopathy (focal, diff use, ischemic, mixed) clinical diabetic vitreopathy i) surgical vitreopathy ii) intermediate vitreopathy iii) non - surgical vitreopathy posterior pole trd 5superior half trd 16tractional macular edema without macular ischemia 15premacular fibrosis 28recurrent vitreous hemorrhages in laser regressed pdr 4, 17secondary rhegmatogenous retinal detachement 27 - 28optic disc traction 27, 28macular heterotropia 41retinal wrinkling 41dense premacular hemorrhage 35 tractional macular edema without macular ischemia 15 premacular fibrosis 28 recurrent vitreous hemorrhages in laser regressed pdr 4, 17 secondary rhegmatogenous retinal detachement 27 - 28 optic disc traction 27, 28 macular heterotropia 41 dense premacular hemorrhage 35 florid neovascularization with /without any of the above indications 26anterior segment neovascularization 24neovascularization non - responsive to laser with/ without any of the above indications 24 - 26large non - resolving vitreous bleeding in laser - treated or untreated pdr 26 florid neovascularization with /without any of the above indications 26 anterior segment neovascularization 24 neovascularization non - responsive to laser with/ without any of the above indications 24 - 26 large non - resolving vitreous bleeding in laser - treated or untreated pdr 26 inferior peripheral trd 16recurrent vitreous hemorrhages in active pdr 28, 36tractional macular edema with ischemia 15, 27 inferior peripheral trd 16 recurrent vitreous hemorrhages in active pdr 28, 36 tractional macular edema with ischemia 15, 27 this classification represents all stages and manifestations of diabetic retinopathy including changes in the vitreous . The classification identifies surgical indications and places different manifestations in accordance with the pathology and indicated treatment . For example, tractional diabetic macular edema is classified as surgical vitreopathy as it is the vitreous traction which is the cause and surgery is the treatment . The classification incorporates the indications for using recent intravitreal anti - vegf drugs also . The study sets the direction for further research and investigations on vitreous changes using modern tools such as ultrasound and oct in diabetics before and after development of diabetic retinopathy and also long - term prospective observations on the retina and vitreous on a larger sample of pdr cases after prp . Changes in the vitreous induced by diabetes mellitus are identified as primary diabetic vitreopathy / subclinical diabetic vitreopathy and changes induced by proliferative retinopathy as secondary diabetic vitreopathy / clinical diabetic vitreopathy . Any indications for vitreous surgery in pdr are produced by vitreopathy and not retinopathy per se . In about two - thirds of pdr cases vitreopathy can be kept under control with adequate prp, the remaining one - third cases may require surgery due to vitreopathy . A new classification is proposed taking into consideration the element of diabetic vitreopathy as well as the clinical use of intravitreal anti - vegf drugs.
We identified isolates for sequencing from 29 invasive gas cases diagnosed in patients in a northern arizona hospital during january july 2015 and randomly selected an additional 99 gas isolates from a repository of> 2,000 arizona gas isolates collected during 20022006 (no isolates from patients in arizona were available for 20072014). Four additional isolates from central arizona identified in 2015 were included in the analysis (technical appendix table). All isolates were grown on 5% sheep blood tryptic soy agar plates (hardy diagnostics, santa maria, ca), and incubated at 37c with 5% co2 . Dna was extracted by using a dneasy blood and tissue kit (qiagen, valencia, ca, usa) following manufacturer s protocol . Genomic dna libraries were prepared by using the nextera xt library prep kit (illumina, san diego, ca) and sequenced with paired - end reads (250 bp) on an illumina miseq instrument, as previously described (9). The finished genome of the emm59 canadian clone mgas15252 (genbank accession no . Cp003116) and high - quality publicly available sequence - read data from 44 us isolates, from ncbi short read archive (bioproject #prjna194066), were included in the subsequent phylogenetic analyses . The final core genome (all nucleotide loci found in all genomes) for single - nucleotide polymorphism (snp) detection was 1,636,024 bp (98.6% of reference). We used nasp snp analysis pipeline (http://tgennorth.github.io/nasp/) for whole - genome snp typing as previously described (10). We used mega version 5.2.2 software (11) to generate maximum parsimony phylogenetic trees . Regions of high snp density were identified as possible regions of recombination and were further analyzed for impact on the consistency index . Gas emm subtypes were assigned by using blast (http://blast.ncbi.nlm.nih.gov/blast.cgi), querying the study genome assemblies against the centers for disease control and prevention s (cdc) emm type - specific sequence database (http://www.cdc.gov/streplab/m-proteingene-typing.html). We resolved dual emm - type hits using cdc s emm typing sanger sequencing primers (http://www.cdc.gov/streplab/protocol-emm-type.html) as a blast query and noting hit locations . We identified 18 of the 29 contemporary northern arizona isolates as subtype emm59; the remaining isolates were composed of 6 additional emm types: emm1 (n = 2), emm5 (n = 2), emm58 (n = 1), emm81 (n = 2), emm83 (n = 1), emm89 (n = 2), and emm94 (n = 1). The 99 historical and 4 contemporary background arizona isolates included 25 distinct emm types (technical appendix table). No emm59 isolates were identified in this background set, and none had been previously reported in arizona . An emm59-only phylogenetic analysis demonstrated the apparent presence of multiple lineages of emm59 in the 2015 arizona isolates (figure 1). A distinct clone consisting of 14 of the 18 emm59 isolates were separated from each other by only 04 snps, genomically supporting the presence of an ongoing outbreak;> 8 of these patients were epidemiologically linked to physical contact, cohabitation, or both with 1 other person (data not shown). The additional emm59 isolates make up additional lineages separated from one other by 828 snps . A relatively large number of snps and indels were seen within an approximate 23-kilobase region (figure 1). This region has been previously reported to contain mutational hotspots associated with virulence (12,13). Considering the presumptive positive selective force on this region, snps within the region phylogenetic single - nucleotide polymorphism (snp) tree of emm59 isolates from a northern arizona hospital displaying distribution of mutations in a 23 kb positively selected region during invasive group a streptococcus outbreak, southwestern united states . Maximum parsimony tree of all snp loci (n = 58) in emm59 isolates (n = 18) from arizona, 2 recent new mexico isolate genomes, and the canadian clone reference isolate mgas15252 . Branch lengths represent numbers of snps between isolates; unit bar is in the figure . Numbered circles distinguish lineages of selected mutations in scpa, enn, sfbl, mga, sfbx, and sof genes in a 23-kb hotspot mutational region . When compared with all other publicly available us emm59 isolate genomes, nearly all the genomes identified in the united states were closely related to each other and to the canadian clone mgas15252; individual isolate snp branch lengths ranged from 0 to 10 (figure 2). The arizona outbreak isolates were separated from 2 new mexico isolates by 4 and 5 snps each; these isolates fell within the overall arizona clade and were subsequently included in the arizona - only phylogenetic analysis (figure 1). Conversley, the isolate from patient m appears more distant from the larger arizona population . The arizona clades, with the exception of that of the isolate from patient m, all appear to arise from the large minnesota polytomy . The previously estimated 1.32.1 snps / year mutation rates for gas (14,15) further support the arizona outbreak as being caused by a single clone, likely originating from new mexico and being spread over 612 months . Phylogenetic single - nucleotide polymorphism (snp) tree of emm59 isolates from arizona during invasive group a streptococcus outbreak in the southwestern united states, previously analyzed us emm59 isolates, and the canadian clone . Maximum parsimony tree of all 177 snp loci (44 parsimony informative snps) in emm59 isolates from arizona (n = 18), minnesota (n = 29), oregon (n = 8), new mexico (n = 3), colorado (n = 2), and california (n = 1) and the canadian clone reference isolate mgas15252 . Tree has regions of recombination removed and is rooted with minnesota isolate srr11574570 . The emm59 subtype of gas, the etiologic agent of a substantial nationwide outbreak of invasive gas in canada during 20062009 (4), is now present in arizona, causing at least 1 outbreak of epidemiologically and genomically linked cases and several additional epidemiologically unrelated cases . The lack of emm59 in background isolates in arizona from the previous decade, along with its low genetic diversity, suggests that emm59 emerged recently in arizona . Following the emm59 epidemic in canada, this subtype was subsequently seen in a few us states; a retrospective analyses of the centers for disease control and prevention active bacterial core surveillance (abcs) system (http://www.cdc.gov/abcs/reports-findings/survreports.pdf) identified 40 us emm59 isolates during 20002009 (6) and an additional 67 isolates during 20102012 (7). Of note, only 5 (of the 40 emm59 isolates from 20002009 (2 from minnesota, 2 from california, and 1 from oregon) appeared to be closely related to the canadian clone (defined by the authors as being separated by <16 snps) (6); in contrast, all of the strains from the 20102012 survey appeared to be more closely related to the canadian clone . The more recent abcs analysis identified an increasing number of southwestern isolates, including 4 from colorado and 6 from new mexico (7), although no outbreaks were specifically described in these states (arizona is not included in the abcs system). (7), in an analysis of 60 mn emm59 isolates from case - patients with identified race, determined that 25 (42%) were from native americans; of 5 isolates from new mexico in that study, 3 were from native americans . Given the apparent distal nature of the arizona / new mexico isolates to the minnesota population in our study, it is reasonable to propose an unidentified epidemiologic relationship between these case populations . However, caution must be used in drawing conclusions regarding the relationships of isolates from disparate geographic regions because only limited comparable sequence data from previous emm59 studies in the united states (7) were publicly available to compare to the arizona isolates . Epidemiologic investigations, along with healthcare provider and patient education activities, are ongoing in arizona to further determine the extent of the current outbreak and the associated risk factors and to help mitigate effects and limit or prevent further spread to at - risk populations.
Primary tumors of the trachea are rare and account for 0.1% of all malignancies . In adults, approximately 90% of the primary tracheal tumors are malignant, whereas in children, the majority of these tumors are benign [2, 3]. In a retrospective study by webb et al ., 55.4% of the patients were male and 77.3% of them were smokers . The frequency of this rare disease has been estimated to be 0.10.4% of all malignancies, with an annual incidence of 2.6 new cases per million per year . Primary tracheal tumors can arise from the respiratory epithelium, salivary glands and mesenchymal structure of the trachea . Squamous cell carcinoma (scc) and adenoid cystic carcinoma (acc) make up about 71.6% of all adult primary tracheal tumors . The remaining portion (28.4%) scc occurs predominantly in men in the sixth and seventh decades, whereas acc is equally distributed between the sexes and peaks in incidence in the fourth and fifth decades . In contrast to acc, scc (the most common type of tracheal tumors) has a more aggressive course . Median survival time ranges from 6 to 14 months, and tumor disappearance is dependent on whether the primary lesion is resected . Sccs spread to the lymph nodes and 1020% of the patients will have distant metastasis at presentation . Carcinoids, lymphomas, granular cell tumors and small cell carcinomas have variable prognoses; yet they seem to behave better than sccs, adenocarcinomas or sarcomas . The majority of the tracheal neoplasms are primary in nature, and other primary sites that can metastasize to the trachea are the lung and esophagus, the latter being the most common . Patients with surgically resectable primary tracheal tumors have a better prognosis than those with tumors that cannot be resected . Preoperative radiation therapy has been attempted in some patients, yet the most compelling evidence for adjuvant radiation therapy comes from postoperative cases . The present study reports a rare case of primary tracheal malignancy treated with surgery and adjuvant radiation therapy . Our 60-year - old diabetic and hypertensive male patient had a 1-month history of cough associated with marked respiratory distress and occasional dyspnea in the right lateral lying position . The patient consulted a chest physician and underwent bronchoscopy, which showed a polypoid growth in the trachea extending to the larynx (fig . A computed tomography (ct) scan revealed irregular soft tissue thickening in the region of the trachea extending up to the adjacent larynx with no soft tissue calcification, and there was no definite lymphadenopathy in the cervical region and no involvement of the underlying bone . A repeat ct scan of the neck (fig . 2) showed evidence of an ill - defined, irregular, nodular, infiltrating soft tissue growth arising from the posterior tracheal wall, bridging the lumen and reaching up to the anterior wall, with an infiltration of about 2.2 1.8 cm . The location was opposite the c7-d1 level involving the 5th or 6th tracheal ring, approximately 7 cm proximal to the carina with no significant evidence of cervical and mediastinal lymphadenopathy . Excision of the tracheal growth through a cervical transverse incision was performed, and a tracheostomy tube was inserted . Soon after surgery, the cough subsided and the patient attended square hospital, dhaka, bangladesh, for postoperative irradiation . Ninety - five percent of the planning target volume (ptv) covered 97.3% of the prescribed dose . The minimum and maximum dose received by the ptv was 50.05 and 63.68 gy, respectively . 3). Three multileaf collimator fields (left anterior oblique, right anterior oblique and posterior) were used for three - dimensional conformal radiation therapy (3dcrt). Complete response was documented, and the patient is still alive without any evidence of disease during 30 months of follow - up . Primary malignant tumors of the trachea are uncommon, and therefore limited data supporting a standard management are available . The vast majority of the primary tracheal tumors in adults are malignant and most of them usually diagnosed at a later stage, which is due to a delayed presentation of specific symptoms like cough, dyspnea and hemoptysis . It is clear that patients who can be resected have a better prognosis than those who cannot, prompting the recommendation of surgical resection for most primary tracheal tumors . All resected patients need postsurgical irradiation, regardless of tumor burden, margin status, histology or nodal status . Grillo and mathisen experienced a median survival of 34 months in scc and 118 months in acc patients who underwent a combined modality like surgery and radiation therapy . Attained a 61-month median survival using a combined surgery and radiation therapy in their study . . Showed that 73% of the patients with a 5-year survival underwent radiation therapy postoperatively . The role of external beam radiation therapy as an adjuvant to surgical resection is better established . Grillo and mathisen advocated maximal (4,5006,500 cgy) radiation therapy after scc and acc resection because of the close margins necessary for resection and the high likelihood of local recurrence . Modern ct - based 3dcrt or intensity - modulated radiation therapy theoretically allows higher and safer doses delivered to the trachea . For postoperative cases, all patients with this tumor in the postoperative or definitive setting should be treated with the 3dcrt technique . An intraluminal boost technique after external beam radiation therapy may decrease the risk of late side effects . There are many studies showing the benefit of postoperative irradiation therapy in primary tracheal malignancies such as sccs and accs . However, little experience has been shared in the case of primary adenocarcinomas of the trachea, the tumor type described in our report . The prognosis of patients with malignant tumors of the trachea remains gloomy, and long - term median survival of tracheal adenocarcinoma patients undergoing combined modalities is unknown to us . Primary adenocarcinoma of the trachea needs to be diagnosed at an early stage, and combined multimodal approaches may be explored to attain an extended median survival . None of the authors has any conflicts of interest regarding the content of this article.
Intractable nausea and vomiting are very rare symptoms of medullary compression and there are approximately less than 10 cases reported in the literature of the aforementioned symptomatology mostly due to tumors . There is one reported case where vertebral artery compression of the medulla led to intractable nausea and vomiting . A 69-year - old woman presented with a 10-month history of intractable nausea and vomiting resulting in a 50 pound weight loss . She had an extensive medical workup at multiple outside hospitals including a comprehensive gastrointestinal workup which was significant for celiac disease . Her pertinent past medical history includes a history of breast cancer, mastectomy, and cholecystectomy . During her evaluation by the gastroenterologists at our hospital, the diagnosis of celiac disease was confirmed, and she was also noted to have a duodenal ulcer . Hematologic and biochemical workup was only significant for anemia attributable to her ulcer and celiac disease . Magnetic resonance imaging / angiography (mri / mra) of the brain and neck, and diagnostic four vessels cerebral angiogram were obtained to further evaluate her dizziness and double vision . The left vertebral artery angiogram revealed a tortuous left vertebral artery with a 9.6 5.6 mm dissecting aneurysm in the v3 segment . This, along with dolichoectasia of the vertebro - basilar arteries, resulted in compression of the medulla oblongata, which was also confirmed in the contrasted mri / mra of the brain and neck [figure 1]. Computed tomography angiography (cta) also confirmed that the vertebral artery was pushing the medulla medially [figure 2]. The patient was offered a microvascular decompression (mvd) of the vertebral artery to attempt to decompress the brainstem and alleviate her intractable nausea and vomiting . Preoperative magnetic resonance imaging (mri) t2 sequenceshowing compression of the medulla by the vertebral artery preoperative computed tomography angiography (cta) showing a tortuous left vertebral artery crossing the midline a left retrosigmoid craniotomy was completed to approach the vertebral artery and lower brainstem . The elongated, ectatic vertebral artery additionally, indentation of the lower part of the medulla by the vertebral artery was also recognized . Apericranial graft was used as a sling and tacked to the dura, to decompress the brainstem . The postoperative course was uneventful, and the patient was kept in the hospital to transition her from gastrostomy tube feedings to regular oral feeding . Postoperative cta demonstrated that the vertebral artery had been mobilized [figure 3] away from the medulla . Postoperative cta showing left vertebral artery away from the medulla the patient was discharged from the hospital without any antiemetic medication and was documented to have started gaining weight . At her 2-year postoperative visit, the patient had a nonfocal neurologic exam without recurrence of her prior nausea or vomiting . Patients with central lesions often wait months or years and undergo countless tests before a central etiology is added to the differential diagnosis because most cases do not present with localizing neurological deficits . Sustained hiccups with vomiting or isolated spontaneous vomiting with negative gastrointestinal symptoms should prompt further workup, including neuroimaging, to search for a central cause . The patient in our case was incidentally found to have dizziness and diplopia during the initial workup, but was not demonstrating positional vomiting or headaches, which would have prompted a neurological workup sooner . The tortuous vertebral artery in this case was found on the diagnostic mri / mra of the head and neck obtained secondary to her history of breast cancer and new onset symptoms of diplopia and dizziness . Without a proper neurological examination this patient's celiac disease and gastrointestinal reflux would have continued to mask the true cause of her symptoms . While compression of the medulla by a dilated vertebral artery is much less common than other brainstem compression syndromes such as trigeminal and glossopharyngeal neuralgias; it is still significant . Vascular compression of the medulla can cause disabling positional vertigo, hypertension, and hemifacial spasm; but there are only a few reports of intractable nausea and vomiting as a symptom of an ectatic vertebral artery . Less than 10 cases of nausea and vomiting due to vertebral artery compression have been reported in the literature . Of the 20 cases of vertebral artery compression of the medulla reported in a meta - analysis by savitz et al ., only one patient experienced nausea and vomiting as a symptom . The majority of patients presented with hemiparesis and cranial nerve dysfunction . Mvd was the choice of treatment for 17 of the patients, including the patient with symptoms of nausea, and it was shown to relieve symptoms in 16 patients . Medullary compression can produce a wide spectrum of signs and symptoms from very few to several . Regardless of the etiology, compressive forces on the neuronal tracts and nuclei that lie within the medulla can produce a number of clinical scenarios . When the etiology is vascular in origin, ischemic injury can produce symptoms such as headache, transient ischemic attacks (tias), or infracts depending on the location of the compression . At the floor of the rhomboid fossa it contains specialized cells consisting of ependymal cells and tanycytes that allow for direct communication of cerebro spinal fluid and blood because it lacks an intact blood brain barrier . As a circumventricular organ, the area postrema is able to detect toxins and drugs in the blood as well as hormones and other humoral signals to help maintain autonomic homeostasis . Stimulation of the area postrema by vagal afferents from the nucleus ambiguous and gastrointestinal system or from emetogenic drugs and cytokines in the blood may trigger vomiting . The nucleus tractus solitarius (nts) lies adjacent to the area postrema, serves as a relay center to collect all afferent signals and activate the appropriate visceral nuclei to coordinate the action of vomiting . Efferent pathways from the area postrema and nts project to the central pattern generator, ventral medulla, and hypothalamus . The central pattern generator is proposed to coordinate the activation of these nuclei within the medulla . The efferent signals stimulate the appropriate parasympathetic and sympathetic neurons, which produce the different phases of vomiting . The signal also diffuses through the brain via microglial messengers and cytokines such as substance p to allow for the cognitive recognition of emesis . A lesion to any of the afferent relay tracts or emetic reflex center proper may produce an abnormal emetic reflex and could produce intractable nausea and vomiting, as was observed in our case . On the preoperative mri, the vertebral artery was seen to be compressing the medulla, where the area postrema is located, and nts at the inferior and posterior limit of the fourth ventricle . The potential disruption of the area postrema in this case may have caused an overstimulation of the emetic reflex . The first mvd was performed in 1966 by dr peter jannetta to relieve the facial pain in a patient with a compressed trigeminal nerve . It is now used to treat compression of the facial and glossopharyngeal nerves, as well as vascular abnormalities . The indications of mvd could potentially be expanded to treat compressive symptomology; including, for example, intractable vomiting due to vascular compression of the medulla as demonstrated in this case report . This case demonstrated the importance of neurological investigations in the isolated intractable vomiting patient without a clear evidence of peripheral cause and including a central nervous system etiology on the differential, and the role of mvd in achieving cure.
Osteoarthritis (oa) is a chronic synovial joint disease, characterized by two main features: (1) progressive damage of articular cartilage, bone remodeling, and new bone formation (osteophytes and subchondral bone sclerosis) and (2) synovial inflammation and fibrosis of ligaments, tendons, menisci, and capsules . All joints may be affected, but the most commonly involved are knees, hands, and hips (fig . 1). While chronic oa used to be regarded as a wear and tear disease, researchers now believe that low - grade inflammation and growth of blood vessels and nerves from the subchondral bone into articular cartilage, as well as metabolic disorders, play a major role in disease pathology . Pharmacological interventions are mostly palliative, focusing on alleviation of symptoms or slowing disease progression until damaged hip or knee joints are eventually replaced . Differences in knee anatomy (narrower femurs, thinner patellae, larger quadriceps angles, and differences in tibial condylar size), previous knee trauma, and genetic and hormonal influences may play a role . Women present for treatment in more advanced stages of oa and have more debilitating pain than men . Women also have less cartilage volume, greater cartilage wear, and overall differences in mechanical alignment . Symptomatic knee oa typically presents with narrowing of the joint space and bone spurs (arrows). (a and b) during the development of oa, articular cartilage breaks down over time and becomes thin . As a result, the bone surfaces rub against each other, further damaging the cartilage and bone and causing pain . (c and d) joints with late - stage oa are often painful, warm to the touch, possibly red, swollen, have subchondral cysts, and notable loss of function . Pain medications currently used to treat the symptoms of oa include acetaminophen, topical capsaicin, topical and oral nonsteroidal anti - inflammatory drugs (nsaids; i.e., naproxen and ibuprofen), and the synthetic opioids tramadol and codeine . However, each of these therapies has potential drawbacks that may limit their widespread use . Analgesics can be addictive, whereas acetaminophen can have serious side effects, such as kidney and liver damage . Treatment with nsaids, which inhibit cyclooxygenases (cox1 and cox2), thereby blocking prostaglandin synthesis, improves quality of life and decreases pro - inflammatory cytokines including interleukin-6 (il-6), vascular endothelial growth factor (vegf), and tumor necrosis factor- (tnf-) in synovial fluid and mitogen - activated protein kinases (mapks) in knee oa . However, nsaids can also cause serious side effects, including upper gastrointestinal (gi) toxicity (dyspepsia, ulcers, perforation, obstructions, and bleeding) and liver dysfunction . As such, they are typically prescribed for the shortest possible duration at the lowest effective dose . To reduce the risk of these upper gi complications, the us fda has approved the use of the nsaid hzt501 (duexis), a drug containing 800 mg ibuprofen, in combination with 26.6 mg famotidine, a histamine h2-receptor antagonist . Alternatively the nsaid celecoxib has less risk of upper gi complications by selectively inhibiting the isoenzyme cox-2, which is specific to inflamed tissue, versus cox-1, which is constitutive in many tissues including the gi tract . It should be noted that daily treatment with celecoxib is more effective in patients with normal body mass index (bmi) than obese patients . Intra - articular injection of corticosteroids (gc) is recommended to relieve inflammation and pain in oa joints . However, gc injections are short acting, prone to adverse side effects, and have limited disease - modifying effects . For patients with knee oa, viscosupplementation with hyularonin may be used to replaces shock absorbing and lubricant material in the joint fluid, but the effects are similarly short - lived . Currently, guidelines for oa management are available from numerous organizations, including the american academy of orthopedic surgeons (aaos), the american college of rheumatology (acr), the american geriatrics society (ags), the american pain society (aps), and the osteoarthritis research society international (oarsi) in the united states and the european league against rheumatism (eular) and the united kingdom s national institute for health and clinical excellence (nice) in europe . Collectively, these guidelines reflect the experience of physicians across a variety of medical disciplines . Whereas all generally use the same data sources (i.e., evidence - based research, expert opinion, patient experience, and cost - effectiveness analysis), they differ in focus . For instance, the aaos and ags guidelines reflect the perspective of specialists in orthopedic surgery, geriatrics, and pain management, whereas the eular and oarsi guidelines primarily emphasize the findings of experts in rheumatology . The nice guidelines are developed jointly by physicians and other health care professionals working in conjunction with a range of clinical researchers . In addition, the scope varies, with some guidelines (e.g., aaos, acr, eular, and oarsi) addressing specific types of oa (i.e., knee, hip, or hand) and others (e.g., ags, aps, and nice) addressing oa more generally . As such, recommendations can vary widely, for instance, guidelines for use of nsaids . Recommended nonpharmacologic interventions range between therapeutic exercises, patient education, transcutaneous electrical nerve stimulation, acupuncture, orthotics and insoles, heat and cryotherapy, patellar tapping, and weight control . In an effort to evaluate these varying guidelines, the appraisal of guidelines research and evaluation (agree ii) scored 17 clinical practice guidelines (cpgs) including eular, nice, oarsi, aaos, and acr, on six different measures: d1, scope and purpose; d2, stakeholder involvement; d3, rigor of development; d4, clarity and presentation; d5, applicability; and d6, editorial independence . The general clinical management recommendations tended to be similar among high - quality cpgs, although interventions addressed varied . New noninvasive, disease - modifying therapies for oa are lacking and needed by millions of patients . A number of prospective new treatments targeting pro - inflammatory mediators, cytokines, bone turnover, and angiogenic and neurogenic factors are being investigated, with varying success in clinical trials and clinical use . Interleukin-1 (il-1) may prove an effective target, as il-1 induces matrix metalloproteinase (mmp) production, resulting in the degradation of aggrecan and other matrix constituents . Il-1 also induces high levels of cox2 and prostaglandin e2 (pge2), which may explain the pain associated with oa degeneration . The drug diacerein, an inhibitor of il-1, may modify both disease symptoms and disease structure in oa . Oral diacerein has proven effective in reducing pain, although evidence from clinical trials and scientific literature suggest that the effectiveness in oa is weak . It can be used in conjunction with nsaids or viscosupplementation therapies for additive effects due to its alternative mechanism of action . The most common side effects of diacerein are gastrointestinal, such as diarrhea, and changes in the color of urine . Meanwhile, the il-1-receptor antagonists anakinra and orthokin are reported to improve western ontario and mcmaster universities arthritis index (womac) scores . Gevokizumab is in phase ii clinical trials for safety and biological activity in the treatment of hand oa . Nerve growth factor (ngf) has also been recognized as an important mediator of chronic pain in oa . Tanezumab, a monoclonal antibody against -ngf receptor tyrosine kinase (trka), inhibits ngf action and reduces pain in patients . Two randomized phase iii clinical trials indicate that tanezumab provides superior pain relief while improving physical function and global disease assessment scores in patients with painful hip oa . Although in most cases tanezumab is well tolerated, the unexpected occurrence of rapid destructive arthropathies suggests there may be safety issues . Alternatively, using the drug adalimumab to inhibit tnf-, which upregulates -ngf, does not improve global disease assessment scores in oa of the hand . Strontium ranelate (srra), an element similar to calcium, is easily taken up by the body and incorporated into bones in place of calcium . The sekoia (srra efficacy in knee osteoarthritis trial) trial, a 3-year randomized, double - blind, placebo - controlled trial, evaluated the efficacy, safety, and disease - modifying effects of srra given at 1 to 2 g / day in patients with knee magnetic resonance imaging (mri) data indicate that srra significantly reduced cartilage volume loss and bone marrow lesion progression . Symptoms also improved in terms of pain and physical function after 6 and 12 months, respectively, although treatment was deemed safe and well tolerated . These data indicate that srra could be a promising new symptom and disease - modifying treatment for oa . Additionally, there is a need for further investigations to establish the optimal dosage and to better clarify the mechanism of action of srra in oa . Several clinical studies have investigated the effects of anti - resorptive therapies such as bisphosphonates on oa symptoms . A study by carbone et al . Found that alendronate (aln) use in oa patients decreased bone abnormalities and attenuated knee pain, yet cartilage degeneration was still present in the mri scans of treated patients . Determined that risedronate use led to significant improvements in womac scores and preservation of knee joint space compared with placebo in a 1-year randomized control trial involving patients with moderate oa . However, a 2-year randomized control trial of risedronate treatment revealed contradictory results, with no significant improvement of womac score or joint space retention in the knee . Similarly, nishii et al . Observed no inhibition of oa progression in treated hip oa patients after 2 years of aln treatment therefore, in spite of the growing body of clinical work investigating the subject, no definitive conclusion can be reached on the practicality of using bisphosphonates to treat patients with oa . Antidepressants have shown promising preliminary results for treatment of pain associated with oa by increasing serotonin levels in the brain . Serotonin - norepinephrine reuptake inhibitors duloxetine (cymbalta) and milnacipran significantly improve pain in oa . An open - label trial also suggested analgesic effectiveness of methotrexate, an anti - inflammatory drug that acts by inhibiting the metabolism of folic acid, demonstrating that up to 20 mg / week for 6 months achieved oarsi responder criteria in knee oa and warranted a randomized controlled trial . The small molecule kartogenin was identified in an image - based high - throughput screen to promote chondrocyte differentiation . It shows chondroprotective effects in vitro and is efficacious in two animal models of oa . Kartogenin induces chondrogenesis by disrupting the interaction between filamin a and the transcription factor core - binding factor b subunit (cbf), thereby altering cbf-runx1 and possibly runx2 transcriptional programs . Autologous injection of platelet - rich plasma (prp) has been used to stimulate cartilage repair and healing in oa patients, but the presence of other growth factors in prp may be problematic . Furthermore, bone morphogenic protein 7 (bmp7), fgf-8, and botulium toxin a (bont - a) are used in the treatment of knee oa . Bont - a has an analgesic effect by temporarily suppressing acetylcholine secretion at presynaptic nueuromuscular junctions and appears to be effective and safe for the management of advanced knee oa . However, these results cannot be generalized to patients with mild knee joint pain or nonspecific soft tissue pain in the knee joint region . Further research is necessary to investigate possible complications such as aggravation of infection, effect on muscle strength, and neuropathic joint degeneration . Recently, a phase i trial was reported in which chondrocytes were modified via intra - articular dna injection to produce tgf-1 in patients with advanced knee intra - articular injection of adipose - derived stem cell (adsc) therapy in a new european program is also under investigation . Adsc induced the release of trophic factors that exerted anti - inflammatory effects on both synoviocytes and chondrocytes, with no mmp1, mmp3, or mmp13 production, suggesting safe and effective use of adscs for clinical applications . However, both treatments need proof - of - concept studies in larger patient populations . Alternatively, intra - articular injection of human mesenchymal stem cells can lead to articular cartilage protection through the sdf-1/cxcr4 axis . Natural products can be safer than prescription medications with less undesirable side effects . Dietary supplements including avocado soybean unsaponifiables (asu), chondroitin sulfate, hyaluronan, and glucosamine sulfate they are used to treat mild to moderate pain and alleviate symptoms to reduce the consumption of nsaids . Several trials for chondroitin sulfate, glucosamine sulfate, and hyaluronan (c14h21no11) are in process . Chondroitin sulfate, glucosamine sulfate, and hyaluronan are building blocks for proteoglycan synthesis, and major constituents of the extracellular matrix in cartilage and synovial fluid . Hyaluronan and hyaluronic acid (hyalgan hylan - gf20/synvisc) can be injected into the knee joint of patients with oa who cannot tolerate nsaids or are awaiting joint surgery . A recent report indicates that viscosupplementation with hylan - gf20 slows type ii collagen degradation and joint inflammation in patients with oa . However, hylan - gf20 was not present in granulomas, an indicator of inflammation, raising the question of clinical significance in pain reduction . Also, viscosupplementation with hyaluronic acid itself does not significantly improve disease outcome, and little is known about long - term effects . The efficacy of glucosamine and/or chondroitin in treating knee oa pain was evaluated in the glucosamine / chondroitin arthritis intervention trial, funded by the national center for complementary and alternative medicine and the national institute of arthritis and musculoskeletal and skin diseases . Patients were treated daily for 24 weeks with glucosamine alone (1,500 mg), chondroitin sulfate alone (1,200 mg), glucosamine and chondroitin sulfate combined (same doses), a placebo, or celecoxib (200 mg), which served as a positive control . Although there were no statistically significant differences between any of the experimental treatments and the placebo overall, patients with moderate - to - severe pain given both glucosamine and chondroitin sulfate did show improvement (79% experienced pain reduction vs. 54% for placebo). Because of the small size of this subgroup, these findings should be considered preliminary and need to be confirmed in further studies . Glucosamine does not appear to slow arthritis progression over the long term and has many potential complications . The most common adverse effects are epigastric pain or tenderness, heartburn, diarrhea, and nausea . Glucosamine also may cause allergic reactions in patients with seafood allergies, as a product of lobster, crab, and shrimp shells . Glucosamine may interact with various pharmaceuticals, such as warfarin (coumadin) and diabetes medications, dangerously modifying their efficacy . Similarly, chondroitin sulfate appears not to provide meaningful benefit for patients with oa, and their combination has not proven effective for either pain management or functional improvement . Oarsi and nice no longer recommend the use of glucosamine or chondroitin sulfate alone, or in combination, if no effects are observed after 6 months or radiographic changes are marginal . A network meta - analysis of 10 trials in 3,803 patients by juni and collaborators in 2012 found no clinically significant improvements in oa pain alleviation or jsn parameters with glucosamine, chondroitin, or combined treatment compared with placebo . Despite these results, many patients believe otherwise, potentially due to the natural course of disease, regression to the mean, or the placebo effect . The authors conclude that such patients should be permitted to use these supplements if they cover the cost themselves, since neither of these preparations was found to be dangerous . Sierrasil is a dietary supplement marketed for joint pain relief that is derived from the mineral - rich clay found in the high sierra mountains in the united states . Clinical trial testing short - term efficacy of sierrasil at doses of 2 and 3 g per day failed to show sustained benefits over placebo, and iron toxicity has been reported . Some oa patients experience pain relief from topical creams containing capsaicin, the active component of chili peppers . However, use of these creams may introduce side effects such as burning, stinging, and redness of the skin and eyes . Avocado / soybean unsaponifiables are natural vegetable extracts made from avocado and soybean oils, consisting of the leftover fraction (approximately 1%) that cannot be made into soap after saponification . Asu is composed of one third avocado and two thirds soybean unsaponifiables (a1s2u). The major components of asu are phytosterols -sitosterol, campesterol, and stigmasterol, which are rapidly incorporated into cells . Asu is a complex mixture of many compounds including fat - soluble vitamins, sterols, triterpene alcohols, and possibly furan fatty acids . The sterol contents of asu preparations are the primary contributors to biological activity in articular chondrocytes . Preclinical in vitro and in vivo studies have demonstrated that asus have beneficial effects on oa . It inhibits the breakdown of cartilage and promotes cartilage repair by inhibiting a number of molecules and pathways implicated in oa (tables 1 and 2). Asu stimulates the synthesis of collagen and aggrecan by inhibiting inflammatory cytokines such as il-1, il-6, il-8, tnf, and pge2 through modulation of nf - kappab . The combination of asu and epigallocatechin gallate (egcg; a major component of green tea catechins) affects an array of inflammatory molecules including expression of cox-2 and production of pge2 in chondrocytes . Cox-2 regulates the production of pge2; both are mediators involved in the process of cartilage breakdown . Asu also inhibits the release and activity of collagenase (mmp2) and stromelysin 1 (mmp3) in cultured chondrocytes, increases tissue inhibitors of metalloproteinases (timp-1), and inhibits il1-induced erk but not p38 or jnk in chondrocytes in vitro . Stimulatory effects of avocado soybean unsaponifiable on anti - inflammatory, anabolic mediators that protect against osteoarthritis . Inhibitory effects of avocado soybean unsaponifiable on inflammatory and catabolic mediators of osteoarthritis . In vitro studies show that asu inhibits fibrinolysis by stimulating the expression of plasminogen activator inhibitor (pai-1). Pai-1 inhibits tissue plasminogen activator and urokinase (upa), thereby blocking plasminogen activation and inhibiting fibrinolysis (the physiological breakdown of blood clots). This fibrinolytic and tissue destructive proteinase cascade may play a role in oa joint inflammation via altered expression of upa receptors . Asus alter growth factor levels implicated in oa pathogenesis, increasing tgf-1 and tgf-2 in the canine knee joint fluid, to repair cartilage and decreasing vegf, which is markedly elevated in synovial fluid of patients . In a study of implant osseointegration in rat tibiae, asu administration improved markers of bone growth, including bone morphogenic protein 2 (bmp-2) and transforming growth factor beta 1 (tgf-1), though histomorphometric analysis of implant osseointegration was only slightly improved . Sixty percent of patients with oa exhibit high levels of oxidized low - density lipoproteins (oldl) in serum, which mediates reactive oxygen species (ros) activity in chondrocytes and oa pathology . Treating patients with a daily dose of 300 mg asu for 3 months decreased oldl levels . At the clinical level, asu reduces pain and stiffness while improving function in joints, resulting in decreased dependence on analgesics . Asu efficacy and safety during and after treatment have been assessed in various randomized, double - blind, multicenter trials in patients with symptomatic knee or hip oa . Two studies conducted over a 3-month period report that standard treatment with 300 mg / day of asu improved indices of pain, stiffness, and physical function, as measured by womac, and decreased analgesic drug demanded in patients with oa . A third trial conducted over 6 months reports similarly improved function compared with placebo, measured by the lequense functional index, with persistent effects after termination of treatment . In a 6-month trial on patients with femorotibial gonarthrosis, asu was as effective as 400 mg of chondroitin sulfate three times per day, as measured by womac . Most recently, a 3-year randomized trial on patients with hip oa, performed under the acr criteria (minimum of 1 - 4 mm hip jsw on the pelvic radiographs), reports excellent safety, but no significant reduction in the mean rate of jsn after 1 year . However, analyzing the results under different parameters reveals a significant 20% reduction in the rate of progression in patients with severe hip oa (p = 0.04), indicating a potential structure modifying effect of asu, as confirmed in the eradias study . In a clinical trial of patients with hip oa, the effects of asu treatment over 3 years were evaluated by radiography to identify joint pathology and disease progression on the structural level . Although jsn was not statistically significant between asu and placebo treatment, secondary analysis of disease progression, measured by jsn (0.5 mm) or total hip replacements, indicated 20% improvement with asu (42.2% vs. 51.4% of placebo group, p = 0.054). Computerized image analysis also showed significant histological differences not detectable by traditional scoring methods . In sheep, asu treatment following cartilage insult improved articular integrity, as measured by toluidine blue staining, after 6 months compared with untreated animals . These improvements were the result of decreased catabolism and increased anabolism of cartilage by asu . Indeed, asu reduces inflammation - mediated cartilage degradation by reducing il-1, pge2, and mmp-3 production, while also inducing proteoglycan, noncollagenous protein (ncp), and collagen synthesis within 72 hours of administration to bovine cells in culture . A recent study in patients with nonspecific dorsalgia demonstrated analgesic effect of piascledine with positive outcome after 1 month however, a randomized, double - blind, placebo - controlled clinical trial carried out in 14 obese adult volunteers over 3 months reports no significant effect on these parameters, as measured by hyperglycemic four double - blind placebo - controlled randomized human clinical trials (rcts) evaluate asu s impact on knee and hip oa . Another found that asu improves lfi compared with placebo over the course of 6 months, and also that improvements took 2 months to take effect, and subside after treatment ended . Alternatively, a long - term study indicated no significant difference in jsn, or other parameters of disease, after 2 years of asu treatment, indicating that the beneficial impact of asu on oa may be limited to short - term effects . However, this study also focused on identifying structure - modifying effects, versus symptom - modifying effects; although these two different measures of oa severity often correlate, asu may affect each uniquely . Evidence for symptom - modifying effects of asu is much stronger, and thus an alternative explanation for these contradictory findings is that while asu does not improve structural damage of oa, as measured in this study, it does improve symptoms such as pain and mobility, as measured in previous studies . In a study of chronic nonspecific back pain, treatment with asu (piascledine) combined with the nsaid artrosiline (320 mg / day) showed significant analgesic effect over nsaid treatment alone ., the eradias trial determined whether asu expanscience treatment slowed the radiological progression of hip oa . As for safety, none of the four rcts reported significant differences in adverse effects between asu and placebo . Factors like bmi, severity of disease, and activity level may influence the effect of asu, as these conditions exacerbate inflammatory conditions and mechanical stresses that contribute to oa . Adipose tissue plays an important role by producing metabolic factors with catabolic and pro - inflammatory properties, including cytokines, chemokines, and adipokines (il-6 and tnf-, il-8, ifn-), which orchestrate pathophysiological processes in oa . Soluble mediators produced by adipocytes may also modulate chondrocyte metabolism and contribute to cartilage degradation . Asu may counteract these inflammatory processes by inhibiting the translocation of the transcription factor nf-b from the cytoplasm to the nucleus, which controls transcription of many pro - inflammatory factors (table 2). As such, asu acts as an anabolic agent in vitro, reducing the production of pro - inflammatory mediators, including il-1, il-6, il-8, macrophage inflammatory protein-1, no, mmp-13, tnf-, and cox2/pge2 from various cell types (table 1). In mice, asu decreases pro - inflammatory interferon- (ifn-) and il-4 production, in the context of parasitic diseases . Although more studies need to be conducted to show the effects of asu in patients with varying bmi, the anti - inflammatory effects of asu are likely to protect cartilage from obesity - associated inflammatory degradation and improve oa symptoms . Indeed, asu significantly decreased the rate of oa progression to 40% compared with 50% in the placebo group in one study . However, this study showed that asu did not influence the rate of oa progression in the obese subset of patients with mean symptom duration 4 and bmi of 27 kg / m . However, excessive inflammation associated with obesity may also impede efficacy, as it does with celecoxib (nsaid) treatment, which is not as effective in obese patients (bmi in excess of 30 kg / m). The influence of obesity, and how it influences asu efficacy, may also depend on the parameters used to measure and define disease . In a study examining the relationship between bmi and oa in patients scheduled to undergo hip replacement, increasing bmi was associated with increasing levels of pain and functional disability, but not radiographic joint damage . This should be taken into account when designing and assessing studies intended to examine the impact of obesity on treatment efficacy . Asu has anti - inflammatory effects in mice when administered in conjunction with the anti - parasitic drug praziquantel, reducing inflammatory cytokines ifn- and il-4, as well as granuloma size, while increasing cidal activity . Asu also protects gingival elastic fibers from degradation by human leukocyte elastase, hypodermatitis, and ischemic damage . A recent electronic database analysis demonstrated the benefits and harms of oral medicinal plant products in treating oa . The authors used standard methods for trial selection and data extraction, and they assessed the quality of the body of evidence using the grade approach for major outcomes such as pain, function, radiographic joint changes, quality of life, withdrawals due to adverse events, total adverse events, and serious adverse events . The asu product piasclidine formed a small and clinically questionable improvement in symptoms, compared with placebo after 3 to 12 months treatment . Radiographic joint changes, as change in joint space width (jsw), did not differ between asu 300 mg treatment and placebo . Moderate - quality evidence from a single study confirmed possible benefits of asu 600 mg over placebo . There is no evidence that piasclidine significantly improves joint structure, and limited evidence that it prevents joint space narrowing . The authors suggest further investigations are required to determine optimum daily doses producing clinical benefits without adverse events . Asu is considered as drug in most countries and is therefore prescribed by physicians . However, in the united states it is classified as dietary supplement and can be purchased as over - the - counter supplements, avoca asu (asu - nmx1000, nutramax laboratories inc ., avoca asu, a combination of asu and glucosamine sulfate, has been shown to suppress tnf-, il-1, cox2, inos, pge2, nf-b activation and nitrite production in articular chondrocytes and monocytes / macrophages, reducing pain and inflammation in oa patients . However, conflicting reports indicate the complete absence of specific asu molecules in avoca asu when compared with piascledine . Questions remain about the efficacy and safety of asus for treatment of oa (table 4). Macaigne and colleagues published a case report in 2004 describing a female with lymphatic colitis associated with piascledine treatment . Further prospective multicenter studies are warranted to investigate whether other microscopic colitis cases are observed in patients treated with piascledine . Avoca asu that contains glucosamine can induce allergic reaction in people with shellfish allergy . Even in very small quantities, these people may experience mild symptoms, such as hives or nasal congestion, or more severe, even life - threatening, symptoms . An alternative asu formulation is arthrocen (pharmin, usa, llc, san jose, ca). Arthrocen is an extract from avocado and soybean oils that does not contain any ingredients of animal origin, artificial flavor, sweetener, preservative, or color . Each capsule contains 100 mg unsaponifiable persea gratissima unsaponifiable (avocado) and 200 mg unsaponifiable glycine max (soybean) extracts, silica, magnesium stearate (e470b manufactured from vegetable oil), and gelatin fines . In general, the fda does not hold dietary supplements to the stringent standards of pharmaceutical manufacture . If asu is to be widely used for the treatment of oa, serious consideration should be given to their current regulatory status in order to ensure potency, purity, and as well as the excipients . Many studies have demonstrated substantial variation between the content listed on the labels of these products and the actual content . The sterols content of asu have been demonstrated to have biological activities in culture and in animal models . 2). We found multiple peaks were present in the piascledine-300 (expanscience) mass spectrometry analysis (agilent 7890 gc system, 7693 auto sampler, agilent vl msd with triple axis detector, brea, ca), compared to the arthrocen 300 mg (pharminusa) or asu300-avocado soy unsaponifiable with sierra 600 mg (maximize, maximum int .) Preparations (fig . Similar results were found for piascledine-300, with mass spectra sterol content of c20h30o2, c20h28o2, sitosterol, stigmasterol, campesterol, squalene, -tocopherol, desmethyl tocopherol, oleic acid docosane, -amyrin, and cholesterol . In two letters to the editor, msika et al . And henroitin claimed that the exact ingredients and preparation of asu - expanscience was an intellectual proprietary, protected by patent . Msika further emphasized that the tocopherols, sterols, and patented specific molecules from avocado contribute to the originality of the product, different from natural avocado unsaponifiables . In contrast, they analyzed dasuquin with msm, dasuquin, and avoca asu (nutramax), and compared them with asu expanscience . The analyses revealed content of products were significantly different from those indicated on the nutramax labels with no citrostadienol, and brassicasterol present in asu expanscience . Contrary to that they found contents included high level of rapeseed oil and unsaponifiables products with very low tocopherol, and without respected ratio of 1:2 for avocado to soybean unsaponifiables . The original asu piascledine 300 pills contains 100 mg of avocado unsaponifiables and 200 mg of soybean unsaponifiables . The difference in sterol content is based on the a / s ratio and avocado - specific modified unsaponifiables obtained by a patented process . Have shown asu effects are best when the asu ratio is 2:1 . In support, these issues and reported adverse effects of asu (table 4) raises concerns about the content and purity of asu supplements on the market, with implications for patient safety . Mass spectrometry analysis of major sterol components of piascledine 300, avocado 300 soy unsaponifiable with sierra 600 mg, and arthrocen 300 . Control sample exhibited characteristic peaks corresponding to 1 = dihydrocholesterol (5-cholestan-3-ol; internal control; sigma aldrich), 2 = brass (brassicasterol), 3 = camp (campesterol), 4 = stigmn (stigmastanol), 5 = -sito (-sitosterol), 6 = stigma (stigmasterol). Improving joint function and patient activity is a central public health concern to improve quality and length of life . The aim is not only to treat pain but also to prevent the onset of disease . There is no cure for oa, and even symptomatic treatment options are scarce, dominated by pain management and surgical intervention . Asus may prove to be an effective treatment option for symptomatic oa, as they have been shown to possess chondroprotective, anabolic, and anticatabolic properties, as well as anti - inflammatory properties . At the clinical level, asus reduce pain and stiffness while improving joint function . Importantly, asus are a natural, slow - acting agent that do not merely address acute pain but actively prevent progression of oa symptoms . Further studies are required to determine the specific mechanisms and target molecules of asu function on oa at the cellular and metabolic level.
In modern society, the fashion of wearing tight clothes for stylish dressed states or comeliness is getting very popular1 . However, specialists reported that excessively pressing certain areas of the human body could cause many problems in the cardiovascular systems and visceral organs1 . In addition to this pressure inflicted on the human body may deform muscles, the skeletal system, and even the overall body type2, 3 . Jeans that are too tight compress a nerve that cuts off sensation to the thighs, and this is consistent with human anatomy and physiology4 . Trousers that are too tight can squeeze a sensory nerve under the hip bone, causing a tingling, burning sensation called paresthesia1, 4, 5 . With this as the background, the present study aimed to understand the hazards of the habit of wearing tightly fitting clothes in relation to deformations of the musculoskeletal system and the movement of the lumbar spine and pelvis for the purpose of providing basic data regarding proper habits in wearing clothes for the prevention of pain in the musculoskeletal systems2, 3 . So the present study evaluated the effect of wearing tight pants on the trunk flexion and pelvic tilting angles in stand - to - sit movement and in a seated posture . This study was performed on nine males aged 2027 years (23.22.0 years, meansd) whose height and weight were 175.13.2 cm and 62.13.4 kg, respectively . Subjects with conditions that might affect trunk mobility, such as injury or neurologic deficits of the hip and lower extremities during the past year, were excluded from study . The study purpose and methods were explained to all the subjects, who provided informed consent according to the principles of the declaration of helsinki before participating . Data were collected at a sampling rate of 100 hz with a motion capture system (vicon mx, oxford metrics, oxford, uk) that consisted of eight infrared cameras . Sixteen reflective markers were attached to the lower body according to the plug - in - gait marker set (oxford metrics) using double - sided tape . The software used for kinematic data collection was nexus 1.4.1 (oxford metrics), and the data were analyzed with the polygon 3.1 software (oxford metrics). The experimental protocol required the completion of two stand - to - sit trials for each of the two pants conditions . The worn pants were made from the same material, cotton, woven into a rugged cotton textile . We used general and tight pants from the same company (g company). The pants were worn under two conditions in this study: (1) general pants, worn with sizes equivalent to 105110% of the subjects hip, thigh, and calf circumferences, and (2) tight pants, worn with sizes equivalent to 9095% of the subjects hip, thigh, and calf circumferences . Each subject was asked to stand up at a self - selected speed from a seated posture and to stand in an erect spine posture . For time normalization, the time required for a complete stand - to - sit movement cycle, that is, from movement onset to completion, was considered to be 100%; values were determined for each 2% of the movement, beginning at 0% . The changes in trunk flexion and pelvic posterior tilting angles were calculated based on the difference between the maximal and initial angles . As the analysis was performed with a withwn - subject design, the paired t - test was conducted to test for differences in pelvic and trunk kinematics values during the maneuver . All significance levels were set at p<0.05, and spss version 12.0 (spss, chicago, il, usa) was used for statistical analyses . The change in the posterior pelvic tilting angle (9.3 4.1) during the stand - to - sit movement when wearing the tight pants increased significantly compared with that when wearing the general pants (7.0 3.3) (p<0.05). The change of the trunk flexion angle (19.3 5.4) during the stand - to - sit movement when wearing the tight pants increased significantly compared with that when wearing the general pants (12.0 3.8) (p<0.05). The change in the posterior pelvic tilting angle (13.3 3.2) in the seated posture when wearing the tight pants increased significantly compared with that when wearing of the general pants (10.3 2.9) (p<0.05). The change in the trunk flexion angle (25.3 6.1) in the seated posture when wearing the tight pants increased significantly compared with that when wearing of the general pants (20.1 5.4) (p<0.05). Park and yoo reported that the tightness of a waist belt might restricts forward movement of the center of mass and that pelvic inclination might be increased as a compensatory mechanism2 . They showed that the wearing a tight belt could interrupt normal lumbo - pelvic coordination, which might contribute to muscle imbalance2 . Elevated abdominal pressure has been shown to cause multidirectional stiffness of the spine5, 6 . A previous study that used a wide belt reported that the intramuscular pressure on the erector spinae could influence spinal stiffness separately from muscle7 . The results of the present study showed that the trunk flexion and posterior pelvic tilting angles during the stand - to - sit movement when wearing tight pants significantly increased when compared with wearing general pants . Also, the trunk flexion and posterior pelvic tilting angles in the seated posture when wearing tight pants significantly increased when compared with those when wearing of general pants . The lumbar and hip rhythm and interaction between the lumbar spine and hip are important kinematic factors that are used not only in experimental research but also in clinical examination8, 9 . Wearing tight jeans induced excessive lumbar flexion during stand to sit movement and in the seated posture . The exaggerated lumbar flexion may overstretch posterior connective tissues, such as the interspinous ligament, apophyseal joint capsule, and thoracolumbar fascia, or increase stress on discs and apophyseal joints5 . Posterior pelvic tilt decreases lordosis via flexion of the lumbar spine, causes posterior movement of the nucleus pulposus, and increases the diameter of the intervertebral foramina5, 8 . Slump sitting reduces the activation of the spinal stabilizing muscles and is associated with increases in loading on the intervertebral disc and connective tissue8, 9 . Therefore, wearing tight pants could produce musculoskeletal disorders via abnormal movement and posture in the lumbar spine and pelvis . So the effects of wearing tight pants need to be investigated in further studies to reveal their direct relationship to musculoskeletal problems . The effects of tight clothes and accessory items on the human body should also be investigated in order to provide basic data for proper use . Further studies will become the starting point for studies regarding the effects of wearing tight clothes and tight fashion accessories on the musculoskeletal system.
In commonly accepted opinion every searching for new, more effective drugs should be rationalized i.e., determined by the low cost and non time - consuming procedures . These procedures are especially useful on the preliminary stage of searching for new chemical structures of potential biological activity (jorgensen, 2004; leeson and springthorpe, 2007; ou - yang et al ., 2012). In general, on this purpose there are employed various correlation qsar methods (dudek et al ., 2006; yang and huang, 2006; shailesh et al ., 2012). However, in particular cases it is more convenient to develop the procedure of selection of the appropriate structures based on more direct and easier interpretatively criteria . It seems that just such a case is a search for effective ligands of 5ht1a, 5ht2a, and d2 receptors since many structural data on their agonist and antagonist as well as the models of these receptors are well - known (klabunde and hessler, 2002; bissantz et al ., 2003; teeter et al ., 1994; chambers and nichols, 2002; homan et al ., 1999). In addition, wide availability of various bases containing a lot of structural data on very active ligands allows to generate pretty accurate pharmacophore patterns (nelson, 1991; bojarski, 2006). Thanks to these all literature data it is possible to estimate the affinity of potential ligand for receptor of interest . The chemical structure of pharmacophore of being selected potential ligand and its affinity to the receptor seem to be sufficiently unambiguous discriminators, on a preliminary stage, in the search for new effective antipsychotics . To verify this hypothesis, the first step includes determination of pharmacophores for two tested compounds of well - known affinity (previously in vitro determined) to the same receptors as well as pharmacophore pertinent to well - known d2 receptor agonists or antagonists and finally comparison of their properties to in vitro binding data . The pharmacophore model of d2 receptor ligands was found on the basis of 15 compounds of high affinity to d2 receptor reported in literature (sowiski et al ., 2011). These two tested compounds were 3-acylamine derivatives of tropane: n-(8-furan-2-ylmethyl-8-azabicyclo[3.2.1]oct-3-yl)-2-methoxybenzamide (compounds i) and n-(8-furan-2-ylmethyl-8-azabicyclo[3.2.1]oct-3-yl)-2.3-dimethoxybenzamide (compound ii) (fig . 1). Their synthesis have been developed and described in the previously published paper on tropane derivatives (sowiski et al . . 1the chemical formulas of compound i and compound ii the chemical formulas of compound i and compound ii the pharmacophores of compounds i and ii were found on the basis of their structures determined by x - ray diffraction method . The ccdc (cambridge crystallographic data centre) numbers of compounds i and ii are: 905689 and 905690, respectively (figs . 2, 3).fig . 3the x - ray diffraction structure of compound ii the x - ray diffraction structure of compound i the x - ray diffraction structure of compound ii the molecular structure of compound i shows an intramolecular hydrogen bond between the o atom of the methoxy group and the nh of the amide function leads to a six - membered ring . The dihedral angle between the least - squares planes of the phenyl and this virtual ring is only 2.50(7). The piperidine moiety adopts a chair conformation . The best plane of the furan ring and the c1/c2/c4/c5 plane make an angle 69.42(9) and the dihedral angle between the planes of the furan and benzene rings is 72.50(8). The compound ii molecule adopts a folded conformation with an angle between the furan and benzene rings of 63.29(8) and between the best plane of the furan ring and the c1/c2/c4/c5 plane of 87.56(9). This conformation is stabilized by an intramolecular n15h15ao25 and c26h26co27 hydrogen bonds . As a result of n15h15ao25 interaction a six - membered ring the piperidine moiety assumes a chair conformation and the substituent at n8 is in an equatorial position . Conformations of both methoxy groups are different . The disposition of these groups with respect to the phenyl ring can be described by the torsion angles c18c19o25c26 of 107.8(2) and c21c20o27c28 of 11.1(3). In consequence, the methyl carbon atom c26 is found to be 1.107(4) out of the phenyl plane, and c28 atom is almost coplanar with this ring . The pharmacophore structure is a reflection template of the geometrical distribution of property centers localized in molecule and determines to large extent its biological activity . It means that even subtle differences in the geometry of structurally similar molecules can significantly impact on their affinity to receptor binding site . The comparative analysis of the studied pharmacophores was intended to find the specific properties and geometrical parameters which are crucial for the strength of binding of potential ligands to the receptors of interest . The second step of the applied procedure devoted to the selection of the potential agonists or antagonists of the studied receptors relies on docking of the reference compounds i and ii to the models of the d2 receptor (sakhteman et al ., 2011). From analysis of in vitro results (table 1) follows that the both studied compounds (i, ii) are very poorly being bounded to 5-ht1a and 5-ht2a receptors . Indeed, the model docking of compounds i and ii to these receptors also showed that such binding cannot take place . The both molecules of compounds i and ii were placed outside the receptor binding pockets . Thus, only docking of compounds i and ii to d2 receptor is detailed analyzed . The most discriminative parameters which distinctly classify the quality of docking are number and strength (equivalently length and geometry) of the hydrogen bonds formed between ligand and specific amino acids not only inside the receptor binding pocket but also, although to a less degree, intermolecular interactions of other types e.g., hydrophobic and edge-to-face.table 15ht1a, 5ht2a, and d2 receptor affinitiesligandreceptor [k(nm)]5ht1a5ht2ad2compound i6,1006,0001,000compound ii3,000744.526.3 5ht1a, 5ht2a, and d2 receptor affinities the used 3d homology model of d2 receptor has been revealed by comparative modeling using the crystal structure of the human 2-adrenergic receptor and the bovine rhodopsin as the templates (sakhteman et al ., 2011; strzelczyk et al ., 2004; the quite recently reported x - ray structure of the human 2-adrenergic receptor opens new possibilities for modeling of the correct structures of the dopamine ones . Currently, the human 2-adrenergic receptor is considered to be more homologous to the dopamine receptors than bovine rhodopsin (cherezov et al ., 2007). All modeling of the pharmacophores as well as docking of the compounds i and ii to the d2 receptor model were done by discovery studio software (accelrys software inc ., discovery studio modeling environment, 2005). Crystals of compounds i and ii suitable for x - ray analysis were grown by slow evaporation from acetate / diisopropyl ether (compound i) and hexane / ethanol (compound ii) solutions . The data were collected on an oxford diffraction km4ccd diffractometer at 293 k, using graphite - monochromated mo k radiation . The unit cell parameters were determined by least - squares treatment of setting angles of highest - intensity reflections chosen from the whole experiment . The structure was solved by direct methods using the shelxs97 program (sheldric, 1990) and refined by the full - matrix least - squares method with the shelxl97 program (sheldric, 1997). The function w(\|fo\| \|fc\|) was minimized with w= [(fo) + (0.0688p)], where p = (fo2 + 2fc2)/3 . = kfc[1 + (0.001fc2/sin2)] (sheldric, 1997) and the extinction coefficient was equal to 0.014(2). The coordinates of the hydrogen atoms were calculated in idealized positions and refined as a riding model with their thermal parameters calculated as 1.2 (1.5 for methyl group) times ueq of the respective carrier carbon atom . Crystals of compounds i and ii suitable for x - ray analysis were grown by slow evaporation from acetate / diisopropyl ether (compound i) and hexane / ethanol (compound ii) solutions . The data were collected on an oxford diffraction km4ccd diffractometer at 293 k, using graphite - monochromated mo k radiation . The unit cell parameters were determined by least - squares treatment of setting angles of highest - intensity reflections chosen from the whole experiment . The structure was solved by direct methods using the shelxs97 program (sheldric, 1990) and refined by the full - matrix least - squares method with the shelxl97 program (sheldric, 1997). The function w(\|fo\| \|fc\|) was minimized with w= [(fo) + (0.0688p)], where p = (fo2 + 2fc2)/3 . = kfc[1 + (0.001fc2/sin2)] (sheldric, 1997) and the extinction coefficient was equal to 0.014(2). The coordinates of the hydrogen atoms were calculated in idealized positions and refined as a riding model with their thermal parameters calculated as 1.2 (1.5 for methyl group) times ueq of the respective carrier carbon atom . The in vitro binding data for compounds i, ii as ligands of 5ht1a, 5ht2a, and d2 receptors are given in table 1 (sowiski et al ., 2011). These experimental binding data unambiguously points at very low affinity of compound i to 5ht1a and 5ht2a receptors and somewhat better to d2 one, yet, compound ii displayed very weak binding activity to 5ht1a, moderate to 5ht2a and very high to d2 receptors . The differences between parameters (geometrical and property types) of the reference pharmacophores and the pharmacophores pertinent to compounds i and ii are expected to reflect the differences in affinity of tested compounds to the receptors of interest . The found structures of pharmacophores described by their specific properties are given on figs . The particular colors denote the following properties: red positive ionization (nitrogen atom), green hydrogen bond acceptor, magenta hydrogen bond donor, pale blue hydrophobic, aromatic, ultramarine hydrophobic, aliphatic, orange aromatic ring (table 2).fig . 4the spatial distribution of pharmacophore properties on a background of compound i x - ray diffraction structure . A green square depicts the plane of a phenyl ring (color figure online)fig . 5the spatial distribution of pharmacophore properties on a background of compound ii x - ray diffraction structure . A green square depicts the plane of a phenyl ring (color figure online)fig . Aliphatic property (color figure online)table 2pharmacophore properties of compound i and iipharmacophore feature / propertycompound icompound iipositive ionization (red)nitrogen atomnitrogen atomhydrogen bond acceptor (hba, green)carbonyl group of amide bondcarbonyl group of amide bondaromatic ring (orange)benzene ring substituted with methoxy groupbenzene ring substituted with two methoxy groupshydrophobic, aromatic (pale blue)furane ringfurane ringhydrophobic, aliphatic (ultramarine)one methyl group in methoxy moiety attached to the benzene ringtwo methyl groups in methoxy moieties attached to the benzene ring the spatial distribution of pharmacophore properties on a background of compound i x - ray diffraction structure . A green square depicts the plane of a phenyl ring (color figure online) the spatial distribution of pharmacophore properties on a background of compound ii x - ray diffraction structure . A green square depicts the plane of a phenyl ring (color figure online) the spatial distribution of pharmacophore properties of d2 receptor ligands . Aliphatic property (color figure online) pharmacophore properties of compound i and ii the geometry of a spatial distribution of pharmacophore properties in obtained models is an exact reflection of the x - ray diffraction structure of compounds i and ii (table 3). It is worthy to note that in spite of the high similarity of chemical structures of these compounds, that their conformations significantly differ each from other . Obviously, it should be taken into account some flexibility of the spatial pharmacophore geometry and possibility of its change during docking of studied compounds to particular receptors . However, such changes are often possible only to small degree or impossible at all on account of the high energetic rotation barriers . In this context, the presence of two separate aliphatic hydrophobic centers in pharmacophore of compound ii takes on a special importance for explanation of very high affinity of this compound, in contrast to compound i, for d2 receptor . It is likely that just second methoxy group in compound ii molecule underlies its high binding to d2 receptor while the same group do not affect the affinity of compound ii to 5-ht1a and 5-ht2a receptors . The comparative analysis of the d2 receptor ligand pharmacophore (fig . 6) and pharmacophores of compounds i and ii also leads to the same conclusion (figs . 4 and 5). The pharmacophore of d2 ligand quite well matches the pharmacophore of compound ii but does not the pharmacophore of compound i (c.f . In addition, specificity of the structural relation between these pharmacophores results from the identical spatial localization of the aliphatic property of d2 ligand pharmacophore and its analog present in pharmacophore of compound ii but absent in pharmacophore of compound i (table 2). Fig . 7superposition of the d2 receptor ligand pharmacophore and pharmacophore of compound iitable 3pharmacophore geometry parameterspharmacophore geometry parameterscompound icompound iidistance between piperidine nitrogen atom and center of the benzene ring7.85 7.76 dihedral angle between benzene ring plane and furane ring plane72.5063.29dihedral angle between piperidine ring (c1/c2/c4/c5) plane and benzene ring plane65.7950.97dihedral angle between piperidine ring (c1/c2/c4/c5) plane and furane ring plane69.4287.56dihedral angle between carbonyl group plane and piperidine ring plane73.5086.72 superposition of the d2 receptor ligand pharmacophore and pharmacophore of compound ii pharmacophore geometry parameters docking of both tested compounds to d2 receptor model turned out to be non discriminative investigation not giving criteria for explanation of difference in ability to the binding of compounds i and ii with d2 receptor . Both compounds docked to d2 receptor interact with its amino acids via the same hydrogen bonds . In case of compound i the hydrogen bonds are: ligand thyrosine 379 (length 2.198), ligand alanine 185 (length 2.315), and compound ii ligand thyrosine 379 (length 2.310), ligand alanine 185 (length 2.139). In addition, both compounds interact similarly with d2 receptor with hydrophobic forces (fig . 8the molecules of compounds i and ii (green) inside binding pocket of d2 receptor . Yellow dashed lines denote hydrogen bonds (color figure online) the molecules of compounds i and ii (green) inside binding pocket of d2 receptor . Yellow dashed lines denote hydrogen bonds (color figure online) the obtained docking results are not unexpected since, purposely, the structurally similar compounds were investigated to point out that even very subtle differences in the chemical structure of compounds, to which docking procedure is insensitive, may impact crucially on their therapeutic activity . Thus, it should be stated that two stages pharmacophore and docking investigations are necessary to estimate properly an affinity of newly designed receptor ligands . On the whole, these studies were intended to prove that postulated two - stages procedure can be applied to verification of the properties of even very similar structurally potential and being designed antipsychotics.
The treatment of lung cancer with radiation has undergone significant improvements over the past decade . First, it is possible to better delineate the target volume through the advancement of imaging such as positron emission tomography (pet) fusion [1, 2]. Second, there has been an increase in the utilization of radiation planning and delivery systems such as intensity - modulated radiation therapy (imrt), stereotactic body radiation therapy (sbrt), and proton therapy that allow more conformal delivery of radiation to achieve dose escalation while minimizing toxicity to normal structures [6, 7]. Finally, the advancement of imaging modalities available during the planning and delivery process has made it feasible to adapt the target volume, both within a given fraction of treatment and between fractions, for such factors as internal motion, tumor response, anatomical changes, and weight loss . With the availability of 4d ct(four - dimensional computerized tomography)based radiotherapy planning and on - board imaging (obi), accurate positioning of the target using daily image guidance may result in many advantages, such as a decreased probability of target miss, smaller setup margins, and less normal tissue exposed to high radiation doses . In addition, novel treatment adaptation algorithms can provide more effective modification of treatment plans to adapt to the changes in a patient's anatomy and organ motion during and/or between (intra- and/or inter-) treatment fractions . Adaptive radiotherapy, and will be the focus of this paper . In this examination of adaptive radiation in the setting of lung cancer, we will assess the impact of this development in sbrt, conventionally fractionated imrt, and proton therapy . We will first define key terms in adaptive radiation, and will then review prior studies assessing the magnitude of intrafractional changes, which we define as changes within a given fraction of radiation, from setup to delivery . This discussion will be followed by the effect of these changes on delivered dose and specific planning techniques that have been utilized to decrease this variation, specifically cone beam computed tomography (cbct) and respiratory gating . We will then focus on interfraction changes that occur during the length of treatment (over a period of several weeks), such as changes in tumor mobility, tumor volume, anatomy, and body weight, and examine specific studies that assess the impact of adaptive planning on improving dose distributions in these setting . Finally, we will appraise future directions of adaptive radiation, including selective dose escalation through the visualization of nonresponsive regions and internal tracking to monitor intrafraction motion in real - time, both of which may be able to allow for further sparing of critical structures while maintaining accurate delivery for treatment of this aggressive disease . 50: . Tumor visible by any imaging modality, to include both the primary tumor and any involved lymph nodes . Lymph nodes greater than 1 cm in size in the shortest axis are generally considered positive for disease, but functional imaging such as pet scanning is critical for target delineation [10, 11]. This region cannot be visualized as a discrete structure with radiographic imaging, and the extent has been defined by surgical series with pathologic correlates, as well as autopsy series . As defined by giraud et al ., based on nsclc surgical specimens, an appropriate gtv to ctv margin for adenocarcinoma is 8 mm, and for squamous cell carcinoma, 6 mm . Another study found that in stage i adenocarcinoma, a margin of 9 mm is sufficient to cover 90% of disease . In terms of mediastinal disease, a gtv to ctv margin has not been rigorously analyzed, but based on an abstract presented at the 2006 meeting of the american society for therapeutic radiology and oncology that found a maximal microscopic extension of lymph nodes is between 0.5 and 8.9 mm, we routinely use margins of 8 mm for nodal disease . Ctv with a margin to account for organ motion; or, in other words, changes in the size, shape, and position of the target during treatment . Delineating the itv from 4d ct images involves assessing the target volume (ctv) on expiratory - phase images and then registering the outline to the images from other phases to create a union of target contours enclosing all possible positions of the target . An alternative method is to create a maximum intensity projection (mip) image by combining data from the multiple ct data sets with data from the whole - breath cycle and modifying the itv by visually verifying the target volume throughout the breathing phases (typically 10). In this process, attention should be paid to irregular breathing and breathing pattern variations during each treatment session and over the entire treatment course, as well as to the effects of these irregularities on the itv margin . Because it is often more straightforward to delineate the boundaries of the gross tumor volume with motion on 4d ct image data sets as opposed to the clinical target volume, we previously proposed the concept of the internal gross tumor volume (igtv), which envelops the gtv motion throughout the whole - breath cycle . In this process, rather than delineating the gtv, expanding to the ctv, and then adding the itv, the gtv is contoured, motion is assessed as outlined above (i.e., through the mip images or through all breathing phases), and this target is expanded to the igtv . Then, the igtv is expanded to the itv (itv = igtv + ctv). This latter method is what is commonly utilized at our institution to account for organ motion in the treatment of nsclc . Ctv plus a margin to account for both organ motion and daily setup . Or, the itv with a margin to account for daily setup . The application and revision of margins for the ptv and itv will be discussed in detail below . Bissonnette et al . Examined cbct images during each fraction of 18 patients receiving sbrt for medically inoperable stage i nonsmall cell lung cancer (nsclc). The cbct images were performed at the beginning, midpoint, and end of each fraction to determine differences in tumor motion amplitude . The authors found that at a mean time of 35 minutes (from the beginning of each treatment to the end), the mean change in tumor amplitude was 0.4, 1.0, and 0.4 mm in the medial - lateral (ml), superior - inferior (si), and anterior - posterior (ap) directions, respectively . These values were not statistically significantly different when compared to the initial respiratory correlated (4d) ct scan, other than in patients in which abdominal compression was used, in which longer times on the couch were hypothesized to increase these differences . In another study, michalski et al . Compared the amount of motion between the three aforementioned directional axes (ml, si, and ap) in 23 patients undergoing 4d ct scans . The authors found that the largest intrafractional extent of motion was in the si direction, with the largest range being 3.59 cm . A similar study from our institution also assessed respiration - induced tumor motion during radiation for lung cancer . This study by liu et al . Assessed 166 tumors in 152 lung cancer patients, 57% of whom had stage iii or iv disease . The authors also found that the largest axis of motion was in the si direction, and that 39% of tumors moved> 0.5 cm in this direction, compared to 1.8% and 5.4% in the ml and ap directions, respectively . Tumor motion was also found to be correlated with the amount of diaphragm motion, the si location of the tumor, and the size of the gtv, with smaller tumors exhibiting a greater degree of intrafractional motion . A study by thomas et al . Demonstrated that mediastinal nodal regions also move substantially with the respiratory cycle, generally posteriorly and superiorly with exhalation, and that inferior nodal stations exhibit a greater degree of motion, such that the same issues with respiratory variation in parenchymal tumors can also be applied to mediastinal disease . Thus, it can be concluded that: (1) inferior tumors move more than superior tumors, (2) the largest axis of motion is in the si direction, and (3) mediastinal lymph nodes are also subject to a significant degree of tumor motion . There are several methods to detect setup error and intrafractional variation during radiation therapy for nsclc . In this technique, two - dimensional images are composed in the treatment position, and the patient is set up on patient anatomical landmarks such as bony anatomy . Historically, megavoltage (mv) imaging had been used, but the images produced with this technique were generally of poor image quality and exposed the patient to high radiation doses . In the past decade, the utilization of kilovoltage (kv) imaging has increased, which has improved image quality and decreased radiation exposure . In this method, a ct image is reconstructed on the treatment table from a set of projection images acquired at multiple angles around the patient . Image reconstruction with this modality differs from conventional ct scans in that rather than a linear array of detectors being back projected to construct a 2d slice, a detector array (e.g., a flat - panel portal imager) is used to reconstruct a 3d data set . (figure 1) it follows that the ct images that are a result of this process are theoretically superior in improving intrafractional error as compared to portal images, and multiple studies have exhibited this improvement, particularly in sbrt where longer treatment times can lead to greater instability and less reproducibility of the setup process . Intrafraction treatment variation has two components in lung cancer: uncertainties in patient positioning and variations in internal motion . Nelson et al . Examined the effect of lung tumor motion and setup uncertainties using implanted fiducial markers . The authors found that systematic and random uncertainties ranged between 4 and 6 mm in all three directions . With daily portal imaging, the authors found in a subsequent study that alignment based on implanted fiducials reduced systematic errors in the left - right and superior - inferior direction each by 3 mm . As a result, daily portal imaging is often utilized to decrease setup error in conventionally fractionated regimens, with a daily setup error of up to 5 mm . Borst et al . Compared cbct setup with portal imaging in daily setup for 62 patients in 524 scans with nsclc, and found that cbct reduced the setup error to less than 5 mm, from 51% of patients with setup errors more than 5 mm to 2% with cbct . Bissonnette et al . Assessed the accuracy of cbct in rt for lung cancer in patients receiving both sbrt for early - stage malignancies and in those patients undergoing conventionally fractionated treatment for locally advanced disease . The couch position was adjusted for discrepancies greater than 3 mm between the initial setup and treatment images . The accuracy of this adjustment was then verified with a second cbct . Without cbct adjustments, positioning errors were found to exceed 5 mm in approximately 55% of patients . However, cbct decreased this error, such that systematic and random setup margin was within 3 mm for 82% of fractions in the sbrt group, and between 76% and 84% of the patients in the conventionally fractionated group . Similar findings were published by grills et al ., who found that cbct image guidance significantly decreased setup margins in sbrt, with calculated precorrection population margins of 913 mm and 1014 mm with a stereotactic body frame and alpha cradle, respectively, while these same margins were 1 - 2 mm and 2 - 3 mm postcorrection, and 24 mm and 25 mm postreatment, respectively . The conclusion of these studies is that consistent cbct imaging can decrease the amount of systematic and random error on a daily basis (to within 3 mm), and thus decrease intrafraction variation . Lung tumors differ from many other treatment sites in that internal motion can account for large variations in tumor position, and as a result, dosimetry . Mechalakos et al . Examined treatment plans for 12 patients receiving radiation therapy for nsclc . The authors found that the dose to 95% of the gross tumor volume (gtv), also known as the d95, changed on an average of only 1.4% when normal breathing effects were incorporated . Therefore, the authors concluded that while the chance of a 10% or greater decrease in d95 was less than 4%, patients with a large degree of respiratory motion could have significant effects, and thus these patients should be identified . A study from the university hospital rotterdam in the netherlands found that with a gtv to planning target volume (ptv) margin of 1.5 cm, approximately 11% of the tumor was not covered in mobile tumors . Engelsman et al . Found that when combined with setup error, respiratory motion reduced the tumor control probability (tcp) by almost 9% (from 50% to 42%) in patients receiving conventionally fractionated radiation to 70 gy in lung tumors . There are several methods in which tumor motion can be taken into account with the delivery of radiation therapy and which are utilized at our institution . First, breathing can be monitored during simulation and the igtv / itv can then be added as a margin to ensure that the tumor is treated adequately throughout all phases of the respiratory cycles as outlined above (e.g., if the tumor is noted to move 1.5 cm during treatment, then a 1.5 cm margin, termed the itv / igtv, is added to account for this respiratory motion . ). If 4d ct imaging is not available at the institution, then an alternative method to account for respiratory motion is through the use of breath - hold spiral ct simulation . In this method, the patient is instructed to hold his breath during simulation, both at end inspiration and expiration . Images are then taken at end - inspiration and end - expiration, such that an itv can be generated by combining the two ctvs from the inspiratory and expiratory scans . A disadvantage to the free - breathing method is that in tumors that have a large magnitude of motion, the amount of normal tissue that is treated can be relatively large, since radiation is being delivered throughout the breathing cycle . Therefore, an alternative method of radiation delivery is to instruct the patient to hold their breath during treatment and activate the radiation while the tumor is in this full inspiratory, fixed position . The disadvantage to this technique (often termed deep inspiratory breath hold, or disb) is that it requires full cooperation of the patient, in that the patient will need to hold their breath for 15 seconds or longer . As an alternative to this method, radiation could be delivered in either a relaxed inspiratory position or in expiration, which is often more reproducible than disb . In patients that are not able to comply with any of these instructions, a third method of delivering radiation in tumors that have a great deal of motion is through a ventilatory - gated approach, during which the radiation beam is coordinated with the respiratory cycle through the placement of externally placed fiducial markers . Or, in other words, the radiation is only delivered at certain phases, most typically full expiration . This type of therapy is generally most useful for tumors less than 5 cm in diameter and for a tumor motion of greater than 1 cm . Figure 2 demonstrates the motion of a tumor in the inferior portion of the lung . It is evident that with timed radiation delivery, a great deal of normal tissue could be spared by reducing the treatment margins . Vlachaki et al . Assessed 10 patients with lung tumors to determine the effect of respiratory gating on dosimetry . Gated images were acquired at full inspiration, full expiration, and at each quartile of respiratory movement . The authors found that gating led to higher minimum target volume doses, and that the v20 was reduced from 35% to 26% for gated plans . The mean lung, heart, and esophageal doses were also lower with gated plans . The authors concluded that gating could improve the dose to normal structures while maintaining target tissue coverage . Underberg et al . Examined 31 patients simulated with 4d ct scans and compared the dose to normal tissue in three different techniques to account for breathing motion in target delineation: (1) standard, population - based margins to account for internal motion, (2) the generation of an itv based on tumor mobility in three consecutive phases, and (3) a ptv generated from respiratory gating . Margins led to unnecessary normal tissue irradiation, and that the risk was best minimized if gating was utilized . A recent study from fox chase cancer center arrived at a similar conclusion, in that 4d ct - based treatment planning maintains target coverage while reducing normal tissue dose . It is important to note that the intrafractional adaptive radiotherapy techniques of cbct and breathing control are not mutually exclusive, but in contrast should be utilized together to optimize the therapeutic ratio, as demonstrated by several studies . Koch et al . Found that the relationship between internal lung motion and skin fiducial motion is complex and unpredictable, and that the ap motion of tumors correlated poorly with skin surface markers . They also found that there is significant intersubject variability . In a follow - up study, liu et al . Found that this variability (and in turn, the target volume margins) could be substantially reduced with the use of respiratory gating techniques . And in a study by nelson et al ., the authors assessed the margins necessary to account for uncertainties in tumor position with respiratory gating, image - guided patient setup, neither, or both . The authors found that utilizing both methods simultaneously allowed for the greatest reduction in margins that completely encompassed the tumor, and therefore concluded that when respiratory motion management is used, it should be used in conjunction with image - guided patient setup in order to reduce the overall treatment margin effectively . Another delivery system of increasingly widespread use is cyberknife therapy, which utilizes a stereotactic guidance system designed primarily for radiosurgery in multiple organ systems . The premise behind this system is that the linear accelerator is mounted on a robotic arm, which can then track the target during treatment . Cyberknife has been reported in single institution studies in the setting of sbrt for lung cancers . For example, le et al . Reported 32 patients with lung tumors treated in a dose escalation study using single fractions, and found a 1-year local control rate of 91% for doses greater than 20 gy and at doses less than 25 gy there was no significant toxicity . Brown et al . Assessed the efficacy of this technique in peripherally located stage i nonsmall cell lung cancers . In a cohort of 31 patients, the authors reported no grade 3 or higher toxicities and 1-year local control rates of 93.2% . Further studies on this technique will continue to define its role in adaptive radiotherapy and the definitive treatment of nsclc . More recently, novalis tx provides stereo x - ray targeting and adaptive gating using exactrac x - ray 6d and snap verification system . Reported 94.4% local control with minimal toxicities with 50 gy delivered in 5 fractions using the novalis / brain lab system . All of these novel systems may provide an optimal treatment for clinical challenging cases such as patients with poor lung function and/or lesions close to critical structures . Several factors change during the course of radiation can affect target and normal tissue dose . These factors include but are not limited to changes in tumor size, alterations in tissue anatomy, variations in respiratory patterns, and reductions in patient weight . In this setting, adaptive radiotherapy refers to repeat assessment of the target volume, either through repeat cbct, 4d imaging, or both . Multiple studies have been performed examining causative factors of interfractional changes and the effect of these variations on dosimetry throughout treatment . Examined 10 patients to determine whether tumor excursion due to respiratory motion was stable when comparing images acquired at the time of simulation with those during treatment . The authors found that while there was interfraction consistency in tumor excursion during treatment, the relationship between the gtv and other anatomic structures between respiratory changes varied with rescanning . The authors therefore expressed caution in relying on surrogate anatomic markers to assess tumor motion throughout treatment . Bosmans et al . Assessed 23 patients with locally advanced nsclc who underwent ct - pet and respiration - correlated imaging prior to treatment, and repeated at the first and second weeks after the start of radiation . The authors observed that while changes in tumor motion were relatively small, there was a great deal of variation in tumor size during therapy . These changes ranged from an increase in greater than 30% to a decrease of the same magnitude . The authors concluded that the changes in tumor size warranted assessment for replanning during the course of therapy . Van zwienen et al . Found that clinically evident regression occurred in approximately 40% of patients undergoing definitive treatment for nsclc, with approximately 10% of patients undergoing> 25% reduction in size by week three and in 24 out of 114 patients by week four . A study from johns hopkins university supported this finding of large variations in the gtv, and thus also recommended an adaptive approach in conventionally fractionated patients . Britton et al . Analyzed the effect of gross tumor volume regression and motion changes during the course of radiation therapy in locally advanced nsclc at md anderson cancer center . The authors found that in 8 patients with weekly 4dct data sets, the tumor volume was reduced by a range of 1571% . There was also noted to be increased tumor mobility in the si and ap directions throughout treatment, without any clear trends in tumor motion . In a follow - up study at the same institution, the authors found that the dose to 95% of the ptv and itv with weekly ct scans changed by approximately 12% and 2.5%, respectively . While the lung v20 and mean lung dose only increased by a mean of 3.1% and 2.2%, respectively, the spinal cord dose changed by an average of 34.3% . These studies together imply that continued assessment of the target volume is recommended and is essential in tumors that lie near the spinal cord . Figure 3 demonstrates the significant reduction in size of a tumor throughout treatment, and the large effect that this reduction has on the dose distribution . A study from denmark demonstrated that anatomical and motion changes persist even with respiratory gating . In a study by juher - nottrup et al ., ten patients receiving 60 gy in 2 gy fractions underwent serial 4d ct scans during treatment . The authors found that the interfractional overlap of lung tumors was only 80%87% when bony landmarks were used with a gating technique, and that this overlap decreased to 70%76% when skin tattoos were present . With mediastinal tumors, it can therefore be concluded that gating does not preclude interfractional adaptive planning in patients with lung or mediastinal tumors . Weight loss can be a factor in changing anatomy and in turn the dose to target volume, such as in the case of a patient that loses a great deal of weight that causes the effective beam path to change . Some investigators have questioned whether weight loss in itself can be a cause of setup error during the course of treatment due to factors such as changes in the position of skin marks . Johansen et al . Attempted to answer this question by evaluating the relationship of interfractional setup errors with body mass index and weight loss . The authors assessed 34 head and neck cancer patients and 20 lung cancer patients who received serial cbct images to evaluate whether there was a change in 3d position between the initial cbct scans and those from the 10th and 20th treatment session . The study did not find a statistically significant correlation between setup error and either patient body mass index or weight loss . Therefore, while it is still recommended that replanning is performed on the basis of weight changes during treatment, interfractional changes as a result of weight loss may be more a function of subsequent anatomical variations rather than true setup error . Similar studies as above quantitating interfractional dosimetric error have been performed in patients being treated with sbrt . Matsugi et al . Examined 4d ct scans for 8 patients being treated with sbrt, to measure interfraction variations in position and the size of target volumes with this technique . In contrast to a conventionally fractionated treatment which is several weeks in duration, the authors found that the size of the gtv did not change significantly during treatment and that variations in motion range and position were also small . . However, the target volume is usually small in sbrt and a large fraction size is delivered in less than 5 fractions; therefore, missing a small volume of target even just in one fraction can cause significant underdosing of the tumor and/or overdosing surrounding normal tissues . We therefore reiterate that we recommend daily volumetric verification for target coverage in sbrt . With the advent of proton therapy, investigators have recently begun to examine the effect of interfractional motion and anatomic changes on dose distribution with this technique . This issue is particularly important with protons because the impact of motion and anatomy changes is more significant in proton compared with photon (figure 3). Hui and chang et al . Acquired weekly 4d - ct scans on 8 patients with locally advanced nsclc treated with imrt . A conformal passive scattering plan was generated for each patient and compared with imrt plan over 7 weeks of radiotherapy . The authors found that normal tissue doses were increased and ctv coverage was significantly compromised in one patient (with a decrease in approximately 8%) with proton therapy but much less significantly in the imrt plan . The authors concluded that proton therapy is more sensitive to motion and anatomy changes compared with photon and interfractional adaptive planning and is indicated in select patients . At our institution, all patients undergo a 4d ct simulation to assess for internal motion . Patients are immobilized with an upper body cradleand t - bar, which has a daily setup uncertainty of approximately 7 mm . At the time of simulation, whose tumor motion is less than 1 cm, a free - breathing technique is typically used, with the creation of an itv or igtv and radiation treatment delivery in all phases of the breathing cycle . If the target volume moves more than 1 cm and the patient can breathe reproducibly, then radiation is either timed with certain phases of the breathing cycle while the patient breathes freely (ventilatory gated technique), or the patient is instructed to hold their breath for at least 15 seconds while radiation is delivered at deep inspiration (disb). We utilize visual and/or audio feedback guidance for patients who can comply with these devices . Patients undergoing standard fractionated regimens are initially set up daily with kv imaging for verification, which reduces the setup margin to 5 mm . All patients are assessed for concordance between bony / skin landmarks and tumor setup . If it is found that these setup parameters are discordant, or that the tumor is changing rapidly, cbct is employed, which reduces the itv to ptv margin to 3 mm . All patients receiving sbrt undergo volumetric verification of set up and motion such as cbct prior to each treatment . Repeat 4d simulations are performed selectively at the physician's discretion in patients with nsclc undergoing conventional fractionation for 6 - 7 weeks and are routinely performed in patients with small cell lung carcinoma undergoing hyperfractionated (twice a day) or dose escalated / accelerated radiotherapy for 3 to 7 weeks . Adaptive 4d replanning is performed if motion / anatomy changes may change the target coverage and/or increase dose to surrounding critical structures . Interfractional adaptive 4d planning is not routinely utilized on patients undergoing sbrt (4 - 10 fractions), as these patients receive daily volumetric imaging . It is notable that it is still unclear if target volume reductions are warranted in the scenario of gtv shrinkage during the course of radiotherapy [5052]. Some physicians advocate an approach in which the target volume remains constant due to concerns for residual microscopic disease . To address these concerns, one option is to deliver at least 50 gy, the standard dose for microscopic disease, to the original target volume, and then to boost the reduced volume to the full dose . New technologies are evolving to improve adaptive radiation therapy, such that the high dose region is focused on the target while sparing critical normal tissues . Treatment planning based on 4d ct images and on - board image - guided adaptive treatment delivery assists the radiation oncologist in tracking tumor motion and targeting the tumor precisely . As advancements in technology have allowed for both intra- and interfraction replanning, investigators have begun to assess whether or not the response of tumor during treatment can lead to changes in total dose to portions of the target volume based on the magnitude of tumor response to the initial phase of treatment (image - guided adaptive treatment). This strategy is particularly relevant because several studies have shown that dose escalation in lung cancer improves local control . Furthermore, prior studies have demonstrated that regions with high suvs and hypoxic areas are more likely to exhibit local failure when treated with radiation [54, 55]. The information thus suggests that the radioresistance and propensity for distant metastasis in gross disease varies based on factors other than histologic type, stage, and tumor grade . It is in this realm that adaptive dose escalation may have a role in addition to the utility of imrt dose painting . Performed a pilot study in which 14 patients with nsclc underwent repeat pet - ct scans prior to the start of radiation therapy and mid - way through treatment . Boost fields were then designed based on residual pet avidity . In this dosimetric study, the authors found that this method allowed for a mean dose escalation of 58 gy or a reduction in normal tissue complication probability (ntcp) of up to 3% in patients with a reduction in tumor size . In a similar study, gillham et al . Performed an additional pet - ct scan in week 5 or 6 of radiotherapy for patients being treated with localized inoperable nsclc to 66 gy in 2 gy fractions . The authors found that there was a median ptv reduction of 20%, and that in this dosimetric study, dose escalation to 78 gy was feasible in four out of ten patients without exceeding normal tissue constraints . Future studies will determine whether this technique of adaptive dose escalation is feasible from a clinical standpoint without increasing toxicity and while maintaining tumor control . An additional technologic advancement in the context of adaptive radiotherapy is the development of real - time tracking of tumor motion so that the treatment can be actively and variably adapted in concordance with intrafractional changes . In addition to cyberknife, which has been used clinically as discussed above, another commercially available system is the calypso medical three- or four - dimensional electromagnetic tracking system . The system has undergone initial assessment in a wide variety of tumor sites and several studies have been published describing the technique . For example, smith et al . Compared dosimetric results with and without real - time tracking . The authors found that the dose profiles were comparable with an idealized gating algorithm while minimizing the uncertainties inherent in the use of anatomical surrogates for target location, with a high level of efficiency . In a follow - up study, the same authors proposed a method for linear accelerator gating with wireless internal fiducial markers without the requirement for ionizing radiation for imaging, leading to improvements in the dose distribution . Adaptive radiotherapy may improve tumor control in lung cancer by reducing target misses, escalating target dose, and minimizing side effects by avoiding critical structures over the course of radiotherapy.
Lipomas are benign, well circumscribed, expansile connective tissue neoplasms predominantly composed of mature white fat cells . They may occur anywhere in the body and usually present as slow - growing, solitary and asymptomatic subcutaneous or superficial lesions . About 20% of cases of lipoma affect the head and neck region with only 1%-5% of these neoplasms involving the oral cavity . Histologically, lipomas are composed of mature adipocytes arranged in lobules that are separated by fibrous connective tissue septa and are occasionally associated with one or more secondary mesenchymal elements . Different variants of lipoma have been described, such as fibrolipoma, angiolipoma, myolipoma, spindle cell lipoma, chondroid lipoma, chondrolipoma and osteolipoma . The buccal mucosa is the most affected intra oral site; and lipomas and fibrolipomas are the most frequently observed histological types in the oral cavity . Among the histopathological variants, lipomas with cartilaginous or osseous metaplasia, called chondrolipomas or osteolipomas respectively, have been described in the subcutaneous and deep soft tissues, particularly in the parosteal localization but are rare in the oral cavity . As per pubmed database, till date only 14 cases of chondrolipoma in oral cavity have been reported in english literature . Herein, we report a case of this rare intraoral variant in a 35-year - old male patient . A 35-year - old indian male patient presented with a painless mass on the postero - dorsal region of tongue since last 20 years . The lesion showed slow growth and the patient reported that there was no increase in size for past many years . Clinically, the lesion was an oval, well - circumscribed nodule measuring approximately 1.0 1.0 cm . On palpation, the mass was firm in consistency with no tenderness or discharge [figure 1]. A clinical diagnosis of fibroma was made and based on the long standing history and benign appearance, an excisional biopsy was performed . The surgical area healed uneventfully and six - month follow - up of the patient revealed no evidence of any recurrence . Figure shows nodular growth on the posterior dorsal surface of tongue gross examination of the excised mass revealed a pale yellowish cut surface with foci of shiny white areas [figure 2]. Microscopically, the tumor was composed of well circumscribed, encapsulated lobular mass of mature adipose tissue separated by fibrous septae . Foci of metaplastic hyaline cartilage were evident within the mass along with delicate capillaries [figure 3]. The cartilage cells as well as the fat cells did not show any mitosis, pleomorphism or any other histological evidence of malignancy . No evidence of lipoblasts or hibernoma - like cells was seen [figure 4]. Note the yellowish color of the cut surface of the lesion with foci of white hyalinized areas (arrows) low power photomicrograph showing stratified squamous epithelium overlying a well - circumscribed mass of adipose tissue, surrounded by a fibrous capsule and containing a focus of hyaline cartilage . (h&e stain, 40) high - power photomicrograph showing cartilaginous foci made up of mature normal appearing chondroid tissue with peripheral rim of spindle shaped lining cells . The first case of oral chondrolipoma was reported by mcandrew and greenspan in 1976 in a 72-year - old male in lower lip and since then only a handful of cases been reported in oral cavity . Many clinical and histopathological characteristics of chondrolipomas of the oral cavity continue to be poorly understood because of the rarity of this tumor, together with the lack of data in the literature . A review of the pubmed database revealed 14 such cases in english literature . We have included only those cases which presented strictly within the oral cavity and histologically showed presence of only cartilaginous tissue in a predominantly mature adipocyte proliferation the clinicopathological features of these cases including the present case have been summarized in table 1 . Clinical features of 14 cases of oral chondrolipoma described in english literature cases of oral chondrolipoma have been diagnosed in patients aged from 14 to 72 years, but it seems to be a tumor of older individuals with majority of cases being diagnosed after the age of 50 years . While these lesions are usually diagnosed in older adults, they may originate at a younger age as some of these tumors including the present case have shown long duration of presentation . There may be an actual bimodal age predilection as substantial number of cases (almost one - third including the present case) seem to originate in the first two decades of life but a larger number of cases will be required to accept or refute this observation . This feature of chondrolipoma slightly differs from a conventional lipoma, which are rarely seen in the first two decades of life and are thought to make their appearance at the age when fat starts accumulating in inactive individuals . Hence, most lipomas including those of oral cavity become apparent between 5 to 7 decades of life . In fact all of the cases of chondrolipoma involving young individuals have been reported in recent literature probably highlighting the fact that awareness about this variant is increasing among histopathologists and possibly some of these lesions may have earlier been overlooked . Similar to other lipomas at other sites, oral lipomas are believed to be more common in men and this seems to be true even for chondrolipomas in the oral cavity . Oral chondrolipomas seem to have a strong predilection for occurrence on tongue but interestingly none of the lesions have been described in buccal mucosa which is the preferential site for conventional lipomas . Histologically, variants of lipoma differ from ordinary lipoma by characteristic microscopic picture and specific clinical setting . This group is chiefly represented by angiolipoma, myolipoma, angiomyolipoma, myelolipoma, chondroid lipoma, spindle cell and pleomorphic lipoma . Chondro lipomas are characterized by the proliferation of mature adipocytes with additional mature cartilaginous tissue formation . Though this term has been used in past, it was first defined by stout for tumors that are composed of two or more mesenchymal elements . On the other hand,, described mesenchymomas as an unencapsulated soft tissue neoplasms composed of two or more mature mesenchymal tissues not normally associated with each other and no single mesenchymal tissue should predominate with respect to the other mesenchymal elements . Usually the cartilage in lipoma represents only a small part of the tumor and hence would not strictly fit to the above criteria . Mature cartilaginous areas in a chondrolipoma should be distinguished from chondroid lipoma, a lipoma variant that is uncommon in the oral cavity and consists of mature adipocytes admixed with multivacuolated lipoblast like cells in a myxohyaline and chondroid matrix . This newly described lesion, having an immature aspect may give a pseudosarcomatous appearance and may be mistaken for lipoblastic or chondroblastic malignancies . On the other hand, in true chondrolipoma the adipose component is entirely composed of mature tissue with lack of any lipoblastic cells . Due to similarity in nomenclature, the two entities are often confused with each other and it is possible they may have been diagnosed interchangeably in past . In this review, we have included only those cases which show typical features of a chondrolipoma and all cases showing immature lipoblast like component or hibernoma - like areas have been excluded . In addition, chondrolipoma may sometimes need to be differentiated from extra - skeletal chondroma specially those occurring in deep submucosal areas and hence surrounded by adipose tissue . Histologically, chondromas are characterized by greater proportion of cartilaginous tissue arranged usually in distinct lobular pattern . Whereas the chondroid component of chondrolipoma is focal and lacks any lobular arrangement . Several hypotheses have been tried to explain the occurrence of cartilage within the mass of adipose tissue . One of the possible explanations could be that this tumor represents a true mesenchymoma i.e. Both the chondroid as well as the adipose components being neoplastic in nature . The initial neoplastic change may take place in the pleuripotent mesenchymal stem cells, present in oral mucosa and these neoplastic stem cells may then differentiate into adipogenic and chondrogenic cells . Presence of pluripotent mesenchymal stem cells, capable of such differentiation, has been demonstrated in the connective tissue of skeletal muscles and dermis and it may be prudent to assume that similar cells may also be present in the lamina propria and submucosal tissues of oral cavity . Ability for multipotential differentiation including chondrogenic potential has been demonstrated in adipose tissue derived stem cells . Hence, it could be possible that some of the neoplastic adipocytes may differentiate into chondroblastic cells, the stimulus being either spontaneous or a metaplastic reaction triggered by long - term irritation . Similar metaplastic chondrogenesis has been described in peripheral fibromas of gingiva . And may be attributed to chronic mechanical stress . An interesting, albeit a little farfetched possibility is that the neoplastic adipose tissue may develop in a pre - existing cartilaginous choristoma . Such choristomas, though rare, are known to occur in the oral cavity with tongue being the most common location . This may also explain the rarity of this lesion and preferential occurrence in tongue as compared to the higher propensity of involvement of buccal mucosa for conventional lipoma . Finally, the lesion may not be neoplastic at all and may result from a combination of hamartomatous and choristomatous growth . Such combined lipomaotus hamartomas and choristomas made up of mature adipose tissue containing cartilage have been described in other sites of the body as well as in inbred laboratory mice . It was suggested that those cases containing additional tissue elements in these chiefly lipomatous growths should be considered choristomas . Even in the murine lipomatous growths additional tissue elements were found more frequently in long standing cases and it was considered that these additional elements like cartilage could be present at an earlier stage but may constitute only a minor volume of the lesion, hence could be easily overlooked and thus require careful complete serial sectioning of the entire growth . Recently, role of factors like sox-9, runx-2, tgf-, bone morphogenic protein (bmp) has been discussed in chondrogenic potential of lipomas . We believe that oral chondrolipomas constitute a heterogenous group of lesions, some of which may represent true neoplasms while others, especially those appearing at a younger age, may be hamartomatous / choristomatous proliferations . The pluripotentiality of adipose derived stem cells and preferential expression and interplay of prochondrogenic molecules either through some genetic alteration or environmental factors are responsible for chondrogenesis in these lesions . Whatever the pathogenesis, lesion is essentially benign and surgical excision is the treatment of choice . The cartilage found in the tumor most likely represents a metaplastic change or harmartomatous / choristomatous proliferation and could be attributed to multipotentiality of mesenchymal stem cells . The characteristics of oral lesions tend to show few deviations from the classical oral lipoma, chiefly being the marked predilection for involvement of tongue and possibly bimodal age of onset and warrants reporting of such cases with detailed clinicopathological evaluation in future.
A retrospective chart review was performed for cases of infectious scleritis seen from january 2007 to august 2011 . The diagnostic criteria for a case of infectious scleritis included the presence of single or multiple inflamed anterior scleral nodules or ulcerations, which revealed a causative organism on microbiological culture . Microbial cultures were considered significant if growth of the same organism was present on more than one solid medium or if there was a confluent growth on inoculation on one solid medium or if growth of one medium was consistent with direct microscopy findings . Data regarding patient demographics, predisposing factors, causative organisms, and clinical features for all 12 patients was extracted . All patients except one were followed up for a period of at least 6 months and, therefore, the treatment and outcome details were analyzed for 11 patients . Data was analyzed by the chi - square test using spss version 11.0.1 to determine the prognostic factors . Twelve eyes of 12 patients were identified to have culture - positive infectious scleritis . Demographic data and clinical features preceding surgical procedures performed included manual small - incision cataract surgery (4), pterygium excision (1), and trabeculectomy (1). None of the patients had a history of fever, joint pain, respiratory illness, skin rashes, etc ., one patient (case 2) had progeria, another had chronic liver disease (case 6), and 2 patients had diabetes (case 8 and 10). Patients with multifocal scleritis were associated with growth of either gram - negative bacilli (gnb) or nocardia on culture an example of which is shown in fig . 2a and b. demographics, clinical features, organisms, management, and outcomes of infectious scleritis (a) multifocal scleral abscesses owing to pseudomonas sp . (b) healed scleritis with minimal scleral thinning 2 months after treatment with topical fortified antibiotics and dexamethasone (case 11) (a) nocardia asteroides: gram stain showing filamentous forms fragmented into bacillary and coccoid forms (magnification = 1000). (b) nocardia asteroides: orange coloured, centrally heaped, dry colony all patients received topical antimicrobials based on the culture and sensitivity report . Seven patients were also treated with topical steroids due to severe intra - ocular inflammation . In addition to medical treatment, 5 patients underwent various surgical procedures in an attempt to decrease the load of infective organism or to preserve the globe as shown in table 1 and fig . Of these, only 2 patients had a good final visual acuity (better than 6/18) on the last follow - up visit . Six months after corneo - scleral patch graft following infectious scleritis post - cataract surgery (case 8) we determined the prognostic factors of infectious scleritis using the chi - square test . A poor visual acuity at presentation (less than counting fingers at 1 m) usually resulted in a worse visual outcome (p = 0.005). The causes of poor visual acuity at last follow - up include corneal scarring following keratitis (1), failed corneal graft (1), secondary angle closure glaucoma and optic atrophy (1), choroidal detachment (1), and endophthalmitis (5). The visual outcome was not dependant on any particular organism (p = 0.099) or the presence of multifocal abscesses (p = 0.209). The addition of surgical intervention did not result in a significantly better visual outcome than medical management alone (p = 0.209), but resulted in a higher rate of globe preservation (p = 0.045). A clinical suspicion of infectious scleritis in all cases of scleral inflammation following trauma has been emphasized previously . However, in our series, 4 cases were preceded by cataract surgery and only 1 by pterygium surgery . Another case series of indian eyes also showed maximum number of their cases had cataract surgery prior to an episode of infectious scleritis . This may be due to the large number of scleral - tunnel small incision cataract surgeries performed in india as compared to clear corneal phacoemulsification performed in developed countries . Pseudomonas aeruginosa is the leading cause of microbial scleritis in reported literature . In our series, 33.33% had gnb, 25% had nocardia, and 25% had fungal infection . One of the fungi was identified to be rhodotorula spp, which we have reported elsewhere . In cases of scleritis following trauma where infection is suspected but routine microbial culture is negative, polymerase chain reaction tests may be used to confirm the diagnosis . If these tests are also negative, the possible diagnosis of surgically induced necrotizing scleritis (sins) should be considered . Sins is a believed to be a delayed - type hypersensitivity response to any form of surgical trauma . Up to 90% of patients who develop sins may have an undiagnosed systemic vasculitis and, therefore, a detailed systemic investigation is warranted . This is often seen as late as 9 months after the surgical intervention and, after ruling out an infectious cause, requires treatment with intensive systemic immunosuppression . When infectious scleritis occurs long after surgery, it has been hypothesized to be due to an underlying sins with secondary microbial infection, which can make diagnosis and treatment difficult . These patients may benefit from a systemic evaluation to rule out an autoimmune vasculitis, which will require appropriate management . Some presented with scleritis and developed secondary intraocular spread, which has been described in other reports . Others had scleritis, uveitis, and endophthalmitis at presentation (e.g., case 7 where the sclera in the area of the bleb was necrotic with vitreous inflammation). The wide use of anti - metabolites at the time of trabeculectomy increases risk of scleral melt and inflammation, which may have resulted in secondary infection and endophthalmitis . Pseudomonas is most often associated with this presentation, which has been attributed to an intra - scleral dissemination facilitated by its proteolytic products and motility . Our series had 6 cases of multifocal scleral abscesses, 3 of which were due to p. aeruginosa, 2 due to nocardia, and 1 due to klebsiella sp . A review of past literature suggests that multifocal scleritis can be caused by several organisms and may be related to factors such as the load of infection, rather than specific organisms . The visual acuity at presentation is the most important prognostic factor determining the final visual acuity, and this has been seen in other studies . This suggests that early diagnosis should help preserve the patient's remaining vision if effective treatment is initiated . There is a lot of discrepancy among researchers regarding the effect of surgical debridement on the outcome of infectious scleritis . A large series showed that despite adjuvant surgery in 46 of 56 eyes, approximately 50% lost functional vision . However, unlike other studies, we did not find any difference in the final vision between the medically and surgically treated groups . In conclusion, manual small incision cataract surgery is a common predisposing factor to infectious scleritis in developing countries . The visual acuity at presentation is the most important prognostic factor determining the final visual outcome . Aggressive surgical intervention improves the chance of globe preservation but does not improve the visual outcome.
Lipoma is a common benign soft tissue neoplasm that sometimes may have mixed tissue components . Ossification of a lipoma was first described in 1959, and it is rarely reported7,16). Several names have been used to describe ossification of lipomas, including secondary calcification, ossified lipoma, ossifying lipoma, or osteolipoma, and some authors have used these terms interchangeably . As with classic lipomas, lipomas with ossifications may be found in any part of the body, but are usually found in the head, neck, oral cavity, and extremities, adjacent to bone . Only two cases of osteolipoma arising in connection with vertebrae have been described . Here, we present a case of an intramuscular osteolipoma on the posterior neck, independent of vertebrae, and review the clinicopathologic features of osteolipoma previously published in the literature . A 51-year - old female presented with a 5-year history of a painless, progressively enlarging mass on the posterior neck . Physical examination revealed a soft, non - tender, uniform mass without surface abnormalities . Computed tomography (ct) and magnetic resonance imaging (mri) showed a circumscribed mass involving the posterior neck muscles in the midline between the c2 and c6 spinous processes with a large calcified component (fig . The ovoid mass was measured 4 cm in width, 6 cm in length, and 3 cm in height, without infiltration of the cervical spine . Subsequently, the patient underwent a whole body bone scan, which revealed an amorphous calcification of the posterior neck without metastasis (fig . The mass had a well - demarcated margin and the outer wall was very firm without adhesion to ligaments or paravertebral muscles . Gross examination revealed a well - circumscribed mass composed of adipose and bony tissue, with red bone marrow visible on the cut surface (fig . 3). Histological study showed a lipomatous lesion with large foci of osseous metaplasia at the periphery of the mass; the bony portion was surrounded by mature adipose tissue (fig . Microscopic examination of the red bone marrow showed a meshwork of bone trabeculae and hematopoietic marrow elements (fig . The patient was diagnosed with an intramuscular osteolipoma of the posterior neck and no recurrence was observed at the 6-month follow - up . A lipoma is a common soft tissue neoplasm, but lipomas with distinct osseous components are rare2 - 4,8,10 - 12,14,15). As noted by heffernan et al.11), in allen's review of 635 cases, less than 1% of lipomas were ossified . Further, as reported by de castro, et al.5), when furlong analyzed histological findings from a group of 125 lipomas of the oral and maxillofacial region, there was no variant that presented as osseous metaplasia . Lipomas presenting with osseous components have been reported as secondary calcification of lipoma or ossified lipoma4,12,14). Secondary calcification of lipoma involves calcification that progresses from the periphery inwards as a result of necrosis after impaired blood supply or repeated microtrauma5,14). An ossified lipoma is defined as a histologic variant of lipoma that has undergone osseous metaplasia, and can be classified into 2 groups according to the predominance of the osseous component: ossifying lipoma and osteolipoma . If the adipose component of the lipoma is more dominant than the osseous component, it is an ossifying lipoma, whereas an osteolipoma presents with a dominant osseous component14). Osteolipomas have been reported in middle aged or elderly patients presenting with large painless masses with a long duration, even years, and they may be found incidentally2,7). They have been reported at various sites located adjacent to bone or periosteum, including upper and lower extremity, oral mucosa, soft tissue of neck, and intracranial regions, including tuber cinereum, hypothalamus, suprasellar cistern, and the interhemispheric area1 - 5,7,10 - 15,17,18). One patient was an 8-year - old female who presented with a progressively enlarging swelling over the lumbar region with a dermal sinus tract12), and other was 20-year - old female with a cervical osteolipoma contained within the spinal canal14). Presented here, the osseous lipoma was located in the deep neck musculature, independent of bony vertebral structures . There are several hypotheses regarding the pathogenetic mechanisms that influence the osseous metaplasia of lipomas, but the details are still not clear . First, these tumours may originate from multipotent mesenchymal cells that can differentiate into lipoblasts, chondroblasts, fibroblasts, angioblasts, and osteoblasts . This could account for the several lipomatous variations of classic lipomas, i.e. Lipoblastoma, lipomatosis, fibrolipoma, angiolipoma, myolipoma, myelolipoma, hibernoma, and atypical lipoma, and mesenchymoma is defined as a rare soft tissue lesion that is composed of fibrous tissue associated with two or more types of well differentiated mesenchymal cells that would not normally be found in the same area 2,7). Second, ossification may also have been induced by poor nutritional supply in the centre of a large lipoma after repetitive trauma, metabolic changes, or ischemia leading to transformation of fibroblasts into osteoblasts2,8). Fritchie et al.8) reported cytogenetic analyses of three osteolipomas and reported that the translocations in all cases were consistent with the karyotypic features of lipoma . Other benign tumors that may contain bone including teratomas or dermoids, masses with secondary ossification due to trauma, liposarcomas with metaplastic changes, or congenital malformations, should be considered in the differential diagnosis . In addition, tumour calcinosis, calcification in a bursa, and other conditions such as ossifying fibromas, myositis ossificans, and osteosarcomas should be taken into consideration . The use of ct scanning provides excellent visualization of the calcified or ossified components of a lipoma and confirmation of proximity to adjacent bone, and mr imaging can also provide detailed information that is useful for further evaluation2,9). Definitive diagnosis of osteolipoma can be made with histopathologic findings after surgical excision, which is usually the recommended treatment . A histopathologic appearance of diffuse, mature ossification within adipose tissue and gross features demonstrating a dominant osseous component confirm the diagnosis . Histologically confirmed osteolipomas are benign neoplasms, as with classic lipomas, and do not recur2,4 - 6,11). To the best of our knowledge, it is the first case report of an intramuscular osteolipoma within the posterior neck independent of adjacent bone . We suggest that although osteolipoma is a rare variant, it is important to keep it in mind when a soft tissue mass of the posterior neck mixed with osseous component is encountered.
The most appropriate way to diagnose airway obstruction is currently the subject of heated debate.110 most national and international chronic obstructive pulmonary disease (copd) guidelines recommend to use a forced expiratory volume in one second / forced vital capacity ratio (fev1/fvc) of 0.70 as a suitable threshold value to define the presence of an obstructive ventilatory defect . Commonly used guidelines include those of the global initiative for obstructive lung disease (gold),11 the american thoracic society / european respiratory society,12 the british thoracic society,13 the canadian thoracic society,14 and the national institute for health and clinical excellence.15 however, there is evidence that the fixed cut - off value of 0.70 for fev1/fvc becomes less specific in males aged> 40 years and females> 50 years, implying a risk of overestimation of airway obstruction . This is due to an age - related decline in pulmonary volumes, particularly in fev1, which is observed even in healthy people with no history of exposure to noxious particles or gases.1619 proposed strategies for reducing the misclassification of airway obstruction include use of the lower limit of normal for fev1/fvc, calculated as the fifth percentile of the normal distribution in a healthy population,2022 or the use of different fev1/fvc thresholds for different age groups (eg, 0.70 for subjects aged <70 years, 0.65 for those aged 7080 years, and 0.60 for those aged> 80 years).19 on this basis, a group of colleagues involved in respiratory research and/or the diagnosis and treatment of lung diseases recently wrote an open letter to members of the gold committee inviting them to change the method by which airway obstruction is defined and asking for retraction of the fixed ratio in favor of the lower limit of normal.1 the aim of the present study was to provide additional information for determination of the most appropriate spirometric criteria for confirming airway obstruction in the elderly, by describing lung function and calculating the lower limit of normal for fev1/fvc in healthy nonsmoking elderly subjects (age> 65 years) who participated in the italian multicenter sara (acronym of salute respiratoria nellanziano, italian for respiratory health in the elderly) study . The degree of potential misclassification relative to use of 0.70 or other proposed fixed thresholds for fev1/fvc was also evaluated . We performed a cross - sectional analysis of data from the sara study, the design of which, along with technical characteristics of instruments as well as training of operators and results of quality control of spirometry, have been described in detail elsewhere.23 briefly, the study involved 24 pulmonary or geriatric institutions distributed throughout italy . A total of 1971 subjects aged 65100 years were recruited as consecutive outpatients referred between january 1996 and december 1997 to the participating centers (see appendix). Lung function was measured using an identical fully computerized water - sealed stead - wells spirometer (baires system, biomedin, padua, italy) by specifically trained and certified personnel supervised by a rigorous real - time control of acceptability and repeatability23 according to american thoracic society recommendations.24 fvc maneuvers were performed with the patient sitting . The fev1/fvc ratio was calculated on the basis of the highest values of individual parameters obtained in the acceptable curves for each subject . Analyses were conducted only on patients with good repeatability of the above - mentioned indices (difference between two best values <150 ml).25 of the 1870 subjects who performed spirometry, we selected only those without any previous or present diagnosis or any sign or symptom suggestive of respiratory diseases according to the modified international union against tuberculosis and lung disease bronchial symptoms questionnaire.25,26 current smokers and previous smokers with a smoking exposure> 5 pack / year were excluded, since the <5 pack / year smoking exposure was not significantly associated with decreased lung function.27 additional exclusion criteria were: severe hepatic failure; severe renal failure; severe cardiac failure; cognitive and/or sensory impairment severe enough to affect a multidimensional assessment; severe kyphoscoliosis with occiput wall distance (distance between the occiput and the wall when the patient stands with heels and shoulder against the wall with the back straight)> 10; occurrence of a major psychosocial event (eg, bereavement) within the past 6 months; and hospitalization for any reason within the past 6 months . We further excluded individuals who had hypertension (diastolic pressure 90 mmhg and/or systolic pressure 160 mmhg), diabetes, and/or major electrocardiographic abnormalities . Statistical analyses were performed using spss (spss inc, chicago, il) and stata (stata corporation, college station, tx) software packages . Regression models were used for testing the relationship between spirometric indices and anthropometric variables . Based on the recommendations from the american thoracic society / european respiratory society task force,22 the lower limit of normal for fev1/fvc was estimated as the fifth percentile of its frequency distribution . To evaluate the effect of aging on spirometric measures independently of body height, fev1 and fvc were normalized for height at the third power.28,29 after applying the above - mentioned selection criteria, our final data set consisted of 367 healthy, nonsmoking subjects . One hundred and one subjects were excluded for lack of availability of lung function testing, a further 709 subjects because of a history of respiratory disease, 445 for significant smoking exposure (> 5 pack / year), 262 for inadequate quality of spirometry, and 87 for the above - mentioned additional exclusion criteria . Tables 1 and 2 show anthropometric and functional data for the sample and distribution of the participants according to age and gender . Although the most advanced ages were less represented, a total of 73 subjects aged 80 years and over were included . The sample consisted of 314 never - smokers (85.6%) and 53 former - smokers with a smoking exposure from 0.15 to 5 pack - years (mean standard deviation, 2.75 1.5). As shown in figure 1, the values of fev1/fvc showed a normal frequency distribution . The mean fev1/fvc was 0.75 0.6 in males and 0.78 0.6 in females, whereas the corresponding fifth percentiles were 0.65 and 0.67, respectively, in males and females . Fev1 and fvc significantly decreased with age (r = 0.38 and 0.35; p <0.001), while fev1/fvc, was not significantly correlated with age or height in either gender group over the considered range of age (lowest p = 0.103). As a consequence, there was no rationale to develop reference equations for fev1/fvc, with age or height as independent variables in this restricted range of age . Figure 2 shows the decline in fev1 and fvc with increasing age observed in the study sample in men and women, after normalization for height . In the sample of healthy subjects, 15% had a fev1/fvc <0.70, and would have been inappropriately classified as obstructed according to gold criteria . Table 3 describes the proportion of participants with the ratio <70 in the different age groups . By applying the fev1/fvc thresholds proposed by hardie et al19 for different age groups (ie, 0.70 for <70 years, 0.65 for 7080 years, and 0.60 for> 80 years), the percentage of obstructed subjects decreased to 6% (men 11%, women 4%). In particular, the proportion of subjects aged 6570 years with a ratio below 0.70 was 11% (men 16%, women 8%); the proportion of subjects between 70 and 80 years of age with the ratio below 0.65 was 5% (men 12%, women 2%), whereas none of the subjects aged 80 years or more had fev1/fvc <0.60 . This study provides additional evidence helpful for determining the most appropriate spirometric criteria to define airway obstruction in elderly subjects . The fifth percentile of fev1/fvc observed in the considered sample of healthy subjects aged> 65 years was lower than 0.70 in both men and women, thus confirming the inadequacy of this threshold for fev1/fvc after the age of 65 years . Moreover, the findings of the present study suggest that, in such a population, fev1/fvc <0.65 and <0.67 (for males and females, respectively) could represent valid criteria that are simple to use and incorporate the known physiological decline in lung function with aging . The present findings are in agreement with observations made by other authors,1618 who have emphasized that the use of the fev1/fvc threshold proposed by gold leads to a risk of overdiagnosis of copd in geriatric subjects . Furthermore, the current results suggest that the inaccuracy of using a threshold of 0.70 for fev1/fvc already exists in subjects aged 65 years and older . This differs to some extent from what has been suggested by the most recent gold guidelines11 (that recognize some imprecision of the threshold of 0.70 for fev1/fvc in people over 70 years of age) and by medbo et al,10 who suggested the use of a threshold of fev1/fvc <0.65 in subjects over the age of 70 years on the basis of prebronchodilator spirometry data from a population - based study in norway . Interestingly, in our study, the rate of decline in the fev1/fvc ratio with aging was not statistically significant in men or women older than 65 years, because of a concomitant decline in both fev1 and fvc . Thus, in contrast with the findings of hardie et al,19 our results do not support the need to decrease the lower limit of normal for fev1/fvc to 0.60 in elderly subjects (> 80 years). For the same reason, no predictive equation for fev1/fvc could be derived; accordingly, the mean value and the fifth percentile of fev1/fvc from this healthy population can be used as the predicted value and lower limit of normal, respectively, for people aged 65 years and over . All reference equations derived from samples with a wide age range describe a progressive decline in the fev1/fvc ratio with aging; the age - related decrease involves both fev1 and fvc, seems to be nonlinear, and accelerates with aging.3032 in 1982, crapo et al33 found that between 20 and 70 years of age, vital capacity decreases to approximately 75% of the best values achieved previously . According to the present observations, presumably in the oldest people the decline of vital capacity accelerates more than in the younger age groups and such a decline is similar to the reduction of fev1, so that the fev1/fvc ratio could undergo minimal variations in the last decades . Recently, langhammer et al34 and falaschetti et al35 observed that fev1/fvc reaches a near plateau phase in elderly subjects . The authors emphasized that, although the sample consisted of subjects with a wide range of height and age, the extremes did not influence the equations . In a reference study specifically designed for elderly residents in madrid (age range 6585 years), garcia et al36 found a significant relationship between fev1/fvc and age in men and between fev1/fvc, age and height in women . Even in this study, the predicted equation for fev1/fvc had a very low r (0.048 and 0.083 for men and women, respectively) and the authors highlighted the strong negative relationship of fvc with age . By applying predictive equations recently derived from kuster et al37 in a swiss population, the lower limit of normal for fev1/fvc does not show important decreases with aging; for example, in men with a height of 170 cm, the lower limit of normal for fev1/fvc ranges from 0.66 at the age of 65 years to 0.64 for people aged 95 years . All these results support a position in favor of almost stable predicted values and lower limits of normal for fev1/fvc in elderly people aged 65 years and over . A clear position in favor of the fixed threshold of fev1/fvc <0.70 has been recently reported by other authors, with the claim that it is easy to use, thus helping to remove barriers to widespread use of spirometry.3,68 probably the use of the two lower fixed values for the elderly, suggested by data from the sara study, does not add elements of particular complexity for physicians involved in interpretation of pulmonary function tests . However, this approach would diminish the number of false diagnoses of copd, with significant cost savings due to reduction of inappropriately prescribed drugs . Thus, more resources could be redirected to primary prevention of copd (smoking cessation) and treatment of more severe copd . Authors advocating retaining fev1/fvc <0.70 often quote the results of mannino et al, who found that such a threshold is very good for identifying patients at risk of death and copd - related hospitalizations.38 however, although this indicates that fev1/fvc <0.70 may recognize a proportion of individuals at risk, it does not mean that this is the best way to diagnose the disease . On the other hand, vas fragoso et al39 found elevated risk of death and respiratory symptoms in adults with fev1/fvc less than the lower limit of normal . Sorino et al40 recently confirmed that fev1/fvc less than the lower limit of normal, fev1/fev6 less than the lower limit of normal, and fev1 less than the lower limit of normal are all significant predictors of all - cause and cardiopulmonary mortality in older individuals . The strongest spirometric predictor of all - cause mortality remains the appropriately named vital capacity, because the majority of deaths in adult smokers, with or without copd, are caused by cardiovascular disease . Two recent studies investigated subjects in between the two definitions of airway obstruction (ie, fev1/fvc <0.70 but lower limit of normal), showing that their clinical profile is characterized by relevant comorbid disease and poor health - related quality of life, but similar exercise, frequency of exacerbations, and indices of systemic effects.41,42 the investigators emphasized that these subjects might be at risk and should be followed carefully; we should be aware that fewer than one in five smokers with mild airway obstruction ever develop clinically important copd, and that today we are not yet able to identify which smokers will be rapid fallers.2,4 the present study has some limitations . First, the cohort was recruited for a cross - sectional investigation, whereas the effect of aging on respiratory function would be better assessed in a longitudinal study . It is plausible that fev1/fvc decreases significantly even after the age of 65 years, but this can be less reliable when derived by a cross - sectional observation of older people . In fact, they could represent individuals who had higher spirometric values at a younger age, and fev1/fvc similar to those of younger subjects at the time of recruitment . Second, subjects participating in the sara project were not randomly selected from the population, but consisted mainly of subjects with nonrespiratory illnesses attending outpatient clinics; this might have resulted in some selection bias, although it would not explain the higher pulmonary volumes than in other studies . The authors wish to emphasize that fev1/fvc less than the lower limit of normal should not be the only criterion used for diagnosis of airway obstruction, but should always be combined with evaluation of fev1 as percent of predicted . Indeed, patients with severe airflow limitation could have an important reduction both in fev1 and fvc, with a sustained fev1/fvc ratio . Thus, in doubtful cases, measurement of residual volume and total lung capacity is recommended . In conclusion, the present findings confirm the inadequacy of fev1/fvc <0.70 for diagnosis of airway obstruction in elderly people, and we propose other easy to remember thresholds for fev1/fvc after 65 years of age, ie, 0.65 in men and 0.67 women . Further studies are needed to assess both the classificatory and prognostic properties of such a threshold as well as epidemiological surveys to confirm it . Coordinators: v bellia (palermo) and f rengo (napoli). Scientific committee members: r antonelli incalzi (taranto), v grassi (brescia), s maggi (padua), g masotti (florence), g melillo (naples), d olivieri (parma), m palleschi (rome), r pistelli (rome), m trabucchi (rome), s zuccaro (rome). Participating centers, principal investigator, and associated investigators (the latter in brackets): divisione di medicina i, ospedale geriatrici inrca, ancona, dl consales (d lo nardo, p paggi); divisione di geriatria, ospedale civile, asti, f goria (p fea, g iraldi, r corradi); cattedra di gerontologia e geriatria, policlinico universitario, bari, a capurso (r flora, s torres, g venezia, m mesto); divisione di geriatria, ospedale malpighi, bologna, s semeraro (l bellotti, a tansella); divisione di medicina generale, ospedale civile, brescia, v grassi (s cossi, g guerini, c fantoni, m de martinis, l pini); clinica pneumologica, fondazione e maugeri, telese, g melillo (r battiloro, c gaudiosi, s de angelis); istituto di medicina interna e geriatria, ospedale cannizzaro, catania, l motta (i alessandria, s savia); istituto di gerontologia e geriatrici, ospedale ponte nuovo, universit florence, florence, g masotti (m chiarlone, s zacchei); divisione di geriatria, ospedale morgagni, forli, v pedone (d angelini, d cilla); divisione di geriatria, ospedale galliera, genova, e palummeri (m agretti, p costelli, d torriglia); grouppo ricerca geriatrica ricerca geriatrica, ospedale richiedei, gussago, m trabucchi (p barbisoni, f guerini, p ranieri); divisione di geriatria, ospedale generale, laquila, f caione (d caione, m la chiara); divisione di geriatria, ospedale san gerardo, monza, g galetti (a cantatore, d casarotti, g anni); cattedra di gerontologia e geriatria, universit federico ii, napoli, f rengo (f cacciatore, ai pisacreta, c calabrese); istituto di medicina interna, ospedale geriatrico, padova, g enzi (p dalla mont, s peruzza, p albanese, f tiozzo); istituto di clinica delle malattie dellapparato respiratorio, ospedale rasori, parma, d olivieri (v bocchino, a comel, n barbarito); istituto di gerontologia e geriatria, policlinico monteluce, perugia, u senin (f arnone, l camilli, s peretti); divisione di geriatria, ospedale israelitico, roma, sm zuccaro (m marchetti, l palleschi); divisione di geriatria, ospedale generale addolorata, roma, m palleschi (c cieri, f vetta); istituto di medicina interna e geriatria, policlinico gemelli, roma, pu carbonin (f pagano, p ranieri); istituto di semeiotica medicina e geriatria, policlinico le scotte, siena, s forconi (g abate, g marotta, e pagni); fondazione san raffaele, cittadella della carit, taranto, r antonelli - incalzi (c imperiale, c spada); cattedra di gerontologia e geriatria, ospedale maggiore, milano, c vergani (g giardini, mc sandrini, i dallera); cattedra di malattie dellapparato respiratorio, ospedale v cervello, palermo, v bellia (f catalano, n scichilone, s battaglia). Coordinating center: dipartimento di medicina, pneumologia, fisiologia e nutrizione umana, universit degli studi di palermo.
Proteomics aims to identify large sets of proteins in defined biological fractions, e.g., tissue or cell extracts, isolated organelles, or protein fractions generated by biochemical preparation procedures . As a first step, protein identifications today are nearly exclusively carried out by mass spectrometry (ms). However, since the mass of a native protein normally does not allow any conclusion on its identity, proteins have to be fragmented by specific endopeptidases before mass analyses to generate defined peptides . These peptide fragments are then searched against a database of theoretically digested proteins using a software package such as mascot, sequest, or x! Many proteome projects use one- or two - dimensional (2d) gel electrophoresis for protein separation . Following this experimental strategy, proteins are first separated, then individually picked from gels, treated with an endopeptidase (trypsin) and finally identified by ms and database interrogation . As a result, proteome reference maps proteome maps were developed in several fields of biology and are greatly appreciated by the scientific community (see for instance gallardo et al . Originally, maps were exclusively presented in scientific publications in the form of images and long lists of identified proteins which are assigned to the spots on the image using arrows and numbers . About a decade ago, the first web - based resources were developed which offer interactive features in the maps: upon clicking on a spot, information on a protein is presented in a small pop - up window which sometimes is linked to further background information in databases (examples at http://seed.proteome.free.fr/, http://gabi.rzpd.de/projects/arabidopsis_proteomics/, http://gene64.dna.affrc.go.jp/rpd/). Recently, the gelmap software package for annotating gel - based proteome data was developed . It offers several new possibilities for the web - based publication of reference maps, e.g., functional annotation of proteins and assignment of multiple proteins to individual spots on a gel (rode et al ., 2011a). In the following sections, we summarize the characteristics of gelmap and introduce novel features which were newly implemented into the gelmap software package (gelmap 2.0). Gelmap is offered as a web tool free of charge at www.gelmap.de . Minimal requirement for building a new map are (a) an image file and (b) a protein table which includes the coordinates of the spots on the gel . (by reading the spot s coordinates from graphics software like photoshop, gimp, or even ms paint) but is usually generated automatically by specialized proteomics software tools like delta2d (decodon, greifswald, germany), decyder (ge healthcare, munich, germany), or progenesis samespots (non - linear dynamics, newcastle, uk). To use gelmap to full capacity, the table should be extended with information on (1) the spot identification number (corresponding to the number on the gel image), (2) the mascot probability score (or the score of another matching software package), (3) coverage of a protein by identified peptides, (4) the accession number of a protein, (5) the database used for protein identification, (6) assignment of a protein to functional categories like a protein complex, a physiological area or subcellular localization, (7) molecular mass (calculated and/or apparent mass on the gel), etc . (table 1). To ensure that gelmap recognizes ids, coordinates, scores, accession numbers, and filters, a second line beneath the line with the column names has to include special keywords (table 1). The third line can contain commentaries for the tooltips in gelmaps spreadsheet view (figure 1). To mark the columns of high importance since gelmap provides a three - level filter tree, the fifth line can define if this column should be used as a (1) root level, (2) second level, or (3) third level filter . This control information is required for a fully functional map and is documented with examples on www.gelmap.de/howto . The two icons given in the header allow access to the protein spreadsheet and the peptide spreadsheet . The image file and the protein spreadsheet file can be directly uploaded at www.gelmap.de/create . The resulting map consists of the gel image, a side menu for functional categories of the identified proteins (right side), and frames for search options (figure 1)., information automatically is displayed in the following way: (i) upon hovering over a spot, all proteins identified within this spot are displayed within a tooltip . Proteins are ordered according to their mascot probability scores (this, on a semi - quantitative level, reflects abundance of the identified proteins within a protein spot). (ii) after clicking a spot, the protein names included in the tooltip are transformed into links, which can be used to get protein - specific information within a second info frame . In addition to the information on the protein accession and the calculated molecular mass of the protein this frame can offer links to an external database (via the accession number) and to the protein spreadsheet (link more protein details). The protein spreadsheet also can be accessed directly by using an icon in the header (figure 1). (iii) proteins assigned to functional categories are accessible via the three - level tree in the side menu . Upon clicking onto a topic included in this menu, all proteins forming part of this category automatically become highlighted by circles on the gel image . At the same time, subcategories become visible for many topics of the main menu, which allow to differentially visualize proteins assigned to the next functional level . For details we recommend visiting one of the established gelmaps, e.g., http://gelmap.de/47 . To display primary ms data for all identified proteins, a second table called peptide spreadsheet was introduced in gelmap 2.0 (figure 1). This table may include information on all identified peptides within a spot . In the case of the arabidopsis thaliana bn / sds map, the table includes information on the amino acid sequences of the peptides, peptide modifications, and positions of peptides with respect to the complete protein sequences (column range: number of the first and the last amino acid of a peptide with respect to the n - terminal methionine). Meta portals which are realized on the basis of peptide position data (see integration of gelmap into the mascp gator). The linked icon for the peptide spreadsheet is located next to the protein spreadsheet icon at the top (figure 1). Alternatively, the peptide spreadsheet is accessible through a text link in the information frames for individual proteins on the gel image . The content of this table can be freely defined as long as the first column contains the hit s i d to link this information to the spots (see http://www.gelmap.de/47 for example). In summary, gelmap offers the following features: proteins of interest can easily be found using the different search options or by browsing / navigating through / using the categories in the side menu . Since all proteins are annotated according to functional criteria, sets of proteins involved in metabolic processes or other functional categories are easily visualized . Detailed information on the identified proteins is provided by the information frames which are accessible via the gel image . Background information and raw data on all proteins is given in the protein spreadsheet and the peptide spreadsheet gelmap allows complete annotation of ms data for all analyzed protein spots: not only the protein with the highest mascot probability score is given, but all identified proteins above a lower boundary score to be defined by the user . The gelmap portal was established for creation and presentation of reference maps . Upon finishing a new map, a user can decide to make it accessible via the official gelmap site and/or via a direct link which can be presented in publications . Gelmap also allows private (password protected) projects to share proteome data in a closed group or analyzing results without sharing them . Currently (february 28th, 2012), four projects are publicly available at the gelmap portal: the cyclamen persicum seed proteome (rode et al ., 2011b). In the frame of this project, identified proteins are assigned to 30 different metabolic pathways within 10 metabolic divisions . Furthermore . In addition to the features mentioned above, this gelmap allows to differentially display proteins of changed abundance between the two compared protein fractions . This illustrates the various extensions which easily can be realized based on the gelmap software package . The a. thaliana mitochondrial proteome separated by 2d blue native / sds page (klodmann et al ., 2011 this map is based on a special protein separation procedure: protein complexes are separated under native conditions on the first gel dimension and the subunits of the protein complexes under denaturing conditions on the second dimension, which is carried out in the presence of sds . On the resulting gels, proteins belonging to the same protein complex form a vertical row of spots . For 2d blue native / sds page, gelmap offers special advantages: selective display of functional categories allows identifying vertical positioning of proteins of low abundance . This led to the discovery of new protein complexes in arabidopsis mitochondria . In the frame of this study, 471 non - redundant proteins were identified and assigned to more than 35 protein complexes . The a. thaliana mitochondrial proteome separated by 2d ief / sds page (taylor et al ., 2011). Recently, the proteomic aggregation utility mascp gator has been established to link proteome databases for the model plant a. thaliana (joshi et al ., 2011). In this platform, protein data is simultaneous displayed from many different international repositories to allow assessment of the global knowledge of a protein or several proteins . So far, projects integrated in the mascp gator exclusively represented shot - gun approaches not based on 2d page . Since september 2011, gelmap offers a limited application programming interface (api) which allows other projects (such as gator) to run automated search queries on the gelmap database and collect the results . The two gelmaps dedicated to the mitochondrial proteome of arabidopsis very recently were the first gel - based projects integrated into the mascp gator . Upon submission of a protein accession, the user of the mascp gator directly can access the reference maps at www.gelmap.de (figure 2). Information on peptides available at gelmap is graphically displayed at the mascp gator site together with peptide information of other databases . In the future integration of the mitochondrial reference maps at gelmap into mascp gator (http://gator.masc-proteomics.org/). Upon submission of a protein accession (here: at2g47260), matching peptides from gelmap are graphically displayed at the gator site . Direct access to gelmap is provided by links in the left side menu of the gator . The gelmap software package allows complete annotation of ms data corresponding to 2d protein separations . Based on its central features functional annotation of proteins and at the same time annotation of complete sets of proteins identified within each spot it allows the comprehensive evaluation of gel - based proteome data sets . In the future, the gelmap platform will be used to annotate further projects . Integration of these projects into the mascp gator might soon allow extensive coverage of the arabidopsis proteome by gel - based analyses . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Brain structure segmentation is the apportionment of brain tissue into gray matter and white matter, based on the appearance of tissue in images produced by magnetic resonance imaging (mri). Because manual tracing of the boundaries between tissues in the brain is labor intensive, difficult, error - prone, and unrealistic for large amounts of data, an automated or semiautomated segmentation technique is needed for either visualization or diagnosis . Different imaging modalities, such as t1-weighted, t2-weighted, or proton density (pd) images, have been used for different segmentation methods . T1-weighted images, because of their good contrast, have been widely tested for various segmentation methods [26]. A t1-map is a parametric image of pure t1 (spin lattice relaxation time), derived from the solution of an equation describing tissue relaxation, and a parametric t1-map which is different from a t1-weighted image . The relationship between t1 and several diseases, such as schizophrenia or sickle cell disease, has been studied [710], and t1 may be used as a possible indicator of pathology . Change in t1 of certain voxels in the brain over time may be an early indicator of possible pathology . Therefore, the segmentation of a parametric brain t1-map may highlight pathology unseen by other imaging approaches . The three tissues white matter (wm), gray matter (gm), and cerebrospinal fluid (csf) constitute the main parts in the brain . Different methods have been proposed to achieve this goal, and they may be classified into various categories: fuzzy segmentation methods [4, 11], markov random field (mrf) methods [12, 13], bayesian methods, active contour methods [1, 5, 6], or the combinations of two or more techniques . Some of these combinations are as follows: leemput et al . Used expectation maximization (em) and mrf, xu et al it is an adaptive version of mumford - shah's model to systematically segment different tissues in the brain . Before the segmentation, a t1-map may be calculated by a rapid method known as the variable nutation method which provides comparable precision but much faster speed over conventional methods . This method requires the acquisition of a set of flip angle images and the t1 information can be extracted therein . [18, 19] proposed methods to determine the optimal flip angles by basically maximizing the signal - to - noise ratio (snr) of a t1-map . Their first work required the knowledge of average tr / t1 in advance though, and their second work introduced a weighted least square method to estimate the angles . We take another approach to determine the flip angles to achieve the trade - off between acquisition time and t1 accuracy . Since a t1-map is generated by a set of flip angle images, alignment of the images is important in order to obtain an accurate t1-map . The registration of flip angle images is usually ignored though because the movement of the head in the coil is minute . However, slight registration errors affect the resulting t1-map dramatically, as will be shown . Radiofrequency (rf)-inhomogeneity is another unavoidable problem encountered in mr imaging . The nonuniform distribution of the rf field can cause the resulting images to have low contrast and inhomogeneous intensity, which makes quantitative description and segmentation of the image difficult . Rf inhomogeneity affects the generation of t1-map in the sense that the spins are not tilted by the predefined nominal angles [17, 2022]. Hence, we focus on calibrating the rf nonuniformity in order to generate an accurate t1-map . Cheng and wright calculated an analytical form of t1 errors induced by rf nonuniformity and allowed simple correction of t1 measurements . We propose a method, which assumes the average t1 value among a transverse plane is the same across the central brain slices, to jointly segment a t1-map and calibrate the rf nonuniformity along the direction perpendicular to the transverse plane . Taking into account that we average out the heterogeneity of t1, and doing so only within few central brain slices, this assumption help calibrate rf - inhomogeneity and generate a quantitative t1-map for segmentation . Section 2 we state our adapted model, with a fast implementation method . In section 3 we first illustrate some necessary preprocessing to generate an accurate t1-map, which includes the registration of flip angle images with the generation of a brain mask and the determination of optimal flip angles, to achieve a trade - off between quality and efficiency . We propose a novel method to jointly segment a t1-map and calibrate rf - inhomogeneity, and in section 5 we show the results of registering the flip angle images, determining optimal flip angles, and segmenting the t1-maps . We also show validations, the resulting t1-map after rf - calibration, and the 3d segmentation for two sets of t1-weighted data . At last in section 6 active contour methods comprise a popular segmentation technique in which an initialized contour is driven by a partial differential equation (pde) to minimize an energy functional designed to attract the contour toward image edges . Active contour methods can be classified into two categories: edge - based [2427, 2734] and region - based [16, 3544] methods . Edge - based methods examine the gradient information of the image, and stop the contour whenever the gradient is high . However, there are many situations when the edge is not clearly characterized by the gradient, and it has been shown that region - based methods outperform edge - based methods [3537, 41, 43, 44]. By examining some statistics of the region inside and outside of the active contour, and optimizing the separation of these two statistics, we may achieve a better segmentation performance, thus making region - based methods more attractive . Our proposed model is a modified momford and shah functional, and falls in the region - based category . Two adaptations of mumford - shah's model constitute the novelty of our proposed technique . First is the incorporation of an information - theoretic view, which characterizes the statistical property of data . The second is a selective weighting, similar to the weight parameter in, which favors erring towards one tissue type or another, and thus make 3-tissue segmentation possible . We use a modified mumford - shah energy functional: (1)e(frin, frout, c)=i = in, outiri{(frig(i))2+fri2}dx+cds, where fri approximate a function g() applied of the image i for region ri, i = in or out . Fri is smooth within each region ri, but not across the boundaries; c denotes the region boundaries, which is the contour of interest in this paper, and 0 i 1 are weights such that in + out = 1 . By minimizing this functional we obtain a function fri which is faithful to the image (first term) and smooth within each region but not across the boundaries (second term), while penalizing excessive length of the boundaries (last term). The first adaptation is the function g() applied on to image i instead of the image itself . Specifically, an information - theoretic approach for maximizing a probabilistic disparity measure, jensen - shannon (js) divergence, was proposed . A constructed function g() characterizes the property of the probability density function (pdf) of the image intensity such as skewness (g(i) = i), or kurtosis (g(i) = i), relative to a gaussian . A proper choice of g() will capture the statistical characteristics of the data and will hence achieve a good segmentation . The second adaptation, similar to that in, is the selective weight i applied on the inside / outside energy terms . This provides a probabilistic assignment to the segmented regions, and also makes 3-tissue segmentation possible, as will be shown in section 3 . Enhancing the weight in is tantamount to penalizing both the error of the difference between the approximated function frin and the data fidelity term g(i), as well as the degree of smoothness of frin this would yield a smaller segmented region which is likely to be more faithful to the image, and of purer tissues . Figure 1 shows examples of white matter (wm) segmentation with different weights . With a larger inside - weight in, the segmented region is smaller and the segmented tissues are purer . Minimizing the mumford - shah energy functional involves solving for the approximating functions frin / out and for the contour c. the joint search for these infinite dimensional unknowns usually entails gradient descent flows . In particular, the approximated functions are typically modeled as a linear combination of a basis set whose dimension equals the number of pixels in the image, that is, each pixel is assumed to be independent . Alvino and yezzi proposed a fast implementation using a significantly smaller basis number to model the approximated functions, while still achieving sufficient resemblance to the obtained functions when using the pixel - by - pixel basis . We adopt their so - called linear heat equation basis with the change from i to g(i) to incorporate the statistical properties of the data from an information - theoretic point of view in the previous section . We hence have, (2)fri=1,ig(i)+2,imean(g(i)), where mean() is the average function, and the coefficients j, in(out), j = 1,2, may be similarly derived in . Our derivations are included in the appendix . Substituting (2) into (1), and using a classical methods of variational calculus, the gradient descent evolution of the curve may be derived as (3)ct={out[(froutg(i))2+frout2] in[(fring(i))2+frin2]}nn,(4)=(outfoutinfin)nn, where denotes the curvature of the contour c, t an artificial time evolution parameter, and n the outward normal of the contour . Fin / out are derived by substituting frin / out from (2) into (3) as shown in the appendix . A brain parametric t1-map is the calculated result (variable nutation method) from several flip angle images . Because the flip angle images are acquired at different times, and because the subject may move during image acquisition, registration should be carried out first to obtain an accurate t1-map . Moreover, in order to achieve a balance between the acquisition time and the resulting t1-map quality, we propose a method to determine optimal flip angles . In the following subsections we will first illustrate how we register the flip angle images and obtain a brain mask as a byproduct, then describe our method to determine a set of optimal flip angles, and at last describe our proposed procedure to segment a t1-map . The value of t1 is traditionally determined by acquisition methods such as inversion recovery (ir) or saturation - recovery (sr). Other rapid methods, such as variable nutation (the despot method) have been proposed, and require acquisition of several flip angle images, and calculation of t1 . Since these flip angle images are acquired at different times, registration must first be accomplished . Even though the interval between consecutive scans may be as short as two minutes, and movement of the subject's head inside the receiver coil may be a few pixels (under the resolution of a 256 256 image), the effects of such off - registration can be significant, as shown (figure 5). Here we describe a method to register flip angle images and jointly obtain a mask, as a byproduct, to get rid of the skull and other structures around the brain . We use a joint segmentation and registration (jsr) technique proposed by yezzi et al . With an additional tuning weight, to achieve registration and to obtain the mask . The theory of jsr technique consists of evolving two contours, with a enforced relationship (ex: rigid or affine transform) between them, in two images according to a partial differential equation (pde) which is a result of optimizing, for example, the sum of two energy functionals . For our particular task, we may choose a region - based energy, such as chan and vese's model incorporating weights, which arises as a special case of (1) with the data term g(i) = i and the approximating function frin / out = mean(i) inside and outside the contour . This model approximates the image i by a simple piecewise constant function, which suits our goal here because it creates a mask that divides the image into two parts- brain region and nonbrain region . We observe that the boundary between the brain and nonbrain is visually more easily distinguished than the boundary between different tissues within the brain, for every flip angle image . We therefore put a very small weight in on the inside energy in (1) to penalize very little the difference between the data inside the contour, i, and the approximated function mean(i). We carry out this registration technique pairwise for all flip angle images, and once the flip - angel images are registered to each other, they are used to generate a t1-map . The determination of the flip angles that yields qualitative t1-map and saves time, is mainly by comparing the difference between the gold standard and the t1-maps generated from different combinatorial flip angles . We begin by acquiring images at a set of 19 flip angles which spans the range of standard angles and will give an optimized t1-map . We call this particular t1-map the gold standard, or t1 g, and denote this set of angles as 19 = [2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90]. We then compare the t1-map generated from all combinations of the subset of 19 with t1 g . Out of these combinations since the data acquisition is slice - based, this study of optimal flip angle focuses on the central slice, which is least affected by rf - inhomogeneity and is routinely selected at the level of the basal ganglia, including both the genu and the splenium of the corpus callosum, and generally shows the putamen and lateral ventricle . We denote the optimal n - angles opt, n, which is a subset of 19, as those that exhibit the smallest difference between t1 g and the t1-map generated by n flip angles: (5)opt, n = arg min n(x, y)brain\|t1g(x, y)t1n(x, y)\|, where n {2,3,, 19}, n19, t1n denotes the t1-map generated by n, and the summation of (x, y) is over the whole brain region at the central slice . Equation (5) therefore gives 18 sets, with the number of elements ranging from 2 to 19, of optimal angles . For the determination of the optimal set of flip angles, in reaching a compromise between efficiency and t1-map quality, we examine the error between t1opt, n and t1 g and compute the error rate . The error rate is defined as (6)e=(x, y)braine(x, y)a(brain), where a(brain) is the area of the brain, and e(x, y) is the error, defined as 1 if the difference between t1g(x, y) and t1opt, n(x, y) is greater than some threshold, and 0 otherwise . The threshold is defined as the minimum of the standard deviation among the t1 values of three brain tissues (wm, gm, and csf) manually segmented by an expert . The summation of e(x, y) is over the whole brain at the central slice . The plot of the error rate versus the number of flip angles is shown in figure 6, where the inflection point of the fitted curve is at 10 flip angles (opt,10 = [2, 5, 55, 60, 65, 70, 75, 80, 85, 90]). Once the brain mask is obtained (section 3.1), it is used to segment away the skull, leaving only three major tissues in the image: wm, gm, and csf . Notice that the curve evolution corresponding to the energy introduced in section 2 always results in a binary segmentation, where we will have regions inside (foreground) and outside (background) the contour(s). We cannot simultaneously segment the three tissues, even though we will later talk about multiphase segmentation . We may, however, tune the weights (section 2.1), to penalize the error between the data term and the approximated function (1) to segment one tissue at a time, with a progression analogous to that of peeling an onion . We illustrate our t1-map segmentation procedure for a hard segmentation of three tissues, and the probabilistic segmentation is obtained by varying the weights around the value of the trained weight (section 5.4). A t1-map segmentation procedure consists of three steps, where the first two steps are to evolve the contours by minimizing the energy in (1) with different weights, and the third step is just a simple subtraction . The procedure is as follows: treat wm as the foreground, everything else as the background; let in in (1) be the trained in, wm, and use it to segment wm in the interior region of the contour . Treat wm and gm as the foreground, csf and everything else as the background; let in in (1) be the trained in, csf, and use it to obtain csf in the exterior region of the curve filtered by the brain mask . The procedure is based on the anatomical observation that gm is enclosed by csf, and that csf is separated from wm [1, 3], such that we may peel off one layer at a time . The choice of function g(i) in (1), which is chosen to better capture the statistical property of t1-map (and for other modality), will be shown in section 5 . The values of in, wm and in, csf are determined through a training process . If re denotes the segmentation region by the expert, and rin denotes the segmentation region by the weight in with some fixed g(i), for some tissue, then the value of in, tissue is determined by minimizing (7)in, tissue = argmin in{1\|rerin\|\|rin\|+\|re\|\|rerin\|}, where \|r\| denotes the number of pixels in region r. the first term inside the bracket is an overlap metric (om) which will be discussed again in section 4 . The strength of the rf field varies within the mr scanner, such that the resulting image may be of low contrast or of nonuniform intensity . In what follows, we will first describe how a t1-map is calculated from a set of flip angle images, then how the t1-map is affected by rf - inhomogeneity and then show our proposed correction method . A t1-map is calculated by variable nutation . Flip - angle images are acquired using a flash sequence at different flip angles, and t1 is calculated from the slope of a least square fitted line to the pair of data [s()/sin, s()/tan], where s() is the signal strength of the flip angle image expressed as a function of the flip angle . Rf - inhomogeneity affects the computation of t1 in that the spins are not tilted by the nominal angle because the strength of the rf field is not as predefined . Calibration of rf - inhomogeneity, or, the correction of flip angles, produces a more accurate t1-map . A spatially dependent scaling factor, therefore, is introduced to adjust the tilted angle [20, 22], that is, replacing by (8)[s()sin(),s()tan()], and t1 is extracted from the slope of the line fitted to the above space . Our imaging setting is slice - based across different transverse planes (figure 2), thus our proposed joint segmentation and rf - inhomogeneity calibration (jsric) method is as an initial step to easily calibrate rf - inhomogeneity vertically . Our method is based on the assumption that the average t1 of wm should be roughly the same across central brain transverse slices . We however average out the heterogeneity of t1, and carry out the method only within few central brain slices . The advantage is that as the segmentation delineates more precisely the wm region, flip angle correction is therefore enhanced, and as flip angle correction is carried out more precisely, which gives better quality t1-map, segmentation is facilitated as well . Our joint segmentation and rf - inhomogeneity calibration (jsric) method requires first taking the average t1 value for segmented wm at the central slice (which has relatively uniform b1 field [51, 53]) as the reference, and then iteratively segmenting and searching for the scaling factor in (8) for all other slices . This is a three - step iterative process (as shown inside the dashed box in flowchart 3). Segment wm for the central slice by the method proposed in section 3.3, compute the average t1 value of wm, and denote the average as m. for slice m, varies in (8), and calculate the corresponding t1-map, denoted as t1(). If goes beyond max, claim the current slice as uncalibratable and repeat this step for the next slice m + 1 . Segment wm for t1(), compute the average t1 of wm, and denote it by l(). If m l(), go back to step 2 and increase, otherwise claim it is done for the current slice . The variables min, max, and in flowchart 3q are all predefined parameters . In this section we show a series of results from segmenting a parametric brain t1-map, which includes the registration of flip angle images, generation of a brain mask as a byproduct, determination of the optimal flip angles, and joint t1-map segmentation and rf - inhomogeneity calibration . In addition, we will apply our proposed segmentation method to another modality, t1-weighted images, in a 3d setting, to show the generality of our segmentation method . The subjects being scanned were recruited from a clinic in the department of psychiatry at the university of north carolina, under an irb - approved protocol to image the brain . A transverse 3d flash sequence using different flip angles was acquired in a 3 t siemens mri scanner with a quadrature head coil . The scan parameters were: tr = 25 msec, te = 2.83 msec, 16 slices, and slice thickness = 5 mm . The center slice used for optimal flip angle study was routinely selected as described (section 3.2). The registration of a set of flip angle images is carried out pair wise, and figure 4 shows two flip angle images and the resulting brain mask . Note that even though the flip angle images are off - registered by no more than four pixels in both the x- and y - directions, the impact is obvious . Figure 5 shows two t1-maps generated by registered and unregistered flip angle images, and we can see that after registration the anatomical structure of the t1-map is more readily distinguished, and the high signal intensity artifact in the upper left of the unregistered t1-map map is reduced . The plot of the error rate e (defined in (6)) versus the number of flip angle is shown in figure 6, and section 3.2 already concluded that a set of 10 flip angles (opt,10 = [2, 5, 55, 60, 65, 70, 75, 80, 85, 90]) is a good compromise between t1 quality and efficiency . Figure 7 shows a comparison of generated t1-maps when using 2 optimized flip angles ([5, 55]), 6 optimized flip angles ([2, 5, 60, 65, 70, 75]) and 10 optimized flip angles, with an error map the t1-map generated by 2 angles exhibits substantially more error than the maps using 6 or 10 angles, as shown by the bright pixels in the error map . We conclude that 10 flip angles is an acceptable choice considering the trade - off between accuracy and scan time . Out of a total of 16 slices, the bottom 4 slices were discarded because they did not cover sufficient wm for segmentation by our jsric method . The method follows the flowchart in the dashed box shown in figure 3 from slice 5 to slice 15 . Slice 16 could not be calculated, possibly because of its proximity to the boundary of the rf field and hence degraded . The function g(i) introduced in (1) was empirically chosen as the cubic function i, which characterizes the skewness of a pdf . Moreover, in, wm and in, csf are obtained by training according to (7) based on an expert's manual segmentation of one subject (training subject). These values are in, wm = 0.93 and in, csf = 0.53 . The same values are then applied to the other subject (testing subject). The segmentation of a t1-map is achieved by evolving contours according to (4). Since the curve evolution is based on a gradient flow, the final result may vary depending upon the initialization . A t1-weighted image histogram is shown (figure 8(a)), and a spectral analysis can be carried out to threshold the image as an initialization [5, 55], to achieve a good segmentation after fine tuning of the contour . The t1-map does not have the same level of contrast as a t1-weighted image (figure 8(b)). We therefore initialize the contours by either placing uniformly spaced squares or by manually seeding (by mouse clicking and dragging on the image). (versus slice number) for two subjects, where the parameter is as defined (section 4.1). The strength of the rf field drops significantly at the top slices (slice 14 and 15) such that the corresponding flip angles have to be rectified by a scaling factor much smaller than 1 . Figure 10 shows two t1-maps generated with and without flip angle rectification for the same subject at slice number 5, 6, 14 and 15 . It is clearly seen that the top 2 slices of the t1-maps with calibration have much better quality than those without . Figure 11 shows some selective segmentation results of wm and gm for the test subject . The commonly examined metrics which determine the performance of a segmentation are tp (true positive), fp (false positive), and om (overlap metric) [1, 5]. The overlap metric is defined for a class assignment as the sum of the number of pixels that both have the class assignment in each segmentation divided by the sum of pixels where either segmentation has the class assignment . This metric approaches 1 for segmentations that are very similar, and is near 0 when they share no similarly classified pixels . Figure 12 shows three overlap metric curves (om versus slice number) of wm and gm segmentation for the training subject (the test subject exhibits a similar result). The first curve corresponds to the segmentation of calibrated t1-maps, with our tuned weights and cubic function g(i) = i, the second corresponds to the t1-maps generated by nonrectified flip angles (using the same segmentation parameters), and the third corresponds to the calibrated t1-map, with function g(i) = i and tuned weights (in, wm = 0.9 and in, csf = 0.7). These results show that calibrated t1-maps enhance the segmentation performance compared to un - calibrated t1-maps, especially at the top two slices (slice 14 and 15) which are most seriously affected by rf - inhomogeneity . The comparison of different functions g(i) shows that the performance of wm segmentation is comparable for the two functions . There is, however, a significant difference for csf segmentation, thus affecting gm segmentation . The cubic function it also demonstrates that the cubic function better characterizes the statistical properties, the skewness, of the data . Figure 13 shows tp and fp for wm segmentation with different weights in around the value of in, wm, to demonstrate the notion of our probabilistic segmentation . Tp = \|rinre\|/\|re\|, and fp = (\|rin\| \|rinre\|)/\|re\|, where re and rin are the expert segmented regions and ours using in respectively, and \|r\| denotes the area of region r. when the weight increases, so does the penalty for the error between the data term and the approximating function (1). Therefore tp and fp decrease correspondingly, and vice versa . In this section we show the generality of our proposed segmentation method in a 3d setting by applying it to a commonly exploited modality t1-weighted images . The same procedures are carried out as in section 3.3, except that now the images are collated into volumes and the active contour is replaced by an active surface . We test it on two open databases accessible online brainweb (http://www.bic.mni.mcgill.ca/brainweb/) and ibsr (http://www.cma.mgh.harvard.edu/ibsr/). The former is a simulated brain mri database, therefore ground truth is provided and the latter is genuine brain mri data which also has been manually segmented by experts . We preprocessed the data to filter out everything except for the three main tissues, wm, gm, and csf . We first tested our segmentation method on two brainweb subjects (a training and a testing subject) using simulated brain t1-weighted data . The images are 1 mm slice thick, with 3% noise level, and 20% rf intensity nonuniformity (inu) and each data set was 217 181 106 pixels . Because the contrast between different tissues was high, we did a histogram analysis and applied the threshold method similar to for initialization . As i, and in, wm and in, csf are obtained by training as 0.3 and 0.2 respectively . The results show that it achieves a high performance of om being around 0.8 for three tissues . The computational time for one subject is less then 1 minute on a laptop with a 1.73 ghz cpu and 1 gb memory . We then tested our segmentation method on 20 data sets of t1-weighted images provided by the center for morphometric analysis at massachusetts general hospital on the ibsr website . The data set for each subject was 256 256 l, where l ranges from 58 to 63 pixels . We arbitrarily chose one subject (subject 1_24) as the training data, empirically chose the function g(i) = i for wm and g(i) = i for csf, frin(out) = meanin(out)(g(i)), in, wm = 0.75, and in, csf = 0.2 . We did not carry out any preprocessing to denoise the data or to decrease the rf - inhomogeneity effect which seriously degraded the data . Therefore the spectral analysis could not be carried out and we used uniformly spaced cubes as the initialization . Out of 20 subjects we however still have 3 particularly unsuccessful cases (specifically subject 2_4, 16_3, and 111_2) due to rf - inhomogeneity which we did not rectify . Including these three subjects, we have an averaged overlap metric around 0.651, and if excluding these 3 outliers, we achieved an averaged om of around 0.707 . The computational time is less then 5 minutes on the same pc as above . The statistics show that our method outperforms most other methods even if the outliers are included; if excluding those, we have wm segmentation slightly poorer but gm segmentation slightly better than the current best segmentation method . To register flip angle images we used the jsr technique, which has the advantage of jointly segmenting, registering, and generating a brain mask . Other techniques such as the information - theoretic method only register the images, and the brain mask has to be otherwise generated . Our jsr has been carried out pair wise on 10 flip angle images, registering 9 images to a reference image . In theory, however, it should be possible to integrate multiple - image in jsr's formulation . By summing the energy functionals of multiple images with a relationship enforced between each contour, that is, e(c1, g2,, gn) = e1(c1) + e2(g2(c1)) + + en(gn(c1)), where ci = gi(c1), i = 1,, n, the evolution of the contours ci and registration parameters gi may be derived similarly . Our segmentation of three brain tissues is based on the tuning of weights, to penalize differently the error of the approximated function, to obtain different regions of tissue . Segmentation uses the anatomical nature of brain tissue which has a layered structure such that we may peel off one layer at a time . Our method uses an active contour that is able to separate an image into two parts: the inside and the outside of the contour . However, multiphase active contour techniques exist [36, 58] and are able to evolve multiple contours simultaneously and segment multiple regions at once . Our method is different in the sense that it has a probabilistic flavor that the tuning weights determine a purity - level of segmented tissues . Our jsric method works by enforcing the average of white matter t1 value to be homogeneous across different transverse planes in the central brain region, to find the scaling factors which affects the flip angles . Even though t1 has regional heterogeneity, by taking the average of wm for the transverse plane we average out this heterogeneity, and doing so only in the central brain region . Wm segmentation and rf - calibration enhance the precision of one another, as shown in the performance plot in the previous section . In conclusion, we propose an adapted mumford - shah type energy functional for segmentation . The two adaptations are: (1) a function g(i) is able to characterize the statistical properties of the data to achieve better segmentation results, and (2) the tuning weights in(out) are able to segment brain tissues in a probabilistic fashion and achieve three - tissue segmentation . We also propose a novel method (jsric) to jointly segment a t1-map and calibrate rf - inhomogeneity . The whole procedure moreover includes the determination of optimal flip angles in achieving the balance between accuracy and efficiency, and joint registration of flip angle images and generation of brain mask . After rf - calibration, the top and bottom slices of t1-maps show better contrast and enhance the segmentation performance . The segmentation method has also been applied to t1-weighted data, to show the generality of our segmentation method, and the results are validated by ground truth and by expert manual segmentations . To derive the gradient flow of our energy in (1) with alvino et al . 's fast mumford - shah implementation, we first restate their result for the approximated function in scale space . Suppose the approximated function fri, i = in or out, is expressed as a linear combination of basis j: (a.1)fri=j=1nj, ij, then the coefficients j, i can be solved by a linear system (a.2)i+ii=i, where (a.3)i=[ri11rin1ri12rin2ri1nrinn],i=[ri1,1rin,1ri1,2rin,2ri1,nrin,n], and each n n matrices must be computed for each region . Also, (a.4)i=[1,i2,in, i], i=[ri1g(i)ring(i)] are each n 1 vectors . Therefore we may replace the basis j by g(i) and mean(g(i)) as in (2), and solve for the coefficients i: (a.5)1i = timisiti+wimisi, 2i = witi+wimisi where (a.6)si=rig(i)dxdy, mi = siridxdy, ti=rig(i)2dxdy, wi=rig(i)2dxdy . Then substituting the coefficients in (2) by the above results and then pluging frin(out) into (3)
Knowledge about dysphonia manifestations and the degree of commitment that this change causes in laryngeal and vocal behavior are important for defining rehabilitation direction . In order to do that, procedures are necessary for clinical assessment . Perceptual and acoustic assessments, among other procedures, are commonly performed . Oral and vocal fold diadochokinesis (ddk) is one of the tests that can be applied to perform acoustic analysis . The ddk, or syllable alternating motion rate, is the ability to perform rapidly opposite contractions of relatively simple patterns . Vocal fold ddk analysis allows evaluation of neuromuscular integrity of the vocal folds and oral ddk analyzes the ability to perform quick repetitions of speech segments . Some studies try to understand the vocal manifestation of dysphonic patients through the perceptual and acoustic analysis of voice . (2008) evaluated voice acoustic parameter changes in patients with parkinson's disease (pd) who were searching for their relationship with motor control . The voices perceptual and acoustic ratings evaluations of 20 pd patients, 12 men and 8 women, were compared with the voices of 20 control subjects of corresponding ages and genders . Patients with pd had smaller values for maximum phonation time and ddk compared to the control group, while jitter, shimmer and average fundamental frequency (f0) were similar between the two groups . Although men have poorer performance in ddk than women, these values were not statistically significant . Thus, only a few significant correlations between vocal parameters and motor control were found . The vocal fold ddk measurement indicates laryngeal gestures of opening and closing of the vocal folds, being that changes in extension and speed movement of vocal folds will reproduce changes in the production rate in patterns of duration and transglottic airflow rate . Therefore, the presence of organic and functional changes in dysphonia cases may relate to ddk results . The ddk evaluation associated with other clinical assessment procedures is an important resource in understanding the manifestations of individual communication disorders . The assessment of oral and vocal fold diadochokinesis (ddk) in individuals with voice disorders may contribute to the understanding of factors that affect balanced vocal production . Scientific studies that make use of this assessment tool support advances in knowledge in this area and reflect the development of more appropriate therapeutic planning . Consequently, the objective of this study was to compare the results of oral and vocal fold ddk of dysphonic women to the results of oral and vocal fold ddk in women without vocal disorders . This study has been approved by the research ethical committee of the school of dentistry of the university of so paulo, bauru campus . The recordings were obtained in an acoustically treated studio, and the patients remained seated with a headset microphone (akg, model - c444pp) positioned laterally between 60 degrees and 5 cm from the labial commissure . The emissions were recorded on a intel pentium 4 computer (cpu 2.040 ghz and 256 mb ram) with a 17 " lg flatron e7015 monitor and a creative audigy ii sound card . The system also used sound forge 7.0 professional audio software (madison, wi, usa), with a sampling rate of 44.100 hz and a 16 bit mono channel . The experimental group was composed of 28 women ranging in age from 19 to 54 years old, diagnosed with dysphonia and referred for a voice assessment by a speech pathologist and otorhinolaryngologist . The control group included 30 nondysphonic women age - matched with the experimental group who were evaluated in previous research with normal adults . The presence of central neurological diseases and the presence of communication disorders were considered exclusion factors for both groups, with the exception of dysphonia for the experimental group . During the oral and vocal fold ddk test isolated emissions were requested; namely, the syllables " pa, " " ta " and " ka, " the sequence " pataka " and then the vowels " a " and " i. " Before the beginning of the test, the patient was requested to repeat the sound as rapidly as possible . The patients practiced the sounds before each recording by repeating as fast as possible, after deep breathing, in a clear and precise way the emissions with a comfortable frequency and intensity in order to understand how to perform the test . Soon thereafter, the subjects were guided to perform the tasks, by receiving a signal with the thumb to begin and to stop the repetition . The " pa, " " ta, " " ka, " " pataka, " " a " and " i " emissions were edited by sound forge 7.0 professional audio editing professional . Software (madison, wi, usa). The beginning and end of each sample were excluded, except for four seconds from the third to the sixth second . Monosyllabic emissions " pa, " " ta " and " ka " and the vowels " a " and " i " were filed at sampling rate of 11.025 hz, 16 bit mono channel and analyzed by advanced motor speech profile program (msp) model 5141 version 2.5.2 (kay's elemetrics corp ., lincoln park, nj, usa), whose parameters are described in figure 1 . The msp program displays a graphical record of emissions showing the horizontal axis (time in seconds) and the vertical axis (energy in db). To do the ddk count, the program drew a line in the central point at the energy scale of the vertical axis in db . The line of analysis was manually positioned in the corresponding value to the intensity average of the ddk sample, provided by the msp program, and moved up or down, so the program could count all emissions when necessary (figure 2). Parameters for diadochokinesis (ddk) analyzed by advanced motor speech profile (msp) program advanced motor speech profile (msp) program graphic, where time (seconds) on the horizontal axis and energy (db) on the vertical axis can be observed the ddk of the " pataka " sequence was analyzed quantitatively through the sound forge 7.0 program . The count of trisyllables per second was performed manually with the auditory and visual waveform support displayed by the program . Average values of oral and vocal fold ddk in dysphonic and nondysphonic women were correlated using the " t student " test and were considered significant when p<0.05 . The experimental group was composed of 28 women ranging in age from 19 to 54 years old, diagnosed with dysphonia and referred for a voice assessment by a speech pathologist and otorhinolaryngologist . The control group included 30 nondysphonic women age - matched with the experimental group who were evaluated in previous research with normal adults . The presence of central neurological diseases and the presence of communication disorders were considered exclusion factors for both groups, with the exception of dysphonia for the experimental group . During the oral and vocal fold ddk test isolated emissions were requested; namely, the syllables " pa, " " ta " and " ka, " the sequence " pataka " and then the vowels " a " and " i. " Before the beginning of the test, the patient was requested to repeat the sound as rapidly as possible . The patients practiced the sounds before each recording by repeating as fast as possible, after deep breathing, in a clear and precise way the emissions with a comfortable frequency and intensity in order to understand how to perform the test . Soon thereafter, the subjects were guided to perform the tasks, by receiving a signal with the thumb to begin and to stop the repetition . The " pa, " " ta, " " ka, " " pataka, " " a " and " i " emissions were edited by sound forge 7.0 professional audio editing professional . The beginning and end of each sample were excluded, except for four seconds from the third to the sixth second . Monosyllabic emissions " pa, " " ta " and " ka " and the vowels " a " and " i " were filed at sampling rate of 11.025 hz, 16 bit mono channel and analyzed by advanced motor speech profile program (msp) model 5141 version 2.5.2 (kay's elemetrics corp ., lincoln park, nj, usa), whose parameters are described in figure 1 . The msp program displays a graphical record of emissions showing the horizontal axis (time in seconds) and the vertical axis (energy in db). To do the ddk count, the program drew a line in the central point at the energy scale of the vertical axis in db . The line of analysis was manually positioned in the corresponding value to the intensity average of the ddk sample, provided by the msp program, and moved up or down, so the program could count all emissions when necessary (figure 2). Parameters for diadochokinesis (ddk) analyzed by advanced motor speech profile (msp) program advanced motor speech profile (msp) program graphic, where time (seconds) on the horizontal axis and energy (db) on the vertical axis can be observed the ddk of the " pataka " sequence was analyzed quantitatively through the sound forge 7.0 program . The count of trisyllables per second was performed manually with the auditory and visual waveform support displayed by the program . Average values of oral and vocal fold ddk in dysphonic and nondysphonic women were correlated using the " t student " test and were considered significant when p<0.05 . Subjects were classified by the types of dysphonia, according to behlau, et al . It was considered primary functional dysphonia when there were no injuries or structural alterations of vocal folds identified by nasolaryngoscopy; secondary functional dysphonia to anatomical inadequacy, in the presence of structural alterations of vocal folds, such as epidermoid cyst, varicosity, stria sulcus; organic functional dysphonia, in the presence of edema, leucoplast or bilateral nodules; and organic dysphonia in cases of gastroesophageal reflux disease or unilateral vocal fold paralysis . Thus, the sample was composed of a participant with primary functional dysphonia, thirteen with secondary functional dysphonia by anatomical inadequacy, seven with organic and functional dysphonia, and three with organic dysphonia, because three women in the sample had no otorhinolaryngologic diagnosis . The results concerning the oral and vocal fold ddk of the two groups were compared using the " t student " test and are described in table 1 . Comparison of oral and vocal fold diadochokinesis (ddk) average and standard deviation, between dysphonic and nondysphonic women, in the emissions of " pa, " " ta, " " ka, " " pataka ", " a, " and " i. " Although the findings did not show a statistically significant correlation of oral and vocal fold ddk between the group of women with and without voice disorders (table 1), some aspects about the performance of diadochokinetic tasks are relevant and should be discussed . The results showed that the ddk speed, the average time between the vocalizations, the ability to keep constant vocalizations and keeping the intensity of these vocalizations constant did not correlate with the presence of dysphonia (table 1). It was expected that no neurological changes related to vocal production would interfere with oral and vocal fold ddk, since for both tasks there was participation of the opening and closing of vocal folds, especially related to " a " and " i " emissions, that involve specifically alternating motions of glottic adduction and abduction, since organic and functional factors may also interfere with the results of the ddk, including not necessarily expressing a disturbance in oral communication . It was observed that in the " pa " emission, the parameter cvi (p=0.061), although not statistically significant, showed a tendency for nondysphonic women to keep the vocalizations more constant as compared to the dysphonic group . This can be explained by the fact that the glottic changes found in dysphonic women, make intensity control difficult, since vocal balance depends on correct coordination between the myoelastic and aerodynamic forces of the larynx; this finding is possibly related to the size of the sample, thus, statistically significant values can be found if the number of subjects were more expressive . The impairment of phonoarticulatory organs can generate vocal consequences, such as changes in vocal quality, impaired laryngeal mobility, and extrinsic muscle tension of the larynx . The interference of articulation with phonation shows that such factors might affect the motor performance of these structures and thus, in ddk . Thus, there is a physiological and anatomical inter - connecting of the structures common to phonation and articulation . Thus, individuals who have dysphonia due to a myofunctional alteration that initiates larynx overload could have poorer ddk performance . Some minor structural alterations have morphological characteristics that change the vibration pattern of vocal folds . Therefore, the sulcus vocalis shows an arched vocal fold, and cysts appear on a bulged vocal fold . This could interfere with the speed of the vocal folds alternating motion rate (ddk). Leeper, heeneman and reynolds (1990) reported that, when ddk involves vowel emission, refined control of vocal fold opening and closing is required . Factors such as rigidity and mass can interfere with the fundamental frequency of voice, beyond subglottic pressure, which reflects on vocal intensity . There are significant differences in the effect of frequency and vocal intensity upon the airflow through the vocal folds . Furthermore, vocal intensity seems to be the primary factor controlling the speed of the ddk and airflow during the production of the vowel " a " . In this study, although no statistically significant correlation was observed, an average frequency of disturbances in the ddk " a " (ddcjiit) were higher in dysphonic subjects than in the control group, and this value was approximately doubled . High frequency values of ddk disturbance may indicate a lower ability to control the vocal folds' motion during phonation . Vocal fold ddk has not been investigated in detail like an analysis of laryngeal subsystem, as has already occurred with oral ddk . Nondysphonic individuals have appropriate adjustments in their laryngeal mechanism that allows for quick glottal opening and closing during the production of the consonants and vowels of syllables . This suggests that these adjustments in laryngeal diseases are inadequate, resulting in an increased effort to compensate . Therefore, information obtained with the ddk can be reference for the interpretation of complex tasks . There is a need for more studies that compare ddk in a control group and in dysphonic individuals, the latter split into distinct groups according to the vocal alteration, thus making it possible to compare diadochokinetic task performance differences in different dysphonias . Although the results don't indicate any difficulties in oral and laryngeal motor control in the dysphonic group, the major instability in vocal fold ddk in the experimental group should be considered, and studies of this ability in individuals with communication disorders must be intensified.
Restless legs syndrome (rls) is a sensorimotor disorder characterized by an urge to move the legs that are associated with unpleasant paresthesias . Usually, the symptoms worsen when at rest and at night and are relieved by movement . Various studies suggest that genetic component, iron - deficiency, disturbances in the dopaminergic neurotransmitter system and abnormality in spinal conduction pathways are associated with the disorder . Transcranial magnetic stimulation (tms), developed in 1985, is a noninvasive technique with the ability to stimulate neurons in the cerebral cortex through the scalp safely and with minimal discomfort . Repetitive tms (rtms) is a technique that delivers long trains of closely spaced pulses to specific brain areas in order to alter cortical activity and connectivity . Previous studies have suggested that low - frequency rtms decreases the excitability of the cortex while high - frequency rtms increases it . Recently, rtms has been widely applied in the treatment of patients with psychiatric disorders, epilepsy, migraine, chronic pain, and neurodegenerative disorders, including parkinson's disease (pd), although its mechanism is not yet well understood . Numerous reports have demonstrated that rtms of the human primary motor cortex induces the release of dopamine in the putamen, which indicates that rtms probably modulates striatal dopaminergic neurotransmission . Currently, an increasing number of studies have provided support for a link between rls and pd . Some studies have found that the prevalence of rls is higher in patients with pd than in the general population . Based on the common mechanism involving disturbances in the dopaminergic neurotransmitter system between rls and pd, we investigated whether rtms application to the cortex was beneficial in patients with rls . We included fourteen idiopathic rls patients treated at the sleep clinic of our hospital between 2011 and 2012 that were diagnosed according to the international criteria of the international restless legs syndrome study group set in 2003 . The exclusion criteria were as follows: (i) all secondary rls stemming from a vitamin deficiency, iron - deficiency anemia, pregnancy, diabetes mellitus, severe metabolic disorders, liver dysfunction, or renal disease; (ii) peripheral neuropathy and radiculopathy; (iii) a history of psychiatric disease; (iv) neuropathic pain; (v) leg cramps or epilepsy; (vi) use of a cardiac pacemaker, vagal nerve stimulator, or any metal implants; (vii) other severe medical diseases . Patients were not placed on any new medications, including dopaminergic agonists, psycholeptics, or benzodiazepines; if they were already taking them for at least 4 weeks prior to the initiation of the study, they continued the medicine throughout the study at the prescribed dosage . All patients provided written informed consent, and the study had the approval of hospital ethics committee . We administered rtms at 15 hz using a magstim system (magstim super rapid stimulator, magstim company, whitland, dyfed, uk) with a figure - eight coil . One rtms train consisted of 75 pulses delivered at 15 hz with an intertrain interval of 10 min . In one session, 600 pulses (8 rtms trains) were delivered to each hemisphere . One session was performed per day for 5 continuous days and stopped for 2 days . The patients were seated in a comfortable chair, and the coil was positioned at the leg representation in the motor cortex of frontal lobe . The optimal stimulation position for the tibialis anterior muscle was located by stimulating the presumed motor cortex at every 1 cm in a 6-cm . The resting motor threshold (rmt) was defined as the minimal stimulus intensity that produced a motor evoked potential in the relaxed muscle with a peak - to - peak amplitude of> 50 mv on 50% of 10 trials . The international rls rating scale (irls - rs), pittsburgh sleep quality index (psqi), hamilton anxiety scale (hama) and hamilton depression scale (hamd) were used to evaluate the severity of rls, quality of sleep, and the severity of anxiety and depression, respectively . The assessments were taken at the baseline (prior to stimulation), at end of 14 session, and at 1- and 2-month posttreatment by a trained clinical neurologist . One - way analysis of variance was used to compare the means of scale scores at different time points . A p <0.05 was considered as statistically significant . We included fourteen idiopathic rls patients treated at the sleep clinic of our hospital between 2011 and 2012 that were diagnosed according to the international criteria of the international restless legs syndrome study group set in 2003 . The exclusion criteria were as follows: (i) all secondary rls stemming from a vitamin deficiency, iron - deficiency anemia, pregnancy, diabetes mellitus, severe metabolic disorders, liver dysfunction, or renal disease; (ii) peripheral neuropathy and radiculopathy; (iii) a history of psychiatric disease; (iv) neuropathic pain; (v) leg cramps or epilepsy; (vi) use of a cardiac pacemaker, vagal nerve stimulator, or any metal implants; (vii) other severe medical diseases . Patients were not placed on any new medications, including dopaminergic agonists, psycholeptics, or benzodiazepines; if they were already taking them for at least 4 weeks prior to the initiation of the study, they continued the medicine throughout the study at the prescribed dosage . All patients provided written informed consent, and the study had the approval of hospital ethics committee . We administered rtms at 15 hz using a magstim system (magstim super rapid stimulator, magstim company, whitland, dyfed, uk) with a figure - eight coil . One rtms train consisted of 75 pulses delivered at 15 hz with an intertrain interval of 10 min . In one session, 600 pulses (8 rtms trains) one session was performed per day for 5 continuous days and stopped for 2 days . Then, another 4 days of stimulation were given . In total, 14 sessions were performed for each patient in our study . The patients were seated in a comfortable chair, and the coil was positioned at the leg representation in the motor cortex of frontal lobe . The optimal stimulation position for the tibialis anterior muscle was located by stimulating the presumed motor cortex at every 1 cm in a 6-cm . The resting motor threshold (rmt) was defined as the minimal stimulus intensity that produced a motor evoked potential in the relaxed muscle with a peak - to - peak amplitude of> 50 mv on 50% of 10 trials . The international rls rating scale (irls - rs), pittsburgh sleep quality index (psqi), hamilton anxiety scale (hama) and hamilton depression scale (hamd) were used to evaluate the severity of rls, quality of sleep, and the severity of anxiety and depression, respectively . The assessments were taken at the baseline (prior to stimulation), at end of 14 session, and at 1- and 2-month posttreatment by a trained clinical neurologist . One - way analysis of variance was used to compare the means of scale scores at different time points . A p <0.05 was considered as statistically significant . Among 14 patients, there were 4 males and 10 females with a mean age of 59.22 10.10 years and a range of 4673 years . The irls - rs scores at the four - time points assessed (baseline, end of 14 session, 1- and 2-month posttreatment) are summarized in table 1 . All of the irls - rs, psqi and hama scores showed continuous and significant improvement posttreatment compared to baseline . The irls - rs, psqi, hama, and hamd scores in the fourteen idiopathic rls patients irls - rs: international rls rating scale; psqi: pittsburgh sleep quality index; hama: hamilton anxiety scale; hamd: hamilton depression scale . * different from baseline, p<0.05 . Our results indicated a significant improvement in irls - rs scores after 14 sessions, from 23.86 5.88 to 11.21 7.23 . This proved the effect of rtms in treating rls and that the effect could last for some time (at least 2 months, according to our study) after the stimulation . Khedr et al . Found that repeated sessions of rtms could produce prolonged changes in enhanced dopamine function that may be responsible for long - lasting clinical effects . After rtms treatment, psqi scores decreased from 15.00 4.88 to 9.29 3.91 and persisted at least 2 months after treatment . The long - lasting change in psqi scores was consistent with the improvement in irls - rs scores . Found rls patients had significantly higher levels of anxiety and depression . In our study, rls patients showed obvious improvements in anxiety, and further improvements occurred in the 2 months after treatment . In addition, the patients depression improved after treatment, although this change was not statistically significant . It is likely that rtms improved symptoms of rls directly rather than its associated symptoms, such as mood disorders, which in turn led to an improvement in symptoms of rls . Many studies have indicated that rls is not associated with the use of antidepressants, and some studies have suggested that antidepressants might exacerbate rls symptoms . In addition, depression did not improve as significantly as the symptoms of rls in our study . Thus, we believe that rtms alleviates rls directly rather than through the treatment of associated symptoms . Found that high - frequency rtms over the supplementary motor area (sma) improved irls - rs scores significantly after 5 and 10 sessions of stimulation . This result is consistent with ours, although the configuration of stimulation parameters differed, and they did not evaluate sleep quality, anxiety or depression . In burcu's study, the stimulus frequency was 5 hz, and the stimulation was centered at points 3 cm anterior to the leg motor area at the sagittal midline . Their parameters were taken from a previous study of pd, in which 5 hz rtms was administered over the sma, resulting in a modest improvement of motor symptoms . A recent study verified connectivity between the sma and the primary motor cortex . Because stimulating either the sma or the primary motor cortex can alleviate rls syndrome, we speculate a common effect of rtms on upstream sma and downstream primary motor cortex . The exact mechanism underlying the treatment of rls with rtms is complex and not clearly understood . Previous studies have revealed a shortened cortical silent period in the anterior tibialis muscle in patients with rls, which indicated a disturbed supraspinal inhibition mediated by decreased excitation of cortical inhibitory interneurons, thus leading to the hyperexcitability of spinal pathways . Another probable mechanism involves the release of endogenous dopamine in the striatum, based on single photon emission computed tomography studies . However, a study conducted in japan suggested that chronic rtms had a limited effect on the dopaminergic system . Hence, there may be multiple mechanisms of action involved in modulating symptoms of rls . Revealing the exact mechanism is interesting and valuable and should be the subject of future study . In addition, no adverse effects were observed during stimulation or after treatment, and all patients showed good compliance . Thus, 15 hz rtms delivered over the leg representation area of motor cortex is a safe treatment for rls . Medications traditionally used to treat rls provide dramatic immediate benefits but may augment rls symptoms over time . Thus, rtms and medications each possess advantages and provide patients with a variety of treatment options . However, our study had a limited sample size and no control group, therefore, a large case - control study is necessary to provide more convincing evidence . In addition, an optimized rtms paradigm should be established . In the future, we plan to compare cortical excitement before and after rtms treatment using neurophysiological and imaging measurements, such as paired pulse tms and functional magnetic resonance imaging, to identify the mechanism underlying rls . In conclusion, our study proves the utility of rtms for the treatment of rls patients despite the study's limited sample size . In the future, a large case - control study should be performed, and the rtms protocol should be optimized.
Malaria control programmes utilising indoor residual spraying are only effective if a high coverage of targeted structures is achieved and an insecticide that is effective against the specific mosquito vector is correctly applied . Ongoing monitoring of spraying operations is essential to assure optimal programme performance and early corrective action, where indicated . Successful development and application of a computerised spraying operations management system in mpumalanga province, south africa during 1998 resulted in its adaptation and introduction in neighbouring maputo province, southern mozambique during 2000 . The structure and components of this computerised management system are described, and its' operational benefit in southern mozambique, where community - based spray operators apply intradomiciliary insecticide, are reviewed . The computerised management system allowed malaria programme management and field supervisors to monitor spraying coverage, insecticide consumption and application rates on an ongoing basis . The system supported a successful transition to community - based spraying, while assuring correct insecticide application and spraying completion according to schedule . In 1946, south africa introduced intradomiciliary spraying with residual insecticides, ddt (dichlorodiphenyltrichloroethane) and bhc (benzene hexachloride), to kill indoor - resting vector mosquitoes and thereby control malaria . This resulted in a 75 percent reduction in the geographic extent of the malaria - affected area, with malaria occurrence limited to summer epidemics in the low - lying northern and eastern border areas with botswana, zimbabwe and mozambique . Similar malaria control programmes were initiated in other southern african countries . In mozambique, spraying operations for malaria control collapsed during the 1970s due to the protracted civil war with a resulting high burden of endemic malaria throughout the country . Recently there has been a resurgence of malaria in southern africa, attributed to a number of factors, including parasite drug - resistance, mosquito insecticide - resistance, climate changes and large - scale population migration . The hiv / aids epidemic has resulted in a simultaneous dwindling of resources available for public health programmes, thus placing an onus on programme managers to ensure optimal efficiency of their activities . An efficient spraying programme is characterized by application of the correct volumes of insecticide on surfaces suitable for mosquito resting . This should be achieved before the onset of peak malaria transmission with a high coverage of targeted structures . A formal spraying management system was introduced in south africa for the first time in 1973, with activity forms being completed by the field officials responsible for each spraying team and a " hut - card " left under the eave of each sprayed dwelling . This was expanded to a set of seven different spraying record forms in 1975 under the auspices of the world health organization . This set consisted of a spray operator record, a daily spray - team report, a weekly report, a monthly report, a locality completion report, a sector completion report and a spraying completion report . Although this manual system was cumbersome it became entrenched, and remained essentially unaltered in south africa for more than 20 years . A major flaw of this data system was that field staff were not trained to use the data for programme monitoring . Thus serious programme deficiencies usually only became apparent at the end of the spraying season when the data were centrally analysed . Mpumalanga province, located on the border with mozambique in the north - east of south africa, began to replace permanently employed spray personnel with temporary spray personnel recruited from each malaria - affected community . One hundred community - based spray personnel were appointed, each accountable to their local community, and the team was responsible for spraying almost 170,000 structures during each annual spraying round . With supervisory support limited to one field manager and six field officers, it was postulated that unless an improved spraying management system was introduced, programme performance and efficiency might deteriorate . A computerised management system was developed in mpumalanga province to enable malaria programme management and field supervisors to monitor, even on a daily basis when required, spraying coverage, individual spray operator's performance, and insecticide consumption and application rates . The success of this computerised management system resulted in it being extended to neighbouring swaziland and also to southern mozambique, where a malaria vector control programme was recently re - introduced through the multilateral lubombo spatial development initiative (lsdi), a partnership between the governments of mozambique, swaziland and south africa . We describe the structure and components of this computerised management system, and provide operational evidence of its value in the southern mozambique malaria control programme . The computerised spraying management system in southern mozambique was designed prior to the first round of spraying early in 2000 . The area targeted for spraying included the rural districts of namaacha and matutuine, and sections of the peri - urban districts of matola and boane in maputo province, covering an area of 7,962 square kilometres with an estimated 200,000 homes . The management system comprises two platforms, a spraying database and a spatial mapping platform . A microsoft access 2000 database was used with front - end data entry and output screens . The data entry screen mirrors the manually completed weekly spraying form, which collates data from daily spraying report forms that are submitted each week by spray operators to their supervisor (figure 1). " Drop - down " menus ensure speed, ease and accuracy of data entry . A number of datasets have been integrated to form the basis of the spraying data base, including a comprehensive listing of place names to be sprayed, number of homes (ministry of health database), demographic data for each electoral area (1995 electoral census by the department of planning and cooperation), spray operator identification codes and details of insecticides used during spraying operations . Each record is automatically allocated a unique number and all information can be linked through a relational database to a specific spray operator, team, time - period or administrative area . Additional information collected includes the number of visits made to a specific house, reasons for not spraying (where applicable), insecticide used by each spray operator and number of spray - can refills . Data is automatically backed up after each day of data entry onto an external zip drive . Data entry screen of the malaria information system the geographic information system (gis) platform required both spatial (roads, clinics, locality boundaries) and attribute data (locality or clinic names, population figures). The gis software package mapinfo professional[mapinfo corporation, 4 global view, troy, new york, 12189, usa .] Was used and maps of southern mozambique (scale 1:250,000) were obtained from the direco nacional de geografia e cadastro in maputo . The south african medical research council digitally captured relevant geographical and cadastral features, including roads, rivers, towns and different administrative level boundaries . The smallest administrative unit boundaries at which data was captured (localities) were drawn onto local maps by the staff of the ministry of health involved in the project and then digitally captured for inclusion in the gis display . Preset queries, to generate standardized management reports, were planned and designed in partnership with field supervisors and programme management staff to meet their specific needs . However, the system was designed to allow user - friendly production of customized additional reports where required . Specific training was provided for programme supervisors on making sense of data and interpreting reports, with a spraying database manual developed for ready reference . A major emphasis in training has been on identifying operational problems and implementing timely remedial action . As field managers enter the summary data at the namaacha field office on a weekly basis, necessary remedial action is taken within a week . The ongoing monitoring of individual spray operator's productivity, in terms of number of structures sprayed each day and volume of insecticide sprayed per structure, has facilitated early detection of operational problems, leading to prompt investigation and supportive corrective action by supervisors . A standardized aid for evaluating individual operators' spraying quality during field visits was designed and implemented . Field managers discuss evaluation results with spray operators on - site in order to eliminate mistakes . The frequency of required supervisory corrective interventions has steadily decreased since the beginning of the programme and it is likely that some of this effect is due to the enhanced monitoring and response made possible by the computer management system . Insecticide application rates (grams per square meter) are constantly monitored for under- and over - application . Under - application reduces insecticide residual activity and thus effectiveness, while over - application is wasteful . Where sub - optimal application rates were detected, investigation included scrutiny of insecticide preparation, nozzle condition, application pressure, distance of nozzle tip from sprayed surface, and spraying rhythm . Spraying coverage exceeded 90% of 222,000 structures in southern mozambique, a level that is considered more than adequate . In areas where the management system repeatedly identified lower coverage than expected, investigations were conducted to determine whether this was the result of diminished community support, absenteeism or poor motivation of the specific spray operator . Maps depicting the coverage achieved by each team proved useful to malaria control programme management for tracking teams' progress, providing ongoing updates to senior health management and encouraging spray personnel to attain their target (figure 2). An example of a spraying progress map by locality in maputo province, mozambique, 2003 an additional benefit was that data captured during the first spraying round during 2000 was used to verify official data sources and provide actual household figures to replace official government estimates . This provided more precise information for malaria control planning and demographic data needed by other government agencies for planning . Competition for ever - dwindling public health resources is a major challenge for malaria control programmes in sub - saharan africa . Constant monitoring and evaluation of spraying activities is obligatory where indoor residual spraying is an important component of malaria control, to ensure effective application and prevent wastage . Computerised management systems are proving useful tools for providing timely information to address potentially deleterious human, social and technical factors that could negatively impact on malaria control . The computerised management system has been in operation for three years supporting the mainly rural vector control programme that now covers an area of 13,770 square kilometres in southern mozambique . Not only has it supported a successful and cost - effective transition to a community - based spraying approach, but it has also ensured that spraying was completed to schedule . The ability to immediately identify problems at the level of an individual spray operator and institute appropriate remedial action assured good coverage and programme efficiency . The computerised management system is a useful method for enhancing malaria control by improving application of existing tools . Note: a powerpoint presentation providing more detail on the malaria management information system and database is available at . Mb, bs, cm, jlg, dd conceptualised, developed and implemented the mpumalanga computerised management system prototype; mb, bs, cm, bm adapted this system for application in maputo province, mozambique . All authors contributed to the review of the system and the preparation of this manuscript.
Tuberculosis (tb) is still a major cause of morbidity and mortality around the world and the second leading infectious cause of death among adult globally . The risk factors for the development of tuberculosis include immigration, low income populations, immunosuppression, human immunodeficiency virus, and living in close contact with patients suffering from tuberculosis [2, 3]. The precise incidence of genital tb cannot be determined with certainty as some cases are asymptomatic and uncovered accidentally during investigation of infertility . In developing countries, genital the symptoms and signs of abdominopelvic tb can mimic peritoneal carcinosis or ovarian malignancy . Based on iranian national health program, it is more than 50 years that all iranian newborn will receive vaccination against tb after delivery and before releasing from the hospital . But in cities close to the country borders, cases of tb have been reported mainly due to immigration from neighborhood countries particularly afghanistan, pakistan and iraq . Although pulmonary tb can be diagnosed by its particular signs and symptoms as well as laboratory and imaging assessment, peritoneal tb has common symptoms with advanced ovarian carcinoma, including pelvic pain, mass, ascites, and elevated serum ca125 levels . Based on literature review some other case reports showed ambiguity in the differentiation between peritoneal tuberculosis and advanced ovarian cancer before operation [2, 57]. Therefore, preoperative diagnosis between these two distinct diseases continues to be a dilemma . We presented three cases of peritoneal tuberculosis mimicking ovarian malignancy to point out the importance of the histopathologic diagnosis before chemotherapy in women suspected having advanced ovarian cancer, especially if there is a family history of pulmonary tb or infertility . Three women with abdominopelvic tb were operated during one year in taleghani hospital with preoperative suspicious of ovarian malignancy . Case 1 a 24-year - old married, nulligravid woman was referred from gasteroentrology ward to the gynecologic ward with the complain of abdominal pain and distension, nausea, on and off vomiting in the preceding 3 months . Her past medical history was unremarkable but in gynecologic history hypomenorrhea was highlighted (figure 1). Family history revealed that her father had been diagnosed with pulmory tb, also her cousin had been involved with genital tb . During physical examination, she was a pale young lady with a distended abdomen and a fixed mass palpated up to the umbilicus . In her paraclinic investigations, among all serum tumor markers related to ovarian cancer, there was only a mild elevation of ca-125 (45 u / ml) hemoglobin was 9.0 mg / dl . As she had a positive family history for tb a chest x - ray and skin tuberculin test have been performed and both were negative for tb.pelvic ultrasonography showed a normal size uterus with endometrial thickness equal to 5 mm . There was a septated multicystic mass of 134 140 81 mm cube in right adnexa with extension toward the left adnexa . Left ovary was also bigger than normal (70 35) mm containing multiple follicles . Sonographic impression was mucinous cystadenoma of ovary or hydatid cyst.abdominopelvic computed tomography showed large amount of ascites in peritoneal cavity with cystic and solid vegetative mass, mild thickening of peritoneal layers in lower abdomen suggesting ovarian mucinous cystadenocarcinoma with peritoneal metastasis was noted . Other possibilities suggested were primary carcinoma of mesenteric, peritoneal like mesothelium, or papillary carcinoma of peritoneum.because of highly suggestion of ovarian malignancy preoperation, the patient underwent exploratory laparatomy with midline incision . Total volume of 400cc ascitic fluid was aspirated and sent for cytology and culture . Adhesions were released . Then left ovary 5 5 6 cm appeared with hemorrhagic cyst and adhesion to the left fallopian tube . A 24-year - old married, nulligravid woman was referred from gasteroentrology ward to the gynecologic ward with the complain of abdominal pain and distension, nausea, on and off vomiting in the preceding 3 months . Her past medical history was unremarkable but in gynecologic history hypomenorrhea was highlighted (figure 1). Family history revealed that her father had been diagnosed with pulmory tb, also her cousin had been involved with genital tb . During physical examination, she was a pale young lady with a distended abdomen and a fixed mass palpated up to the umbilicus . In her paraclinic investigations, among all serum tumor markers related to ovarian cancer, there was only a mild elevation of ca-125 (45 u / ml) hemoglobin was 9.0 mg / dl . As she had a positive family history for tb a chest x - ray and skin tuberculin test have been performed and both were negative for tb . Pelvic ultrasonography showed a normal size uterus with endometrial thickness equal to 5 mm . There was a septated multicystic mass of 134 140 81 mm cube in right adnexa with extension toward the left adnexa . Left ovary was also bigger than normal (70 35) mm containing multiple follicles . Abdominopelvic computed tomography showed large amount of ascites in peritoneal cavity with cystic and solid vegetative mass, mild thickening of peritoneal layers in lower abdomen suggesting ovarian mucinous cystadenocarcinoma with peritoneal metastasis was noted . Other possibilities suggested were primary carcinoma of mesenteric, peritoneal like mesothelium, or papillary carcinoma of peritoneum . Because of highly suggestion of ovarian malignancy preoperation, the patient underwent exploratory laparatomy with midline incision . Adhesions were released . Then left ovary 5 5 6 cm appeared with hemorrhagic cyst and adhesion to the left fallopian tube . Case 2 a 39 year old married woman gravida 6, para 6 has been transferred to the gynecologic ward from the gasteroentrology ward with the complain of abdominal pain, weight loss and fever in the last 4 months (figure 2). Her past medical history was unremarkable, and in her surgical history, last 2 deliveries were through cesarean section and she performed the tubal ligation . It is worth to mention that her normal menstrual pattern has been changed to oligomenorrhea since last 6 months . She had also a positive family history; her mother had been diagnosed with pulmonary tb . As case 1, due to positive family history for tb, a chest x - ray and skin tuberculin test has been performed and both were negative for tb.profiling of serum tumor markers and revealed a high level of ca-125: 207 u / ml . Hemoglobin was 10 mg%.pelvic ultrasonography disclosed uterus and adnexa as a large complex cystic mass shifting to the right with extensive adhesion to the adjacent structures . As the preoperation assessment was in favour of ovarian malignancy, the patient underwent exploratory laparatomy with a midline incision . There was no ascites, but miliary seeding all over the peritoneum and small bowel and large bowel, uterus, fallopian tubes and ovaries were seen, which were biopsied . A confluent mass containing uterus, ovaries and fallopian tube was seen which was necrotic . The frozen sections of the biopsied specimens showed granulation tissue, but as the family was completed and the mass was necrotic, hysterectomy and bilateral salpingo oophorectomy have been done . The permanent result of pathology showed many granulomatous lesion comprised epithelial cells, histiocytes, giant cells . Right ovary and right tube were unremarkable, left ovary, fallopian tube and uterus had granulomatous lesion consistent with tuberculosis . Old married woman gravida 6, para 6 has been transferred to the gynecologic ward from the gasteroentrology ward with the complain of abdominal pain, weight loss and fever in the last 4 months (figure 2). Her past medical history was unremarkable, and in her surgical history, last 2 deliveries were through cesarean section and she performed the tubal ligation . It is worth to mention that her normal menstrual pattern has been changed to oligomenorrhea since last 6 months . She had also a positive family history; her mother had been diagnosed with pulmonary tb . Her abdominopelvic examination revealed a fixed and tender mass in the pelvis . As case 1, due to positive family history for tb, a chest x - ray and skin tuberculin test has been performed and both were negative for tb . Profiling of serum tumor markers and revealed a high level of ca-125: 207 u / ml . Pelvic ultrasonography disclosed uterus and adnexa as a large complex cystic mass shifting to the right with extensive adhesion to the adjacent structures . As the preoperation assessment was in favour of ovarian malignancy, the patient underwent exploratory laparatomy with a midline incision . There was no ascites, but miliary seeding all over the peritoneum and small bowel and large bowel, uterus, fallopian tubes and ovaries were seen, which were biopsied . A confluent mass containing uterus, ovaries and fallopian tube was seen which was necrotic . The frozen sections of the biopsied specimens showed granulation tissue, but as the family was completed and the mass was necrotic, hysterectomy and bilateral salpingo oophorectomy have been done . The permanent result of pathology showed many granulomatous lesion comprised epithelial cells, histiocytes, giant cells . Right ovary and right tube were unremarkable, left ovary, fallopian tube and uterus had granulomatous lesion consistent with tuberculosis . Case 3 a 57-year - old married woman, gravida 9, para 8, consulted with the gynecologic ward by gasteroentrology consultant and then admitted complaining abdominal pain and distension since last two months (figure 3). She lost 10 kg during these 2 months which is highly suggestive of a serious problem . Ultrasonography showed normal liver, spleen, kidneys, and uterus, but showed cystic masses in both adnexa with septation in favour of malignancy . So laparatomy with midline incision was done with impression of ovarian malignancy.about one liter ascites aspirated and omental cake with miliary seeding, which were seen all over the peritoneum and pelvic organs and over the liver and spleen, has been biopsied . Ovarian masses were removed and sent for frozen sections along with the other biopsied specimens with the result of granulomatosis . Because of necrotic and hemorrhagic appearance of uterus and ovaries as well as the patient's age and condition total abdominal hysterectomy and bilateral salpingooophorectomy were done to prevent postoperation complications . Right ovary with granulomatous inflammation in favour of tb ascitic fluid cytology showed inflammatory cells . Patient was discharged with antituberculosis drugs.all three cases were in very good status in 6 months followup with normal pelvic exam, normal pelvic ultrasonography and ca125 measurement . A 57-year - old married woman, gravida 9, para 8, consulted with the gynecologic ward by gasteroentrology consultant and then admitted complaining abdominal pain and distension since last two months (figure 3). She lost 10 kg during these 2 months which is highly suggestive of a serious problem . Ultrasonography showed normal liver, spleen, kidneys, and uterus, but showed cystic masses in both adnexa with septation in favour of malignancy . About one liter ascites aspirated and omental cake with miliary seeding, which were seen all over the peritoneum and pelvic organs and over the liver and spleen, has been biopsied . Ovarian masses were removed and sent for frozen sections along with the other biopsied specimens with the result of granulomatosis . Because of necrotic and hemorrhagic appearance of uterus and ovaries as well as the patient's age and condition total abdominal hysterectomy and bilateral salpingooophorectomy were done to prevent postoperation complications . Right ovary with granulomatous inflammation in favour of tb ascitic fluid cytology showed inflammatory cells . All three cases were in very good status in 6 months followup with normal pelvic exam, normal pelvic ultrasonography and ca125 measurement . Peritoneal tb with nonspecific symptoms mimicking ovarian malignancy is a serious problem especially in developing countries . Diagnosis of peritoneal tb before operation is not easy, there is no particular laboratory or imaging assessment to differentiate this disease from advanced ovarian cancer . In our 3 cases diagnosed with abdominopelvic tb, two were in reproductive age (cases 1 and 2) and one postmenopausal (case 3). All the cases were admitted firstly to gasteroentrology due to abdominopelvic pain and distension and then transferred to gynecology oncology ward with the diagnosis of advanced ovarian malignancy . Adnexal mass suspicious of malignancy in ct scan and/or sonography and elevated ca125 were seen in all cases . Family history for pulmonary tb was positive in cases 1 and 2 and genital tb for case 1 . The first two cases had a history of oligomenorrhea and hypomenorrhea in the last year before surgery . Due to the positive family history, peritoneal as previous case reports [7, 8] in all our 3 cases non - invasive tests, such as acid - fast stain and culture of the ascitic fluid, ultrasonographic features of the abdomen and pelvis, ct, as well as serum ca125 level were nonspecific for differentiating abdominopelvic tb from ovarian malignancy . In our 3 cases, because of ambiguity of the results of non invasive tests, explorative laparotomy with a midline incision has been done before treatment . Ascites or peritoneal washings were sent for cytology as well as bacteriologic examination which was negative for both malignant cell as well as tb miliary deposits all over . The omentum or peritoneal surfaces were seen and assessed by frozen - section analysis, which detected granulamotous inflammation with central necrosis highly suggestive of tuberculous peritonitis . Therefore, although tuberculin skin test may be helpful but might be negative, as in our cases, it might be positive because vaccination is a national program in our country . The ca125 level, which is elevated in more than half of early and two thirds of advanced epithelial ovarian malignancy, can be increased in peritoneal tb [9, 10]. Predictive value, specificity, and sensitivity of this marker to reveal epithelial ovarian malignancy are less in premenopausal than postmenopausal due to other benign conditions [11, 12]. Therefore, in areas where tb is endemic, in premenopausal women with elevated ca125, this infection should be considered . Some studies assessed new diagnostic tools to differentiate abdominopelvic tb from malignamcy before treatment . (1998) studied the polymerase chain reaction for mycobacteria but they reported a negative results in cases of peritoneal tuberculosis mimicking advanced ovarian cancer . Tinelli (2008) suggested that abdominopelvic laparoscopy with histopathological findings plus enzyme - linked immuno - spot can confirm the suspect of ap - tb . Although we were unable to perform the last two investigations, their validity is still under investigation . Consequently, we suggest a laparatomy and biopsy of involved area as the gold standard method for definite differentiation between advanced ovarian malignancy and peritoneal tb especially in endemic areas for tb with limitations of using new or expensive technology . Although some studies suggest laparoscopic biopsy for histologic diagnosis, we preferred a minilaparatomy to decrease the complications of laparoscopic entrance to the abdomen with extensive adhesions between loop of intestines, abdominal wall, and pelvic organs seen in abdominopelvic tb . Although this report was not a case control study but it seems that removing the infected pelvic masses from the field of the operation before beginning the medical therapy is beneficial . In conclusion, cytology or histopathologic verification of ovarian malignancy before beginning of aggressive therapy including debulking and or adjuvant chemotherapy is mandatory . Based on these cases, even in the presence of symptoms and signs highly suggestive of ovarian malignancy, the presence of familial history of pulmonary and/or peritoneal tb along with complaining of oligomenorrhea or hypomenorrhea with low socioeconomic situation are enough markers for the physician to rule out abdominopelvic tb . Women living on the border of our country, close to immigrants campus are mostly at risk to develop the tb . Therefore, taking a precise personal and family history as well as geographic and socioeconomic status of the patients will be helpful . Considering this, we suggest that all families, friends, and people living in the vicinity of women diagnosed with tb should be called through a national health program to be tested for tb and treated accordingly.
Stroke is the second leading cause of death and causes permanent disability in one - third of survivors1, 2 . Many stroke patients suffer from motor impairments that limit muscle control, activities of daily living, and locomotion activity . In addition, stroke patients have increased double support duration, decreased cadence, shorter stride, decreased stance time, and shorter lower limb joint range of motion compared to able - bodied persons3,4,5 . Rhythmic auditory stimulation (ras) uses musical stimuli to improve the gait performance of patients who have neurological conditions (e.g., parkinson s disease, cerebral palsy, traumatic brain injury, stroke)6 . Because music includes temporal characteristics such as anticipation and predictability7, ras may act on the indicators that inform the gait - phase sequence . Stroke patients have shown better performance when engaging in conventional gait training with ras versus conventional gait training only6 . For example, a stroke group that received ras combined with gait training showed greater improvements in gait velocity, cadence, stride length8, and hip joint motion symmetry and decreased vertical center of mass displacement9 . Lee et al.10 reported improvements in gait symmetry, velocity, and cadence in stroke patients when gait training by matching the footfall of the affected side with ras and applying baseline and> 30% baseline speed . Compared to the control group, the chronic stroke patients who had trained with home - based auditory stimulation showed significant improvements in affected side stride length, unaffected side stride length, stride length ratio, affected side single support time, single support time ratio, and gait velocity11 . Recently, the results of studies with auditory cueing systems for gait rehabilitation training have been reported . Nagano, japan)12, 13, which was developed to simultaneously support locomotion compensation and gait rehabilitation training using ras methods . This device consists of a transmitter that pairs with three - axis acceleration sensors worn on each ankle and wireless headphones . Muto et al.13 reported a significant decrease in ground - contact timing asymmetries and gait tempo fluctuation during gait training with the walk - mate device in stroke hemiplegic patients . In another study, casamassima et al.14 described inertial measurement units (imu) based on a wearable auditory feedback system that monitors gait training with parkinson s disease patients . This system provides real - time audio feedback and estimates spatiotemporal gait parameters via gait event data detected from imu sensors mounted on both shoes . The advantages of the application include its portability, simple configuration, inclusion of various auditory stimuli, and ease of saving real - time data . This device type must have the capacity to be used during gait training anywhere at any time . In this study, two aspects of our smartphone - based ras application were investigated: (1) the effects of various ras tempos on stroke gait patterns and (2) the effects of short - term gait training on spatiotemporal gait parameters in stroke patients . The inclusion criteria were as follows: at least 6 months since stroke, no visual or auditory deficits, and independent gait> 10 meters . The general characteristics of the study participants are detailed in table 1table 1.participant informationparticipants (n=15)gender (m / f)11/4age (years)56.0 (7.4)height (cm)167.7 (7.7)weight (kg)68.3 (8.2)paretic side (left / right)8/7since onset (months)81.9 (87.8)mmse - k28.4 (2.1)mbi96.3 (4.4)values are means (sd). Mmse - k: mini mental state examination - korean version, mbi: modified barthel index . The institutional review board of the korea national rehabilitation center approved the study protocol . The purpose and procedures of the study were fully explained, and all participants were required to provide informed consent . Mmse - k: mini mental state examination - korean version, mbi: modified barthel index gait training with ras was performed in an unobstructed space and additional auditory stimuli were blocked in order for subjects to concentrate on the ras sound . Each ras condition was practiced for 10 minutes . A 3-minute adaptation period and a 7-minute gait - training period all subjects walked at a comfortable speed three times, after which their mean cadence (baseline) was calculated and converted to 10%, 5%, 0%, + 5%, and + 10% of baseline . The mean cadence was calculated by averaging three 10-meter walk individual times, while subtracting the acceleration and deceleration periods . The ras application has seven sound sources (c - e - g, c - f - a, a - d - g, clap, click, gun, and robot) that could be selected and applied based on the user s preference . To adapt to the ras sounds, participants listened to the rhythm of the application and were instructed to tap their fingers in their lap or stamp their feet when in a seated position . Then, subjects were asked to listen carefully to the rhythm of the ras application and heel strike to the sounds . Ten - minute intervals between trials eliminated any remaining effects that could influence the next trial . Imu sensors (shimmer3, shimmer, dublin, ireland) were attached above both ankle joints for heel - strike detection . These sensors were connected to the ras smartphone application via bluetooth, and heel - strike detection information was shown on the display as a red dot . Thus, gait training focused on the timing of heel strikes on the ground and patients were asked to execute heel strikes in tandem with the ras sound . A validated wireless inertial sensing device (g - walk, bts s.p.a . The sensor was attached to subjects waists (l45 intervertebral space) using a semi - elastic belt to determine the acceleration values for three anatomical axes: anterior - posterior, medial - lateral, and vertical17 . Park and woo18 suggested that the g - walk represents a valid method for assessing the effectiveness of clinical therapeutic interventions of gait analysis . Their comparative study proved that there is a significant and high correlation between gait velocity and cadence and the foot pressure sensor system (gaitrite, cir systems, inc ., spatiotemporal parameters such as gait speed, cadence, stride length, gait cycle duration, and step length on the affected and unaffected sides were recorded . The spatial symmetry ratio was calculated by recording the step length on the affected and unaffected sides . For each trial, the subjects were asked to walk a level gait way (16 m). To eliminate acceleration and deceleration effects, only the middle distance (10 m) was used for calculations . Data collection was repeated three times with 3 minutes of rest between trials . To determine the effect of various ras tempos on gait patterns, the kolmogorov - smirnov test was used firstly to check if the data were normally distributed . Secondly, a one - way repeated measures analysis of variance (anova) and least significant difference (lsd) post - hoc analysis were performed . Data were analyzed using spss statistics for windows, version 21.0 (ibm corp ., armonk, ny, usa) parametersbaseline10%5%0%+5%+10%after ras trainingspeed(m / min)70.53(12.36)57.84(8.27)64.30(9.74)68.53(10.89)77.22(16.07)77.53(15.21)79.53(15.85)cadence(steps / min)51.89(6.23)46.30(5.35)48.99(6.03)51.44(6.07)53.73(6.50)55.32(6.79)53.30 (7.22)stride length(m)1.37(0.22)1.27(0.17)1.32(0.18)1.35(0.18)1.44(0.22)1.42(0.24)1.50(0.22)gait cycleduration (s)1.18(0.16)1.32(0.17)1.25(0.17)1.18(0.16)1.13(0.16)1.10(0.15)1.15(0.18)step lengthaffected side (m)0.69(0.11)0.65(0.09)0.67(0.10)0.67(0.09)0.73(0.12)0.72(0.12)0.74(0.09)step lengthunaffected side (m)0.68(0.13)0.61(0.10)0.65(0.11)0.67(0.10)0.70(0.13)0.68(0.13)0.75(0.15)symmetry ratio(step length)1.04(0.17)1.06(0.14)1.04(0.15)1.02(0.15)1.06(0.16)1.08(0.15)1.02(0.14)values are means (sd). * significantly different from baseline (without ras) (p<0.05). Significantly different from the 10% ras condition (p<0.05). Significantly different from the 5% ras condition (p<0.05) significantly different from the 0% ras condition (p<0.05). Significantly different from the + 5% ras condition (p<0.05). * * significantly different from the + 10% ras condition (p<0.05). Significantly different from after ras training (without ras) (p<0.05). In the 10% ras condition, m / min, f=34.219), cadence (46.30 5.35 steps / min, f=114.253), stride length (1.27 0.17 m, f=12.082), step length on the affected side (0.65 0.09 m, f=14.026), and step length on the unaffected side (0.61 0.1 m, f=13.066) decreased significantly compared to baseline (p <0.05). Furthermore, gait cycle duration (1.32 0.17 s, f=150.236) increased significantly compared to baseline (p <0.05). In the 5% ras condition, m / min, f=34.219) and cadence (48.99 6.03 steps / min, f=114.253) decreased significantly, while gait cycle duration (1.25 0.17 s, f=150.236) increased significantly compared to baseline (p <0.05). In the + 5% ras condition, gait speed (77.22 16.07 m / min, f=34.219) and cadence (53.73 6.5 steps / min, f=114.253) increased significantly, while gait cycle duration (1.13 0.16 s, f=150.236) decreased significantly compared to baseline (p <0.05). In the + 10% ras condition, gait speed (77.53 15.21 m / min, f=34.219) and cadence (55.32 6.79 steps / min, f=114.253) increased significantly, while gait cycle duration (1.10 0.15 s, f=150.236) decreased significantly compared to baseline (p <0.05). M / min, f=34.219), cadence (53.30 7.22 steps / min, f=114.253), stride length (1.50 0.22 m, f=12.082), step length on the affected side (0.74 0.09 m, f=14.026), and step length on the unaffected side (0.75 0.15 m, f=13.066) increased significantly compared to baseline (p <0.05). Gait cycle duration (0.75 0.15 s, f=150.236) decreased significantly compared to baseline (p <0.05). Values are means (sd). * significantly different from baseline (without ras) (p<0.05). Significantly different from the 5% ras condition (p<0.05). Significantly different from the 0% ras condition (p<0.05). Significantly different from the + 5% ras condition (p<0.05) * * significantly different from the + 10% ras condition (p<0.05). Significantly different from after ras training (without ras) (p<0.05) ras is an increasingly popular form of rehabilitative exercise therapy for neurological gait disorders . Auditory - motor synchronization in the reticulospinal tract is the key concept of ras19 . Timing cues in gait motion have the capacity to improve movement control and change temporal gait parameters7 . Several studies have demonstrated the effects of ras on neurological gait disorders such as stroke, parkinson s disease, and cerebral palsy . These studies reported improvements in gait velocity, stride length, and cadence in response to ras rehabilitative therapeutic methods8, 19, 20 . However, most studies of stroke patients have used ras with constant speed- or time - based stimulation increases . Cha et al.21 studied the immediate effects of four ras tempos (10%, 0%, + 10%, and + 20%) on the gait patterns of hemiplegic stroke patients . The authors reported that a faster ras tempo increased gait velocity, which indicated that there was potential to immediately improve walking abilities . Suh et al.22 reported improvements in gait velocity, stride length, cadence, and standing balance in a group of hemiplegic stroke patients who received gait training with 5% and 10% increased ras compared to a group that did not receive ras . In this study, the effect of various ras tempos on gait patterns was examined and the effectiveness of short - term gait training with a smartphone - based ras application was confirmed . The results showed significant differences in spatiotemporal gait parameters after ras training compared with those before training . In the 10% ras condition, gait speed, cadence, stride length, and step length on the affected and unaffected sides decreased significantly, while gait cycle duration increased significantly . In the 5% ras condition, gait speed and cadence decreased significantly, while gait cycle duration increased significantly . In the + 5% and + 10% ras conditions, gait speed and cadence increased significantly, while gait cycle duration decreased significantly . When the ras tempo increased, all spatiotemporal gait parameters appeared to increase gradually, while the gait cycle duration decreased . Gait speed, cadence, and gait cycle duration showed the highest significant improvement in the + 10% ras condition . After gait training with ras, gait speed, cadence, stride length, and step length on the affected and unaffected sides increased significantly; gait cycle duration decreased significantly compared to that before ras gait training . It is especially noteworthy that step length increased significantly not only on the affected side but also on the unaffected side . Increased gait speed, which is determined by cadence and stride length23, is often an outcome measure when evaluating gait function . The improvement in movement patterns activates an internal time - keeping mechanism, which leads to movement synchronization24 . In our study, therefore, the spinal motor system was optimized for movement patterns following the reticulospinal pathway at the brainstem level25 . Suteerawattananon et al.26 reported that auditory cues can increase spinal motor neuron excitability via the reticulospinal tract and decrease muscle reaction time in response to motor commands, thus improving gait speed . Therefore, the auditory information provides stimulation that bypasses the damaged anatomy, leading to good muscle performance or timing control, and modulates a motor timing - control signal that is received by the brain s compensatory network . This has a useful effect on the neurological function and movement - processing centers of the brain27 . In the 0% ras condition prassas et al.9 reported no significant changes in stride length when baseline speed was applied with stroke patients . Another study also reported no significant differences in gait parameters such as gait velocity, cadence, or stride length in hemiplegic stroke patients when 0% ras was applied21 . Therefore, it may be inferred that rhythmical tempo variations have a positive impact on the gait of stroke patients . However, the equivalent rhythm to gait speed did not significantly affect the gait of stroke patients . Symmetry is an important factor during exercise or therapeutic treatment, particularly after stroke . Patterson et al . Recommended that step length, stance time, and swing time be used as gait parameters when determining the symmetry ratio28 . In the present study, step length as it pertained to spatial symmetry was examined . Symmetry ratio appeared to improve at 0% and around the 5% ras condition as well as after ras training . These trends are likely related to the fact that patients walked with symmetrical auditory cueing that adjusted to a comfortable walking cadence . Although statistically significant results were not obtained, the tendencies of the spatial symmetry ratio were confirmed . Further studies involving more subjects and temporal symmetry factors such as stance time and swing time will be needed . The present study showed that hemiplegic stroke patients spatiotemporal gait parameters change with various ras tempos . Moreover, significant improvements in gait speed, cadence, stride length, and step length on the affected and unaffected sides were observed after gait training . Previous studies of gait training with ras over the course of three weeks reported significant improvements in gait velocity, stride length, cadence, and symmetry8 as well as standing balance22 for stroke patients . Future studies need to evaluate more patients and the long - term effects after a certain period of ras gait training . A longer period of intervention could result in a higher number of positive results . In the future, we plan to combine our ras system with a wearable lower - limb rehabilitation robot . Zanotto et al.29 examined how different auditory feedback modalities in healthy subjects may affect gait modification through short - term gait training using an exoskeleton robot . The authors asserted that a combination of kinetic and visual guidance might be as effective as combined kinetic guidance and rhythmic cues . We also hope to conduct comparative analyses with robotic devices for various tempos . In this study, imu sensors were attached to both ankle joints to gather heel - strike information . The acquired information, which was displayed on a smartphone, was used by the participants to time their heel strikes to match the ras sound during gait training . We plan to update the ras application for use not only in gait training but also during real - time gait evaluation (via calculations based on heel - strike information, including gait symmetry). We expect the application to be useful during gait rehabilitative training and that chronic stroke survivors in any environment may find it useful.
The ultimate goal of periodontal therapy includes not only the arrest of progressive periodontal disease but also the restitution of those parts of the supporting apparatus which have been destroyed by disease . Regenerative procedures have been evaluated in several studies using grafting materials, root surface conditioning, guided tissue regeneration and application of growth factors . Different materials have been introduced for bone defect filling, i.e., autografts, xenografts, allografts, and alloplasts . Among the various subgroups of alloplastic bone grafts, bioactive glass (bg) is a type of bioactive ceramic with the ability to improve bone formation that has been assessed in experimental models . Previous studies have shown that bg particles, when implanted in vivo, incorporate into the connective tissue and undergo chemical transformation increasing the ph of the site to approximately 10 . This process leads to the formation of a silica gel on the surface of the particles and subsequently an amorphous calcium phosphate layer is formed by the interaction between calcium and phosphate from the bg and the surrounding medium . Hench and polak (2002) have shown that the release of ions (na, ca, and si) from bg materials controls the cell cycle leading to the differentiation and proliferation of bone cells, modulating the expression of genes that regulate osteogenesis, and the synthesis of growth factors . Growth factors are vital modulators during this process which can induce the migration, attachment, proliferation and differentiation of periodontal progenitor cells . Platelet - rich plasma (prp) is a preparation of autologous plasma that contains a higher platelet concentration, allowing it to deliver a greater concentration of autologous growth factors such us platelet - derived growth factor (pdgf), transforming growth factor beta (tgf-), vascular endothelial growth factor (vegf), insulin - like growth factor (igf - i), and epithelial growth factor (egf), that regulate cell proliferation, chemotaxis and differentiation . Some specific platelet growth factors, like pdgf and tgf-, could promote the growth and differentiation of the periodontal ligament and alveolar bone cells and could be responsible for the clinical improvement observed in experimental sites . Other interesting feature of prp is its sticky consistency due to its high fibrin content . The fibrin component of prp may work as a haemostatic agent aiding in stabilizing the graft material and the blood clot . Blood clot immobilization has been suggested as an important event in the early phases of wound healing of periodontal regenerative procedures . Studies using histological techniques suggested that prp preparations may enhance local bone formation, while others did not confirm these findings . Clinical studies have reported that treatments with a combination of prp and demineralized freeze - dried bone graft allograft (dfdba) improve clinical parameters in intrabony periodontal defects . In the other hand did nt find additional benefit in the reduction of pocket depth, clinical attachment gain and defect filling using prp with bg in the treatment of intrabony defects . The use of prp associated with biomaterials is an alternative treatment approach that may favor the process of bone tissue regeneration and should be investigated . Therefore, the goal of this study was to evaluate, histomorphometrically, the healing of surgically created intrabony defects in dogs treated with prp+bg . Nine adult female mongrel dogs (mean weight=15 kg) were included in the experiment . The study protocol was approved by the institutional animal research committee, state university of campinas (protocol #686 - 1). The surgical procedures were performed under general anesthesia with intravenous injection of a 3% sodium pentobarbital solution (0.5 ml / kg). The mandibular second and fourth premolars had been previously extracted and the extraction sites had been allowed to heal for 2 months . After this period, buccal and lingual mucoperiosteal flaps were elevated and 3-wall intrabony defects (4x4x4 mm) were created at the mesial and distal aspect of the left and right mandibular first molar (figure 1). Following root planning, aiming cementum removal, a reference notch was made with curettes on the root surface at the cement - enamel junction . Clinical view of the surgically created 3-wall intrabony defect (4x4x4 mm) at the mesial aspect of the first mandibular molar before that the gingival flaps have been positioned and sutured, a tofflemire matrix involved by cotton ligature was adapted over the tooth surface and the remaining defect was filled with gutta - percha (figure 2). So, the defects were exposed to plaque accumulation for a period of one month before the treatment . After that, scaling and root planing was performed and a regimen of daily brushing plus topical application of 0.1% chlorhexidine gluconate was instituted for 7 days prior to the surgical procedures . Clinical view of the insertion of gutta - percha in the defect blood was obtained several min before the administration of anesthesia . Four 5-ml tubes containing 0.5 ml of 3.2% sodium citrate solution (an anticoagulant) were drawn from each dog . The blood was thus separated into 3 basic parts: red blood cells (at the bottom of the tube), platelet enriched plasma (a discrete grey line in the middle of the tube) and platelet poor plasma (at the top of the tube). The portion corresponding to the platelet poor plasma was discarded from each tube and the remaining content was centrifuged again at 1,200 rpm for 15 min . Four hundred microliters of the middle portion, corresponding to the platelet enriched plasma, were pipetted from each tube . In order to obtain the gel, 30 l of 10% calcium chloride was added to prp and heated in a water bath at 37c . The gel was achieved after a period of 10 to 15 min . A tube with a sample of whole blood as well as a sample of prp, retrieved before adding calcium chloride of 10%, was sent to a clinical veterinary laboratory to perform a platelet counting . In the moment of the defect treatment, an apical reference notch was made with curettes on the root surface at the remaining bone level . Each defect in each animal (a total of 4 defects/ animal) was randomly assigned to one of the following treatments (the treatments with prp were located at the same side that was randomly determined): control: the defect was naturally filled with coagulum; bg (perioglas, us biomaterials, alachua, fl, usa): bg particles were applied, filling the defect; prp: after obtaining the prp gel, it was immediately applied on the defect; prp+bg: bg was incorporated to the prp (1:1) and this assemblage was immediately taken to a water bath at 37c for formation of a mixture of the gel and the bg graft . Primary, tension - free wound closure was accomplished following defect treatment, with the gingival flaps positioned and sutured with interrupted sutures (vicryl, ethicon inc, so jos dos campos, sp, brazil) at their presurgical position . Immediately after the surgeries, an intramuscular injection of penicillin (1.5 ml 150,000 iu) was given to the animal . Postoperative plaque control was performed by irrigation with a solution of 1% chlorhexidine gluconate (every other day). After 90 days, under general anesthesia, the animals were sacrificed with a perfusion of 10% neutral formalin solution . The jaws were dissected and the blocks containing the experimental specimens were removed, and decalcified in a solution of equal parts of 50% formic acid and 20% sodium citrate for 5 months . The decalcified specimens were washed in running water, dehydrated and embedded in paraffin . Five sections representing the midbuccal portion of each defect were selected for the histometric procedures . The following distances were measured: total epithelium (sulcular and junctional epithelium): from the gingival margin to the apical border of the junctional epithelium . Connective tissue adaptation (without cementum): from the apical border of the junctional epithelium to the coronal end of new cementum . New cementum: from the apical notch to the most coronal part of new cementum . New bone (bone position): from the apical notch to the most coronal extent of new bone . Negative values would be assigned if the bony crest was located apically to the apical notch . New bone area: area of newly formed bone filling the defect area (limited by the distance between the two notches in an apico - coronal direction and 4 mm away from the root surface in a lateral direction). The area of new bone was measured with an image analysis system that allowed the placement of a lattice on the image of the created bone defect . The intersection lines localized on bone were marked to calculate the new bone area whereas intersections lines localized on bg particles were not marked and therefore were not included for this purpose . The measurements were performed using a microscope (zeiss axioskop 2, carl zeiss instruments, gottingen, lower saxony, germany) with a 1.25/.035 objective associated with a video camera / computer / software (image pro plus version 3.0, media cybernetics, silver spring, md, usa). The mean values for all groups were determined using the individual means from the 9 dogs . The hypothesis that there were no differences among the groups was tested by one - way anova . If a statistical difference was detected, tukey s pairwise multiple comparison test was used . Four 5-ml tubes containing 0.5 ml of 3.2% sodium citrate solution (an anticoagulant) were drawn from each dog . The blood was thus separated into 3 basic parts: red blood cells (at the bottom of the tube), platelet enriched plasma (a discrete grey line in the middle of the tube) and platelet poor plasma (at the top of the tube). The portion corresponding to the platelet poor plasma was discarded from each tube and the remaining content was centrifuged again at 1,200 rpm for 15 min . Four hundred microliters of the middle portion, corresponding to the platelet enriched plasma, were pipetted from each tube . In order to obtain the gel, 30 l of 10% calcium chloride was added to prp and heated in a water bath at 37c . The gel was achieved after a period of 10 to 15 min . A tube with a sample of whole blood as well as a sample of prp, retrieved before adding calcium chloride of 10%, in the moment of the defect treatment, an apical reference notch was made with curettes on the root surface at the remaining bone level . Each defect in each animal (a total of 4 defects/ animal) was randomly assigned to one of the following treatments (the treatments with prp were located at the same side that was randomly determined): control: the defect was naturally filled with coagulum; bg (perioglas, us biomaterials, alachua, fl, usa): bg particles were applied, filling the defect; prp: after obtaining the prp gel, it was immediately applied on the defect; prp+bg: bg was incorporated to the prp (1:1) and this assemblage was immediately taken to a water bath at 37c for formation of a mixture of the gel and the bg graft . Primary, tension - free wound closure was accomplished following defect treatment, with the gingival flaps positioned and sutured with interrupted sutures (vicryl, ethicon inc, so jos dos campos, sp, brazil) at their presurgical position . Immediately after the surgeries, an intramuscular injection of penicillin (1.5 ml 150,000 iu) was given to the animal . Postoperative plaque control was performed by irrigation with a solution of 1% chlorhexidine gluconate (every other day). After 90 days, under general anesthesia, the animals were sacrificed with a perfusion of 10% neutral formalin solution . The jaws were dissected and the blocks containing the experimental specimens were removed, and decalcified in a solution of equal parts of 50% formic acid and 20% sodium citrate for 5 months . The decalcified specimens were washed in running water, dehydrated and embedded in paraffin . Five sections representing the midbuccal portion of each defect were selected for the histometric procedures . The following distances were measured: total epithelium (sulcular and junctional epithelium): from the gingival margin to the apical border of the junctional epithelium . Connective tissue adaptation (without cementum): from the apical border of the junctional epithelium to the coronal end of new cementum . New cementum: from the apical notch to the most coronal part of new cementum . New bone (bone position): from the apical notch to the most coronal extent of new bone . Negative values would be assigned if the bony crest was located apically to the apical notch . New bone area: area of newly formed bone filling the defect area (limited by the distance between the two notches in an apico - coronal direction and 4 mm away from the root surface in a lateral direction). The area of new bone was measured with an image analysis system that allowed the placement of a lattice on the image of the created bone defect . The intersection lines localized on bone were marked to calculate the new bone area whereas intersections lines localized on bg particles were not marked and therefore were not included for this purpose . The measurements were performed using a microscope (zeiss axioskop 2, carl zeiss instruments, gottingen, lower saxony, germany) with a 1.25/.035 objective associated with a video camera / computer / software (image pro plus version 3.0, media cybernetics, silver spring, md, usa). Mean values for each parameter were obtained per defect . The mean values for all groups the hypothesis that there were no differences among the groups was tested by one - way anova . If a statistical difference was detected, tukey s pairwise multiple comparison test was used . All groups presented a similar healing pattern characterized mainly by the presence of new cementum covering most of the root surface associated with the defect and new bone, up to the top of the defect, with a small amount of connective tissue and epithelium (figure 3). The periodontal ligament was characterized by the presence of collagen fibers obliquely oriented to the root surface, extending between the new cementum and the newly formed bone . Photomicrographs of the treated 3-wall intrabony defect showing new bone and cementum formation extending coronally to the apical notch (h&e, original magnification x20). A) control; b) prp; c) bg; d) bg+prp . Note the remaining particles of bg surrounded by new bone and connective tissue (c and d). Arrows indicate areas with new bone in contact with bg particles in the defects treated with bg, it was possible to observe that part of the material seemed to be resorbed and replaced by bone, while some of the remaining bg particles showed dissolution of their central part with newly formed bone inside of them . Some defects presented bone in direct contact and/or surrounding the bg particles (figure 4). Almost all defects that received bg presented a dense connective tissue surrounding the remaining particles and areas suggesting the formation of new bone . New bone formation observed in the 3-wall intrabony defect treated with bg (h&e, original magnification x100). Arrows indicate bone in direct contact with bg particles the histometric results are shown in table 1 . A superior area of new bone was observed in the sites treated with prp+bg and bg (13.802.32 mm and 15.632.64 mm, respectively). In the other groups, the new bone area was 8.191.46 mm (control group) and 8.811.47 mm (prp). Although the difference in the new bone area, no significant difference was found in the linear parameter new bone, e.g. Bone position: from the apical notch to the most coronal extent of new bone (4.370.44 mm, 4.240.68 mm, 4.370.46 mm and 4.440.42 mm, for control group, bg, prp and prp+bg, respectively; p=0.85). Mean and standard deviation (sd) for the parameters evaluated after all the treatments (control, bg, prp and bg+prp). Means followed by different letters are statistically different at 5% data analysis showed no significant differences among the groups regarding the initial defect extension (4.550.17 mm, 4.480.20 mm, 4.480.28 mm and 4.570.32 mm, for control group, bg, prp and prp+bg, respectively; p=0.74). In addition, intergroup analysis demonstrated that no significant difference was observed among the groups in the following parameters: total epithelium extension (2.240.58 mm, 1.940.37 mm, 1.970.37 mm and 1.810.61, for control group, bg, prp and prp+bg, respectively; p=0.45), connective tissue extension (0.900.28 mm, 0.840.41 mm, 1.070.27 mm and 1.150.32 mm, for control group, bg, prp and prp+bg, respectively; p=0.59) and new cementum extension (2.630.70 mm, 2.560.36 mm, 2.370.38 mm and 3.100.47 mm, for control group, bg, prp and prp+bg, respectively; p=0.85). The results from the histometric measurements, i.e., epithelial length (the portion of epithelium located on the root), connective tissue adaptation and new cementum formation, expressed as the percentage of the distance between the coronal and the apical notch are presented in figure 5 . No undesirable events like inflammation and foreign body reactions were observed . All groups presented a similar healing pattern characterized mainly by the presence of new cementum covering most of the root surface associated with the defect and new bone, up to the top of the defect, with a small amount of connective tissue and epithelium (figure 3). The periodontal ligament was characterized by the presence of collagen fibers obliquely oriented to the root surface, extending between the new cementum and the newly formed bone . Photomicrographs of the treated 3-wall intrabony defect showing new bone and cementum formation extending coronally to the apical notch (h&e, original magnification x20). A) control; b) prp; c) bg; d) bg+prp . Note the remaining particles of bg surrounded by new bone and connective tissue (c and d). Arrows indicate areas with new bone in contact with bg particles in the defects treated with bg, it was possible to observe that part of the material seemed to be resorbed and replaced by bone, while some of the remaining bg particles showed dissolution of their central part with newly formed bone inside of them . Some defects presented bone in direct contact and/or surrounding the bg particles (figure 4). Almost all defects that received bg presented a dense connective tissue surrounding the remaining particles and areas suggesting the formation of new bone . New bone formation observed in the 3-wall intrabony defect treated with bg (h&e, original magnification x100). A superior area of new bone was observed in the sites treated with prp+bg and bg (13.802.32 mm and 15.632.64 mm, respectively). In the other groups, the new bone area was 8.191.46 mm (control group) and 8.811.47 mm (prp). Although the difference in the new bone area, no significant difference was found in the linear parameter new bone, e.g. Bone position: from the apical notch to the most coronal extent of new bone (4.370.44 mm, 4.240.68 mm, 4.370.46 mm and 4.440.42 mm, for control group, bg, prp and prp+bg, respectively; p=0.85). Mean and standard deviation (sd) for the parameters evaluated after all the treatments (control, bg, prp and bg+prp). Means followed by different letters are statistically different at 5% data analysis showed no significant differences among the groups regarding the initial defect extension (4.550.17 mm, 4.480.20 mm, 4.480.28 mm and 4.570.32 mm, for control group, bg, prp and prp+bg, respectively; p=0.74). In addition, intergroup analysis demonstrated that no significant difference was observed among the groups in the following parameters: total epithelium extension (2.240.58 mm, 1.940.37 mm, 1.970.37 mm and 1.810.61, for control group, bg, prp and prp+bg, respectively; p=0.45), connective tissue extension (0.900.28 mm, 0.840.41 mm, 1.070.27 mm and 1.150.32 mm, for control group, bg, prp and prp+bg, respectively; p=0.59) and new cementum extension (2.630.70 mm, 2.560.36 mm, 2.370.38 mm and 3.100.47 mm, for control group, bg, prp and prp+bg, respectively; p=0.85). The results from the histometric measurements, i.e., epithelial length (the portion of epithelium located on the root), connective tissue adaptation and new cementum formation, expressed as the percentage of the distance between the coronal and the apical notch are presented in figure 5 . The use of different regenerative approaches in the treatment of periodontal defects has demonstrated a variable clinical result . Some techniques have been used in association, aiming to increase their effect on periodontal regeneration . The present study was designed to evaluate, histomorphometrically, the healing process of 3-wall intrabony defects treated with bg, prp and their association . Prp has become a focus of current studies due to its potential to accelerate wound healing . It has been previously shown that prp stimulated pdl cells and fibroblastic cell proliferationbut suppressed epithelial cell division in vitro . Consequently, by ordering these cellular responses into a series of related events, prp may facilitate wound - healing and tissue regeneration . The claimed ability to suppress epithelial cell proliferation seems advantageous for periodontal regeneration by favoring the formation of a new connective tissue attachment and new cementum on the root surface . The results of the present study failed to demonstrate that the use of prp can promote an additional effect in terms of cementum formation when treating 3-wall intrabony defects . Cementum formation for bg and prp followed the same trend observed for the control group (bg=2.5 mm/57%, prp=2.4 mm/53%, control=58%) while the prp+bg showed a slightly greater cementum formation (prp+bg=3.10 mm/68%), but the differences among the groups were not statistically significant . In this respect, it should be recognized that a non - contained osseous defect might have favored the discrimination of quantitative differences among the treatments . In a previous histological study, kim, et al (2004) evaluated the healing patterns following flap surgery in experimentally induced 1-, 2- and 3-wall intrabony defects . According to the authors, one- and 3-wall intrabony defects can be considered pre - clinical models that are relatively easy to manage and from which homogeneous healing outcomes may be expected . They suggested that these defects appear to be reproducible models to evaluate candidate technologies for periodontal regeneration . Regarding the 3-wall intrabony defect, they reported a mean cementum formation of approximately 70% of the initial defect extension . In the present study, a period of 1 month of plaque accumulation was allowed as a way to obtain some chronification of the defect . The control group showed 2.6 mm (58%) of the original defect covered with new cementum . This result confirms the high regenerative potential of this type of defect that provides favorable anatomic characteristics for clot stabilization and protection . It is well accepted that healing of intrabony defects is positively correlated to the number of bone walls limiting the defect . Therefore, the 3-wall intrabony defect used in the present study provides ideal extent and location of tissue resources, cells and vascularity circumscribing the defect, which might have favored bone formation . The linear measurement of the new bone height revealed an impressive coronal bone formation for all the groups, with no significant differences among the treatments . The claimed osteoinductive properties of prp could not be supported by the results of the present study . It has been suggested that prp could be used to increase the bone deposition rate and the quality of bone regeneration when augmenting sites prior to or in conjunction with dental implant placement . Nevertheless, more recent animal studies could not find increased bone regeneration when prp was used . Recently, bioactive glass has been used as a way to achieve bone and periodontal regeneration based on its osteoconductive properties . The need for an alternative to autologous and allogenic bone grafts have encouraged the search of a material that could be safe, surgically convenient and predictably promote regeneration with a reduced morbidity . In this study, the use of bg was not associated with any foreign body reaction, showing biocompatibility with periodontal tissues . Histological observations after treatment of periodontal defects with bg in nonhuman primates showed a significant increase of new cementum and inhibition of downgrowth of junctional epithelium . When bg is implanted in vivo, the ph of the site increases, a layer rich in silica gel is formed on the surface of the particles and subsequently an amorphous calcium phosphate layer is formed by the interaction between calcium and phosphate from the bg and the surrounding medium . Its superficial bioactivity may stimulate a rapid formation of a connective tissue seal that is supposed to have the ability to block the epithelium migration and allow for the repopulation of the previously contaminated area by periodontal ligament cells . In the present study, almost all defects that received bg presented a dense connective tissue surrounding the remaining particles in accordance with observations of previous studies . In the groups treated with bg, the area of new bone was 13.802.32 mm (prp+bg) and 15.632.64 mm (bg), respectively . In the groups that did not receive this material, the new bone area was 8.191.46 mm (control group) and 8.811.47 mm (prp). Based on the significant differences observed in the area of newly formed bone when groups with and without bg were compared the present study corroborates the hypothesis that this material could act as an osteoconductive graft . In addition, it was observed that some remaining bg granules showed dissolution of their core with newly formed bone, as described in the literature . Aiming to increase the periodontal and bone regeneration, some studies evaluated the additional effect of prp, combined with different types of grafting materials, on the treatment of intrabony defects . In spite of some clinical results showing a greater reduction of pocket depth and gain in clinical attachment level, other clinical studies failed to demonstrate an additional effect . In 3-wall periimplant defects in dogs treated with prp associated to xenogenic bone graft, the same authors also reported no significant effect with the addition of prp to xenogenic bone grafts on bone mineral density or graft maturity . These observations are in agreement with the results of the present study, which could not demonstrate an additional effect of prp in periodontal and bone regeneration of 3 wall intrabony defects . The histomorphometric findings demonstrated that the sites treated with prp+bg showed a similar amount of new bone (13.802.32 mm) when compared with the sites that only received bg (15.632.64 mm). The ideal platelet and growth factor concentration to promote periodontal regeneration has not yet been established . To the date, there is insufficient information about growth factor interactions and how they influence the activations of gene expression and protein production . In the present study, the achieved percentage mean of platelet concentration was 227.02% (mean of 463.015 platelets / ml) in relation to blood platelets count . (2003) reported that growth factors may act at specific times and at appropriate concentrations and this may be other explanation for the different results obtained in studies evaluating the use of prp . The great variability in the outcomes of the studies with prp may also be explained by the diversity of methods to obtain it . The technique to produce prp in the present study was used in other animal studies by our research group . This technique enables the production of a platelet concentration generally four times greater than the whole blood . According to landsberg, et al . (1998), thrombin promotes the release of antibodies to coagulation factors v and vi, which could be a potentially life - threatening event . These antibodies cross - react with human factor v thereby, causing a factor v deficiency that can be sufficiently severe to induce excessive bleeding and even death . Considering that, the use of thrombin in this type of study is not allowed in our country . In the present study, instead of thrombin, 10% calcium chloride was added to prp and heated in a water bath at 37c . (2003) measured the concentrations of bfgf, vegf, pdgf - bb and tgf-1 released from platelet concentrate and whole blood, before and after the addition of calcium alone, thrombin alone and various concentrations of calcium and thrombin . Those authors observed that calcium chloride, regardless of the use of thrombin, released platelet growth factors . In this study, the concentration of growth factors that was released was sufficient to promote endothelial cell proliferation in vitro . Recently, the tissue engineering science has provided a new possibility of cell therapy as a future alternative for the use of conventional regenerative techniques . This strategy that exploits the regenerative capacity of stem cells grown in a three - dimensional scaffold and subsequently implanted into the defect, in the presence of molecule modulators, may help overcoming several limitations of current regeneration modalities . In an in vitro study, the application of growth factors found in prp (tgf-1 and igf - i) in cultures of periodontal ligament cells, increased their mitotic activity and their capacity for adhesion on the root fragments surface . The use of platelet growth factors associated with cell therapy could be an alternative favoring the process of periodontal regeneration . Within the limits of this animal model study, it may be concluded that the association of prp with bg did not exert an additional effect to periodontal regeneration of 3-wall intrabony defects in dogs . Further studies are necessary to investigate the advantages and limitations of these materials in the treatment of periodontal defects.
The prevalence of diabetes is increasing globally, associated with an increase in obesity and in sedentary lifestyle [13]. This is associated with morbidity and mortality due to the effects of hyperglycemia related complication and by its association with atherosclerosis - heart disease and stroke, as the leading causes for mortality in diabetics . Hospitalization for acute internal and coronary disorders is therefore high among patients with diabetes; estimates from several studies show that the percentage of patients with diabetes admitted is up to three times the percent of diabetes in the relevant population . The increase in the prevalence diabetes and obesity is, however, not distributed evenly between different racial and ethnic groups, with some groups showing higher susceptibility to environmental changes brought by immigration or rapid socioeconomic changes . The arab population in israel has recently been observed to have an alarming high prevalence of diabetes, reaching 50% in women above 50 years old . This high prevalence seems to be higher than observed in arab countries or in arab immigrants to the usa or western europe . The unique situation of this population calls for both studies and intervention on a national level to promote health and prevent the consequences of the metabolic perturbation . Considering the high prevalence of diabetes in the arab population in israel, we hypothesized that we will find substantial number of patients in the internal and the intensive coronary unit (icu) wards diagnosed and undiagnosed diabetic patients . Seeking such a high risk population will enable the characterization of demographic, clinical state and diabetes related parameters . This data will serve to form intervention strategy in order to enhance health related outcomes . The study was performed at the internal medicine wards and icu of the holy family (hfh) hospital and the nazareth hospital in nazareth . The hfh hospital (established 1882) has 32 beds in the internal ward and icu . The nazareth hospital was established by the edinburgh medical mission society in 1861 in which 40 beds are in the internal ward and the icu . The rural population has shifted from mainly an agrarian society toward an urban like society . Both hospitals serve mainly the arab population in northern israel with an estimated population of 253,000 . Diabetes was defined as either known or unknown diabetes . Known diabetes: pervious diagnosis and/or treatment with diabetes related medication . Unknown diabetes: two measurements of blood glucose> 200 mg% and/or hba1c> 6.5% . Dwelling was defined as urban residing in the areas of major cities nazareth and acre; rural - residing in the areas of smaller towns and villages . Data on all patients hospitalized in the internal medicine and icu units in hfh and nazareth hospitals between 1.1.2009 and 31.12.2009 were reviewed . Charts of all diabetics were probed for data concerning demographics and previous diagnosis, and data pertaining to the hospitalization cause and the outcomes . All patients who were hospitalized during the months: january, april, july, and october served as a control group . All measured variables and derived parameters were tabulated by descriptive statistics . For descriptive statistics summary tables were provided giving sample size, absolute and relative frequency for categorical variables and sample size, arithmetic mean, standard deviation, median, minimum, and maximum for continuous variables . The following statistical tests were used in the analysis of the data presented in this study . Chi - square test was applied for testing the statistical significance of the differences in frequency of categorical variables between the study groups . The two - sample t - test was applied for testing the statistical significance of the differences of continuous variables between the study groups . Analysis of covariance (anova) was applied for comparing the differences of continuous variables between the outcomes of patients . Logistic regression was applied using patient's death as outcome and glucose on day 1 as predictor variable . Multiple regression was applied using days of hospitalization as outcome and glucose on day 1 as predictor variable . Chi - square test was applied for testing the statistical significance of the differences in frequency of categorical variables between the study groups . The two - sample t - test was applied for testing the statistical significance of the differences of continuous variables between the study groups . Analysis of covariance (anova) was applied for comparing the differences of continuous variables between the outcomes of patients . Logistic regression was applied using patient's death as outcome and glucose on day 1 as predictor variable . Multiple regression was applied using days of hospitalization as outcome and glucose on day 1 as predictor variable . All tests applied were two - tailed, and p value of 5% or less was considered statistically significant . The data was analyzed using the sas version 9.1 (sas institute, cary, nc). The number of all patients hospitalized one or more during the study year was 3784 . Thus, the proportion of diabetic patients hospitalized for one or more times during the study year was 39% . Patient demographics show the preponderance of the arab patients admitted: 92.8% of diabetic and 90.7% in the control group (p = nonsignificant (ns)). The minority were jews (3.45, 5.1%, resp .) Or other ethnic groups (p = ns). More patients with diabetes reported residence in rural versus urban areas than in the control group (table 1). Clinical characteristics at admission differed between patients with diabetes and controls (table 2). Diabetic patients admitted were significantly older than control 66.53 12.72 years and 54.26 21.53 years, respectively (p <0.0001). Of the diabetic patients admitted, 52.9% were women in comparison to 45.0% of the control group (p = 0.0003). Weight did not differ between groups 82.39 49.12 versus 85.08 19.08 (p = 0.47). The indications for hospitalization were different between diabetes and controls and between genders: the main difference was in the higher occurrence of hospitalization due to atherosclerotic related diseases both cardiovascular disease and strokes in the diabetic group versus controls (37% versus 27%, resp . Significant differences in the indications for hospitalization were maintained within the gender in each group . While only 18.4% of control women were hospitalized for cardiac indications, 32.9% of diabetic women were hospitalized for cardiac reasons a similar rate as in the nondiabetic men (34.2%). In comorbidities, we also detected significant difference between groups with a noticeable higher renal disease in the diabetic group (16.6% versus 7.9%) (table 3). Hospitalization: duration of hospitalization was significantly longer for the diabetic patients than for control patients: 3.71 2.99 days versus 3.27 2.97 days, respectively (p = 0.0008) (table 2). No significant differences in discharge status were noted between the two groups with the majority of patients being home discharged and with a low rate of in - hospital death2.6% control and 2.3% diabetic (table 4). One - year outcome was poorer in the diabetic group but without reaching statistical significance with a major difference in readmission rates in the diabetic group between the two hospitals . Mortality during the first year following discharge was 11.5% in the control versus 13.2% in the diabetic group (p = 0.48). A high readmission rate for both groups 19.9% and 20.3% control and diabetic group respectively, but there was an inequality between the two hospitals with one hospital demonstrating a significant difference between the two groups in the rate of readmission being significantly higher in the diabetic group during the first year after discharge in comparison to the control readmission rate . Average daily glucose levels during hospitalization as described in figure 2 demonstrate a decline in values from day one 211.4 (118.1) mg / dl to less than 200 mg / dl and thereafter stable levels while glucose levels were stable in the control group (figure 2). The glucose level at admission was a significant and an independent predictor for longer hospitalization and death in the nondiabetic but not in the diabetic group (data not shown). As a group, indications for hospitalization differed between patients with diabetes and controls . For both groups, the leading indication for hospitalization was cardiovascular disease, but cardiovascular disease and urinary tract infection were more prevalent as an indication for hospitalization in the diabetic group (36.8% versus 27.1% and 7.7% versus 6.9%, resp .) While chest infections were significantly less prevalent in the diabetic group versus control group (12.5% versus 16.9%, resp . ). The glucose lowering treatment for the patients with diabetes, prior to their hospitalization, was diet alone in 13.1%, oral therapy in 60%, insulin in 19%, and combination therapy in 7% of the patients . At discharge, there was no significant shift in the treatment paradigm in the total group (table 5). This study sheds light on the characteristics of hospitalized patients with diabetes in hospitals serving a high risk for diabetes population . We found high percentage of diabetes among the patients hospitalized in the internal medicine wards and cardiac intensive units, reflecting the high prevalence of diabetes in the population . 39.3% of total patient admitted to the internal medical ward and intensive care unit were diabetic . There was a female preponderance among patients admitted with diabetes 52.9% while only 45.0% of patients without diabetes, hospitalized, were women (p = 0.0003). All this reflects the alarming prevalence of obesity and diabetes among adult arab women in northern israel . It has been recently reported that the prevalence of overweigh and obesity among arab woman over 40 years of age who attend primary health clinic is 74% [79] with diabetes near 50% . This novel observation is seminal by bringing attention to this specific group of women with high morbidity risk and now observing high rate of hospitalization . Interestingly, diabetic women tend to have similar prevalence of coronary heart disease as main indication for admission, as for nondiabetic men (32.9% versus 34.2%, resp . ). As cardiovascular disease and urinary tract infection were more prevalent as an indication for hospitalization in the diabetic group, prevention of cardiovascular disease and urinary tract infection should therefore be reinforced particularly in the group of arab diabetic women within diabetic population . Patients with diabetes had a significant greater length of stay than hospitalized patients without diabetes, similar to data described in previous publications [1012]. The reasons might be multifactorial, and it is important to note that interventions with chronic disease management programs for outpatients and diabetes team consultation during hospitalization have been proved to be an effective means to reduce the likelihood and duration of hospitalizations for individuals with diabetes . Implementing similar strategies in this high risk group and evaluating the impact of this intervention on outcomes of rate and recurrence of hospitalization and hospital length of stay is therefore of paramount importance . In this study, we can additionally identify areas for intervention; as is evident in table 5, the stay in the hospital did not affect the patients treatment regimens, although glycemic levels at discharge were above the target (mean glucose levels at discharge> 190 mg / dl). Furthermore, in only a minority of patients, an hba1c measurement was performed during hospitalization . These issues reflect the current practice and constraint on hospitalization length and on the performance of blood tests, pushing toward shorter hospital stay and cutting back on performing blood tests not directly relevant to the immediate hospital care . It will be of interest to examine whether interventions as described above will cause changes in treatment regimens with improvement in long - term outcomes and readmissions . The glycemic control goals in nonsurgical hospitalized patients continue to be debated, but a consensus defining upper limits below 180 m / dl in noncritical patients is well based on outcomes studies and is therefore recommended by recent position papers . Average glucose levels during hospitalization were above 180 mg% throughout the hospital stay (figure 2); therefore, as noted there is a need for team consultation during hospitalization tailored to hospitals that provide medical care to populations in high risk for diabetes . Results of this study show the impact of the high prevalence of diabetes on the hospitalization of arab patients in northern israel, with specific emphasis on the burden of diabetes of the arab women . It calls attention to the need of in - hospital consultation team and postdischarge plan . Information from the cohort described within this study might be applicable to other medical centers providing large population of arab descent [1517].
The ivy sign might be suggestive of diffuse leptomeningeal collaterals found on post - contrast t1-weighted mr images in patients with moyamoya disease11,19). It has also been defined as a continuous linear leptomeningeal high - signal intensity along the cortical sulci and subarachnoid spaces on mr fluid - attenuated inversion recovery (flair) images13). Its proliferation is suggestive of a retrograde slow vascular flow that is highly correlated with the existence of a decreased cerebrovascular reserve (cvr)2,4,5,10,18). Accordingly, recent studies have examined the usefulness of mr flair images as a follow - up study after superficial temporal artery (sta)-middle cerebral artery (mca) anastomosis in patients with moyamoya disease6,9). Kawashima et al.9) reported that the ivy sign on mr flair images was a useful indicator in detecting brain hemodynamic changes at a follow - up in patients with moyamoya disease who underwent sta - mca anastomosis . But, most of these studies have been conducted in younger patients with a mean age of 20 - 29 years . Considering the age distribution, the higher incidence of moyamoya disease was observed in adults 45 to 49 years of age and the second in children 5 to 9 years of age, their reliability would be questionable on aspect of age1). Given the above background, we conducted this study to examine the usefulness of the ivy sign on mr flair images as compared with single photon emission computerized tomography (spect) and mr perfusion studies in detecting brain hemodynamic changes between preoperatively and postoperatively in adult patients (above 20 years) with moyamoya disease3,12,17). From sep 2010 to dec 2012, twelve adult patients with moyamoya disease visited our medical institution . In these patients (average age, 45 years; range, 23 - 64 years), moyamoya disease was incidentally found on a regular check - up without notable symptoms or it was detected on examinations for transient ischemic attack (tia) or cerebral infarction . The suzuki grade of moyamoya disease was analyzed and then compared with the mean ivy score . Twelve patients and thirteen cases (one having surgery at both hemispheres) underwent sta - mca anastomosis . Our patients had a mean age of 45 years and they underwent sta - mca anastomosis on preoperative and postoperative mr flair images . In each patient, such imaging studies as a mri, a ct angiography, and a spect were performed both preoperatively and postoperatively . A board - certified specialist in radiology reviewed the images and rated the ivy sign scores . Mr perfusion imaging represents a form of functional imaging that assesses alterations in blood flow with additional information on metabolism and regional measures of a specific tracer . This technique can be used to find salvageable ischemic brain tissue which represents the " schemic penumbra " . Patients identified with a larger penumbra are more likely to benefit from recanalization therapies making it crucial to properly recognize them23). Mr perfusion image was performed on 1.5 and 3 t machine (magnetom skyra, siemens, germany) with spin echo - planar imaging sequences by using the following parameters: repetition time (tr)/echo time (te), 1950/30 ms; flip angle, 90; matrix, 128128; field of view 220220; section thickness 5 mm; and intersection gap, 1 mm in all patients . Sixty sections and 25 images per section in all patients were obtained before, during and after the administration of contrast agent . Contrast agent 0.2 mmol gadopentetate dimeglumine (multihense, patheon, italy) per kilogram of body weight at a rate of 3 ml / sec was injected by using an mr imaging - compatible power injector (spectrics; medrad, pittsburgh, pa, usa). This was followed by a 20 ml bolus of saline administered at the same injection rate . Perfusion maps of cbv were generated after eliminating the effect of contrast agent recirculation by using gamma - variate curve fitting . We utilized software program (numaris/4, version syngo mr d13) for evaluation of cbv . Mr flair was also performed on a 3 t machine (magnetom skyra, siemens, germany) with spin echo - planar imaging sequences . Protocol imaging included axial flair sequence (tr: 9000 ms; te: 95 ms; field of view 199220; section thickness 5 mm; flip angle, 90; matrix size: 512278). In studies of cerebral vascular reserve by spect with the use of n - isopropyl-4-iodoamphetamine (123i - imp), it is necessary to measure cerebral blood flow at rest and after acetazolamide (acz) challenge . The spect scanner e - cam (siemens, germany) and image processor e - soft (siemens) were used . The collimator used was a low - energy, ultra - high resolution, fan - beam collimator (83 cps / mbq). Conditions for acquisition were as follows: 5-min acquisition (7.5 s / step, 3/step, continuous), matrix of 128128 (3.56 mm / pixel), and radius of rotation of 13 cm . A filtered backprojection method using a ramp filter was utilized to reconstruct images from 5-min data . As a 3d post - filter, a low - pass filter (cutoff 0.4 cycles / cm; order 5.0) was used . The corticosubcortical area of cerebral hemisphere was divided into the following four regions on each hemisphere: the anterior cerebral artery (aca), the anterior half of the middle cerebral artery (ant - mca), the posterior half of the mca (post - mca), and the posterior cerebral artery (pca)16) (fig . 1). The central sulcus is a landmark that separates between the ant - mca regions and the post - mca ones . We compared both flair images obtained preoperatively and postoperatively based on the ivy sign scores, for which we used a 3-grade system: grade 0 (negative; no ivy sign), grade 1 (minimal; the ivy sign is defined as minimal or equivocal high signal intensity) and grade 2 (positive; the ivy sign is obviously linear and punctate high signal intensity). The spect results were demonstrated using 20 levels of color ranging from black to white . We give the score from 0 to 100 points, it means that each color was given 5 points . This scoring system was possible because the 20 levels of color spectrum is exactly specified in the spect image and this result was confirmed the radiologist . When there was a sufficient cerebral perfusion, it was shown as red colors with an intensity of 60 to 80 . We applied cvr which results of post - administration of acetazolamide to this study because that it is significantly associated with an increased risk of stroke recurrence in patients with symptomatic cerebral infarction8). We analyzed the relationship between the ivy sign on mr flair images and cvr on spect ones and then compared them between prior to and following the sta - mca anastomosis . The statistical analysis was performed with the student t - test for correlations between patients with an ivy sign on mr flair and cvr on spect . The difference was considered to be significant if the p value was less than 5% (p<0.05). From sep 2010 to dec 2012, twelve adult patients with moyamoya disease visited our medical institution . In these patients (average age, 45 years; range, 23 - 64 years), moyamoya disease was incidentally found on a regular check - up without notable symptoms or it was detected on examinations for transient ischemic attack (tia) or cerebral infarction . The suzuki grade of moyamoya disease was analyzed and then compared with the mean ivy score . Twelve patients and thirteen cases (one having surgery at both hemispheres) underwent sta - mca anastomosis . Our patients had a mean age of 45 years and they underwent sta - mca anastomosis on preoperative and postoperative mr flair images . In each patient, such imaging studies as a mri, a ct angiography, and a spect were performed both preoperatively and postoperatively . A board - certified specialist in radiology reviewed the images and rated the ivy sign scores . Mr perfusion imaging represents a form of functional imaging that assesses alterations in blood flow with additional information on metabolism and regional measures of a specific tracer . This technique can be used to find salvageable ischemic brain tissue which represents the " schemic penumbra " . Patients identified with a larger penumbra are more likely to benefit from recanalization therapies making it crucial to properly recognize them23). Mr perfusion image was performed on 1.5 and 3 t machine (magnetom skyra, siemens, germany) with spin echo - planar imaging sequences by using the following parameters: repetition time (tr)/echo time (te), 1950/30 ms; flip angle, 90; matrix, 128128; field of view 220220; section thickness 5 mm; and intersection gap, 1 mm in all patients . Sixty sections and 25 images per section in all patients were obtained before, during and after the administration of contrast agent . Contrast agent 0.2 mmol gadopentetate dimeglumine (multihense, patheon, italy) per kilogram of body weight at a rate of 3 ml / sec was injected by using an mr imaging - compatible power injector (spectrics; medrad, pittsburgh, pa, usa). This was followed by a 20 ml bolus of saline administered at the same injection rate . Perfusion maps of cbv were generated after eliminating the effect of contrast agent recirculation by using gamma - variate curve fitting . We utilized software program (numaris/4, version syngo mr d13) for evaluation of cbv . Mr flair was also performed on a 3 t machine (magnetom skyra, siemens, germany) with spin echo - planar imaging sequences . Protocol imaging included axial flair sequence (tr: 9000 ms; te: 95 ms; field of view 199220; section thickness 5 mm; flip angle, 90; matrix size: 512278). In studies of cerebral vascular reserve by spect with the use of n - isopropyl-4-iodoamphetamine (123i - imp), it is necessary to measure cerebral blood flow at rest and after acetazolamide (acz) challenge . The spect scanner e - cam (siemens, germany) and image processor e - soft (siemens) were used . The collimator used was a low - energy, ultra - high resolution, fan - beam collimator (83 cps / mbq). Conditions for acquisition were as follows: 5-min acquisition (7.5 s / step, 3/step, continuous), matrix of 128128 (3.56 mm / pixel), and radius of rotation of 13 cm . A filtered backprojection method using a ramp filter was utilized to reconstruct images from 5-min data . As a 3d post - filter, a low - pass filter (cutoff 0.4 cycles / cm; order 5.0) was used . The corticosubcortical area of cerebral hemisphere was divided into the following four regions on each hemisphere: the anterior cerebral artery (aca), the anterior half of the middle cerebral artery (ant - mca), the posterior half of the mca (post - mca), and the posterior cerebral artery (pca)16) (fig . 1). The central sulcus is a landmark that separates between the ant - mca regions and the post - mca ones . We compared both flair images obtained preoperatively and postoperatively based on the ivy sign scores, for which we used a 3-grade system: grade 0 (negative; no ivy sign), grade 1 (minimal; the ivy sign is defined as minimal or equivocal high signal intensity) and grade 2 (positive; the ivy sign is obviously linear and punctate high signal intensity). The spect results were demonstrated using 20 levels of color ranging from black to white . We give the score from 0 to 100 points, it means that each color was given 5 points . This scoring system was possible because the 20 levels of color spectrum is exactly specified in the spect image and this result was confirmed the radiologist . When there was a sufficient cerebral perfusion, it was shown as red colors with an intensity of 60 to 80 . We applied cvr which results of post - administration of acetazolamide to this study because that it is significantly associated with an increased risk of stroke recurrence in patients with symptomatic cerebral infarction8). We analyzed the relationship between the ivy sign on mr flair images and cvr on spect ones and then compared them between prior to and following the sta - mca anastomosis . The statistical analysis was performed with the student t - test for correlations between patients with an ivy sign on mr flair and cvr on spect . The difference was considered to be significant if the p value was less than 5% (p<0.05). Thirteen cases of twelve patients, one patient to undergo surgery, bilateral hemispheres, were diagnosed with moyamoya disease on angiography and they underwent sta - mca anastomosis (table 1). With that as a result of the sum of cvr in all patients, post - contrast image, on the spect images was 3035 preoperatively and 3480 postoperatively (p<0.05), the surgery was successful in all patients . Among these patients, the cerebral hemisphere was divided into four regions: the aca, ant - mca, post - mca, and pca . Thus, 4 regions of 13 cases, a total of 52 regions were selected . We compared changes in the sum of cvr between preoperatively and postoperatively in each 4 regions . We aimed to proof of the ivy sign could be another marker of hemodynamic change, so the analysis of preoperative and postoperative change of ivy sign is the most important topic in this reports . But ivy sign was not observed on whole region of hemisphere, so we selected the slice of mr flair images which demonstrated the typical ivy signs and compared that with cvr on the same slice of spect images . This showed that cvr was increased from 745 to 815 in the aca region (p<0.05), from 720 to 885 (p<0.05) in the ant - mca region, and from 710 to 895 (p<0.05) in the post - mca region . These results indicate that there was a significant increase in the sum of cvr in all of these three regions . In the pca region, however, it was increased from 860 to 885 but this difference did not reach a statistical significance . To evaluate the usefulness of ivy sign at a follow - up, we compared ivy sign changes on mr flair images between preoperatively and postoperatively with cvr changes on spect images between prior to and following the sta - mca anastomosis7,19). All of the patients who showed the minimal or positive ivy sign were identified in 18 of 24 hemispheres (75%). Preoperative and postoperative mr flair images were taken at 46.7 days on average (range, 8 - 99 days) and at 196.6 days (range, 33 - 368 days), respectively . Minimal or positive ivy signs were found in 8 aca regions, 13 ant - mca regions, 19 post - mca regions and 3 pca regions . Of these regions, the ivy signs were predominantly recorded from the mca territory; their prevalence was the highest in the post - mca region . By the corticosubcortical area of cerebral hemisphere after sta - mca anastomosis, ivy score was 8 and 4 in the aca regions, 13 and 4 in the ant - mca regions and 19 and 9 in the post - mca regions . All of these differences reached a statistical significance (p<0.05). In the pca regions, however, there was no significant difference in the sum of ivy score between preoperatively (3) and postoperatively (3). In particular, the sum of ivy score was significantly higher in the post - mca region as compared with other regions and the degree of the reduction in the sum of ivy score was also the highest in the post - mca region . We compared these results with cvr scores between preoperatively and postoperatively on spect images and checked these correlation outcomes reached a statistical significance (p<0.05) (table 2). We compared ivy sign changes on mr flair images with mr perfusion images between prior to and following the sta - mca anastomosis . We selected the same number of the slice of mr perfusion images with the slice of mr flair image, same methods as compared mr flair with spect, which demonstrated the typical ivy signs . We compared on mr perfusion images, the degree of change of mean transit time (mtt) was higher in the regions with positive or minimal ivy sign as compared with those with negative one . After sta - mca anastomosis, it was also demonstrated that the hemodynamic profile was improved in the regions both spect (fig . 4) and perfusion mri images where the ivy sign was missing or decreased (fig . We divided into three groups on patients clinical states; asymptomatic groups, tia and complete stroke group and checked the change of ivy score by clinical symptoms . The mean preoperative ivy score was 2 points in the asymptomatic group, 3 points in the tia group and 5.2 points in the complete stroke group, thus indicating that higher ivy score was noticed on the patients with more severe clinical symptoms . To date, numerous studies have evaluated brain hemodynamic changes . Since brain hemodynamic changes were first measured based on co2 reactivity in a published study, the spect has been regarded as a standard diagnostic modality where the cvr is measured based on the iatrogenic metabolic effect by using an intravenous administration of acetazolamide20,21,23). In addition, the results of mtt on mr perfusion are also useful to determine the hemodynamic change in moyamoya patients12,13). Ohta et al.19) first reported that the ivy sign is a diffuse leptomeningeal enhancement found on t1-weighted mr images of patients with moyamoya disease in childhood . Based on published studies supporting the applicability of ivy sign on t1-weighted mr images in measuring brain hemodynamic changes have been conducted up to present11). But, there are some reports that indicated the side effects occur during the spect or mr perfusion, t1-weighted mri including discomfort to patients . According to settakis et al.22), side effects such as headache and discomfort may occur as a result of the activation of acz . In patients with a low hemodynamic state, there is also a possibility that the cbf might be further reduced with the administration of acz because of the vascular steal phenomenon in the adjacent area where the cvr is relatively preserved . Special attention should therefore be paid to the administration of acz in patients with severe ischemic symptoms . Mr perfusion and t1-weighted mri also requires administration of contrast agent which eventually require the patient should be evaluated via an intravenous route in a fasting state, so patients should keep the empty stomach for several hours . According to recent studies, the ivy sign on mr flair images showed a new diagnostic value in detecting brain hemodynamic changes10,13). Several studies had shown that the ivy sign is also seen on mr falir images and it is suggestive of brain hemodynamic status in patients with moyamoya disease . We assume that mr flair would be more useful in performing a follow - up evaluation as compared with spect or perfusion mri, t1-weighted mri study because it causes less discomfort to patients with moyamoya disease15,22). The mr flair study is advantageous in that it can be easily performed in a clinical setting . As compared with spect or mr perfusion, t1-weighted mri study, the mr flair imaging is more advantageous in that it takes less than ten minutes, causes a less side effects, do not need to feel hunger and is a non - invasive modality . Based on the published studies about the usefulness of the ivy sign on mr flair images in evaluating brain hemodynamic changes, further studies have been conducted to examine whether it is also a useful indicator at a follow - up in patients who underwent sta - mca anastomosis . In 2010, kawashima et al.9) used the ivy sign on mr flair images for a follow - up study of patients with moyamoya disease who underwent sta - mca anastomosis, thus reporting that the ivy sign was a useful indicator in evaluating the preoperative and postoperative hemodynamic status of brain . Ideguchi et al.6) reported that the ivy sign is a non - invasive indicator in detecting brain hemodynamic changes through a comparison between spect and mr flair study . However, it should be noted that the above studies enrolled relatively younger patients; kawashima et al . Enrolled patients with a mean age of 25 years and ideguchi et al . Did those with a mean age of 24 years . The detection rate of moyamoya disease per year was 0.94 patients per 100000 people, and prevalence was 10.5 patients per 100000 people . The incidence of ischemia concerned with the disease was 0.53 patients per 100000 people - years and hemorrhage was 0.2 patients per 100000 people - years . And, considering the age distribution, the highest incidence of moyamoya disease was observed in adults 45 to 49 years of age and the second in children 5 to 9 years of age, their reliability would be questionable on aspect of age, the above two studies have the limitation of subject selection in appropriate age1). We performed mr flair study to evaluate the diagnostic value of the ivy sign in adult patients (average age, 45 years; range, 23 - 64 years). Thus, our results support that it can be considered as a useful indicator for adult patients with moyamoya disease . We underwent sta - mca anastomosis in thirteen cases of twelve patients (one patient having surgery on bilateral hemispheres) who were diagnosed with moyamoya disease on angiography . All of the surgery was successful as a result of the sum of cvr on the spect images was 3035 preoperatively and 3480 postoperatively (p<0.05). Based on the reports by ideguchi et al.6), we compared the ivy signs between preoperatively and postoperatively in each region . Our results showed that it was no significant different in hemodynamic state evaluation between mr flair images and spect ones, and based on these results, we conclude that the ivy sign on mr flair images is a useful indicator in detecting brain hemodynamic changes between preoperatively and postoperatively as much as cvr on spect ones . To assess the accuracy of mr flair, we separated the results by four regions . Of four areas (aca, ant - mca, post - mca, pca), results were significant changes in the ivy sign on mr flair images and the cvr on spect ones postoperatively as compared with preoperatively in both the aca and mca territories and the degree of changes in both indicators was the highest in the mca territory (p<0.05). But, there were no significant differences in two indicators between preoperatively and postoperatively in the pca territory (table 2). These findings can be explained based on the relative preservation of posterior circulation by pca in patients with moyamoya disease, as reported by fujiwara et al.23). Compared to other reports, although it is not absolute comparison, our reports showed that 75% of regions of cerebral hemispheres were positive for the ivy sign; it was higher than 64.7% reported by kawashima et al . Our report was consistent the report by mori et al.14,22), ivy sign was a significant correlation with the severity of symptoms . The sum of the ivy scores was 5.2 points in the complete stroke group, 3 points in the tia group and 2 points in the asymptomatic group . These results are different from other studies indicating that the ivy sign on mr flair is also a useful indicator in detecting brain hemodynamic changes between preoperatively and postoperatively in adult patients (above 20 years) with moyamoya disease . Also, mr flair was not performed at constant tesla, some patients have taken mri at 1.5 tesla and others at 3 tesla . Further studies are warranted to compare the ivy signs between preoperatively and postoperatively in patients presenting with clinical symptoms . If there is a significant correlation between the ivy sign and the symptoms, this would be useful to evaluate their prognosis . The ivy sign on flair images was present in the regions where both the cerebral perfusion and the cvr were decreased . After sta - mca anastomosis, the ivy sign was disappeared or its intensity was decreased in the hemisphere . As compared with conventional diagnostic modalities such as spect or mr perfusion, the ivy sign is considered as a useful indicator in detecting brain hemodynamic changes between preoperatively and postoperatively . Our results also show that it is also a useful indicator in detecting brain hemodynamic changes in adult patients . Furthermore, the degree of ivy sign was higher in patients with a greater severity of symptoms . Further studies are warranted to examine whether the ivy sign is also useful in identifying the improvement of hemodynamic status and assessing the effectiveness of sta - mca anastomosis in adult patients with moyamoya disease.
Although alzheimer's disease (ad) and its main brain histopathology, that is, senile plaques and neurofibrillary tangles (nfts), were described a century ago, significant research advances in the disease began only a few decades ago . The discoveries of the major protein components of senile plaques as amyloid -peptide [1, 2] and of nfts as abnormally hyperphosphorylated tau [3, 4] in the 1980s initiated a new era of ad research . Since then, much research has focused on the molecular mechanisms of initiation and formation of the senile plaques and nfts and their roles in the pathogenesis of ad . Evidence accumulated in the last two decades indicates that malprocessing of both tau and -amyloid precursor protein, which produces -peptide, is pivotal, if not central, to the molecular mechanism of ad . The severity of dementia symptoms in ad strongly correlates to the number of nfts, but not of senile plaques, in ad brains [59], suggesting that tau pathology might be associated with the disease mechanism more directly . Abnormal hyperphosphorylation of tau and its deposits in the brain is also seen in several other neurodegenerative diseases that are collectively named tauopathies (for review, see [10, 11]). The discovery of tau mutations that cause hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (ftdp-17) [1214] further indicates that tau abnormality alone is sufficient to produce dementia . Therefore, for developing rational therapeutic treatment of ad, it is essential to understand the molecular mechanism by which tau abnormalities lead to neurofibrillary degeneration . Because tau aggregated in the brain of ad and all other tauopathies is always abnormally hyperphosphorylated, numerous studies have focused on the roles of the abnormal hyperphosphorylation and the mechanism leading to tau hyperphosphorylation . Recent studies demonstrate that it is the abnormal hyperphosphorylation that makes tau lose its normal function to stimulate microtubule assembly, gain toxic activity, and aggregate into nfts [1523]. In addition to tau, several other brain proteins such as neurofilaments, microtubule - associated protein (map) 1 b, -tubulin, and -catenin are also found to be hyperphosphorylated [2427], suggesting that the protein phosphorylation / dephosphorylation system might be dysregulated in ad brain . Because abnormally hyperphosphorylated tau is pivotal to ad and has been extensively studied, this review focuses on tau hyperphosphorylation . Prevention and reversal of abnormal hyperphosphorylation of tau as a potential promising therapeutic strategy is also discussed . Tau was first discovered by weingarten et al as a microtubule - associated protein that stimulates microtubule assembly . There was not much research interest in tau protein until a decade later, when it was found to make up the paired helical filaments (phfs) that form nfts in ad brain [3, 4, 29]. Human tau gene was found on the long arm of chromosome 17 (position 17q21) and was found to contain 16 exons . This single tau gene encodes six tau isoforms in adult human brain as a result of alternative splicing of its mrna . The six isoforms of tau differ from each other by the presence or absence of one or two inserts (29 or 58 amino acids) in the n - terminal part and by the presence of either three or four repeats in the c - terminal half . The repeats in the c - terminal half of tau are the domains that bind to microtubules [3234]. The region upstream of the microtubule - binding domains contains many proline residues and, hence, is called the proline - rich region . The best - known biological functions of tau are to stimulate microtubule assembly and to stabilize microtubule structure . Tau binds to microtubules via its microtubule - binding domains located at the c - terminal half of the molecule [3234]. The n - terminal part projects from the microtubule surface, where it may interact with other cytoskeletal elements and the plasma membrane [35, 36]. Each of the six tau isoforms possibly has its particular physiological roles and differential biological activities, because they are differentially expressed during development and have different activities to stimulate microtubule assembly [37, 38]. Only the shortest isoform of tau is expressed in fetal brain, whereas all six isoforms are seen in adult brain [39, 40]. In addition to stimulating microtubule assembly, several studies have suggested that tau may have other physiological functions . It appears to interfere with binding of kinesin and kinesin - like motors to microtubules, leading to a preferential inhibition of plus - end - directed axonal transport . Overexpression of tau inhibits kinesin - dependent trafficking of vesicles, mitochondria, and endoplasmic reticulum . This may explain the symptoms of amyotrophic lateral sclerosis with neurofilament accumulation in motor neurons of several transgenic models of tau overexpression [4346]. Tau has been found to interact with mitochondria, plasma membrane, and nucleic acids [48, 49], suggesting that it may act as a mediator between microtubules and these organelles . Tau also appears to interact with src - family nonreceptor tyrosine kinases such as fyn [50, 51] and phospholipase c- [52, 53] via its proline - rich region . These data suggest that tau may also play a role in the signal transduction pathways involving src - family tyrosine kinases and phospholipase c-. However, the physiological significance of these interactions remains to be elucidated . As early as 1977, tau was found to be a phosphoprotein . In 1984, it was demonstrated that phosphorylation of tau negatively regulates its activity in promoting microtubule assembly . Because tau is abnormally hyperphosphorylated in ad and other tauopathies, tau phosphorylation has been studied extensively . Normal brain tau contains 2 or 3 moles of phosphates per mole of tau [5658]. Studies on human brain biopsy tissue indicated that several serine and threonine residues of tau are normally phosphorylated at substoichiometrical levels [59, 60]. A normal level of phosphorylation appears to be required for tau's optimal function, whereas the hyperphosphorylated tau loses its biological activity [15, 16, 6169]. The discovery that tau aggregated in ad brain is abnormally hyperphosphorylated has stimulated many studies on the extent and sites of tau hyperphosphorylation and their role in the pathogenesis of ad . The phosphorylation level of tau isolated from autopsied ad brains is 3- to 4-fold higher than that from normal human brains [5658]. In addition, the hyperphosphorylated tau is accumulated in both brains [70, 71] and cerebral spinal fluid [7280] of individuals with ad . All six isoforms of tau are aggregated into phfs in the abnormally hyperphosphorylated forms in ad brains [3, 4, 31, 81]. To date, at least 37 serine and threonine residues have been found to be phosphorylated in phf - tau (for review, see). These residues include thr39, ser46, thr69, thr123, ser137, thr153, thr175, thr181, ser198, ser199, ser202, thr205, ser208, ser210, thr212, ser214, thr217, thr231, ser235, ser237, ser238, ser241, ser262, ser285, ser305, ser324, ser352, ser356, ser396, ser400, thr403, ser404, ser409, ser412, ser413, ser416, and ser422 . Many of these residues are also phosphorylated in normal human brains without nfts at smaller extents, but they are rapidly dephosphorylated during postmortem delay and tissue processing [59, 60]. However, the phosphate groups at these sites are not readily dephosphorylated during the postmortem period and tissue processing of ad brain, probably because of the deficient protein phosphatase activities [8389]. Some of the phosphorylation sites seen in phf - tau are not phosphorylated at all in normal brains . Because all of the previously identified phosphorylation sites of normal tau and phf - tau are at either serine or threonine residues, it was thought that tau was phosphorylated only at serine and threonine residues . However, recent studies suggest that tau in developing brain and in ad brain is also phosphorylated at tyrosine residues . The src - family nonreceptor tyrosine kinase fyn can bind to and phosphorylate tau in vitro and in transfected cells [50, 51, 93]. Tyrosine phosphorylated tau at this position is also seen immunohistochemically in the brain of transgenic mice that express mutated human taup301l . Williamson et al demonstrated that in primary human and rat brain cortical cultures tau is phosphorylated at tyr 29 upon treatment with a. the tyrosine phosphorylation of tau appears rapid and transient . Interestingly, antibodies specific to tyrosine phosphorylated tau labeled purified phf - tau, but not normal tau, suggesting that phf - tau is phosphorylated at the tyrosine residues [93, 94]. In addition, tyr394 was also found to be phosphorylated in phf - tau and in tau from fetal brains, and the phosphorylation at this site is catalyzed by another nonreceptor tyrosine kinase c - abl . It is not clear if the phosphorylation at any of the above tyrosine residues is stoichiometrically significant . Therefore, whether the tyrosine phosphorylation of tau has any pathophysiological relevance remains to be elucidated . Numerous studies have demonstrated the important role of abnormal hyperphosphorylation of tau in its aggregation into nfts and in alzheimer's neurofibrillary degeneration . In cultured cells, hyperphosphorylation of tau after treatment with phosphatase inhibitors impairs its activity to bind to microtubules and induces filamentous aggregation of tau . Pseudohyperphosphorylated tau that simulates abnormally hyperphosphorylated tau by mutation of serine or threonine residues into glutamate at selected ad - related sites exerts a cytotoxic effect, whereas wild - type tau is neutral . In contrast, neurons from tau - knockout mice are resistant to a-induced neurotoxicity . Overexpression of human tau in combination with phosphorylation by drosophila gsk-3 homologue shaggy, but not tau overexpression alone, exacerbates tau - induced neurodegeneration and results in the formation of nft - like filamentous tau aggregates . This study shows a causal relationship between tau hyperphosphorylation and neurofibrillary degeneration in vivo . A study in disabled-1 (an adapter protein) knockout mice further demonstrates that tau hyperphosphorylation causes early death of the animals . Most importantly, tau in inclusions of all tauopathies in human and animal models is always hyperphosphorylated (for reviews, see [11, 98]). Abnormal hyperphosphorylation of tau appears to precede its aggregation into nfts in ad brain [57, 99101]. Taken together, these studies suggest that the abnormal hyperphosphorylation of tau is crucial to neurofibrillary degeneration in ad and other tauopathies . The largest isoform of human brain tau (441 amino acids) contains 80 serine and threonine residues and five tyrosine residues . Phosphorylation at nearly half of these residues has been reported in phf - tau (see for review). Many studies have demonstrated that phosphorylation of tau at different sites has different impacts on its biological function and on its pathogenic role . For instance, a quantitative in vitro study demonstrated that phosphorylation of tau at ser262, thr231, and ser235 inhibits its binding to microtubules by 35%, 25%, and 10%, respectively . In cultured cells, phosphorylation of tau at ser214 and ser262 decreases its binding to microtubules and appears to inhibit its assembly to filaments . In vitro kinetic studies of the binding between hyperphosphorylated tau and normal tau suggest that phosphorylation of tau at ser199/ser202/thr205, thr212, thr231/ser235, ser262/ser356, and ser422 are among the critical phosphorylation sites that convert tau to a toxic molecule to sequester normal maps from microtubules . Further phosphorylation at thr231, ser396, and ser422 promotes self - assembly of tau into filaments . Similarly, tau mutated at ser396 and ser404 (changing ser into glu) to mimic phosphoserine is more fibrillogenic than wild - type tau, and a tau construct in which ser422 is mutated to glu shows a significantly increased propensity to aggregate . Consistent with these observations is that mutation of ser422 to ala prevents a-induced tau aggregation . These results suggest that phosphorylation of ser422 may play a key role in tau filament formation in vivo . An important question is, by what mechanism is the tau abnormality involved in the pathological cascades that lead to neurodegeneration in ad and other tauopathies . Does a hyperphosphorylation - induced defect in its activity to stimulate microtubule - assembly contribute to cell dysfunction? Although tau loses its activity to stimulate microtubules, lack of overt phenotype of tau knockout transgenic mice suggests that it is very unlikely that tau abnormality contributes to neurodegeneration via loss of normal function due to its hyperphosphorylation . By a series of studies, we have found that both the abnormally hyperphosphorylated tau isolated from ad brain and in vitro hyperphosphorylation tau gain a toxic activity to sequester normal tau and other maps, such as map1 and map2, and cause microtubule disassembly [16, 18, 66, 108]. Upon dephosphorylation polymerization of the hyperphosphorylated tau into phfs also abolishes this toxic activity (alonso a et al, unpublished observation). Hence, we speculate that the abnormal hyperphosphorylation of tau causes neurodegeneration by gain of toxic activity rather than by loss of normal activity that can be compensated for by other maps and that formation of phfs / nfts from the hyperphosphorylated tau in neurons is a defense mechanism by which neurons aim to reduce the toxic activity of the abnormally hyperphosphorylated tau . Conditional overexpression of gsk-3 in the transgenic mouse brains induces tau hyperphosphorylation and neurodegeneration, but no tau aggregation . In contrast, there are nfts but no memory loss in several lines of tau transgenic mice (for review, see). This phenomenon is probably common to other diseases characterized by abnormal protein aggregates such as huntington disease and cardiomyopathy, in which the abnormal, nonfibrillar protein oligomers, rather than the aggregates themselves, appear to be pathogenic [111, 112]. To understand the mechanism leading to abnormal hyperphosphorylation of tau in ad, protein kinases and phosphatases that regulate tau phosphorylation level must be identified first . In the last two decades, numerous studies aimed to the identification of tau kinases and phosphatases have been carried out . It was found that in vitro, dozens of phosphoseryl / phosphothroenyl protein kinases and most of the major protein phosphatases could act on tau protein at various phosphorylation sites (for reviews in detail, see [82, 113, 114]). Tau appears to be a universal substrate for protein kinases and phosphatases in vitro . This may not be surprising, because nearly 20% of the amino acid residues of tau molecule are serines and threonines, and nearly 50% of these residues are phosphorylated to certain degrees in ad brain (see for review). However, it is unlikely that all these enzymes that act on tau in vitro catalyze tau phosphorylation / dephosphorylation in vivo . Immunohistochemical studies also have shown a colocalization of more than a dozen protein kinases and several protein phosphatases with nfts of ad brain . As we now know that nfts are very sticky structures that can be stained immunohistochemically by antibodies to numerous antigens, immunohistochemical colocalization with nfts can only support other data that indicate a role of the specific protein or enzyme in the formation of nfts, but itself cannot indicate such a role . Further studies in cultured cells, in situ, and especially in vivo suggest that a few protein kinases and phosphatases may be involved in regulation of tau phosphorylation in the brain . The kinases that most likely play a role in phosphorylation of tau in the brain include glycogen synthase kanase-3 (gsk-3), cyclin - dependent kinase 5 (cdk5), camp - dependent protein kinase (pka), stress - activated protein kinases, and calcium / calmodulin - dependent kinase ii (camk - ii). The sites of tau phosphorylation by these kinases, except stress - activity protein kinases, have been summarized in our recent review . Among protein phosphatases, pp2a has been shown to be the major tau phosphatase in the brain [69, 116120]. In a recent study, we compared the catalytic kinetics of tau dephosphorylation by various major brain protein phosphatases and determined the relative contributions of these phosphatases to the regulation of tau phosphorylation quantitatively . We found that pp2a accounts for 70% of the total tau phosphatase activity, whereas pp1, pp2b, and pp5 each accounts for only 10% of the total tau phosphatase activity . Because pp2b activity is upregulated rather than downregulated in ad brain, it is unlikely that it regulates tau phosphorylation in vivo . Accumulated evidence indicates that tau phosphorylation is regulated by several protein kinases and that more than one kinase might be involved in abnormal hyperphosphorylation of tau in ad brain . Interestingly, gsk-3 phosphorylates tau at both prime sites (ie, tau needs to be primed by phosphorylation with other kinases at other sites) and unprimed sites [122126]. In a cotransfection study, cho and johnson found that a gsk-3 mutant (gsk-3-r96a) that only phosphorylates unprimed sites has no negative impact on tau's ability to bind to microtubules, in contrast to wild - type gsk-3, which significantly impairs tau's ability to bind to microtubules . Further studies demonstrate that primed phosphorylation of tau at thr231 by gsk-3 plays a critical role in decreasing tau's ability to both bind to and stabilize microtubules . In rat brains, activation of pka not only induces primed phosphorylation of tau by gsk-3, but also impairs the spatial memory of rats [124, 127]. Gsk-3 appears to be regulated by both phosphoinositol-3 kinase and protein kinase c pathways [128131]. An obvious approach to understanding how tau becomes abnormally hyperphosphorylated in ad is to study whether tau kinase(s) or tau phosphatase(s) are dysregulated in ad brain . Several studies have focused on whether the activities and expression of these enzymes are altered in ad brain . Among protein kinases, cdk5 was reported to be upregulated in ad brain by one laboratory, but this result was challenged by others [133136]. On the other hand, both the activity and the expression of pp2a as well as the activities of pp1 and pp5 are decreased in the selected areas of ad brain [8389]. Consistent with this finding, several other neuronal proteins such as neurofilaments, map1b, -tubulin, and -catenin are also hyperphosphorylated in ad brain [2427]. Hence, it appears that downregulation of the phosphatases, especially of pp2a, might underlie the abnormal hyperphosphorylation of tau and other proteins in ad brain . Studies of metabolically active rat brain slices and transgenic mice suggest that the downregulation of pp2a may produce hyperphosphorylation of tau, not only by the deficient dephosphorylation of tau, but also through the activation of several pp2a - regulated protein kinases, including pka, camk - ii, map kinases, and stress - activated protein kinases [139141]. Nevertheless, inhibition of pp2a activity in animal brain could only induce hyperphosphorylation of tau at some of the hyperphosphorylation sites seen in phf - tau, but does not result in nfts . Attempts to produce massive tangles of phfs in animal models merely via alteration of tau phosphatase and/or kinase activities have not yet been successful . These observations suggest that the downregulation of tau phosphatases in ad brain may be only partially responsible for the abnormal hyperphosphorylation of tau . The causes leading to decreased pp2a activity in ad brain are not well understood . Downregulation of pp2a expression and upregulation of pp2a endogenous inhibitor proteins i1pp2a and i2pp2a in ad brain may both contribute to the downregulation of pp2a activity . Because the activities of pp1 [83, 88] and pp5 [88, 89], which contribute to regulation of tau phosphorylation to a much smaller extent than pp2a, are also decreased in ad brain, there might be a common factor that downregulates the activities of the major brain protein phosphatases in ad brain . In addition to tau kinases and phosphatases, alterations of tau itself, the substrate of these enzymes, may also play an important role in its abnormal hyperphosphorylation and conversion into phfs . Tau is also modified post - translationally by -n - acetylglucosamine (glcnac) via a glycosidic bond at the hydroxyl groups of serine and/or threonine residues, and this modification is called o - glcnacylation [143145]. Because o - glcnac could modify the same serine or threonine residues of tau as phosphate does and a reciprocal relationship between o - glcnacylation and phosphorylation has been seen in many proteins (for review, see), o - glcnacylation could affect phosphorylation of tau . Recent studies in various systems found that tau phosphorylation is indeed regulated by o - glcnacylation inversely [144, 145, 147]. Most interestingly, fasting of mice induces downregulation of tau o - glcnacylation, which relies on glucose metabolism to supply udp - glcnac as a donor for protein o - glcnacylation, and in turn leads to hyperphosphorylation of tau . These findings led to the novel hypothesis that impaired glucose uptake / metabolism in ad brain, which was well established decades ago, contributes to the disease pathogenesis via downregulation of tau o - glcnacylation and, consequently, upregulation of tau phosphorylation that leads to neurofibrillary degeneration . Classical n - linked glycosylation is a modification of protein at asparagine residues by oligosaccharides, which normally modifies only membrane proteins and secreted proteins . Tau in ad brain, but not in normal human brain, was found to be modified by n - glycosylation [68, 149, 150], and this aberrant tau modification appears to precede and facilitate abnormal hyperphosphorylation of tau [150152]. There is no doubt that the abnormality of tau plays a central role in neurofibrillary degeneration in ad and other tauopathies . A critical review of the literature accumulated in the last two decades sheds light onto the probable mechanism of neurofibrillary degeneration of ad (figure 1). Tau is the major microtubule - associated protein of mature neurons where it stimulates microtubule assembly and stabilizes microtubule structure . The phosphorylation level of tau is regulated by tau kinases and tau phosphatases, as well as by the alteration of tau itself . In ad and probably also in other tauopathies, metabolic and genetic abnormalities lead to dysregulation of signal transduction pathways, which in turn causes an imbalance of the phosphorylation / dephosphorylation system, that is, downregulation of pp2a in the brain . This imbalance results in increased phosphorylation (ie, hyperphosphorylation) of tau . The impaired brain glucose uptake / metabolism that precedes ad also facilitates hyperphosphorylation of tau via downregulation of tau o - glcnacylation . Aberrant n - glycosylation of tau in ad brain also makes tau a more favorable substrate for major tau kinases and less favorable for tau phosphatases [151, 152], thereby facilitating tau hyperphosphorylation . The abnormally hyperphosphorylated tau resulting from any of the above causes not only loses its biological activity to stimulate microtubule assembly, but also becomes a toxic molecule, sequesters normal tau, map1, and map2, and causes disassembly of microtubules . The breakdown of the microtubule network in the affected neurons compromises axonal transport and leads to retrograde degeneration, which in turn results in neuronal death and dementia . On the other hand, the abnormally hyperphosphorylated tau detached from microtubules is not only easier to polymerize into phfs as a result of hyperphosphorylation, but it also causes increased intraneuronal soluble tau concentration due to sequestration of normal tau from microtubules, which further facilitates tau aggregation into phfs . The polymerized abnormal tau is further modified by ubiquitination, glycation, polyamination, nitration, and truncation (for review, see), and forms mature phfs / nfts . Unlike the unpolymerized hyperphosphorylated tau that is toxic, phfs / nfts appears to be inert (alonso a et al unpublished observation), but these lesions grow in size with disease progression and eventually might physically choke the affected neuron to death . Because neurofibrillary degeneration plays a central role in the pathogenesis of ad, one of the most attractive therapeutic targets of ad is to inhibit neurofibrillary degeneration . As outlined in figure 1, the most promising approaches to achieve this goal are to inhibit the abnormal hyperphosphorylation of tau and to inhibit its sequestration of normal maps . The former approach is more effective since it should both rescue the disruption of microtubule and axoplasmic flow and prevent further deposition of nfts . Several academic groups and pharmaceutical companies have been investigating this approach by restoring pp2a activity or inhibiting tau kinase activity in the brain . Memantine, a low - to - moderate - affinity antagonist of nmda receptor, which improves mental function and the quality of daily life of individuals with moderate to severe ad [153, 154], reverses the okadaic - acid - induced inhibition of pp2a activity and prevents tau hyperphosphorylation in hippocampal slice cultures from adult rats . The restoration of pp2a activity to normal level by memantine also leads to restoration of the expression of map2 in the neuropil and a reversal of hyperphosphorylation and accumulation of neurofilaments . Wang's group has demonstrated that treatment of brain slices and rats with melatonin can restore pp2a activity that is inhibited by okadaic acid or calyculin a and reverse hyperphosphorylation of tau and neurofilament proteins as well as cytotoxicities [156158]. Melatonin also prevents tau hyperphosphorylation and aggregation induced by overactivation of gsk-3 or pka [131, 159]. These are examples showing that inhibition of dysregulation of protein phosphorylation / dephosphorylation is a promising target to treat ad . Further investigation of new compounds that can inhibit abnormal hyperphosphorylation of tau will likely provide new treatments for ad . Maps, microtubule - associated proteins; mts, microtubules; phfs, paired helical filaments; nfts, neurofibrillary tangles.
Noninfectious rhinitis (nir) affects 40% of the adult population, has a significant impact on health - related quality of life, and results in substantial health economic costs to the society.13 nir is characterized by symptoms of upper airway inflammation, such as nasal obstruction, secretion, itching and sneezing, and represents several pathological entities, including allergic rhinitis, vasomotor rhinitis, and chronic rhinosinusitis.4 the disease has previously been linked to asthma, smoking, occupational exposure to gas, fumes, or dust, and also to gastroesophageal reflux disease, but for many patients with nir, the pathophysiology remains unknown.1,3,5,6 nir has been reported in individuals with chronic obstructive pulmonary disease (copd), but epidemiological evidence is lacking.7 copd is an inflammatory, progressive airway disease with persistent airflow limitation . The airflow limitation in copd is caused by a combination of small airway disease (obstructive bronchiolitis) and the destruction of lung parenchyma (emphysema). Exposure to noxious particles and gases, particularly tobacco smoking, but also occupational exposure to gas, fumes, or dust, is related to the development of copd.8,9 interestingly, the same risk factors have also been associated with nir.1,6 copd occurs predominantly in patients aged> 40 years as a direct result of the slow initial development of the disease.1012 furthermore, new research has shown paranasal sinus opacification to be associated with copd and self - reported respiratory symptoms.13 the estimated prevalence of copd in the nordic countries, defined according to the global initiative for chronic obstructive lung disease stage i and higher, varies from 9.4% to 18.8%.1418 to date, only a few studies have evaluated a possible link between nir and copd . This is surprising, as both nir and copd are common inflammatory conditions in the respiratory tract and could potentially be linked through a generalized airway inflammation in accordance with the united airways concept introduced to explain the link between upper and lower inflammatory airway disease in asthma and nir, even though other inflammatory mechanisms are likely to be involved.7 the diagnosis and classification of copd is based on a clinical assessment and spirometry is mandatory.8 to the authors knowledge, there are no large population - based studies of the relationship between rhinitis and copd based on spirometry data . In the present study, data on spirometry and respiratory symptoms were combined from a large longitudinal population - based cohort, followed for 5 years . The main hypothesis of the study was that copd is related to the development of nir . This study is a part of the longitudinal population - based study called adonix (adult onset asthma and nitric oxide). Adonix was approved by the regional ethical review board of gothenburg (dnr 092 - 01) and has previously been described in detail.5 the adonix study was designed to evaluate risk factors for airway disease such as nir, copd, and asthma . A total of 6,665 subjects, randomly selected, aged 2575 years, from the municipal register of gothenburg, sweden, were included at baseline between 2001 and 2003 . The subjects performed spirometry and answered a questionnaire with questions on upper and lower respiratory symptoms and diseases . The subjects also underwent a measurement of fractional exhaled nitric oxide (feno) and a laboratory analysis of specific immunoglobulin e antibodies for atopy status . After 5 years, the included subjects were asked to answer a follow - up questionnaire . This study evaluates the risk of developing new - onset nir in relation to having copd during a 5-year observation period . The questions used for new - onset nir and associated risk factors, such as smoking, educational status, and occupational exposure to gas, fumes, or dust, are shown in table 1 . The questions on occupational exposure to gas, fumes, or dust, and educational status were added in 2005 and were therefore not answered by all the participants at baseline . For this reason subjects who reported nir at baseline were excluded from the study population, along with subjects who did not answer the nir question at follow - up . To avoid misclassification between asthma and copd, all the subjects who reported asthma during the past 12 months at baseline were also excluded . As a result, 3,612 subjects were included in the analyses . Spirometry was performed with a dry wedge spirometer (vitalograph, buckingham, uk). According to the guidelines available at the time of the study,19 the best of a minimum of three similar maneuvers the ratio of the forced expiratory volume in 1 second (fev1) divided by forced vital capacity (fvc) was calculated and a cut - off value for the fev1/fvc ratio of <0.7 defined copd in accordance with the global initiative for chronic obstructive lung disease guidelines,8 apart from the fact that no reversibility test was performed . Feno was measured before spirometry using a chemiluminescence analyzer and has been extensively described elsewhere.20 a cut - off value of 50 ppb or more in the exhaled no concentration was used to indicate a high likelihood of asthma in patients with copd.21 atopy was defined as a positive phadiatop test (pharmacia, uppsala, sweden). Class 0 was regarded as negative and class 1 as positive (atopic).22 data on age, gender, and body mass index (bmi) were also collected (table 2). Analyses were performed with the sas statistical software version 9.4 (sas institute inc ., cary, nc, usa). Univariate analyses included using t - tests and chi - square tests for continuous and categorical variables respectively . A multivariate logistic regression model was used to calculate odds ratios and 95% confidence intervals . This study is a part of the longitudinal population - based study called adonix (adult onset asthma and nitric oxide). Adonix was approved by the regional ethical review board of gothenburg (dnr 092 - 01) and has previously been described in detail.5 the adonix study was designed to evaluate risk factors for airway disease such as nir, copd, and asthma . A total of 6,665 subjects, randomly selected, aged 2575 years, from the municipal register of gothenburg, sweden, were included at baseline between 2001 and 2003 . The subjects performed spirometry and answered a questionnaire with questions on upper and lower respiratory symptoms and diseases . The subjects also underwent a measurement of fractional exhaled nitric oxide (feno) and a laboratory analysis of specific immunoglobulin e antibodies for atopy status . After 5 years, the included subjects were asked to answer a follow - up questionnaire . This study evaluates the risk of developing new - onset nir in relation to having copd during a 5-year observation period . The questions used for new - onset nir and associated risk factors, such as smoking, educational status, and occupational exposure to gas, fumes, or dust, are shown in table 1 . The questions on occupational exposure to gas, fumes, or dust, and educational status were added in 2005 and were therefore not answered by all the participants at baseline . For this reason, subjects who reported nir at baseline were excluded from the study population, along with subjects who did not answer the nir question at follow - up . To avoid misclassification between asthma and copd, all the subjects who reported asthma during the past 12 months at baseline were also excluded . As a result, spirometry was performed with a dry wedge spirometer (vitalograph, buckingham, uk). According to the guidelines available at the time of the study,19 the best of a minimum of three similar maneuvers the ratio of the forced expiratory volume in 1 second (fev1) divided by forced vital capacity (fvc) was calculated and a cut - off value for the fev1/fvc ratio of <0.7 defined copd in accordance with the global initiative for chronic obstructive lung disease guidelines,8 apart from the fact that no reversibility test was performed . Feno was measured before spirometry using a chemiluminescence analyzer and has been extensively described elsewhere.20 a cut - off value of 50 ppb or more in the exhaled no concentration was used to indicate a high likelihood of asthma in patients with copd.21 atopy was defined as a positive phadiatop test (pharmacia, uppsala, sweden). Class 0 was regarded as negative and class 1 as positive (atopic).22 data on age, gender, and body mass index (bmi) were also collected (table 2). Analyses were performed with the sas statistical software version 9.4 (sas institute inc ., cary, nc, usa). Univariate analyses included using t - tests and chi - square tests for continuous and categorical variables respectively . A multivariate logistic regression model was used to calculate odds ratios and 95% confidence intervals . The comparison between subjects with new - onset nir and no nir at follow - up is presented in table 2 . As shown, there was no significant difference in mean age, gender, or bmi . The prevalence of copd was 8% in the total study population and 9.2% for subjects aged over 40 years . New - onset nir was significantly associated with copd (10.8 vs 7.4 p=0.005). New - onset nir was also related to smoking (p=0.004) and atopy (p<0.001) in the univariate analyses . In the logistic regression analysis adjusted for age, gender, bmi, copd, smoking, and atopy, copd, smoking, and atopy remained independently associated with new - onset nir (figure 2). The interaction between copd and smoking in relation to new - onset nir was calculated and was found to be nonsignificant (data not shown). There was no association between atopy and copd, indicating a low risk of asthma misclassification (19.1% vs 19.2%, p=1). As shown in figure 3, the vast majority of the subjects with copd were aged> 40 years, as expected . Only 4.3% (11 of 257 subjects with copd) displayed a feno value> 50 ppb, ie, values that indicate the presence of atopy and asthma . The sub - analysis of occupational exposure to gas, fumes, or dust (n=2,257), and educational status (n=2,256) introduced in 2005 revealed a significant association with new - onset nir for gas, fumes, or dust (p=0.006) but not for educational status (p=0.75) (data not shown). In this longitudinal population - based study of a large cohort, the risk of developing nir, over a 5-year period increased in individuals with copd . Smoking and atopy were also risk factors for nir . The results indicate that there is a link present between upper and lower respiratory inflammation in nir and copd, and it is therefore important to assess nir in patients with copd . Nir is identified by the presence of nasal symptoms, but identifying individuals with copd requires spirometry . The authors are not aware of any previous studies of nir in copd, including spirometry data from a large general population sample . In this study, this could have resulted in misclassification between copd and asthma . In an attempt to minimize the risk of misclassification to eosinophilic inflammation and asthma, all subjects reporting asthma within 12 months at baseline were excluded from the analyses . Also, atopy in subjects with copd was analyzed and no significant association with copd was identified . Furthermore, only 4.3% of the subjects in the copd group had feno> 50 ppb, which indicates a low prevalence of eosinophilic inflammation in this group . Therefore, the subjects in this study with an fev1/fvc of <0.7 are believed to be a representative of copd . The prevalence of copd in this general population sample was 8.8%, which is consistent with previous estimates ranging between 9.4% and 18.8%.1418 the prevalence was also higher in subjects aged 40 and above, which is in accordance with other studies of copd.11 the main finding in this study was that the 5-year cumulative incidence of nir was higher in the subjects with copd, both in the univariate analyses and the logistic regression analyses . Smoking and atopy also remained independent risk factors for new - onset nir in the logistic regression analyses . Although a previous study based on questionnaire data alone has indicated a relationship between rhinitis and copd in a large population sample, this study adds vital information on lung function that is needed for a copd diagnosis and confirms that there is a relationship between the two diseases.7 although the study shows a link between upper and lower respiratory inflammation in copd and nir, it is important to note that the findings do not imply a causal relationship between copd and nir . In spite of this, both diseases share common features that could be of importance for disease development in the airway at different levels . A similar inflammatory response can be elicited in both the upper and lower airways following exposure to inhaled irritants and sensitizers in the common airstream . Gas, fumes, or dust are regarded as risk factors for copd,9 as well as for nir.6 individual vulnerability in the airway mucosa to irritants or deficits in systemic inflammatory responses could also explain why copd and nir can more commonly be found in the same subjects . The humoral spread of inflammatory mediators via the blood stream to both the upper and lower airways as seen in rhinitis and asthma is another possibility.23 tobacco smoke is an airborne irritant that is significantly associated not only with the development of copd but also with nir.1,11,12,24 in terms of the temporal relationship between the onset of copd and nir, it is important to recognize that the airway inflammation in copd and nir is likely to develop over time . In the absence of histological specimens from the airway, it is not possible to determine when and where inflammation starts and symptoms may begin later than signs of airway inflammation . Differences in the concentrations to which the upper and lower airways are exposed may also explain different patterns of symptom debut . Tobacco smoke is mainly inhaled through the mouth directly to the lungs and the tobacco load on the nasal mucosa is likely to be lower . To be able to establish plausible common pathophysiological mechanisms two possible risk factors for new - onset nir were analyzed separately, due to their late introduction in the questionnaire that missed a part of the study population, ie, educational status and occupational exposure to gas, fumes, or dust . Educational status was not associated with new - onset nir, despite having previously been considered to be a risk factor for copd.25 occupational exposure to gas, fumes, or dust was not included in the logistic regression due to missing data, but in the sub - analyses it showed an association with new - onset nir . In developing countries, this is a major risk factor for copd due to cooking over open fires and it may also be important for nir, but the exposure in developed countries, such as sweden, is expected to be low . The main strength of the present study is its longitudinal population - based design with a large general population, including the availability of spirometry data for 93% of the subjects . Nir includes several phenotypes of rhinitis, both allergic and nonallergic, as well as chronic rhinosinusitis . As a result, the nir question does not enable us to link copd to a specific phenotype of rhinitis . The interval of 5 years in the follow - up questionnaire may have introduced recall bias when answering the nir question . Furthermore, this epidemiological study did not include a clinical assessment of copd by a doctor and therefore have no information on the severity of the disease . The copd definition that was used may also have introduced misclassification to other lower airway diseases showing a similar spirometry pattern, but, as previously discussed, the risk of including asthmatics in this study was minimized . There is an established inflammatory link between asthma and rhinitis, which many regard as the concept of generalized airway inflammation the united airways, helping to define and understand the disease.3 this study provides new evidence linking nir to copd, supported by the fact that both diseases involve inflammation of the airway mucosa and that they share common risk factors, such as exposure to tobacco smoke and cooking fumes . In contrast to the link between allergic rhinitis and asthma, the authors are aware of no common inflammatory pathway in copd and nir, such as the allergic inflammation that could explain the coexistence of the two diseases . To be able to confirm a pathophysiological relationship, further studies are needed, but the present study shows that copd should be regarded as a co - factor in the development of nir . The link between nir and copd also suggest that the diagnosis and treatment of the two diseases in the same patient could be of importance for patient outcome in terms of early diagnosis, disease control, and patient satisfaction, which has to be addressed further . This longitudinal population - based study of a large cohort shows that copd is a risk factor for developing nir . The results indicate that there is a link present between upper and lower respiratory inflammation in nir and copd.
Immunocompromised patients might bear a variety of presentations such as rhino - orbito - cerebral, pulmonary, gastrointestinal, cutaneous, central nervous system and disseminated form . Rhino - orbito - cerebral and pulmonary mucormycosis are the most common forms; the latter progresses rapidly to devastating diseases . Presentation of pulmonary mucormycosis may be confused with several infectious and non infectious diseases; therefore its diagnosis is difficult . Chest ct scan of a patient with pulmonary mucormycosis might demonstrate such presentations as air crescent sign, ct halo sign, solitary or multiple pulmonary nodules or masses, bronchopulmonary fistula and pulmonary artery pseudo aneurysm . The occurrence of pneumothorax and its subsequent rhs along with sinusitis, supported by radiology and pathology in a 33 years old patient, is considered as a rare case of pulmonary mucormycosis . Following is the treatment history of a man, 33 years old in july 2014 (day + 14), when he died in our hospital as a result of multiorgan failure . He was a farmer, married, with no history of alcohol consumption, illicit drugs use and extramarital relationships . On day 330: because of edema he visited a medical center in another city, where he was admitted and membranous glomerulonephritis was diagnosed . Consequently the followings administered; prednisolone and cyclosporine for 2 months, cellcept for 1 month, prednisolone and cyclophosphomide for 7 months . On day 75: because of progressive generalized edema he was admitted again . He was discharged under treatment with prednisolone (50 mg / daily) and tacrolymus (1 cap / bd). During the next 2 months he was feeling fine . On day 28, he travelled to village for a few days and he had close contact with sheep . On day 14 the patient reported fever, cough and dyspnea with mild erythema and pain in right eye . Anorexia, weakness, dyspnea and conjunctivitis developed and persisted despite of outpatient antibiotic treatment . In the emergency department (day 0): the patient suffered from dyspnea, weakness and right eye pain . On examination, he was agitated but completely conscious with diaphoretic skin . The vital signs have been as follows; temperature, 36.7 c; blood pressure, 100/60 mmgh; pulse rate, 99 beats per minute; respiratory rate, 22 beats per minute and oxygen saturation in air room, 99% . In right eye, there was moderate conjunctivitis with eyelid erythema; its movement was normal . The first day chest ct scan revealed large pleural effusion with pneumothorax in the left side and a shift of mediastinum to the right side . 1). Consequently drainage with chest tube was administered by the surgeon immediately . Because of the immunocompromised state of the patient, parenteral trimethoprim- sulfamethaxazol (160 mg / iv / tds) and meropenem (1 g / iv / bd) were administered for pneumocystis jiroveci and multi drug resistant organisms . We also started fluconazole for oral lesions . On day + 3, right eye erythema was extended with small size necrosis in right nasal ala without bleeding (fig . With doubt of rhino - orbital mucormycosis, biopsies of tongue and nasal septa were carried out and amphotericin b deoxycholate (1 mg / kg / daily) was administered . On day + 4, paranasal sinuses (pns) ct scan revealed soft tissue density lesion in left ethmoid sinus and nasal passage with extension to left orbit (fig ., he was transferred to intensive care unit (icu) because of respiratory distress and was intubated after 48 h; his conscious level was 14+t/15 . Smear of sputum for acid fast bacilli, stain and culture of sputum for pneumocystis jiroveci were negative . Hiv antibody test, d - dimer enzyme -linked immunosorbent assay (elisa) and collagen vascular diseases screening tests were negative (table 1). Pleural fluid cytologic examination and polymerase chain reaction (pcr) for fungi were negative . On day + 6, laboratory results revealed pancytopenia (table 1), however in peripheral blood smear there was no schistocyte or immature cells . Bone marrow biopsy which could help in finding the cause of pancytopenia was nevertheless postponed due to the patient's poor condition and low platelet count . Meanwhile his blood pressure was decreased and we started inotropic drugs.hemodynamic instability of the patient prohibited further attempts such as bronchoscopy and ct guided lung biopsy to identify the causative agent . On day + 9, section of tounge biopsy showed ulcerated squamous epithelial mucosa with subepithelial fibro fatty tissue and skeletal muscle fibers of tongue that is on site of ulceration . The inflammatory granulation tissue and inflammatory multinucleated giant cells were also seen in gimsa staining, but no cyst and trophozoite of pneumocystis jiroveci were identified . Pulmonary mucormycosis is a devastating infection when it affects immunocompromised patients such as diabetes mellitus, hematologic malignancies, chronic renal failure, post transplantaion and immunosuppressed persons . Herein, we present a rhino - orbital mucormycosis with pulmonary involvement . Reticulation inside the rhs, presence of pleural effusion, multiple nodules and concomitant rhino - orbital mucormycosis as well as documented pathology, collectively were considered as indicating that pulmonary involvement has been due to mucormycosis . The pathological finding is achieved by tongue tissue; lung biopsy has not been possible due to poor condition of the patient . Our case was an immunocompromised patient with documented rhino - orbital mucormycosis (figs . 3, 5 and 6). Chest ct scan showed bilateral pleural effusion, multiple nodules and rhs with thin reticulation (figs . 1 and 4). Although histopathology findings are usually needed to establish the diagnosis of pulmonary mucormycosis, using ct imaging and clinical presentation we can avoid the invasive procedures such as lung biopsy . There are few case reports of pneumothorax in pulmonary mucormycosis, some of which are iatrogenic during mechanical ventilation and bronchoscopic procedure [911]. Combined mucormycosis and methicillin - resistant staphylococcus aureus (mrsa) were found to be responsible for life threatening pneumothorax in an immunocompromised patient . Mucormycosis and aspergillus have been recognized as leading to pneumonia and pneunothorax in a dialysis patient . However, spontaneous pneumothorax in pulmonary mucormycosis that is followed by rhs seems to be a unique case . Based on patient's background and clinical signs, amphotericin b deoxycholate (1 mg the lower dose of antifungal drug has been due to the patient's renal insufficiency . Ambisome would have certainly been a better choice than amphotericin b; however, the former has not been available in iran . However the lack of bone erosion in sinus ct scan, delay in pathologic result and patient's poor clinical condition, lead into the reluctance of surgeon for extensive debridement that had negative impact on patient's prognosis . Pneumothorax which is due to mucormycosis can be fatal particularly in immunocompromised persons, so clinicians should take immediate measures (follow up imaging and pathology) to confirm or reject the presence of invasive fungal infection . If confirmed, antifungal therapy is necessary as soon as possible.
Ovarian follicular fate (i.e. Development / ovulation versus atresia) in the mammals is determined by the fate of the cells within the follicles and is regulated by complex interactions of endocrine (gonadotropin and steroid hormones) and locally produced (growth factors and cytokines) factors (hirshfield, 1991). Although follicular atresia can occur at all the stages of follicular development, a high incidence of granulosa cell apoptosis, a well recognized cellular basis for this degenerative process, is predominantly observed at the early antral stage of follicular development during the ovarian cycle (kim et al ., 1998). Fas antigen (fas), a member of the tnf receptor family, and its ligand fas ligand (fasl), have been identified as cell death mediator and factor, respectively in a variety of cell types (watanabefukunaga et al ., 1992; suda et al ., 1993). Their involvement in the induction of granulosa and luteal cell apoptosis has been demonstrated in several mammalian species (quirk et al ., 1995; hakuno et al ., 1996; kondo et al . Fas and fasl expression is up - regulated in granulosa cells undergoing apoptosis during early stage of follicular development (kim et al ., 1998) and p53-mediated granulosa cell apoptosis is associated with the activation of fas / fasl system during follicular atresia (kim et al ., 1999). In addition, follicle stimulating hormone (fsh) is an important negative regulator of the fas / fasl system and a key cell survival factor during the follicular development and the role of the fas / fasl system in granulosa cell apoptosis appears more prominent at early than at late stages of follicular maturation . Interferon gamma (ifn-) is a member of a family of heterogeneous regulatory proteins having antiviral, immunomodulatory, antiproliferative, and cytodifferentiation regulatory activities (pestka et al ., 1987). It increases adrenal steroidogenesis (blalock and harp, 1981), increases iodide uptake by thyroid cells (friedman et al ., 1982), and suppresses hcg - induced testosterone production by leydig cells (orava et al ., 1989). At physiological concentrations, ifn- inhibits granulosa cell differentiation and steroidogenesis (gorospe et al ., 1988; xiao and findlay, 1992). In addition, ifn- increases fas mrna expression in mouse lymphoma cells and bam3 cells (watanabefukunaga et al ., 1992) and fas content in human granulosa / luteal cells (quirk et al ., 1995). Apoptosis in ovarian surface epithelial cells is mediated by increased fas expression induced by ifn- (quirk et al ., 1997). Moreover, ifn- induces fas - mediated apoptosis in differrentiated mouse granulosa cells which could be potentiated by the presence of tnf (quirk et al ., 1998). Although the importance of ifn- in undifferentiated or poorly differentiated granulosa cells is well established, the presence of this cytokine in the ovary throughout follicular development has not been investigated . In addition, the regulation of fas system in undifferentiated granulosa cells by ifn-, subsequent fas - mediated apoptotic response as well as their modulation by gonadotropin in vivo remain unclear . The purpose of the present study was to examine the role of ifn- in regulation of fas expression and apoptosis in granulosa cells at different stages of follicular development and granulosa cell differentiation in vitro . In addition, we assessed the presence of ifn- in the ovary during follicular maturation and studied the relationship between the presence of ifn-, fas / fasl system and apoptosis in vivo . [p]-ddatp and dntps were obtained from amersham pharmacia biotech (piscataway, nj). Horse radish peroxidase (hrp)-conjugated goat anti - rabbit secondary antibody was purchased from bio - rad laboratories (hercule, ca). Rabbit anti - rat interferon- (ifn-) was obtained from biosource international (camarillo, ca). Biotinylated 16-dutp, in situ cell death detection (fluorescein) kit, proteinase - k and terminal deoxynucleotidyl transferase (tdt) were purchased from boehringer - mannheim (indianapolis, in). Recombinant rat ifn- was from genzyme (cambridge, ma). Fetal bovine serum (fbs), minimal essential medium (mem), non - essential amino acids (naa), penicillin and streptomycin were from gibco / brl (burlington, on, canada). Fitc - conjugated mouse anti - hamster igg, hamster anti - mouse fas monoclonal antibody (clone jo2) and hamster igg were obtained from pharmingen (san diego, ca). Avian myeloblastosis virus (amv) reverse transcriptase, oligo(dt)15 primer, rrnasin ribonucleotide inhibitor and taq dna polymerase were from promega (madison, wi). Rabbit igg, rabbit peroxidase kits, rabbit polyclonal anti - mouse fas (sc-716) and anti - rat fasl (sc-834) antibodies, and neutralization peptides (fas; sc-716p, fasl; sc-834p) were from santa cruz biotechnology (santa cruz, ca). Agarose, bovine serum albumin (bsa; fraction v), diethylstilbesterol (des), equine chorionic gonadotropin (ecg), methyl green, normal goat serum, phenylmethylsulfonyl fluoride (pmsf) and veronal acetate were purchased from sigma chemical co. (st louis, mo). To obtain ovaries consisting mainly of follicles synchronized at the preantral / early antral and medium / large antral stages in immature (21 - 23 days old) sprague - dawley rats (charles river canada; quebec, canada or samtako bio - korea; osan, korea), des (1mg / day, sc, for 3 consecutive days and sacrificed 24 hr after last injection) and ecg (15 iu, ip and sacrificed 48 hr thereafter) were administered, respectively . A 14 h light:10 h dark photo - cycle was maintained with lights - on at 0600 h. all procedures were performed in accordance with protocols approved by the dong - a university animal care and use committee . Following removal of connective tissues, the ovaries were washed in pbs (ph 7.4) to remove excess blood and were either immediately fixed in 10% neutral buffered formalin (ph 7.4) for histological processing or used for granulosa cell isolation by follicle puncture (rao et al ., granulosa cells (2.510) from ovaries of des- and ecg - primed rats were plated for 6 hr (5% co2, 37) in 60 mm falcon plastic culture dishes (becton dickins, lincoln park, nj) in mem supplemented with 2% fbs, 1x naa, penicillin (100 u / ml) and streptomycin (100 g / ml). The medium was then replaced with mem supplemented with 0.1% bsa, 1x naa, penicillin (100 u / ml) and streptomycin (100 g / ml) in subsequent culture . Non - viable (floating) cells were removed at this medium - replacement step . For induction of fas mrna and protein expression, granulosa cells were cultured in the presence of recombinant rat ifn- (0 - 1,000 u / ml of the medium) for 18 hr . Floating and attached cells harvested by centrifugation and trypsinization respectively, were combined and washed with ice - cold pbs, pending protein and rna extraction, and flowcytometric analysis . For immunocytochemistry and tunel, sterilized cover glasses (2222 mm) were added onto the bottom of the culture dishes prior to cell plating . At the end of the culture period, the cover glasses were removed, washed briefly with ice - cold pbs and processed for immunocytochemistry . To assess the influence of ifn- on fas expression and subsequent granulosa cell apoptotic response, cells pretreated for 18 hr with / without the cytokine were incubated for an additional 6 h with either agonistic fas monoclonal antibody (hamster anti - mouse fas mab, igg; 1 g / ml) or hamster igg (control; 1 g / ml). The effect of the treatment was assessed by phase - contrast microscopy, tunel and dna fragmentation analysis . Paraffin embedded whole ovarian sections (4 - 5 m) were used for ifn-, fas and fasl immunohistochemistry . The deparaffinized and hydrated sections were quenched in h2o2 (0.3%; 30 min) and rinsed thoroughly with pbs (315 min). The sections were blocked with normal goat serum in pbs [1.5%; room temperature (rt), 1 hr], and then incubated (rt, 45 min) with rabbit polyclonal anti - rat ifn- (0.8 g / ml) or rabbit polyclonal anti - mouse fas (0.3 g / ml), or rabbit polyclonal anti - rat fasl antibodies (0.3 g / ml) in pbs containing normal goat serum (1.5%). The sections were incubated at rt with biotin - conjugated goat anti - rabbit igg (1:200; 1 hr), avidin - biotin - peroxidase complex (santa cruz rabbit peroxidase kit; 1 hr) and dab solution (1 - 5 min), with nuclei lightly counterstained with hematoxylin . For negative controls, rabbit igg (1 g / ml) instead of the primary antibodies was added to the reaction . In this experiment, antibody specificity was confirmed by antibody preabsorption test, using recombinant rat ifn- (2 - 3 g) and fas and fasl neutralizing peptides (1 g) in the respective primary antibody reaction . For localization of fas protein in cultured granulosa cells, the cells were fixed for 10 min in ice - cold paraformaldehyde in pbs (2%; ph 7.4). After several washes with pbs, the cells were incubated (20 min) in pbs containing normal goat serum (10%) to suppress nonspecific igg binding, washed with pbs (35 min) and incubated for 1h with rabbit anti - mouse antibody [0.1 g / ml in pbs containing normal goat serum (1.5%)]. Following a pbs wash, the cells were incubated (30 min in the dark) with fluorescein - conjugated goat anti - rabbit antibody [1 g / ml pbs with normal goat serum (1.5%)], mounted with 90% glycerol in pbs and observed under fluorescence microscope . (1992) was used to localize apoptotic cells in paraffin whole ovarian sections mounted on positively charged slides (probeon plus; fisher scientific ltd, ottawa, on). Briefly, the sections were deparaffinized, hydrated, treated with proteinase - k (10 g / ml ., 30 min . ; 37). Endogenous peroxidase activity was removed by immersing the sections in methanol containing h2o2 (0.3%, 30 min ., the sections were soaked in the tdt buffer [tris - hcl (25 mm), sodium cacodylate (200 mm), cobalt chloride (5 mm), bsa (250 g / ml), ph 6.6; 15 min), incubated in 50 l of tdt buffer containing tdt (10 u) and biotinylated 16-dutp (1 nmol) [60 min ., 37], reacted with avidin - biotin - peroxidase complex (santa cruz rabbit peroxidase kit; 30 min) and subsequently with dab solution (1 - 5 min), as per manufacturer s instruction . If required, the nuclei were counterstained with methyl green (5%; in 0.1 m veronal acetate, ph 4.0). Tdt enzyme or biotinylated 16-dutp in the labelling reaction were omitted in the negative control slides . For in situ detection of apoptosis in cultured cells, cells were fixed (10 min, 0) in fresh paraformaldehyde (4% in pbs) and an in situ cell death detection (fluorescein) kit (boehringer mannheim; quebec, canada) was used, as per manufacturer s instructions . Total dna was extracted from cultured granulosa cells (floating cells + trypsinized attached cells) according to the modified procedure of gross - bellard et al . (1973), as previously described (li et al ., 1998). For biochemical assessment of dna fragmentation, dna was radiolabeled at the 3-ends according to an established method (tilly et al ., 1993). Briefly, one microgram of dna was incubated (37; 60 min) with 50 l of the labelling buffer [tris - hcl (25 mm), sodium cacodylate (200 mm), cobalt chloride (5 mm), bsa (250 g / ml), ph 6.6] containing terminal deoxynucleotidyl transferase (tdt; 25 u) and [p]-ddatp (5 mci, 3,000 ci / mmol). The labeled dna were resolved on 1.8% agarose gel (3.5 h, 60 v) in tae buffer . The gel was dried and subsequently exposed to x - ray film at -70. total rna was isolated from cultured granulosa cells using the qiagen rneasy mini rna isolation kit (valencia, ca). Briefly, the cells attached to the culture dishes were lyzed with lysis solution (buffer rlt) provided by the manufacturer, and the lysate was passed through the qiagen qia shredder (valencia, ca). To ensure the extracted rna for pcr was free of dna, two micrograms of total rna was reverse - transcribed (42, 15 min) with oligo (dt)15 primers (0.5 g/g rna) and avian myeloblastosis virus (amv) reverse transcriptase (15 u/g rna) in a 20 l reaction mixture containing 10x reverse transcription buffer [tris - hcl (100 mm) (ph 8.8), kcl (500 mm), triton x-100 (1%)], mgcl2 (25 mm), dntp mixture (10 mm) and rrnasin ribonucleotide inhibitor (2 u). The reaction was terminated by inactivating the reverse transcriptase (heating at 99, 5 min). Gene - specific oligonucleotide primers (46.1% gc content) were designed based on published rat fas cdna sequence [22; genbank accession no . D26112] and synthesized using a beckman oligo 1000 m dna synthesizer (fullerton, ca). Primers for rat -actin (nudel et al ., 1983) were obtained from clontech (palo alto, ca). The primers used for pcr amplification for fas were 5'-tgcacctcgtgtggacttgaag-3'[479 - 500; forward primer] and 5'-ccagtcttcccgtgagattgatac-3' [888 - 865; reverse primer]. Polymerase chain reactions were carried out with a pct-100 programmable thermal controller (mj research, inc ., watertown, ma). The pcr reaction mixture included the oligonucleotide primers (5 pmol each), and taq dna polymerase (0.5 u) for each reaction . The total volume was brought up to 50 l with 1x pcr buffer [tris - hcl (50 mm), kcl (20 mm), mgcl2 (3 mm), dmso (0.5%)] and dntp (20 m). Amplification was carried out for 35 cycles under the annealing temperature of 60 (45 sec), primer extention at 72 (1 min) and denaturation at 94 (30 sec). The amplified dna was electrophoresed on 2% agarose gel, and the gel was stained with ethidium bromide for visualization under uv light . At initial stage of this experiment, the amplified fas product was confirmed by southern hybridization (sambrook et al ., 1989), using the rat fas cdna probe . The intensities of the bands were densitometrically scanned and each fas amplified product was normalized by -actin . Harvested granulosa cells (210 cells) were resuspend in ice - cold wash buffer [nan3 (0.1%), fbs (0.5%) in pbs] and centrifuged (500 g; 5 min, 4). The cells were immediately incubated in primary antibody solution [hamster anti - mouse fas (0.5 g / ml) in wash buffer)] at 4 for 30 min ., washed (500 g, 25 min in wash buffer), and incubated (30 min ., 4 in dark), with fitc - conjugated mouse anti - hamster igg (1.0 g / ml). The cells were then washed twice with the wash buffer, filtered through a 35 m nylon mesh to remove cell clumps and aliquoted for appropriate cell concentration (final concentration, 110 cells in 500 l) for flow cytometric analysis (coulter epics profile ii, hialeah, fl). As negative controls, nonspecific binding in flowcytogram was determined in the absence of primary antibody . Pcr and flowcytometry data presented herein were expressed as the meansem of three separate experiments . The presence and relative abundance of ifn- protein in the follicles during development were screened by immunohistochemistry, using a rat specific ifn- antibody (fig . 1a) exhibit immunoreactivity for ifn-, however, the intense and aggregated signals were detected in granulosa cells (arrows) as well as oocyte (including its nucleus). In contrast to its intense immunoreactivity in the cytoplasm, the nuclei of oocytes of secondary and tertiary preantral follicles showed considerably lower immunostaining . 1b), granulosa cells in preantral follicles exhibited immunopositivity for ifn-, although with lesser intensity and aggregated characteristics than those in primordial and early stage of preantral follicles . 1d) antral follicles was much less than that seen in the preantral follicles . Generally, the immunereactivity in theca cells was minimal in the antral follicles . A, secondary (preantral) follicles; b, preantral follicle; c, medium antral follicle; d, large antral follicle . Gc, granulosa cells; tc, theca cells; it, interstitial cells; cc, cumulus cells; o, oocyte; n nucleus; at, antrum . Arrows indicate intense and aggregated immunoreactivity for ifn-. Magnification: 400. scare bar: 50 m . Based upon the immunohistochemical evidence for the presence of inf- at different stage of follicular development in vivo (fig . 1), the potential role of ifn- in the regulation of fas mrna and protein in relatively undifferentiated (des - primed) and differentiated (ecg - primed) granulosa cells was investigated in vitro, using rt - pcr (fig . 3). In cultured des - primed granulosa cells, ifn- up - regulated fas mrna in a concentration - dependent manner (fig . High concentration (> 100 u / ml) of ifn- (1,000 u / ml) resulted in the loss of effectiveness of the cytokine in inducing fas mrna and protein expression (fig . 2). A, analysis of fas mrna expression by rt - pcr; b, densitometric quantification of fas mrna expression obtained from panel a. , p<0.05; * *, p <0.001; ###, p<0.001 vs. control group (0 u / ml). Insert in each flowcytogram shows representative fluorescence microphotograph of fas immunocytochemistry (arrows and arrowheads indicate immunoreactivity in cytoplasm and membrane, respectively . Numbers 1 and 2 in each flowcytogram indicate nonspecific (partially overlapped with low intensity of fas - positive portion) and specific fas - positive (high fluorescence intensity) portion, respectively . Although cultured ecg - primed granulosa cells also expressed fas mrna, its abundance was considerably lower than that in des - primed cells . In addition, while fas mrna also increased in a concentration - dependent manner in the presence of ifn-, significantly higher concentrations of the cytokine (100 - 1,000 u / ml, p<0.01) were required to elicit a significant increase in fas expression than those in des - primed cells (fig . The fas protein was localized in the des- and ecg - primed granulosa cells after ifn- treatment and the expression levels were subjected to fas antibody - conjugated immuno - flowcytometric analysis (fig . 3). As shown in the inserts within each representative flowcytogram (fig . 3), fas protein was localized in both the cellular membrane (arrow head) and the cytoplasm [peri - nuclear region (arrow)]. In addition, intense and aggregated immuno - fluorescence was observed in both des- (fig . 3) granulosa cells after ifn- exposure . Whereas a significant population of the control group in the des - primed granulosa cells exhibited the high intensity of immunofluorescence for endogenous fas immunoreactivity [fig . 3a, portion 2 (184.3%)], the ecg - primed control granulosa cells showed relatively smaller population with high immunofluorescence intensity for fas [fig . Ifn- treatment (100 u / ml) of des - primed granulosa cells significantly increased high intensity (337.5%) portion of fas immunofluorescene (fig . However, treatment with high concentration of ifn- (1,000 u / ml) resulted in a lower number of cells exhibiting immunofluorescence positivity for fas in high intensity (252.3%) portion than those with 100 u / ml but higher than that of the control group . In ecg - primed cells, however, only small increases in immunofluorescence were detected with ifn- at concentrations of 100 and 1,000 u / ml when compared to controls (fig . 3e and 3f). The functional role of ifn--induced fas expression in induction of apoptosis were tested in undifferentiated (des - primed) and differentiated (ecg - primed) granulosa cells in vitro was determined by assessing their the ability to undergo apoptosis following a challenge with an agonistic effect of fas mab . While typical cellular morphology and apoptosis in des - primed granulosa cells are illustrated in fig . 4, differences in these features between des- and ecg - primed groups are summarized in table 1 . In des - primed (undifferentiated) granulosa cells, ifn- had minimal effects on cellular morphology (fig . However, addition of fas mab to the cell cultures resulted in the appearance of apoptotic cellular morphology and characteristics (fig . 4c & 4 g), which could be enhanced with pretreatment with ifn- (fig . 4d & 4h). In ecg - primed (differentiated) granulosa cells, however, ifn- pretreatment failed to potentiate the pro - apoptotic effects of agonistic fas mab, which were observed prominently in des - primed cells (table 1). Left and right panels illustrate cellular morphology (phase - contrast microscopy) and in situ apoptotic cell detection (tunel), respectively . A and e, control group [ifn- vehicle (medium) (24 hr) + igg (6 hr)]; b and f, ifn- group [ifn- (24 hr) + igg (6 hr)]; c and g, fas mab group [ifn- vehicle (medium) (24 hr) + fas mab (6 hr)]; d and h, ifn- and fas mab group [ifn- (24 hr) + fas mab (6 hr)]. Arrowheads in left and right panels indicate floating cells and tunel - positive cells, respecttively . Magnification: 400. inserts in right panels show representative apoptotic dna fragmentation pattern in each treatment group . -: rare, +: occasional, + +: frequent, + + +: extensive, + + + +: very extensive to further examine the colocalization of ifn-, fas and fasl expression and occurrence of apoptosis in vivo, follicles at very early stage of antral formation (estimated between preantral and early antral stages) were analyzed serially by immunohistochemistry and in situ tunel method (fig . 5). Immunoreactivity for ifn- was distributed in the granulosa but not the thecal layers (fig . Likewise, the most intense and aggregated form of fas immuno - positive signals were detected in the loosely attached granulosa cells (fig . Although fasl immunoreactivity could be colocalized in fas - positive cells, he most aggregated and intense (arrows) immunoreactivity was present in granulosa cells mainly lining the antrum (fig . 5c). Similarly, apoptotic (tunel - positive) granulosa cells were also detected in antral granulosa cells (fig . A, ifn-; b, fas; c, fasl; d, tunel . Gc, tc and it represent granulosa cells, theca cells and interstitial cells, respectively . In the present study, we have localized rat ifn- in the rat ovary during the follicular development and tested its role on granulosa cell death via induction of the fas / fasl system in granulosa cells at different stage of differentiation . We have found that the responsiveness of granulosa cells to ifn- in terms of fas receptor expression was significantly compromised with follicular maturation . The immunoreactivity of ifn- was most intense in early stages of folliculogenesis, such as in preantral follicles . Up - regulation of fas by ifn- and the subsequent increased responsiveness to an apoptotic death signal (e.g. Fas activation by agonistic fas mab) were more evident in granulosa cells from the preantral follicles than those from antral follicles previously exposed to gonadotropins . The importance of intraovarian interaction between immune cells (lymphocytes and macrophages) and granulosa cells in ovarian physiology via the synthesis and actions of cytokines (e.g.tnf and il) has been studied extensively (adashi, 1992; terranova et al ., 1993). Although the modulatory roles of ifn- in granulosa cell differentiation and steroidogenesis have been demonstrated in vitro (gorospe et al ., 1998; xiao and findlay, 1992), ifn- is detectable in the follicular fluids (grasso et al ., 1988) and lymphocytes present in the ovary have been suggested to be an important contributor to the follicular ifn- (adashi, 1992). Immune cells (lymphocytes / macrophages) are believed to be mainly present in atretic antral follicles (best et al ., 1996),, 1994a; brannstrom et al ., 1994b) where they remove damaged and dead cells by secreting cytotoxic cytokines and phagocytosis . The presence of immune cells during early stage of follicular development has not been reported and the possibility that immune cells are the only the source of ifn- in preantral follicles cannot be excluded . In this study, ifn- was localized predominantly in granulosa cells and oocytes throughout the follicular development but more abundantly in those at the early follicular (preantral) stage . It has been demonstrated that tnf- and fas ligand are expressed in granulosa cells and oocytes (chen et al . Although we have not studied ifn- gene expression in ovarian cells in the current study, it is possible that oocytes and granulosa cells in the preantral follicles may be an important source of ifn- in the ovary and that this cytokine plays an autocrine and/or paracrine role in regulating the expression and actions of other cytokines involved in cell removal process in the degrading follicles . Ifn- is an important modulator of granulosa cell differentiation during the early stage of follicular development (gorospe et al ., 1988; xiao and findlay, 1992), although its functional role in differentiated granulosa cells in late follicular development (i.e., preovulatory follicles) remains to be further established . Granulosa cell apoptosis is frequently observed in early antral (penultimate) stage of follicular development, it is only assumed that up - regulation of fas receptor by ifn- in granulosa cells of preantral follicles is prerequisite for fasl ligation and that the latter could be activated by intra - ovarian factor(s) following antrum formation . It has been suggested that ifn- facilitate apoptosis in luteinized granulosa cells by decreasing endogenous igf activity via stimulation of igfbps rather than of fas activation (cataldo et al ., 1998). However, the concentrations of ifn- used in above studies could have been pharmacological and cytotoxic to the cells . Quirk et al (1995) demonstrated fas - mediated apoptosis in human granulosa - luteal cells and suggested that ifn- up - regulates fas mrna and protein expression and that pretreatment of cells with ifn- alone or in combination with hcg facilitates fas mab - induced cytotoxicity . Therefore, it is unlikely that the physiological concentration of ifn- could induce fas expression in fully differentiated luteinized (or luteinizing) granulosa cells . Recently, quirk et al (1998) showed that combined ifn- and tnf treatment was more effective in inducing fas expression and potentiating fas mab - induced apoptosis in cultured granulosa cells (mostly from large preovulatory follicles in ecg - primed mice) than by either ifn- or tnf alone . The results suggest that the mechanism(s) of induction of apoptosis and responsiveness against apoptotic insult (e.g. Ifn-) in differentiated granulosa cells might be much more complex and resistant than those of undifferentiated granulsoa cells, as also seen in the present study . The possible mediatory role of the fas / fasl system in granulosa and luteal cell apoptosis during follicular atresia and luteal regression, respectively have been investigated in several mammalian species (quirk et al ., 1995; previous reports have demonstrated that increased granulosa cell fas and fasl protein content is associated with apoptosis at the penultimate (early) stage of follicular development in vivo (kim et al ., 1998), and that p53-mediated granulosa cell apoptosis involves the activation of the fas / fasl system (kim et al ., 1999). These studies also suggested that fsh is a key inhibitory factor on granulosa cell death as well as the involvement of the fas / fasl system in follicular atresia . The nature of the regulation of fas receptor expression throughout the follicular development, however, remains unclear . Although the regulation of fas receptor expression by ifn- has been demonstrated in human granulosa - luteal cells in vitro (quirk et al ., 1995), the potential regulatory role of ifn- in fas mrna and protein expression in undifferentiated granulosa cells during early stages of folliculogenesis has not been thoroughly investigated . In the present studies, we have studied the possible role of ifn- in the regulation of granulsoa cell fas expression during follicular development . While increased fas expression in granulsoa cells from preantral or early antral follicles could be induced with low concentrations of ifn- (10 - 100 u / ml), higher concentrations (1,000 u / ml) were required in ecg - primed cells . Our present immuno - flowcytometric analysis also confirmed that undifferentiated (des - primed) granulsoa cells were more responsive to ifn- in the induction of fas receptor than cells pretreated with gonadotropin . These findings are not only consistent with the studies by quirk et al (1995) which showed increased expression of fas protein in gonadotropin - primed human granulosa cells following exposure to very high concentration (> 10,000 u / ml) of ifn- in vitro, but extended their observations to suggest a role for the cytokine in the ovary at physiological concentrations . It is well established that fsh is an important cell survival factor in the mammalian ovarian follicle (chun et al . Whereas stimulation of immature rats with ecg resulted in suppressed basal granulosa cell fas / fasl levels and apoptosis, decreased atresia and enhanced follicular growth, withdrawal of gonadotropic support with the injection of a neutralizing antibody in vivo during early antral development led to increased granulosa cell fas / fasl expression and apoptotic cell death, and eventual follicular demise (kim et al ., 1998). In the present studies, we have demonstrated that while ifn- effectively up - regulated fas expression in granulosa cells from preantral and early antral follicles and sensitized them to the pro - apoptotic action of agonistic fas antibody, cells isolated from large antral follicles previously exposed to the gonadotropin in vivo were more differentiated but less responsive to ifn-. These findings suggest that the anti - apoptotic role of fsh in the granulosa cells may in part involve its modulatory influence on ifn- action, and are consistent with the concept that ovarian follicles beyond the penultimate stage of development are protected physiologically from pro - apoptotic insult and destined to ovulate (chun et al ., 1996; boone et al ., 1997). The mechanism(s) by which the gonadotropin modulates ifn--induced fas expression granulosa cells is unclear . It has been demonstrated that fas expression is regulated by the newly identified fas - regulatory gene tdag51, the expression of which is pkc - dependent (wang et al ., 1998). Since ifn-, like tnf (sancho tello and terranova, 1991) and il-3 (farrar et al ., 1985), appears capable of activating pkc (ostrowski et al ., 1988), it is possible that the reduced responsiveness of differentiated granulosa cells to ifn- might be due to pka activation by fsh . In addition, activation of pka pathway is known to inhibit pkc - dependent mechanism(s) in a variety of biological systems including the granulosa cell (shinohara et al ., 1985). In this context, differential responsiveness of granulosa cells at different cytodifferentiation to ifn- may be related to possible interactions between signal transduction pathways of ifn- and gonadotropin . It is possible that the efficient fas gene expression in undifferentiated granulosa cells may involve activation of pkc pathway by ifn-. Fas receptor signaling is a complex process and continues to capture considerable attention and research efforts . Fas activation initiated by ligation and oligomerrization results in recruitment of fadd (fasassociated death domain - containing protein) (chinnaiyan et al ., 1995). Fadd then activates procaspase8 (muzio et al ., 1996), which appears to be the first step of a proteolytic cascade that activates other caspases such as caspases3, 6, and 7 (hirata et al ., 1998) ifn- significantly increased the population of granulosa cells exhibiting high fluorescence intensity of fas immunoreactivity . While this increase might merely represent an increase in the cell population with enhanced fas expression, it is tempting to speculate that the high intensity of fluorescence reflects the oligomerization of fas receptor proteins, and that fas oligomerization in response to ifn- in undifferentiated granulsoa cells may be more efficient and functional than in differentiated granulosa cells . Whether this indeed is the case requires further investigation and confirmation . In this context, intense and aggregated immunoreactive fas protein has consistently been detected in cytoplasm as well as cellular membrane of cultured granulosa cells of the present study . These results support our previous observation suggesting the presence of aggregated fas immunostaining in the granulosa cells of atretic early antral follicles in the ovary (kim et al ., 1998). Moreover, recent studies have indicated that fas is also localized in the peri - nuclear region of the cytoplasm and that fas - trafficking from golgi apparatus to membrane is p53-dependent and required for fas - mediated apoptosis (bennett et al ., 1998). In light of our recent demonstration of the role of p53 in the regulation of fas expression in rat granulosa cells (kim et al ., 1999) and their intense fas immunostaining following ifn- challenge, investigations into whether this cytokine plays a role in fas trafficking in the granulosa cell would provide important clues on the cellular mechanism involved in the control of fas - mediated granulsoa cell apoptosis during early folliculogenesis . In summary, we have demonstrated that ifn- plays a pivotal role in regulating granulosa cell differentiation and apoptosis by potentiating fas function, a process modulated by gonadotropin (i.e. Fsh). The role of ifn- is most prominent in undifferentiated granulosa cells during early stage of follicular development.
Colorectal cancer is the third most common incident cancer among men and the second most common cancer among women worldwide . Incidence rates vary 10-fold in both sexes worldwide, and the highest rates are estimated in more developed regions, such as north america and western europe, whereas the lowest rates are estimated in africa (except southern africa) and south - central asia . In asia, colorectal cancer is the fourth most common incident cancer among men and the fifth most common cancer among women . Although incidence rates are relatively low in asian countries, those for east asian countries are relatively high . In korea, annual percentage changes in age - standardized incidence rates were 6.2% in men and 6.8% in women between 1999 and 2009 using the world standard population as a standard population . In 2009, 24,986 new colorectal cancer cases (15,068 men and 9,918 women) were diagnosed, accounting for 13.0% of all cancer occurrences . Colorectal cancer is the second most common cancer after stomach cancer among men and the third most common cancer after cancers of the thyroid and breast among women . There have been several reports on differences in patterns of colorectal incidence trends according to age group, sex, and anatomical location [3 - 5]. However, it has not been properly investigated whether colorectal cancers of a specific age group, sex, or anatomical location show more rapid increases in the korean population . The aim of the current study was to demonstrate changing trends of colorectal cancer incidence according to age group, sex, and anatomical location . The korea central cancer registry (kccr), a nation - wide, hospital - based cancer registry, was initiated by the ministry of health and welfare, korea in 1980 . The registry collected information on approximately 80 - 90% of cancer cases from more than 150 training hospitals across the country, and, in 1999, the kccr expanded cancer registration to cover the entire korean population under the population - based regional cancer registry program . Age (five - year intervals) and sex - specific incidence rates and the number of cases of colorectal cancer between 1999 and 2009 were obtained from the korea national cancer incidence database . Anatomical subsites were defined based on the tenth version of the international statistical classification of diseases and related health problems, 10th revision (icd-10). We defined the proximal colon as the cecum (c18.0), appendix (c18.1), ascending colon (c18.2), hepatic flexure (c18.3), transverse colon (c18.4), and splenic flexure of the colon (c18.5); the distal colon was defined as the descending colon (c18.6) and sigmoid colon (c18.7). Overlapping lesions of the colon (c18.8) and colon not otherwise specified (c18.9) were not included in the subsite analysis . Age - standardized rates (asrs) were calculated using the middle - year population of 2,000 as the standard population . Annual percent changes (apcs) for the incidence rates were calculated using a linear model, according to the following formula; (exp(b)-1)100, where b is the slope of the regression of the natural logarithm of the asr in a calendar year . The 95% confidence intervals (cis) were obtained with a standard error from the fit of the regression and the t - distribution function . All analyses were conducted by sex, subsite, and age group (10-year intervals). Male - to - female incidence rate ratios (irr) were calculated using the numbers of cancer patients for each site and the sex - specific population structure for each year using stata / se 10.0 for windows (stata corp lp, college station, tx). Statistical significance in difference between irrs the asrs and apcs overall and for each subsite of colorectal cancer by sex are shown in tables 1 and 2 and fig . 1 . Among men, the asr for colorectal cancer was 27 per 100,000 in 1999 and increased to 50.2 in 2009 (apc, 6.6%). The asr for colorectal cancer among women was 17.2 in 1999 and 26.9 in 2009 (apc, 5.1%). The rectum was the most common cancer site among both men and women in 1999 and 2009 . Cancer of the distal colon showed the highest apc (10.8% among men and 8.4% among women), followed by the proximal colon (7.9% among men and 6.6% among women) and rectum (5.2% among men and 2.4% among women). As a result, the proportion of rectal cancer decreased from 51.5% in 1999 to 47.1% in 2009 among men, and from 50.5% in 1999 to 42.8% in 2009 among women, whereas the proportion of proximal and distal colon cancers increased among both men and women (appendix 2). In all subsites, significantly higher apcs were observed among men, compared with women . As a result, the male - to - female irrs showed a significant increase between 1999 and 2009 for overall colorectal cancer (1.20 in 1999 to 1.51 in 2009), distal colon cancer (1.32 to 1.60), and rectal cancer (1.22 to 1.67), whereas the irr for proximal colon cancer did not show a significant change (1.09 to 1.16) (table 3). The asrs and apcs for colorectal cancer subsites by age group are shown in tables 4 and 5 . For overall colorectal cancer, men who were in their 60s showed the highest increase, whereas, for women, those in their 80s showed the highest increase . Distal colon cancer consistently showed the highest apc in most age groups among both men and women, except for women in their 80s, who showed the highest apc for proximal colon cancer . Among women, for almost every subsite, the most notable increases in incidence were observed for the oldest age group . Proximal colon cancer in men also showed the highest apc in the oldest age groups, whereas apcs were significantly higher only in the 60s for distal colon cancer and 40s and 60s for rectal cancer . However, the apcs showed narrow ranges of between 10.1 and 11.7 for distal colon cancer and between 4.7 and 5.8 for rectal cancer among men over 40 years old . The highest male - to - female irrs for overall colorectal cancer were observed for the oldest age groups, whereas the lowest irrs were observed for younger age groups (table 6). However, significant increases in male - to - female irrs were observed in their 50s and 60s between 1999 and 2009 . In the subsite - specific analyses, the changes in male - to - female irrs between 1999 and 2009 showed a statistically significantly increase for rectal cancer among men and women in their 50s, and 60s and for distal colon cancer among those in their 60s . Incidence of colorectal cancer has increased in most countries, except for the united states and some areas of japan . The most significant increases have been observed in eastern european countries and most asian countries . Although the korean population has experienced a rapid increase in colorectal cancer incidence, it has not been properly investigated whether the increasing pattern differs according to age group, sex, or anatomical location . There have been suggestions that colorectal cancer incidence by subsite differs according to race, sex, age group, and time period . In our study, rectal cancer accounted for the highest proportion among all subsites and ranged from 41% to 51% of all colorectal cancers . This result is consistent in part with a report from the us, which showed that the rectum (c19.9 and c20.9) was the most common subsite for male asians and pacific islanders living in the us (35%), whereas the proximal colon was the most common site for whites and blacks among both men and women, as well as female asians and pacific islanders . However, in the current study, the apc for rectal cancer was the lowest among the subsites included, thus, it appears that the proportion of rectal cancer has decreased among both men and women in korea . Instead, colon cancer, particularly distal colon cancer, has shown a rapid increase . We expect that the proportion of rectal cancer will continue to decrease in the future, and the proportion will eventually reach a level similar to that of western countries . One study suggested that incidence of colorectal cancer has increased among younger age groups in the us population, although overall incidence of colorectal cancer has declined . The increases observed in the younger population were mainly attributed to rectal cancer . In our study, we did not find clear evidence for a more rapid increase in overall incidence of colorectal cancer or rectal cancer in younger age groups, compared to older age groups in women . In men, however, it is worthy of mention that, unlike proximal colon cancer and distal colon cancer, where the apcs were lowest in their 30s and 40s, the apcs of rectal cancer in men in their 30s and 40s were statistically higher than those for men in their 70s and 80s . It is notable that although the incidence was higher among men than among women, the apcs were also higher among men than among women for most subsites . As a result, the male - to - female irr for overall colorectal cancer increased from 1.20 in 1999 to 1.51 in 2009 . A high male - to - female irr was observed for most subsites across ethnic groups in the us population . Our study also showed that the lowest male - to - female irr was observed for proximal colon cancer, indicating that the proportion of proximal colon cancer was higher among women than men . This result is consistent with results reported from the us, germany, and japan . Traditionally, differences in the distribution of colorectal cancer subsites between men and women have been explained by the role of female hormonal factors . Rapid increases in colorectal cancer incidence and the stabilization of colorectal cancer mortality in the korean population could be explained by introduction of colorectal cancer screening . Colorectal cancer screening programs were introduced in 2004 as a part of the national cancer screening program for medical aid recipients and national health insurance beneficiaries in the lower income bracket . The fecal occult blood test (fobt) is provided free of charge as a primary modality for men and women aged 50 years or older . Fobt - positive individuals were provided follow - up by either colonoscopy or double - contrast barium enema . The participation rate was only 10.5% in 2004, however, it increased to 21.1% in 2008 . According to the korea national cancer screening survey, which covers organized and opportunistic cancer screening programs, lifetime screening rates for colorectal cancer were 25.3% in 2004 and 54.2% in 2010, whereas screening rates by recommended guidelines were 19.9% in 2004 and 35.5% in 2010 . In the early phase of screening, however, the introduction of colorectal cancer screening cannot completely explain the differential increase in colorectal cancer incidence by subsite . Fobt usually has a higher sensitivity for advanced neoplasia, including colorectal cancer and advanced colorectal adenomas, in the left vs. right colon . This explains in part the increase in the rate of distal colon cancer, but not the low increase in rectal cancer . In addition, the national cancer screening program guidelines recommend colono - scopy or double - contrast barium enema as a follow - up modality for fobt positive patients . Sigmoidoscopy, which is used as a screening tool for an average - risk population in the us, is not considered as a follow - up modality in korea . Therefore, there is little possibility that screening - detected cancers are more likely to be distal colon and rectal cancer . A transition in risk factors may explain the differential increase in colorectal cancer according to incidence according to subsite . In a large insurance database - based study, frequent alcohol consumption and high consumption amount were more strongly associated with risk of distal colon cancer among men and risk of rectal cancer risk among women . High body mass index (bmi25 kg / m) was associated with increased risk for distal colon cancer among men and for proximal colon cancer among women . In addition, frequent meat intake was associated with proximal colon cancer risk among men and risk of proximal colon and rectal cancer among women . Daily alcohol consumption in korea increased from 10.6 g in 1999 to 17.3 g in 2007 among men and from 1.4 g in 1999 to 2.9 g in 2007 among women . The proportion of adult men with a bmi of 25 kg / m and was 25.1% in 1998 and increased to 35.3% in 2008 . Daily per capita meat consumption was 6.6 g in 1969 and increased to 95.1 g in 2005 . In contrast, the cigarette smoking rate in the adult population decreased from 75.1% in 1992 to 43.1% in 2009 among men and from 5.1% in 1992 to 3.9% in 2009 among women . Cigarette smoking has shown a stronger association with risk of rectal cancer than colon cancer . In our subsite analysis, we excluded overlapping lesions of the colon (c18.8) and colon not otherwise specified (c18.9). The proportions of c18.8 did not change between 1999 and 2009 (1.0% for men and 0.8% for women), however, the proportions of c18.9 during the same period decreased from 9.8% to 5.5% among men and from 11.1% to 5.9% among women . Improved accuracy in topology classification may explain in part the increase in the incidence of distal colon cancer, however, the changes exceeded the portion that might be explained by improved accuracy in topology coding . The rapid increase in colorectal cancer incidence in korea between 1999 and 2009 is mainly attributed to increases in colon cancer, especially distal colon cancer . Increases in the proportion of colon cancer may be explained by a transition in risk factors for subsites and the effect of colorectal cancer screening programs . The male - to - female irrs were higher for distal colon and rectal cancer and increased between 1999 and 2009 due to more rapid changes in male colorectal cancer incidence.
They may be the first manifestation of metastatic spread of an internal malignancy or herald cancer recurrence long after treatment of a primary tumor . Breast cancer, colorectal cancer, and melanoma frequently metastasize to the skin in women . In men; melanoma, lung cancer, and colorectal cancer are the most common sources of cutaneous metastases . However, such patterns may differ geographically as a study in taiwan found internal malignancies metastasize to the skin with different frequencies and at rates differing from primarily caucasian populations . An extensive literature review was conducted using pubmed from may 26, 2011-july 16, 2013 relating cutaneous metastases . Articles chosen for reference were queried with the following prompts: cutaneous metastases, clinical presentation, histological features, and further searches included treatment and management options for metastatic breast, metastatic colorectal, metastatic melanoma, metastatic lung, and hematologic cancers . We review the literature on clinical presentation, diagnosis, and advances in the management . Cutaneous metastases may be asymptomatic or be associated with pain and tenderness [figure 1a]. They frequently present as a rapidly growing painless dermal or subcutaneous nodules with intact overlying epidermis [figures 1b d], but can also mimic an inflammatory dermatosis . Metastases may also present as macules, infiltrated or indurated plaques, discoid lesions, tumor nodules with telangiectasias, bullous or papulosquamous lesions, scarred plaques, or pigmented tumors . While there is no single diagnostic characteristic which predominates; a few patterns have been recognized . (a) gingival metastases from a primary chondrosarcoma. (b) firm nodule at the base of the ear from a primary lung cancer . (c) two subcutaneous firm nodules in the clavicular fossa from metastatic lung cancer . (d) isolated firm nodular lesion from metastatic colon cancer metastases originating from breast cancer tend to appear in the anterior chest wall, either from direct extension of underlying tumor or by lymphatic spread [figures 2a and 3a]. Extensive cutaneous involvement of metastatic breast cancer can simulate cellulitis (carcinoma erysipeloides) or a breast - plate of armor (" en cuirasse " pattern). Interestingly, a subset of breast cancer patients has superior prognosis, even among breast cancer patients with stage iv disease . (a) cellulitis - like appearance of metastatic breast cancer. (b) plaque - like area with multiple nodules from a primary breast cancer . (c) massive recurrence of breast cancer on the chest wall and abdomen of the en cuirasse type . (d) breast cancer, metastatic to the thoracic spine with extension to the skin, and lceration secondary to radiation therapy (a) cellulitic - type appearance with multiple nodules in metastatic breast cancer of the shoulder and upper back . A cam 5.2 stain reveals perinuclear staining (original magnification, 400). (c) metastatic squamous cell carcinoma of the skin reveals neoplastic cells in the dermis (cytokeratin 5/6 stain; original magnification, 100). A her2/neu stain is positive and decorates the neoplastic cells in the dermis indicating possible metastasis from breast carcinoma (original magnification, 200) while most cancers present as solitary nodules, melanoma and carcinomas with an unknown primary frequently present as multiple nodules . Locoregional melanocytic metastases may be explained by angiotropism, an extravascular migratory phenotype, and an important marker and prognostic factor of metastasis in melanoma . The face and scalp are most commonly affected by metastases, suggesting that blood vessels and patterns of innervation may influence the spread of metastases . These patterns can also been associated with cancers of gastric, pulmonary, prostatic, ovarian, laryngeal, palatine - tonsillar, pancreatic, colorectal, parotid, thyroid, or uterine origin and may suggest local treatments that work for melanoma may also work for other metastatic cutaneous malignancies . Although metastases may be comprised of cells which are more undifferentiated than the primary tumor, careful examination usually reveals important clues . For example, melanoma is associated with pigment in at least some of the neoplastic cells in most cases . Squamous cell carcinoma is associated with keratin pearl formation, and adenocarcinomas usually exhibit gland formation . Thyroid cancer may exhibit colloid bodies, colon cancer may be associated with mucinous cells, and other tumors may exhibit telltale clues as to their origin . When confronted with a possible metastasis, comparison with the histologic appearance of any prior malignant neoplasms is usually one of the first steps that may help identify the source . Additionally, studies have indicated the majority of metastatic cancers to the skin are adenocarcinomas . When tumors are poorly differentiated or anaplastic, screening immunohistochemical studies can be very helpful . When neoplastic cells stain with cytokeratin markers such as ae1/ae3 and cam 5.2, a carcinoma is likely [figure 3b]. Staining with cd45/leukocyte common antigen (lca) indicates that neoplastic cells are of lymphoid origin, while s-100, melan - a, mart-1, hmb-45, and sox-10 decorate most melanomas . More detailed immunohistochemical studies [table 2] can be performed when screening studies and clinical correlation have not been fruitful in identifying a tumor of origin . Although advances in immunohistochemistry are very promising, there is still much work that needs to be done to characterize metastases . A recent letter to the editor highlights the pitfalls that can occur when differentiating primary cutaneous apocrine carcinoma from a metastasis . Although cytokeratin 5/6 is associated with cutaneous apocrine carcinoma, this staining pattern has also been noted with metastatic squamous cell carcinoma [figure 3c]. Similarly, cytokeratin 7 usually stains metastases diffusely, but is associated with only focal staining in primary adnexal carcinoma, but this pattern is not absolute . Mammaglobin is of interest as it is associated with more widespread staining of metastases than primary apocrine carcinoma, but the results are still preliminary . Cyclooxygenase 2 is associated with tumors of the mid - gut (ileum and appendix), gut, colorectal adenocarcinoma as well as gastroesophageal adenocarcinoma . Staining of adenocarcinoma of the lung and endometrioid adenocarcinoma can also occur, highlighting the fact that all immunochemical marker studies must be interpreted in the context of other findings . When an epithelial carcinoma is identified, cytokeratin 7 and 20 stains can provide important useful characterization of tumors . Additional testing can include thyroid transcription factor-1 (ttf-1) and other markers as needed . When clinical appearance, clinical history, and the histologic findings on biopsy are all taken into account unfortunately, there are still many cases that elude precise diagnosis regarding whether a tumor is primary or metastatic . Recent reports have proposed a diagnostic algorithm for metastatic tumors of unknown primary . Until better methods are developed we recommend excising metastases when surgically feasible and when this will result in a significant decrease in total tumor burden, improve quality of life, or result in increased functionality . Novel treatments have had a profound effect on prolonging the survival and improving quality of life with metastatic cancer . Oftentimes, treatment of the primary cancer with a successful regimen will cause cutaneous lesions to lessen . Interestingly, the abscopal response represents an unusual response to radiation therapy in which radiation of a tumor causes untreated distant skin lesions to dissipate without recurrence . The pathophysiology and mechanism of the abscopal effect have not been well - defined, but one study used monoclonal antibodies to analyze infiltrating lymphocytes and suggested that the abscopal effect was caused by activated cellular immunity . The abscopal response has been reported with treatment of melanoma, merkel cell carcinoma, hematologic, and solid tumors . The study of effective local treatments for melanoma may serve as a model for other malignancies that disseminate via lymphatics and the blood stream to the skin . For melanomas, we typically excise isolated metastases with a 1 cm margin of normal skin, understanding that there are no evidence based studies looking at the utility of margins for metastatic disease . In areas of high cosmetic importance or when therapy is limited to palliation, we sometimes perform excisions with very limited margins . Therapy for patients with widespread cutaneous and subcutaneous metastases of advanced melanoma that are nonresectable is limited to other types of palliative therapy . Options include radiotherapy, systemic chemotherapy, polychemotherapy, isolated limb perfusion, interferon alpha injections, cryotherapy, laser ablation, or radiofrequency ablation . Electrochemotherapy is a novel therapeutic option based on enhanced drug delivery into cancer cells using externally applied electrical fields that increase cell membrane permeability . Using targeted electric pulses on tumor - specific areas, cytotoxicity of cisplatin increased 80 times and bleomycin 8,000 times with a large reduction in systemic side effects . A study comparing tumor nodules treated with electrochemotherapy with cisplatin versus cisplatin alone found at a 124-week follow - up, 77% of control rate of tumor nodules treated with electrochemotherapy was observed compared to 19% for those treated with cisplatin only . Electrochemotherapy is well - tolerated by patients, highly effective with low doses of cisplatin or bleomycin, has a short duration of treatment sessions, insignificant side effects, and is more cost - effective than isolated limb perfusion, radiotherapy, interferon - alpha, and hyperthermia associated with radiotherapy and chemotherapy . Oncogene - targeted therapy for metastatic melanoma using vemurafenib (plx4032) for melanoma associated with a mutation of the braf gene v600 mutation results in complete or partial tumor regression in the majority of patients with an estimated median progression - free survival of more than 7 months . Side effects include arthralgia, rash, nausea, photosensitivity, fatigue, cutaneous squamous - cell carcinoma, pruritus, and palmoplantar dysesthesia . Imiquimod 5% cream has also been used successfully in the treatment of metastatic melanoma that is difficult to remove by surgery . In one report, a 58-year - old woman with a superficial spreading melanoma with a breslow thickness 0.75 mm was treated with wide local excision followed by flap reconstruction . Melanoma reemerged 14 months later that was treated with wide local excision followed by split - thickness graft repair . The patient was enrolled in a melanoma peptide vaccine trial 5 months later without improvement . Twenty - one months later, the patient was treated with imiquimod 5% cream and histopathologic evaluation revealed regression of the widespread cutaneous metastases after 6 months of imiquimod . Any change in the skin should raise the suspicion of metastases in the correct clinical context . Biopsy is mandatory in patients with a history of cancer or those with at high risk of cancer . Immunohistochemical screening methods continue to improve and assist in diagnosing the primary tumors of origin . Immunohistochemical findings can also help direct treatment options . With advances in novel therapies that prolong survival,
Ampullary adenomas arise from the ampulla of vater, the confluence of the pancreatic and common bile ducts, located at the major duodenal papilla in the second part of the duodenum . Based upon autopsy studies, ampullary adenomas however, the vast majority of these patients will remain asymptomatic [1, 2, 3, 4]. As with colonic adenocarcinoma, ampullary adenomas also follow the adenoma - carcinoma sequence and may thus evolve from localised premalignant lesions into widespread carcinoma . Such lesions may be removed endoscopically or via surgical excision, with either a local resection or an extended procedure, for example, a pancreaticoduodenectomy . This may occur as an idiopathic process, as is more common in the paediatric population, or it may be due to a structural pathology, such as colonic malignancy . As other gastrointestinal adenomas, ampullary adenomas assume tubular, tubulovillous or villous forms . They can occur either sporadically or in patients with a genetic predisposition, such as familial adenomatous polyposis or gardner's syndrome, and such lesions may therefore be identified via endoscopic surveillance programmes . Indeed, previous studies have suggested a further genetic link to other disorders, such as adult polycystic kidney disease, whilst no specific environmental factors such as diet have been identified in their development . Clinically, they are usually asymptomatic and found incidentally at endoscopy; however, some patients may present with symptoms caused by obstruction of the main gastrointestinal tract and/or the distal biliary / pancreatic ducts . The most common presenting symptoms are gastric outlet obstruction or gastrointestinal bleeding [8, 9]. However, rarer presenting complaints may include acute pancreatitis, biliary obstruction or, as in this case, intussusception . Indications for the excision of such lesions are essentially two - fold: to attempt resolution of immediate symptoms and to prevent malignant progression . The management of such lesions may be endoscopic surveillance, endoscopic excision or surgical resection . Surgical resection has the advantage of lower recurrence rates as opposed to endoscopic resection; however, with the obvious caveat of a much higher risk of morbidity and mortality . In this article, we present a case where a female patient developed recurrent chest infections and gastric outlet obstruction secondary to an intussuscepting ampullary adenoma . Due to the size and location of the mass, it was surgically removed by pancreaticoduodenectomy . To the best of our knowledge, this is the first such presentation worldwide, and we hope that it may be of educational value to both physicians and surgeons alike . A 49-year - old nigerian female presented to her local hospital with a 1-year history of recurrent chest infections . She had no relevant past family history of any genetic disorders, in addition to no recent foreign travel or contact exposure to tuberculosis . When she initially presented, plain chest radiographs were performed and demonstrated multiple lung lesions suggestive of an infective / inflammatory process . She grew pseudomonas on sputum culture, but despite treatment for atypical pneumonia with multiple courses of antibiotics, she failed to adequately improve . Chest ct was consequently performed and demonstrated multiple cavitating lung lesions (fig 1). During this time, she had intermittent vomiting with an associated 15-kg weight loss over a 12-month period . These symptoms were initially attributed to her recurrent chest infections and were not further investigated . A working diagnosis of aspergillus infection was established, and the patient remained under the care of the respiratory physicians . At the time of chest ct, however, an incidental finding was noted in the second to the fourth part of the duodenum . Concurrently, she developed subacute bowel obstruction with persistent vomiting and was unable to tolerate oral intake . A dedicated ct scan of the abdomen and pelvis was therefore performed, demonstrating an obstructing polypoid mass at d2 (fig 2). This lesion was confirmed on upper gastrointestinal endoscopy, and a biopsy demonstrated a tubulovillous adenoma with high - grade dysplasia . Nasojejunal tube insertion was attempted at her local hospital; however, this failed to bypass the obstructing lesion . Her case was discussed at the regional hepatopancreaticobiliary multidisciplinary team meeting at the royal london hospital . As her biopsy demonstrated high - grade dysplasia, in addition to her symptomatic presentation, it was felt that a resection was required . Based upon her cross - sectional and endoscopic imaging, it was deemed that this would not be amenable to an endoscopic excision, and she was likely to require surgical resection and should be transferred to the hpb unit at the royal london hospital . Interestingly, despite this large lesion, she never became jaundiced and maintained a normal bilirubin level . On arrival at the hospital, she was admitted to the surgical ward, and although she had persistent vomiting and was cachectic, she was haemodynamically stable . G / l (normal range 3550) due to the failed nasojejunal tube insertion . Consequently, a picc line was inserted by interventional radiology, and in an attempt to optimise her for major abdominal surgery, total parenteral nutrition was administered . However, due to these ongoing nutritional problems and a failure to adequately improve, she underwent a semi - urgent conventional pancreaticoduodenectomy . Intraoperatively, a large mass at d2 with a duodeno - duodenal intussusception and distal duodenal ischaemia was identified (fig 3). Histological analysis reported a polypoid tumour at the ampulla of vater, 75 28 30 mm in size, with a reduced intussusception and an ischaemic distal duodenum (fig 3). These findings were in keeping with a duodenal ampullary low - grade tubular adenoma, with intussusception and complete resection margins . There was no evidence of any high - grade dysplasia, in contrast to her previous diagnostic biopsy, or invasive malignancy . The patient was admitted to the high - dependency unit postoperatively and was stepped down to the surgical ward on postoperative day 2 . She progressed well with early mobilisation, and her nutritional status greatly improved during this time, with due care not to induce a refeeding syndrome . She did not develop any exocrine pancreatic insufficiency or any derangement from her baseline glycaemic control . To date, the patient has not suffered any long - term complications, and remains clinically well on follow - up . Intussusception in adults is rare and is responsible for approximately 15% of all adult intestinal obstructions . These can be classified by location into enteroenteric, colocolonic, ileocolic and ileocaecal, respectively . The presenting symptoms are usually non - specific and chronic, with pain and symptoms of intermittent intestinal obstruction, such as vomiting, bloating and/or diarrhoea or constipation being the major complaints . The classic paediatric triad of cramping abdominal pain, bloody diarrhoea and a tender abdominal mass is rare in adults . As opposed to the paediatric population, where the aetiology is normally idiopathic, in adults, the cause is usually a structural pathology, with two - thirds of colonic cases being a malignant neoplasm, as opposed to one - third of those occurring in the small intestine [11, 12, 13]. Abdominal ct is now regarded as the modality of choice for investigating such lesions . Due to the preponderance for a structural aetiology, the management of such cases is almost always via laparotomy, with no role for the use of air insufflation as utilised in the paediatric population . Intraoperative reduction of the intussuscepted bowel is possible; however, there are associated risks such as tumour seeding and anastomotic complications due to the pathological bowel tissue . It may, however, be carried out if the lesion has been confirmed as benign in the preoperative period, and can be performed by milking the lesion in a distal to proximal direction, followed by a limited resection [15, 16]. A literature search revealed 13 cases of duodenal / ampullary adenomas causing duodeno - duodenal intussusception, and to the best of our knowledge, no case of an ampullary adenoma causing symptomatic intussusception with recurrent chest infections was previously reported . Incidental duodenal polyps are reported in the literature at a rate of 0.34.6% and have a variety of pathological origins, including adenomas, submucosal tumours and hamartomas amongst others . Ampullary adenomas are thought to be precursors to adenocarcinomas, as such tissue is present in 90% of the resected ampullary adenocarcinoma specimens, whilst based on autopsy studies, they may occur at a rate of 0.040.12% in the general population [1, 2, 3]. Due to their location, ampullary adenomas may clinically manifest via obstruction of the main gastrointestinal tract or the biliary and/or pancreatic ducts . However, they are usually asymptomatic and found incidentally at endoscopy, whilst some patients may present with symptoms, most commonly gastric outlet obstruction or gastrointestinal bleeding [8, 9]. Rarer presenting complaints may include acute pancreatitis, biliary obstruction or, as in this case, intussusception . Depending on the clinical urgency of definitive treatment, such lesions may be further investigated using a wide range of modalities, including ultrasonography, ct, endoscopic ultrasonography (eus), magnetic resonance cholangiopancreatography and endoscopic retrograde cholangiopancreatography . Eus has proven to be a vital technique in the local assessment of periampullary tumours and has a higher sensitivity to both us and ct when detecting such lesions and assessing muscular (suggesting invasion, and therefore malignancy) and vascular involvement (to assess resectability). However, ercp is also extremely useful and has a similar sensitivity to eus in detecting ampullary tumours . There are three broad management strategies for ampullary adenomas: surveillance, endoscopic resection and surgery . Endoscopic resection may be possible in adenomas with low - grade dysplasia, which are <1 cm in size and demonstrate benign characteristics at endoscopy . However, chini and draganov suggest that they believe endoscopic resection to be an appropriate course of action even in the presence of high - grade dysplasia if the two above criteria are met . The presence of an ampullary adenocarcinoma warrants surgical resection if the candidate is fit . However, there have been sporadic case reports where such malignancies have been removed endoscopically [22, 23, 24, 25, 26]. However, debate surrounding their management exists, as some groups speculate that the progression from adenoma to carcinoma is a low percentage, whilst others argue that the quality of forceps biopsy via endoscopy may miss occult foci of adenocarcinoma within the adenoma . Asymptomatic patients may be treated with endoscopic management with endoscopic surveillance as follow up; however, there is no current consensus as to the ideal surveillance post endoscopic resection . Clinicians must ensure that patients who are previously well and present recurrently with chest infections are fully investigated and that they consider the possibility of a lesion causing gastric outlet obstruction . Clinicians should consider dedicated abdominal imaging, and if a mass is identified, undergo endoscopy to further characterise the lesion and develop an on - going management plan with appropriate specialist input . The index of suspicion should of course be higher if the patient goes on to further develop signs of an obstructing lesion, such as persistent vomiting.
This is a nonrandomized prospective comparative case study on 57 eyes seeking refractive surgery who were not indicated for laser corneal refractive surgery . Patients with refraction error between 2.00 d and 14.50 d who could use both artiflex and artisan, were free to choose the type of the lens . Other patients underwent artisan implantation . Ophthalmic examinations before surgery included cyclo - refraction, uncorrected visual acuity (ucva), best - corrected visual acuity (bcva), topography, keratometry, endothelial cell count, pupil diameter, and anterior chamber (ac) depth measurement . According to patient history and examination, patients who had the following conditions underwent piol implantation surgery: insufficient corneal thickness for refractive corneal surgery, inappropriate corneal curvature and topography, high myopia more than 8 d which could not undergo corneal refractive surgery, negative history of any previous refractive surgery, sufficient internal ac depth (3 mm or greater), no history of intraocular pressure raise and endothelial cell density of 2500 cell / mm or more . Lens dioptric power was calculated by van der heijde formula, using refractive error, refractive cylinder power, ac depth, and topographically derived keratometry values . Emmetropia was the target power of the surgery, when the precise emmetropic lens was not available our choice was to a slight residual myopia . All surgeries were performed under general anesthesia, for artisan lens implantation, a superior 5 or 6 mm length corneal incision was created at the 12 o'clock position, followed by performing two stab incisions located at 2 and 10 o'clock . The lens was inserted into the ac under protection of intracameral ophthalmic viscoelastic material injection . Artiflex lens implantation was performed similarly, although the lens was inserted through a 3.2 mm incision . Other steps were carried out same as artisan implantation; finally, the wound was closed by stromal hydration without the necessity of suturing . One year after surgery, cyclo - refraction, bcva, ucva, cs, hoa and patient satisfaction were assessed . Cs was determined by csv-1000 chart, and refraction was obtained with topcon auto refractometer and then modified by subjective refraction . Night vision, day vision, halo, photophobia, dryness, pain, and patient satisfaction were assessed by questionnaire with numerical scales (0 = weak, 1 = moderate, 2 = good, 3 = excellent for day and night vision 0 = none, 1 = mild, 2 = moderate, 3 = severe for halo, photophobia, dryness, and pain). The chart consists of four rows corresponding to spatial frequencies of 3, 6, 12, and 18 cycles per degree (cpd). The distance between the patient and the chart was 2.5 m. the test was performed with the patients monocular bcva to eliminate the effects of sia and residual post - operative refraction . The aberrations analyzed were classified in terms of hoa, vertical trefoil, vertical coma; horizontal trefoil, horizontal coma, quatrefoil, second astigmatism, and fourth order spherical aberration . Comparison between artisan and artiflex data were analyzed using parametric paired student's t - test, p <0.05 was considered statistically significant . This is a nonrandomized prospective comparative case study on 57 eyes seeking refractive surgery who were not indicated for laser corneal refractive surgery . Patients with refraction error between 2.00 d and 14.50 d who could use both artiflex and artisan, were free to choose the type of the lens . Other patients underwent artisan implantation . Ophthalmic examinations before surgery included cyclo - refraction, uncorrected visual acuity (ucva), best - corrected visual acuity (bcva), topography, keratometry, endothelial cell count, pupil diameter, and anterior chamber (ac) depth measurement . According to patient history and examination, patients who had the following conditions underwent piol implantation surgery: insufficient corneal thickness for refractive corneal surgery, inappropriate corneal curvature and topography, high myopia more than 8 d which could not undergo corneal refractive surgery, negative history of any previous refractive surgery, sufficient internal ac depth (3 mm or greater), no history of intraocular pressure raise and endothelial cell density of 2500 cell / mm or more . Lens dioptric power was calculated by van der heijde formula, using refractive error, refractive cylinder power, ac depth, and topographically derived keratometry values . Emmetropia was the target power of the surgery, when the precise emmetropic lens was not available our choice was to a slight residual myopia . All surgeries were performed under general anesthesia, for artisan lens implantation, a superior 5 or 6 mm length corneal incision was created at the 12 o'clock position, followed by performing two stab incisions located at 2 and 10 o'clock . The lens was inserted into the ac under protection of intracameral ophthalmic viscoelastic material injection . Artiflex lens implantation was performed similarly, although the lens was inserted through a 3.2 mm incision . Other steps were carried out same as artisan implantation; finally, the wound was closed by stromal hydration without the necessity of suturing . One year after surgery, cyclo - refraction, bcva, ucva, cs, hoa and patient satisfaction were assessed . Cs was determined by csv-1000 chart, and refraction was obtained with topcon auto refractometer and then modified by subjective refraction . Night vision, day vision, halo, photophobia, dryness, pain, and patient satisfaction were assessed by questionnaire with numerical scales (0 = weak, 1 = moderate, 2 = good, 3 = excellent for day and night vision 0 = none, 1 = mild, 2 = moderate, 3 = severe for halo, photophobia, dryness, and pain). The chart consists of four rows corresponding to spatial frequencies of 3, 6, 12, and 18 cycles per degree (cpd). The distance between the patient and the chart was 2.5 m. the test was performed with the patients monocular bcva to eliminate the effects of sia and residual post - operative refraction . The aberrations analyzed were classified in terms of hoa, vertical trefoil, vertical coma; horizontal trefoil, horizontal coma, quatrefoil, second astigmatism, and fourth order spherical aberration . Comparison between artisan and artiflex data were analyzed using parametric paired student's t - test, p <0.05 was considered statistically significant . A total of 57 eyes were included in the study; of which, 24 eyes were in artisan group and 33 eyes were in artiflex group . The preoperative spherical equivalent (se) and bcva were obtained and compared, no statistically significant difference was noticed in the mean preoperative se (10.39 8.43 d in artisan group and 10.39 2.29 d in artiflex group; p = 0.999) and bcva of two groups (0.25 0.21 logarithm of the minimum angle of resolution [logmar] for artisan group and 0.19 0.18 logmar for artiflex group; p = 0.56). The mean postoperative se was 0.93 1.28 d in the artisan group and 0.54 0.82 in the artiflex group, no significant difference was seen in in postoperative se between two groups (p = 0.19). One year after the operation, 54% of artisan - treated eyes (15/24) and 75.7% (25/33) of artiflex treated eyes were within 1.00 d of intended emmetropia [figs . 1 and 2]. In the artisan group, 37.5% of the treated eyes (9/24 eyes) had three or more snellen line improvement of postoperative bcva, 12.5% (3/24 eyes) had two snellen line improvement and 25% (6/24 eyes) had loss of two or more snellen lines . In the artiflex group, 51.5% of the treated eyes (17/33 eyes) had three or more snellen line improvement of bcva, 15.1% (5/33 eyes) had two line improvement, and no eyes had loss of two or more snellen lines of bcva [fig . 3]. The bar graph shows the postoperative defocus equivalent 1 year after artisan (a) and artiflex (b) implantation bar graph of change in best - corrected visual acuity from the preoperative examination to postoperative examination in terms of the number of snellen line change . Artisan (a), artiflex (b) mesopic contrast sensitivity at spatial frequencies of 3, 6, 12 and 18 degree per cycles in this study, total hoa in artisan - treated eyes was significantly greater than artiflex - treated eyes (p = 0.044) with a mean hoa of 0.44 0.15 root mean square (rms) for artisan and 0.35 0.15 rms for artiflex . There was no significant difference in the vertical trefoil, vertical coma, horizontal trefoil, horizontal coma, quatrefoil, second astigmatism, and fourth order spherical aberration . The evaluation of cs in mesopic conditions showed higher cs in artiflex - treated eyes at three spatial frequencies 6, 12, and 18 cpd (p = 0.003, p = 0.007, and p = 0.00, respectively), and no significant difference was seen between two lenses at 3 cpd spatial frequency [table 1 and fig . Contrast sensitivity data after phakic intra - ocular lens implantation subjective comparison of day and night vision in artisan versus artiflex treated eyes the mean of sia was 1.65 0.82 d in artisan group, and 1.31 1.05 in artiflex group, which showed no statistically significant difference between the groups (p = 0.19). In a subjective evaluation of day vision, all patients of the artiflex group reported good and excellent vision, in artisan group, 71% of patients reported good and excellent vision, whereas 7.1% complained of weak day vision . In the assessment of night vision, 50% of patients treated with artiflex reported good and excellent vision and no one complained of weak night vision . In artisan group, 71.4% reported good and excellent vision, whereas 21.4% reported weak night vision [fig . Subjective comparison of halo, photophobia, pain and dryness in artisan versus artiflex treated eyes among artiflex treated eyes, 50% reported moderate halos and in the artisan group 40% reported moderate to severe halos . About 12.5% of artiflex treated eyes experienced moderate photophobia, whereas 35.7% of artisan treated eyes experienced moderate to severe photophobia . In the artiflex group, only 12.5% of treated eyes caused mild pain, whereas 36.7% of artisan treated eyes caused mild to moderate pain . Moderate to severe dryness was reported in 25% of eyes treated with artiflex, whereas no artisan treated eye experienced moderate or severe dryness [fig . 6]. The bar graph of patient's satisfaction in artisan versus artiflex groups satisfaction more than 60% was reported in 100% of patients in artiflex group versus 60% of patients in artisan group [fig . The bar graph presents the postoperative spherical equivalent 1 year after artisan (a) and artiflex (b) implantation . As it is well - confirmed, the recently food and drug administration approved pmma artisan piol is an effective mean for the correction of refractive errors and have been shown to have acceptable safety and efficacy . However, studies have revealed instances of significant induced astigmatism, attributed to the larger incisions performed in the implantation of the artisan lens . Artiflex iris - fixated piol, a foldable version of artisan, is an improvement of the iris - supported piol concept, with a lower incidence of sia . Artiflex piol haptics are made of pmma and the foldable optical zone is made of silicon . The main purpose of this investigation is to evaluate the differences in optic quality between rigid piol versus flexible piol by comparing wavefront aberration, cs and patient satisfaction in patients who underwent artisan and artiflex iris - fixated iol implantation . In this study, mean postoperative residual se were 0.93 1.28 d and 0.54 0.82 d in the artisan and artiflex group, respectively, no significant difference was seen in postoperative se between two groups (p = 0.19). About 54% of artisan group and 75.7% of artiflex group were within 1.00 d of intended emmetropia . In a previous study, 58% and 83.9% of artisan and artiflex group had a residual postoperative se within 1.00 d, which was significantly better in artiflex group . Another study reported that emmetropia (1.00 d) was obtained in 60% and 91.7% of artisan and artiflex group, respectively, the difference was statistically significant [table 2]. Percentage of postoperative refraction within1.00 d of intended emmetropia in a retrospective comparative case series on 27 eyes, postoperative se was within 0.5 d in 76.2% and 85.7% of artisan and artiflex treated eyes, respectively . In a study of evaluating the outcome of iris - claw piol in fifty high myopic eyes, postoperative se was evaluated, which showed that 38% and 68% of the eyes were in the range of 0.5 d and 1.00 d, respectively . Mean postoperative ucva was 0.34 0.25 logmar in artisan group and 0.12 0.15 logmar in artiflex group . Mean postoperative bcva was 0.21 0.13 and 0.06 0.08 logmar in the artisan and artiflex groups, respectively [table 3]. The differences of ucva and bcva in two groups were statistically significant (p = 0.00). In another study, postoperative bcva between artisan and artiflex groups was not significantly different (p = 0.9). Postoperative visual acuity (logarithm of the minimum angle of resolution) in artisan group versus artiflex group in this study, although vertical trefoil, vertical coma, horizontal trefoil, horizontal coma, secondary astigmatism, quatrefoil and fourth order spherical aberration were not significantly different between the artiflex and artisan groups, total hoa was higher in artisan group, that may be due to the slight differences in each component, which totally increases the hoa (p = 0.044). In a previous study, assessment of hoas after implantation of rigid versus foldable iris - fixated lenses, showed a significant decrease in postoperative spherical aberration among artiflex group and a significant increase in postoperative spherical aberration in artisan group . In both groups, vertical trefoil and spherical aberration were higher in artisan group, p = 0.039 and p = 0.001, respectively . Assessment of cs in mesopic condition showed no significant difference between two groups at 3 cpd spatial frequency . Cs was significantly higher in artiflex - treated eyes at three spatial frequencies 6, 12 and 18 cpd (p = 0.003, p = 0.007, and p = 0.00, respectively). A previous investigation showed that mesopic cs was slightly better in artiflex group in comparison with artisan group, but the difference was not statistically significant, they also measured photopic cs function and found that both groups had poorer performance compared to the normal range . Performance with the artisan lens was slightly better than performance with artiflex under photopic conditions, but worse under mesopic conditions; neither differences were statistically significant . In this study, we have used artisan iol for patients not eligible for the available artiflex (six myopic eye more than 14.5 d and one hyperopic eye), comparison of this two groups may have some biases because of other probable conditions such as retinal involvement or amblyopia, which can alter the results, this was one of our study limitations . In this study, 25% of patients with artisan lens lost two or more lines of bcva, we called the patients back and examined them again carefully, we recognized that in one patient both eyes had refractive error postoperatively in spite of implanting a lens with the same power of his preoperative se improvement in bcva was not seen with correction of this refractive error by trial - frame . In the other patients, we could not find any reason for the decrease in the bcva, unless considering pigment deposition on the iol as a reason for the visual loss.
Chronic lymphocytic leukemia (cll) is the most commonly found leukemia in the adult population of the western world and of clinical interest because of its prevalence . It is a neoplasm of monomorphic small round b lymphocytes which can be observed in peripheral blood, bone marrow, and/or lymph nodes . It is likely that cll has a multifactorial mode of inheritance with both genetic and environmental components . However, of all hematologic neoplasms, cll is reported to have the highest genetic predisposition and like many other hematologic malignancies, development of cll is found to be much higher in males than in females with a corresponding m / f sex ratio of at least 1.5 or higher . Although the clinical (phenotypic) m / f sex ratio for cll has been well documented, the genetic (genotypic) m / f sex ratios associated with abnormal fluorescence in situ hybridization (fish) probes have not . For these reasons, cll represents an attractive cancer entity in which to evaluate what genetic impact, if any, gender may have on its development . Genetic studies of sex ratio disparities in human neoplasms have been few, perhaps, because of the relative inaccessibility of investigational materials including appropriate databases which could be informative . It follows that the genetic basis for this phenomenon remains largely unknown and our understanding of it very limited . Since our laboratory has collected clinical and laboratory data over many years on major categories of cancers including cll, review and analyses of these data presented an opportunity to examine certain aspects of this question . In order to better study the nature of the multifactorial components in cll, we determined the representation of genetic abnormalities detected by four defined fish probes, with respect to male and female cll patients . By this approach, our study addressed not only what the m / f ratio is in cll patients having the clinical phenotype, but also the m / f ratio in patients who have a genotype which included specific fish abnormalities . These results provided a genetic basis for the notion that the fish abnormalities found underlie the phenotypic m / f sex ratio and also that they may be sex chromosomes (x and/or y) influenced . Cll fish results in the database are derived from testing done on cytological preparations made from peripheral blood or bone marrow specimens submitted for cll evaluation . Fish analyses were conducted using a fish panel of dna probes specific for the atm gene (11q22.3-q23.1), chromosome 12 centromere (d12z3 locus), the d13s319 locus (13q14.3), and the tp53 gene (17p13.1) commercially available from vysis (an abbott company), downers grove, il . The prognostic value and characterization of these dna probes have been reported in the literature . The technical protocols for fish testing (hybridization of probes and detection of hybridization signals) were those recommended by the manufacturer . Microscopic studies were conducted by experienced technologists, then reviewed and interpreted by board - certified cytogeneticists . These fish test results were then archived in a computer - based database from which data were retrieved and statistically analyzed . Data of fish results from all integrated oncology laboratories, a business unit of esoterix genetic laboratories, llc, using the same dna fish probes to evaluate cll patients were collected, reviewed, and statistically evaluated . The data contained entries of cll - fish panels performed during the period of 11/13/2005 through 10/20/2009 . These panels were specifically used for cll studies, and results were used only when the entire cll panel was applied in order to clearly identify only those patients having or suspected of having cll . Also, cll panel probe results were those used only for the initial fish evaluation of newly diagnosed cll patient - and did not involve repeat studies on the same patient . This investigation was conducted in a fashion in which laboratory results had no patient - identity associations, in order to conform to privacy guidelines and patient confidentiality . This data was then imported into sas9.2 and formatted by sas data step and analyzed by sas procedures for one side binomial test and chi - square test . The one - side binomial tests were used to evaluate the sex ratio with respect to the abnormalities found for each of the probes used . The first category represented all abnormal panels having only a single abnormality per panel, for each probe . The second category included those having any abnormality with respect to a given probe whether it appeared as a single abnormality or in combination with other abnormalities . The binomial test took into account the frequency of an abnormality or abnormalities (according to the categories describe above) in a pool of data (total number of normal and abnormal panels) for each of the fish probes considered, and compared the results obtained for the male group with results obtained for the female group . Essentially, the mean value for positive results (all having the same abnormality +) versus total results (positive results plus normal having 0 abnormalities), was compared between males and females for each of the probe categories described above . The odds ratio (or) and 95% confidence interval (ci) values were derived from the one side binomial test while the p - values were determined by the chi - square tests . Cll fish results in the database are derived from testing done on cytological preparations made from peripheral blood or bone marrow specimens submitted for cll evaluation . Fish analyses were conducted using a fish panel of dna probes specific for the atm gene (11q22.3-q23.1), chromosome 12 centromere (d12z3 locus), the d13s319 locus (13q14.3), and the tp53 gene (17p13.1) commercially available from vysis (an abbott company), downers grove, il . The prognostic value and characterization of these dna probes have been reported in the literature . The technical protocols for fish testing (hybridization of probes and detection of hybridization signals) were those recommended by the manufacturer . Microscopic studies were conducted by experienced technologists, then reviewed and interpreted by board - certified cytogeneticists . These fish test results were then archived in a computer - based database from which data were retrieved and statistically analyzed . Data of fish results from all integrated oncology laboratories, a business unit of esoterix genetic laboratories, llc, using the same dna fish probes to evaluate cll patients were collected, reviewed, and statistically evaluated . The data contained entries of cll - fish panels performed during the period of 11/13/2005 through 10/20/2009 . These panels were specifically used for cll studies, and results were used only when the entire cll panel was applied in order to clearly identify only those patients having or suspected of having cll . Also, cll panel probe results were those used only for the initial fish evaluation of newly diagnosed cll patient - and did not involve repeat studies on the same patient . This investigation was conducted in a fashion in which laboratory results had no patient - identity associations, in order to conform to privacy guidelines and patient confidentiality . This data was then imported into sas9.2 and formatted by sas data step and analyzed by sas procedures for one side binomial test and chi - square test . The one - side binomial tests were used to evaluate the sex ratio with respect to the abnormalities found for each of the probes used . The first category represented all abnormal panels having only a single abnormality per panel, for each probe . The second category included those having any abnormality with respect to a given probe whether it appeared as a single abnormality or in combination with other abnormalities . The binomial test took into account the frequency of an abnormality or abnormalities (according to the categories describe above) in a pool of data (total number of normal and abnormal panels) for each of the fish probes considered, and compared the results obtained for the male group with results obtained for the female group . Essentially, the mean value for positive results (all having the same abnormality +) versus total results (positive results plus normal having 0 abnormalities), was compared between males and females for each of the probe categories described above . The odds ratio (or) and 95% confidence interval (ci) values were derived from the one side binomial test while the p - values were determined by the chi - square tests . A characterization of the study population evaluated by the cll - fish panel is given in table 1, and a summary of general observations are provided in table 2 . For example, table 2 shows that there were a total of 2773 (59.0%) individual abnormal fish panels in the 4698 total fish panels applied to the cll patient study group . Of the abnormal panels, 1711 were male and 1062 were female for an m / f sex ratio of 1.61 . Other comparisons of the genders concerning data collected by this study are also reported in table 2 . Table 3 shows the m / f ratios of fish results for the study population of cll patients in categories of abnormal results (either as a single abnormality or as a single abnormality plus multiple abnormalities), normal results and total results (normal and abnormal) with respect to gender . There were 2035 results that appeared as only single abnormalities, and 3606 results that appeared as single plus other abnormalities combined (artificially inflated since abnormalities were used multiple times). The most frequently found single abnormality was the deletion of 13q14.3 (1327 abnormal results: 65.2%), followed by trisomy 12 (472 abnormal: 23.2%), then deletion of 11q22.3 (138 abnormal: 6.8%), and least frequent was deletion of 17p13.1 (98 abnormal: 4.8%). The sex ratio values for the fish probes ranged from a low value of 1.39 to a high value of 2.54 . With the exception of the sex ratio found for the atp gene (2.54), the rest of the sex ratios for fish abnormalities ranged from 1.39 to 1.54, clustering around 1.5 . From table 3, it should be noted that the ab / nor ratio for each of the probes was not significantly different from each other except for the atm probe . This suggest that proportionality of abnormal results reflect the phenotypic representation of cll clinically in our study population except for abnormalities of the atm gene which is over - represented in male from that expected . Figure 1 presents the distribution of patients ages in the cll study population in which the age range for males and females is very similar . At early (less than 30 years) and late age groups (greater than 80 years) the sex ratios between male and females do not appear to be significant . Results from the chi - square tests and odds ratio analyses are presented in table 4 . Of the four fish probes, only the deletion of the 11q22.3 (atm gene) demonstrated statistical significance in the chi - square test (p - value of 0.0049). The or value of 1.7045 is also significantly different from the or values found for the rest of the fish probes, indicating the male has 70.45% or 64.35% higher risk to have abnormal fish test results for the atm - fish probe . This was true for deletions of the atm gene when found as a sole abnormality (or=1.7045) or in combination with other abnormalities which included the deletion of the atm gene (or=1.6435). Both these two categories of single and multiple probe abnormalities strongly indicated that the deletion of the atm gene was disproportionately over - represented in male cll patients than in female cll patients, and that this difference could not be explained by chance alone . These data lead to the conclusion that this mutation was not only found in a higher percentage in males compared to females, but that it was distinct from the m / f sex ratio of ~1.5 found for the other fish probes [trisomy 12, del(13q14.3), del(17p13.1)]. The or values for the m / f sex ratios of trisomy 12, deletion 13q, and deletion 17p (tp53 gene) fish probe abnormalities in either single or multiple combinations were not significantly different from 1.0 and could be explained by chance alone . Therefore, the m / f sex ratio for each of the cll - fish probe abnormalities appeared to be about the same and not significantly different from an m / f sex ratio of 1.5 . Although sex ratios in lymphoid neoplasms and other tumors show male predominance, there remains an incomplete understanding of these observations . Our study makes this m / f ratio determination based on a study population of patients clinically diagnosed with, or suspected of having cll for whom fish studies were conducted . Based on this clinical assignment, a ratio of 1.51 was found, which is congruent with previous studies, indicating similar values or higher . These observations suggest that cll may have a sex chromosomeinfluenced component determining its transmission . We also examined the question of what the m / f ratio is in those cll patients who had fish abnormalities detected by specific genetic probes and found that 3 of the 4 probes had values close to 1.5 and that 1 probe (atm gene) had a significantly higher m / f ratio of 2.54 . The coincidence that, like the clinical m / f ratio, the genetic m / f ratio for 3 probes may simply suggest that in the cll study population which already has a disproportionately high number of male patients, the likelihood of detecting a genetic abnormality is equally high (in males) and that it may be sex chromosome influenced . However, for the atm - fish probe, the m / f ratio was significantly higher (~2.5) than the ~1.5 ratio found for the other probes and suggests that for this mutation, a special set of conditions necessary for the development of cll are more effectively enhanced in male than in female patients . M / f values vary depending on the type of cancer and the age of the patient . There are three possible categories of cancers (hematologic and non - hematologic) with respect to altered m / f ratios having values ranging from less than 0.50 to over 20.0 . Generally, these categories include cancers in which: i) males have a higher risk, ii) females are more susceptible, and iii) males and females are equally represented . However, these reported m / f values are phenotypic m / f values which do not take genetic aspects into account . Our survey confirms that in our cll study population, as indicated in earlier reports the phenotypic m / f sex value is very close to 1.5 . M / f sex ratio corresponds to cancers in the first category in which common mechanisms of cancer development may exist . The purpose for using the fish data from the cll study population was to initiate a characterization of what the genotypic m / f sex ratio is for each of the fish probes used . Surprisingly, very much like the phenotypic m / f ratio of ~1.5 found, our study demonstrated a genotypic m / f sex ratio higher than 1.0 (1.39 - 2.5) with respect to all cll fish probes . It is important to note that the genotypic m / f sex ratio of ~2.5 found for the deletion of the atm gene, is markedly skewed in males, and suggests a special mechanism which may involve atm gene functions which could include dna fidelity, homologous recombinational repair and chromosomal stability . Losses and mutations of the atm gene have been demonstrated in a number of neoplasms including t - lymphocytic leukemia (t - pll), b - cell chronic lymphocytic leukaemia (b - cll) and in mantle cell lymphoma (mcl). These reports suggest that loss of a tumor suppressor gene [or loss of heterozygosity (loh)] may be one of many steps leading to a cancerous state in complex diseases with multifactorial contributions . Gender dependent susceptibility to complex diseases could include polygenic mechanisms, epigenetic modulations, and sex - chromosome linked genes . Two possible explanations can account for the skewed m / f ratios found in our study (including the atm fish probe showing especially high level of deletions in male cll patients). Explanation #1 involves the non pseudoautosomal regions (non - par) of the x and y chromosome, and explanation #2 which concerns the pseudoautosomal region (par) of the sex chromosomes . Explanation 1: the genotypic m / f sex ratio of ~1.5 was found for trisomy 12, deletion of d13s319 at chromosome 13q14.3, and deletion of the tp53 gene . These hypothetical x - chromosome linked genes appear as a single dose in male cells (hemizygous) and as two (non - par, and not x - chromosome inactivated) doses in female cells . If there is a predisposing mutation for any of the three fish autosomal markers in either males or females, the cll - x gene product will continue normal function with no cll development . However, if the cll - x gene is subsequently mutated in males (hemizygous), gene function of the cll - gene markers would also be impaired resulting in cll . However, in females (heterozygous) the second non - mutated cll - x gene salvages the gene dosage requirement with no development of cll . Mutations of both cll - x genes in females would be necessary for subsequent cll development . This explanation is consistent with the observation that cll typically has an adult age onset, that relatively fewer females succumb to cll given the salvaging effect of the double doses of the xlinked cll genes they possess, and offers a plausible biological / genetic basis for the ~1.5 sex ratio found for these three important cll genetic markers . Since atm loss is considerably higher in males than females, with a genotypic sex ratio of ~2.5, additional factors are implicated . Other highly skewed phenotypic m / f sex ratios reported in hematological, nonhematological cancers, and in certain solid tumors, can be as high as 28.7 such as for kaposi sarcoma . For these entities it may be that mutations are not necessarily disabled by 100% loss of function . In males after the first atm mutation, a partially functioning cll - x gene might contribute to the loss of the 2 atm gene . However, in females the two partially functioning cll - x genes may be result in sufficient dna repair to prevent atm loss at a higher level than that found in males . This secondary salvage pathway would provide for a lower rate of atm gene loss and may represent a relative protective mechanism favoring females . There may also be other models that could explain gender ratio distortions involving multiple dna - repair related gene families . Our hypothesis of x - linked dna repair - related genes is supported by recent mapping of dna repair genes (which include the ape1, cetn2, trex2, ube2a, and rpa4 genes) on the x chromosome . Explanation 2: the par is homologous in the human x and y chromosomes where they pair during meiosis, and where there are 2 doses for every gene found in this region . Compelling evidence shows that the par plays a role in the m / f ratio found in mantle cell lymphoma (mcl) which may also apply to cll . The study demonstrated male predominance in mantle cell lymphoma (mcl) with loss of the y chromosome and homozygous deletions within the par . In all except 3 of the 21 mcl cases studied, the loss of the y chromosome was demonstrated . Furthermore, 2 of the 3 cases (which showed no y chromosome loss) had biallelic losses of par1 in xp / yp, and of 16 cases in which there was y chromosome loss, an additional case showed biallelic loss of par1 . The genotypic 1.5 m / f ratio for the 3 probes used in our study could also be ex plained by mutations / losses of the par region of the x and y chromosomes . A similar linkage to the par has been proposed for hodgkin s lymphoma in which the risk for brothers of affected males or sisters of affected females was higher than the risk for siblings of the opposite gender . Also supporting this explanation is that chemokines and chemokine receptors may play a role in b - cell malignancies, and that related cytokine - receptor genes map to the par of sex chromosomes . Increasingly, it appears that gender plays a major role in defining not only the identity and nature of some neoplasms but also the mechanisms involved in their origin and progression.
Number of diseases comes under the category of silent killers as they gradually consume one without causing any serious symptoms in the early stages . It is defined as a localized resorptive process that commences on the surface of the root below the epithelial attachment and the coronal aspect of the supporting alveolar process, namely the zone of the connective tissue attachment . There is a homeostatic balance of pulp and periodontal ligament preventing clastic insults, which is due to an intact cementoblast and odontoblast cell layer . Any breach in these layers may down - regulate osteoprotegrin and up - regulate receptor activator of nuclear factor ligand causing resorption . The severance of the protective layer may be commonly due to orthodontic treatment, dental trauma, or bleaching . A study has also revealed that an interleukin-1 polymorphism significantly increases the risk external resorption . Heithersay classified icr into four classes according to the extent and severity of the lesion within the tooth . Class 1 - 3 has a better prognosis, while class 4 is not amenable for treatment and might require extraction and replacement with implant retained crown restoration . The lesion classically presents as an asymmetrical radiolucency with ragged or irregular margins in the cervical region of the tooth . Early lesions might be radiolucent; however, more advanced lesions might have a mottled appearance caused by the osseous nature of the advanced lesions . Unless proper treatment is initiated, the resorption continues unabated leading to irreversible destruction of tooth structure . Early diagnosis, elimination of the resorption and restorative management are the keys to a successful outcome . This article discusses an icr managed by endodontic treatment, surgical management of resorptive defect by reverse sandwich technique and periodontal regeneration using platelet rich fibrin (prf), prf membrane and bone graft . A 35-year - old non - smoking male patient presented for consultation at the endodontic department with a chief complaint of swelling and pus discharge in the upper left canine [figures 1a and 2a]. After performing sensitivity tests, tooth #11 was diagnosed as having pulpal necrosis . Clinical examination revealed localized gingival recession and gingival enlargement in relation to tooth #11 . Probing pocket depth on the mesial surface of the tooth #11 was 10 mm with a high bleeding score . Based on the clinical and radiological finding, we came to the diagnosis of localized chronic periodontitis and pulpal necrosis with external inflammatory root resorption . The treatment plan was informed to the patient, which included patient motivation, oral hygiene reinforcement, scaling and root planning, endodontic procedure and lastly surgical intervention . Patient and his parents were informed of possible discomforts and potential risks concerning the planned treatment procedure and prognosis . (a) pre - operative clinical photograph showing swelling and pus discharge in the upper left canine . (b) surgical exploration revealed that two lesions were present on the mesial surface of upper left canine and there was no palatal intercommunication . (c) the resorptive defects were restored with microfilled composite to a thickness of about 1 mm and light cured for 20 s. the rest of the defects were restored with resin modified glass ionomer cement . (d) the bony defect was filled with freeze - dried bone allograft and platelet rich fibrin . (f) follow - up after 2 years shows normal probing depth, no gingival recession, and no loss of clinical attachment loss (a) pre - operative intraoral periapical showing bone loss in the mesial aspect of upper left canine with two resorptive regions . (d) at 2 years follow - up showing significant bone fill in phase one therapy oral prophylaxis was performed . Endodontic access cavity was prepared after placing a rubber dam and disinfecting the area with 2% of chlorhexidine digluconate (calypso, septodont, india). Working length was determined by using the electronic apex locator root zx (j. morita mfg . Corporation, kyoto, japan) and file in - radiograph with 15 number k - files . The root canal was cleaned and shaped by rotary ni - ti protaper system along with glyde (dentsply maillefer company, usa) using the crown down technique . The root canal was copiously irrigated with 2.5% sodium hypochlorite (novo dental product, india). Access cavity was temporized with calcium hydroxide and temporary endodontic restorative material (term). Root canal was irrigated again with normal saline and dried using paper points . Before obturation, master points were seated to test their suitability to canals and radiograph was taken . The canals were obturated with selected master gutta - percha cone (variable taper) and ah - plus sealer (dentsply maillefer company, usa). The coronal gutta - percha cones were sheared off using heated instrument and vertical compaction was done using the heated pluggers at the canal orifices . The access cavity was restored with a composite (clearfil majesty; kuraray, osaka, japan). The surgical procedure was performed under local infiltration anesthesia (2% of lidocaine with 1:100,000 adrenaline) on vestibular mucosa . An intrasulcular incision was made from the distal surface of the maxillary left central incisor to the distal surface of the maxillary left canine and full - thickness mucoperiosteal flap was elevated . Surgical exploration revealed that two lesions were present on the mesial surface of upper left canine and there was no palatal intercommunication [figure 1b]. Granulomatous tissue within the defect area was removed and 90% of trichloroacetic acid was applied to the resorptive defect for coagulation necrosis . The resorptive defects were restored with microfilled composite (durafill vs; heraeus kulzer gmbh, dormagen, germany) to a thickness of about 1 mm and light cured for 20 s. the rest of the defects were restored with resin modified glass ionomer cement (rmgic) (fuji ii lc; gc corporation, tokyo, japan) to a smooth finish with a cervical matrix and light cured for 20 s [figure 1c]. A test dose of 12 ml sample of whole blood was drawn intravenously from the patient's right antecubital vein and centrifuged (remi model r-8c with 12 ml 15 ml swing out head) under 3000 rpm for 10 min to obtain the prf, which was jelly like in consistency . Prf clot started to release its serum (prf - clot exudates) and was ready for compression into the membrane . The bony defect was filled with freeze - dried bone allograft (lifenet, virginia beach, va) and prf [figure 1d]. Without delay autologous prf membrane was placed [figure 1e] and the flaps were secured with 4 - 0 polyglactin 910 sutures (vicryl, ethicon, inc ., piscataway, nj). After the surgery, the patient was prescribed amoxicillin 1 g twice a day for 1 week and 0.2% of chlorhexidine mouthwash 15 ml twice a day for 2 weeks . The desired gingival contour was achieved; the patient was asymptomatic 1 week after the surgery and the sutures were removed . Patient was followed - up for 6 months, [figure 2b] 1 year [figure 2c] and 2 year . At 2-year follow - up period, periodontal status of related tooth demonstrated mild mobility with normal probing depth, no gingival recession and no loss of clinical attachment [figures 1f and 2d]. Endodontic access cavity was prepared after placing a rubber dam and disinfecting the area with 2% of chlorhexidine digluconate (calypso, septodont, india). Working length was determined by using the electronic apex locator root zx (j. morita mfg . Corporation, kyoto, japan) and file in - radiograph with 15 number k - files . The root canal was cleaned and shaped by rotary ni - ti protaper system along with glyde (dentsply maillefer company, usa) using the crown down technique . The root canal was copiously irrigated with 2.5% sodium hypochlorite (novo dental product, india). Access cavity was temporized with calcium hydroxide and temporary endodontic restorative material (term). Root canal was irrigated again with normal saline and dried using paper points . Before obturation, master points were seated to test their suitability to canals and radiograph was taken . The canals were obturated with selected master gutta - percha cone (variable taper) and ah - plus sealer (dentsply maillefer company, usa). The coronal gutta - percha cones were sheared off using heated instrument and vertical compaction was done using the heated pluggers at the canal orifices . The access cavity was restored with a composite (clearfil majesty; kuraray, osaka, japan). The surgical procedure was performed under local infiltration anesthesia (2% of lidocaine with 1:100,000 adrenaline) on vestibular mucosa . An intrasulcular incision was made from the distal surface of the maxillary left central incisor to the distal surface of the maxillary left canine and full - thickness mucoperiosteal flap was elevated . Surgical exploration revealed that two lesions were present on the mesial surface of upper left canine and there was no palatal intercommunication [figure 1b]. Granulomatous tissue within the defect area was removed and 90% of trichloroacetic acid was applied to the resorptive defect for coagulation necrosis . The resorptive defects were restored with microfilled composite (durafill vs; heraeus kulzer gmbh, dormagen, germany) to a thickness of about 1 mm and light cured for 20 s. the rest of the defects were restored with resin modified glass ionomer cement (rmgic) (fuji ii lc; gc corporation, tokyo, japan) to a smooth finish with a cervical matrix and light cured for 20 s [figure 1c]. A test dose of 12 ml sample of whole blood was drawn intravenously from the patient's right antecubital vein and centrifuged (remi model r-8c with 12 ml 15 ml swing out head) under 3000 rpm for 10 min to obtain the prf, which was jelly like in consistency . Prf clot started to release its serum (prf - clot exudates) and was ready for compression into the membrane . The bony defect was filled with freeze - dried bone allograft (lifenet, virginia beach, va) and prf [figure 1d]. Without delay autologous prf membrane was placed [figure 1e] and the flaps were secured with 4 - 0 polyglactin 910 sutures (vicryl, ethicon, inc ., after the surgery, the patient was prescribed amoxicillin 1 g twice a day for 1 week and 0.2% of chlorhexidine mouthwash 15 ml twice a day for 2 weeks . The desired gingival contour was achieved; the patient was asymptomatic 1 week after the surgery and the sutures were removed . Patient was followed - up for 6 months, [figure 2b] 1 year [figure 2c] and 2 year . At 2-year follow - up period, periodontal status of related tooth demonstrated mild mobility with normal probing depth, no gingival recession and no loss of clinical attachment [figures 1f and 2d]. Treatment depends on the severity, location, whether the defect has perforated the root canal system and the restorability of the tooth . As smaller lesions offer the most favorable long - term outcome, endodontic treatment followed by surgical treatment the true nature of the defect could be assessed only after surgical exploration . To treat the icr lesions, it is usually necessary, to curette away the granulomatous tissue from the adjacent periodontium to sever the blood supply to the resorbing cells and fills the defect with an inert filling material . Periodontal regeneration is the next aim, which includes reconstruction of lost or injured tissue so that the form and function of the lost structures are restored . The removal of granulomatous tissue the surgical defect was filled by the reverse sandwich technique microfilled composite as a liner followed by lamination with rmgic . Microfilled resin was used because they tend to flex with the tooth rather than debond and rmgic was biocompatible to periodontal tissues . The development of subgingival plaque over rmgic is prevented by achieving a smooth finish using the cervical matrix . The regenerative process is a complex biologic process in itself, requiring intricately regulated interaction between cells, signaling molecules (local and systemic) and extra cellular matrices in which these entities interact . Prf is a scaffold that not only acts as carriers for growth factors and proteins, but also allow cellular infiltration and subsequent integration of the newly formed tissue within the native one . It is biocompatible, non - cytotoxic and non - immunogenic to prevent adverse effects on recruited cells and neighboring tissue . This scaffold material along with growth factors and cells pave the way to accelerate bone and periodontal regeneration . Prf can up - regulate phosphorylated extracellular signal - regulated protein kinase expression and suppress osteoclastogenesis by promoting the secretion of osteoprotegerin in osteoblasts cultures . Prf membrane has a very significant slow sustained release of key growth factors for at least 1 week and up to 28 days, which means that the membrane stimulates its environment for a significant time during wound healing . Hydroxyapetite (ha) was added to prf in our study, this was because it has been demonstrated that the treatment of intrabony defects with prf results in significant improvements of pocket depth, clinical attachment level and bone fill compared with baseline and secondly, that ha increases the clinical effects observed with prf in the treatment periapical defects . Significant bone fill was obtained in our case after a 2-year follow - up period; thanks to the synergistic combination of prf and ha.
Due to industrial development and the consequent increase in daily comfort, physical activities have gradually decreased, leading to increased body weight with reduced physical strength, as well as more sedentary lifestyles and significantly decreased amounts of exercise . Due to incorrect exercise habits, even among those who exercise regularly, bad posture over extended periods, and inappropriate daily life and work habits, the resulting continuous back tension has increased the number of patients with low back pain1 . Mennell2 presented the term joint dysfunction for arthrokinematic dysfunction in the absence of pathological changes in the joints, including capsules and ligaments . He also attributed muscular pain and muscle spasm to difficulties with normal arthrokinematic mobilization in joint capsules, which limit joint movement when patients attempted to move joints suffering from the symptoms of joint dysfunction . Joint mobilization can be performed to achieve a neurophysiological effect to reduce muscle pain and guarding, and a mechanical effect such as stretch or burst of contracted tissues . One study reported increased active exercise by patients as a result of joint mobilization3 . The physiological effects of joint mobilization, which is aimed at increasing the range of joint motion and pain reduction, can be explained by the gate control theory proposed by melzack and wall4 . The vicious cycle of muscle pain and spasm can be broken by closing the gate where the pain stimulus is largely transmitted through thin filaments, which have slow stimulus conduction velocity, while proprioceptive neurons of thick filaments are stimulated . The present study aimed to determine the effect of an 8-week program of joint mobilization on changes in pelvic obliquity and pain level in seventeen female university students aged in their 20 s with sacroiliac joint dysfunction by dividing them into two groups: a joint mobilization group, and a control group . Seventeen subjects were selected from female university students aged in their 20 s attending n university in cheon - an city, korea . The subjects had sacroiliac joint syndrome, but experienced no problems with daily living and had no previous experience of joint mobilization exercise . The subjects were randomly assigned to a joint mobilization group of eight, and a control group of nine who performed joint mobilization exercise . The mobilization with movement (mwm) group was 21.131.46 years old, 158.596.91 cm tall, and 59.5912.93 kg in weight . The control group was 23.202.15 years old, inbody 720 (biospace, korea) was used to measure the subjects body composition while 4d - formetric (germany) was used for pelvic analysis . Body fat and lean body mass were measured using inbody 7.0 (biospace, korea). The direct segmental multi - frequency bioelectrical impedance analysis method (dsm - bia) was used for body composition measurement . A pressure footstool (pedoscan, diers, germany) and a trunk measurement system (formetric 4d, diers, germany), a 3d image processing apparatus with high resolution for the vertebrae used in the studies of lippold and colleagues5 and schroder6, were used to measure 3d trunk images of vertebrae and pelvis obliquity, as well as static balance ability . The posterior innominate and anterior innominate methods were used for joint mobilization (mobilization with movement). These two methods have been well described in the literature by mulligan7, both procedures were repeated 10 times for one set, and for three sets (figs 1 and 2fig . Posterior innominate of mwm anterior innominate of mwm spss pc for windows (version 18.0) was used for data processing . In order to analyze the inter - group effect, two - way anova with repeated measures if a main effect was found, the paired t - test was conducted to test the difference between the groups, and one - way anova was conducted to determine the difference of each parameter between the groups . All the subjects understood the purpose of this study and provided their written informed consent prior to their participation in the study in accordance with the ethical principles of the declaration of helsinki . The pelvic obliquity dl - dr decreased by 3.25 from 4.88 before the intervention to 1.63 after the mobilization exercise in the control mwm group, but increased by 0.22 from 1.89 to 2.11 in the control group . The pelvic torsion l - r decreased by 0.5 from 2.50 to 2.00 in the mwm group, but increased by 0.66 from 1.56 to 2.22 in the control group . The trunk length increased by 8.38 mm from 440.25 mm to 448.63 mm in the mwm group, but decreased by 1.89 mm from 451.67 mm to 449.78 mm in the control group (table 1)table 1 . Change of pelvis malpositiongrouppre - testpost - testpelvic obliquity dl - dr ()mwm4.882.481.631.92*control1.891.692.111.76pelvic l - r torsion ()mwm2.500.762.000.53control1.561.012.221.20*trunk length (mm)mwm440.2517.62448.6315.62control451.6716.82449.7815.93**p<0.05, * p <0.01: paired t - test . ##p<0.01: independent t - test . * p<0.05, * * p <0.01: paired t - test . ##p<0.01: independent t - test the mean velocity decreased by 0.28 mm from 1.26 mm to 0.98 mm in the mwm group, but increased by 0.1 mm from 1.13 mm to 1.23 mm in the control group . The sway area decreased by 0.14 cm from 0.21 cm to 0.07 cm in the mwm group, but increased by 0.07 cm from 009 cm to 0.16 cm in the control group . The mean frequency decreased by 0.42 hz from 0.97hzto 0.55 hz in the mwm group, but was unchanged at 0.66 hz in the control group (table 2table 2 . Change of static stabilitygrouppre - testpost - testmean velocity (cm / s)mwm1.260.270.980.12control1.130.131.230.14sway area (cm)mwm0.210.220.070.03control0.090.040.160.11mean frequency (hz)mwm0.970.520.550.28control0.660.260.660.18p<0.05, * p <0.01: paired t - test . ##p<0.05: independent t - test). * p<0.05, * * p <0.01: paired t - test . ##p<0.05: independent t - test the sacroiliac joint refers to the posterior joint of the bony pelvis between the sacrum and the ilium of the pelvis . With its extremely limited mobility and small joint mobilization, this joint rarely causes any pathological problem except for body imbalance due to sacroiliac joint obliquity and changes in iliac, ischium, and length of the ilium8 . The analysis of the pelvis obliquity showed the intervention of this study provided the greatest statistically significant interaction effect on left and right pelvis obliquity (p<0.001). Furthermore, a group performing combined joint mobilization and functional exercise showed statistically significant differences between before and after the exercise whereas the control group performing simple joint mobilization did not (p<0.001). In a previous study, yang9 reported the measurement results of changes around the sacroiliac joint after 12 weeks of rolling massage for patients with chronic low back pain . He found that ilium deviation was reduced by about 2.37 mm, sacroiliac joint deviation by about 2.25 mm, and ischium deviation by about 2.5 mm, resulting in a significant difference overall in the sacroiliac joint - related areas (p<0.001). However, lee10 reported that changes in low back pain found in experimental and control groups after manipulative therapy administered to patients with sacroiliac joint dysfunction with muscle imbalance differed significantly between the two groups before and after the experiment (p<0.001), whereas changes in pelvis rotation between the experimental and control groups showed significant difference between before and after the experiment (p>0.05), in contrast to our study s results . We attribute difference in these study results to the difference between our joint mobilization and lee s manipulative therapy, and to the lack of any functional exercise . For pain control, it is advantageous to discover and treat specific lesions causing the pain (trigger point, over - loaded muscle, weakened or abnormal movement types, or joint dysfunction), not only to reduce the symptoms (pain) but also to induce functional recovery11 . The results for the joint mobilization intervention used in this study show that two groups of showed statistically significant decreases of pain compared to the control group (p<0.001). In a previous study, lee12 reported significant reductions of low back pain, functional disorder level, and low back instability in patients with chronic low back pain after lower extremity strengthening exercise along with low back stabilization exercise (p<0.05). He also claimed that a program of combined exercise performing low back stabilization exercise and lower extremity strengthening exercise was more effective at decreasing low back pain, functional disorder level, and low back instability than a stabilization exercise alone . Han13 reported that a combination of functional exercises resulted in significant pain relief from 4.61 to 1.94 on a subjective pain index (vas), while simple exercise resulted in non - significant pain relief from 3.93 to 1.57 . Furthermore, im14 reported that vas showed a significant difference between before and after chuna therapy and spinal stabilization exercises for 16 weeks . Significant differences were also found between two groups: a group of single treatment with chuna therapy and a group of combined treatment of chuna therapy and spinal stabilization exercise.
This article traces the growth in the use of swing - bed services by medicare beneficiaries from 1984 through 1987 . In the context of the medicare program, swing beds are beds that can be used by small rural hospitals to furnish both acute and post - acute care . To be covered under medicare, the post - acute services must meet the same level of care requirements applied to the reimbursement of services by skilled nursing facilities (snfs). States have the option of also covering swing - bed services at the intermediate care level under their medicaid programs . The swing - bed concept was incorporated into the medicare program by the provisions of the omnibus reconciliation act of 1980 (public law 96 - 499). The law authorized the medicare and medicaid programs to cover swing - bed services furnished by rural hospitals with fewer than 50 beds . The provisions of the law were based on the experiences gained in demonstration projects that began in rural hospitals in utah during the early 1970s and later expanded to iowa, south dakota, and texas . The program takes advantage of the declining acute care occupancy rates and the surplus bed capacity that became increasingly common among rural hospitals during the 1970s . It provided these hospitals a means of obtaining additional revenues without incurring significant additional costs . At the same time, it provided greater access to post - acute nursing care services in rural areas where such services tend to be thinly dispersed . The regulations governing medicare coverage of post - acute services furnished in swing - bed hospitals were issued by the health care financing administration in july 1982 . The method of paying for skilled nursing care services furnished by a swing - bed hospital was based on the assumption that these hospitals incur a relatively low incremental cost to provide post - acute care . They use the personnel, equipment, and facilities already in place to serve acute care patients . Additional service requirements to meet the special needs of nursing care patients (e.g., patient activities, discharge planning) would not require a major expansion of staff . Accordingly, the per diem reimbursement rate for the routine care component of post - acute services covered under medicare in a swing bed was set at a rate equal to the average paid by the medicaid program to snfs for skilled nursing care during the prior calendar year in the state where the hospital is located . The period following the issuance of the swing - bed regulations was marked by intense federal efforts to contain the rise of hospital costs to the medicare program . The tax equity and fiscal responsibility act (tefra) was passed in september 1982; the social security amendments of 1983 instituted the prospective payment system (pps) for hospital reimbursement; and the deficit reduction act (defra) of 1984 reinstated a new version of the medicare separate reimbursement limits for hospital - based and freestanding snf care that had been eliminated under tefra . This rapid pace of change in the bases by which medicare reimbursed hospitals for acute and post - acute care induced uncertainty among rural hospitals as to whether it was worthwhile electing the swing - bed option . This was reflected in the initial slow rate of applications by eligible hospitals for certification as a swing - bed facility . However, as the incentives provided by pps at the acute and post - acute interface became clearer, the rate of election increased . This is reflected in table 1 that shows the rate at which hospitals became certified to furnish swing - bed services . By the end of 1983, about 18 months following the issuance of the regulations, only 149 of an estimated 2,236 hospitals eligible to elect the swing - bed option had done so . By mid-1987, the proportion was approaching the halfway point . The increasing participation of hospitals in the provision of post - acute skilled nursing care services resulted in swing beds gaining an increasing share of the medicare snf market . As summarized in table 2 and detailed in table 3, admissions to swing - bed hospitals for snf services increased from 3.0 percent of all medicare snf admissions in 1984 to 9.7 percent in 1987 . The swing - bed share of medicare - covered snf days increased from 1.5 to 6.0 percent during the same period . Reimbursements for swing - bed care increased from 2.0 percent of snf reimbursements in 1984 to 6.2 percent in 1987 . Shaughnessy, schlenker, and silverman (1988) reported findings that help to interpret the data in table 3 . They found that swing - bed patients have substantially shorter stays and greater rehabilitation potential than do nursing home patients . Swing - bed patients, in greater proportion than nursing home patients, were found to need intense medical and skilled care for such problems as recovery from surgery, hip fractures within the past 6 weeks, shortness of breath, and the need for intravenous catheters . Nursing homes tend to treat patients with problems more typically seen in institutional long - term care settings; such as, incontinence, impaired cognitive functioning, and dependence in carrying out activities of daily living (e.g., feeding self, dressing). Each type of facility seems particularly suited to care for patients who can be, respectively, characterized as needing intense subacute care or as the traditional long - term care patient . The evaluation concluded, at the subacute phase, the quality of services furnished by hospitals was found to be better overall than those services furnished by nursing homes . On the other hand, nursing homes provide higher - quality, traditional, long - term care services . In addition to providing a partial explanation for the differences in length of stay, case - mix explains some of the differences in covered charges . The evaluation report estimates (based on 1985 data) that the more intense but shorter term care required by swing - bed patients results in costs about 20-percent higher per day than the average nursing home patient ., swing - bed covered charges averaged $185 per day compared with $169 for all snf days . Reimbursement of routine swing - bed services based on the state medicaid program's average per diem reimbursement to skilled nursing facilities for routine care services during the previous year kept the difference in reimbursement per day to only $2 in 1987 ($79 to $77). A second report evaluated the impact of medicare's prospective payment system (pps) on the swing - bed program (shaughnessy et al ., 1988). This evaluation found that, despite higher per diem costs for post - acute swing - bed services the overall costs for an episode of illness tended to be lower for patients discharged from a swing - bed hospital patients discharged from acute care in hospitals with swing - bed programs were more likely to receive swing - bed care than patients discharged from comparison hospitals . Such patients also received less medicare nursing home (snf) and home health care . Subsequent acute care use and cost also tended to be lower for patients discharged from acute care in swing - bed hospitals . The overall result was a slightly lower total cost of care (both excluding and including the cost of the initial acute care episode) for patients discharged from acute care in swing - bed hospitals . One factor that may explain the narrowing gap from 1984 to 1987 in the medicare reimbursement per day is the decreasing average length of covered stay in all snfs, including skilled nursing services furnished by swing - bed hospitals (table 3). As shown in table 3, this average decreased from 26.6 days in 1984 to 21.5 days in 1987 . This would reflect the decrease in snfs, since during the period 1984 - 87, the average length of nursing care stay increased in swing - bed hospitals . The shorter length of stay decreases the proportion of payment to snfs made by beneficiaries because of the coinsurance kicking in on the 21st day . Thus, medicare payments averaged over fewer coinsurance days increases the average medicare payment per covered day . Another factor narrowing the difference in the average reimbursement per day may be the method of reimbursing for post - acute routine care services by swing - bed hospitals . Ancillary services which include: supplies, operating room use, drugs, laboratory and radiology services, and anesthesia, are reimbursed at cost . The per diem amount that swing - bed hospitals receive for routine care services is based on the state medicaid program's average per diem reimbursement to skilled nursing facilities for routine care services during the previous year . For the purposes of the ensuing discussion routine care charges are usually characterized as room and board charges, but embedded in the cost base on which the charges are established are allocations for such overhead costs as general and nursing administrative services, maintenance and repairs, operation of the physical plant, laundry and linen, housekeeping, dietary services, central services and supply, medical records, and social services . The per diem average amounts charged to medicare from 1985 through 1987 by swing - bed facilities and snfs for accommodations and ancillary services to skilled nursing care patients are shown in table 4 . The average per diem routine care charges by swing - bed hospitals increased by about one - half the rate of increase of the snfs (table 4). Average per diem charges for ancillary services furnished by snfs increased at more than double the rate of swing - bed hospitals although the latter was still 50-percent higher in 1987 . The latter relationship is not unexpected, given the characteristics of post - acute swing - bed patients described earlier and the greater access to ancillary services generally available in hospitals . In interpreting these figures, the reader should bear in mind that from 1985 through 1987 total covered days of care furnished by snfs decreased . Based on the data available for this analysis, it is not possible to apportion reimbursements to routine care or ancillary services . Assuming there is a concomitancy between costs and charges, it is clear that reimbursements per day to snfs have been rising in closer consonance to the rise in covered charges than has been the case for swing - bed hospitals (table 3). This suggests that the current method of paying for routine swing - bed services may not be keeping up with the rate of increase in the hospital's costs of providing routine swing - bed services . However, in light of increasing participation in the swing - bed program, it may be supposed that swing - bed hospitals were still recovering the marginal cost of furnishing post - acute routine swing - bed services in 1987 . Based on 1984 data, the evaluation report estimated that, on average, swing - bed hospitals incurred an incremental cost per day for routine post - acute care of about $33 to $34 . The average routine care revenues received exceeded the costs by $8 to $10 per day . The 1987 data suggest that the difference between marginal routine care costs and revenues may be narrowing . However, given full cost reimbursement for ancillary services, the marginal revenue for otherwise empty beds seems to be attractive for eligible hospitals . The geographic distribution of the use of and medicare payments for swing - bed services in 1987 in relation to all snf services is shown in table 5 . As expected, the number of swing - bed hospitals and the use of swing - bed services were concentrated in the north central and south census regions which contain large expanses of rural areas . Of the 1,058 hospitals that submitted a bill for swing - bed services, almost one - half (504) were located in the north central states . Only 16 hospitals in the northeast region were certified to furnish swing - bed services: 9 in new hampshire, 4 in vermont, and 3 in pennsylvania . Of the 179 hospitals certified in the west to furnish swing - bed services, 131 (73 percent) were in the mountain states . In the north central states, 18 percent of all admissions for snf services were to swing - bed hospitals . In the south, in the largely urbanized northeast, less than 1 percent of the admissions for snf services were made to swing - bed hospitals . However, new hampshire and vermont are notable exceptions to the patterns of the northeast . In these two states, more than one - fourth of the admissions for snf services were to swing - bed hospitals . Admissions to swing - bed hospitals are based on the residence of the patient . Where admissions to swing - bed hospitals are noted in states with no swing - bed facilities, admission to a facility in a neighboring state the west census region presents a dichotomy between the mountain states and pacific coast states . In the mountain states, almost 12 percent of the admissions for snf services were to swing - bed hospitals . In four of the mountain states (montana, idaho, wyoming, and new mexico), more than 20 percent of the admissions for snf services were to swing - bed hospitals with wyoming having almost 60 percent going to swing - bed hospitals . The remaining mountain states show less than 10 percent of the admissions for snf services going to swing - bed hospitals . Only 1 percent of the admissions for snf services in the pacific coast states went to swing - bed hospitals; alaska, with 21 percent, was the only pacific coast state with more than 7 percent using swing - bed hospitals for snf care . The states showing more than 50 percent of the admissions for snf services going to swing - bed hospitals were: north dakota, south dakota, kansas, mississippi (the highest at 89 percent), and wyoming . Delaware and the district of columbia were the only jurisdictions with no admissions for swing - bed services . Figure 1 displays the geographic patterns of admissions to swing - bed hospitals as a percent of all snf admissions . For the individual states, the relationship among admissions, covered days of care, charges, and reimbursement is about that indicated for 1987 in table 2 . As previously mentioned, about 89 percent of the admissions for snf services in mississippi went to swing - bed hospitals . Swing - bed hospitals accounted for almost 91 percent of the covered snf days of care and received 82 percent of snf reimbursements . Mississippi was the only state in which the average length of snf stay in a swing - bed hospital (18.2 days) exceeded the statewide average (17.9 days). The data presented in this article and the findings of the evaluation indicate that the rural hospital swing - bed program has been working as might have been anticipated: swing - beds have assumed the provision of a significant portion of post - acute care services in many states with large rural areas . The post - acute case mix in swing - bed hospitals represent more short term, intense level of care requirements than those in snfs . Swing - bed hospitals seem better suited to meeting nursing care needs of these types of patients than do rural snfs, which seem more suited to meeting the needs of the traditional long - term care nursing home patients . Higher average total charges per day for swing - bed patients suggest that they tend to be more expensive to care for than are the patients in snfs; especially in the use of ancillary services . Per diem reimbursements for swing - bed services have been growing at an average annual rate of about one - third of that for snfs . The latter finding raises question as to whether the current basis for reimbursing for post - acute routine care services in swing - bed hospitals causes per diem revenues to rise at a slower rate than per diem costs . The current difference between marginal costs and revenues seem sufficient to attract increasing participation by rural hospitals with fewer than 50 beds . However, given the different behavior of the overhead as well as the direct cost components of the costs for routine care services in hospitals and snfs, the current method of paying for routine swing - bed services may require re - examination some time in the future . This may become more apparent when the experiences of the larger rural swing - bed hospitals brought into the program by the omnibus budget reconciliation act of 1987 (public law 100 - 203) are analyzed . Under this legislation, the swing - bed option was extended to rural hospitals with fewer than 100 beds . Providing an incentive to small rural hospitals to continue rendering swing - bed services may require re - examination of the bases on which payment for these services are made.
Patients with univentricular heart malformations are at increased risk of suffering from thromboembolic events (te). At least 20% of patients with univentricular hearts have reported to experience te, of which 25% are fatal . Despite the high incidence of te, no consensus has been reached regarding the role of long - term anti - thrombotic treatment in this group of patients . Here, we present a case of a 19-year - old woman with a univentricular heart who suffered a major stroke . A 19-year - old woman born with a univentricular heart was found unconscious in her bed in the morning . She was respiratory and circulatory stable with no fever at admission to the local hospital . The glasgow coma scale score was 5 (eyes, 1; verbal, 1; motor, 3). An electrocardiogram showed sinus rhythm, left axis deviation, and left - sided hypertrophy, but was otherwise normal . A test of arterial blood gasses revealed a fully compensated metabolic acidosis with ph 7.37 (normal range 7.377.45) and base excess 7.7 mm (3.0 to 3.0 mm). Venous blood tests showed raised plasma lactate 3.7 mm (0.72.1 mm), plasma myoglobin 280 g / l (1949 g / l), plasma glucose 8.6 mm (4.27.2 mm), plasma fibrin d - dimer 1.0 mg / l (0.00.5 mg / l), and inr 1.5 (<1.2), whereas the remaining standard tests were all normal, including hemoglobin, leucocyte differential count, electrolytes, c reactive protein, liver and pancreas enzymes, renal parameters, plasma ethanol, plasma paracetamol, and plasma salicylate . Shortly after admission, the patient developed babinski reflexes and a pronounced decorticate posture with spontaneous flexion of the arms over the chest and extended legs with feet turned inward . After a tracheal tube was inserted and assisted ventilation was initiated, the patient was transferred to the neurological intensive care unit at a tertiary hospital . A repeated computed tomography scan and magnetic resonance imaging of the head were performed, as well as a computed tomography angiography of the head and the neck . These scans unveiled a major stroke located in the left cerebral (figure 1) and cerebellar hemispheres, corresponding to the areas supplied by the left middle and posterior cerebral arteries and the left superior cerebellar artery . A segmental occlusion in the top of the basilar artery was identified (figure 3). Finally, a transthoracic echocardiography was performed, revealing a well - functioning univentricular heart with no detectable thrombi . Warfarin was prescribed and the patient gradually regained consciousness . However, a severe right - sided hemiparesis persisted and the patient was transferred to a local neurorehabilitation unit . Later, a magnetic resonance imaging scan of the thorax and upper abdomen was performed (figure 4). The patient had been referred to a cardiologist at the age of 5 months due to shortness of breath and failure to thrive . Cardiac ultrasound and catheterization had revealed a double inlet left ventricle (figure 5) and a hypoplastic right ventricle without transposition of the great arteries . Symptoms were caused by heart failure due to high pulmonary flow, which was treated by pulmonary artery banding . At the age of 6 years, a total cavopulmonary connection (tcpc), including a lateral tunnel with fenestration to the right atrium, was established . The surgical procedure markedly improved the patient s well - being, and, at the age of 13 years, the fenestration was closed . Life - long prophylactic antithrombotic treatment with salicylic acid was prescribed . During the last surgical intervention, the patient suffered mild brain damage and was now described as behaving at the level of a 12-year - old . Postoperative echocardiography revealed good systolic function of the left ventricle and mild right atrioventricular valve regurgitation . Since no cardiac thrombi were identified, it is not known whether the described stroke was caused by a cardiac - derived embolus or spontaneous cranial thrombi . Spontaneous thrombi in 19-year old patients are, however, extremely rare . Hence, seen in the light of a known cardiac malformation, the probability of a cardiac - derived cerebral embolus is very high . The incidence of univentricular hearts is reported to be between 0.5 and three cases per 10,000 live births.1 today, patients with univentricular heart conditions are usually treated surgically with a three - stage tcpc / fontan procedure,2 which separates the systemic and pulmonary venous return a precondition for adequate oxygenation of the arterial blood entering systemic circulation . The technique has evolved from the classic fontan3 (right atrium - to - pulmonary artery connection) through an intracardiac lateral tunnel procedure, and it is currently performed in many centers as an extracardiac tunnel procedure by insertion of a tube graft between the inferior vena cava and the pulmonary artery.4 te, both systemic venous and arterial, are a major cause of early and late mortality in tcpc patients . The reported incidence of te in these patients varies from 3% to 25%, depending on study design, imaging technique, and follow - up period duration . Studies with longer follow - up periods and more sensitive imaging studies suggest an incidence of at least 20%, of which the mortality rate is 25%.59 asymptomatic pulmonary emboli have been detected in 16% of tcpc patients . Although the etiology for the high risk of te is not well defined, possible explanations include abnormal blood flow in the univentricle, arrhythmias, venostasis, dehydration, protein - losing enteropathy, and coagulation abnormalities . No stratification of te risk has been made between the different types of surgical intervention . Such data would be very useful and should be compared against the efficacy of each treatment . Procoagulant factors (factors ii, v, vii, ix, and x; plasminogen; and fibrinogen) and anticoagulant factors (protein c and antithrombin iii) are lower than normal controls prior to stage two or three of tcpc completion.10,11 increased platelet reactivity prior to tcpc completion has also been shown . A recent report, however, found no significant differences in thromboelastography (a global whole - blood assay of coagulation) in pediatric tcpc patients compared with healthy children.12 despite the high incidence of te, no consensus has been reached about the role of long - term antiplatelet or anticoagulation therapy in those patients who remain in stable sinus rhythm . Some studies advise against routine anticoagulation,13 whereas others recommend routine antiplatelet14 or anticoagulation therapy.15,16 one recent study suggests that antiplatelet and anticoagulation therapy are equally effective in preventing te.17 te reduce quality of life18 and cause sudden death19 in adult patients with fontan circulation . Therefore, in these patients, the benefit of long - term prophylactic antiplatelet or anticoagulation therapy must be carefully considered and weighed against the risk of detrimental hemorrhagic side effects . Disagreement about antithrombotic therapy in tcpc patients warrants future study that compares the different therapeutic strategies . Future studies should most likely be observational case - control studies due to the practical and ethical problems associated with randomized controlled trials . The need for such studies is emphasized by the presented case, which might suggest that more aggressive antithrombotic strategies should be routinely introduced.
Patients with underlying or comorbid medical condition, psychotic depression, and those who were already on psychotropic medicines were excluded from the study . Cases having contraindication for imipramine and/or lithium treatment were also excluded from the study . The aim of the study and the method adopted was explained to each patient and his / her cooperation was solicited . Findings of physical examination, mental status evaluation, sociodemographic data were recorded on a specially designed pro forma . All baseline investigations including electrocardiogram and thyroid function were carried out as per standard guidelines . All patients were randomly assigned to one of the two groups: group a patients were given tablet imipramine and placebo . Group b patients were administered tablet imipramine and tablet lithium carbonate as per schedule given below . The assessment of psychopathology was done by structured interview of the hamilton depression / melancholia scale designed by williams . The investigator had reviewed the records maintained at this center and found that clinical response was recorded in almost all cases at the dose range of 100150 mg . Barring unforeseen developments, each patient was to be brought up to dose of 150 mg by day 14 . Lithium carbonate was administered in such a way as to achieve a serum lithium concentration in the range of 0.60.8 meq / l by day 7 . Lithium concentration was measured by atomic absorption spectrophotometry . The nursing staff who administered the drugs was blind to the nature of regimen given to the patients . Students t - test was utilized to test the significance of the difference of means of scores at weekly intervals while categorical data were put to chi - square test, using the statistical package for social sciences - version 16.0 (spss 16.0 . The investigator had reviewed the records maintained at this center and found that clinical response was recorded in almost all cases at the dose range of 100150 mg . Barring unforeseen developments, each patient was to be brought up to dose of 150 mg by day 14 . Lithium carbonate was administered in such a way as to achieve a serum lithium concentration in the range of 0.60.8 meq / l by day 7 . Lithium concentration was measured by atomic absorption spectrophotometry . The nursing staff who administered the drugs was blind to the nature of regimen given to the patients . Students t - test was utilized to test the significance of the difference of means of scores at weekly intervals while categorical data were put to chi - square test, using the statistical package for social sciences - version 16.0 (spss 16.0 . Demographic and clinical details of depressed patients in both groups did not show any significant differences [table 1]. The two groups did not differ in duration for their current depressive episode [table 2]. The patients did not differ in terms of frequency a particular symptom [table 3]. Depression scores of group a and group b patients at baseline and percentage reduction of scores at weekly intervals is shown in tables 4 and 5, respectively . Comparison of two groups in depression ratings at baseline and at weekly intervals [table 6]. There is no difference between the two groups at baseline, but the difference is significant at the end of 1 week, 2 week, 3 week, and 4 week . Demographic and clinical details of depressed patients duration of index episode at outset most common symptoms at the time of initial assessment depression scores of group a patients at baseline and at weekly intervals depression scores of group b patients at baseline and at weekly intervals comparison of two groups in depression ratings at baseline and at weekly intervals (unpaired t - test) mean percentage reductions in depression ratings at weekly intervals for both the groups revealed a larger reduction in depression ratings for group b as compared to group a at end of 1, 2, 3, and 4 week [figure 1]. Difference between the baseline scores and at scores at weekly intervals for groups a and b is shown in table 7 . There are significant reductions in depression ratings for both groups at 1 week, 2 week, 3 week, and at 4 week intervals . However, the decline in scores is more for group b as compared to group a. the mean serum lithium level for the group achieved was 0.555 (standard deviation: 0.186). The plasma serum levels of lithium correlated with the clinical response positively at 1 week but had no correlation at 4 weeks [table 8]. Mean percentage reductions in depression ratings at weekly intervals for both the groups revealed a larger reduction in depression ratings for group b as compared to group a at the end of 1, 2, 3, and 4 week difference between the baseline scores and at scores at weekly intervals for groups a and b (student's paired t - test) mean serum lithium levels and percentage response at 1 week and at 4 weeks for group b patients majority (65%) of patients of group b showed at least 25% improvement in depression ratings by the end of the 1 week . Although the sample size in nonresponder group (i.e., <25% by 1 week) is small, but the trend shows a positive correlation of response with female gender, married status of the patient, absence of an enduring psychosocial problem, and a positive family history for a mood disorder [table 9]. Difference in variables associated with response at 1 week within group b between partial responders (i.e., having response> 25% and) and nonresponders (i.e., having response <25%) the most common side effect for group a was dry mouth whereas it was digital tremor for the group b patients . Other side effects encountered were constipation, postural hypotension, foul taste, blurred vision, urinary problems, palpitations, and impotence . Manic switch for 2 patients in group a and dysarthria and arrhythmia in one patient each for group b were exclusively group specific [table 10]. The last century was often termed as the century of anxiety . In contrast, 21 century can perhaps be described as the age of depression as evidenced by the fact that it is one of the most common scourges causing distress and disability . Although remarkable advances in somatic and psychological interventions have brought in salutary change, but patients are still obliged to endure the anathema at least for a few weeks until the administered drugs start to take effect . Ongoing research holds promise of rationalizing and optimizing drug therapies so as to provide maximum benefit to the patient . The current study was a step in this direction wherein an attempt was made to curtail the lag period of tca response by the addition of lithium from the outset and comparing it with the same tca monotherapy in a double - blinded randomized controlled study . The means are comparable to a similar study where mean age was about 39 years (39.5 for imipramine and 38.5 for imipramine + lithium group). A total of five patients in group a and 1 patient of group b [table 1] shared a positive family of a mood disorder . A total of 6 patients of group a and 5 patients of group b [table 1] had a history of mental or neurological illness . However, none of the patients had a concurrent medical or surgical illness and were not on any psychotropic medications (exclusion criteria). The trial was extended to include unipolar, recurrent depression as well as dysthymic patients [table 1] against an earlier study where they restricted the sample to only bipolar depressed with melancholic features . Duration of the index depressive episode was identical for both the groups [table 2]. The symptom profile for patients of both the groups at baseline [table 3] is in agreement with a similar study, which suggests higher prevalence of somatic rather than cognitive symptoms in depressed subjects of this country . In the current study, patients receiving lithium and imipramine combination responded more rapidly and completely than the imipramine - placebo groups [tables 4, 5 and figure 1]. The differences in response between the two groups at the end of 1 week, 2 week, 3 week, and 4 weeks were both statistically significant and clinically meaningful . The mean percentage change in depression ratings [figure 1] after 1 week (38.37%) for the lithium + imipramine group was higher than the imipramine + placebo group (14.97%). Although the difference between the two groups [table 6] was not significant at baseline (t = 0.5891, p> 0.05), but it was significant at week 1 (t = 3.8747, p <0.01), week 2 (t = 3.6895, p <0.01), week 3 (t = 2.8153, p <0.01), and at week 4 (t = 2.2682, p <0.05). The important clinical relevance in the finding is that the combination proves its superiority over monotherapy in that it brought a faster onset of action which persisted during the duration of the study . The mean percentage reduction at 4 weeks [figure 1] of depression ratings was higher (96.2%) for group b (imipramine + lithium combination) than that of 60.5% for group a (imipramine + placebo combination) implying that the combination brought a more complete remission . The findings are supported by a similar study who found better efficacy at 6 weeks rather than at 4 weeks . The principal hypothesized mechanism of action of imipramine is its ability to inhibit reuptake of both serotonin and noradrenaline . The exact mechanism of action of lithium remains a mystery though recent research points toward its salubrious effect in stabilizing ionic and molecular transmission . Lithium is also known to produce striking enhancements in some aspects of serotonergic functions, which is also caused by imipramine . Although the exact pharmacodynamics and pharmacokinetics were not the principal foci of this study, it appears that the superior response to the combination may have been obtained because of two separate actions concomitantly by imipramine (in monoamine enhancement at the synaptic cleft) and lithium (in altering intra - neuronal signal pathways) at molecular level . The strategy is not new to medical profession and is an accepted norm in the treatment of malignancies and chronic infections such as tuberculosis and hiv . Lithium is not known to interfere with pharmacokinetics of imipramine, and the combination has been described to be a safe and effective one . At the outset, it was not known as to what degree patients would be tolerating the combination, and the emergence of side effects (in the form of coarse digital tremor) at the predefined serum levels of 0.60.9 meq / l was underestimated . Emergence of this side effect in the combination group warranted a more cautious approach and serum lithium targets were revised from 0.60.9 the strategy helped in restricting the tremor to only a mild form, which was acceptable to the patients . The use of other antitremor agents such as clonazepam or propranol was of course considered but was not required as tremors reached acceptable levels by just lowering the mean serum lithium levels . That the therapeutic effect was obtained with this much concentration is supported by earlier studies, which suggest that lower concentrations may be as effective as higher concentration for augmentation purpose . Moreover, correlation studies at 1 week and at 4 weeks [table 8] suggest a mildly significant positive correlation at 1 week and an insignificant correlation at 4 weeks . Lithium was administered for 4 weeks because the intent was to augment imipramine and interest was in early response . How far is the combination likely to be beneficial beyond 4 weeks is subject to further evaluation . However, existing research supports its use, and it may be an alternative in difficult cases . On further analysis, it is seen that 14 out of 20 patients in group b (i.e. 70%) showed a higher than 25% response in depression ratings by the end of 1 week [table 9]. A search was made to study the variables associated with difference in response of more than 25% and that of <25% . The variables of age, occupation, educational status, interpersonal relations, past history of mental or neurological illness, depression typology, or duration of index depressive episode did not significantly influence the outcome though the variables of gender (of being females), married status, absence of enduring psychosocial problem, and a positive family history for a mood disorder predicted a better response . It appears fairly reasonable to presume at this stage that the response to lithium plus imipramine combination was quicker and superior than tricyclic monotherapy alone and it was seen across majority of depressed subjects . The limiting factor of the study was small sample size and a narrow spectrum of depressive disorders which were studied . Concurrent administration of lithium and imipramine from the outset produced quicker antidepressant response in unipolar depression and the effect was evident in 70% of the patients by the end of the 1 week.
The subject of ideals in topological space has been studied by kuratowski and vaidyanathaswamy . An ideal on a topological space (x,) is a nonempty collection of subsets of x which satisfies (i) a and b a implies b and (ii) a and b implies a b . Given a topological space (x,) with an ideal on x and if (x) is the set of all subsets of x, a set operator (): (x) (x), called a local function of a with respect to and, is defined as follows: for a x, a(,) = {x xu}, for every {u (x)} where (x) = {u x u}. A kuratowski closure operator cl () for a topology (x,), called the -topology, finer than is defined by cl(a) = a a(,); when there is no chance for confusion, we will simply write a for a(,) and for (,). If is an ideal on x, then (x,,) a of an ideal space (x,,) is said to be -open if a int(a). A subset a of an ideal space (x,,) is said to be pre--open if a int(cl(a)). The family of all pre--open sets in (x,,) is denoted by po(x,) or simply po(x). Clearly po(x). The largest pre--open set contained in a, denoted by pint(a), is called the pre--interior of a. the smallest pre--closed set containing a, denoted by pcl(a), is called the pre--closure of a. a subset a of an ideal space (x,,) is said to be --open if a int(cl(int())). The family of all --open sets is a topology finer than . We will denote the --interior subset of a of x by int(a). A subset a of an ideal space (x,,) is -dense if cl(a) = x. a space (x,,) is -submaximal if every -dense subset of x is open . Let (x,,) be an ideal space and let a be a subset of x. then pint(a) = aint(cl(a)). Let (x,,) be an ideal space and let a be a subset of x. then pint(a) = aint(cl(a)). A pre--open set a of a space (x,,) is said to be pre--regular pre--open if a = pint(pcl(a)). The complement of a pre--regular pre--open set is called pre--regular pre--closed set, equivalently a = pcl(pint(a)). A subset a of a space (x,,) with an ideal is said to be pre--regular if it is pre--open and pre--closed . A is pre--open which implies a = pint(a) = pint(pcl(a)). Hence a is pre--regular pre--open . But consider the ideal space (x,,) where x = {a, b, c}, = {, {a}, {b}, {a, b}, x}, and = {, {a}}. Here {a} is pre--regular pre--open but not pre--closed . Consider the ideal space (x,,) where x = {a, b, c}, = {, {a}, {b}, {a, b}, x}, and = {, {a}}. Here {a} is pre--regular pre--open but not pre--closed . Moreover, the intersection of two pre--regular pre--open sets is not pre--regular pre--open in general as example 4 shows . Consider the ideal space (x,,) where x = {a, b, c}, = {, {b, c}, x}, and = {, {a}}. Here po(x) = {, {b}, {c}, {a, b}, {a, c}, {b, c}, x}. Thus, a = {a, b}, b = {a, c} are both pre--regular pre--open . Consider the ideal space (x,,) where x = {a, b, c}, = {, {b, c}, x}, and = {, {a}}. Here po(x) = {, {b}, {c}, {a, b}, {a, c}, {b, c}, x}. Thus, a = {a, b}, b = {a, c} are both pre--regular pre--open . Consider the space (x,) with an ideal as in example 4 . Here consider the space x = {a, b, c} and = {, {a}, {b}, {a, b}, x}, = {, {a}}. Here {a} is pre--regular pre--open but not -open . Observe that every pre--regular pre--open set is pre--open but the converse need not be true . Here let (x,,) be an ideal space and let a, b be any subsets of x. then the following hold. (a)if a b, then pint(pcl(a)) pint(pcl(b)). (b)if a pio(x), then a pint(pcl(a)). (c)for every a po(x), pint(pcl(pint(pcl(a)))) = pint(pcl(a)). (d)if a and b are disjoint pre--open sets, then pint(pcl(a)) and pint(pcl(b)) are disjoint. (e)if a is a pre--regular pre--open, then pcl(x a) is pre--regular pre--closed. (f)if a is pre--regular pre--open, then pint(a) is pre--regular pre--open . Let (x,,) be an ideal space and let a, b be any subsets of x. then the following hold. (a)if a b, then pint(pcl(a)) pint(pcl(b)). (b)if a pio(x), then a pint(pcl(a)). (c)for every a po(x), pint(pcl(pint(pcl(a)))) = pint(pcl(a)). (d)if a and b are disjoint pre--open sets, then pint(pcl(a)) and pint(pcl(b)) are disjoint. (e)if a is a pre--regular pre--open, then pcl(x a) is pre--regular pre--closed. (f)if a is pre--regular pre--open, then pint(a) is pre--regular pre--open . If a b, then pint(pcl(a)) pint(pcl(b)). If a pio(x), then a pint(pcl(a)). For every a po(x), pint(pcl(pint(pcl(a)))) = pint(pcl(a)). If a and b are disjoint pre--open sets, then pint(pcl(a)) and pint(pcl(b)) are disjoint . If a is a pre--regular pre--open, then pcl(x a) is pre--regular pre--closed . Proof (a)suppose a bpcl(a) pcl(b). Therefore pint(pcl(a)) pint(pcl(b)). (b)suppose that a po(x,)a = pint(a) pint(pcl(a)). (c)it is obvious that pint(pcl(a)) po(x,), so by (b) we have pint(pcl(a))pint(pcl(pint(pcl(a)))). On the other hand pint(pcl(a)) pcl(a) which implies pcl(pint(pcl(a)))pcl(pcl(a)) = pcl(a). Therefore, pint(pcl(pint(pcl(a))))pint(pcl(a)). Hence pint(pcl(pint(pcl(a)))) = pint(pcl(a)). (d)since a and b are disjoint pre--open sets, we have ab = which implies apcl(b) = apint(pcl(b)) = . Since pint(pcl(b)) is pre--open, pcl(a)pint(pcl(b)) = . Hencepint(pcl(a))pint(pcl(b)) = . (e)given that a is pre--regular pre--open, a = pint(pcl(a)) implies x a = x pint(pcl(a)) = pcl(pint(x a)). Therefore, pcl(x a) = pcl(pint(pcl(x a))). Hence pcl(x a) is pre--regular pre--closed. (f)by (e) if a is pre--regular pre--open, then pcl(x a) is pre--regular pre--closed . Hence x (pcl(x a)) is pre--regular pre--open that implies pint(a) is pre--regular pre--open . (a)suppose a bpcl(a) pcl(b). Therefore pint(pcl(a)) pint(pcl(b)). (b)suppose that a po(x,)a = pint(a) pint(pcl(a)). (c)it is obvious that pint(pcl(a)) po(x,), so by (b) we have pint(pcl(a))pint(pcl(pint(pcl(a)))). On the other hand pint(pcl(a)) pcl(a) which implies pcl(pint(pcl(a)))pcl(pcl(a)) = pcl(a). Therefore, pint(pcl(pint(pcl(a))))pint(pcl(a)). Hence pint(pcl(pint(pcl(a)))) = pint(pcl(a)). (d)since a and b are disjoint pre--open sets, we have ab = which implies apcl(b) = apint(pcl(b)) = . Since pint(pcl(b)) is pre--open, pcl(a)pint(pcl(b)) = . Hencepint(pcl(a))pint(pcl(b)) = . (e)given that a is pre--regular pre--open, a = pint(pcl(a)) implies x a = x pint(pcl(a)) = pcl(pint(x a)). Therefore, pcl(x a) = pcl(pint(pcl(x a))). Hence pcl(x a) is pre--regular pre--closed. (f)by (e) if a is pre--regular pre--open, then pcl(x a) is pre--regular pre--closed . Hence x (pcl(x a)) is pre--regular pre--open that implies pint(a) is pre--regular pre--open . Suppose a bpcl(a) pcl(b). Therefore pint(pcl(a)) pint(pcl(b)). It is obvious that pint(pcl(a)) po(x,), so by (b) we have pint(pcl(a))pint(pcl(pint(pcl(a)))). On the other hand pint(pcl(a)) pcl(a) which implies pcl(pint(pcl(a)))pcl(pcl(a)) = pcl(a). Therefore, pint(pcl(pint(pcl(a))))pint(pcl(a)). Hence pint(pcl(pint(pcl(a)))) = pint(pcl(a)). Since a and b are disjoint pre--open sets, we have ab = which implies apcl(b) = apint(pcl(b)) = . Since pint(pcl(b)) is pre--open, pcl(a)pint(pcl(b)) = . Hencepint(pcl(a))pint(pcl(b)) = . Given that a is pre--regular pre--open, a = pint(pcl(a)) implies x a = x pint(pcl(a)) = pcl(pint(x a)). Therefore, pcl(x a) = pcl(pint(pcl(x a))). Hence pcl(x a) is pre--regular pre--closed . By (e) if a is pre--regular pre--open, then pcl(x a) is pre--regular pre--closed . Hence x (pcl(x a)) lemma 6 . For an ideal topological space (x,,) the following are equivalent . For an ideal topological space (x,,) the following are equivalent . Proof(a) (b): let a be a -dense set that implies cl(a) = x which implies int(cl(a)) = x, so that a int(cl(a)) = x. by (a) every pre--open set is open and hence a is open. (b) (a): let b be a pre--open subset of x, so that b int(cl(b)) = g, say . B) = cl(x g) cl(b) = (x g) cl(b) = x, and thus (x g) b is -dense in x. thus (x g) b is open . Now b = ((x g) b)g is the intersection of two open sets, so that b is open . (a) (b): let a be a -dense set that implies cl(a) = x which implies int(cl(a)) = x, so that a int(cl(a)) = x. by (a) every pre--open set is open and hence a is open . (b) (a): let b be a pre--open subset of x, so that b int(cl(b)) = g, say b) = cl(x g) cl(b) = (x g) cl(b) = x, and thus (x g) b is -dense in x. thus (x g) b is open . Now b = ((x g) b)g is the intersection of two open sets, so that b is open . If a space (x,) with an ideal is -submaximal, then any finite intersection of pre--open set is pre--open . If a space (x,) with an ideal is -submaximal, then any finite intersection of pre--open set is pre--open . Prooffrom lemma 6, every pre--open set is open and hence a finite intersection of pre--open set is pre--open . From lemma 6, every pre--open set is open and hence a finite intersection of pre--open set is pre--open . If a space (x,) with an ideal is -submaximal, then any finite intersection of pre--regular p--open set is pre--regular pre--open . If a space (x,) with an ideal is -submaximal, then any finite intersection of pre--regular p--open set is pre--regular pre--open . N} be a finite family of pre--regular pre--open sets . Since the space x is -submaximal, then by lemma 6, {oii = 1,2, therefore, {oii = 1,2,, n}pint(pcl(oi)). Also, for each i = 1,2,, n, oi oi which implies pint(pcl(oi))pint(pcl(oi)). Also, each oi is pre--regular pre--open that implies oi = pint(pcl(oi)) which implies pint(pcl(oi))oi and so oi = pint(pcl(oi)). N} be a finite family of pre--regular pre--open sets . Since the space x is -submaximal, then by lemma 6, {oii = 1,2,, therefore, {oii = 1,2,, n}pint(pcl(oi)). Also, for each i = 1,2, also, each oi is pre--regular pre--open that implies oi = pint(pcl(oi)) which implies pint(pcl(oi))oi and so oi = pint(pcl(oi)). It should be noted that an arbitrary union of pre--regular pre--open set is pre--regular pre--open . But the intersection of two pre--regular pre--closed sets fails to be pre--regular pre--closed as shown by example 9 . Consider the ideal space (x,,) as in example 3 . Clearly, {a, c}, {a, b} are pre--regular pre--closed but their intersection is not pre--regular pre--closed . Consider the ideal space (x,,) as in example 3 . Clearly, {a, c}, {a, b} are pre--regular pre--closed but their intersection is not pre--regular pre--closed . The following hold for a subset a of a space (x,,). (a)if a is pre--closed, then pint(a) is pre--regular pre--open. (b)if a = pint(a), then pcl(a) is pre--regular pre--closed. (c)if a and b are pre--regular pre--closed sets, then a b if and only if pint(a) pint(b). (d)if a and b are pre--regular pre--open sets, then a b if and only if pcl(a) pcl(b). The following hold for a subset a of a space (x,,). (a)if a is pre--closed, then pint(a) is pre--regular pre--open. (b)if a = pint(a), then pcl(a) is pre--regular pre--closed. (c)if a and b are pre--regular pre--closed sets, then a b if and only if pint(a) pint(b). (d)if a and b are pre--regular pre--open sets, then a b if and only if pcl(a) pcl(b). If a is pre--closed, then pint(a) is pre--regular pre--open . If a = pint(a), then pcl(a) is pre--regular pre--closed . If a and b are pre--regular pre--closed sets, then a b if and only if pint(a) pint(b). If a and b are pre--regular pre--open sets, then a b if and only if pcl(a) pcl(b). Proof(a) since a is pre--closed, a = pcl(a).now, pint(pcl(pint(a))) = pint(pint(a)) = pint(a). Hence pint(a) is pre--regular pre--open. (b) now pcl(pint(pcl(a))) = pcl(pcl(a)) = pcl(a). Hence pcl(a) is pre--regular pre--closed. (c) given that a and b are pre--regular pre--closed sets, therefore, a = pcl(pint(a)) and b = pcl(pint(b)). Clearly, pint(a) pint(b) if a b.conversely, pint(a) pint(b). Now a = pcl(pint(a)) pcl(pint(b)) b. hence a b. (d) given that a and b are pre--regular pre--open, therefore, a = pint(pcl(a)) and b = pint(pcl(b)). Therefore, pcl(a) pcl(b).conversely, pcl(a) pcl(b). Now a = pint(pcl(a)) pint(pcl(b)) b. (a) since a is pre--closed, a = pcl(a). (b) now pcl(pint(pcl(a))) = pcl(pcl(a)) = pcl(a). Hence pcl(a) is pre--regular pre--closed . (c) given that a and b are pre--regular pre--closed sets, therefore, a = pcl(pint(a)) and b = pcl(pint(b)). Clearly, pint(a) pint(b) if a b. conversely, pint(a) pint(b). Now a = pcl(pint(a)) pcl(pint(b)) b. hence a b. (d) given that a and b are pre--regular pre--open, therefore, a = pint(pcl(a)) and b = pint(pcl(b)). Now a = pint(pcl(a)) pint(pcl(b)) b. a subset a of an ideal topological space (x,,) is said to be -rare if it has no interior points in . Theorem 11 . Then the following hold. (a)the empty set is the only subset which is nowhere dense and pre--regular pre--open. (b)if a is pre--regular pre--closed, then every -rare set is pre--open then the following hold. (a)the empty set is the only subset which is nowhere dense and pre--regular pre--open. (b)if a is pre--regular pre--closed, then every -rare set is pre--open . The empty set is the only subset which is nowhere dense and pre--regular pre--open . If a is pre--regular pre--closed, then every -rare set is pre--open . Then a = pint(pcl(a)) = pcl(a)int(cl(pcl(a))), by lemma 1 . Therefore, a pcl(a)int(cl(cl(a)))pcl(a)int(cl(a)) = pcl(a) = . (b) suppose a is pre--regular pre--closed . Then a = pcl(pint(a)) = pint(a)cl(int(pint(a)))pint(a) cl(int(a)) = pint(a) = pint(a). Then a = pint(pcl(a)) = pcl(a)int(cl(pcl(a))), by lemma 1 . Therefore, a pcl(a)int(cl(cl(a)))pcl(a)int(cl(a)) = pcl(a) = . (b) suppose a is pre--regular pre--closed . Then a = pcl(pint(a)) = pint(a)cl(int(pint(a)))pint(a) cl(int(a)) = pint(a) = pint(a). Hence a is pre--open . An ideal space (x,,) is called extremally pre--disconnected if the pre--closure of every pre--open set is pre--open . Theorem 12 . For a topological space (x,,) the following are equivalent. (a)(x,,) is extremally pre--disconnected. (b)every pre--regular pre--open subset is pre--regular . For a topological space (x,,) the following are equivalent. (a)(x,,) is extremally proof(a) (b): assume (x,,) is extremally pre--disconnected . Hence a is pre--closed which implies a is pre--regular. (b) (a): suppose a is pre--open . Then pcl(a) is pre--regular pre--closed which implies x pcl(a) is pre--regular pre--open . (a) (b): assume (x,,) is extremally pre--disconnected . Then pcl(a) is pre--regular pre--closed which implies x pcl(a) is pre--regular pre--open . Let (x,,) be an extremally pre--disconnected space and a x. then the following are equivalent:(a)a is pre--regular,(b)a = pcl(pint(a)),(c)x a is pre--regular pre--open,(d)a is pre--regular pre--open . Let (x,,) be an extremally pre--disconnected space and a x. then the following are equivalent:(a)a is pre--regular,(b)a = pcl(pint(a)),(c)x a is pre--regular pre--open,(d)a is pre--regular pre--open . Then a is pre--open and pre--closed and so a = pint(a) and a = pcl(a). Hence a = pcl(pint(a)). (b) (c): let a = pcl(pint(a)). Then x a = x pcl(pint(a)) = pint(pcl(a)) so x a is pre--regular pre--open. (c) (d) is clear. (d) (a) follows from theorem 12 . Then a is pre--open and pre--closed and so a = pint(a) and a = pcl(a). Then x a = x pcl(pint(a)) = pint(pcl(a)) so x a is pre--regular pre--open . (d) (a) follows from theorem 12 . An ideal space (x,,) is called locally pre--indiscrete if every pre--open subset of x is pre--closed (or) if every pre--closed subset of x is pre--open . Then the following are equivalent. (a)(x,,) is locally pre--indiscrete. (b)every pre--open subset is pre--regular. (c)every pre--open subset is pre--regular pre--open. (d)pint(pcl({x})), for every x x. (e)the empty set is the only nowhere dense subset of x. let (x,,) be an ideal space . Then the following are equivalent. (a)(x,,) is locally pre--indiscrete. (b)every pre--open subset is pre--regular. (c)every pre--open subset is pre--regular pre--open. (d)pint(pcl({x})), for every x x. (e)the empty set is the only nowhere dense subset of x. (x,,) is locally pre--indiscrete . Pint(pcl({x})), for every x x. the empty set is the only nowhere dense subset of x. proof(a) (b): assume that (x,,) is locally pre--indiscrete . Hence a is pre--regular. (b) (c): if a is pre--open, then a = pint(a). Also by hypothesis, hence a is pre--regular pre--open. (c) (d): since {x} is preopen, {x} is pre--open . By (c), {x} is a pre--regular pre--open set . Therefore, {x} = pint(pcl({x})). (d) (e): by theorem 11, in any space, the empty set is the only subset which is nowhere dense and pre--regular pre--open. (e) (a): suppose that a is a pre--closed set . Now int(cl(a pint(a))) = int(cl(a (aint(cl(a))))) = int(cl(a(x a)(int(cl(x a))))) = int(cl(a(x a)(a int(cl(a))))) = int(cl(a int(cl(a))))int(cl(a) int(cl(a))) = int(cl(a) cl(int(cl(a))))int(cl(a) (int(cl(a)))) = . Therefore a pint(a) is nowhere dense which implies a = pint(a), and so a is pre--open . Hence is a locally pre--indiscrete . (a) (b): assume that (x,,) is locally pre--indiscrete . Let a be a pre--open subset of x. by hypothesis, a is pre--closed . Hence a is pre--regular . (b) (c): if a is pre--open, then a = pint(a). Also by hypothesis, (c) (d): since {x} is preopen, {x} is pre--open . By (c), {x} is a pre--regular pre--open set . (d) (e): by theorem 11, in any space, the empty set is the only subset which is nowhere dense and pre--regular pre--open . (e) (a): suppose that a is a pre--closed set . Now int(cl(a pint(a))) = int(cl(a (aint(cl(a))))) = int(cl(a(x a)(int(cl(x a))))) = int(cl(a(x a)(a int(cl(a))))) = int(cl(a int(cl(a))))int(cl(a) int(cl(a))) = int(cl(a) cl(int(cl(a))))int(cl(a) (int(cl(a)))) = . Therefore a pint(a) is nowhere dense which implies a = pint(a), and so a is pre--open . Hence is a locally pre--indiscrete . An ideal space (x,,) is said to be pr - door if every subset of x is either pre--regular pre--open or pre--regular pre--closed . Let (x,,) be a pr - door space; then every pre--open set in the space is pre--regular pre--open . Let (x,,) be a pr - door space; then every pre--open set in the space is pre--regular pre--open . Prooflet a be a pre--open subset of x. since x is pr - door, a is pre--regular pre--closed and so a = pint(pcl(a)) which implies that pinta = pint(pint(pcl(a))). Let a be a pre--open subset of x. since x is pr - door, a is pre--regular pre--closed and so a = pint(pcl(a)) which implies that pinta = pint(pint(pcl(a))). A subset a of x is both --open and --closed; then a is a pre--regular pre--open set a subset a of x is both --open and --closed; then a is a pre--regular pre--open set . Prooflet a be an --open and --closed set . Then a is a pre--open and pre--closed set and hence a is a pre--regular pre--open set . Let a be an --open and --closed set . Then a is a pre--open and pre--closed set and hence a is a pre--regular pre--open set . Let (x,,) be an ideal space . A subset a of x is --open and pre--regular pre--open; then a = int(cl(int(a))). A subset a of x is --open and pre--regular pre--open; then a = int(cl(int(a))). Then a int(cl(int(a))). And a is pre--regular pre--open which implies a = pint(pcl(a))pint(cl(int(pcl(a))))pint(cl(int(a)))int(cl(int(a))). Then a int(cl(int(a))). And a is pre--regular pre--open which implies a = pint(pcl(a))pint(cl(int(pcl(a))))pint(cl(int(a)))int(cl(int(a))).
Lichen sclerosus et atrophicans (lsa) is a chronic inflammatory muco - cutaneous disorder, characterized by sclerotic and atrophic lesions, most commonly found in adult women . It affects the anogenital area in 8598% of the cases and less frequently the extrgenital area . The most commonly affected extragenital areas are the neck and the shoulders, but also the inner thighs, the submammary area, the wrist and occasionally the oral mucosa . The involvement of the scalp is not frequent and its outcome could be similar to scarring alopecia, which could be the result of different diseases . The etiology of lsa is still unknown . Besides the genetic and local factors (koebner phenomenon) and the autoimmune hypothesis, supported by the association of different autoimmune disorders, especially of thyroid origin (30% of cases), an infectious hypothesis has also been proposed . A 57-year - old caucasian woman presented with a history of asymptomatic frontoparietal lesion . Such lesion, which had been developing over the 3 previous years, was initially erythematous and became progressively atrophic and sclerodermic . The patient had been living in a highly endemic area for borrelia burgdorferi, but she could not recall any tick bite or erythema chronicum migrans . She also reported the simultaneous onset of migrating diffused myoarthralgias involving knees, hands, ankles, elbows, shoulders, as well as short - term memory impairment for two years, worsened previous seasonal insomnia for one year . Moreover, she presented migrating paresthesias involving the left side of the body for one month . The dermatological examination revealed an atrophic - sclerodermic lesion of about 10 cm in length and 3 cm in width, from the scalp to the centre of the forehead as reported in figure 1 . The skin was thin, inelastic, mother - of - pearl in shade, with erythematous margins of the lesion with subsequent scarring alopecia as well (figure 1). Figure 1lichen sclerosus et atrophicans with frontoparietal distribution, mimicking scleroderma en coup de sabre . A skin biopsy was obtained from the scalp and after histological examination three different pathologists confirmed independently the diagnosis of lsa . It consisted of epidermal atrophy, oedema with superficial layer, hyperkeratosis and collagen production with cell rarefaction . Lymphoid infiltrate was observed even in the dermal - subepidermal junction indicating a possible later evolution of the lsa toward morphoea (figure 2). Figure 2h&e stained section of the lesion (a) 2,5 magnification of the entire histological section, (b) 20 magnification of the lichen sclerosus et atrophicans features (c) 40 magnification of the lymphatic infiltrate . H&e stained section of the lesion (a) 2,5 magnification of the entire histological section, (b) 20 magnification of the lichen sclerosus et atrophicans features (c) 40 magnification of the lymphatic infiltrate . Serological igm and igg for borrelia burgdorferi with enzyme - linked immunoassay test (elisa) confirmed by western blot analysis was negative . Anti - nuclear antibodies (ana), extractable nuclear antigen antibodies (ena), anti - native dna antibodies (n - dna), anti - neutrophil cytoplasmic antibodies (anca) and erythrocyte sedimentation rate were negative . Pcr analysis for the detection of borrelia burgdorferi was performed on dna obtained from formalin - fixed paraffin - embedded skin biopsy, blood and urine as previously reported . The diagnosis of lyme borreliosis was made on the basis of clinical data, supported by the pcr positivity for borrelia genome . The patient underwent antibiotic treatment with 2 cycles ceftriaxone 2 gr / day i.v for 21 days . After about 6 months since the beginning of antibiotic therapy, the lesion had not progressed, all other clinical symptoms improved and blood pcr resulted negative . The patient received vitamin e 400 mg 2x / day per os for 3 months and applied topic vitamin e prior to uva-1 therapy . Thanks to this combined therapy a remarkable regression of the atrophic - sclerodermic lesion was observed . This report describes a case of lsa, which was unusual for the involvement of the scalp . The lesion was mimicking scleroderma en coup de sabre, which is a frontal or frontoparietal linear morphoea characterized by a linear band of depressed atrophy on skin and scalp . The classical features of lsa are represented by hypopigmented papules that coalesce into white plaques with epidermal atrophy . Although anogenital lsa is associated with a risk of 45% of squamous cell carcinoma, extragenital lesions do not seem to carry any risk of malignant degeneration . The isolated linear frontoparietal involvement was described in few cases and may clinically simulate scleroderma en coup de sabre, requiring careful histopathological recognition . In this case it has already been reported that overlap of histologic features between lsa and morphoea may occur, however in the reported case the clinical and the histologic features were not ambiguous of lsa . The possible later evolution toward morphoea in this case, due to the migration of the lymphatic infiltrate, is not unusual since morphoea and lsa may be closely related such that the latter could be considered the superficial expression of the same disease process which results in morphoea . Regarding the possibility of an infectious etiology of lsa, since the first proposal by aberer and stanek in 1987 several european studies some atrophic skin diseases have been proposed as manifestation of lyme borreliosis with contradictory results . The detection methods, the examined specimens, such as sera, skin biopsies and urine, together with the different geographic region could explain the conflicting results on the association of borrelia with morphoea and lichen sclerosus et atrophicus . Moreover in the manifestation of long standing infection of borrelia the paucity of microorganisms could lead to a low detection rate by pcr, especially when the analysis is performed on archival biopsies . No decision can be made to date as to whether bb plays a role as causative agent of different types of circumscribed scleroderma and lsa . With regard to the disparate findings in different geographic areas, it can be speculated that lsa may be caused in some cases by bb genotypes which are present in that area only . To support this infectious etiology in endemic regions, the fact that in this case borrelia dna was detected both on dna obtained from the biopsy and from blood and urine, strongly supports the hypothesis that borrelia has a causative role on the onset of this unusual lsa . Moreover, the geographical location, middle europe, has been highly associated with borrelia prevalence . Indeed, it is well known that there are significant geographic differences in borrelia infections with a higher prevalence in areas in middle europe . To further support this theory, lyme disease affects mostly the skin: about 80% of all lyme borreliosis cases present skin manifestations . We recognise that classical dermatological events include erythema chronicum migrans (ecm), lymphadenosis benigna cutis (labc) or borrellial lymphocytoma (bl) and acrodermatitis cronica et atroficans (aca), but there is growing evidence that some cases of other cutaneous manifestations could be related to borrelial late infection, mostly borrelia afzelii . Common laboratory tests are not usually revealing for the diagnosis of lyme borreliosis and serologic tests support the diagnosis, but are not always essential in this regard . In this case of lsa mimicking scleroderma en coup de sabre, an associaton with lyme borreliosis is proposed . To our knowledge, this is the first case of lsa, mimicking scleroderma en coup de sabre, which was associated with lyme borreliosis . Our findings indicate an association between this particular form of lsa and borrelia, suggesting that borrelia burgdorferi itself could represent a causative agent of this atypical form of lsa, however it cannot be excluded that borrelia could be only one of the predisposing agent triggering it . In conclusion we propose that tissue pcr for dna of borrelia should be performed in patients with lsa in endemic area, because it could represent a rare manifestation of borreliosis, and in those cases lsa should be treated with proper antibiotic therapy in order to eradicate the microorganism.
With regard to the importance and notable role of human power in an organization, investigation of the elements, which increase staff's function and reduce absenteeism and desertion and ultimately lead to an increase in efficiency, is of great importance for researchers and experts . Nursing managers should design an attractive workplace which can absorb new nurses in addition to preserving the existing staffs in the system . Therefore, high quality of work life has been suggested as an important issue in many organizations including the world health organization (who) from 1970s . Quality of work life was suggested in early 70s and was investigated from different angles during several past decades . Walton is one of the experts who have investigated work life in eight dimensions (fair and adequate payment, safe working environment, provision of opportunities for continued growth and security, rule of law in organization, social ties, life work, overall living space, integrity of the organization, and development of human capabilities). In the 80s, american and european managers pointed to quality of work life as one of the most interesting methods to cause motivation and as a solution for designing and job enrichment, as well as a tool to solve the problems and organizational gordian knot . Quality of work life was used in the nursing context by attridge and challahan from 1990 . The last version of nurses work life quality model was suggested by brooks and anderson in 2001, in which nurses quality of work life was considered in four dimensions . Work life home life work life / home life dimension reveals the nurses life experience at work and home . Work world dimension describes vast social impacts as well as the effects of changes on the functioning of nursing profession . Nurses as a giant group of health providers who handle human lives should have an appropriate work life in order to take care of the clients properly . Results of a study on quality of work life in nurses of tehran university of medical sciences in 2006 showed that 70% of nurses were not satisfied with their work life quality, and complained of most of their work life dimensions . Research conducted in hospitals in tehran in 2010 showed an inverse correlation between nurses level of anxiety and quality of work life . This is why the human resources section should try to improve its personnel's quality of work life . Improvement of personnel's quality of work life has been mentioned as one of the important issues to guarantee health system stability, as high work life quality is essential to absorb and preserve the staffs . Results of a study conducted in 2010 on the association between work life quality and organizational commitment among fire fighters in malaysia showed a significant association between the two factors . Workplace is one of the factors affecting the quality of given care, retaining nurses, and cost efficacy . Research on the necessity of nurses work life improvement in 2003 showed that an increase in nurses work life quality leads to an improvement in patients care and nurses communication with patients families . Work life has a major share in satisfaction with other life dimensions like family, leisure, and health . Results of a study conducted in 2008 on the association between occupational stress and work life quality in army nurses showed an inverse association between the two factors . One of the duties of the managers in a health services organization is to take action for improvement of work life quality and education of coping strategies, as some stressful elements like workplace violence are inevitable in these organizations and prevention of their psychological and behavioral effects is essential . Workplace violence is one of the factors that lead to a decline in nurses work life quality and satisfaction and has a negative effect on the quality of patients care and satisfaction as well as nurses efficiency and competency . Its real level is unknown yet, as what we see is just the tip of an iceberg . Violent behaviors in workplace cause the staffs to experience anxiety, stress, fatigue, and depression, and reduce job satisfaction and organizational commitment . Higher frequency of exposure to workplace violence leads to major mental hazards and negatively affects the victim's behavior . Negative atmosphere, created after workplace violence, affects patient - staff communication and results in lower responses of nurses to patients needs, and consequently, the patients are less satisfied with the quality of health care . Many studies showed that nurses were dissatisfied with their job security, so they were worried about their unsafe workplace . Results of a study conducted in 2011 on 384 employees of kerman bahonar copper company showed an inverse correlation between work life dimensions and employees aggression . The researchers of the present study aimed to define and conduct a study on the quality of work life and its association with workplace violence of nurses in the emergency departments, with regard to the effective role of nurses in health services efficiency and patients and families satisfaction . The obtained results can make nursing managers determined to make a more proper background for improvement of the function and work life of nurses exposed to workplace violence, as well as patients care through control and management of workplace violence and making necessary changes in the working conditions . This is a descriptive correlational study conducted in the emergency wards of selected hospitals in isfahan in 2012 . The number of nurses with at least 1 year of work experience in the emergency ward (n = 360) ok was determined by referring to nursing offices of the selected hospitals . To calculate the sample size, modified cochran formula was adopted in which existence or absence of violence was considered 50 . Total number of nurses with bs and at least 1 year of work experience in the research environment was 360 . Total number of subjects was estimated to be 186 and the sample size of each hospital was randomly allocated by quota sampling through proportion . In the second stage, the questionnaires were completed by qualified nurses meeting the inclusion criteria (having a bs degree in nursing, being mentally balanced, and having at least 1 year of work experience in the emergency ward and working in this ward at the time of study). The nurses who defectively completed the questionnaire were left out of study and sampling went on until the required number of subjects was selected . Demographic information (10 questions), 2 . Investigation of workplace violence exposure in a 1-year period (4 questions), and 3 . Each item was scored 1 - 6 based on likert's scale (absolutely disagree = 1; disagree = 2; relatively disagree = 3; agree = 4; relatively agree = 5; and absolutely agree = 6). Quality of nursing work life(qnwl) questionnaire was designed by brooks and anderson in 2001 and its validity was confirmed . All the participants were given verbal and written information about the purpose of the study . Written informed consent was obtained from all nurses and they were free to withdraw from the study at any time . Its reliability was calculated by cronbach's alpha (= 0.93, = 0.917) which showed an acceptable value . Questionnaire of exposure to workplace violence was a researcher - made brief form of a standard questionnaire which was designed by the who, international nursing association, and public services association in 2003, whose questions were modified to four questions related to goals of the present study . Content validity was used for assessing its validity, wherein the questionnaire was given to 10 academic members of the nursing faculty after preparation of the primary draft, and then, their indications were applied to the questionnaire . The data in the present study were quantitative and qualitative (nominal and ordinal). Descriptive (mean, sd) and inferential (pearson correlation coefficient) statistical tests were used to analyze the data through spss version 16 . Subjects mean age was 33.76 (7.13) years; 70.4% of nurses were married and 29.6% were single . About 26.9% were males and 73.1% were females, 32.3% had work experience of 1 - 5 years, 30.1% had 610 years, and 37.7% had> 10 years work experience in the emergency ward . The highest number of exposures to verbal violence (41.4%) was more than four times, and for physical violence (9.1%), it was two times . About 76.9% of the nurses were exposed to verbal violence and 26.9% to physical violence . Subjects work life quality and each of its dimensions and statistical indexes have been separately presented in table 1 . Association between the number of nurses exposed to verbal and physical workplace violence and their work life quality and its dimensions have been presented in table 3 . Frequency distribution and mean scores of work life quality and its dimensions in emergency nurses responses of nurses in emergency wards to work life items association between the number of nurses exposure to verbal, physical workplace violence and work life quality and its dimensions improvement of work life quality is counted as a long - term and practical way to absorb and preserve human resources, which should be considered by health care managers . Nurses work life quality dimensions and their association with the number of workplace violation exposures are discussed as follows . Most of the nurses were dissatisfied with this dimension and mentioned the reasons as family's needs, working hours, and low energy after doing their daily tasks . Nurses reported that they spend a long time at their workplace, so they have little energy after work and cannot fulfill their families needs, which is consistent with the results reported in previous studies . Disproportionate salary and reward was one of the reasons for nurses dissatisfaction with their work life quality . Behavioral theories like mallow and herzberg behavioral theories showed that fulfillment of primary needs is essential as the individuals cannot concentrate on higher needs if their primary needs are not met . In the present study, 93.5% of nurses believed that their salary was not balanced with the inflation rate in market, which is in line with previous studies . Meanwhile, 57% of nurses in the us believed their salary was balanced with their expenses . Nurses low income is one of the major reasons for their job dissatisfaction and desertion . About 81.2% of the nurses believed that they had high workload, which is consistent with previous studies . On the other hand, 67.2% of the nurses believed they were not independent in taking care of the patients, which concords with former studies in which nurses reported they had low autonomy in decision making about patients care . About 88.7% of the nurses believed there were not adequate nursing personnel in their work environment and 64.5% believed that they were given extra non - nursing tasks . Shortage in human resources and increase of nurses workload act as pressure factors among nurses, which lead to professional and organizational desertion . Despite the shortage in human resources, these dimensions of malutilization of nursing force can increase the shortage of nursing force in a vicious cycle and affect nurses skills and experiences . Such challenges may impose a notable pressure on nurses and negatively affect nurses perception of work life . Managerial methods act as one of the problems in this dimension, which include lack of managers supervision, feedback, participation in decision making, higher level of managers respect toward nurses, inefficient nursing strategies and policies concerning facilitation of work, and modification of nurses concerns so that they think their struggles are not officially noted by nursing managers . Previous studies on quality of work life for nurses show that nurses recognition and function directly affect their intention to stay in nursing profession . Load of work in nurses, without authorities reward, leads to an increase in nurses intention to leave their profession . About 58.5% of nurses believed they were not able to communicate with their supervisors and nurse managers . In a study on the quality of work life among nurses in the us, 72% of nurses reported to have proper communication with their nursing managers and supervisors, which is not consistent with the results of the present study . Communication with supervisors and other colleagues is among the factors which are associated with job satisfaction . About 84.9% of nurses believed that the security section did not make a secure environment for the nurses, and about 87.6% believed that their workplace was not physically, mentally, and verbally safe . The findings of the present study showed that 76.9% and 26.9% of nurses were exposed to verbal and physical violence, respectively, in the year prior to study, which shows a high prevalence and is in line with a study conducted in babol university of medical science in 2009 . As the staffs in health care system are exposed to workplace violence, prevention of violence and providing education of the necessary interventions against violence should be followed at all levels of an institute . The authorities should also help promotion of staffs services, especially that of nurses, by making a secure workplace . There is a negative correlation between the number of exposures to verbal and physical violence and work life quality and its dimensions, which has not been studied so far . Most of the nurses were dissatisfied with this dimension and mentioned the reasons as family's needs, working hours, and low energy after doing their daily tasks . Nurses reported that they spend a long time at their workplace, so they have little energy after work and cannot fulfill their families needs, which is consistent with the results reported in previous studies . Disproportionate salary and reward was one of the reasons for nurses dissatisfaction with their work life quality . Behavioral theories like mallow and herzberg behavioral theories showed that fulfillment of primary needs is essential as the individuals cannot concentrate on higher needs if their primary needs are not met . In the present study, 93.5% of nurses believed that their salary was not balanced with the inflation rate in market, which is in line with previous studies . Meanwhile, 57% of nurses in the us believed their salary was balanced with their expenses . Nurses low income is one of the major reasons for their job dissatisfaction and desertion . About 81.2% of the nurses believed that they had high workload, which is consistent with previous studies . On the other hand, 67.2% of the nurses believed they were not independent in taking care of the patients, which concords with former studies in which nurses reported they had low autonomy in decision making about patients care . About 88.7% of the nurses believed there were not adequate nursing personnel in their work environment and 64.5% believed that they were given extra non - nursing tasks . Shortage in human resources and increase of nurses workload act as pressure factors among nurses, which lead to professional and organizational desertion . Despite the shortage in human resources, these dimensions of malutilization of nursing force can increase the shortage of nursing force in a vicious cycle and affect nurses skills and experiences . Such challenges may impose a notable pressure on nurses and negatively affect nurses perception of work life . Managerial methods act as one of the problems in this dimension, which include lack of managers supervision, feedback, participation in decision making, higher level of managers respect toward nurses, inefficient nursing strategies and policies concerning facilitation of work, and modification of nurses concerns so that they think their struggles are not officially noted by nursing managers . Previous studies on quality of work life for nurses show that nurses recognition and function directly affect their intention to stay in nursing profession . Load of work in nurses, without authorities reward, leads to an increase in nurses intention to leave their profession . About 58.5% of nurses believed they were not able to communicate with their supervisors and nurse managers . In a study on the quality of work life among nurses in the us, 72% of nurses reported to have proper communication with their nursing managers and supervisors, which is not consistent with the results of the present study . Communication with supervisors and other colleagues is among the factors which are associated with job satisfaction . About 84.9% of nurses believed that the security section did not make a secure environment for the nurses, and about 87.6% believed that their workplace was not physically, mentally, and verbally safe . The findings of the present study showed that 76.9% and 26.9% of nurses were exposed to verbal and physical violence, respectively, in the year prior to study, which shows a high prevalence and is in line with a study conducted in babol university of medical science in 2009 . As the staffs in health care system are exposed to workplace violence, prevention of violence and providing education of the necessary interventions against violence should be followed at all levels of an institute . The authorities should also help promotion of staffs services, especially that of nurses, by making a secure workplace . There is a negative correlation between the number of exposures to verbal and physical violence and work life quality and its dimensions, which has not been studied so far . With regard to the above - mentioned negative correlation, it can be noted that workplace violence is a negative element reducing nurses work life quality . As the work life quality of nurses working in selected hospitals in isfahan is less than moderate, managers and authorities in hospitals should make policies for promotion of nurses work life quality through the following interventions: hospital managers should consider improvement of working conditions and making a supportive, friendly, and intimate environment for all the staffs, involving nurses in decision making and respecting their viewpoints, and designing a payment system based on nurses real function and nursing managers and supervisors more efficient humanistic communications.nurses work life quality is influenced by social, executive, managerial, and specific cultural conditions, and the present study revealed a negative correlation between the number of nurses exposed to violence and work life quality . With respect to the outcomes of workplace violence and its effect on work life quality, managers and authorities of these hospitals should think of solutions for the same . As the work life quality of nurses working in selected hospitals in isfahan is less than moderate, managers and authorities in hospitals should make policies for promotion of nurses work life quality through the following interventions: hospital managers should consider improvement of working conditions and making a supportive, friendly, and intimate environment for all the staffs, involving nurses in decision making and respecting their viewpoints, and designing a payment system based on nurses real function and nursing managers and supervisors more efficient humanistic communications . Nurses work life quality is influenced by social, executive, managerial, and specific cultural conditions, and the present study revealed a negative correlation between the number of nurses exposed to violence and work life quality . With respect to the outcomes of workplace violence and its effect on work life quality as the work life quality of nurses working in selected hospitals in isfahan is less than moderate, managers and authorities in hospitals should make policies for promotion of nurses work life quality through the following interventions: hospital managers should consider improvement of working conditions and making a supportive, friendly, and intimate environment for all the staffs, involving nurses in decision making and respecting their viewpoints, and designing a payment system based on nurses real function and nursing managers and supervisors more efficient humanistic communications.nurses work life quality is influenced by social, executive, managerial, and specific cultural conditions, and the present study revealed a negative correlation between the number of nurses exposed to violence and work life quality . With respect to the outcomes of workplace violence and its effect on work life quality, managers and authorities of these hospitals should think of solutions for the same . As the work life quality of nurses working in selected hospitals in isfahan is less than moderate, managers and authorities in hospitals should make policies for promotion of nurses work life quality through the following interventions: hospital managers should consider improvement of working conditions and making a supportive, friendly, and intimate environment for all the staffs, involving nurses in decision making and respecting their viewpoints, and designing a payment system based on nurses real function and nursing managers and supervisors more efficient humanistic communications . Nurses work life quality is influenced by social, executive, managerial, and specific cultural conditions, and the present study revealed a negative correlation between the number of nurses exposed to violence and work life quality . With respect to the outcomes of workplace violence and its effect on work life quality
It is well established that the human body generates a wide variety of volatile organic compounds (vocs) some that are present in exhaled breath, emitted through the skin and released by urine samples . As human - specific signatures, these vocs can be considered as non - invasive biochemical probes that can track normal and abnormal metabolic processes in the body, bacterial and inflammatory processes, and provide invaluable information on exposure to environmental pollutants and/or toxins [17]. Volatile aldehydes are widespread in human tissue and fluids and play important roles in functional processes . They have been reported to be common constituents of human urine [810], exhaled breath, and present in skin emanations [1317]. Some members of this chemical class have been detected in human blood and found to be released by in vitro human cell cultures [1921]. In the medical context, volatile aldehydes have been suggested to be biomarkers of lung cancer [2,19,20,2225], liver cancer, and breast cancer (see table 1). Some aldehydes are thought to be cytotoxic intermediates with several functions, such as signal transduction, gene regulation, and cellular proliferation . Recently, efforts have been made to employ volatile aldehydes in safety and security applications [9,15,3032]. Thus, there is growing evidence provided by a number of studies suggesting that chemical analysis of human odor could considerably improve effectiveness of search and rescue operations (usar) organized after disasters resulting in building collapse (e.g., earthquakes, tropical storms, explosions). Although the origin of some aldehydes in human organisms is unclear, several sources could explain their occurrence . These include (i) alcohols metabolism [3335], (ii) reduction of hydroperoxides by cytochrome p450, (iii) oxidative stress, (iv) diet and (v) environmental exposure (e.g. Tobacco smoking). Within this framework, a precise and reliable identification, and ultimately quantification, of volatile aldehydes proton - transfer reaction mass spectrometry (ptr - ms) is frequently employed in biological, medical, and environmental studies for detecting and quantifying volatile organic compounds [4350]. Its applicability stems from its versatility, excellent sensitivity (low pptv concentration levels), and real - time response . The application of a time - of - flight (tof) mass analyser in ptr - ms instruments notably improves their resolving power and, thereby, the discrimination between isobaric compounds . The recent employment of additional precursor (reagent) ions such as no, o2, and kr instead of the usual h3o (creating a selective reagent ionization time of flight mass spectrometry (sri - tof - ms)) has further enhanced the analytical possibilities of this technique . The primary goal of the present work was to investigate the product ion distributions for the reactions with no ions of 22 aldehydes involved in human physiology and pathophysiology using a sri - tof - ms, a variant of the well - established ptr - ms technique . The reactions of no with vocs in sri - tof - ms are relatively poorly known, inhibiting their use for trace gas analysis, but it is certain that for most such reactions multiple product ions will result, as can also happen using h3o reagent ions in ptr - ms . An interesting advantage of no as a reagent ion is that different chemical classes of vocs have their typical reactions with no (charge / electron transfer, hydride ion (h), transfer, hydroxide ion (oh) transfer, alkoxide ion (or) transfer, and no / analyte molecule association), as has been summarized in a detailed review paper . This ion chemical variability is of potential value because it allows in some cases the separation of functional isomers . 22 aldehydes were selected for inclusion in the present study as guided by the available literature that reports their presence in human urine, breath, blood, and skin emanation, as summarized in table 1 . Single - compound standard mixtures were prepared from liquid aldehydes, the majority of which were purchased from sigma aldrich (austria); acetaldehyde (99%), n - propanal (97%), n - butanal (99%), n - pentanal (97%), n - hexanal (98%), n - heptanal (95%), n - octanal (99%), n - undecanal (97%), 2-methyl propanal (99.5%), 3-methyl butanal (97%), 2-ethyl hexanal (96%), 2-methyl 2-propenal (95%), and (e)-2-butenal (99%). Moreover, n - nonanal (95%), n - decanal (95%), 2-propenal (95%), and benzaldehyde (99%) were obtained from fluka (switzerland), whereas, 2-methyl butanal (90%), 3-methyl 2-butenal (97%), (e)-2-methyl 2-butenal (97%), (e)-2-undecenal (90%), and furfural (98%) were provided by safc (usa). The compound purities are also given in table 2 for assistance in the interpretation of the product ion distributions of the no / aldehyde reactions . First, single - compound primary standards were prepared in 1-l glass bulbs (supelco, canada). Before usage, each bulb was thoroughly cleaned with methanol and dried at 70 c under the flow of high - purity nitrogen for at least 12 h to remove potential contaminants . The bulb was then evacuated using a membrane vacuum pump and approximately (0.51) l of liquid analyte was injected through a rubber septum . Next, the bulb was heated to 100 c for 30 min to ensure complete evaporation and then the pressure was balanced to ambient pressure with high - purity cylinder air . The desired standard mixtures were prepared by transferring appropriate volumes of the primary standard into 3-l volume transparent tedlar bags (skc inc ., usa) filled with predefined amounts of purified and humidified air, the latter being produced by a gaslab calibration mixtures generator (breitfuss messtechnik, germany). Effectively, for each compound the product ion distribution was investigated using 3 distinct concentration levels of each aldehyde in air ranging approximately from 25 to 150 ppbv and at two different absolute humidity levels of essentially 0 and 3.5% . The no / aldehyde reactions were studied using an ionicon analytik (innsbruck, austria) type 8000 sri - tof - ms instrument, a variant of the familiar ptr - ms flow - drift tube instruments . The no precursor / reagent ions were generated by the ionization mechanism extensively described elsewhere, essentially by charging the hollow cathode discharge ion source with high purity dry air . The settings of the ion source were chosen as follows: ion source current 5 ma, source voltage (us) 20 v, source - out voltage (uso) 70 v, and source valve opening 40% . With these settings the major parasitic impurity ions, as detected downstream by the analytical tof - ms, were h3o, o2, and no2 at relative levels (parasitic ion / no) of 0.30.6%, 11.5%, and 12%, respectively in the air carrier / buffer gas . The no / aldehyde reactions occurred in the carrier / aldehyde sample gases in the flow / drift tube at a total pressure of 2.23 mbar and a gas temperature of 60 c . Moreover, the voltage along the drift section was set to 600 v leading to an e / n ratio of approximately 130 td . The high resolution realized by the tof analyzer ranged from m / z 1 to 500 and were acquired at a time of 30 s by co - adding 750,000 single 40-s long tof - ms extractions recorded at a sampling frequency 1/t = 10 ghz . This corresponds to a theoretical upper limit m/m of 90,000 at m / z 100 (the flight time of these ions being 18 s). However, the actual mass resolution obtained from the detected peaks was 4000 at m / z 100 . This high resolution allows the separation of ions at nominally the same integer mass, for example, the nominally isobaric ions c3h7 and ch3co ions that are sometimes produced simultaneously in the analysis of gaseous matrices containing hydrocarbons, aldehydes and ketones . The mass calibration was based on three impurity peaks always present in the spectra: h3o (19.0178), no (30.9945), and no2 (45.9924). The standard mixtures entered the flow / drift tube of the sri - tof - ms instrument at a steady flow rate of 10 ml / min via a two - meter - long, heated (40 c) teflon transfer line . The total duration of a single measurement was 5 min, which corresponds to 10 mass spectra acquired per concentration level . Effectively, the average of these 10 spectra was used to determine the percentages of the product ions resulting from each no / aldehyde reaction . Single - compound standard mixtures were prepared from liquid aldehydes, the majority of which were purchased from sigma aldrich (austria); acetaldehyde (99%), n - propanal (97%), n - butanal (99%), n - pentanal (97%), n - hexanal (98%), n - heptanal (95%), n - octanal (99%), n - undecanal (97%), 2-methyl propanal (99.5%), 3-methyl butanal (97%), 2-ethyl hexanal (96%), 2-methyl 2-propenal (95%), and (e)-2-butenal (99%). Moreover, n - nonanal (95%), n - decanal (95%), 2-propenal (95%), and benzaldehyde (99%) were obtained from fluka (switzerland), whereas, 2-methyl butanal (90%), 3-methyl 2-butenal (97%), (e)-2-methyl 2-butenal (97%), (e)-2-undecenal (90%), and furfural (98%) were provided by safc (usa). The compound purities are also given in table 2 for assistance in the interpretation of the product ion distributions of the no / aldehyde reactions . First, single - compound primary standards were prepared in 1-l glass bulbs (supelco, canada). Before usage, each bulb was thoroughly cleaned with methanol and dried at 70 c under the flow of high - purity nitrogen for at least 12 h to remove potential contaminants . The bulb was then evacuated using a membrane vacuum pump and approximately (0.51) l of liquid analyte was injected through a rubber septum . Next, the bulb was heated to 100 c for 30 min to ensure complete evaporation and then the pressure was balanced to ambient pressure with high - purity cylinder air . The desired standard mixtures were prepared by transferring appropriate volumes of the primary standard into 3-l volume transparent tedlar bags (skc inc ., usa) filled with predefined amounts of purified and humidified air, the latter being produced by a gaslab calibration mixtures generator (breitfuss messtechnik, germany). Effectively, for each compound the product ion distribution was investigated using 3 distinct concentration levels of each aldehyde in air ranging approximately from 25 to 150 ppbv and at two different absolute humidity levels of essentially 0 and 3.5% . The no / aldehyde reactions were studied using an ionicon analytik (innsbruck, austria) type 8000 sri - tof - ms instrument, a variant of the familiar ptr - ms flow - drift tube instruments . The no precursor / reagent ions were generated by the ionization mechanism extensively described elsewhere, essentially by charging the hollow cathode discharge ion source with high purity dry air . The settings of the ion source were chosen as follows: ion source current 5 ma, source voltage (us) 20 v, source - out voltage (uso) 70 v, and source valve opening 40% . With these settings the major parasitic impurity ions, as detected downstream by the analytical tof - ms, were h3o, o2, and no2 at relative levels (parasitic ion / no) of 0.30.6%, 11.5%, and 12%, respectively in the air carrier / buffer gas . The no / aldehyde reactions occurred in the carrier / aldehyde sample gases in the flow / drift tube at a total pressure of 2.23 mbar and a gas temperature of 60 c . Moreover, the voltage along the drift section was set to 600 v leading to an e / n ratio of approximately 130 td . The high resolution realized by the tof analyzer ranged from m / z 1 to 500 and were acquired at a time of 30 s by co - adding 750,000 single 40-s long tof - ms extractions recorded at a sampling frequency 1/t = 10 ghz . This corresponds to a theoretical upper limit m/m of 90,000 at m / z 100 (the flight time of these ions being 18 s). However, the actual mass resolution obtained from the detected peaks was 4000 at m / z 100 . This high resolution allows the separation of ions at nominally the same integer mass, for example, the nominally isobaric ions c3h7 and ch3co ions that are sometimes produced simultaneously in the analysis of gaseous matrices containing hydrocarbons, aldehydes and ketones . The mass calibration was based on three impurity peaks always present in the spectra: h3o (19.0178), no (30.9945), and no2 (45.9924). The standard mixtures entered the flow / drift tube of the sri - tof - ms instrument at a steady flow rate of 10 ml / min via a two - meter - long, heated (40 c) teflon transfer line . The total duration of a single measurement was 5 min, which corresponds to 10 mass spectra acquired per concentration level . Effectively, the average of these 10 spectra was used to determine the percentages of the product ions resulting from each no / aldehyde reaction . The channel procentages were calculated using the signal intensities corrected for the mass dependent discrimination of the instrument (transmission). Only product ions with abundance greater than 0.5% of the total signal were included in the table unless they clearly originated from the species under study (e.g., the small adduct ion in the benzaldehyde reaction), although the stated purities of the aldehydes (see section 2.1), which range from 90 to 99%, means that product ions percentages should be considered in the light of the specific purities of each aldehyde . The abundance percentages listed for each reaction are the averages of values obtained for 3 distinct mean concentrations (from 10 spectra) of the aldehydes in the dry air and the humid air, as indicated in table 2 . At these low interaction energies the reactions are generally considered to occur by an intimate ion molecule interaction forming an excited intermediate complex, in the present reactions (nom) , which can either spontaneously undergo unimolecular decomposition back to the reactants or forward to fragmentation product ions, or be partially or totally stabilized by a third body collision (with the bath gas atoms and/or molecules in which the reaction occurs) resulting in the stable nom adduct ion . Looser or longer range interactions can occur, usually at higher interaction energies as in ion - neutral beams via the process of exothermic charge / electron transfer, but heavy particle exchange, such as hydride ion, h, transfer, rarely occurs at long distance . In all the reactions in the present series a significant, often dominant reaction mechanism is seen to be hydride ion transfer generating (mh) ions, as indicated by the percentage product ions given as bold in table 2 . This is in accordance with earlier studies carried out under the thermalized condition of the sift and sift - ms [6264]. This strongly implies that these no / aldehyde reactions in the thermalized sift - ms, and even the suprathermal sri - tof - ms reactors, do proceed via close interactions and the initial formation of the intermediate (nom) excited complexes . In the sift experiments, the observed product ions for the saturated alkanals mostly result from h transfer, occasionally the partial formation of nom adduct ions in the case of the unsaturated alkenals, and rarely ions that result from chemical rearrangements within the excited complex however, it is known and expected from mechanistic considerations that the lifetimes of these (nom) * complexes decreases with increasing interaction energy and gas temperature and so their lifetimes are expected to be shorter in sri - tof - ms flow / drift reactors than in thermalized sift - ms flow tube reactors (carrier gas temperature typically 25 c). This will diminish the probability of seeing stabilized nom adduct ions in the sri - tof - ms reactor at similar carrier / buffer gas pressures but at the higher carrier gas temperature of 60 c (see below). In the present flow / drift tube experiments, significant percentages of fragment ions are also seen for both the alkanals and alkenals reactions, most often as hydrocarbon ions cnhm, and this must be due to the elevated ion molecule interaction energies in the flow / drift tube and the possible injection from the ion source of energetic ions such as n2 that will not be seen downstream by the analytical mass spectrometer because of reactive loss . Thus, fragmentation occurs in addition to hydride ion transfer for all these aldehyde reactions, as can be seen by the multiple product ions in many reactions, with the exception of the 2-undecenal and furfural reactions, seen in table 2 . Consider first the no / acetaldehyde reaction that results in only the ch3co product ion and an hno neutral molecule . It is straightforward to show that this reaction is exothermic by 0.7 ev (electron volts), yet this reaction is known to be relatively slow even under sift - ms conditions; it is uncertain what its rate will be under ptr - ms conditions, but this would need to be ascertained if the no / acetaldehyde reaction is to be exploited for analysis . The fraction of (mh) to the total product ions in the no / propanal reaction indicated in table 2 and fig . 1 is 100% for moist carrier / sample gas and <100% in dry air when obviously additional product ions are seen:(1a)no + c2h5cho c2h5co + hno(1b) c2h5 + co + hno(1c) c2h3 + (ch2o + hno) hydride ion transfer (1a) is exothermic by 1.1 ev and is thus more exothermic than in the analogous acetaldehyde reaction . But what are the neutral products of reactions (1b) and (1c)? The fragmentation channel (1b) leading to a closed shell hydrocarbon ion is calculated to be endothermic by> 2 ev to produce two radical neutrals hco, and no, and endothermic by only 1 ev to produce the closed shell molecules co and hno . Reaction (1c) is very endothermic by> 4 ev to produce three neutral products cho, h2 and no and this is very unlikely, but it is less endothermic at 3.2 ev to produce the two products indicated as ch2o and hno . Thus, it is problematic to identify the neutral products of these apparently very endothermic reactions, especially so because of the uncertainty in the kinetic energy distribution of the ions in the flow / drift tube (but which must depart from maxwellian and is probably skewed toward the high energy tail), the vibrational state of the reactant no ions, and the interaction energies of the reactant ions and aldehyde molecules . What is certain is that the interaction energies will be elevated above the thermal energies prevailing in sift studies and this could drive endothermic reactions as apparently is the case for several of the reaction channels indicated for other reactions listed in table 2 . In support of the likely elevated interaction energies in the flow / drift tube reactors is the detailed analysis of hydrated hydronium ions, h3o(h2o)n, in ptr - ms flow / drift tube reactors by de gouw et al ., which indicates that the monohydrate ion h3oh2o, which has an h3o h2o binding energy of 1.4 ev, is entirely dissociated at an e / n of 130 td as adopted in the present experiments . This implies that ions like c2h5co, such as may be formed in reaction (1a), could fully dissociate to c2h5 and co. even so, it is difficult to see how reactions that are apparently endothermic by much more than 1 ev for ground vibronic state no ions can occur . It is therefore futile to attempt to define the reactions fully or even to guess the neutral products for the minor channels of these reactions when their origins are so uncertain . In summary, for the c3c7 alkanal reactions, co elimination is observed as a common fragmentation mode of the hydride ion product (mh) leading to the observed closed shell hydrocarbon ions cnh2n+1 . For c6c11 n - alkanals the hydride ion transfer process may be followed by the elimination of an h2o molecule (or formation of dihydroxyamine h3no2 as a single neutral product), but this could only be substantiated by detailed consideration of the energetics of the reactions; this uncertainty is indicated by bracketing the probable neutral products of each reaction in table 2 . The energy required for fragmentation of the primary product ion (mh) to eliminate co or h2o molecules can be acquired in multiple collisions with the n2 and o2 molecules that excites internal vibrational modes of (mh) ions, since at an e / n of 130 td the effective temperature of the interaction can be 500800 k . Interestingly, the possible h2o molecule elimination is apparently not occurring for the branched alkanal reactions for which the major mechanism can be interpreted as the elimination of cnh(2n1)o groups and the formation of hydrocarbon ions, c3h7 and c4h9 (see table 2). In the case of n - alkanals, the percentage (branching ratio) of the (mh) hydride ion transfer products exhibits an interesting dependence on the number of carbon atoms in the molecule (see fig . 1) that varies from 100% for the acetaldehyde reaction to a minimum percentage (13%) for the n - hexanal reaction . For instance, the percentage of (mh) ions for the n - octanal reaction is 27%, whereas for the n - undecanal reaction it is about 85% . This phenomenon may assist identification of heavier alkanals in the sri - tof - ms(no) instrument, but it would be complicated for the analysis of real matrices that contain mixtures of aldehydes and other trace compounds . Why does the plot of (mh) ion percentage signal levels have the form shown in fig . 1? The answer must lie to some extent in the nature and the energetics of individual product channels of the no / m reactions, specifically the lifetime of the intermediate (nom) * excited complexes and the accessibility of the other reaction channels . The production of (mh) ions is exothermic for all the reactions, but others of the observed channels may also be exothermic, especially so for those reactions where (mh) production is relatively small fraction of the total product ions . The lifetimes of the (nom) * excited complexes are generally assumed to be shorter for small m and to increase with molecular weight of m, as a consequence of the increasing number of vibrational modes of the complex in which the binding energy can be temporarily dispersed . A combination of these parameters could explain the increased efficiency of (mh) production within the longer - lived complexes of the larger alkanals . The presence of a side - chain in the aldehyde molecule promotes greater fragmentation of isomeric aldehydes, as can be seen in table 2 . However, this effect seems to occur only when the methyl (or ethyl) group is located at the second position of the main carbon chain . For example, the abundance of the (mh) product ion of n - pentanal and 3-methyl butanal reactions is very similar at 1415%, whereas the fraction of this ion in the 2-methyl butanal reaction is only 2% . A similar effect can be seen by comparing the n - butanal and the 2-methyl propanal reactions (18% vs 4%), and the n - octanal reaction with the 2-ethyl hexanal reaction (27% vs 2%). The same holds true for the alkenal reactions (e.g., 96% for 2-butenal vs 26% for 2-methyl 2-propenal). Consequently, the identification of 2-methyl(ethyl) aldehydes by sri - tof - ms(no) could be problematic in biological matrices using only this information . A further interesting point is that in the reactions of no with the largest alkanal, n - undecanal, in some unsaturated aldehydes, the heterocyclic furfural, and the aromatic benzaldehyde, small stable nom association product ions are observed, as indicated in italics in table 2 . The percentages are only small, but greatest for the furfural and 2-undecenal reactions where they reach approximately 10% of the total product ions . This reaction product channel has been seen previously for some unsaturated aldehydes in sift studies and is attributed to the proximity of the ionization energies (ie) of the aldehydes with ie of no, which results in charge transfer complexing that prolongs the lifetime of the (nom) * intermediate complexes . This interesting phenomenon is especially efficient in the association of no ions with ketones in sift reactors . It can also be reported here that a recent spot investigation of the no / n - undecanal reaction in a (thermalized) sift experiment significantly revealed a 40% no / undecanal adduct ion together with a 60% hydride ion channel and no significant fragmentation channels . Amongst all the aldehydes included in this study, charge transfer was only observed in the furfural reaction (50% of the total product ions), this process being marginally exothermic because the ionization energy, ie, of the furfural molecule at 9.22 ev is just lower than the ie of no (9.26 ev). This charge transfer efficiency will be assisted by the increased no / furfural molecule interaction energy in the sri - tof - ms reactor . The presence of water molecules in the sample / carrier gas has little or no effect on the product ion distributions for all these no / aldehyde reactions except for the n - propanal reaction (see table 2) for which in humid air the only observed product ion is (mh). This is probably due to the fact that the product c2h5 and c2h3 ions of the no / propanal reaction can rapidly react with water molecules in moist air and thus be eliminated from the reactor and not be seen by the downstream mass spectrometer . This will generally be the case for product ions of the type mh, where m has a proton affinity (pa) lower than the pa of water molecules (691 kj mol). This applies to protonated acetylene c2h3 (pa = 641.4 kj mol) and protonated ethylene c2h5 (pa = 680.5 kj mol), as appear in reactions (1b) and (1c). This observation is consistent with an analogous phenomenon in sift - ms reported by francis et al . . Significantly, the abundance of the c3h5 product ion for other aldehyde reactions reduced somewhat in the spectra obtained for the humid sample / carrier gas (see e.g., this product ion for the 2-methyl propanal and the 3-methyl butanal reactions). Although, the chemical structures of the particular c3h5 product ions are not established, three c3h4 underlying structural isomers are possible, all three having higher pa than water molecules (allene: 775 kj mol, propyne: 748 kj mol, and cyclopropene: 818.5 kj mol). Whilst the differences in these pa and that of water molecules is rather large and so proton transfer will be inhibited, it could be partially promoted by the elevated energies of the ion molecule interactions in the sri - tof - ms flow / drift reactor and because of the very large number density of the h2o molecules in the sample / carrier gas at an absolute humidity of 3.5% . The results of the present study of the reactions of no with some 22 aldehydes, compromising several n - alkanals, several branched chain alkanals, and several alkenals, indicates that most of the reactions result in multiple ion products, but common (mh) product ions are seen that result from hydride ion transfer with the fractional abundance of these ions increasing with the alkanal molecular mass . A series of hydrocarbon ions, cnhm, appear at widely differing percentages of the total ion products at m / z values that reflect the complexity (atomicity) of the aldehyde molecules . More specifically, alkanals exhibited a higher degree of fragmentation than the alkenals and the branched aldehydes dissociated most commonly at the position 2 . It must be emphasized that these ion product distributions will surely depend on the actual value of e / n used in the sri - tof - ms instrument . Whilst increasing the humidity of the sample / carrier gas had little effect on the product ion branching ratios, such serious fragmentation limits the value of sri - tof - ms using no reagent ions for the identification of aldehydes, especially when more than one of these compounds occur together in real samples such as exhaled breath and the headspace of biological fluids . Analytical sensitivity is diminished when multiple ion products of the analytical reactions occur unless such is carefully accounted for . It is also imperative that the rate constants for the analytical reactions be determined under the actual conditions of the reactor in order to obtain reliable quantification . This is especially so for these no reactions occurring in the flow / drift tube, because minor association channels are seen which is a clear warning of rate constants that can vary (usually decrease) with increasing interaction energy . In this regard, it is worthy of note that the rate constant for the no / acetaldehyde is much less than the collisional rate constant even under the thermal conditions of the sift system and would most probably decrease with increasing e / n in the flow / drift tube of a sri - tof - ms system.
Considerable research attention over the past several decades has focused on isolating the physical and psychological effects of induced abortion [15]. The majority of abortions in the united states are performed early in pregnancy and most of the research pertaining to indicators of postabortion health has logically involved the study of women who have undergone 1st trimester abortions . However, it is significant to note that 12%-13% of the annual 1.2 million u.s . Abortions are performed after the first trimester [68] and this translates out to approximately 144,000 per year, with 3.7% or 36,000 taking place at 1620 weeks and 1.3% or 15,600 occurring beyond the 20th week of pregnancy . Although empirical data is in short supply, a few large scale research efforts have revealed that 2nd trimester (1324 weeks) and 3rd trimester (2536 weeks) abortions pose more serious risks to women's physical health compared to 1st trimester abortions [9, 10]. The abortion complication rate is 3%6% at 12 - 13 weeks gestation and increases to 50% or higher as abortions are performed in the 2nd trimester . Data spanning the years from 1988 to 1997, bartlett and colleagues reported the following rates of abortion - related mortality: 14.7 per 100,000 at 1315 weeks of gestation, 29.5 per 100,000 at 1620 weeks, and 76.6 per 100,000 at or after 21 weeks . Based on the potential for serious consequences associated with late - term abortion, a first step toward reducing the numbers of late - term abortions is to gain a comprehensive understanding of why women decide to terminate as opposed to continue a pregnancy once they have allowed the pregnancy to progress for several months . According to the guttmacher institute, the most frequently endorsed reasons for late - term abortions include the following: (1) not realizing one is pregnant (71%), (2) difficulty making arrangements for an abortion (48%), (3) fear of telling parents or a partner (33%), and (4) feeling the extended time is needed to make the decision (24%). In the guttmacher study, only 8% of the women sampled indicated pressure not to have an abortion from someone else was part of the reason for delay and fetal abnormalities were identified as factoring into only 2% of all late - term abortion decisions . For example, decision ambivalence is often characteristic of women who undergo abortions in the 2nd trimester and beyond [1214]. Further, women who obtain 2nd trimester abortions have reported more deficient social supports and more energy expended toward assessing the resources available to help them keep a child compared to women who obtain 1st trimester abortions [14, 15]. Research suggests that 30% of women who delay an abortion beyond 16 weeks are afraid to tell those closest to them about the pregnancy . When compared to women obtaining earlier abortions, women who obtain late - term abortions are more likely to experience stronger attachment to the fetus, have more moral or religious objections to abortion, and concede to an abortion based on the wishes of others [15, 16]. Finally, women who seek late - term abortions (after 16 weeks) are significantly more likely to be under age 18, black, unemployed, and/or poor . As indicated above there is not an extensive published literature on the physical effects of late abortions; however there are even fewer published studies on women's mental health outcomes after 2nd trimester abortions . Nevertheless, it is logical to anticipate more serious mental health problems in response to abortions occurring later in pregnancy compared to earlier terminations for various reasons: (1) awareness that the fetus has developed more fully prior to the termination, (2) women have more opportunity to bond with the developing fetus, (3) there may be more active desire to maintain the pregnancy, and/or (4) pressure from others to abort may be more pronounced . In fact, most of the established predictors of late - term abortion described above, including decision ambivalence and dissatisfaction, lacking support to carry to term, a strong attachment to the fetus, timing during adolescence, and low income are predictors of poor postabortion psychological adjustment in the general abortion literature [1722]. In a study of british women who had prostaglandin - induced abortions between 2024 weeks gestation and felt fetal movements, further, sderberg et al . Reported that 37.5% of women who underwent 2nd trimester abortions experienced extreme postabortion emotional problems . Although these studies indicate that late - term abortions are more likely to initiate psychological problems, they were both very small scale and did not involve direct comparisons to women undergoing 1st trimester abortions with logical controls for variables likely to discriminate between the two populations . Empirical evidence of a link between 1st trimester abortion and ptsd symptoms has accumulated in recent years [2630]. In fact 1220% of women with an abortion history meet the full diagnostic criteria for ptsd with considerably higher percentages of women experiencing some trauma symptoms, while not meeting the full criteria [2830]. Even when the full criteria are not met, the more ptsd symptoms present, the greater the risk of psychological impairment and suicidal ideation . In the current study, comparisons were made between women who had an early elective abortion (up to 12 weeks gestation) and women who had undergone a later elective abortion (13 weeks gestation or later) based on individual ptsd symptoms, ptsd symptom subscale scores (intrusion, avoidance, and hyperarousal), total ptsd scores, and the degree to which ptsd symptoms met the full dsm - iv diagnostic criteria . As an anxiety disorder, ptsd is initiated by exposure to a psychosocial stressor which is perceived to be traumatic . This disorder is comprised of three stressor - related criteria not present before the trauma: (1) intrusion which involves persistent and unwanted reexperiencing of the traumatic event in the form of recurrent and distressing memories, flashbacks, and hyperreactivity to associated stimuli; (2) avoidance which pertains to persistent and deliberate efforts to avoid recalling the traumatic event using various forms of denial, dissociation or detachment; and (3) hyperarousal which is a general uneasiness or jumpiness that may include insomnia, the tendency to startle easily, feelings of impending danger or disaster, trouble concentrating, extreme irritability, and possibly violent behavior . Although no previous studies have been published comparing the mental health of women undergoing early and late term abortions, the evidence reviewed above is sufficient to hypothesize that abortions occurring across the 2nd trimester and into the 3rd trimester would be associated with higher levels of ptsd symptomatology than 1st trimester abortions . Since 2nd and 3rd trimester abortion are less common than 1st trimester abortions and women may be more reticent about acknowledging such abortions due to stronger social prohibitions against later terminations, web - based data collection was deemed a useful method in that it affords a high level of anonymity . The obvious drawback to this methodology is the risk for selection bias wherein more women who have struggled with an abortion experience may be more inclined to participate due to the increased salience of the experience and a possible quest for answers . Established benefits of internet data collection include time and cost efficiency [32, 33], access to difficult to reach populations [34, 35], and enhancement of participant comfort and engagement [36, 37]. A review by skitka and sargis, indicated that as early as the years 2003 to 2004, 21% of apa journals had published at least one study with online data collection, suggesting this is rapidly becoming an established mode of data collection . The most frequently cited criticism of web - based surveys is that they are comprised of convenience samples, rendering generalization difficult [39, 40]. However, even this shortcoming engenders benefits such as clarity and thorough responses that are less inclined to be contaminated by social desirability biases and underreporting [4143]. Several published papers indicate that online data collection is equivalent to more traditional data collection methodologies in terms of reliability, validity, and representativeness [4447]. The primary goal of the present exploratory study was to compare the mental health status of a sample of women who experienced a 1st trimester abortion (up to 12 weeks gestation) to women who had a 2nd or 3rd trimester abortion (13 weeks and beyond). In the analyses, sociodemographic and personal history factors, particularly those related to significant life stressors such as exposure to abuse, that may systematically vary across the two groups (early and late) were controlled in order to more accurately examine the independent effects of abortion timing . A secondary goal was to provide additional descriptive data on women who delay abortion decisions until the 2nd and 3rd trimesters with a focus on variables pertaining to the abortion decision . Surveys were posted on an internet website from april, 2005 through august, 2008 . Although the time frame was chosen for convenience, the goal was to collect data until a minimum of 50 women who experienced a late - term abortion had responded in order to insure adequate statistical power . All respondents who had experienced an abortion and completed at least 95% of the items on the survey were included . Participants were informed that submission of the survey constituted consent to participate and they were told they could withdraw at any point . Informational website links offering support or counseling services recruitment occurred through email requests to us - based crisis pregnancy centers and to a few additional organizations that offered postabortion counseling . Questions comprising the survey addressed five sociodemographic characteristics (age, race, education, marital status, and number of children), meaningfulness of religious affiliation, abortion history, reasons for abortion, perceived adequacy of preabortion counseling, partner agreement in abortion decision - making, political opinion regarding abortion at time of procedure, relationship status with the partner, mental health history, abuse history, trauma symptoms related to abortion, abortion - related anger, relationship problems, sexual problems, and general stress attributed to abortion . The majority of items measuring demographic characteristics, personal history, and the circumstances surrounding the abortion were dichotomous (yes / no). The precise nature of the items and response options are easily identified by the data in tables 1 and 2 . Posttraumatic stress disorder symptoms were assessed with the ptsd checklist - civilian version (pcl - c). For the purposes of the present investigation, a score of 1 was entered for each item endorsed on the scale at the level of moderately, quite a bit, or if the respondents indicated not at all or only a little bit she was not identified as having the corresponding symptom . Subscale score ranges were from 0 to 5 for intrusion or reexperience (5 items), 0 to 7 for avoidance (7 items), and 0 to 5 for hyperarousal (5 items). Respondents were considered to have met the symptom criteria for a diagnosis of ptsd based on dsm - iv criteria: (1) one or more endorsements of intrusion, (2) three or more endorsement(s) of avoidance symptoms, and (3) two or more endorsements of hyperarousal symptoms not present prior to the abortion . Reliability and validity evidence for the pcl is provided by weathers and colleagues . With the current sample, internal consistency reliability estimates using cronbach's alpha for the full scale and for the intrusion, avoidance, and hyperarousal subscales were equal to .92, .82, .80, and .82, respectively . All analyses were conducted using spss software and included both basic descriptive statistics and inferential statistical tests to examine the hypotheses and conduct secondary tests of the data . Specific analyses conducted included independent t - tests, analyses of variance (anova), analyses of covariance (ancova), multivariate analyses of variance (manova), multivariate analyses of covariance (mancova), and logistic regression . In the analyses wherein statistical controls were employed, the following variables were entered: race, marital status at the time of the abortion, number of years of formal education, number of abortions, number of years since the target abortion, having received mental health counseling before the abortion, having been hospitalized for emotional problems before the abortion, meaningfulness of the respondent's religion, and a childhood or adult history of physical or sexual abuse . Surveys were completed by 374 women with 81% of the respondents indicating u.s . Citizenship . Additional respondents identified the following countries of citizenship: england (4%), canada (6.4%), and australia (2.7%), with smaller percentages from france, ireland, norway, romania, czechoslovakia, germany, sweden, new zealand, south africa, kenya, mexico, nicaragua, brazil, nepal, and south korea . The average age of the respondents was 38 years at the time the survey was completed (sd = 11.1). Also, at the time of the survey, 48.0% were married for the first time, 10.5% were married for the second time, 12.1% were divorced and single, 2.2% were separated, and 26.4% had never married . Religious affiliations were indicated to be 81.6% christian, 3% jewish, 9.5% other, and 8.6% none at the time of the survey . Most of the sample endorsed liberal views of abortion at the time of the procedure, with 24% believing abortion should be legal for any reason throughout pregnancy and an additional 36% contending abortion should be available for any reason during the 1st trimester . Only 24% felt it should be legal under various circumstances and 16% believed it should never be legal . Approximately 14% (n = 52) reported having undergone an abortion between 13 and 30 weeks gestation (m = 16.87 weeks; sd = 4.24) and 86% reported abortions up to 12 weeks gestation (m = 8.23 weeks; sd = 2.39) the women reported an average of 15 years (sd = 11.8) had passed since the abortion and again no significant differences were observed between the early and late groups . Table 1 provides the full sample frequencies for respondents' race, education, marital status, number of children, number of abortions, meaningfulness of religion, mental health counseling prior to the abortion, hospitalizations for emotional reasons before the abortion, and the experience of physical and sexual victimization in childhood and adulthood . Use of independent t - tests and logistic regression analyses revealed no significant differences between the early and late abortion groups relative to age, ethnicity, marital status at the time of the abortion, religious orientation, meaningfulness of religion, marital status at the time the survey was completed, education, number of children, number of abortions, mental health counseling prior to the abortion, preabortion hospitalizations for emotional problems, and the experience of sexual abuse in childhood or adolescence . However, the women who obtained late abortions were more likely to report having been the victim of physical abuse in childhood (t (373) = 2.37, p <.001), to have been the victims of physical abuse in adulthood (t (273) = 2.05, p = .044), and to have been the victim of sexual abuse in adulthood (t (373) = 2.94, p = .005). Table 2 provides data regarding the percentage of study respondents from the early and late abortion groups who indicated agreement with various parameters surrounding the abortion decision . As indicated by the data presented, logistic regression analyses revealed that women who had experienced a late - term abortion were significantly more inclined to report the following: (1) the pregnancy was desired by their partner: (2) someone other than their partner pressured the decision: (3) the respondent left her partner before the abortion: (4) the respondent's partner did not know about the abortion until afterwards: and (5) physical health concerns factored into the abortion decision . In contrast, women who secured earlier abortions were more likely to report abortion decision agreement with their partner and they were more likely to report that mental health, financial, and educational concerns factored into their decision to abort . In order to test the hypothesis that 2nd and 3rd trimester abortions would be more stressful than 1st trimester abortions, several analyses were conducted that enabled the researchers to determine if women experiencing late - term abortions are generally more at risk for ptsd symptoms . A manova was conducted using timing of the abortion (early versus late in pregnancy) as the independent variable and the three subscales of the pcl as the dependent variables . The multivariate effect was significant, f (3,322) = 2.69, p = .046 . In addition, the univariate effect for the intrusion subscale was significant, f (1,324) = 7.49, p = .007, with the means for the early and late abortion groups equal to 2.42 (sd = 1.63) and 3.13 (sd = 1.64), respectively . Similarly, the difference between the two abortion timing groups was significant for the hyperarousal subscale, f (1,324) = 4.76, p = .030, with the mean for the early group lower (m = 2.22; sd = 1.78) than the mean for the late group (m = 2.85; sd = 1.95). The variables in this first analysis were then entered into a mancova with controls for several demographic and personal experience variables (race, marital status at the time of the abortion, number of years of formal education, number of abortions, number of years since the target abortion, having received mental health counseling before the abortion, having been hospitalized for emotional problems before the abortion, meaningfulness of the respondent's religion, and a childhood or adult history of physical or sexual abuse). The results revealed only one significant univariate effect, for the intrusion subscale, f (1, 260) = 4.91 . The adjusted mean for the early group was equal to 2.51 (se = .103) and the adjusted mean for the late group was equal to 3.15 (se = .26). An anova was conducted with abortion timing groups again employed as the independent variable and total pcl scores as the dependent variable . The initial unadjusted effect was significant, f (1, 342) = 5.89, p = .016, with the late group identifying significantly more trauma symptoms (m = 10.52; sd = 5.13) than the early group (m = 8.65; sd = 4.81). However, when the covariates listed above were entered into an ancova with these same independent and dependent variables, the result was not significant, f (1, 260) = 3.16, p = .077 . A series of logistic regressions were computed to assess the extent to which a late - term abortion was associated with an increased risk of having met the dsm - iv criteria for the intrusion, avoidance, and hyperarousal subscale scores and for ptsd generally . Although the data presented in table 3 indicates a higher percentage of late - term abortion respondents meeting the ptsd criteria for the subscales and for the full scale, the results of the logistic regression analyses were not significant . A second manova and a second mancova were conducted using timing of abortion groups as the independent variable and the 17 items on the ptsd scale as separate dependent variables in an effort to identify specific trauma symptom differences between the two groups . However, several univariate tests were significant in each analysis, and the results are provided in table 4 . After applying the control variables, the late - term group was significantly more likely to report repeated disturbing dreams, reliving the abortion, and trouble falling asleep . Without the controls, the same items were significant as were feeling emotionally numb, super alert or watchful, and feeling jumpy or easily startled . Exploratory analyses were performed to examine the extent to which ptsd scores varied as a function of the reasons that factored into women's abortion decisions . In these analyses, the seven reasons for abortion (mental health, physical health, finances, education, career, family size, and social concerns) listed in table 2 operated as independent variables . In one test, a mancova, the scores on the three pcl subscale scores served as the dependent variables; whereas in the second test, an ancova, total pcl scores were employed as the dependent measure . All the control variables used in the previous analyses were included in these analyses as well . Due to power concerns, these exploratory analyses could only be conducted with the full sample rather than exclusively focusing on the late - term group . The results of the exploratory mancova indicated that only the multivariate effect for social reasons was significant, f (3,176) = 4.14, p = .007, with the univariate tests for the intrusion f (1,178) = 12.86, p = .007, avoidance f (1,178) = 7.70, p = .006, and hyperarousal subscales f (1,178) = 4.40, p = .007 also significant . In each case, higher means were observed for women who identified social reasons compared to those who did not cite social reasons as relevant to their decisions to abort . Specifically the means on the intrusion, avoidance, and hyperarousal subscales were equal to 1.74 (se = .26), 3.16 (se = .34), and 1.74 (se = .29), respectively, for the group who did not report social reasons as relevant to their decision to abort . In contrast, the means on the intrusion, avoidance, and hyperarousal subscales were equal to 2.99 (se = .21), 4.41 (se = .27), and 2.53 (se = .23), respectively, for the group who did report social reasons as relevant to their decisions . In the ancova with total pcl scores examined, again only the univariate effect for social reasons was significant, f (1,178) = 9.04 p = .003 . The mean pcl total score for the group that did not indicate social reasons were part of their decision to abort was 6.63 (se = .74); whereas the mean for the group that noted said social reasons were relevant to their decision was considerably higher, 9.93 (se = .59). Finally, a logistic regression with the same statistical controls employed indicated that women who noted social reasons were relevant to their decision to abort were 186% more likely to experience ptsd symptoms consistent with meeting the dsm - iv diagnostic criteria, or = 2.86 (ci = 1.57 5.23; p = .001). The purpose of this study was to test the hypothesis that women who undergo 2nd and 3rd trimester abortions would be more traumatized than their peers who experience 1st trimester abortions as evidenced by significantly higher rates of ptsd symptoms . After instituting statistical controls for race, marital status, formal education, number of abortions, number of years since the abortion, mental health counseling and hospitalizations for emotional problems before the abortion, meaningfulness of the respondent's religion, and a childhood or adult history of physical or sexual abuse, all the group differences were in the hypothesized direction but only a few were statistically significant . Specifically, the difference in intrusion subscale scores was statistically significant and when individual ptsd items were examined, the late - term group was found to report more disturbing dreams, more frequent reliving of the abortion, and more trouble falling asleep . The first two of these items are intrusion subscale symptoms and the last is a symptom from the hyperarousal subscale . Differences between the groups were few due to a large percentage of women from both the early and late abortion groups reporting symptoms of ptsd . In fact, 52.5% and 67.4% in the early and late abortion groups, respectively, met the dsm - iv symptom criteria, a considerably higher percentage than in earlier published reports [2830]. First, for this particular sample of women, a great deal of time had elapsed since the abortion (an average of 15 years) and the symptoms could conceivably have developed later in this extended time frame . For example, developmental changes including both normative and nonnormative life events could have been quite stressful and added to the salience of the abortion (e.g., difficulties conceiving a wanted pregnancy, a miscarriage, or relationship problems, etc . ). Second, the fact that the current sample was characterized by high rates of exposure to potentially traumatizing physical and sexual abuse in childhood and adulthood may have yielded a sample with increased susceptibility to experiencing the abortion as a trauma . Third, because the data were collected anonymously and voluntarily, women who had been more negatively affected may have been more interested in the study and were therefore more willing to participate than women who were less adversely impacted . Fourth, in this study the participants were asked if they had experienced the symptoms on the pcl at any point after the abortion and as a result of the abortion, but they were not asked to indicate the symptom duration . Therefore, the high numbers meeting the symptom criteria for ptsd evidenced in this investigation should be viewed cautiously and future studies should incorporate more comprehensive assessments of ptsd symptoms, ideally using clinician administered diagnostic tools . Finally, more than a quarter of the current sample had experienced more than one abortion and the effects may have been cumulative . There is evidence that repeated abortion increases the risk for mental health problems [49, 50]. Specifically, freeman reported that repeated abortion patients exhibited significantly higher distress scores on measures of interpersonal sensitivity, paranoid ideation, phobic anxiety, and sleep disturbance when compared to women who had only one abortion . In that study, somatization, hostility, and psychoticism were likewise elevated in the group of women who had more than one abortion . Similarly, niemela and colleagues found that finnish women seeking a second abortion rated lower on impulsivity, emotional balance, realism, self - esteem, life stability, and in the capacity for positive personal relationships compared to women with only one abortion . A secondary objective of this study was to identify possible differences in the context of abortion decision - making associated with an early versus late abortion . When compared to a 1st trimester abortion the following differences pertaining to the context of the abortion decision were observed in those who had 2nd or 3rd trimester abortions: (1) the abortion was more likely to have been desired by the partner, (2) the abortion was less likely to have been desired by both parties, (3) there was more pressure from someone other than the partner to abort, (4) male partners were less likely to have been informed of the abortion until afterwards, and (5) women were more inclined to have left the partner before undergoing the procedure . In general, these results are indicative of more ambivalence and conflict surrounding the decision and the likelihood of less stable partner relationships among women who obtain later abortions . Logically, women who are unsure about how to proceed with an unplanned pregnancy are more likely to put off the decision to abort, perhaps hoping that their circumstances will improve and enable them to carry to term . Other significant differences between the early and late - term groups related to reasons for the abortion . In particular, mental health, financial, and educational concerns figured into decisions to abort more often with 1st trimester abortions compared to later abortions . The only reason that was more frequently reported by the women undergoing later abortions compared to the women who had early abortions was a general category of physical health concerns with nearly 30% of the late abortion group voicing these concerns compared to approximately 15% of the early group . Unfortunately no data were gathered pertaining to the specific nature of the concerns and future research should examine the extent to which these factors were based on preexisting conditions or concerns introduced by the pregnancy . Interestingly, social concerns such as embarrassment were the most frequently reported concern, with endorsement by 62% of the late term group and 69.1% of the early group . This may have been an additional reason why so many women in the sample experienced stress afterwards as they likely chose the abortion to preserve their reputations despite ambivalence or actually desiring to continue the pregnancy . Some of the women may have also come to feel the abortion was not sufficiently justified . The least commonly identified reason for the choice to abort was family size with approximately 13% of the women in each group indicating this concern entered into their decision . The results of the exploratory analyses employing the full sample of women, both those who had early and late abortions, indicated that social reasons for an abortion were strongly related to pcl total scores and to intrusion, avoidance, and hyperarousal subscale scores . With a large percentage of the participants reporting that social reasons entered into their decisions, this could be an additional explanation for why the ptsd rates were so high in the sample . These data suggest that women who postpone their abortion into the 2nd or 3rd trimester experience elevated risk for certain forms of unwelcome re - experience of the abortion procedure, likely requiring active professional intervention . Relief from intrusive memories whether in the form of flashbacks or disturbing dreams may only occur if the individual learns to effectively integrate memories of the traumatic experience with other aspects of her past as well as with contemporary experiences and emotions . This is a process that ordinarily involves not only uncovering painful memories, but also transforming them in a personally meaningful way in order to bring relief . Verbalizing traumatic experiences and sharing with others is an essential component of this complex healing process and sadly due to the guilt and related shame which frequently occur in conjunction with an abortion experience, many women may never confide in others and are unable to effectively integrate traumatic memories . Over time, the triggers that initiate the intrusion may become more generalized and debilitating as well . For example, a woman may initially experience intrusive symptoms whenever she sees a nurse if most of her interaction was with a nurse during the abortion and then with time, contact with any health care professional in a uniform may initiate an episode of reliving the abortion . Social reasons for both early and late abortions were related to numerous ptsd symptoms; therefore, professionals should examine the extent to which women are selecting abortions for social reasons, such as to protect their reputation or to avoid embarrassment . In such cases the abortion may, over time, feel unwarranted or frivolous to the woman and incite deep feelings of guilt or remorse that can trigger trauma symptoms . In addition, under such circumstances there is the possibility that the woman desired the pregnancy but decided not to go through with it for social reasons . The research cited previously has shown that pregnancy wantedness is a predictor of postabortion distress . Based on employment of a convenience sample, the relatively small number of respondents with late - term abortion experience, and reliance on self - report, this study is most appropriately viewed as a pilot for future analyses of the psychological impact of later - term pregnancy termination procedures . Nevertheless, securing a sample of any size is not an easy prospect when the focus is on late - term abortion and there are very few published reports on this seriously understudied and potentially quite fragile population . Based on societal interest in the morality and legality of late - term abortion, data related to the psychological sequelae of late - term abortion is needed in order to protect women's mental health . (1) do women who have later abortions tend to experience intrusive trauma - related symptoms for a longer duration than women who have earlier abortions due to the nature of late - term abortion procedures? (2) are women with abuse histories who may be primed for trauma more prone to experiencing serious trauma symptoms after an abortion and are they less likely to seek needed help and achieve inner peace due to compromised self - esteem, self - blame, or shame? (3) are there other mental health problems, such as depression, anxiety, and substance abuse that tend to occur more frequently after later abortion compared to early abortion? (4) assuming differences are detected and late - term abortions are identified to be more emotionally taxing on women, who are most at risk for mental health problems and what specific characteristics of 2nd and 3rd trimester abortions make them more traumagenic? As long as 2nd and 3rd trimester abortions are medical services that are freely available to women residing in the u.s, we have an ethical obligation to more fully understand the mental health risks involved and to convey this information in a sensitive manner to women as they struggle with difficult abortion decisions.
Denosumab (dmab) is a fully human monoclonal antibody against the receptor activator of nuclear factor-b ligand (rankl), a molecule that is crucial for the formation, function, and survival of osteoclasts.1 dmab binds rankl with high affinity and specificity, and inhibits the rankl / rank interaction, thus reversibly reducing osteoclast - mediated bone resorption . In phase 1 and phase 2 studies, dmab was demonstrated to decrease bone turnover and to increase bone mineral density (bmd),25 and in the freedom trial (fracture reduction evaluation of denosumab in osteoporosis every six months, nct00089791), a randomized placebo - controlled phase 3 study, the subcutaneous administration of dmab 60 mg every 6 months for 36 months significantly reduced the risk of vertebral and nonvertebral fractures, and reduced the risk of hip fracture in postmenopausal women with osteoporosis.6 moreover, in the open - label extension of this study, dmab therapy beyond the third year of treatment was associated with a further reduction in nonvertebral fracture rate, and was associated with a continued low vertebral fracture rate that persisted through 8 years of continuous administration, with an overall safety profile that remained consistent over time.7 on the basis of available evidence, in 2010, dmab was approved for the treatment of postmenopausal osteoporosis, thus becoming a further therapeutic option for the reduction of fracture risk in addition to the other available antiresorptive therapies (ie, bisphosphonates and selective estrogen receptor modulators) and the anabolic teriparatide.8 the available studies comparing the effect on bmd and bone turnover of dmab and bisphosphonates, which are the most frequently used agents for the management of osteoporosis, showed significantly greater gains in bmd at all measured skeletal sites913 and greater reduction in bone turnover912 with dmab compared to bisphosphonates with a similar safety profile . However, both bisphosphonates and dmab, in association with calcium and vitamin d, appear to be about equally effective in clinical trials in reducing the risk of fragility fractures,14 which represent a considerable problem of public health, considering the increasing fracture - related morbidity, mortality, and medical costs in many regions of the world.15 it must be considered, however, that any therapy, even if proved to be effective in clinical trials, requires adherence to achieve successful treatment outcomes . Persistence is the duration of time from initiation to discontinuation of therapy, while compliance is the degree to which a patient takes the medication as prescribed.16 accordingly, nonpersistence and noncompliance are usually defined as a gap in therapy greater than 90 days and a medication taken less than 80% of possible treatment days, respectively.16 adherence to osteoporosis treatments is particularly challenging for health care professionals treating osteoporosis . Indeed, persistence and compliance with osteoporosis therapies are generally poor, thus leading to a significant reduction in their antifracture efficacy,17 which in turn leads to increased human and economic costs.18 in order to understand the extent of the problem, it is worth explaining that previous studies showed that one - third to one - half of treated patients are not adherent to oral bisphosphonate treatment,19 and that the majority of patients discontinue oral bisphosphonate treatment within 1 year,17,19 with a mean persistence of only 184 days.17 in comparison with oral dosing regimens, persistence seems to be greater with an intravenous bisphosphonate administered less frequently, like the annual infusion of zoledronic acid, but it is anyway suboptimal . Indeed, a variable proportion of patients from one - third to two - thirds across studies did not receive a second administration of the drug, often because of adverse effects (postinfusion syndrome).2022 these findings are due to the fact that treatment adherence among patients with chronic diseases like osteoporosis depends on various factors, among which difficult dosing regimens, high dosing frequency, and the occurrence of side effects play a significant role in reducing compliance and persistence . Moreover, patient perception about the necessity of the prescribed medication to treat osteoporosis and their concerns about potential adverse effects are important and potentially modifiable determinants of adherence, especially if clarified and addressed at the beginning of the treatment . Finally, understanding patient preference may be a strategy to improve adherence to osteoporosis therapy, since a lower treatment satisfaction is associated with an increased risk of discontinuing or switching the ongoing osteoporosis medication, as compared with a higher treatment satisfaction.23 in this review, we will focus on the results of studies that investigated patient preference and adherence to dmab for the treatment of postmenopausal osteoporosis in comparison with alternative osteoporosis therapies, especially bisphosphonates, in order to establish who can take more advantage of dmab therapy, to understand the possible factors that influence medication - taking behavior, and to discover potential strategies for improving adherence . Patient preference to and satisfaction with a specific drug are important determinants of adherence to therapies for chronic diseases, including osteoporosis.23,24 preference is a relative index of desirability, and it can be measured as a choice between alternatives or scaled as a degree of desirability,25 while treatment satisfaction measures the degree to which patient expectations with different features of the ongoing treatment (eg, perceived efficacy, presence and severity of side effects, convenience, and bother with treatment) are met.25 available studies typically compared patient preference to and satisfaction with dmab versus bisphosphonates, especially alendronate, which is usually the first - line medication for the treatment of postmenopausal osteoporosis.2628 since existing questionnaires assessing preference to and satisfaction with osteoporosis treatments were considered inadequate for the comparison between a weekly oral tablet and a 6-monthly subcutaneous injection, a new tool, the preference and satisfaction questionnaire (psq), was developed to compare dmab and alendronate.25 the psq consists of 34 items that explore preference (the treatment choice made by a patient), satisfaction (the degree to which the features of a specific drug actually meet the patient expectations), and finally, bother (the degree to which the patient is disturbed by certain features of the treatment).25 in the determining efficacy: comparison of initiating denosumab versus alendronate (decide) trial and the study of transitioning from alendronate to denosumab (stand), two international, double - blind, double - dummy, randomized, phase 3 head - to - head trials comparing dmab with alendronate,9,10 psq was completed after 12 months of treatment or upon study discontinuation.26 among the subjects who expressed a preference, significantly more patients, who were blinded to their treatment assignment, preferred the injection over the tablet, and were more satisfied overall and with the dosing frequency of a 6-monthly injection over a weekly tablet after 12 months of treatment . Moreover, more patients indicated that they would choose the 6-monthly injection, which was better fitted to their lifestyles, for long - term use or continuation of treatment . Finally, among patients who expressed bother with treatments, more patients found that the weekly tablet was more bothersome than the 6-monthly injection.26 a subsequent multicenter, randomized, open - label, 2-year crossover trial, the denosumab adherence preference satisfaction (daps) study,28 enrolled drug - nave postmenopausal women with low bmd, who were randomized in one of two treatment sequences: dmab subcutaneously every 6 months for 1 year followed by alendronate orally once weekly for 1 year, or vice versa . At each follow - up visit, subjects completed questions about preference, satisfaction, and bother, which were taken from the psq . At baseline and at 6 months, subjects reported lower mean scores concerning preference for alendronate than for dmab, at 12 months significantly more subjects treated with dmab than with alendronate reported to be either satisfied or quite satisfied with the dosing frequency, route of administration, convenience, and expressed overall satisfaction with the ongoing dmab treatment.27 the final results from both years of the daps study further confirmed the data obtained before the crossover: at the end of the study, 92.4% of subjects preferred subcutaneous dmab injections over alendronate tablets, and 91.2% of subjects said that they would choose dmab injections for long - term treatment . In addition, at 24 months, regardless of the treatment sequence, a greater proportion of subjects reported that they were quite / very satisfied with the attributes of dmab compared with those of alendronate.28 a recent study evaluated the change in treatment satisfaction in women with postmenopausal osteoporosis who were suboptimally adherent with prior daily or weekly bisphosphonate therapy and who were shifted to subcutaneous 6-monthly dmab or monthly oral bisphosphonate (ibandronate or risedronate).29 in such study, a post hoc analysis of the results of two international, multicenter, randomized, open - label studies that had bmd and bone turnover variations as primary endpoints,12,13 was performed . The change in treatment satisfaction was assessed using the treatment satisfaction questionnaire for medication (tsqm), a tool validated for the measure of patient satisfaction with treatments of different chronic diseases and which consists of 14 items to assess an individual s perception of four domains of treatment satisfaction: 1) effectiveness, 2) side effects, 3) convenience, and 4) global satisfaction.30 the results of the study showed that osteoporotic postmenopausal women sub - optimally adherent with oral daily or weekly bisphosphonate therapy, who switched to dmab or monthly bisphosphonate treatment, reported greater satisfaction in all four domains of tsqm in both treatment groups at 6 and 12 months, but that these positive changes were significantly greater in patients in the dmab group compared to those in patients in the monthly bisphosphonate group at all post - baseline time points.29 whereas patient preference to 6-monthly dmab injections versus oral weekly or monthly bisphosphonates was not surprising in relation to the more acceptable route of administration and the less frequent dosing regimen of the 6-monthly treatment option, patient preference between dmab and another long - acting injectable therapy, such as zoledronic acid, could be less obvious . However, while several studies clearly demonstrated that patients preferred once yearly intravenous infusion of zoledronic acid rather than oral weekly bisphosphonates,3133 a direct comparison in terms of patient satisfaction between dmab and zoledronic acid for the treatment of postmenopausal osteoporosis is lacking . A recent retrospective study on a limited cohort of patients reported a statistically similar patient satisfaction between a group of patients treated with dmab and another one treated with zoledronic acid,34 but the small sample size and the design of the study (ie, each patient experienced only one of the two treatments without any experience of the other treatment) other parameters closely related to treatment satisfaction and preference, which could influence patient medication - taking behavior, are patient perceptions about a therapy in terms of the perceived necessity of the prescribed medication to treat a specific condition and concerns about potential adverse effects . A validated tool to assess these beliefs and concerns can be found in the beliefs about medicines questionnaire (bmq), which consists of 22 questions in the following major domains: 1) the necessity of the prescribed medication to treat osteoporosis in that moment or in the future; 2) concerns about potential side effects of taking the prescribed medication; and 3) preference for one drug over the other.35 at baseline in the daps study,27,28 when women were nave to therapy, necessity and concerns scores were similar between groups . Subsequently, subject beliefs about the necessity for the prescribed treatment were significantly higher for dmab than for alendronate at 6 months, but not at the following visits . Subject concerns about potential side effects were significantly lower for dmab than for alendronate at the follow - up visits after the cross - over, when patients had experienced both forms of treatment administration, but not at previous time points.27,28 these variations in subject perceptions about treatment resulted in a significantly higher necessity concerns differential (ncd) (ie, how much treatment necessity outweighs treatment concerns) for dmab compared with alendronate at 6 months for both treatment years.36 finally, the bmq survey in the daps study provided significantly lower mean preference scores for alendronate than for dmab at every visit, consistent with the preference scores of the psq.27,28 many of the studies, which investigated preference for and satisfaction with dmab, also evaluated adherence to the treatment, overall or in comparison with oral bisphosphonates, especially alendronate . Unfortunately, studies specifically designed to compare adherence to dmab versus zoledronic acid are still lacking . In the decide and the stand studies, where participants were strictly followed up every 3 months, compliance at 12 months (both injections received and 80% of the oral tablets) was 93% and 94%, respectively, with dmab and 91% and 94%, respectively, with alendronate.9,10 in the daps study, adherence, ie, both compliance (both dmab injections 6 months 4 weeks apart or 80% of alendronate tablets taken) and persistence (both dmab injections or greater than two alendronate doses in the last month and completion of the treatment period), was assessed separately for each treatment year.27,28 by the end of the first 12 months, 88.1% of postmenopausal women were adherent to dmab and 76.6% of patients were adherent to alendronate, while after the crossover, the adherence rate was 92.5% for dmab and 63.5% for alendronate . A 46% and 80% relative risk reduction of nonadherence was calculated with dmab compared to alendronate in the first and in the second year, respectively.27,28 the increase of nonadherence for alendronate - treated subjects after the crossover from dmab, and conversely, the further decrease of nonadherence for dmab - treated subjects after the crossover from alendronate, suggest a possible treatment sequence effect: a weekly dosing frequency may be more difficult to follow after a biannual dosing frequency than the opposite treatment sequence . Daps investigators also examined whether the subjects perception of their osteoporosis treatment and the treatment preferences influenced adherence.36 they found that at the beginning of the second year of treatment participant perception, as measured by bmq scores, was a significant predictor of nonadherence . Indeed, as necessity scores increased, the odds of nonadherence decreased, and conversely, as concerns scores increased, the odds of nonadherence increased . Indeed, higher ncd scores were significantly associated with lower odds of nonadherence, thus suggesting that positive perceptions of treatment positively influence medication - taking behavior.36 by extension, it seems feasible that understanding the factors that influence patient perceptions of osteoporosis treatments may result in an improved educational effort to increase adherence . In relation to this hypothesis, a successive study, although with several limitations, showed that in osteoporotic patients starting with a first dmab injection, a positive feedback given to the patient already 6 months thereafter, based on the demonstration through a careful medical explanation of a rapid and highly significant bmd increase and on a good safety profile, was able to guarantee in 99% of patients the willingness to accept a second injection, thus reinforcing the role of patient perceptions and of their assessment during doctor patient interactions on adherence to treatment.37 several studies on medication - taking behavior of patients receiving dmab are now ongoing, and data available so far confirm a high adherence to this 6-monthly subcutaneous treatment.38,39 the 12-month interim results of a european noninterventional study involving germany, austria, greece, and belgium showed that 82.7%89.3% of patients received a second dmab injection within 6 months 4 weeks, and therefore, these patients were considered adherent to treatment,38 a proportion of patients significantly greater than that observed in similar studies on bisphosphonates.17,19 in all four countries, these percentages increased as the permissible time window was extended, and up to 95.3% of patients received a second administration within 6 months 8 weeks.38 at baseline, all participants completed the morisky 8-item medication adherence scale (mmas-8) questionnaire, a tool used to measure the probability of adherence.40 however, although the majority of patients had a low or medium score for adherence to prior treatments, their adherence to dmab was high anyway, suggesting that some features of dmab, such as the dosing schedule, may have positively influenced adherence behavior . No baseline variables were found to be significantly associated with persistence in the four countries, probably because of the high percentages observed and because of the different health care systems in the individual countries; nevertheless, in some countries, several significant associations were identified . Indeed, parental history of hip fracture was associated with higher persistence, while increased age, decreased mobility, and increased distance to the clinic were correlated with lower persistence.38 finally, interim results of a 24-month multicenter, prospective, single - arm observational study in the us and canada showed that at 12 months, 81.9% of patients were persistent with dmab (ie, they received a second dmab injection within 6 months 8 weeks).39 as already described in another study,38 this percentage changed as the window was modified (from 74.8% of patients with a 4-week window to 84.8% of patients with a 12-week window). In this population, several baseline variables were found to be significantly associated with persistence among us patients and others among canadian patients.39 in particular, it is worth signaling that us patients with greater ncd obtained by means of bmq had a higher odds ratio for persistence,32 further confirming the role of patient perceptions of a specific medication in influencing medication - taking behavior, as already described in previous studies.23,36,37 firstly, participants enrolled in a clinical trial may differ from patients seen in real - life clinical practices . This difference may be observable to a greater extent in randomized clinical trials, where patients are regularly followed up by a skilled health care professional according to a precise study protocol . However, the participation in any prospective study, even observational, may influence patients behavior, potentially leading to an overestimation of adherence to and preference for a specific treatment . This observation is supported by the finding that the rates of bisphosphonate adherence observed in the studies cited so far are meaningfully higher than rates observed in previous retrospective observational reports (less than half in the first year).17,19 the main reasons that may explain this phenomenon are the willingness of subjects to participate in a study and thus to accept the treatment offered, and the awareness of patients that their medication - taking behavior is being monitored . These two factors, differently from real - world behaviors, eliminate primary nonadherence patient behaviors in trials (ie, the refusal of a medication at first prescription). Moreover, in the clinical trials, participants are selected according to given inclusion criteria, and this aspect may limit the generalizability of results . Secondly, in many of the cited studies,2629,3639 a conflict of interest cannot be excluded, and the provision of a drug to study participants by the study sponsor may have concealed the possible influences of treatment cost and accessibility on patient preference and adherence . Nevertheless, although several studies suggested that dmab is cost - effective as compared to bisphosphonates,4145 it must be considered that the effect of these aspects may be very difficult to assess since health care and reimbursement systems vary extensively between countries, and thus studies performed in different regions of the world become scarcely comparable . Thirdly, the use of 1- or 2-year treatment periods in the cited trials27,28,38,39 may limit conclusions about long - term adherence to treatment, although a previous meta - analysis on bisphosphonate treatment suggests that nonadherence usually occurs shortly after treatment initiation.19 in spite of these limitations, available data suggest that, in comparison with bisphosphonates, postmenopausal women report greater preference to and satisfaction with dmab, both overall and with its dosing frequency and route of administration, and they would choose dmab over bisphosphonates for long - term osteoporosis treatment.2629 moreover, patient beliefs about the necessity of osteoporosis treatment and patient concerns about potential adverse events appear higher and lower, respectively, with dmab than with bisphosphonates . This situation results in greater ncd scores, indicating that in patient perceptions, treatment necessity clearly outweighs concerns about dmab.27,28,36 furthermore, clinical trials showed that adherence among women treated with dmab was consistently> 80% across studies;9,10,13,27,28,3739 therefore, significantly higher adherence was shown in women treated with dmab than in women treated with bisphosphonates, even when bisphosphonates were administered once monthly or intravenously.17,1922 this different medication - taking behavior appears to be even more pronounced in patients previously treated, often sub - optimally, with bisphosphonates.12,13,28,38 although in clinical trials an overestimation of adherence is conceivable, it must be considered that the adherence to dmab requires only a 6-monthly visit, while the adherence to oral bisphosphonates requires the patient self - administration of tablets according to a correct dosing schedule and in the correct way . Therefore, it is possible that in clinical practice, the differences between dmab and oral bisphosphonates in term of compliance and persistence may be even higher, although additional real - life studies are needed to confirm this assumption . Moreover, patient perception about treatment seems to influence medication - taking behavior,36,39 and during treatment follow - up, positive reinforcement based on the evidence of actual successful treatment outcomes, such as the bmd increase913 or bone turnover suppression,912 could help to further improve patient adherence.37 indeed, due to the asymptomatic nature of osteoporosis, until a fracture occurs or even later, the patient could easily underestimate the importance of osteoporosis medication, resulting in poor adherence and therefore an increased risk of fracture . In conclusion, current evidence underlines the necessity to personalize osteoporosis treatment, taking patient preference into account, especially in regards to frequency and route of administration . Current evidence also draws attention to patient beliefs at the initiation of therapy and during follow - up . These efforts are addressed to improve adherence to osteoporosis treatment and, as a consequence, to achieve more successful treatment outcomes, thus positively impacting on the cost - effectiveness of the chosen drug.45 from this perspective, according to the data demonstrating a better adherence to dmab compared to other osteoporosis treatments, especially bisphosphonates, dmab may represent a reasonable and effective alternative to bisphosphonates, particularly for osteoporotic women in whom a suboptimal or even poor adherence to oral treatments is expected . Further studies are required in the future to assess long - term adherence and preference to dmab in real - world clinical practices, to evaluate its long - term safety, and to assess its effectiveness as compared head - to - head with bisphosphonates.
The motor system is a dynamic network of cortical and subcortical areas interacting through excitatory and inhibitory circuits, modulated by somatosensory input . Modifications of cortical excitability enable recovery and reorganization of the remaining motor areas both in animal models [4, 5] and in humans [1, 6]. Transcranial magnetic stimulation (tms) and magnetoencephalography (meg) have both been applied in stroke patients to reveal cortical excitability changes . Motor threshold (mt), that is, the minimal tms intensity eliciting motor evoked potentials (meps), is related to membrane excitability as it is influenced by drugs affecting neuronal ion channels . Paired pulse tms (pp - tms) reveals short - interval intracortical inhibition (sici), related to the activation of gaba - a receptors and intracortical facilitation (icf) attributed mainly to glutamatergic nmda receptor activation (for references, see). In acute stroke, the mt is increased in the lesioned hemisphere (lh) whereas in the nonlesioned (nh) hemisphere it is normal or decreased one month after stroke . Sici is decreased in both hemispheres early after stroke; icf is stronger in nh in severe than mild strokes [1, 6]. Finger movements and median nerve or finger stimulation modify spontaneous meg oscillations over the sensorimotor cortex in the beta band (1525 hz). They are suppressed at 100300 ms after tactile stimulation (event - related desynchronization; erd), reflecting increased excitability, and increased at 5001000 ms (event - related synchronization; ers), reflecting removal of excitation [12, 13] or reduced excitability . Inhibitory gaba - a agonist diazepam increases meg beta activity [15, 16]. Combined meg and magnetic resonance spectroscopy showed a linear relationship between the beta ers strength and gaba concentration . Beta ers has been shown to be significantly weaker in the lh than nh; stronger ers amplitude was correlated with a better affected hand function up to 3 months after stroke . Reduction of beta ers, however, correlated with clinical improvement after physiotherapy of patients with chronic stroke . Movement - related beta erd was significantly reduced in lh of stroke patients . The hand representation in the somatosensory cortex (s1), estimated by somatosensory evoked fields (sefs), is the largest one month after stroke and its increase was correlated with the affected hand function . In tms mapping, hand motor representation expands in the lh up to 1 month . In animal models, somatosensory reorganization is activated immediately after the lesion by diminished gaba - a - related inhibition and by glutamatergic activation after one month . We hypothesized to see correlations between erd and mt related to the early cortical excitability and between ers and sici, both attributed to gaba - a - related processes . Moreover, we expected that icf, reflecting glutamatergic activity, would correlate with the somatosensory modifications in meg, as glutamate is associated with late somatosensory plasticity . Meg and ntms were recorded from thirteen patients (age 67.3 11 years, 8 women), with first - ever ischemic stroke in the middle cerebral artery territory one (t1) and three months after stroke (t2). Six patients had a subcortical and seven patients had a subcorticocortical or cortical stroke (table 1). At t1, one patient used amitriptyline 10 mg / day and another used citalopram 10 mg / day . One patient used zopiclone 7.5 mg for occasional sleeplessness . At t2, citalopram 10 mg / day was used by one patient . Data from these patients has been presented previously [18, 21, 23, 24]. As the patients having both tms and meg recordings are a subset of the previous patient groups, we recalculated the group - level electrophysiological parameters to show that the present patient group is sufficiently similar to those reported previously (see supplementary tables 1 and 2 in supplementary material available online at http://dx.doi.org/10.1155/2015/309546). An eximia navigated magnetic stimulator with a coplanar figure - of - eight coil of 70 mm wing radius (nexstim ltd ., helsinki, finland) was used for ntms . Patients' mris, required for navigation, were scanned at t1 and used also at t2 . The site was then stimulated by single tms pulses at 110% of mt and by the pp - tms at 90% for conditioning and 110% of mt for test pulses . Fifteen single pulses or pairs of conditioning and test pulses were delivered in each stimulation session . The interval between stimuli was 3.3 s and the intersession interval varied between 1 and 3 min . The isi selected for the paired - pulse stimuli was 2 ms for sici and 12 ms for icf . The peak - to - peak amplitudes of meps elicited by pp - tms were normalized by dividing them by the corresponding single - pulse mep amplitude to simplify subject - to - subject comparisons . Meg during rest and tactile stimulation of the thumb (d1), index (d2), and little finger (d5) were recorded with a 306-sensor neuromagnetometer (elekta neuromag, helsinki, finland) in biomag laboratory, right before the ntms measurement . Time - frequency representations (tfr) in the 1030 hz band were calculated to define the frequency range of beta modulation, which was quantified by the temporal spectral evolution method (tse) from signals of 2 to 4 meg sensors showing the strongest reactivity . Only the contralateral beta modifications (the affected hand stimulation for the lh and the unaffected hand for nh) were analyzed . Onset and offset of the erd and ers were defined as a time point when the signal differed 2 sds from the baseline . The absolute erd and ers strengths were calculated from the peak amplitudes and converted into relative values in relation to the 300 ms prestimulus baseline . For sefs, about 120 responses were averaged for stimulation of d2 (isi 3005 ms), and d1 and d5 (isi 1005 ms) in separate sessions . The size of the hand representation in the si was determined by calculating the euclidean distance in xyz - space between the equivalent current dipoles (ecds) of the earliest responses to d1 and d5 stimulation . Multiple comparison correction was carried out according to the number of tests (n = 32) suggested by the four prior hypotheses (t1 and t2 were tested separately; lh and nh variations lead to four tests in each case; n = 442 = 32). We also present significances of correlation values without multiple comparisons and supply all correlation coefficients and corresponding p values (cf . [1, 9, 28] for a similar approach and for its statistical aspects). The differences between ntms and meg values obtained at t1 and t2 were tested with student's t - test . In the lh, meps were found in 11 patients both at t1 and at t2 and were present in the nh in all patients . Mt was higher for lh than nh in 9 patients at t1 (p <0.05, binomial test) and in 10 patients at t2 (p <0.05, binomial test). The mts in the lh and nh correlated strongly both at t1 (r = .82, p <.01) and at t2 (r = .78, p <.01). Pp2ms stimulations of nh did not inhibit meps (disinhibition; diminished sici) in 5 patients at t1 and in 3 patients at t2 . Pp12ms stimulation of lh facilitated meps (icf) in 10 out of 11 patients at t1 and in all patients at t2 . In nh mep amplitudes elicited by pp12ms stimulations were correlated between the lh and nh at t1 (r = .62; p <.05) but not at t2 (supplementary table 2). Erd started 140 10 ms after tactile stimulation and peaked at 250 10 ms . The subsequent ers started at 520 40 ms and peaked at 900 70 ms . At t1, erd was absent in one patient and ers in two patients in the lh; ers was missing from the nh in one patient . At t2, ers was smaller in the lh than nh at t1 (46 31% versus 63 32%; p <.05); at t2, the difference was nonsignificant . Sefs from both hemispheres were detected in 12 patients at t1 and in all patients at t2 . They were smaller in the lh than nh at t1 (25 nam versus 32 nam; p <.04) but not at t2 . The si hand representation area was larger in the lh than nh at t1 (12 3 mm versus 10 3 mm p <.003) but not at t2 (supplementary table 3). The plots of the most relevant correlations are depicted in figure 1 to show that they were not driven by outliers . All correlations are displayed in table 2 to enable evaluation of significance of our hypotheses against general effects of the lesions . The mt and erd were correlated in the lh at t1 (r = .66, p <.03), indicating that small erd was associated with a high mt (figure 1(a)). At t2, this correlation was weaker (r = .58, p <.06). However, the mt of the lh correlated with the erd of nh (r = .62, p <.04), and the mt of the nh correlated with erd of the lh (r = .65, p <.02), suggesting that high mt was associated with a small erd in the opposite hemisphere at t2 (table 2). Sici and the ers did not correlate at t1 or in lh at t2 . In the nh, high ers was associated with a strong sici (r = .59, p <.04; figure 1(b)). In addition to hypothesized correlations, sici of the nh and erd of the lh at t2 were correlated (r = .82, p <.001), indicating that strong erd in the lh was associated with a strong sici in the nh . Sici in the lh was correlated also with the si amplitude of the lh (r = .64, p <.04), indicating that small si amplitude was associated with a weak sici (table 2). Icf and sef parameters did not correlate at t1 . At t2, icf in the lh correlated with the s1 amplitude of lh (r = .65, p in addition, icf in the nh correlated (r = .82, p <.001) with the si finger representation area of the lh; this correlation remained significant also after bonferroni correction (table 2). Moreover, icf in the lh at t1 was correlated (r = .83; p <.002) with the si finger representation area of lh at t2; high icf at t1 resulted in a small hand representation area at t2 (figure 1(c)). Our study is the first to compare meg and ntms excitability parameters during stroke recovery . Navigated tms, not applied previously in longitudinal studies of stroke patients, enabled the precise replication of the stimulus site between separate measurements, adding reliability to the follow - up . We found correlations between cortical excitability estimates derived from ntms and meg . As expected, we found correlations between mt and erd, but in only one of the four possible intrahemispheric and two out of four interhemispheric conditions . The sici and beta ers, both attributed to gabaergic mechanisms, were correlated in one of their four possible intrahemispheric conditions (in the nh at t2). One reason for this may be that various gaba - a receptor subtypes contribute to sici . Nonselective gaba - a receptor activators modify sici whereas the gaba - a1 receptor specific zolpidem did not . Nonselective gaba - a agonist zopiclone increased meg beta activity whereas zolpidem suppressed beta activity in the vicinity of stroke lesion . Moreover, in healthy subjects, diazepam increased meg erd but did not affect ers when the increase of baseline beta activity was taken into account . This nonspecificity could contribute to the correlations of sici with the erd and mt as well . Correlations between ntms parameters and meg erd / ers were stronger at t2 than at t1 . Analogously, most tms intracortical excitability measures did not correlate with the hand function acutely but did so 3 months after stroke . Recovery of sensorimotor fmri activation to digit stimulation from 1 to 3 months was correlated with final motor function, emphasizing the importance of this time period for stroke recovery . Is attributed mainly to glutaminergic mechanisms, glutamate may contribute to stroke - induced plasticity . Somewhat surprisingly, high icf at t1 correlated with small si hand area at t2 (figure 1(c)); thus, the narrowing towards normal hand representation size may be supported by glutaminergic activity . Icf did not correlate with the meg erd / ers, and the si hand area and beta erd / ers were not correlated; this suggests different mechanisms underlying sici and icf (see for a detailed discussion). For example, high mt was associated with a small erd in the opposite hemisphere, and strong erd in the lh was associated with a strong sici in the nh . Increased excitability within the unaffected motor cortex may cause imbalance between the homologous cortical motor areas and worsen also the ipsilesional hand coordination (for references, see). Thus, some functional correlations may relate to the modified general excitability properties of the motor system, instead of effects in the immediate vicinity of the stroke . Tms results give direct information of the changes in the motor output and the immediate effects of tms are relatively local . However, also subcortical and spinal processes affect the meps used to evaluate the tms effects . Meg reveals the activity of the whole cortical mantle and enables mapping of network effects generated by stroke . Meg source analysis suggests mainly motor cortex origin of beta ers [13, 33, 34]. However, in electrocorticography, recorded directly from the cortex, beta erd and ers appear outside of pre- and postcentral gyri, in supplementary motor cortex, or broadly from pre- and postcentral gyri, frontolateral and medial cortex [37, 38]. The widespread cortical generation of the erd and ers may make them resilient to small cortical strokes . Multitude of generators may contribute to considerable variability of source locations of beta erd in stroke patients (cf . ). Multiple sources underlying meg signals may also explain resilience of auditory evoked fields after small strokes . Stronger correlations between icf and si parameters than between mt and sici and ers / erd may, in part, result from spatially more limited source areas of s1 responses than those of erd / ers . It can be expected that meg and ntms produce complementary information about the effects of stroke on cortical networks . Moreover, meg parameters in the affected hemisphere and ntms indices in the unaffected hemisphere were correlated with the motor performance of the affected hand (cf . [18, 21, 24]). Erd in the 822 hz band may reflect downregulation of intracortical inhibition in the human motor cortex, as tms delivered during erd is associated with increased mep amplitudes and reduced sici . However, 1 hz repetitive tms over the motor cortex reduces meps to subsequent single tms pulses, indicating inhibition, but decreases postmovement beta ers, and intermittent theta burst tms facilitates meps but increases postmovement beta ers . Beta ers is reduced in patients with myoclonus epilepsy, indicating increased cortical excitability . In line, sici is decreased in myoclonus epilepsy patients; however, mt is increased [44, 45], and the silent period after the tms pulse, reflecting motor cortical postsynaptic inhibition, is prolonged, indicating prevailing inhibitory cortical tonus . Thus, only some aspects of the cortical excitability may be shared in excitability estimates obtained by tms and meg . Our results suggest that some tms and meg excitability measures reflect the activity of the same transmitter systems . However, high mt and absence of erd / ers may also correlate because of severely affected sensorimotor connections between the periphery and the cortex . We detected sefs in 12/13 patients and meps in 11/13 patients already at t1, indicating that both somatosensory and motor pathways were conveying signals . Motor function can be maintained despite significant damage to the corticospinal tract, as estimated from mt of stroke patients . Moreover, we observed fewer correlations at t1, when the sensorimotor pathways probably were more affected, than at t2 . However, such spurious correlations should remain stable or decrease during recovery from t1 to t2 but not increase, as in our data . The limitations of our study include the small size of the patient group as the precise features of structural and functional changes may differ among the patients . Cortical excitability is modified differently in cortical and subcortical strokes [46, 47]. This, however, should not alter our correlations between the tms and meg, as both were recorded from the same patients . Possible effects of medication on excitability should go in parallel for meg and ntms, as the patients were tested sequentially during the same day . The patients were not tested in the acute stage with tms, and meg recordings showed most dramatic erd and ers modifications between the acute phase and t1 . Although mt in the lh is correlated with the paretic hand function in acute stroke, this correlation, however, weakens during recovery, and tms intracortical excitability parameters correlated with the clinical performance best at 3 months . The 2 ms or 12 ms isis, selected for our paired - pulse stimuli, produce clear sici and icf in healthy subjects and in stroke patients, but we did not test other parameters, which could have produced stronger correlations between ntms and meg . Icf and si response amplitude and area size, mt and the erd of the hemisphere harboring the stroke lesion, and sici and ers of the nonlesioned hemisphere are correlated in stroke patients . Numerous correlations of the excitability parameters between the lh and nh emphasize the importance of the hemispheric balance of the excitatory - inhibitory properties of the sensorimotor system in analyzing the stroke - related dysfunction during stroke recovery.
Although surgery is the current standard treatment for localized surgically operable lesions, many patients with liver metastases cannot undergo surgical resection because of associated comorbidities, concerns about their age, the extent of the disease, or the patient's wishes . Alternative treatment approaches for unresectable liver metastasis and primary liver cancer include chemoembolization, radiofrequency ablation, cryotherapy, and the oral multikinase inhibitor sorafenib . Treatment choice is guided by the barcelona clinic liver cancer staging system and recommended treatment strategy . Stereotactic body radiotherapy (sbrt) is a technique that allows the delivery of a precise dose of radiation to a tumor while sparing adjacent normal tissues . However, the movement of intra - abdominal organs due to respiration has hampered the use of sbrt . The insertion of a fiducial marker near to the tumor before radiotherapy allows respiratory motion to be tracked, thus enabling accurate dose delivery while the patient breathes freely . Recently, the percutaneous insertion of fiducial markers has been described [4, 5], but experience of such procedures is still limited . This marker is made from gold, which makes it biocompatible and ensures it exhibits good contrast on x - ray images . In this study, we describe our initial experience with this new fiducial marker and evaluate the technical feasibility, clinical efficacy, and safety of sbrt using this marker . This study was part of a prospective sbrt study in which the cyberknife g4 was used to treat liver tumors and was approved by the institutional review board . Written informed consent was obtained from all patients . Since the patient accrual for this sbrt study was relatively slow, we report our initial experience with a gold flexible linear fiducial marker (visicoil, radiomed corporation, barlett, tn, usa) in this article . Between july 2012 and february 2015, 18 patients underwent percutaneous fiducial marker placement under computed tomography (ct) fluoroscopic guidance or ultrasonographic guidance before sbrt for liver tumors . Their median age was 68 years (range, 4483 years), and all of them were inpatients . First, all patients underwent diagnostic ct scans of their abdomen composed of 35 mm - thick contiguous axial tomographic sections . After reviewing these preliminary images, an appropriate puncture site and the optimal needle guidance method, i.e. Ct fluoroscopic or ultrasound guidance, was determined in advance . The marker placement was performed during breath - holding under ct fluoroscopic guidance in 8 cases and under ultrasound guidance in 10 cases . The imaging parameters during ct fluoroscopy included a ct beam width collimated to 3 mm . One patient needed contrast - enhanced ct fluoroscopy during the procedure to delineate the tumor clearly . Ultrasonography was performed with a convex probe (25 mhz). A gold flexible linear marker containing an 18-gauge coaxial needle (0.75 mm in diameter and 5 mm in length, fig . Local anesthesia was achieved via the subcutaneous administration of 1% lidocaine . After confirming that the needle tip had reached the target lesion, after the procedure, ct or cone - beam ct was performed to determine whether any complications such as pneumothorax or bleeding had occurred . (a) an 18-gauge coaxial introducer needle containing the gold flexible linear fiducial marker . (b) ct image shows the percutaneous insertion of the gold flexible linear fiducial marker . (c) ct image shows the successful placement of the gold flexible linear fiducial marker (arrow) in the tumor . The gold flexible linear fiducial marker (visicoil). (a) an 18-gauge coaxial introducer needle containing the gold flexible linear fiducial marker . (b) the gold flexible linear fiducial marker . A 73-year - old man with metastasis from carcinoma of the ampulla of vater . (b) ct image shows the percutaneous insertion of the gold flexible linear fiducial marker . (c) ct image shows the successful placement of the gold flexible linear fiducial marker (arrow) in the tumor . The target site was chosen by consensus by an interventional radiologist and a radiation oncologist before the procedure . Clinical success was defined as the completion of sbrt without the marker dropping out of position . Marker position was checked by ct or cone - beam ct images taken immediately after placement and planning ct images taken before radiotherapy . Migration of a marker before radiotherapy was defined as its displacement exceeding 3 mm from the initial position on ct images; the marker position within or relative to the tumor was evaluated . During radiotherapy, migration of a marker was evaluated by comparing abdominal plain x - ray images at an inspiratory phase in the supine position taken after marker placement with fluoroscopy images taken at an inspiratory phase of the treatment system; thereby the marker position was evaluated in relation to the rib bones and diaphragm . It was difficult to evaluate subtle displacements of the marker by fluoroscopy images alone, especially once radiotherapy was started, because the size of the tumor and the surrounding tissue could change with treatment . Therefore, when migration of the marker that could influence treatment accuracy (e.g.> 3 mm) was suspected by fluoroscopy, ct scanning was scheduled for further evaluation to determine whether the displacement was significant or not . First, all patients underwent diagnostic ct scans of their abdomen composed of 35 mm - thick contiguous axial tomographic sections . After reviewing these preliminary images, an appropriate puncture site and the optimal needle guidance method, i.e. Ct fluoroscopic or ultrasound guidance, was determined in advance . The marker placement was performed during breath - holding under ct fluoroscopic guidance in 8 cases and under ultrasound guidance in 10 cases . The imaging parameters during ct fluoroscopy included a ct beam width collimated to 3 mm . One patient needed contrast - enhanced ct fluoroscopy during the procedure to delineate the tumor clearly . Ultrasonography was performed with a convex probe (25 mhz). A gold flexible linear marker containing an 18-gauge coaxial needle (0.75 mm in diameter and 5 mm in length, fig . Local anesthesia was achieved via the subcutaneous administration of 1% lidocaine . After confirming that the needle tip had reached the target lesion, the fiducial marker was deployed, and then the needle was removed (fig . After the procedure, ct or cone - beam ct was performed to determine whether any complications such as pneumothorax or bleeding had occurred . (a) an 18-gauge coaxial introducer needle containing the gold flexible linear fiducial marker . (b) ct image shows the percutaneous insertion of the gold flexible linear fiducial marker . (c) ct image shows the successful placement of the gold flexible linear fiducial marker (arrow) in the tumor . The gold flexible linear fiducial marker (visicoil). (a) an 18-gauge coaxial introducer needle containing the gold flexible linear fiducial marker . (b) the gold flexible linear fiducial marker . A 73-year - old man with metastasis from carcinoma of the ampulla of vater . (b) ct image shows the percutaneous insertion of the gold flexible linear fiducial marker . (c) ct image shows the successful placement of the gold flexible linear fiducial marker (arrow) in the tumor . The target site was chosen by consensus by an interventional radiologist and a radiation oncologist before the procedure . Clinical success was defined as the completion of sbrt without the marker dropping out of position . Marker position was checked by ct or cone - beam ct images taken immediately after placement and planning ct images taken before radiotherapy . Migration of a marker before radiotherapy was defined as its displacement exceeding 3 mm from the initial position on ct images; the marker position within or relative to the tumor was evaluated . During radiotherapy, migration of a marker was evaluated by comparing abdominal plain x - ray images at an inspiratory phase in the supine position taken after marker placement with fluoroscopy images taken at an inspiratory phase of the treatment system; thereby the marker position was evaluated in relation to the rib bones and diaphragm . It was difficult to evaluate subtle displacements of the marker by fluoroscopy images alone, especially once radiotherapy was started, because the size of the tumor and the surrounding tissue could change with treatment . Therefore, when migration of the marker that could influence treatment accuracy (e.g.> 3 mm) was suspected by fluoroscopy, ct scanning was scheduled for further evaluation to determine whether the displacement was significant or not . The characteristics of patients, tumors and fiducial marker placement are summarized in table 1 . The 18 tumors consisted of 5 hepatocellular carcinomas (28%) and 13 liver metastases (72%). The target sites for marker insertion were as follows: inside the tumor in 11 cases (61%) and near the tumor in 7 cases (39%). Two patients underwent radiofrequency ablation of another lesion after fiducial marker placement; therefore, their hospitalization period was 5 days . Sbrt was successfully performed in all 18 cases, and none of the markers was judged to have dropped out of its position . The median period between marker implantation and sbrt was 16 days (range: 031). Table 1.summary of patients, tumors and fiducial marker placementpatients (n = 18) male / female12/6median age (range)68 (4483)tumors (n = 18) size (mm) median of maximum length (range)35.5 (1188)type hepatocellular carcinoma5 liver metastasis (colon / gastric / others)13 (6/2/5)site s1/4/6/7/8/1&8/3&4/4&8/7&81/3/1/1/7/2/1/1/1fiducial marker placement (n = 18) ct fluoroscopic / us guidance8/10 inside / near the tumor11/7s1 = caudate lobe, s3 = ventrolateral segment of the left lobe, s4 = medial segment of the left lobe, s6 = posteroinferior segment of the right lobe, s7 = posterosuperior segment of the right lobe, s8 = anterosuperior segment of the right lobe . Summary of patients, tumors and fiducial marker placement s1 = caudate lobe, s3 = ventrolateral segment of the left lobe, s4 = medial segment of the left lobe, s6 = posteroinferior segment of the right lobe, s7 = posterosuperior segment of the right lobe, s8 = anterosuperior segment of the right lobe . No major complications, such as bleeding or marker migration, occurred (0%, 0/18). One patient developed mild pneumothorax; however, the sbrt was performed as planned because the pneumothorax disappeared after a few days' observation . In sbrt for liver lesions, techniques for controlling tumor motion are required because the bowel and stomach, which have a perforation risk, lie close to the liver . There are two main methods for reducing the uncertainty regarding the positions of liver tumors . One is to minimize tumor motion via the inhalation of oxygen, abdominal compression, learning of regular respiratory patterns, or breath - holding techniques [79]. The other technique, which is more sophisticated, is target gating or target chasing, during which the movements of the skin surface or other markers are monitored [10, 11]. The placement of the gold flexible fiducial markers near or inside a tumor is considered to be the most direct version of this method . In the present study, fiducial markers were inserted before tumor - tracking sbrt (using a cyberknife g4) during abdominal compression to improve the accuracy of localization . Percutaneous fiducial marker placement has been widely performed, but some complications such as marker migration (which might cause delayed or inappropriate treatment) have been reported [4, 5]. For example, a previous study described cardiac embolization due to the migration of a nester embolization coil that was used as a fiducial marker . Unlike traditional cylindrical gold seed fiducial markers, for which the migration rate was reported to be 5%, the marker used in this study is flexible and has a coiled design, which might reduce the incidence of fiducial migration . In our study, no marker migration was observed; thus, this gold flexible linear fiducial marker seems to be superior . However, pneumothorax occurred in one case, and a previous report found that percutaneous insertion of the gold flexible linear fiducial marker into the lungs is associated with a high risk of pneumothorax . In our experience, the needle included with the gold flexible linear fiducial marker is not sharp enough, and it is more difficult to penetrate the pleura and tumors with this needle than with a biopsy needle . Ohta et al . Previously reported that the transarterial placement of a fiducial marker resulted in a low complications rate (2%) and a high technical success rate (100%). However, complications such as femoral pseudoaneurysms have also been reported to occur after angiography . In addition, some of the transarterial marker procedures failed, and the outcomes of such procedures were considered to depend on the tumor site and the anatomy of the hepatic artery . Celiac artery stenosis makes it difficult to identify the hepatic artery [16, 17]. Therefore, the tumor site and the anatomy of the hepatic artery should be confirmed with ct and angiography . On the other hand, reported that the complications rate for percutaneous fiducial marker placement in the abdomen or pelvis was 4.3% (8/188); there were five minor complications (small hematomas in four cases, pneumothorax in one case) and three major complications (bleeding in two cases, sepsis in one case). The main benefit of percutaneous marker placement is that it is easy to place the marker in the target position and does not take much time . Therefore, there is a clear need for additional research to address this issue . At this time, the fiducial marker placement method should be decided based upon a consensus being reached between radiation oncologists and interventional radiologists after consideration of the tumor location and the anatomy of the hepatic artery, which should be confirmed with ct and angiography . However, the deep part of the liver cannot be clearly observed using ultrasound . Furthermore, the skill of the operator and the efficiency of the ultrasound machine vary among hospitals . Therefore, the implantation method should be discussed on an individual basis . Three or more fiducial markers were used in the cases described in the literature . In our study, however, only one marker was used based on the consensus of an interventional radiologist and a radiation oncologist . Complications such as pneumothorax might occur more frequently when multiple fiducial markers are inserted percutaneously . One marker is considered to be sufficient because the gold flexible linear fiducial marker is less likely to migrate . In this study, no signs of migration were observed, and all patients underwent radiotherapy without any problems . In future, the complications rate and the frequency of recurrence after radiotherapy should be analyzed with respect to the number of markers employed . In conclusion, the new gold flexible linear fiducial marker is useful for percutaneous insertion because of its good stability . In this study, we were unable to determine the local control rate of patients undergoing sbrt with this fiducial marker due to the short follow - up periods involved . It is therefore necessary to observe our patients for a longer period to evaluate the clinical usefulness of this marker . Funding to pay the open access publication charges for this article was provided by each of the co - authors in this search.
Quite often, we come across diseases in various medical specialties that are catalogued in biomedicine as idiopathic, that is to say, diseases with no apparent or known cause . Normally, these idiopathic diseases are treated with symptomatic remedies, whose objective is to improve the symptoms that have negative impacts on the normal lifestyle of patients, but without dealing with or solving the causes that provoke them . Biomedicine is always searching for the causes for diseases inside the organism, without finding them, when the causes and pathogenesis tend to be outside of the organism, in the environment . Often, treatment consists of social modifications that attempt to resolve these pathological processes . Environmental factors influence all diseases, but in idiopathic processes are crucial . Given the limitations of biomedicine to give clear explanations, and consequently a cure or remedy for certain diseases, a new paradigm is needed that can explain the causes of these pathologies that are considered idiopathic . To this end, it is essential that we integrate different elements through the formation of collaborative groups or health teams, defined by the world health organisation in 1973 as a nonhierarchical group of people with different professional backgrounds but a common objective, which is to provide the most comprehensive care possible to patients and their families, in any situation . Currently, collaborative work teams can be found in several different medical specialties such as oncology, geriatrics, and forensic medicine, whose health teams are primarily composed of health care professionals . One striking exception is radiology and traumatology, in which other specialties are starting to be incorporated such as biomedicine, physics, and engineering . Within otorhinolaryngology, a medical / surgical specialty that is concerned with the prevention, diagnosis, treatment, and rehabilitation of diseases of the ear and upper respiratory / digestive tracts (mouth, nose, pharynx, and larynx), and the functions derived from these structures (hearing, respiration, olfaction, taste, swallowing, and phonation: voice and speech) as well as the cervical and facial structures connected or related to these pathologies and sociology, a science dedicated to the empirical and theoretical analysis of social processes and structures . More specifically, it is the close collaboration between otology, which involves the biological study of diseases and abnormalities of the ear, and health sociology that directly collaborates with doctors and other health professionals, in addition to the syncretic integration of other disciplines such as anthropology and social / clinical psychology . In this manner, the joint labour of otology and sociology gives way to otosociology, a discipline dedicated to the study, intervention, and prevention of organic and functional pathologies of the auditory system with special emphasis on the influence of social factors . In the following sections, we describe how otosociology is capable of explaining both the social consequences and causes of certain diseases identified by otology as idiopathic . In the following section (diseases, ischaemia, and alterations), we describe the process of passing from identification of audiovestibular diseases recognised by otology to discuss these abnormalities as symptoms from the viewpoint of otosociology . In the third section (otosociology), we explain both the training and work focus of otosociologists and the methodologies employed by this new perspective, justifying its use in daily otorhinolaryngological practice . Until now, audiovestibular pathologies have been treated by otological medicine, which identifies them exclusively as biological diseases, attempting to situate the aetiopathogenesis in the audiovestibular organ itself, and as a result, the causes and consequences remain hidden, making any treatment strategy strictly palliative in nature . In contrast, otosociology views and treats these pathologies as symptoms of a social problem that affects the biological part of the subject . Otosociology, by identifying social problems that cause these symptoms and alterations in the patient's environment, can apply effective medical treatment and directly address the social consequences (table 1). The most important otological pathologies are sudden deafness, mnire disease, benign paroxysmal positional vertigo, tinnitus, and hyperacusis, commonly grouped within the category of audiovestibular diseases . These diseases cause patients to seek otological care and are immediately ascribed to the ear and are produced by the ear and are treated as exclusively otic pathologies . Many examples of the aetiology, incidence, diagnosis, treatment, and prognosis of these audiovestibular diseases are available in the medical literature and are discussed here . Its otological definition is sensorineural or perceptive hearing loss, usually in one single ear, of sudden onset, with a loss of over 30 db, at least three consecutive frequencies, with no previous otological background . Otology attempts to discern the causes of sudden deafness in the ear, and several aetiologies have been proposed such as rupture of the cochlear membrane, microangiopathic processes in the ear, viral ear infection, autoimmune diseases of the inner ear, mnire disease, vestibular schwannoma, or meningioma, although none of these theories sufficiently clears up the issue, nor can be applied in all cases . The incidence of sudden deafness has increased over time and is estimated to reach 160 cases per year per 100 000 inhabitants . In japan, where sudden deafness is registered, probable causes of the increase in sudden deafness include increased general awareness of this disease in the japanese population and the presence of diseases correlated with lifestyle, such as hypertension, diabetes, hyperlipidemia, and heart disease, associated with vascular pathologies, with the conclusion that vascular pathologies derived from hypertension and diabetes can lead to alterations in cochlear microcirculation, which leads to sudden deafness from cellular stress . This diagnosis is reached through clinical symptoms, audiometry, and a magnetic resonance of the internal auditory canal through which the auditory nerve passes . Medical treatment, which is an idiopathic process, is based on corticosteroids, vasodilators, and antioxidants . The patients with the worst potential prognosis for recovery are those with old age, severe initial hearing loss, vestibular symptoms, late treatment and time to recovery (the longer it takes to recover, the greater the chance that the patient never will), and the presence of tinnitus (table 2). In otology, mnire disease is defined as an internal ear disorder that affects both balance and hearing, characterised by an abnormal sensation of movement or rotatory vertigo, loss of hearing in one or both ears, tinnitus, sensation of aural fullness, and hyperacusis and occurs in recurring crises . Mnire in 1861 described in his mmoire sur des lssions de l'oreille interne donnat lieu des symptmes de congestion crbrale apopectiforme the findings from an autopsy of a woman, in which he observed damaged semicircular canals full of a red, plastic material, resembling a sort of bloody exudation that was only marginally present in the vestibule and nonexistent in the cochlea . Seven years after the death of mnire, his student politzer (1867 cited by rizzi in 2000) published these symptoms as mnire disease in the archives fr ohrenheilkunde . Twelve years after the death of mnire, charcot (1874 cited by baesly and jones, 1996) popularised the name of mnire disease for the symptoms of vertigo, deafness, and tinnitus . Mnire disease affects the inner ear with an unknown aetiology, characterised by a dilation of the membranous labyrinth due to increased endolymph (endolymphatic hydrops) of an unknown cause . The incidence of this disease ranges between 17/100 000 in japan and 205/100 000 in italy . Mnire disease is clinically diagnosed when the patient develops recurrent crises of rotatory vertigo, low - frequency fluctuating sensorineural hearing loss, hyperacusis, and a sensation of blockage in the ear or aural fullness . The committee on hearing and equilibrium of the american academy of otolaryngology - head and neck surgery (1995) put together a guideline based on clinical histories with four stages: (1) possible mnire disease (episodes of vertigo with no hearing loss, fluctuating or fixed sensorineural hearing loss, with disequilibrium but no definitive episodes, excluding other possible causes). (2) probable mnire disease (one episode of vertigo; audiometrically documented hearing loss on at least one occasion; tinnitus or otic pressure), (3) definite mnire disease (two or more episodes of vertigo lasting at least 20 minutes; audiometrically documented hearing loss on at least one occasion; tinnitus or aural fullness of the affected ear), and (4) certain mnire disease (established disease with histological confirmation). Since biopsy is not possible without destroying the inner ear, confirmation is only possible through autopsy; that is to say, no living patient has been diagnosed with certain mnire disease . In mnire disease, the worst symptom for the patient is vertigo, requiring medical treatment with corticosteroids, benzodiazepines, dimenhydrinate, thiethylperazine, or sulpiride . If these medical treatments fail, drugs such as corticosteroids and gentamycin can be administered directly into the inner ear . Another therapeutic option is pressotherapy which places pressure on the middle ear that in turn affects the inner ear and can improve the vertigo by affecting the pressure exerted on the liquids in the inner ear . The final alternative for the treatment of vertigo can involve a neurectomy of the vestibular nerve, a labyrinthectomy, or drainage of the endolymphatic sack . Benign paroxysmal positional vertigo is defined as a situation in which brief episodes of vertigo are produced by movements of the head . The incidence of this condition is estimated at 4681 cases per 100 000 inhabitants and increases by 38% for every decade of life . The idiopathic variety is twice as common in women as in men and occurs between the ages of 50 and 70 years [15, 16]. When the aetiology is trauma or vestibular neuritis it may go unnoticed in daily life and only is recognised when undergoing diagnostic tests . Schuknecht (1962) and schuknecht (1969) proposed the theory of cupulolithiasis to explain how this vertigo is produced within the inner ear . According to this theory, this vertigo is caused by microscopic stones composed of calcium carbonate and proteins, otoliths, which move within the utricle of the otic vestibular system, that is to say, the interior of the equilibrium centre . For their part, hall and colleagues (1979) proposed the theory of canalithiasis, stating that these minute particles circulate improperly through the canals of the inner ear labyrinth, altering balance and producing vertigo . Dix and hallpike (1952) [2224], who had thoroughly researched vertigo of the ear, developed a diagnostic test for this process, the dix - hallpike test . Thus, once the otic mechanisms of this vertigo and how to diagnose it were established, semont and colleagues in 1988 established the treatment of a repositioning manoeuvre to place the calcium deposits in their original place in order to halt the vertigo, known as the semont manoeuvre . In a similar manner, in 1992, epley described another repositioning manoeuvre for the posterior canal, known as the epley manoeuvre . Recently, hilton and pinder (2002, 2004) [15, 16] performed a review in the prestigious cochrane organisation in which they demonstrate that the epley manoeuvre is effective at repositioning the calcium deposits in the inner ear . The prognosis, from an otological point of view and as its definition indicates, is benign, recurrent but benign . As has been shown, the diagnosis of benign paroxysmal positional vertigo has a perfectly defined set of signs and symptoms, which are always produced, diagnosed, and treated within the inner ear (table 4). Ischaemia is a deficit of blood flow, whether transient or definitive, in an organ or part of it . The concept of ischaemia allows us to make a significant conceptual advancement since the medical viewpoint of the condition starts to look outside of the ear when focusing on its vascularisation, although other factors may be interacting . In recent years, several scientific advancements have been made in this field . With regard to sudden deafness, the ischaemic processes of the inner ear have arisen as a mechanism of pathogenesis . In mnire disease, proposed a model based on haemodynamic disequilibrium that produces transient ischaemia and could have an effect on ph and the proton pump of the inner ear . In this manner, under conditions of ischaemia and metabolic acidity, the activity of the proton pump would create an overload of anions in the endolymph with the result of increased osmolality . This, in turn, leads to the formation of hydrops, an increase in pressure in the endolymph fluid, which causes the fluctuating deafness, vertigo, and tinnitus that are characteristics of mnire disease . It has also been established that endolymph hydrops can be produced without causing vertigo [2933]. Similarly, vertebrobasilar ischaemia is starting to be considered as the pathogenesis of benign paroxysmal positional vertigo . To conclude, it is interesting to point out that currently, several authors consider ischaemia of the nervous system as the pathogenic mechanism of tinnitus [3541], a symptom produced in the majority of cases of sudden deafness and mnire disease . This simple change in perspective allows us to extend our focus outside of the auditory organ, a structure specific to the field of otology, in order to offer explanations of these otic pathological processes, favouring the examination of ischaemia as a pathogenic factor and passing from an otological viewpoint to that of otosociology . Figure 1 compiles the different aetiopathogenic mechanisms of ischaemia from the stress generated by interactions between the patient's social and cultural environment and the vulnerability or resiliency of a person . Blood analysis of the following substances: endothelin-1, prolactin, cortisol, adrenaline, noradrenaline, and cholesterol provides the means of an objective analysis of the patient's evolution . In light of the available literature on the subject that affirms that patients diagnosed with idiopathic sudden deafness have a higher probability of suffering a stroke in the next 5 years [42, 43], an otosociological viewpoint would indicate that otology lacks the necessary tools to consider these conditions from a more global perspective . With a mind - set focused on the ear, it is impossible to contemplate the triggering factors or causes that, of course, are not located within the ear, and so they continue to cause damage . Medicine attempts to improve a patient's health and environment in order to reach a better biological / psychological / social well - being, and so the expanded focus provided by otosociology comprehends the global reality of the patient much better, offering more chances of recovery . Otosociology understands that the causes of audiovestibular symptoms that patients describe are produced outside of the ear and even outside of the patient; that they originate in the patient's social environment . The social environment in which we carry out our lives implies situations that provoke states of anguish, anxiety, preoccupation, and irritability, as well as the sensation or perception of not being able to successfully confront these situations . This is what we commonly refer to as stress (figure 2), which can be measured directly through blood levels of certain substances (endothelin-1, prolactin, cortisol, adrenaline, noradrenaline, and cholesterol), which allows for the objective analysis of its evolution . As we have been discussing, the otologist treats sudden deafness, mnire disease, and benign paroxysmal positional vertigo exclusively as otic diseases, whereas the otosociologist considers them to be symptoms of a cerebral ischaemic pathogenesis produced by psychosocial stress . That is to say, three different symptoms will all lead to the same diagnosis: audiovestibular stroke, with transient ischaemic auditory different studies have started to highlight the importance of lifestyle and stress in the pathophysiological mechanisms of audiovestibular symptoms . Stress leads to vasoconstriction, haemoconcentration, and occlusion of the microcirculation that occurs in the inner ear . Stress and stressful lifestyles, such as those related to work, social life, and emotional conflicts, as well as personality types that tend towards greater stress, have been correlated with audiovestibular symptoms . More specifically, studies relate situations of psychosocial stress with sudden deafness [4749], mnire disease, benign paroxysmal positional vertigo [5154], tinnitus [5564], and hyperacusis [6567]. This theory is supported by the findings that social stress can extend itself farther than simply one's social situation and can even produce physiological damage . In other fields of medicine, many diseases such as gastric ulcer [68, 69], diabetes, hypertension, and acute myocardial infarction have a social component in their origins, whether from previous conditions or sustained stress through time (chronic stress). After providing a detailed description of how otology defines, diagnoses, and treats that which it considers to be audiovestibular diseases, we have introduced the variable of ischaemia as an aetiopathogenic explanation, from the point of view of otosociology, of the true causes of these alterations . Alterations, not diseases, that have their origins outside of the ear and are external to the individual because they belong to the social environment . In this manner, we are now ready to expound upon and develop the specific field of otosociology in the following section . We have already established that otosociology is a discipline dedicated to the study, intervention, and prevention of organic and functional pathologies of the auditory system, with special emphasis on the influence of social factors . As we can appreciate, otosociology stems from a holistic viewpoint that not only comprehends the dysfunction of an organ or body part, but rather the person in his / her entirety, including social and cultural environments . As such, it is evident that otosociology does not treat patients or subjects, but people . In this sense, otosociology involves a new theoretical framework with different names, concepts, and research processes from those used in the otological approach . The history of human knowledge is full of situations in which two different people or disciplines come to similar conclusions, in the same period of time, given a certain problem . Serendipity, destiny, and chance have been used to describe this phenomenon which, in this case, brought together two different disciplines that researched a common subject; audiovestibular alterations, but with completely different objectives, methodologies, and points of view . They could even appear to be completely devoid of any connection to each other . In the case of medicine, the issue to be researched is based on finding an explanation for the causes of these alterations with the goal of elucidating an effective treatment; since viewed solely through the lens of biomedical methodology, the diagnosis is idiopathic and the treatment of symptoms alone is not sufficient to resolve this pathology . In the case of sociology, the common point consists of an unexpected finding in the study of the processes of exclusion of people with sudden deafness . In both cases, researchers were faced with a one - way alley: the causes of audiovestibular alterations were due to factors that were external to the biological states of individuals . They were due, in reality, to the stress produced by the individual's social and cultural environment . Collaboration for field work revealed the same conclusions that had been gained by observing the same individuals with different objectives . Thus commenced a scientific dialogue, with the end result of otosociology the tight collaboration between two disciplines that differ significantly in their language, methods, and perspectives requires searching for points of common ground throughout the research process . Constant questions and clarifications arise regarding concepts, methodologies, and points of focus that are integral to one discipline but foreign to the other . This tension produced upon the collaboration of these two disciplines has the result of generating new ideas, but also highlights the fact that, in the majority of cases, the two groups are discussing the same issue but with different perspectives and languages . In any case integration of other ways of thinking and analysing problems based on common ground also requires significant effort and a learning curve from both sides, along with an expanded overall perspective and, above all, the possibility of doing the same things in different ways to reach results that, done in a different manner, would not have been achieved . Of course, during the process, frustration can arise as often as satisfaction, and many occurrences come to pass that are worth remembering . Starting with the very first interviews performed with patients diagnosed with sudden deafness for sociological research, the collaboration commenced with a case by case discussion, providing different points of view and integrating new perspectives and viewpoints with each study interview, testing hypotheses . Case by case, and during a year and a half, weekly sessions lasting a mean of 5 hours gave way to the moulding and shaping of what was to become otosociology, which will be described in the following sections . Otosociologists must have a thorough understanding of the ear, but more importantly, must properly situate the ear within its surroundings . In this manner, an otosociologist is an otologist with training in sociology that takes into account the influence of social factors in dealing with patients . This involves performing not just an otological examination, but also a biosocial exploration of the patient and his / her condition . Training in otosociology, which is given by otologists, sociologists, and psychologists, is directed towards doctors specialised in otorhinolaryngology through a focus on otosociology at the undergraduate or postgraduate level, using b - learning methodologies from sociology faculties, including study materials that delve into sociological theory, methodologies, and research techniques in addition to central themes related to audiovestibular processes . As has been established, medicine involves its own scientific research methods and otologists utilise a series of protocols that they apply rigorously to their daily practice (figure 3). Sociology boasts a qualitative method for investigation involving in - depth interviews, participant observation, and group discussions . It is evident that the protocol currently used by biomedicine does not actually cure patients of what in otosociology we call audiovestibular alterations . It is also evident that the time allotted for a medical consultation is insufficient for applying the traditional qualitative methods employed in sociology . As such, otosociology utilises a hybrid method that combines the clinical and social condition of the patient, which the otosociologist can apply during the time allotted for a consultation . To this end, a pilot study lasting two months tested this hybrid methodology, which has allowed us to elaborate a specific, simplified protocol to be used in daily clinical practice by otosociologists . For the most complicated cases in which the protocol may be insufficient for detecting the causes that have led to the audiovestibular alterations, the otosociologist can seek the express collaboration of a sociologist that is also an expert in otology for an in - depth interview and later analysis . In this pilot study, it was shown that an otologist with proper training in otosociology is capable, in an initial consultation lasting 20 minutes, of performing the normal clinical examination and applying otosociological protocols for arriving at the proper diagnosis and treatment . In cases that were harder to solve or in which the otosociologist had doubts regarding the causes of the alterations, a collaborating sociologist performed an in - depth interview and later compared conclusions with the otosociologist . In all cases, the response from the patient was positive with regard to the medical attention received, the explanations offered, the diagnosis made, and the treatment proposed . The quality of the medical care given undergoes a substantial and objective improvement, and the perception of the patient is very positive . If we assess medical costs, these are considerably reduced by removing the need to prescribe unnecessary medication and repeated consultations . In terms of patient quality of life, we find that this improves immediately upon the consultation . In the patients' self - evaluation of quality of life, one primary factor is that the person stops feeling like a patient, a sick person, and starts feeling like an individual capable of controlling the alterations that provoked the consultation . Additionally, the subject feels released from the years of dependency on medications and medical care, and finally, both he / she and his / her close friends and relatives understand what has occurred and its causes, facilitating over time a recovery from the alterations, whether partial or complete . Above all, intervening on the social and cultural causes of the issue removes the possibility that these same causes could repeat themselves in this or any other organ of the body . We have reached a new mutual intellectual understanding through fluid communication and reciprocity . To this end, a positive disposition has been necessary for including other points of view and recognising complementary abilities, that is to say, being open to learning and adapting, maintaining the principle of respect for the capacities and contributions from both sides to concrete points as a whole . Secondly we can also affirm that the collaboration between otologists and sociologists in health centres is possible in several different combinations . In an otorhinolaryngology department with one sociologist for every given number of otorhinolaryngologists, the otosociologist can resolve these cases by collaborating with the sociologist in those situations in which the social and cultural causes of the issue and proper interventions to treat it are not clear . Thirdly, the integration of a sociologist and the training of otologists in otosociology will benefit all parties involved . The health system will benefit because its principles are centred on the needs of the patient, and the objectives therein are orientated towards promoting health and preventing disease, whether in conditions of treatment, recovery, or palliative care . We cannot forget either the economic efficiency in medication, diagnostic tests, and decreased repetition in doctor visits . The objective of the doctor is to cure, but this is often impossible in idiopathic diseases, which constitutes a source of frustration for medical practice . The otosociologist has the opportunity to elucidate the sociocultural causes that produces audiovestibular alterations and can carry out successful interventions to treat it, increasing doctor satisfaction and prestige . For the patient within the health system, otosociology provides high - quality medical care, demedicalisation of the issue, and a consequent recovery of the patient's identity as an individual, with substantial improvement in his / her condition . This all leads to improved perception of the multidimensional concept referred to as quality of life . By improving a person's concept of health, to conclude, otosociology has arisen due to the inability of otology to provide effective solutions to the aforementioned audiovestibular symptoms . With this in mind, otosociology provides an aetiopathogenic explanation based on the cultural and social environment of the patient, delivering a correct diagnosis and definitive treatment.
Crohn's disease (cd) is a chronic inflammatory disorder of the gastro - intestinal tract [1, 2, 3]; its exact aetiology and pathogenesis is not known . Current models of pathogenesis include interactions between genetic factors, environmental factors, and the intestinal flora which lead to dysregulation of the immune response and to inflammation of the wall of the gastro - intestinal tube [4, 5]. Exacerbations are due to flares of inflammation of the wall of the gastro - intestinal tube . Inflammation in cd is transmural and segmental [1, 4, 6]; thus it spares certain regions and leaves healthy mucosa between the affected, ulcerated sites . Transmural inflammation leads to the development of sinus tracts in the organ's wall, which can lead to phlegmon, fistulas to neighbouring structures, and abscesses . Cd can affect any part of the gastro - intestinal tract from the mouth to the anus, but it most often affects the terminal ileum and the colon [3, 4]. Over 70% of patients have small bowel involvement, usually of the terminal ileum . About 40% of patients have ileo - colic disease, and 30% present with small intestinal disease . Cd of the upper gastro - intestinal tract (oesophagus, stomach, and duodenum) is much less frequent and most often associated with involvement of more distal parts of the gastro - intestinal tract [1, 7, 8]. Further, upper gastro - intestinal disease may be associated with progression and recurrence of intestinal disease [6, 9]. The literature shows variable data regarding the prevalence of upper gastro - intestinal involvement ranging from 0.2 to 16% [1, 3, 4, 5, 6, 7, 8, 9]. However, isolated oesophageal cd is a very rare condition [1, 2, 3, 7, 10, 11]. The clinical presentation and endoscopic and histologic findings of oesophageal cd are mostly non - specific and share features of more common diseases of the oesophagus . Therefore, accurate diagnosis of oesophageal cd can be very challenging [9, 10, 11] and is often made late in its course [2, 10]. In the following we report the case of a relapsing para - oesophageal abscess posing a great diagnostic challenge . A 42-year - old male patient with a para - oesophageal abscess and a fistula into the distal oesophagus was referred to our gastroenterology department in september 2012 for further evaluation and treatment . The past medical history consisted of chronic back pain in the context of a lumbar disc herniation, for which he underwent spinal fusion surgery . [non - steroidal anti - inflammatory drug (nsaid)] and esomeprazole 40 mg q.d . Our patient complained of progressive epigastric pain that did not improve after the nsaid had been withdrawn and esomeprazole was increased to 40 mg b.i.d . On admission he was febrile (38.3c), palpation of the epigastric region was tender, and laboratory studies showed inflammatory changes with a leucocyte count of 15.6 10/l (normal value 410 10/l), a left shift of neutrophils of 28% (normal value <16), and an elevated c - reactive protein (crp) level of 282 mg / l (normal value <5). Thoraco - abdominal computed tomography (ct) revealed a para - oesophageal abscess with a maximal extension of 6 cm adjacent to the oesophago - gastric junction and a fistula into the distal part of the oesophagus (fig . 1). Upper endoscopy showed the fistula's porus at 39 cm from the tooth row; otherwise the mucosa of the oesophagus, stomach, and duodenum was normal . We performed an endoscopic ultrasound (eus) that revealed an asymmetrical thickening of the oesophageal wall adjacent to the abscess, which caused a narrowing of the lumen . Histologic specimens taken from the oesophagus revealed chronic inflammation with a preponderance of granulocyte infiltration . We continued treatment with the ppi and inserted a naso - duodenal tube for enteral nutrition . Further, we started an intravenous antibiotic therapy with amoxicillin 2,000 mg and clavulanic acid 200 mg t.i.d . Endoscopy combined with eus confirmed the complete resolution of the fistula and the abscess, whereas the thickening of the oesophageal wall and the enlarged lymph nodes persisted . Because of a possible malignancy, a follow - up endoscopy was performed 2 and 5 months after discharge . Only a little pseudo - diverticula remained at the site of the former fistula, the thickening of the oesophageal wall had resolved completely, no other mass could be detected, and the mucosa appeared normal . However, more distally, a partial stenosis remained, impeding the passage of the eus endoscope . As the cause of the abscess remained unclear, we supposed the previous treatment with the nsaid as a possible trigger . Oesophageal contrast examination revealed stenosis at the oesophago - gastric junction with a lack of relaxation of the lower oesophageal sphincter documented by high - resolution manometry . Two balloon dilations (30-mm savary balloon) achieved good control of the complaints in the following months . After a symptom - free period of 18 months, the patient was referred to our emergency department due to a rapidly progressive odynophagia impeding oral intake of food or fluids . On admission the patient was afebrile (36.8c) and in a poor general state; blood pressure (117/75 mm hg) and heart rate (80 bpm) were within the normal range . Bowel sounds were clearly audible, and palpation of the epigastric region provoked tenderness, but there were no signs of peritonism . Otherwise, the clinical examination showed normal findings . Laboratory studies revealed inflammatory changes with a leucocyte count of 13.2 10/l and an elevated crp level of 139 mg / l . Ct scanning of the thorax and the upper abdomen detected a para - oesophageal air - containing fluid collection of 3.5 cm with an enhancing wall at the same location as the previous abscess had been . The histologic specimen of the oesophagus revealed acute - on - chronic inflammation with ulceration and granulation . Again, there was no evidence of an eosinophilic oesophagitis, a tumorous growth, or a fungal infection . In the stomach, minimal non - active unspecific inflammation was detected, and the histology of the duodenum had a normal aspect . Culture showed a mixed growth of aerobe and anaerobe bacteria as well as some yeast . A serologic examination for human immunodeficiency virus (hiv) was negative (hiv-1/2 antigen - antibody chemiluminescent microparticle immunoassay). Studies for cytomegalovirus (cmv) revealed undetectable cmv igm, and cmv igg was 138 ae / ml (normal value <6). Also for varicella zoster virus (vzv), igm was negative and vzv igg was 740 serologic studies for herpes simplex virus (hsv) types 1 and 2 showed a raised hsv1/2 igg titre of 41,000 (normal value <230) and an indeterminate testing for hsv1/2 igm . Thus the results regarding hsv types 1 and 2, vzv, and cmv were consistent with a past infection . Anti - saccharomyces cerevisiae antibody igg and iga were positive (igg 10 u / ml, iga 11 u / ml; normal value <7). Titre of anti - neutrophil cytoplasmic antibody was normal (normal value <1: 20), whereas titre of anti - nuclear antibody was slightly raised (> 1: 80; normal value <1: 80). Helicobacter pylori serology was within normal range (igg <10 e / ml; normal value <10). The histologic specimens of the terminal ileum, colon, and rectum did not show evidence of a chronic inflammatory disease . Mri of the small intestine also showed normal findings . Based on the current endoscopic, histologic, and also serologic findings and preclusion of other diseases, we made the diagnosis of cd with isolated involvement of the oesophagus complicated by a recurrent para - oesophageal abscess . We started intravenous antibiotic treatment with amoxicillin 2,000 mg and clavulanic acid 200 mg t.i.d . For 20 days and then changed to an oral regimen of amoxicillin 500 mg and clavulanic acid 125 mg t.i.d . The symptoms resolved rapidly, and the leucocyte count and crp level returned to normal . Upper endoscopy with eus performed after 2 weeks of treatment showed only a small residual of the abscess . After 3 days . Currently, the patient is treated with azathioprine alone and is still in remission . There are only limited data available regarding the prevalence of oesophageal cd, and the prevalence of upper gastro - intestinal involvement is estimated to be 0.216% in patients with co - existing ileo - colic cd [1, 3, 4, 5, 6, 7, 8, 9]. Not all patients with cd undergo upper endoscopy; therefore its prevalence might be underestimated [8, 9, 11]. On the other hand, different study populations are analysed, and also different diagnostic criteria are used to define cd involvement of the upper gastro - intestinal tract and oesophagus . Furthermore, the prevalence of upper gastro - intestinal involvement has increased since the 1990s compared to older reports [1, 5, 12]. This is probably due to wider use of endoscopy for clinical staging and research as well as better diagnostic tools [7, 12]. The clinical presentation of oesophageal cd varies, ranging from asymptomatic [1, 7, 12] to serious illness [2, 3, 7, 8, 9, 10, 13]. Complications include strictures and stenosis [1, 7, 10, 11, 14], abscesses, and fistulas to neighbouring organs [2, 11] such as the respiratory tract [1, 13]. As reported by our patient, symptoms are often unspecific in oesophageal cd, resembling those of other oesophageal diseases . Most often, patients complain of dysphagia, odynophagia, retrosternal pain, or discomfort [1, 2, 3, 4, 7, 8, 10, 11, 13]. Since most cases of oesophageal cd occur in patients with known cd or concurrent intestinal cd, diagnostic procedures may lead towards the correct diagnosis . The diagnostic challenge in our case was the fact that there were no findings to support the diagnosis of cd at the time of first presentation . Typical symptoms of intestinal cd (e.g., crampy lower abdominal pain or change of bowel habits such as diarrhoea or bloody stool). At first presentation, upper endoscopy revealed only the fistula's porus; apart from that there was no evidence of a diffuse inflammatory process . Almost 3 years later, disseminated oesophageal ulcerations became evident for the first time and raised a high suspicion of cd . The endoscopic and macroscopic findings of oesophageal cd described in the literature range from mild mucosal hyperaemia and superficial aphthous ulcers [3, 8] in early disease stages to deep ulcerations and erosions, fistulas, and cobblestone appearance in more advanced disease stages [1, 3, 5, 7, 8, 9, 10, 11, 14]. Differential diagnosis of oesophageal cd is broad and includes other causes of oesophageal disease like gastro - oesophageal reflux disease and eosinophilic oesophagitis, drug - induced oesophagitis, viral and fungal infections, tuberculosis, epidermolysis bullosa acquisita, connective tissue disease, and vasculitis as well as malignancy [1, 4, 8, 11]. In our case non - caseating granulomas are considered to be specific to cd; however, they are found in less than 40% of biopsies taken from the oesophagus [3, 4, 5, 7, 8, 11]. The histologic findings did therefore not allow endorsing or excluding the diagnosis of cd; however, chronic and acute - on - chronic inflammation is, although not specific, associated with oesophageal cd [1, 2, 3, 7, 8, 9, 10]. On the other hand, histology allowed us to exclude other possible causes . Malignancy and but since the thickening of the oesophageal wall had resolved, no other mass became evident, and repeated histologic specimens never provided evidence of malignant cells or eosinophilic infiltration, malignancy as well as eosinophilic oesophagitis seemed very unlikely to be the underlying cause . Our immunologic examinations for hsv, cmv, vzv, and hiv did not show active infection, and histologically there was no evidence of fungal infection or viral cytopathic effects . Signs of a multi - system disease such as connective tissue disease or vasculitis were not evident . Ileo - colonoscopy and mri - enterography did not reveal chronic intestinal inflammation; only some cicatrized sites in the terminal ileum were found . Whether they represented the residual of a past, somehow asymptomatic or subclinical inflammation remains unclear . In view of the clinical history of relapsing abscess formation and fistulization, the macroscopic aspect of diffuse ulcerative lesions, histologically chronic and acute inflammation, the positive anti - saccharomyces cerevisiae antibody titre as well as the lack of other aetiologies, we established the diagnosis of isolated oesophageal cd . Regarding the treatment of oesophageal cd there are no randomized controlled trials; evidence is based on case series [1, 4, 7, 12]. The european crohn's and colitis organization (ecco) proposes ppi for the treatment of oesophageal and gastroduodenal cd . Depending on the severity of the disease, ppi can be combined with systemic corticoids, thiopurines, or methotrexate . Most authors propose dilation with or without steroid injections for strictures [1, 8, 9, 11, 12], and surgery remains an option for refractory strictures as well as fistulas and abscesses which cannot be managed otherwise . Some concerns exist that ppi as sole treatment might not be sufficient, since the inflammatory process is not inhibited [8, 15]. Our patient had been under ppi treatment even before he developed the abscess and the stricture . Diagnosis of isolated oesophageal cd can be challenging [3, 8, 9, 11], since clinical findings often are non - specific . The diagnosis has to be made by integration of symptoms as well as endoscopic, histologic, and serologic findings [3, 8, 9, 11]. Other, more frequent diseases (e.g., malignancy, eosinophilic oesophagitis, and viral or fungal infections) must be ruled out.
Complete debridement and effective disinfection of the root canal space is an important prerequisite for achieving long - term success of nonsurgical endodontics . Chemomechanical instrumentation reduces majority of infecting bacteria, together with their principal substrate of necrotic pulp debris but retention of microorganisms within the dentinal tubules is thought to be a source of persistent endodontic infection . The use of an intracanal medicament helps in the elimination of bacteria that remain even after cleaning and shaping, thereby making the environment conducive for periapical tissue repair . It is probable that the physiologic state of the cells, particularly in retreatment cases, is closest to the starvation phase . Recent studies have also shown that e. faecalis is highly resistant to commonly used intracanal medicaments, such as calcium hydroxide . Even 2% chlorhexidine gluconate has been used as an irrigant and intracanal medicament in endodontics . Chlorhexidine has a broad spectrum antimicrobial activity targeting both gram - positive and gram - negative microbes . Hence, the combination of chlorhexidine and calcium hydroxide as an intracanal medicament has also been tried to achieve the properties of both medicaments but the antimicrobial action of chlorhexidine was found to be reduced . Morinda citrifolia, commercially known as noni, is famous as an important folk medicine and as a health drink . The juice of m. citrifolia has a broad range of therapeutic effects, including antibacterial, antifungal, antiviral, antitumor, antihelminthic, analgesic, hypotensive, anti - inflammatory, and immune - enhancing effects . The effectiveness of m. citrifolia with sodium hypochlorite and chlorhexidine gluconate to remove the smear layer from the canal walls of endodontically instrumented teeth was compared by murray et al . And it was concluded that 6% m. citrifolia can be used as an endodontic irrigant (as per article after usage of irrigant regime it was concluded). A gel based on papain, a proteolytic cysteine enzyme, exhibits significant antibacterial and anti - inflammatory properties . It acts only on affected tissues, which lack the 1-antitrypsine plasmatic antiprotease that inhibits proteolysis in healthy tissues . In addition to papain, the chloramines present have the potential of dissolving carious dentin by means of chlorination of the partially degraded collagen . Cosmetic and some medicinal products are made from the mucilaginous tissue in the center of the aloe vera leaf and is called as aloe vera gel . There is no study to comparatively evaluate the antimicrobial activity of natural extracts of m. citrifolia, papain, and aloe vera (all in gel formulation), 2% chlorhexidine gel and calcium hydroxide, against enterococcus faecalis . Hence, this study was undertaken to evaluate the disinfection of dentinal tubules when contaminated with e. faecalis using m. citrifolia gel, papain gel, and aloe vera when compared with calcium hydroxide and chlorhexidine gel . A rotary diamond disk was used to decoronate the teeth 5 mm below cementoenamel junction and the apical part of the root to obtain 6 mm of the middle third of the root . 3 (mani inc, tachigi - ken, japan) in a slow speed handpiece was used to standardize the internal diameter of the root canals . The blocks were treated in an ultrasonic bath of 17% ethylene diamine tetra acetic acid for 5 min followed by 3% naocl for 5 min to remove the organic and inorganic debris . The traces of chemicals used were removed by immersing the blocks in an ultrasonic bath containing distilled water for 5 min . The first cycle was at 121c and the second was with the blocks immersed in 1 ml of tryptone soya (ts) broth in individual microcentrifuge tubes . All the blocks were coated externally with paraffin wax . The test organism used for this study was e. faecalis, which is a gram - positive facultative anaerobic bacterium . E. faecalis (atcc 29212) was grown in ts agar for 24 h. the culture was suspended in 5 ml of ts broth and incubated for 4 h at 37c and its turbidity was adjusted to 0.5 mcfarland standard . Each dentin block was placed in presterilized microcentrifuge tubes containing 1 ml of the ts broth . Fifty microliters of the inoculum containing the e. faecalis was transferred into each of the microcentrifuge tubes . At the end of 24 h purity of the culture was checked by subculturing 5 l of the broth from the incubated dentin blocks in ts broth on ts agar plates . Contamination of the dentin blocks were carried out for a period of 21 days . At the end of 21 days the blocks were irrigated with 5 ml of sterile saline to remove the incubation broth . Group 1: saline (negative control) group 2: calcium hydroxide group 4: m. citrifolia gel group 5: aloe vera gel group 6: 2% chlorhexidine gel calcium hydroxide (sigma aldrich, mumbai, india) was mixed with sterile saline in a ratio of 1.5:1 (wt / vol) to obtain a paste - like consistency . Ltd, gujarat, india .) Was used as a thickening agent in the ratio of 2:1 (vol / wt) for group 3 (papain raw extract taken from fruit), group 4 (m. citrifolia raw extract taken from fruit), group 5 (aloe vera raw extract taken from leaf), and group 6 (chlorhexidine). Hydroxyethyl cellulose is a nonionic, highly inert, and water - soluble agent and has been used in various studies for gel formation . The medicaments were placed inside the canals and sealed at both the ends with paraffin wax . They were incubated in an anaerobic environment for 37c . At the end of 1, 3, and harvesting of the dentin was carried out at 2 depths (200 and 400 m) with gates glidden drills no . 4 and 5, respectively . The collected dentin shavings were transferred into 1 ml of sterile ts broth and incubated in an anaerobic environment at 37c for 24 h. after 24 h, the contents of each tube was serially diluted, 100 l of the broth in 100 l of sterile saline for 5 times . Fifty microliters of the dilution was then plated on ts agar plates and incubated for 24 h. colonies were counted and readings were tabulated as shown in table 1 . Mean colony counts for different intracanal medicaments at 200 and 400 m depths at different time intervals the data were statistically analyzed with one - way analysis of variance followed by tukey multiple comparison means to check the difference in bacterial inhibition between the groups (p <0.05). The paired t test was used to check for differences in growth at different time intervals within groups and for differences at the 2 depths (p <0.05). A rotary diamond disk was used to decoronate the teeth 5 mm below cementoenamel junction and the apical part of the root to obtain 6 mm of the middle third of the root . 3 (mani inc, tachigi - ken, japan) in a slow speed handpiece was used to standardize the internal diameter of the root canals . The blocks were treated in an ultrasonic bath of 17% ethylene diamine tetra acetic acid for 5 min followed by 3% naocl for 5 min to remove the organic and inorganic debris . The traces of chemicals used were removed by immersing the blocks in an ultrasonic bath containing distilled water for 5 min . The first cycle was at 121c and the second was with the blocks immersed in 1 ml of tryptone soya (ts) broth in individual microcentrifuge tubes . The test organism used for this study was e. faecalis, which is a gram - positive facultative anaerobic bacterium . E. faecalis (atcc 29212) was grown in ts agar for 24 h. the culture was suspended in 5 ml of ts broth and incubated for 4 h at 37c and its turbidity was adjusted to 0.5 mcfarland standard . Each dentin block was placed in presterilized microcentrifuge tubes containing 1 ml of the ts broth . Fifty microliters of the inoculum containing the e. faecalis was transferred into each of the microcentrifuge tubes . At the end of 24 h purity of the culture was checked by subculturing 5 l of the broth from the incubated dentin blocks in ts broth on ts agar plates . At the end of 21 days the blocks were irrigated with 5 ml of sterile saline to remove the incubation broth . Group 1: saline (negative control) group 2: calcium hydroxide group 4: m. citrifolia gel group 5: aloe vera gel group 6: 2% chlorhexidine gel calcium hydroxide (sigma aldrich, mumbai, india) was mixed with sterile saline in a ratio of 1.5:1 (wt / vol) to obtain a paste - like consistency . Ltd, gujarat, india .) Was used as a thickening agent in the ratio of 2:1 (vol / wt) for group 3 (papain raw extract taken from fruit), group 4 (m. citrifolia raw extract taken from fruit), group 5 (aloe vera raw extract taken from leaf), and group 6 (chlorhexidine). Hydroxyethyl cellulose is a nonionic, highly inert, and water - soluble agent and has been used in various studies for gel formation . The medicaments were placed inside the canals and sealed at both the ends with paraffin wax . They were incubated in an anaerobic environment for 37c . At the end of 1, 3, and harvesting of the dentin was carried out at 2 depths (200 and 400 m) with gates glidden drills no . 4 and 5, respectively . The collected dentin shavings were transferred into 1 ml of sterile ts broth and incubated in an anaerobic environment at 37c for 24 h. after 24 h, the contents of each tube was serially diluted, 100 l of the broth in 100 l of sterile saline for 5 times . Fifty microliters of the dilution was then plated on ts agar plates and incubated for 24 h. colonies were counted and readings were tabulated as shown in table 1 . Mean colony counts for different intracanal medicaments at 200 and 400 m depths at different time intervals the data were statistically analyzed with one - way analysis of variance followed by tukey multiple comparison means to check the difference in bacterial inhibition between the groups (p <0.05). The paired t test was used to check for differences in growth at different time intervals within groups and for differences at the 2 depths (p <0.05). Contamination of the dentin blocks was confirmed when debris samples harvested from the saline group (negative control) yielded positive growth . Table 1 shows the antibacterial activity, measured at 2 depths (200 and 400 m) and at 3 time intervals (1, 3, and 5 days). The inhibition of growth in all the groups was statistically significant in comparison to the control group (saline). Group 6 (2% chlorhexidine gel) was the most effective against e. faecalis to the depth of 400 m on all days of incubation . Intergroup comparison of inhibition between groups 4 and 5 (m. citrifolia gel and aloe vera gel) showed no statistical difference on day 1, but on days 3 and 5 there was a statistically significant difference . Inhibition in group 4 (m. citrifolia gel) was also statistically significant compared with groups 3 and 2 (papain gel and calcium hydroxide, respectively) on all days (1, 3, and 5). Inhibition in group 3 (papain gel) was statistically better than group 2 (calcium hydroxide) on day 3, whereas on days 1 and 5 they had similar efficacy . The inhibition of growth of e. faecalis at 200 and 400 m was uniform with no statistically significant difference for all the groups . The inhibition of growth of e. faecalis at the end of days 1, 3, and 5 was varied with different medicaments . Group 2 (calcium hydroxide) showed sufficient antibacterial activity on day 1, which decreased on day 3 and again increased by day 5.group 3 (papain gel) showed a decrease in antibacterial activity at the end of day 5.group 4 (m. citrifolia gel) showed constant antimicrobial efficacy on all the days (1, 3, and 5 days).group 5 (aloe vera gel) showed comparable antibacterial activity to m. citrifolia gel on first day, but there was a gradual decrease in the efficacy by the end of day 5 . Group 2 (calcium hydroxide) showed sufficient antibacterial activity on day 1, which decreased on day 3 and again increased by day 5 . Group 3 (papain gel) showed a decrease in antibacterial activity at the end of day 5 . Group 4 (m. citrifolia gel) showed constant antimicrobial efficacy on all the days (1, 3, and 5 days). Group 5 (aloe vera gel) showed comparable antibacterial activity to m. citrifolia gel on first day, but there was a gradual decrease in the efficacy by the end of day 5 . To summarize the results, the overall percentage inhibition at 2 depths and different time intervals was, 100% with chlorhexidine gel, 86.02% with m. citrifolia gel, 78.9% with aloe vera gel, followed by 67.3% papain gel and 64.3% with calcium hydroxide [figure 1]. (abbreviations: mc, morinda citrifolia; chx, chlorhexidine gel; ca(oh)2; calcium hydroxide) currently, the use of natural extracts in dentistry has gained importance both to patients and endodontic professionals as the shift is toward natural health remedies . Hence the use of an intracanal medicament made of natural extracts is of great significance . The use of a biocompatible intracanal medicament possessing antimicrobial properties between appointments may reduce or eliminate bacteria in the root canal system and significantly increase the success of root canal treatment . The in vitro model developed by haapasalo and rstavik has been used to assess the efficacy of endodontic medicaments in the disinfection of dentinal tubules . Lynne et al . Modified this model to include quantitative analysis of bacteria in the dentin tubules to define a percentage of reduction in colony forming units in infected dentin before and after the application of intracanal medication preparations . E. faecalis was chosen as the test organism because it has long been implicated in persistent root canal infections and more recently has been identified as the species most commonly recovered from root canals of teeth with post - treatment disease . This study reveals the effect of newer organic intracanal medicaments (when used as raw extracts) against e. faecalis . On an average, chlorhexidine gel showed maximum inhibition of e. faecalis at depths of 200 and 400 m followed by m. citrifolia gel (86.02%), and aloe vera gel (78.9%). Levels of inhibition of papain (67.3%) and calcium hydroxide (64.3%) were somewhat comparable . In the present study, chlorhexidine gel showed 100% inhibition at depths of 200 and 400 m from day 1 to 5 . The reason could be due to the bactericidal dosage of 2% and increased diffusion of the medicament into the dentinal tubules . Showed that 2% chlorhexidine gel produced a better antimicrobial action as compared to 0.2% chlorhexidine gel or calcium hydroxide mixed with 0.2% chlorhexidine . M. citrifolia gel showed 86.02% inhibition at depths of 200 and 400 m from day 1 to 5 . Claim that the presence of l - asperuloside and alizarin may be responsible for the antibacterial and antimicrobial property of m. citrifolia . This improved performance could be attributed to the increased contact time and physical contact of the gel . These results are in accordance with another study conducted by wang et al ., wherein 2% chlorhexidine solution when converted into gel form performed better than the solution alone . Further studies are needed to determine the optimum concentration of m. citrifolia required when used in gel form as an intracanal medicament against e. faecalis . Aloe vera showed 78.72% and 80% inhibition at depths of 200 and 400 m from day 1 to 5 . First day results of aloe vera gel were comparable to that of m. citrifolia gel, but the values declined over day 3 and 5 . The possible reason for antimicrobial action of aloe vera could be the presence of 75 potentially active constituents: vitamins, enzymes, minerals, sugars, lignin, saponins, salicylic acids, and amino acids . The pharmacologic actions of aloe vera, as studied in vitro or in animals (in most cases the total leaf extract was used) include anti - inflammatory, antibacterial, and hypoglycemic effects . Gel based on papain showed 68.05% and 67.28% inhibition at depths of 200 and 400 m from day 1 to 5 . This was probably due to proteolytic cysteine enzyme present in papain, which exhibits antibacterial and anti - inflammatory properties . Calcium hydroxide showed 63.82% and 66.22% inhibition at depths of 200 and 400 m from day 1 to 5 . Gomes et al . Reported that e. faecalis present in the dentinal tubules were resistant to calcium hydroxide intracanal dressing over 10 days . Calcium hydroxide had decreased efficacy on day 3, but had an increased antimicrobial efficacy at day 5 . Chlorhexidine gel showed the maximum antimicrobial activity against e. faecalis, whereas calcium hydroxide showed the least . Among the natural intracanal medicaments, m. citrifolia gel consistently exhibited good inhibition up to the day 5 followed by aloe vera gel and papain gel.
Antiphospholipid syndrome (aps) is an autoimmune disease characterized by thrombosis and morbidity, specifically in pregnancy, due to antiphospholipid antibodies . About half of the cases of aps occur as a primary disorder, while the rest arise in association with other autoimmune diseases, such as systemic lupus erythematosus (sle). Some diseases, such as pulmonary thrombosis and pulmonary hypertension, are known to be complicated by aps; however, aps with pleural effusion is extremely rare . Here, we present a case of aps complicated by unilateral pleural effusion that responded well to oral corticosteroid therapy . A 75-year - old japanese man was admitted to our hospital for spreading erythema on his trunk and extremities, as well as dyspnea . One year prior to admission, he visited us with a 1-year history of erythema and purpura on his legs, accompanied by intermittent fever . Results of laboratory examinations for antiphospholipid antibodies, lupus anticoagulant (using the phospholipid neutralization test), and anticardiolipin antibody had been positive 12 weeks apart . In addition, he showed positive antinuclear antibody (1:80, homogeneous pattern), but was negative for anti - dsdna antibody, anti - sm antibody, anti - rnp antibody, anti - ss - a antibody, anti - ss - b antibody, antitopoisomerase i antibody, and anticentromere antibody . Mpo - anca, pr3-anca, and cryoglobulin were negative . Given the diagnosis of aps, we initiated combination therapy with aspirin (100 mg daily) and warfarin (target international normalized ratio, 2.03.0), but the skin lesions continued to gradually worsen . Violaceous erythema, purpura, and pigmentation were widely noted on his trunk and extremities (fig . 1); they were associated with low platelets (93,000/l) and elevated partial thromboplastin time (48.4 s). A biopsy specimen revealed marked thrombosis in the dermal and subcutaneous small vessels (fig . 2). There were interface changes of the dermo - epidermal junction and mild inflammatory infiltrates in the perivascular area of the dermis, but mucin deposition and thickening of the basal layer of the epidermis were not apparent . In addition, a chest x - ray and computed tomography demonstrated a large pleural effusion in the left lung (fig . 3), without evidence of large vessel thrombus . Electrocardiogram and echocardiogram were normal . Despite serial thoracenteses, effusion recurred . Bacterial and fungal cultures, as well as cytology analyses for malignant cells, were all negative . After excluding infectious diseases, malignancies, pulmonary thrombosis, and heart failure, we added oral prednisolone (30 mg daily) to his prior anticoagulant regimen . The skin lesions and the pleural effusion improved rapidly, eventually disappearing without complication (fig . 4). On follow - up clinical examinations, no symptoms related to sle or other collagen diseases were noted . Common causes of pleural effusion include malignancies, infectious diseases, pulmonary embolism, collagen vascular disease, and heart failure . Aps - related pleural effusion has rarely been reported, and those cases that have been reported appeared to be complications of accompanying pulmonary embolism, sle, or catastrophic aps [4, 5, 6]. Pleuritis, which can induce pleural effusion, is the most common pleuropulmonary manifestation of sle . In the present case, after excluding these differential diagnoses, aps was determined to be the direct cause of the pleural effusion . However, a strong possibility still exists that the pleural effusion may be associated with occult collagen vascular disease, particularly sle or lupus - like disease (lld) heretofore undiagnosed . A long - term follow - up study in 128 patients with primary aps demonstrated that 11 patients (8%) developed sle, while 6 (5%) developed lld during a median follow - up period of 8.2 years (range, 114 years). The results of this study suggest that the pleural effusion may be attributed to a coexisting condition like lld, although our patient has not fulfilled american college of rheumatology diagnostic sle criteria to date . This may be supported by the fact that oral corticosteroid therapy was a remarkably effective treatment of the pleural effusion that had previously been unsuccessfully treated by anticoagulant therapy and repeated drainage . Corticosteroids and immunosuppressants continue to be the treatment of choice for severe sle complications, including pleural effusion . Furthermore, the clinical manifestations of primary aps and aps associated with sle are similar, which makes it more difficult to differentiate these diseases . As pleural effusion can be life - threatening corticosteroids might be an effective choice of treatment for intractable pleural effusion in aps patients.
Surgically treated patients with hepatocellular carcinoma (hcc), which represent a highly selected group, have higher survival rates compared to those of medically treated patients at a comparable stage . However, long - term prognosis remains unsatisfactory because of the high incidence of tumor recurrence and metastasis after hepatectomy . Thus, identification of markers of poor prognosis is important in order to provide the opportunity for timely intervention . High proliferation rate, a classic hallmark of cancer, is due to the self - sufficiency of growth signals, insensitivity to anti - growth signals, and limitless replicative potential . A variety of methods, including analysis of proliferating cell nuclear antigen, bromodeoxyuridine, argyrophilic nuclear organized regions, ki-67 nuclear antigen, and phosphorylated histone h3, are used in evaluation of proliferative activity [5 - 7]. However, many of these methods cannot be applied in daily clinical practice . In contrast, the mitotic index, which is a useful and simple method for analysis of cell proliferation, can be easily applied to routine clinical practice . The prognostic role of mitotic index in patient survival has been confirmed in several cancers . In addition, mitotic index has been incorporated in the american joint committee on cancer (ajcc) seventh tumor staging system for malignant melanoma, gastrointestinal tumor, and neuroendocrine tumors of the gastrointestinal tract . In hccs, previous studies indicated a potential role of high mitotic index as an adverse prognostic indicator in cohorts of fewer than 200 patients . However, the practical utility of mitotic index as a predictor of prognosis in patients with hcc has not been determined . In this study, we evaluated mitotic index as a possible prognostic marker in a large cohort of 282 patients with primary hcc who received long - term follow - up for 120 months . We also attempted to determine the cutoff value for mitotic index that showed the most significant prognostic role in hcc patients . A total of 290 patients who were pathologically confirmed to have primary hcc and underwent curative resection at samsung medical center, seoul, korea between july 2000 and may 2006 were enrolled in this study . Eight patients who received preoperative treatments, including transcatheter arterial chemoembolization, radiofrequency ablation, and radiation therapy, were excluded; therefore, 282 patients were included in this study . Curative resection was defined as complete resection of all tumor nodules with clear microscopic resection margins and no residual tumors on computed tomography scans performed 1 month after surgery . Clinical parameters, including age, gender, date of surgery, serum -fetoprotein (afp), and serum albumin, were obtained by reviewing the medical records . When the tumor was less than 3 cm in size, all tumors were sectioned and embedded . When the tumor was larger than 3 cm in size, at least four sections were taken for the pathologic examinations and the mean number of blocks was one for 1 cm of tumor diameter . Histopathologic features of hccs, including histologic differentiation, microvascular invasion, major portal vein invasion, intrahepatic metastasis, multicentric occurrence, and non - tumor liver pathology, were reviewed by two pathologists (s.y.h . And c .- k.p . ). Intrahepatic metastasis and multicentric occurrence were determined according to the criteria of the liver cancer study group of japan . Hcc recurrence within the first two years following surgery is mainly due to intrahepatic metastasis, whereas late recurrence usually results from multicentric occurrence . Using 2 years as a cutoff, all patients were staged according to the ajcc staging system and barcelona clinic liver cancer (bclc) staging classification . During follow - up, serum afp levels were monitored and three phase dynamic computed tomography scan or magnetic resonance imaging was performed every 3 months after surgery . The median follow - up period was 120 months (range, 14 to 151 months) for survivors . Recurrence - free survival (rfs) was measured from the date of surgery until detection of tumor recurrence . Disease - specific survival (dss) was defined as the interval between the date of surgery and the date of hcc - related death, which was defined as: (1) the tumor occupying more than 80% of the liver, (2) portal venous tumor thrombus proximal to the second bifurcation, (3) obstructive jaundice due to the tumor, (4) distant metastases, and (5) variceal hemorrhage with portal venous tumor thrombus proximal to the first bifurcation . Two liver pathologists (s.y.h . And c .- k.p .) Counted the number of mitotic cells in 10 high - power fields (hpfs) of hematoxylin and eosin - stained slides, and found areas containing the most mitotic figures, the so - called hot spot . After counting the mitoses in the hot spot, if no hot spot could be found and mitoses were sparse and randomly scattered throughout the lesion, a representative mitosis was chosen and the count began with that field and was extended to adjacent fields . According to the criteria of mitotic figures defined by baak, mitotic cells were counted only if there was complete concordance between the two pathologists using a multi - head microscope . The x - tile statistics package (yale university, new haven, ct) was used to determine the optimal cutoff value with the highest level of statistical significance related to patient survival; cases were dichotomized into low and high mitotic index groups based on the established cutoff value . Analysis of the association between mitotic index and clinicopathologic parameters was performed using the chi - square test, fisher exact test, or cochran armitage test . Differences in survival rates were assessed using the log - rank test or breslow test . The cox proportional hazard regression model was used to assess the association between clinicopathologic factors and survival time . We examined the proportional hazard assumption graphically to determine whether variables in the cox proportional hazard model were constants that do not depend on time . A total of 290 patients who were pathologically confirmed to have primary hcc and underwent curative resection at samsung medical center, seoul, korea between july 2000 and may 2006 were enrolled in this study . Eight patients who received preoperative treatments, including transcatheter arterial chemoembolization, radiofrequency ablation, and radiation therapy, were excluded; therefore, 282 patients were included in this study . Curative resection was defined as complete resection of all tumor nodules with clear microscopic resection margins and no residual tumors on computed tomography scans performed 1 month after surgery . Clinical parameters, including age, gender, date of surgery, serum -fetoprotein (afp), and serum albumin, were obtained by reviewing the medical records . When the tumor was less than 3 cm in size, all tumors were sectioned and embedded . When the tumor was larger than 3 cm in size, at least four sections were taken for the pathologic examinations and the mean number of blocks was one for 1 cm of tumor diameter . Histopathologic features of hccs, including histologic differentiation, microvascular invasion, major portal vein invasion, intrahepatic metastasis, multicentric occurrence, and non - tumor liver pathology, were reviewed by two pathologists (s.y.h . And c .- k.p . ). Intrahepatic metastasis and multicentric occurrence were determined according to the criteria of the liver cancer study group of japan . Hcc recurrence within the first two years following surgery is mainly due to intrahepatic metastasis, whereas late recurrence usually results from multicentric occurrence . Using 2 years as a cutoff, all patients were staged according to the ajcc staging system and barcelona clinic liver cancer (bclc) staging classification . During follow - up, serum afp levels were monitored and three phase dynamic computed tomography scan or magnetic resonance imaging was performed every 3 months after surgery . The median follow - up period was 120 months (range, 14 to 151 months) for survivors . Recurrence - free survival (rfs) was measured from the date of surgery until detection of tumor recurrence . Disease - specific survival (dss) was defined as the interval between the date of surgery and the date of hcc - related death, which was defined as: (1) the tumor occupying more than 80% of the liver, (2) portal venous tumor thrombus proximal to the second bifurcation, (3) obstructive jaundice due to the tumor, (4) distant metastases, and (5) variceal hemorrhage with portal venous tumor thrombus proximal to the first bifurcation . Two liver pathologists (s.y.h . And c .- k.p .) Counted the number of mitotic cells in 10 high - power fields (hpfs) of hematoxylin and eosin - stained slides, and found areas containing the most mitotic figures, the so - called hot spot . After counting the mitoses in the hot spot, if no hot spot could be found and mitoses were sparse and randomly scattered throughout the lesion, a representative mitosis was chosen and the count began with that field and was extended to adjacent fields . According to the criteria of mitotic figures defined by baak, mitotic cells were counted only if there was complete concordance between the two pathologists using a multi - head microscope . The x - tile statistics package (yale university, new haven, ct) was used to determine the optimal cutoff value with the highest level of statistical significance related to patient survival; cases were dichotomized into low and high mitotic index groups based on the established cutoff value . Analysis of the association between mitotic index and clinicopathologic parameters was performed using the chi - square test, fisher exact test, or cochran armitage test . Differences in survival rates were assessed using the log - rank test or breslow test . The cox proportional hazard regression model was used to assess the association between clinicopathologic factors and survival time . We examined the proportional hazard assumption graphically to determine whether variables in the cox proportional hazard model were constants that do not depend on time . The median patient age was 53 years (range, 17 to 76 years); 234 patients were males, and 48 were females . Two hundred and eighteen patients (77.3%) were infected with hepatitis b virus, and 26 (9.2%) with hepatitis c virus . Two hundred and three patients (72.0%) suffered from tumor recurrence; 153 patients (54.3%) from early recurrence, and 50 patients (17.7%) from late recurrence . Twenty - nine of the 127 deaths were due to non - hcc related causes . Seventeen of the 29 deaths were due to hepatic failure; eight were due to non - hepatic causes, and four were due to unknown causes . The mean mitotic index was 7.75 (95% confidence interval, 6.47 to 9.03) and the median value was 3.00 (range, 0 to 60). Using the x - tile package, mitotic index was graded as low (4 or less mitoses per 10 hpfs) or high (5 or more mitoses per 10 hpfs) (fig . High mitotic index showed significant association with younger age (p <0.001), larger tumor size (p=0.022), higher edmondson grade (p <0.001), microvascular invasion (p <0.001), major portal vein invasion (p=0.026), intrahepatic metastasis (p <0.001), higher afp level (p <0.001), hepatitis b virus etiology (p=0.012), and liver cirrhosis (p=0.016). As the ajcc t - stage or bclc stage increased, the frequency of high mitotic index also showed a significant increase (p <0.001 and p <0.001, respectively). The 3-, 5-, 7-, and 9-year rfs rates for 282 hcc patients were 43.5%, 37.2%, 31.1%, and 30.0%, respectively . The 3-, 5-, 7-, and 9-year dss rates were 75.1%, 67.1%, 59.6%, and 53.4%, respectively . Patients with high mitotic index had shorter dss (p <0.001) and tended to have shorter rfs (p=0.112) (fig . 2). By applying the breslow test, which attributes greater weight to earlier events, patients with high mitotic index had shorter rfs (p=0.003). In subgroup analysis among patients with a larger tumor size (> 5 cm), microvascular invasion, intrahepatic metastasis, higher ajcc t - stage, and higher blcl stage, high mitotic index showed unfavorable influences on dss (p=0.001, p=0.008, p=0.003, p=0.012, and p <0.001, respectively) (fig . Larger tumor size, edmondson grade iii, microvascular invasion, major portal vein invasion, intrahepatic metastasis, higher ajcc t - stage, higher bclc stage, lower albumin level, and higher afp level showed unfavorable influences on both rfs and dss . In addition, we found that high mitotic index showed an unfavorable influence on dss (p <0.001) (table 2). Because ajcc t - stage and bclc stage were associated with vascular invasion, to avoid potential bias we did not perform multiple analyses using these indices . In multivariate analysis, intrahepatic metastasis and lower albumin level larger tumor size and high mitotic index (p=0.004) were found to be independent predictors of shorter dss . Patients with high mitotic index were more likely to suffer from disease - specific death compared to those with low mitotic index (hazard ratio, 1.818) (table 3). Multivariate analysis of rfs with mitotic index could not be performed, because the survival curves according to mitotic index were crossed and the proportional hazard assumption was violated . The median patient age was 53 years (range, 17 to 76 years); 234 patients were males, and 48 were females . Two hundred and eighteen patients (77.3%) were infected with hepatitis b virus, and 26 (9.2%) with hepatitis c virus . Two hundred and three patients (72.0%) suffered from tumor recurrence; 153 patients (54.3%) from early recurrence, and 50 patients (17.7%) from late recurrence . Twenty - nine of the 127 deaths were due to non - hcc related causes . Seventeen of the 29 deaths were due to hepatic failure; eight were due to non - hepatic causes, and four were due to unknown causes . The mean mitotic index was 7.75 (95% confidence interval, 6.47 to 9.03) and the median value was 3.00 (range, 0 to 60). Using the x - tile package, mitotic index was graded as low (4 or less mitoses per 10 hpfs) or high (5 or more mitoses per 10 hpfs) (fig . High mitotic index showed significant association with younger age (p <0.001), larger tumor size (p=0.022), higher edmondson grade (p <0.001), microvascular invasion (p <0.001), major portal vein invasion (p=0.026), intrahepatic metastasis (p <0.001), higher afp level (p <0.001), hepatitis b virus etiology (p=0.012), and liver cirrhosis (p=0.016). As the ajcc t - stage or bclc stage increased, the frequency of high mitotic index also showed a significant increase (p <0.001 and p <0.001, respectively). The 3-, 5-, 7-, and 9-year rfs rates for 282 hcc patients were 43.5%, 37.2%, 31.1%, and 30.0%, respectively . The 3-, 5-, 7-, and 9-year dss rates were 75.1%, 67.1%, 59.6%, and 53.4%, respectively . Patients with high mitotic index had shorter dss (p <0.001) and tended to have shorter rfs (p=0.112) (fig . 2). By applying the breslow test, which attributes greater weight to earlier events, patients with high mitotic index had shorter rfs (p=0.003). In subgroup analysis among patients with a larger tumor size (> 5 cm), microvascular invasion, intrahepatic metastasis, higher ajcc t - stage, and higher blcl stage, high mitotic index showed unfavorable influences on dss (p=0.001, p=0.008, p=0.003, p=0.012, and p <0.001, respectively) (fig . Larger tumor size, edmondson grade iii, microvascular invasion, major portal vein invasion, intrahepatic metastasis, higher ajcc t - stage, higher bclc stage, lower albumin level, and higher afp level showed unfavorable influences on both rfs and dss . In addition, we found that high mitotic index showed an unfavorable influence on dss (p <0.001) (table 2). Because ajcc t - stage and bclc stage were associated with vascular invasion, to avoid potential bias we did not perform multiple analyses using these indices . In multivariate analysis, intrahepatic metastasis and lower albumin level larger tumor size and high mitotic index (p=0.004) were found to be independent predictors of shorter dss . Patients with high mitotic index were more likely to suffer from disease - specific death compared to those with low mitotic index (hazard ratio, 1.818) (table 3). Multivariate analysis of rfs with mitotic index could not be performed, because the survival curves according to mitotic index were crossed and the proportional hazard assumption was violated . In the current study, we elucidated the prognostic significance of mitotic index in a large cohort of patients with primary hcc who received long - term follow - up . We attempted to establish a cutoff value for mitotic index that showed the most significant prognostic role in hcc patients . Low mitotic index was defined as four or fewer mitoses per 10 hpfs, and high mitotic index as five or more . High mitotic index showed correlation with larger tumor size, higher edmondson grade, microvascular invasion, major portal invasion, intrahepatic metastasis, higher ajcc t - stage, higher bclc stage, higher afp level, hepatitis b virus etiology, and liver cirrhosis . In addition, patients with high mitotic index had shorter dss and tended to have shorter rfs . The mean time gap between recurrence and death in the high mitotic index group was significantly lower than in the low mitotic index group (30.5 months vs. 43.8 months, p=0.004). Treatment modality after recurrence did not differ statistically between low versus high mitotic index group (p=0.38) (supplementary table 1). It is assumed that the high mitotic index group had poorer response to the salvage treatment after recurrence than the low mitotic index group . Only a few studies on the prognostic role of mitotic index in hcc haratake et al ., who divided mitotic index into three groups (0 - 4/10 hpfs, 5 - 9/10 hpfs, 10 and more/10 hpfs), observed a better prognosis for patients with hcc who exhibited low mitotic activity . They reported that longer survival periods were observed in some patients with larger tumors if the mitotic indices were low . Ouchi et al . Showed that mitotic index was an independent significant variable, influencing the overall survival of 40 patients with hcc following hepatic resection . In addition, they found that hcc with a high mitotic index (5/10 hpfs) was associated with multiple tumors and advanced tumor stage . They also reported a highly significant correlation between mitotic index and ki-67 labelling index (r=0.870). Nanashima et al . Also reported correlation of high mitotic index (5/10 hpfs) with overall survival in 81 patients with hcc by univariate analysis . They also found a significant association of high mitotic index with vascular invasion, poor histologic differentiation, and recurrence rate . Interestingly, the criteria of mitotic index used in previous studies are the same as those applied in our study, which were determined statistically for the strongest prognostic effect . To the best of our knowledge, this is the first report to show that high mitotic index is an independent predictor of shorter dss . Hcc recurrence within the first 2 years following surgery is mainly due to intrahepatic metastasis, whereas recurrence after 2 years following surgery usually results from multicentric occurrence of hcc . It is assumed that the high mitotic index group did not show shorter rfs after 48 months following surgery, because recurrence after 48 months following surgery was mainly due to newly developed hcc . The most outstanding finding of this study is that subgroup analysis among patients with larger tumor size, microvascular invasion, intrahepatic metastasis, higher ajcc t - stage, and higher blcl stage showed unfavorable influences of high mitotic index on dss . Counting mitotic cells is a simple, rapid, and inexpensive method which can be routinely performed in daily practice . Our data show, for the first time, that high mitotic index is an independent predictor of shorter dss in surgically resected hccs . Prospective studies are needed to further investigate the role of mitotic index as a prognostic factor in hcc.
Prostate cancer is the most common cancer diagnosed in new zealand (nz) males and the third most common cause of male cancer deaths.1 generally prostate cancer is a slow growing cancer with relatively good prognosis, with 80% of patients with localised disease being still alive at 15 years.2 around 70% of men in nz are identified with low - grade prostate cancer with a good prognosis.3 unfortunately, some men present with advanced disease and their first symptoms may be due to metastases . The stage and grade of cancer will obviously influence treatment options, as will the presence of various co - morbidities.4 men with metastatic prostate cancer may be offered pharmacologic androgen deprivation therapy (adt), specific chemotherapeutic medication or be treated with orchidectomy.5 in new zealand, mori men are less likely to be diagnosed with prostate cancer but have a 70% increased risk of dying compared with non - mori men.6 moreover, mori men diagnosed with non - localised prostate cancer have a threefold risk of dying from the disease compared with non - mori men.7 variation in treatment may be one of the reasons for the observed survival disparities . In the uk, increased use of adt has been linked to the trend of decreasing mortality.8 it is possible that variation in the use of adt also contributes to the survival differences between mori and non - mori men with non - localised prostate cancer . However, little information is available on the use of adt and chemotherapeutic agents in prostate cancer patients in new zealand . The aim of this study was to ascertain the patterns of dispensing adt, including anti - androgens and luteinising hormone - releasing hormone (lhrh) analogues, and chemotherapeutic agents in new zealand men within the first year after prostate cancer diagnosis . We also explored the effect of age, ethnicity, year of diagnosis and orchidectomy on pharmacologic adt use . This nationwide audit of androgen deprivation therapy and chemotherapy treatment for prostate cancer was undertaken in new zealand, a nation of 4.5 million people with a universally subsidised health system that includes free public hospital and pharmaceutical care . We identified a cohort of men diagnosed with prostate cancer between 1 january 2006 and 31 december 2011 from the new zealand cancer registry (nzcr, http://www.health.govt.nz/nz-health-statistics/national-collections-and-surveys/collections/new-zealand-cancer-registry-nzcr), which collects data on all new cases of malignant cancers in nz excluding squamous cell carcinoma and basal cell carcinoma of the skin . For each patient nzcr data included date of diagnosis, extent of disease at diagnosis, age at diagnosis, and ethnicity . The extent of cancer at diagnosis is coded in the nzcr as follows: b (localised), c (invasion of adjacent tissues or organs), d (invasion of regional lymph nodes), e (distant metastases), and f (unknown). For the purpose of our study, only about one - quarter of prostate cancer cases have an extent at diagnosis listed in the nzcr, and the accuracy of the extent has not been assessed yet . Therefore, the extent of prostate cancer at diagnosis used in this study needs to be understood as that recorded in the nzcr, and may potentially differ from the actual extent at diagnosis . Data for the cohort of men identified from the nzcr were linked to the pharmaceutical collection by a unique encrypted number derived from the national health index (nhi) number, which is unique for every public health system user in new zealand . The pharmaceutical collection is an administrative claims database that contains information from pharmacists on dispensing subsidised medications . Once a funded prescription is dispensed in new zealand the data are collected in a national repository and available for analysis . In addition to prescriber details, the medication name, strength, quantity and dosage are recorded . For our study, data were extracted on androgen deprivation therapy, including anti - androgens (flutamide, bicalutamide, cyproterone) and lhrh analogues (goserelin, leuprorelin), and also on chemotherapeutic agents (doxorubicin, epirubicin, paclitaxel, mitozantrone, docetaxel). The information included chemical i d, indicating the primary active chemical ingredient, and the therapeutic group level 1 - 3 (more detail on http://www.pharmac.health.nz/tools-resources/pharmaceutical-schedule). In addition, registration data were linked to the national minimum dataset (national collection of public and private hospital discharge information on inpatients and day patients) to identify men treated with orchidectomy . Men with prostate cancer morphology not consistent with adenocarcinoma (67), men with unknown ethnicity (1478) and those diagnosed at death (374) were excluded from the analysis . In addition, 17 men were excluded because their domicile was listed as overseas . We examined the frequency of adt and chemotherapy use in the first year after the initial diagnosis by patients' age (<60 years, 60 - 69 years, 70 - 79 years, 80 + years), ethnicity (mori, pacific and non - mori / non - pacific), and extent of disease at diagnosis . Differences between distributions were tested using the or fisher exact test (when sub - group sample sizes were small). Probability (p) multivariate logistic regression models were constructed to assess the likelihood of use of adt for patients with advanced (regional spread and metastatic) prostate cancer, adjusting for age, ethnicity, year of diagnosis and orchidectomy . The final study population included 15,947 men diagnosed with prostate cancer in new zealand in the six years between 2006 and 2011 . Table 1 summarises the demographic information (age and ethnicity) by extent of prostate cancer at diagnosis . Most men were diagnosed between the ages of 60 and 79 years (68.2%). There were 908 (5.7%) mori men, 445 (2.8%) pacific men, and 14,594 (91.5%) non - mori / non - pacific men in the sample . The proportion of mori men in the 2006 census total nz male population of 50 + years (since most prostate cancer cases occur in men aged 50 +) was 7%, while pacific males comprised 3%, and non - mori / non - pacific men 90% . In total, 15.0% of men were recorded as having localised extent at diagnosis, 7.6% regional spread, 5.8% metastases, and 71.7% were recorded with unknown extent . Androgen deprivation therapy (flutamide, bicalutamide, cyproterone, goserelin, leuprorelin) or chemotherapeutic agents (doxorubicin, epirubicin, paclitaxel, mitozantrone, docetaxel) were dispensed for 4978 (31.2%) men in the first year following their initial diagnosis . Most of the patients received doxorubicin (11), with docetaxel being the second most common agent used (5). Due to such a small sample size, patients with chemotherapy were not considered in the regression analysis . Within the first year post - diagnosis, pharmacologic adt was dispensed for 47 patients with localised prostate cancer at diagnosis (1.9% of all men with localised disease recorded in the nzcr), 266 patients with regional spread (22.1%) and 664 patients with distant metastases (71.8%). Due to the small number and proportion of patients with localised disease who received adt within one year post - diagnosis, further analysis focused on patients with regional and metastatic prostate cancer . Figures 1 and 2 show the frequency of types of pharmacologic adt by age, ethnicity and extent of disease at diagnosis (regional spread, distant metastases, and all extent (including localised, regional, distant and unknown extent). In patients with metastatic cancer, anti - androgens (60.1%) were used more commonly than lhrh analogues (50.1%; p<0.0001). By contrast, overall (all extents), more patients received lhrh analogues (25.5%) than anti - androgens (20.6%; p<0.0001) as did patients with regional spread (18.8% v. 14.8%; p=0.008). Men younger than 70 years overall and specifically those diagnosed with regional prostate cancer were less likely to receive adt compared with older men (21.3% v. 44.5%; p<0.0001; 17.0% v. 37.8%; p<0.0001, respectively). However, in men diagnosed with metastatic cancer those aged under 70 were more likely to receive adt than older men (80.4% v. 69.0%; p=0.001). Overall, adt was less likely to be dispensed for non - mori / non - pacific men than for mori and pacific men (30.5% v. 38.5%; p<0.0001, and v. 38.9%; p<0.0001, respectively). In men with regional disease, pacific men were more likely to receive adt compared to non - mori / non - pacific men (44.0% v. 21.4%; fisher exact test p=0.01) and they were also more likely to receive anti - androgens than non - mori / non - pacific and mori men (40.0% v. 14.1%; fisher exact test p=0.002, and v. 16.4%; fisher exact test p=0.03, respectively). In men with metastatic prostate cancer, mori men were more likely to receive anti - androgens than non - mori / non - pacific men (72.5% v. 58.2%; fisher exact test p=0.02). Similarly to anti - androgens and lhrh analogues, orchidectomy can be used to achieve reduction of testosterone levels and thus reduce prostate cancer growth.5 in our sample, 3.3% of patients (165 out of 4968 who were prescribed anti - androgens or lhrh analogues) underwent orchidectomy within the first year after initial diagnosis . The majority of these men (77.6%) received either anti - androgens or lhrh analogues but not both in that year . In addition, there were 202 men who underwent orchidectomy but did not receive pharmacologic adt (1.8% of men with prostate cancer not on pharmacologic adt) in the first year post - diagnosis . Since our further analyses focus on men with advanced (regional spread or metastatic) prostate cancer, table 2 shows distribution of orchidectomy by age, ethnicity, and pharmacologic adt use separately for all patients and for those with advanced cancer . Men older than 70 years with advanced cancer were treated more commonly by orchidectomy only (10.0% v. 1.8%; fisher exact test p<0.0001), and they were also more likely to undergo orchidectomy overall compared with men younger than 70 (7.8% v. 3.2%; fisher exact test p<0.0001). Mori men with any extent were more likely to be treated by orchidectomy compared to non - mori / non - pacific men (3.4% v. 2.2%; p<0.0001). In order to assess the use of adt from the clinical point of view, patients with advanced cancer were categorised into three groups, i.e. Those who received anti - androgens only, those who received lhrh analogues only and those who received both anti - androgens and lhrh analogues within the first year post diagnosis . In men with advanced cancer, 53.2% (out of all men on adt) received both anti - androgens and lhrh analogues within the first year post - diagnosis, followed by those who received anti - androgens only (25.8%) and those who received lhrh analogues only (21.0%). Table 3 shows the distribution of anti - androgens and lhrh analogues use individually and in combination in men with advanced disease . A significantly larger proportion of men older than 70 years at diagnosis received anti - androgens only compared with men younger than 70 (29.4% v. 19.4%; fisher exact test p=0.001), while a significantly larger proportion of non - mori / non - pacific men received lhrh analogues only compared with mori and pacific men (22.7% v. 11.7%; fisher exact test p=0.03, and v. 6.5%; fisher exact test p=0.009, respectively). Multiple logistic regression models were used to assess the effect of age at diagnosis, ethnicity, year of diagnosis, and orchidectomy on the use of adt (anti - androgens and lhrh analogues alone or combined) in men with advanced cancer at diagnosis . Six different models were built: model i included age only; model ii age and ethnicity; model iii age, ethnicity and year of diagnosis; model iv age and orchidectomy; model v age, ethnicity and orchidectomy; and model vi included age, ethnicity, year of diagnosis and orchidectomy (table 4). In all models, age was a contributing factor, with older men with advanced cancer being more likely to receive adt . Mori and pacific men (compared with non - mori / non - pacific men) were approximately 2.1- and 3.1-fold more likely to receive pharmacologic adt when adjusting for age and combinations of age, year of diagnosis and orchidectomy . However, year of diagnosis and orchidectomy in the same year did not seem to pose as confounders and did not significantly contribute to the models . The aim of this study was to assess the frequency of use of adt and chemotherapeutic agents for nz men in the first year after cancer diagnosis, particularly for metastatic patients for whom adt should be prescribed immediately.9 therefore, we have not followed up the cohort for a longer period of time . Seventy two percent of men recorded as having metastatic disease at diagnosis received pharmacologic adt (anti - androgens and/or lhrh analogues). Whilst a small number of men with prostate cancer had an orchidectomy (2%), it seems that a quarter of men with advanced prostate cancer did not receive hormonal treatment . Since management guidelines for locally advanced and particularly metastatic prostate cancer clearly include use of androgen deprivation therapy as part of the treatment pathway5,10, there is a need for improvement in this area in new zealand . In comparison, in the usa 95% patients diagnosed between 1994 and 2002 with stage iv disease received either surgical or pharmacologic adt, while 16% received chemotherapy.11 in our sample, chemotherapeutic agents were used very rarely in the first year post - diagnosis . Some of the chemotherapeutic agents, such as docetaxel were first subsidised in 2011, so they would not appear in the pharmaceutical collection previously . There seems to be a greater potential for the use of these agents and newer treatments in men with hormone - refractory advanced prostate cancer.5 older men with metastatic disease in particular were less likely to receive pharmacologic adt . A larger proportion of men older than 70 years at diagnosis received anti - androgens only compared to men younger than 70 . Contrary to our expectations mori and pacific men were more likely to receive adt than non - mori / non - pacific men . Mori men were more likely to be treated with orchidectomy, while they were less likely to receive lhrh analogues when compared with non - mori / non - pacific men . The dispensing of pharmacologic adt and use of orchidectomy for reduction of testosterone levels in advanced prostate cancer was influenced mainly by patients' age and ethnicity . Men with advanced prostate cancer were more likely prescribed both anti - androgens and lhrh analogues in the first year as opposed to anti - androgens or lhrh analogues alone . Data from other countries showed that physician preference has an important influence on the use of adt4, as do the presence of subsidies12, patient's age and tumour grade at diagnosis.13 thus the solution to improved dispensing is likely to involve a greater understanding of the barriers to prescribing from the physicians' perspective but also of patients' views on adt use . The strength of this study is that we used data linkage to assess use of adt nationally . Men registered with prostate cancer in the new zealand cancer registry were identified and this information was linked to the national dataset of pharmaceutical dispensings as well as to the national minimum dataset to ensure that the use of orchidectomy did not introduce a bias into our analysis . This allowed us to undertake a large - scale pharmaco - epidemiological study on the dispensing of adt and chemotherapeutic agents, which is quite unique internationally . Studies from other countries investigated the use of adt in certain patient groups, such as medicare beneficiaries or patients visiting specialist services, but to our knowledge none were population - based.11,14 - 18 in addition, several of the most recent studies were restricted to prostate cancer patients with localised disease . These studies highlighted the benefits of using adt as primary or adjuvant therapy, particularly in high - risk patients.14 - 17 the main weakness of our study is that 72% of men registered with prostate cancer had the extent of disease recorded as unknown . This may have reduced the power of the study and introduced bias if men with advanced disease with known extent in the nzcr were treated differently than men with advanced disease at diagnosis whose extent was recorded as unknown . Prostate cancer is similar to bladder and liver cancer with respect to the low recording of extent at diagnosis . Although the proportion of incident prostate cancer cases with known extent at diagnosis slightly improved from 25.7% to 28.1% between 2006 and 2010, for further research into prostate cancer on national level it will be essential to at least achieve proportions of known extent similar to colorectal and breast cancer, where more than 80% of cases have known extent recorded.6 the new zealand cancer control council is currently reviewing the reporting of all cancers in an effort to improve the availability of data in the nzcr, including extent at diagnosis.19 another weakness may be potentially incomplete information linkage either due to incomplete coverage of nhi numbers or deficiencies in reporting . However, the use of nhi numbers across the country was on average 95% during the study period, and more than 95% of all prescriptions recorded in the pharmaceutical collection have had an nhi attached . In addition, the reporting of pharmaceutical cancer data by district health boards was voluntary until july 2008, which may have affected data linkage in the first years of the study . However, the frequency of adt use in the first year after diagnosis was 70.2% for men diagnosed with distant metastases in 2006 - 2007 and 70.9% for those diagnosed in 2009 - 2010 . Therefore, even if changes in data recording within this period might have had an impact, it does not appear to be substantial . This audit showed that there is a clear under - utilisation of adt and particularly chemotherapeutic agents in nz men with advanced prostate cancer . Particularly in men with distant metastases
To describe the surgical technique and initial experience with a single - port retroperitoneal renal biopsy (sprrb). A single 1.5 cm incision was performed under the 12th rib at mid - axillary line, and an 11 mm trocar was inserted . After lower pole exposure, a laparoscopic biopsy forceps was introduced through the nephroscope working channel to collect a renal tissue sample . The mean operative time was 32 minutes, and mean estimated blood loss was less than 10 ml . The hospital stay of all patients was two days because they were discharged in the second postoperative day, after remaining at strict bed rest for 24 hours after the procedure . Sprrb is a simple, safe and reliable alternative to open and videolaparoscopic approaches to surgical renal biopsy . Image - guided percutaneous renal biopsy is the most widely used method to sample renal parenchyma for the evaluation of malignancy or diffuse renal disease . The risks of this procedure are minimal and the overall success rate of all renal biopsies varies from 70 to 100% (1). Its major indications rest on diagnosis and follow - up of several systemic and nephrological conditions that lead to glomerular damage and renal function impairment, providing useful data for treatment and prognosis . It may also be used for evaluation of solid renal masses and cystic renal lesions (1). However, this method has absolute and relative contraindications that may hamper or preclude it, such as the presence of a solitary kidney, uncontrolled arterial hypertension, coagulation disorders, renal artery aneurysm, previous percutaneous needle biopsy failure and obesity . Bleeding and inadequate amounts of renal tissue for diagnosis are not infrequent, and constitute potential disadvantages of the procedure . In addition, children may be unable to cooperate, requiring general anesthesia . In these settings, open and laparoscopic approaches are well - established alternatives and should be considered, although with a higher level of invasiveness and complexity . In search for an alternative that could minimize surgical aggressiveness of these procedures and hence spread its use, we outlined a renal biopsy technique through a single retroperitoneal laparoscopic access using standard urological instruments . The aim of this paper is to describe the technique now standardized in our institution and our initial experience with the single port retroperitoneal renal biopsy (sprrb). After receiving general anaesthesia, orogastric and bladder catheterization, the patient is usually positioned in the left flank position, as the kidney is more easily accessible at the right side due to its lower position . A 1.5 cm incision is carried out just below the tip of the 12th rib, at the mid - axillary line, and is followed by blunt access to the retroperitoneum space . An initial digital dissection is done aiming to identify the lower renal pole, while also displacing the peritoneum anteriorly . During this step, care must be taken in order to avoid peritoneal tearing, as the pneumoperitoneum resulting from gas insufflation would hamper the maintenance of adequate retroperitoneal working space . Next, a rubber balloon is positioned between the kidney and the posterior abdominal wall, and is filled with 300 - 400 cc of saline, creating a virtual cavity . The saline is drained after a few minutes, to achieve hemostasis, and the balloon is then removed . An 11 mm trocar is inserted and carbon dioxide is used to maintain pneumoretroperitoneum at 12 to 15mmhg . Retroperitoneal inspection and identification of the psoas muscle and the lower pole of the kidney are now performed with a standard 26 french nephroscope, as shown in figure-1 . It is frequently possible to expose the renal surface bluntly, by using gentle movements of the tip of the scope to drag the perirenal fat away from the intended site of biopsy . Alternatively, standard laparoscopic surgical aspirator, scissors or hooks can be inserted through the working channel of the nephroscope, and then be used to dissect, cut and coagulate nearby structures, allowing a clear renal surface to be assessed . Once the biopsy site is cleared from fat, one or two samples are taken with the aid of a toothed biopsy forceps, also through the nephroscope (figure-2). Bleeding is expected to be negligible, as the injury caused by the forceps is shallow (figure-3), but the parenchyma can be coagulated with the same instruments, and a cellulose hemostatic bolster can be applied, if needed . Finally, the pneumoretroperitoneum is evacuated and, if no bleeding is observed, the trocar is removed and the access port is closed . Figure 2sampling renal parenchyma with a toothed biopsy forceps, through the nephroscope working channel . Figure 3aspect of kidney surface after a tissue sample was taken, with only minimal bleeding . At our institution, laparoscopic retroperitoneal renal biopsy is currently often performed for pediatric patients with nephrological conditions (2). As the surgical team s experience progressed and the procedure was standardized, however, we felt that it should be even less invasive, especially for this very young population . Additionally, in order to spread and popularize its execution, we devised how to use instruments that are already present in a regular urological operating room, such as the nephroscope and laparoscopic scissors and forceps, in a different fashion . A similar approach has been described previously, in pediatric surgery, for appendectomies and varicocelectomies, but with only one case of renal biopsy (3). Between january and april/2013, five children underwent sprrb in our hospital, referred from the nephrology clinic for renal biopsy . Informed consent was previously obtained from parents, respecting our institution s ethics committee recommendations and approval . The procedure was successfully performed with the technique above described, by a supervised resident in - training . The overall mean operative time was 32 minutes, and mean estimated blood loss was less than 10 ml . No open conversion was needed . The hospital stay of all patients was two days because they were kept in absolute bed rest for 24 hours after the procedure, before being discharged home . Pain and analgesics use were low, and there were no significant detected complications . Regarding the obtained samples, the average number of glomeruli present in the specimens was 31, and the histopathological findings showed focal proliferative lupus glomerulonephritis in two cases, diffuse mesangial proliferative glomerulonephritis in another two, and nephritis related to henoch - schnlein purpura in one child . These results are comparable to those previously shown by us, with laparoscopic renal biopsy in children, regarding operative time, blood loss, hospital stay and success in obtaining adequate samples (2). Table 1clinical features of patients submitted to single - port retroperitoneal renal biopsy.patientgenderage (years)bmi (kg / m)ot (min. )bl (ml)gndiagnosiscomplications1m0723.625423diffuse mesangial proliferative glomerulonephritisnone2f0924.5371338nephritis related to henoch - schnlein purpuranone3f1121.827530focal proliferative lupus glomerulonephritisnone4m1024.0401743diffuse mesangial proliferative glomerulonephritissmall skin ecchymosis5f1232.031621focal proliferative lupus glomerulonephritisnoneaverage-9.825.1832931 - - bmi = body mass index (kg / m); ot = operative time (minutes); bl = blood loss (milliliters); gn = number of glomeruli per biopsy . Bmi = body mass index (kg / m); ot = operative time (minutes); bl = blood loss (milliliters); gn = number of glomeruli per biopsy . Although it is likely that the same approach could be used in adult patients as well . Our experience with this very initial group was composed entirely of children, and sprrb has been shown to be a very simple, safe and reliable alternative to other laparoscopic approaches . The use of a nephroscope, instead of a regular laparoscope, obviates the need to place an additional trocar for using an auxiliary instrument to dissect the perirenal fat, as is the standard practice (4, 5). Its working channel finely substitutes that, sparing one incision, the cost of another trocar and also surgical time to place it . Because a second trocar traditionally would be only 5 mm wide, it may seem that the benefit here is not strongly relevant in terms of postoperative pain or cosmetic results, but it is our understanding that no technical difficulty was added whatsoever, by using only one access . Moreover, especially children could benefit the most even of a small effect, and coincidently they constitute the majority of patients requiring a surgical renal biopsy in our hospital . Mini - perc nephroscopes are not available at our institution at this time, but its use could be a step forward, in this regard, and further decrease the required size of the access port incision . Additionally, the ease for urologists in using regular urological equipment, and the possibility that the surgeon simultaneously controls both the camera and laparoscopic scissor / biopsy forceps, are other advantages of this alternative method . In our hospital, retroperitoneal laparoscopy is the procedure of choice for renal biopsy in children and the sprrb is an even less invasive option for these patients, performed through a single incision and with very satisfactory results and only minor pain.
Cystic fibrosis (cf) is the most common autosomal recessive disorder among caucasians with an incidence rate of 1 in 2,500 individuals . The epithelial cells of several organs, including respiratory tract, exocrine pancreas, intestine, vas deferens, hepatobiliary system and also exocrine sweat glands are involved in cf . Therefore several clinical features, including suppurative lung disease, pancreatic insufficiency, neonatal bowel obstruction (meconium ileus), multifocal biliary cirrhosis, absent vas deferens to malabsorbtive condition and growth retardation could be seen in affected patients [2, 3]. High sweat electrolytes (chloride and sodium) concentration, which is seen in cf patients became basis for sweat chloride test since 1949 . The measurement of sweat electrolyte concentrations was established as a standard procedure for diagnosis of cf and remained the gold standard test for the diagnosis of cf . The diagnosis of cf could easily be made in the majority of cases based on typical clinical features and abnormal sweat chloride values . In such situations, genetic analysis of cystic fibrosis transmembrane conductance regulator (cftr) gene is not necessary . However, it may be useful in confirming the diagnosis, which also enables carrier testing and prenatal diagnosis [6, 7]. The universally accepted reference intervals for sweat chloride concentrations regardless of age or sex are> 60 mmol / l, which is considered diagnostic of cf; 4060 mmol / l is considered borderline, whilst <40 in addition to sweat chloride concentrations, sweat sodium is also usually measured, as there is little difference between sweat sodium and chloride concentrations [810] in order to ensure accurate results from a quantitative sweat test using filter paper, a minimum sweat rate of 1g / m / min corresponding to 75 mg collected in 30 minutes is required (for a 22 inch filter paper). Conventionally, it takes around 45 minutes to collect proper volume (50400 mg) sweat for accurate test, which makes the classic sweat test as a time consuming procedure . Indeed many local laboratories in developing countries have not been approved to do the test for both techniques of sweat gathering and also electrolytes measuring . Forming of salt crystallization of perspiration described by ferre calvete et al could be considered as a useful and alternative test for easy detecting cf patients in these regions . Therefore, we designed this study to compare the results obtained by these two methods . In this study, 60 children with clinical signs suggestive of cf, who were referred to the children's medical center, pediatrics center of excellence in iran, were investigated . The study protocol was approved by the research ethics board of the childrens medical center, tehran university of medical sciences . Classic sweat tests (gibson and cooke sweat test) and crystallization test were performed for each subject for at least two times in the referral laboratory of the hospital . Localized sweating was produced by iontophoresis of pilocarpine nitrate (gibson and cook method) using wescor gel discs [5, 12] a copper electrode was then attached and a weak electrical current of about 3 milli - amperes (ma) was generated using a 9-volt battery for 5 minutes to stimulate sweating . Immediately following stimulation, a preweighed filter paper was placed directly over the site of the positive electrode . At the end of the collection about one hour later, the filter paper was removed and the weight was determined . The test was repeated for two or three times in all subjects to confirm the results . Meanwhile one drop of each sample was put on lamella and heated by the light of microscope for 5 minutes . Sixty children (29 females and 31 males) with age range of 9 months to 2 years had taken part in this study . Meq / l, while it was 49.81meq / l in the male group without any significant difference between genders (p>0.05). Cf was diagnosed for the remaining 29 patients who showed positive values of sweat classic test . 1), which provided the test with 100% sensitivity (ci: 93.1100). Only one of 31 subjects without cf (17 males and 14 females; aged 9 months to 2 years) had positive crystallization test, which provided the test with 96.7% specificity (95%ci: 92.9100). Crystal formation in sweat sample of cystic fibrosis patients cystic fibrosis (cf) is one of the most frequent (1 in 2500) autosomal recessive diseases characterized by substantial clinical heterogeneity recent studies have begun to identify chromosomal locations that identify specific genes contributing to this variation . Over the past several decades, there has been substantial progress toward diagnostic tools of cf . Determination of chloride concentration in sweat is the current diagnostic gold standard for cf . The conventional sweat test with elevated sweat sodium and chloride concentration after iontophoresis of pilocarpine is the standard laboratory test for cf . An accurate sweat test relies on coordination of several factors . Technical error of instrument calibration and result reporting the tests should be performed by expert personnel to ensure sufficient sweat volumes and proper use of equipment . The centers doing such tests should follow standard guidelines to reduce complexity in interpreting a variety of result ranges [14, 15] advanced equipments and experienced personnel which are necessary for accurate classic sweat test made this test unavailable for many centers, especially in developing countries . Although sweat studies became standard diagnostic strategy for diagnosis of cf, it has some limitations whilst it may be unreliable due to not enough collected sweat or borderline values . Indeed genetic studies to detect cftr mutation(s) take time and may even find no useful information . Therefore selecting the best cost - benefit method with high sensitivity and specificity is needed for diagnosis of cf . For this reasons nanoduct as a new analyzing system measuring conductivity which requires only 3 microliters of sweat and gives results within 30 minutes is also introduced as a reliable diagnostic tool . Nanoduct has a failure rate comparable to other sweat tests and can be used as a simple bedside test for fast and reliable exclusion, diagnosis or suspicion of cf . Sands and his colleagues indicated that simultaneous bilateral sweat testing with two different methods (concentration and conductivity or nonoduct) provides an extra quality control system, allowing more time efficient organization of the diagnostic and training procedures . Forming of salt crystallization of perspiration seems as an attractive and alternative test for easy detecting cf patients . In this study, we have shown that looking for salt crystals in just one drop of sweat could diagnose cf, since crystal formation of sweat under light microscope was detected in a significant number of cf patients . Comparing these two methods of sweat test showed good positive predictive value of 96.7% and the negative predictive value of 100% with specificity and sensitivity of 96.7% and 100%, respectively . Therefore, the test could be a very useful alternative test, whenever the classic test is not accessible . Since the classic sweat test measuring chloride levels with the use of acceptable methods (gibson - cooke or wescor macroduct) should be performed in centers that conduct sweat tests frequently with properly documented controls, we recommend sweat crystallization test as an alternative test for cf diagnosis at least in areas where neither classic sweat test nor genotyping are accessible . Limitations: there were some limiting factors to consider in interpreting the study's result . First, this study was conducted on a relatively small sample size . Ideally, a larger and more popular sample size would perhaps delineate more suitable differences between the two methods of cf diagnosis in children with cystic fibrosis . Second, this study compared two kinds of test in children whose first presentation was compatible with cystic fibrosis, although this would not be statistically a problem . As further study, comparing two methods of the mentioned tests between cf patients and normal children could be more helpful . This study demonstrates the validity of sweat crystal formation test to support a diagnosis of cf in children whenever conventional sweat test is unavailable . In this study, 60 children with clinical signs suggestive of cf, who were referred to the children's medical center, pediatrics center of excellence in iran, were investigated . The study protocol was approved by the research ethics board of the childrens medical center, tehran university of medical sciences . Classic sweat tests (gibson and cooke sweat test) and crystallization test were performed for each subject for at least two times in the referral laboratory of the hospital . Localized sweating was produced by iontophoresis of pilocarpine nitrate (gibson and cook method) using wescor gel discs [5, 12] a copper electrode was then attached and a weak electrical current of about 3 milli - amperes (ma) was generated using a 9-volt battery for 5 minutes to stimulate sweating . Immediately following stimulation, a preweighed filter paper was placed directly over the site of the positive electrode . At the end of the collection about one hour later, the filter paper was removed and the weight was determined . The test was repeated for two or three times in all subjects to confirm the results . Meanwhile one drop of each sample was put on lamella and heated by the light of microscope for 5 minutes . Sixty children (29 females and 31 males) with age range of 9 months to 2 years had taken part in this study . Meq / l, while it was 49.81meq / l in the male group without any significant difference between genders (p>0.05). Cf was diagnosed for the remaining 29 patients who showed positive values of sweat classic test . 1), which provided the test with 100% sensitivity (ci: 93.1100). Only one of 31 subjects without cf (17 males and 14 females; aged 9 months to 2 years) had positive crystallization test, which provided the test with 96.7% specificity (95%ci: 92.9100). Cystic fibrosis (cf) is one of the most frequent (1 in 2500) autosomal recessive diseases characterized by substantial clinical heterogeneity recent studies have begun to identify chromosomal locations that identify specific genes contributing to this variation . Over the past several decades, there has been substantial progress toward diagnostic tools of cf . Determination of chloride concentration in sweat is the current diagnostic gold standard for cf . The conventional sweat test with elevated sweat sodium and chloride concentration after iontophoresis of pilocarpine is the standard laboratory test for cf . Technical error of instrument calibration and result reporting are major factors that affect the results . The tests should be performed by expert personnel to ensure sufficient sweat volumes and proper use of equipment . The centers doing such tests should follow standard guidelines to reduce complexity in interpreting a variety of result ranges [14, 15] advanced equipments and experienced personnel which are necessary for accurate classic sweat test made this test unavailable for many centers, especially in developing countries . Although sweat studies became standard diagnostic strategy for diagnosis of cf, it has some limitations whilst it may be unreliable due to not enough collected sweat or borderline values . Indeed genetic studies to detect cftr mutation(s) take time and may even find no useful information . Therefore selecting the best cost - benefit method with high sensitivity and specificity is needed for diagnosis of cf . For this reasons nanoduct as a new analyzing system measuring conductivity which requires only 3 microliters of sweat and gives results within 30 minutes is also introduced as a reliable diagnostic tool . Nanoduct has a failure rate comparable to other sweat tests and can be used as a simple bedside test for fast and reliable exclusion, diagnosis or suspicion of cf . Sands and his colleagues indicated that simultaneous bilateral sweat testing with two different methods (concentration and conductivity or nonoduct) provides an extra quality control system, allowing more time efficient organization of the diagnostic and training procedures . Forming of salt crystallization of perspiration seems as an attractive and alternative test for easy detecting cf patients . In this study, we have shown that looking for salt crystals in just one drop of sweat could diagnose cf, since crystal formation of sweat under light microscope was detected in a significant number of cf patients . Comparing these two methods of sweat test showed good positive predictive value of 96.7% and the negative predictive value of 100% with specificity and sensitivity of 96.7% and 100%, respectively . Therefore, the test could be a very useful alternative test, whenever the classic test is not accessible . Since the classic sweat test measuring chloride levels with the use of acceptable methods (gibson - cooke or wescor macroduct) should be performed in centers that conduct sweat tests frequently with properly documented controls, we recommend sweat crystallization test as an alternative test for cf diagnosis at least in areas where neither classic sweat test nor genotyping are accessible . Limitations: there were some limiting factors to consider in interpreting the study's result . Ideally, a larger and more popular sample size would perhaps delineate more suitable differences between the two methods of cf diagnosis in children with cystic fibrosis . Second, this study compared two kinds of test in children whose first presentation was compatible with cystic fibrosis, although this would not be statistically a problem . As further study, comparing two methods of the mentioned tests between cf patients and normal children could be more helpful . This study demonstrates the validity of sweat crystal formation test to support a diagnosis of cf in children whenever conventional sweat test is unavailable.
An important aspect of any research is the use of appropriate methodologies either to control or to reduce the effects of potential confounding factors . A matter of great concern that can influence the results of epidemiological studies of dental caries is the variation in disease diagnosis between two or more examiners (interexaminer error) and for the same examiner in two or more occasions (intraexaminer error). Therefore, it is very important that data collection measures are standardized in order to minimize measurements variations1 . The calibration process including the determination of reliability, with both previous and ongoing epidemiological survey, is a basic step to understand and standardize the examination criteria, and also to evaluate the interexaminer variability in order to ensure accurate results15,17 . The assessment of reliability is the most employed measure in dental caries surveys during the examiners' calibration . Reliability is related to the extent to which examiners agree in their evaluations16 . The most used measures to assess reliability in epidemiological studies of dental caries are the overall percentage of agreement and the kappa statistics11 . Kappa test is a measurement of reliability that takes into consideration the agreement among raters by chance, providing better evaluation of interexaminer disagreement during calibration processes5 . The purposes of this study were: a) to evaluate interexaminer reliability in caries detection considering different diagnostic thresholds and b) to indicate, by using kappa statistics, which is the best way of measuring interexaminer agreement during the calibration process in dental caries surveys . The epidemiological examinations were initiated after approval of the study design by the research ethics committee of the dental school of piracicaba, state university of campinas, brazil (protocol no . The volunteers' parents signed an informed consent form authorizing the enrollment of the children in the study . Dentists with previous experience in epidemiological surveys examined schoolchildren at baseline, 3 and 6 months after initial training, using two diagnostic thresholds on dental caries: who criteria, traditionally used in epidemiological surveys17 and who+il including the diagnostic of initial caries lesions (il, white spot lesions), after being calibrated by a " gold standard " examiner, a dentist who routinely uses the who criteria for exams and had been previously trained and calibrated in il diagnosis . Eleven dentists with previous experience in epidemiological surveys of dental caries were invited to participate in this study . Schoolchildren aged 6 - 7 years from two public schools in piracicaba, sp, brazil, were selected by a dentist, according to their caries activity . The dentists used mirror, cpi probe, air - drying for examination after the children brushed their teeth . The exclusion criteria were: use of fixed orthodontic device, presence of severe fluorosis and/or hypoplasias, and severe systemic diseases . For each training or calibration period of training, 10 to 13 different children were selected . Two diagnostic thresholds were used to record dental caries: 1) who threshold (dmft index) following the who codes and criteria17, in which a tooth is considered as decayed when a cavitation is present; 2) who+il threshold, in which active initial lesions were also recorded following criteria adapted from nyvad, et al.13 and fyfee, et al.6 . An il was defined as an active carious lesion which, upon visual assessment by a calibrated examiner, presented intact surface, no clinically detectable loss of dental tissue, with a rough, whitish / yellowish colored area of increased opacity presumed to be carious (when the cpi probe was used, its tip should be moved gently across the surface). Il locates close to gingival margin in smooth surfaces or extending along walls of fissure in occlusal surfaces . Each examiner was helped by a recorder during the study . A benchmaker examiner (" gold standard ") conducted the training processes with both theoretical and practical activities, which lasted 20 hours; and the calibration exercises, which lasted 8 hours at each phase: baseline, 3 and 6 months after initial training . The training and calibration processes were conducted by the gold - standard examiner using two diagnostic thresholds on dental caries, as described in previous studies2,3 . During theoretical discussions, the benchmaker examiner showed the examiners some photographic slides with clinical examples of each criterion that would be used in the study, in order to determine the examiners' knowledge about epidemiological diagnosis, to instruct them on the criteria and examination method to be used, and finally, to achieve an initial standardization among them . The clinical training consisted of 4 periods of 4 hours each, and was conducted in an outdoor setting . Each dentist examined 10 to 13 children, with distinct caries activity and prevalence, per period . During this phase, examiners discussed clinical diagnosis, study codes and criteria, recording and other errors in order to reach an acceptable level of agreement (kappa>0.8517). The calibration exercises, in which the examiners did not discuss their findings, were carried out in 2 periods of 4 hours each, with a 1-week interval . These were also undertaken after 3 and 6 months, after the first calibration phase (baseline). The epidemiological examinations were carried out in an outdoor setting, under conditions such as natural light, with dental mirror and ball - ended cpitn probes with a diameter of 0.5 mm (to remove debris, assess the presence of fissure sealants and, in case of doubt, to check the surface texture of il). Toothbrushing with fluoridated dentifrice was performed by the children, under supervision of a dental hygienist, following the bass modified technique during approximately two minutes . Tooth air - drying (approximately 5 seconds per tooth) was performed by using compressed air delivered by a dental compressor (wetzel: medical line 3.6/30 0.5 hp). A program using microsoft excel has been developed by the department of community dentistry of fop - unicamp to calculate the interexaminer reliability by means of the kappa statistics7 that has been recommended by the who17 and the british association of community dentistry15 for evaluation of agreement among examiners in oral health surveys . The code recorded for each dental unit or surface was entered for each examiner, in accordance with the different diagnosis thresholds (who; who+il) used in the three calibration phases . The objective of the statistical analysis was to assess interexaminer reliability under different caries diagnostic thresholds and different ways of analysis . Thus, interexaminer reliability was calculated by using kappa statistics, according to two diagnosis thresholds (who; who+il) and considering: a) the entire dentition; b) upper / lower jaws; c) sextants: upper / lower right / left, upper / lower anterior; d) each teeth individually . For each evaluation, codes from examination made by each examiner were compared to those of other examiners, (example: 1x2, 1x3 1x11; 2x3 . 2x11; 10x11). The epidemiological examinations were initiated after approval of the study design by the research ethics committee of the dental school of piracicaba, state university of campinas, brazil (protocol no . The volunteers' parents signed an informed consent form authorizing the enrollment of the children in the study . Dentists with previous experience in epidemiological surveys examined schoolchildren at baseline, 3 and 6 months after initial training, using two diagnostic thresholds on dental caries: who criteria, traditionally used in epidemiological surveys17 and who+il including the diagnostic of initial caries lesions (il, white spot lesions), after being calibrated by a " gold standard " examiner, a dentist who routinely uses the who criteria for exams and had been previously trained and calibrated in il diagnosis . Eleven dentists with previous experience in epidemiological surveys of dental caries were invited to participate in this study . Schoolchildren aged 6 - 7 years from two public schools in piracicaba, sp, brazil, were selected by a dentist, according to their caries activity . The dentists used mirror, cpi probe, air - drying for examination after the children brushed their teeth . The exclusion criteria were: use of fixed orthodontic device, presence of severe fluorosis and/or hypoplasias, and severe systemic diseases . For each training or calibration period of training, 10 to 13 different children were selected . Two diagnostic thresholds were used to record dental caries: 1) who threshold (dmft index) following the who codes and criteria17, in which a tooth is considered as decayed when a cavitation is present; 2) who+il threshold, in which active initial lesions were also recorded following criteria adapted from nyvad, et al.13 and fyfee, et al.6 . An il was defined as an active carious lesion which, upon visual assessment by a calibrated examiner, presented intact surface, no clinically detectable loss of dental tissue, with a rough, whitish / yellowish colored area of increased opacity presumed to be carious (when the cpi probe was used, its tip should be moved gently across the surface). Il locates close to gingival margin in smooth surfaces or extending along walls of fissure in occlusal surfaces . Each examiner was helped by a recorder during the study . A benchmaker examiner (" gold standard ") conducted the training processes with both theoretical and practical activities, which lasted 20 hours; and the calibration exercises, which lasted 8 hours at each phase: baseline, 3 and 6 months after initial training . The training and calibration processes were conducted by the gold - standard examiner using two diagnostic thresholds on dental caries, as described in previous studies2,3 . During theoretical discussions, the benchmaker examiner showed the examiners some photographic slides with clinical examples of each criterion that would be used in the study, in order to determine the examiners' knowledge about epidemiological diagnosis, to instruct them on the criteria and examination method to be used, and finally, to achieve an initial standardization among them . The clinical training consisted of 4 periods of 4 hours each, and was conducted in an outdoor setting . Each dentist examined 10 to 13 children, with distinct caries activity and prevalence, per period . During this phase, examiners discussed clinical diagnosis, study codes and criteria, recording and other errors in order to reach an acceptable level of agreement (kappa>0.8517). The calibration exercises, in which the examiners did not discuss their findings, were carried out in 2 periods of 4 hours each, with a 1-week interval . These were also undertaken after 3 and 6 months, after the first calibration phase (baseline). The epidemiological examinations were carried out in an outdoor setting, under conditions such as natural light, with dental mirror and ball - ended cpitn probes with a diameter of 0.5 mm (to remove debris, assess the presence of fissure sealants and, in case of doubt, to check the surface texture of il). Toothbrushing with fluoridated dentifrice was performed by the children, under supervision of a dental hygienist, following the bass modified technique during approximately two minutes . Tooth air - drying (approximately 5 seconds per tooth) was performed by using compressed air delivered by a dental compressor (wetzel: medical line 3.6/30 0.5 hp). A program using microsoft excel has been developed by the department of community dentistry of fop - unicamp to calculate the interexaminer reliability by means of the kappa statistics7 that has been recommended by the who17 and the british association of community dentistry15 for evaluation of agreement among examiners in oral health surveys . The code recorded for each dental unit or surface was entered for each examiner, in accordance with the different diagnosis thresholds (who; who+il) used in the three calibration phases . The objective of the statistical analysis was to assess interexaminer reliability under different caries diagnostic thresholds and different ways of analysis . Thus, interexaminer reliability was calculated by using kappa statistics, according to two diagnosis thresholds (who; who+il) and considering: a) the entire dentition; b) upper / lower jaws; c) sextants: upper / lower right / left, upper / lower anterior; d) each teeth individually . For each evaluation, codes from examination made by each examiner were compared to those of other examiners, (example: 1x2, 1x3 1x11; 2x3 . For both diagnostic thresholds, high mean values of kappa were obtained (tables). Moreover, interexaminer agreement was constant when considering the entire dentition, jaws and sextants (tables 1 and 2). Kappa values above 0.85 were obtained by analysis of sextants according to who threshold . However, when considering the who+il threshold, the values for posterior sextants decreased (tables 1 and 2). The results of interexaminer reliability considering each tooth individually showed that the main difficulty was related to caries diagnosis in posterior teeth, especially the permanent first molars, for both thresholds with, in general, lower values for the who+il threshold (tables 3 and 4). E1 = exercise at baseline; e2= exercise after 3 months; e3= exercise after 6 months . E1=exercise at baseline; e2= exercise after 3 months; e3= exercise after 6 months . The process of examiners' calibration is an important aspect in planning and conducting oral health surveys . Brazilian surveys4,10 have shown that training and calibration of examiners have been an aspect of great concern for measuring interexaminer agreement, according to the recommendations of the who, which has indicated the use of kappa statistics17 . Kappa test provides a better evaluation of disagreement among examiners during calibration processes since it is a measurement of adjusted agreement by taking into consideration the ratio of chance agreement5 . Analysis of variance and post - hoc tests, such as scheff, have also been used to assess significant differences in caries indices among examiners9 . The present study showed, in general, high means of interexaminer reliability for both diagnosis thresholds when considering the entire dentition, the upper / lower jaws and sextants (tables 1 and 2). On the other hand, lower kappa values were observed for dental units (each tooth individually), especially when considering the most sensitive diagnosis threshold (who+il) (tables 3 and 4). As a consequence, good interexaminer agreement for the entire dentition may not be as real as if one considers separately the posterior teeth, in which cavitated and non - cavitated carious lesions are concentrated8 . Moreover, considering the current epidemiological profile of dental caries, the higher number of sound teeth (fewer errors in diagnosis) in comparison to carious teeth (more errors in diagnosis) may dilute the errors attributed to carious teeth, leading to a positive vision of the results achieved in examiners' calibration14 . Therefore, one may speculate that the analysis of kappa values considering the entire dentition rather than each tooth individually may not be the best way to evaluate interexaminer reliability, especially in areas with low caries prevalence . It may be suggested the need for future reformulations in conducting examiners' calibration, paying more attention to diagnosis of posterior teeth and selecting children of distinct caries activity and prevalence1 . Low kappa values under the who+il threshold can also be explained by the inherent difficulty in diagnosing il, especially in surveys1 . Although the examinations were carried out in sunny days under high luminosity conditions, the use of artificial light could generate an increase in interexaminer agreement by facilitating the view of posterior teeth . Further studies are needed to determine the relevance of using artificial light in dental caries surveys, mainly for detecting initial lesions . In general, the use of more detailed measures to determine interexaminer agreement, such as the evaluation by dental unit (each tooth individually), improves the calibration process by showing which teeth are leading to great disagreements and indicating the possible need for greater efforts in training examiners14 . However, it must be emphasized that the method of evaluating interexaminer agreement also depends on the study design and objectives, the desired degree of accuracy and the available resources . As an example, the calibration process using more rigorous statistical measures, such as the analysis by dental units, would be indicated in either clinical trials or case - control studies, in which the effect of preventive measures on the reduction of caries levels, including the detection of initial lesions, must be evaluated . Therefore the kappa statistics considering reliability values according to each code / clinical condition can be employed12 . On the other hand, in order to know and evaluate the epidemiological profile of dental caries in an underprivileged community, for instance, these more robust measures could be dispensed . It is important to mention that the present study, which is part of a 12-month longitudinal study on examiners' calibration, presents some limitations, such as lack of validity results by comparing the examiners' results to those from the gold - standard examiner, and lack of intraexaminer errors . Such measures were not taken into consideration because the main goal of this study was to evaluate interexaminer reliability by using different diagnostic thresholds for caries detection as well as to verify the behavior of kappa statistics in order to indicate the most adequate way to measure reliability during the examiners' calibration process in dental caries surveys . The results of this study showed that the interexaminer reliability and its maintenance for six months were possible, under both caries diagnosis thresholds . Nevertheless, great disagreement was observed for the posterior teeth, especially when the who+il criteria were used . The analysis considering dental elements individually was the best way of detecting disagreements among examiners during the calibration sections.
Dna microarray analysis has gained widespread application and a wide variety of methods have been developed to analyze the large and complex datasets generated by this technology (1). Due to the sheer volume of data, and the high number of sources of potential error, quality control and quality assurance since error may be the result of many factors at multiple steps, a number of qc / qa measures have been proposed to monitor specific sources of error . Most of these methods focus on individual spots or spots within a single array (3). Other methods monitor an individual array as a whole during the experimental process using classification methods (4) or by comparison of the statistical features of an array with the same statistical features based on historical data (5). Here we report an r function (www.rproject.org) named microarray outlier filter (mof), which was designed to screen outliers at the whole array level by using the arrays from the current experiment and those from the historical archive that meet defined criteria . Our lab now routinely applies this software to the qa of our microarray experiments (6). In essence, mof examines the consistency of arrays in a large scale experiment (multiple arrays). Arrays, as a whole, are inspected to reveal those arrays that are obviously different from most others . These few abnormal arrays are most likely failed arrays that may contain unreliable data . This analysis is based on two assumptions: first, that all samples are similar in gene expression profile (that is, technical or biological replicates) and that they have been subjected to the same experimental procedure (in principle, the results should be similar between arrays since expression levels should be uniform for the majority of the probes); second, that data from most of the arrays are of good quality, resulting in only a few unusual arrays being labeled as potentially failed ones . Otherwise, it is possible for a systematic experimental error to result in the few good arrays being suspected as failures because they are identified as abnormal by mof . Note that historical arrays satisfying these two criteria can be included to generate a larger dataset for analysis of the outliers . An outlier data point is defined in the context of all the data points for the same specific probe, across all the arrays . The resistant z - score is used to tag an outlier data point, which is defined as: zi = xixswhere x and s are the median and median absolute deviation values, respectively, for each probe across all arrays . A data point is designated as an outlier if its resistant z - score falls outside a preset threshold, which may be 3, 4 or 5 in our experience . The percentage of outliers among all data points in consideration from an array is used as an indicator of the quality of the array . This is based on our observation that an outlier array, which is largely different from the majority of comparable arrays in its gene expression profile, tends to have more data points distributed at extremes . The other statistical index is the pearson correlation coefficient, expressed as: r=i=1k(xix)(yiy)(n1)sxsywhere x and y are two variables of the signal intensities from the two arrays, n is the number of probes included, x and y stand for means, sx and sy stand for standard deviations of two variables, respectively . The correlation coefficient between two arrays is computed by a function provided in the r package . Each array is represented by a collection of data points in the same order of probes . Thus, the correlation coefficient reflects the similarity of the two arrays in regard to the expression levels for all the probes collectively . If an array displays apparently low correlation or even reverse correlation to many other arrays, it is flagged as an unusual array merit closer review for failure . Therefore, mof can be used to assist in the detection of potentially failed arrays . As an r function, mof is a ready - to - use tool for listing the arrays by the possibility of failure . It does not mean that there must be failed arrays in each batch of arrays . Thus it is very important for the user to further verify that the outlier arrays are actually failed arrays by other means, such as scatter plots, clustering, etc ., starting from the worst outlier arrays . In our experience, the two lists determined by the mof indices often show the same unusual arrays, thus confirming each other . However, since these two indices do not reflect exactly the same properties of the data, they may detect different abnormalities in the arrays and thus complement each other . Again, additional validation is critical to identify the truly failed arrays and the underlying causes . The input text file is a matrix containing preprocessed microarray data with rows as probes and columns as arrays . For maximum reliability, data points with expression levels close to the background or within the scanner saturation range should be discarded . The output is three text files and two heat maps . In the first text file, two lists of arrays are ordered according to the average correlation coefficient to the rest of the arrays and percentage of outlier spots, respectively . A correlation coefficient table containing the correlation coefficients for all pairs of arrays is found in the second text file . A heat map is generated to provide a general visualization of this table (figure 1a) as a guide for the utilization of the data for additional detailed analysis . Similarly, percentages of outlier spots for all the arrays are also given as a table in a text file and a corresponding heat map (figure 1b). In essence, mof examines the consistency of arrays in a large scale experiment (multiple arrays). Arrays, as a whole, are inspected to reveal those arrays that are obviously different from most others . These few abnormal arrays are most likely failed arrays that may contain unreliable data . This analysis is based on two assumptions: first, that all samples are similar in gene expression profile (that is, technical or biological replicates) and that they have been subjected to the same experimental procedure (in principle, the results should be similar between arrays since expression levels should be uniform for the majority of the probes); second, that data from most of the arrays are of good quality, resulting in only a few unusual arrays being labeled as potentially failed ones . Otherwise, it is possible for a systematic experimental error to result in the few good arrays being suspected as failures because they are identified as abnormal by mof . Note that historical arrays satisfying these two criteria can be included to generate a larger dataset for analysis of the outliers . An outlier data point is defined in the context of all the data points for the same specific probe, across all the arrays . The resistant z - score is used to tag an outlier data point, which is defined as: zi = xixswhere x and s are the median and median absolute deviation values, respectively, for each probe across all arrays . A data point is designated as an outlier if its resistant z - score falls outside a preset threshold, which may be 3, 4 or 5 in our experience . The percentage of outliers among all data points in consideration from an array is used as an indicator of the quality of the array . This is based on our observation that an outlier array, which is largely different from the majority of comparable arrays in its gene expression profile, tends to have more data points distributed at extremes . The other statistical index is the pearson correlation coefficient, expressed as: r=i=1k(xix)(yiy)(n1)sxsywhere x and y are two variables of the signal intensities from the two arrays, n is the number of probes included, x and y stand for means, sx and sy stand for standard deviations of two variables, respectively . The correlation coefficient between two arrays is computed by a function provided in the r package . Each array is represented by a collection of data points in the same order of probes . Thus, the correlation coefficient reflects the similarity of the two arrays in regard to the expression levels for all the probes collectively . If an array displays apparently low correlation or even reverse correlation to many other arrays, it is flagged as an unusual array merit closer review for failure . Therefore, mof can be used to assist in the detection of potentially failed arrays . As an r function, mof is a ready - to - use tool for listing the arrays by the possibility of failure . It does not mean that there must be failed arrays in each batch of arrays . Thus it is very important for the user to further verify that the outlier arrays are actually failed arrays by other means, such as scatter plots, clustering, etc ., starting from the worst outlier arrays . In our experience, the two lists determined by the mof indices often show the same unusual arrays, thus confirming each other . However, since these two indices do not reflect exactly the same properties of the data, they may detect different abnormalities in the arrays and thus complement each other . Again, additional validation is critical to identify the truly failed arrays and the underlying causes . The input text file is a matrix containing preprocessed microarray data with rows as probes and columns as arrays . For maximum reliability, data points with expression levels close to the background or within the scanner saturation range should be discarded . The output is three text files and two heat maps . In the first text file, two lists of arrays are ordered according to the average correlation coefficient to the rest of the arrays and percentage of outlier spots, respectively . A correlation coefficient table containing the correlation coefficients for all pairs of arrays is found in the second text file . A heat map is generated to provide a general visualization of this table (figure 1a) as a guide for the utilization of the data for additional detailed analysis . Similarly, percentages of outlier spots for all the arrays are also given as a table in a text file and a corresponding heat map (figure 1b). Using mof requires setting proper thresholds for the two statistics to flag problematic arrays . In our practice we set the cut - offs at 0.8, 3, and 6% for pearson correlation coefficient, z - value, and the outlier percentage, respectively . Then, common arrays on the two lists reported by mof satisfying the thresholds respectively can be considered primary candidates of problematic arrays . Another way to set the threshold is to take the top 15%20% worst arrays as indexed in the two lists, and then identify common arrays . We would like to caution that the user needs to adjust the threshold based on their experience and the available resources . For reference, we report here two scenarios in using mof, illustrated with experimental data generated by our core facility . In one scenario, a couple of problematic arrays are so different from the rest of the arrays that the two statistics effectively singled them out . For example, we have a dataset composed of 35 arrays using the universal human reference (uhr) rna sample (stratagene, la jolla, usa), both indices flagged the same 3 arrays as outlier arrays (figure 1). Each of the 3 arrays had a distinctly lower average correlation coefficient and higher percentage of outlier spots than other arrays . In details, pearson correlation coefficients were 0.22, 0.30, and 0.49 for the top 3 arrays, and> 0.76 for the rest; taking \|z\|=3, the same 3 arrays contained outliers of 28%42%, with the fourth showing 11% and the rest showing 5% or less . Two of these three arrays were confirmed by the laboratory as failed experiments with reasons identified . In another scenario, there is a lack of outstanding problematic arrays and on the two lists reported by mof the statistical index changes relatively smoothly, from low to high for pearson correlation coefficient or from high to low for z - score reported outlier percentages . For example, in a dataset composed of 185 arrays of human blood samples (30 controls and 155 patients with breast disease), following the thresholds we have about 30 arrays from either reported list to work on . 12 arrays were common in the top 30 arrays on both lists and were later verified to have obvious problems by scatter plots . Mof has been successfully applied to our in - house microarray data as a routine qa measure (6). However, users should note that in order to apply mof to any microarray datasets, the two assumptions mentioned above must be met . In our practice, when applying mof to a mouse dataset from different tissue types, tissue specific clusters were generated apparently due to the differences in gene expression profiles between tissue types . In such a case, the first of the two assumptions was not satisfied and thus mof was not applicable . As an additional note, the percentage of outliers changes when using different z - scores . Selecting different z - scores may make some arrays to stand out with apparently higher percentages of outlier data points . Therefore, the relative level of percentage of outliers is a more meaningful characteristic for judgment than the absolute value itself . Users are encouraged to identify more realistic cut - offs in their specific settings using our thresholds as guidance . Although a minimal of three arrays is required for correlation coefficient to pick out an outlier array, we recommend that mof should be used on datasets comprised of ten or more arrays . Historical data obtained with the same technology on the same tissue type can be used in the analysis to increase the normal array base.
Tsp-1 is the best - studied member of the thrombospondin (tsp) family, which consists of five extracellular calcium - binding multifunctional proteins: tsp-1, tsp-2, tsp-3, tsp-4, and tsp-5 . Tsp-1 and tsp-2 are structurally similar, and they are expressed on the cell surface during physiological events . A variety of normal cells, including endothelial cells, fibroblasts, adipocytes, smooth muscle cells, monocytes, macrophages, and transformed cells such as malignant glioma cells, secrete tsp-1 [2, 3]. Tsp-1 binds to protein components of the extracellular matrix, such as fibronectin . By this way, tsp-1-specific domains bind to proteoglycans, membrane proteins such as integrins, and other matrix proteins expressed by a variety of cells [4, 5]. Tsp-1 contains an n - terminal globular domain that binds heparin, the type i, type ii, and type iii repeats, and a terminal globular domain . The nh2-terminal, heparin - binding domain of tsp-1 interacts with low - density lipoprotein receptor - related protein (lrp1). This tsp-1 domain also binds heparin sulfate proteoglycans and a number of integrins that have an important function in angiogenesis, chemotaxis adhesion, and cell motility . All five members of the tsp family have the repeat domains type ii and iii, but only tsp-1 and tsp-2 contain the type i repeats . Type i repeats, also called thrombospondin structural homology repeats (tsrs), inhibit angiogenesis by activating cd36 and inducing apoptosis in endothelial cells . Cd36 (also known as fatty acid translocase, fat) is a glycosylated protein member of the class b scavenger receptor family . It plays an important role in multiple processes such as fatty acid and glucose metabolism . Cd36 is found on the surface of diverse cell types and binds to many ligands, including tsp-1 [10, 11]. It has been reported that upon binding with tsp-1, cd36 dimerizes, becoming actively involved in signal transduction . However, activation of cd36 as a monomer has also been reported . The adhesive and antiangiogenic functions of tsp-1 have been mainly attributed to its interaction with cd36 . Other domains of tsp-1 can, however, impact these functions by interacting with other key receptors as it will be discussed in succeeding sections . Tgf1 mediates wound healing, cell proliferation, extracellular matrix formation, and the immune response . This multifunctional cytokine is secreted to the extracellular matrix in its inactive form, by virtue of its noncovalent association with the latency - associated peptide (lap). The activating function of tsp-1 is due to the amino acid sequence rfk located in the tsr [1416]. Tsp-1 releases tgf1 from its latent form when it interacts with the n - terminal region of lap and binds the mature tgf1 . This interaction results in the formation of a complex that involves conformational changes in tgf1, making it accessible to its receptor . Lap is crucial for tgf1 activation and regulates many of its functions; additionally, lap has functions in inflammation independently of tgf1, such as the induction of chemotaxis of monocytes to injured tissues . They contain amino acid sequences that interact with the neutrophil elastase, and upon this binding these repeats activate neutrophils [18, 19]. These type 3 repeats also inhibit the binding of fibroblast growth factor to endothelial cells, reducing angiogenesis . The cooh - terminal domain of tsp-1 binds to cd47, also known as integrin - associated protein . This domain also interacts with integrins such as 1 and v6 integrins and actively binds to proteoglycans allowing cell adhesion and spreading . These and other interactions significantly affect angiogenesis, cell proliferation, and immune responses . Tsp-1 binding with cd47 also regulates nitric oxide (no), a biogas, quite important in both normal and pathological events . By modulating the effects of no, the carboxy - terminal domain of tsp-1 has important function in vasodilation and chemotaxis . This receptor inhibits no as well as all its vascular functions even when tsp-1 is present at very low (physiological) concentrations . Analysis of wound bed vascularity at 72 hours after skin grafting from tsp-1 and cd47 null mice shows significant increased numbers of blood vessels . Most recently it has been reported that cd47 associates with the receptor of vascular endothelial growth factor, vegfr2 . However, the binding of cd47 with tsp1 or other ligands inhibits vegfr2 phosphorylation and further angiogenesis . This paper focuses on well - known interactions of tsp-1 with key receptors and growth factors during the initial inflammatory events throughout the chronic inflammatory processes . New developments are also herein discussed, showing the involvement of tsp-1 in pivotal transcriptional pathways related to inflammation and inflammation - induced carcinogenesis . The inflammatory acute process begins when cells sense the injury, and they release chemical mediators called cytokines . Local macrophages express surface membrane receptors called toll - like receptors (tlr) that recognize specific types of antigens . Once activated, tlr triggers the release of more cytokines promoting inflammation and attracting white blood cells . Cytokines will promote leukocytosis by inducing factors favoring the rapid release of neutrophils from the red bone marrow . Neutrophils enter the blood stream, and by diapedesis they emigrate outside the blood vessels . Chemotactic agents accelerate the migration of leukocytes to the site of injury such as monocytes, which later become macrophages, engulfing any on - site cell debris or pathogens . In addition, mast cells (producing histamine), injured tissue cells, phagocytes, lymphocytes, basophils, and blood proteins are all sources of inflammatory mediators . Tsp-1 is transiently released early during the acute phase of inflammation, and multiple factors seem to modulate the release of tsp-1 during this process . Tsp-1 is strongly expressed in neutrophils, inducing an intense chemotactic response to injured tissues . Tsp-1 is secreted in response to inflammation, promoting the resolution of the inflammatory process and facilitating phagocytosis of damaged cells [25, 26]. Thus, enhanced production of tsp-1 could be a compensatory mechanism for controlling the immune response and protecting tissues from excessive damage . This receptor is coexpressed with tsp-1 in macrophages and endothelial cells, and, by binding with cd36 (figure 2), tsp-1 induces apoptosis in endothelial cells . By activating cd36, tsp-1 also controls blood flow and leukocyte infiltration modulating the action of the no pathway in injured tissues . No is a gas produced when l - arginine is converted to l - citruline by the enzyme nitric oxide synthase (nos). There are four different isoforms of nos, neuronal (nnos), endothelial (enos), mitochondrial (mtnos), and the inducible isoform (inos). The first two are secreted during normal physiological events, but only inos is expressed upon inflammatory stimuli . The effects of no in inflammation have been extensively recognized in a variety of studies . No can modulate leukocyte adhesion in a dose - dependent manner . At low doses, no is anti - inflammatory and antiangiogenic but, after inflammatory stimuli, high levels of no are secreted promoting angiogenesis and leukocyte adhesion to the endothelium . Tsp-1 could inhibit the soluble guanylyl cyclase system in endothelial cells and consequently the activation of no by interacting with cd36 and cd47 . Through this mechanism, tsp-1 inhibits inflammation by blocking adhesion and activation of leukocytes to the endothelium and diminishing angiogenesis [22, 30, 31]. Another factor interacting with tsp-1 during early inflammation is the peroxisome proliferator - activated receptor (ppar). Ppar greatly enhances the proapoptotic effects of the tsp-1-derived peptide abt510 (abbott laboratories) [33, 34]. This peptide corresponds to the tsr of tsp-1 and induces vascular apoptosis in vitro and in vivo through its interaction with cd36 . By using a ppar agonist, the expression of cd36 in endothelial cells the tsp-1 receptor cd47 is critical for the migration of leukocytes through endothelial and epithelial barriers . Cd47 is strongly expressed in polymorphonuclear cells, and its activation enhances the expression of tsp-1 in leukocytes . Cd47 can directly cause apoptosis through mitochondrial mechanisms, or by activation of the fas / cd95 pathway . Expression of cd47 in apoptotic granulocytes can influence the phagocytic functions of the macrophages in inflammatory sites suggesting a critical role of this factor in the resolution of the process (figure 2). Acute inflammation could advance to a resolution, progress to the formation of an abscess, walling off by fibrotic capsule, or evolve as scar upon tissue destruction, fibrin and collagen deposition . Chronic inflammation is characterized by infiltration of mononuclear cells, macrophages, lymphocytes, and plasma cells . Chronically inflamed tissues have fibroblast proliferation, angiogenesis, tissue destruction, and fibrosis . They invade the injured area during the acute process but, if the cause is not eliminated, infiltration by macrophages persists for long periods of time . The continued secretion of chemotactic factors allows the constant supply of monocytes from the blood and their conversion to macrophages . These cells are key for further lymphocyte infiltration, fibroblast proliferation, tissue destruction, and fibrosis . Lymphocytes arise from the hemoblasts of the bone marrow, and later they develop immunocompentence and self - tolerance . Plasma cells or b lymphocytes produce antibodies against antigens persisting in the area and therefore provide humoral immunity . Included in this group are dendritic cells (dcs), which internalize antigens and present antigenic determinants on their surface for recognition by t lymphocytes . They are part of the adaptive immune system that recognizes something as foreign and acts to immobilize and remove it . During the early stages of injury and inflammation, high levels of tsp-1 can modulate inflammation by inhibiting or enhancing the secretion of the cytokine interleukin 10 (il10), by this way, tsp-1 can also regulate the functions of dc . In addition, after adding il-6, il-10, or tgf1 to cultured dc, they become immune tolerant and show upregulation of intracellular tsp-1 . Tsp-1 also inhibits the function of apc by suppressing their capacity to sensitize t - cells in the host . This is demonstrated in a corneal transplantation model, in which most of the corneal tsp-1 null allografts are rejected . Cd47 has also a crucial role in t - cell activation [42, 43]. Interaction of tsp-1 with cd47 promotes the activation of thymus - derived cd4 + cd25 + t regulatory cells (tregs). Through this mechanism suppression of cd47 or tsp-1 expression in dc by using small interfering rna (sirna) technique actually protects newborn mice against bacterial (escherichia coli) meningitis . Again, the loss of cd47 activity prevents the maturation of the dcs and the production of inflammatory cytokines . In conclusion, cd47 seems to have pivotal functions in inflammation and provides a major mechanistic pathway for the functions of tsp-1 in that process . Cd36 mice exhibit an impaired early proinflammatory response to infection, elevation of cytokines, and higher mortality [45, 46]. These findings suggest that cd36 is quite critical for the recognition and clearance of pathogens in acute and chronic infections . By binding to this receptor, tsp-1 could modulate the inflammatory process by activating macrophages and favoring phagocytosis . During chronic inflammation, these adaptive immune mechanisms provide defense against disease and are regulated by cellular interactions and cytokines . B lymphocytes secrete antibodies that bind to infectious agents and label them for destruction or elimination . Once inside a cell, a pathogen becomes inaccessible to those antibodies and cytotoxic t cells could destroy them by inducing apoptosis of the cell host . Regulatory t cells can modulate the secretion of cytokines enhancing the functions of macrophages and b - lymphocytes . Tsp-1 has been reported to decrease immune responses by inhibition of t - cell effectors, or by directly inducing t cell apoptosis [47, 48]. In addition, by binding to 41 integrin tsp-1 promotes t - cell adhesion and chemotaxis . Tgf1 activation is a crucial element in intestinal homeostasis . In the intestinal tract, an abnormal response to the normal gut flora is a characteristic of the pathogenesis of inflammatory bowel disease (ibd). Mucosal t cells from patients with ibd express high levels of smad7, an inhibitor of tgf1 signaling . By this mechanism, tgf1 mediates intestinal healing and susceptibility to injury [5053]. However, by activating tgf1, tsp-1 also enhances fibrosis and compromises organ function [54, 55] (figure 3). During the immune response, leukocytes produce reactive oxygen species (ros) that include free radicals and peroxides . Ros are quite important for the killing of pathogens, but they can also produce cell damage . Tgf1 favors the formation of ros, and, as a cycle, ros can also activate tgf1 promoting apoptosis and fibrosis . Angiogenesis is an active component of chronic inflammatory diseases such as rheumatoid arthritis, atherosclerosis, diabetic retinopathy, airway inflammation, and others . Tsp-1 exerts a powerful antiangiogenic effect, and this function has a significant impact in chronic inflammation . Activated endothelial cells secrete cytokines, chemokines, matrix metalloproteinases, and growth factors that can greatly influence the inflammatory reaction and promote carcinogenesis . Tsp-1 deficient mice display extensive acute pneumonia, leukocytosis, pancreatitis, and inflammatory infiltrates in the lacrimal glands [26, 58, 59]. This phenotype suggests that tsp-1 has a significant role in inflammation, a role that has not been exhaustively analyzed . Tsp-1 has anti - inflammatory and proinflammatory effects observed in several diseases and animal models . These contrasting functions in inflammation are possible due to interactions with multiple receptors or to the presence of specific matricellular proteins in injured tissue . In addition, tsp-1 might act by a biphasic or dose - dependent mechanism . Tsp-1 enhances fibrosis and renal damage by its interaction with tgf1, while lskl, a peptide antagonist of tsp-1, reduces renal interstitial fibrosis in a rat experimental model of kidney disease . This effect is attributed to the competitive properties of lskl that prevents tsp-1-mediated activation of tgf1 . Tsp-1 is also expressed in glomerulopathies and is considered an early marker of inflammation and fibrosis . The development of diabetic nephropathy is attenuated in tsp-1-deficient mice as demonstrated by a significant reduction of glomerulosclerosis, glomerular matrix accumulation, podocyte injury, renal infiltration with inflammatory cells, and alterations of renal functional parameters . As an activator of tgf1, tsp-1 could modulate the functions of tgf1 in cardiovascular diseases, atherosclerosis, and obesity . Inflammatory cells secrete tsp-1 during the acute phase of the healing process in myocardial infarction . In addition, tsp-1 is selectively expressed in the infarcted border suggesting that tsp-1 might inhibit the expansion of the inflammation by activating tgf1 . In the inflammatory processes leading to atherosclerosis, tsp-1 deficiency enhances inflammation by accelerating plaque maturation and necrosis in apoe - deficient mice . However, tsp-1 expression is increased in response to high glucose in the wall of large vessels and accelerates atherosclerosis and other pathological events observed in diabetes . This correlation is explained by the tsp-1-dependent tgf1 activation that leads to upregulation of plasminogen activator inhibitor 1 (pai-1) gene expression . Elevated circulating pai-1 levels mice lacking tsp-1 or the integrin 6 subunit (tsp-1 and itgb6 mice, resp .) The inflammation is not as severe as the one found in tgfb1 mice, which develop marked infiltrates of activated t cells in multiple organs and die soon after weaning . Tsp-1 null mice have a more severe course of acute induced colitis; they display increased bleeding and colonic inflammation compared to wt mice controls . Tsp-1-deficient mice under multiple cycles of 2.5% of dextran sodium sulfate for induction of colitis die before the cycles are completed . These mice show high incidence of megacolon and peritonitis due to the deeper infiltration of leukocytes into the muscularis and intestinal perforation . In order to increase survival, the use of the tsp - mimetic peptide abt510 drastically reduces intestinal inflammation and angiogenesis and enhances the expression of cd36, which suggests that cd36 modulates inflammation in this model . Additionally, the interaction of tsp-1 with the no pathway seems to be involved in the antiangiogenic mechanisms mediated by the abt510 in cancers [31, 69], and it might also explain the anti - inflammatory effects of this peptide in the colitis model . Similarly, in a rat model with bleomycin - induced lung injury, a cd36 peptide also decreases inflammation and fibrosis in the lungs . A synthetic peptide derived from the tsp-1 type 3 repeats (figure 1) also diminishes inflammation and angiogenesis in an experimental model of erosive arthritis . This peptide decreases angiogenesis, leukocyte infiltration, and thickening of the synovial lining of the joint . In the spleen and liver, this peptide significantly reduces the formation of granulomas . However, tsp-1 is upregulated in monocytes, and tissues from patients with rheumatoid arthritis (ra) [74, 75] and high plasmatic levels of tsp-1 are correlated with proinflammatory cytokines in these patients . Tsp-1 also exerts proinflammatory effects in certain types of myositis [77, 78]. The chemotactic effect of tsp-1 in leukocytes by its binding to cd47 perpetuates the muscle inflammation in response to high levels of tnf - alpha . In transplantation research, most of the corneal tsp-1 null allografts are rejected, suggesting a protolerogenic function [41, 79]. Interestingly, when tsp-1 is inhibited by sirna - transfection in pancreatic islet cells, improved revascularization and function are observed in the pancreatic grafts . Antiangiogenic compounds, such as tsp-1, might be useful for achieving a better perfusion and oxygenation in organ transplants . It is involved in many biological processes such as inflammation, immunity, differentiation, cell growth, tumorigenesis, and apoptosis . Phosphorylation and subsequent degradation of ib releases nf-b and allows it to be translocated to the nucleus . Once inside the nucleus, this transcription factor induces its target genes, thereby inhibiting apoptosis, promoting inflammation, angiogenesis, and carcinogenesis . Importantly, nf-b activates the transcription of genes such as tnf - alpha and il-6, which have major roles in regulating the immune response . Recently, it has been reported that blocking the activation of nf-b upregulates tsp-1 expression in rat granulation tissue . Tsp-1 has a major role in wound healing, and a significant delay of the healing process is observed in tsp-1 mice . The link between tsp-1 and nf-b seems to be closely related to mechanisms of angiogenesis and carcinogenesis . Tsp-1 enhances the binding of nf-b to dna inhibiting angiogenesis, and these events are reverted by blocking the nf-b pathway . Activation of nf-b by oxidized low - density lipoprotein (oxldl), a specific ligand of cd36, is reduced in macrophages of patients with cd36 deficiency . Studies in prostate cancers indicate that the combined decrease of nf-b and increase of tsp-1, modulated by the expression of the androgen receptor, exert antitumor effects . The tsp-1-derived peptide angiocidin has antitumor effects and induces the differentiation of monocytes to macrophages by activating the nf-b pathway . In response to cytokines and growth factors, members of the stat family are phosphorylated by receptor - associated kinases forming homo- or heterodimers . The lack of stat3, the most important member of this family, actually leads to embryonic lethality . Stat3 mediates the expression of a variety of genes in response to cell stimuli and thus plays a key role in many cellular processes such as cell growth and apoptosis . The binding of il-6 to the gp130 receptor triggers the phosphorylation of stat3 by jak2 [90, 91]. It has been reported that downregulation of tgf1 in epithelial cells inhibits the secretion of il-6 . By this mechanism furthermore, activated stat3 has been recently reported as crucial for intestinal carcinogenesis in colitis - associated cancer . In addition, protein profiling of head and neck squamous carcinomas shows a significant decrease of several proteins such tsp-1 and tgf1 with concomitant increase of stat3 . In a model of angiogenesis, higher levels of il-6 are secreted by tsp-1 aortic explants, and similar results are found in a mouse model of colitis . The higher levels of il-6 are lowered upon activation of tgf1 only in wt aortas . The activity of il-6 was specifically examined through the stat3 pathway in colonic tissues evaluating the status of the phosphorylated forms of stat3 (p - stat3). Interestingly, p - stat3 expression is highly expressed in tsp-1 colons and almost abolished in colons from mice treated with a new tsp-1-mimetic abt-898 (abbott laboratories). These data suggest that this mimetic peptide directly binds to tsp-1 receptors such as cd36 or indirectly interacts with other membrane domains to downregulate stat3 pathway, thereby depressing the immune response . Therefore, activation of this pathway is critical for further activation of stat3 in colitis . Tsp-1 interacts with a variety of factors in a synergistic way, playing a crucial role in many stages of the inflammatory response . We discussed herein the interactions of tsp-1 with well - known partners such as cd36, cd47, no, and tgf1 . We also reviewed recent developments explaining how tsp-1 and its derived peptides directly or indirectly regulate inflammatory events in animal models and human diseases . Leukocytes migrate to areas of injury or infection in elevated numbers; similarly, tsp-1 is secreted at high levels in some areas in order to activate specific mechanisms for regulating the inflammatory response . In some cases finally, new insights about key signaling pathways such as nf-b and stat3 were discussed . Because chronic inflammation is linked with tumor development, further studies analyzing the role of tsp-1 in these pathways could elucidate the similar contradictory functions of tsp-1 in cancer . The unraveling of these mechanisms will make possible the development of new and more effective therapies for controlling inflammation and blocking carcinogenesis.
Dentistry is a mostly a social interaction between a dentist and the patient in their limited job setting and with personal characteristics . A healthy dentist is one of the most important components in a successful dental practice . Like all other professionals, dentists are exposed to occupational health hazards which predispose them to develop a multitude of health problems . Maintaining the steady hand and posture by the dentist comes at a cost to the health of the dentist . High frequency of musculoskeletal disorders (msds) in dentists has been reported in the literature . A recent review of the literature that examined the prevalence and risk factors of msds in dentists reported that the prevalence of general musculoskeletal pain in dental professionals ranges between 64% and 93% . There are many factors that contribute to msds in dental professionals, including repetitive motion, pinch - grasp, vibration, force, and awkward positions, sitting for a long period of time, operator position, poor posture, lack of flexibility and strength, poor ergonomics, and insufficient work breaks . It is generally agreed that the physical posture of the dentist should be relaxed while they work . Dentists can, and do, experience illnesses and problems that can disrupt or impair their practice . Career and job satisfaction are the indicators that may have an influence on career longevity . Change in the work environment of the dentist might increase his / her career longevity . Complementary and alternative medicine (cam), as defined by national centre for complementary and alternative medicine (nccam), is a group of diverse medical and health care systems, practices, and products that are not presently considered to be part of conventional medicine . A large number of patients using cam are those who suffer from chronic musculoskeletal pain . Many studies have reported cam therapies, including yoga, ayurveda, homeopathy, reiki, acupressure, massage, prayers, and acupuncture, to be effective in managing chronic musculoskeletal pain and other discomforts in the general population . There are currently no reports that link musculoskeletal pain, cam, and career satisfaction among dentists working in western india . Since a large number of dentists all over the world report msds, this study was conducted in western india with an aim to determine if dentists are using cam therapies to manage their msds and, if so, to determine if cam therapies are associated with their job satisfaction and longevity, compared with conventional therapy users . The survey was conducted with the approval of the institutional review board of teerthankar mahaveer university (tmu). Dentists registered under indian dental association (ida) were recruited to complete an 21-item questionnaire under 5 domains . A pilot study was conducted among dentists working in the teerthankar mahaveer dental college and research centre . Following these pilot tests, the questionnaire was further modified and was uploaded on the web - based survey software . All dentists of western india who are current members of the ida were recruited to participate . The final version of the questionnaire was formatted using web - based survey software for electronic distribution . This study included all registered dentists residing in western india with their e - mail addresses (n = 2166). Dentists were sent the link to their e - mail address for competing the survey . Dentists who participated in the pilot study, dental students, members of the general public, dental hygienists, dental assistants, and others who were not registered dentists were excluded . The questionnaire consisted of five domains: demographic profile of the dentist, experience with musculoskeletal pain, use of conventional therapies or use of cam therapies for its management, opinions about cam and conventional therapies, and job / career satisfaction related to cam . Univariate and bivariate analyses were performed to determine the demographic information, frequently reported areas of location of pain, the number of respondents that used cam or conventional therapies, types of cam or conventional therapies frequently used, work disruption caused by msds, and job satisfaction by using cam and conventional therapies . Association between conventional therapy and cam use in relation to career variables was assessed using odds ratio (or). Independent samples t - tests were used to determine the opinions about cam and conventional therapies for msd management . Univariate and bivariate analyses were performed to determine the demographic information, frequently reported areas of location of pain, the number of respondents that used cam or conventional therapies, types of cam or conventional therapies frequently used, work disruption caused by msds, and job satisfaction by using cam and conventional therapies . Association between conventional therapy and cam use in relation to career variables was assessed using odds ratio (or). Independent samples t - tests were used to determine the opinions about cam and conventional therapies for msd management . A total of 2166 survey e - mails were sent electronically, with a response rate of 73% (n = 1581). The nonrespondents were assumed to be similar to the respondents based on the notion that the group under study was somewhat a homogeneous group . Findings of the demographic status of the dentists showed that a majority of the study population was males (75.7%) and worked primarily in their own private dental clinics (85.7%). A total of 79% (n = 1249) reported having msds, with the mean duration of pain being 8.3 years (median = 3.5). Neck and lower back were the most common sites, followed by shoulders, upper back, wrist, elbow / arm, knee, hips, and legs . Demographic characteristics of the dentists percentage of dentist reported pain by location figure 2 shows work disruption among dentists as a result of msds . When comparison was made between individuals who used cam therapies or conventional therapy alone and those individuals who used both cam and conventional therapies, the latter group had 4 times lower odds of temporarily quitting work for longer than 1 month [or = 3.4, 95% confidence interval (ci) = 1.7 - 17.8]. Work disruption among dentist due to msd figure 3 depicts the use of cam modalities by dentists . About 81% (n = 1012) dentists reported using both cam and conventional therapies most frequently to manage msds . Also, of the 1249 individuals who reported msds, 31% (n = 388) used cam therapies alone, 19% (n = 238) used conventional therapies alone, and 3% (n = 38) did not use any therapy . Dentists degree of pain improved significantly after using cam therapies versus conventional therapies (p = 0.004). Dentists who suffered from musculoskeletal pain agreed 3 times more that cam therapies were acceptable for msd management (or = 3.7, 95% ci = 2.7 - 3.9) than those with no pain, and were 3 times more likely to use cam therapies for msd management (or = 3.4, 95% ci = 1.4 - 5.9). Table 2 depicts the job / career satisfaction among dentists who used cam therapies and conventional therapies . Dentists who believed in cam therapies and used them alone had significantly higher odds of agreeing that they were satisfied with their career as a dentist (or = 3.2, 95% ci = 1.5 - 5.7) and that it contributed to career longevity (or = 1.92, 95% ci = 1.4 - 7.3), increased overall health (or = 1.67, 95% ci = 1.11 - 6.1), and improved the working efficiency (or = 2.37, 95% ci = 1.1 - 7.3), and thus were satisfied with the job (or = 1.51, 95% ci = 1.7 - 7.3) when compared to users of conventional therapies . Older dentists had significantly high rate of cam usage than younger dentists (or = 2.17, 95% ci = 1.157 - 1.007). Dentists who never used cam reported poorer general health when compared to cam users (or = 1.16, 95% ci = 1.06 - 2.4). There were no statistically significant differences when controlling for race, type of degree earned, and number of years of practice . Cam use among dentist with msd association between conventional therapy and cam usage with job / career satisfaction many different types of cam therapies, including whole medical systems (homeopathic and naturopathic medicine), mind body medicine (meditation, prayer, and mental healing), biologically based practices (ayurveda / herbal products), manipulative and body - based medicine (chiropractic care and massage), and energy medicine (reiki and therapeutic touch) are practiced worldwide . Many plants are used in various formulations such as decoctions, powders, medicated oils, paste, etc . Ayurvedic drugs like zingiber officinale, (shng jing), ricinus communis, commiphora mukul, boswellia serrata, nyctanthes arbor - tristis, etc . Modern research scientists have conducted many scientific researches to assess the safety, efficacy, and anti - inflammatory potential of these drugs . Deep breathing exercises involve slow, deep breaths through the nose for 10 sec, followed by a complete exhalation for 10 sec for at least 5 cycles . Meditation is a practice in which an individual attempts to keep the mind clear and free from any other thoughts . Massage therapist uses the technique of massage to adjust the muscles, which helps in relaxation . Yoga practitioners had less muscle weakness than compared to non practitioners .. acupuncture may also be sometimes used for treating msds . It involves using thin, metallic needles to penetrate the skin at different anatomical points of the body . Even in optimal seated postures, more than one - half of the body's muscles are contracted statically and there is little movement of the vertebral joints . This may result in damaging physiological changes that can lead to back, neck, or shoulder pain or msds . The term musculoskeletal disorder is used to describe a wide range of injuries to the tendons, ligaments, nerves, and supporting structures . Chronic musculoskeletal pain appears after only a few years of clinical practice, or even during second or third year of undergraduate training . Cam therapies have been effective in reducing the risk of and treating musculoskeletal pain in the general population for several decades . In our study, individuals who used cam therapies alone were less likely to report temporarily quitting work . Therefore, a dentist who uses cam therapies may reduce work interruptions caused by musculoskeletal pain . According to the present study, dentists who do not suffer from musculoskeletal pain experience higher job satisfaction when compared to those who suffer from msds (dentist using cam therapies alone had greater career satisfaction compared to those who used conventional therapies alone . Therefore, dentists using cam therapies for msd management experienced higher satisfaction and longevity when compared to those using conventional therapies . The most favored cam therapies among participants in the current study were massage, herbal supplements, and yogic exercises . Therefore, no effective gender comparison can be made . To date, no studies have examined the use of cam for msds among dentists and its association with career satisfaction; therefore, the results of this study cannot be compared with any other similar study . Similar to other studies, our study also reported maximum musculoskeletal complaints in the neck and lower back region . Most of the dentists (81%) reported use of both cam and conventional therapies in a complementary way for the treatment of musculoskeletal problems . The hectic schedule of the dentists leaves them with no time to practice mind body techniques and other cam - related modalities to treat their musculoskeletal pain . Various researches have been conducted on the characteristics of cam users and the determinants of cam use . Some researches show that utilization of cam is influenced by an individual's personality, family and friends, and socioeconomic factors such as race / culture, education, and economic level . Dentists must practice some sort of exercise in their day - to - day routine, including yoga, to be relieved from the problem of musculoskeletal pain . Dentists using cam therapies reported they had greater overall health and more life satisfaction compared to conventional therapy users . Cam education should be incorporated in the dental curriculum to train the budding dentists about better management of msds . Education and additional research are needed to promote an understanding of the complexity of the problem and to address the problem's multifactorial nature . Knowledge and training of alternative therapies is very much required among professionals, especially dentists, so that they can use these therapies as a preventive and treatment modality for chronic pain emerging in day - to - day life and prolong their career.
Transcatheter aortic valve implantation (tavi) has gained wide acceptance for the treatment of severe aortic stenosis among patients deemed to be at increased risk for surgical aortic valve replacement . Expansion of tavi to lower risk patients is critically dependent on the refinement of earlygeneration devices to further reduce the risk of paravalvular regurgitation, device malposition, atrioventricular (av) conductance disturbances, accesssite complications, and periinterventional bleeding . Newergeneration devices feature external cuffs or internal skirts to seal the prosthesis to the aortic annulus and reduce the risk of paravalvular regurgitation . Atraumatic, precurved, and steerable delivery catheter systems aim for a reduction of plaque embolization, and smaller catheter diameters mitigate the risk of access site complications and bleeding.1 the lotus valve system (boston scientific, natwick, ma) is a novel, fully repositionable tavi prosthesis that permits evaluation of the final configuration of the deployed valve, the degree of aortic regurgitation, as well as reduced coronary flow before detachment . Singlearm registries of the lotus valve showed high rates of procedural success and suggested substantially lower rates of paravalvular regurgitation compared with earlygeneration devices2, 3, 4; conversely, rates of av conduction disturbances were relatively high, resulting in permanent pacemaker implantation in 1 in every fourth patient up to one in every third patient.2, 3 the safety and effectiveness of the fully repositionable lotus valve system as compared with other newergeneration tavi devices have not been evaluated to date . We therefore performed an adjusted comparison of the lotus valve system with the balloonexpandable edwards sapien 3 prosthesis in patients with aortic stenosis undergoing tavi within the nationwide swiss transcatheter aortic valve implantation registry (nct01368250). All patients undergoing tavi procedures performed in switzerland are consecutively captured in a nationwide, prospective cohort study (clinicaltrials.gov nct01368250).5 for the purpose of the present analysis, we investigated all patients with severe aortic stenosis treated with the edwards sapien 3 prosthesis or the lotus valve system . Selection of tavi candidates, device allocation, and periprocedural management was left to the discretion of the operators . All data were recorded in a webbased database held at the clinical trials unit of the university of bern, switzerland . The swiss tavi registry has been approved by the local cantonal ethics committee and the institutional review boards of all participating sites . The lotus valve system consists of a single nitinol wire that is braided into a stent frame upon foreshortening and mechanical expansion . The prosthesis is attached to the delivery system with 3 coupling fingers; buckles at the distal end connect to posts located at the commissures of the 3 leaflets upon shortening, and lock the valve in its final configuration . The stent frame accommodates a bovine pericardial valve and comes in 3 prosthesis sizes (23 mm, 25 mm, and 27 mm) fitting an annulus diameter ranging from 20 mm to 26 mm . An adaptive seal in the distal portion of the prosthesis and an outer sleeve have been designed to reduce paravalvular regurgitation . The lotus prosthesis is fully repositionable and allows for an assessment of the final result before detachment of the valve from the coupling fingers of the delivery system . The precurved delivery catheter has a diameter of 18 f to 20 f and is not steerable . The valve can be implanted without rapid ventricular stimulation and predilatation is not necessary in all cases . The lotus valve system received ce mark approval on october 28, 2013, and on july 14, 2014, for its 23/27 mm and 25 mm prosthesis, respectively, and since then has become available for commercial use and implantation in switzerland . The edwards sapien 3 valve (edwards lifesciences, irvine, ca) is the fourth iteration of the first balloonexpandable transcatheter aortic valve prosthesis . The stent frame houses a valve made of 3 modified pericardial tissue leaflets, and accommodates annulus sizes from 18 mm to 28 mm using 3 device sizes (23 mm, 26 mm, 29 mm). An outer sealing skirt in the distal portion of the prosthesis complements the inner pet skirt and aims at a reduction of paravalvular aortic regurgitation . The prosthesis is loaded on the delivery balloon in the abdominal aorta rendering the delivery catheter compatible with 14 f to 16 f. the commander delivery catheter is steerable and has a wheel to fineadjust valve positioning.6 the edwards sapien 3 valve was introduced in switzerland for implantation on january 27, 2014, and completely replaced the previously available edwards sapien xt prosthesis . Patients underwent transthoracic echocardiography before hospital discharge, and were contacted for clinical followup at 30 days . Standardized interviews, documentation from referring physicians, and hospital discharge summaries were used for the collection of clinical end points . All end points were defined according to the updated version of the valve academic research consortium (varc2) definitions.7 an independent clinical event committee adjudicated all events . The prespecified end point was the varc2 early safety outcome, a composite of allcause mortality, stroke, lifethreatening bleeding, acute kidney injury stage 2 or 3, coronary obstruction requiring intervention, major vascular complication, and valverelated dysfunction requiring repeat procedure . Continuous data are reported as meansd, and categorical variables are reported as number (percentage) of patients . Events are reported as counts of first occurrence per (sub)type of event within 30 days of followup (% of all patients). Event rates at 30 days were compared for patients treated with the lotus versus the edwards sapien 3 bioprosthesis using cox regressions, censoring patients at death or lost to followup . Reported are crude hazard ratios (hrs; with 95% cis) with p values from wald chisquare tests, or continuity correct risk ratios with p values from fisher exact tests in case of zero events . Multiple imputation of missing data was performed using chained equations (n=20 data sets generated) before the adjusted analyses . Reported are adjusted hrs (95% cis), with the two valves compared, adjusting for age, dyslipidemia, peripheral vascular disease, aortic regurgitation moderate or severe, aortic valve area, new york heart association class iii or iv, and society of thoracic surgeons (sts) predicted risk of mortality score . The estimates of adjusted hrs from 20 data sets after multiple imputation of missing values were combined using rubin's rule and presented with adjusted p values . Twosided p values <stratified analyses of the following subgroups were performed: age (83 years versus <83 years median), sex (female versus male), left ventricular ejection fraction (40% versus> 40%), peripheral vascular disease (yes versus no), sts risk score (> 4 versus 4), and p value for the interaction between subgroups and valve type . All analyses were performed with stata version 14 (statacorp, college station, tx). All patients undergoing tavi procedures performed in switzerland are consecutively captured in a nationwide, prospective cohort study (clinicaltrials.gov nct01368250).5 for the purpose of the present analysis, we investigated all patients with severe aortic stenosis treated with the edwards sapien 3 prosthesis or the lotus valve system . Selection of tavi candidates, device allocation, and periprocedural management was left to the discretion of the operators . All data were recorded in a webbased database held at the clinical trials unit of the university of bern, switzerland . The swiss tavi registry has been approved by the local cantonal ethics committee and the institutional review boards of all participating sites . The lotus valve system consists of a single nitinol wire that is braided into a stent frame upon foreshortening and mechanical expansion . The prosthesis is attached to the delivery system with 3 coupling fingers; buckles at the distal end connect to posts located at the commissures of the 3 leaflets upon shortening, and lock the valve in its final configuration . The stent frame accommodates a bovine pericardial valve and comes in 3 prosthesis sizes (23 mm, 25 mm, and 27 mm) fitting an annulus diameter ranging from 20 mm to 26 mm . An adaptive seal in the distal portion of the prosthesis and an outer sleeve have been designed to reduce paravalvular regurgitation . The lotus prosthesis is fully repositionable and allows for an assessment of the final result before detachment of the valve from the coupling fingers of the delivery system . The precurved delivery catheter has a diameter of 18 f to 20 f and is not steerable . The valve can be implanted without rapid ventricular stimulation and predilatation is not necessary in all cases . The lotus valve system received ce mark approval on october 28, 2013, and on july 14, 2014, for its 23/27 mm and 25 mm prosthesis, respectively, and since then has become available for commercial use and implantation in switzerland . The edwards sapien 3 valve (edwards lifesciences, irvine, ca) is the fourth iteration of the first balloonexpandable transcatheter aortic valve prosthesis . The stent frame houses a valve made of 3 modified pericardial tissue leaflets, and accommodates annulus sizes from 18 mm to 28 mm using 3 device sizes (23 mm, 26 mm, 29 mm). An outer sealing skirt in the distal portion of the prosthesis complements the inner pet skirt and aims at a reduction of paravalvular aortic regurgitation . The prosthesis is loaded on the delivery balloon in the abdominal aorta rendering the delivery catheter compatible with 14 f to 16 f. the commander delivery catheter is steerable and has a wheel to fineadjust valve positioning.6 the edwards sapien 3 valve was introduced in switzerland for implantation on january 27, 2014, and completely replaced the previously available edwards sapien xt prosthesis . Patients underwent transthoracic echocardiography before hospital discharge, and were contacted for clinical followup at 30 days . Standardized interviews, documentation from referring physicians, and hospital discharge summaries were used for the collection of clinical end points . All end points were defined according to the updated version of the valve academic research consortium (varc2) definitions.7 an independent clinical event committee adjudicated all events . The prespecified end point was the varc2 early safety outcome, a composite of allcause mortality, stroke, lifethreatening bleeding, acute kidney injury stage 2 or 3, coronary obstruction requiring intervention, major vascular complication, and valverelated dysfunction requiring repeat procedure . Continuous data are reported as meansd, and categorical variables are reported as number (percentage) of patients . Events are reported as counts of first occurrence per (sub)type of event within 30 days of followup (% of all patients). Event rates at 30 days were compared for patients treated with the lotus versus the edwards sapien 3 bioprosthesis using cox regressions, censoring patients at death or lost to followup . Reported are crude hazard ratios (hrs; with 95% cis) with p values from wald chisquare tests, or continuity correct risk ratios with p values from fisher exact tests in case of zero events . Multiple imputation of missing data was performed using chained equations (n=20 data sets generated) before the adjusted analyses . Reported are adjusted hrs (95% cis), with the two valves compared, adjusting for age, dyslipidemia, peripheral vascular disease, aortic regurgitation moderate or severe, aortic valve area, new york heart association class iii or iv, and society of thoracic surgeons (sts) predicted risk of mortality score . The estimates of adjusted hrs from 20 data sets after multiple imputation of missing values were combined using rubin's rule and presented with adjusted p values . Twosided p values <stratified analyses of the following subgroups were performed: age (83 years versus <83 years median), sex (female versus male), left ventricular ejection fraction (40% versus> 40%), peripheral vascular disease (yes versus no), sts risk score (> 4 versus 4), and p value for the interaction between subgroups and valve type . All analyses were performed with stata version 14 (statacorp, college station, tx). Between february 4, 2014, and september 29, 2015, 140 patients were treated with the lotus valve system and 815 patients with the edwards sapien 3 prosthesis in 12 centers across switzerland . Age, sex, medical history, and cardiovascular risk factors were well balanced between the two treatment arms . Compared with patients treated with the lotus valve system, patients treated with the edward sapien 3 prosthesis more commonly had peripheral vascular disease (15.5% versus 7.9%, p=0.01) and higher estimated surgical risk as assessed by the logistic euroscore (18.914.8% versus 15.08.6%, p=0.018) and sts score (5.03.8% versus 4.12.4%, p=0.005). Baseline characteristics values are expressed as means with sds (p value from t tests) or counts (% of all patients; p value from fisher or chisquare tests). Ccs indicates canadian cardiovascular society; nyha, new york heart association; sts, society of thoracic surgeons . Procedural characteristics are shown in table 2 . Although procedure time was comparable, the amount of contrast media was greater with the lotus valve system compared with the edwards sapien 3 prosthesis (17777 ml versus 15393 ml, p=0.004). Patients treated with edwards sapien 3 more commonly underwent femoral surgical access (12.6% versus 5.7%, p=0.018) and predilatation with balloon valvuloplasty (81.8% versus 31.4%, p<0.001). Device success was 77.1% among patients treated with the lotus valve and 75.7% among patients treated with the edwards sapien 3 prosthesis (p=0.713). There were no significant differences between the two devices with regards to transprosthetic gradient, patient prosthesis mismatch, or postprocedural aortic valve area, respectively (table 3). Patients treated with the lotus valve more commonly had no aortic regurgitation after intervention (71.4% versus 53.2%, difference 18.3%; 95% ci, 9.427.1) (table 2). Whereas 7 patients (0.9%) treated with the edwards sapien 3 prosthesis underwent valve in series implantation due to malpositioning, no case of valve malpositioning was reported in the lotus cohort (p=0.271). Procedural characteristics values are expressed as means with standard deviations (p values from t tests) or counts (% of all patients; p values from fisher tests or chisquare tests). Clinical outcomes at 30 days depicted are the number of first events within 30 days with percentage of all patients . Cox regressions reporting hazard ratios (hrs; with 95% cis) or continuity corrected risk ratios (95% cis) in case of zero events with fisher exact p values . Adjusted hr from cox regressions, adjusting for age, dyslipidemia, peripheral vascular disease, aortic regurgitation moderate or severe, aortic valve area, new york heart association class iii or iv, and society of thoracic surgery risk score (combining the estimates of 20 data sets using rubin's rule because of missing data). Multiple imputation of missing data was performed using chained equations (n=20 data sets generated). Mi indicates myocardial infarction; tia, transient ischemic attack; varc2, valve academic research consortium . The early varc2 safety end point occurred in 14.3% of patients treated with the lotus and 14.6% of patients treated with the edwards sapien 3 prosthesis with no difference in crude (hr, 0.97; 95% ci, 0.611.56 [p=0.915]) and adjusted (hr, 1.03; 95% ci, 0.641.67 [p=0.909]) analyses (figure 1). Allcause mortality at 30 days was 2.2% among patients treated with the lotus valve system, and 2.8% among patients treated with the edwards sapien 3 valve (adjusted hr, 0.75; 95% ci, 0.222.51 [p=0.636]). Estimated and observed mortality are illustrated in figure 2 . There were no significant differences between the two devices with regard to mortality, cerebrovascular accidents, myocardial infarction, vascular access site, and bleeding complications . While none of the patients in the lotus group experienced periprocedural myocardial infarction, 7 patients treated with the edwards sapien 3 prosthesis did (0.9%) (hr, 0.39; 95% ci, 0.026.79 [p=0.602]). Meier estimates of the valve academic research consortium 2 (varc2) early safety composite outcome at 30 days . The blue line relates to the lotus valve system; the red line relates to the edwards sapien 3 valve . Bar graph of estimated and observed mortality at 30 days . Society of thoracic surgeons (sts) risk scores were used to estimate mortality at 30 days . Despite a higher amount of contrast used in patients treated with the lotus valve the number of permanent pacemaker implantations was higher in patients treated with the lotus (34.3%) as compared with the edwards sapien 3 prosthesis (14.1%) (hr, 2.76; 95% ci, 1.973.87 [p<0.001]) (figure 3). In a stratified analysis for the varc2 early safety outcome, there were no significant interactions across major subgroups, with the exception of a positive effect for treatment with the lotus valve among patients 83 years and older (p for interaction=0.030) (figure 4). The blue line relates to the lotus valve system; the red line relates to the edwards sapien 3 valve . Stratified analysis for the valve academic research consortium 2 early composite safety outcome (based on crude hazard ratios). Between february 4, 2014, and september 29, 2015, 140 patients were treated with the lotus valve system and 815 patients with the edwards sapien 3 prosthesis in 12 centers across switzerland . Age, sex, medical history, and cardiovascular risk factors were well balanced between the two treatment arms . Compared with patients treated with the lotus valve system, patients treated with the edward sapien 3 prosthesis more commonly had peripheral vascular disease (15.5% versus 7.9%, p=0.01) and higher estimated surgical risk as assessed by the logistic euroscore (18.914.8% versus 15.08.6%, p=0.018) and sts score (5.03.8% versus 4.12.4%, p=0.005). Baseline characteristics values are expressed as means with sds (p value from t tests) or counts (% of all patients; p value from fisher or chisquare tests). Ccs indicates canadian cardiovascular society; nyha, new york heart association; sts, society of thoracic surgeons . Procedural characteristics are shown in table 2 . Although procedure time was comparable, the amount of contrast media was greater with the lotus valve system compared with the edwards sapien 3 prosthesis (17777 ml versus 15393 ml, p=0.004). Patients treated with edwards sapien 3 more commonly underwent femoral surgical access (12.6% versus 5.7%, p=0.018) and predilatation with balloon valvuloplasty (81.8% versus 31.4%, p<0.001). Device success was 77.1% among patients treated with the lotus valve and 75.7% among patients treated with the edwards sapien 3 prosthesis (p=0.713). There were no significant differences between the two devices with regards to transprosthetic gradient, patient prosthesis mismatch, or postprocedural aortic valve area, respectively (table 3). Patients treated with the lotus valve more commonly had no aortic regurgitation after intervention (71.4% versus 53.2%, difference 18.3%; 95% ci, 9.427.1) (table 2). Whereas 7 patients (0.9%) treated with the edwards sapien 3 prosthesis underwent valve in series implantation due to malpositioning, no case of valve malpositioning procedural characteristics values are expressed as means with standard deviations (p values from t tests) or counts (% of all patients; p values from fisher tests or chisquare tests). Clinical outcomes at 30 days depicted are the number of first events within 30 days with percentage of all patients . Cox regressions reporting hazard ratios (hrs; with 95% cis) or continuity corrected risk ratios (95% cis) in case of zero events with fisher exact p values . Adjusted hr from cox regressions, adjusting for age, dyslipidemia, peripheral vascular disease, aortic regurgitation moderate or severe, aortic valve area, new york heart association class iii or iv, and society of thoracic surgery risk score (combining the estimates of 20 data sets using rubin's rule because of missing data). Multiple imputation of missing data was performed using chained equations (n=20 data sets generated). Mi indicates myocardial infarction; tia, transient ischemic attack; varc2, valve academic research consortium . The early varc2 safety end point occurred in 14.3% of patients treated with the lotus and 14.6% of patients treated with the edwards sapien 3 prosthesis with no difference in crude (hr, 0.97; 95% ci, 0.611.56 [p=0.915]) and adjusted (hr, 1.03; 95% ci, 0.641.67 [p=0.909]) analyses (figure 1). Allcause mortality at 30 days was 2.2% among patients treated with the lotus valve system, and 2.8% among patients treated with the edwards sapien 3 valve (adjusted hr, 0.75; 95% ci, 0.222.51 [p=0.636]). There were no significant differences between the two devices with regard to mortality, cerebrovascular accidents, myocardial infarction, vascular access site, and bleeding complications . While none of the patients in the lotus group experienced periprocedural myocardial infarction, 7 patients treated with the edwards sapien 3 prosthesis did (0.9%) (hr, 0.39; 95% ci, 0.026.79 [p=0.602]). Kaplan meier estimates of the valve academic research consortium 2 (varc2) early safety composite outcome at 30 days . The blue line relates to the lotus valve system; the red line relates to the edwards sapien 3 valve . Society of thoracic surgeons (sts) risk scores were used to estimate mortality at 30 days . Despite a higher amount of contrast used in patients treated with the lotus valve the number of permanent pacemaker implantations was higher in patients treated with the lotus (34.3%) as compared with the edwards sapien 3 prosthesis (14.1%) (hr, 2.76; 95% ci, 1.973.87 [p<0.001]) (figure 3). In a stratified analysis for the varc2 early safety outcome, there were no significant interactions across major subgroups, with the exception of a positive effect for treatment with the lotus valve among patients 83 years and older (p for interaction=0.030) (figure 4). The blue line relates to the lotus valve system; the red line relates to the edwards sapien 3 valve . Stratified analysis for the valve academic research consortium 2 early composite safety outcome (based on crude hazard ratios). The key findings of our analysis can be summarized as follows . In a nationwide prospective registry of patients undergoing tavi, we found no differences for the primary end point, the early composite safety end point within 30 days between patients treated with the fully repositionable lotus valve system versus the balloonexpandable edwards sapien 3 prosthesis.rates of device success were comparable for both devices.more than mild residual aortic regurgitation was exceedingly low with both devices.patients treated with the lotus valve system had a 2 to 3fold increased risk of permanent pacemaker implantation compared with patients treated with the edwards sapien 3 prosthesis . In a nationwide prospective registry of patients undergoing tavi, we found no differences for the primary end point, the early composite safety end point within 30 days between patients treated with the fully repositionable lotus valve system versus the balloonexpandable edwards sapien 3 prosthesis . Patients treated with the lotus valve system had a 2 to 3fold increased risk of permanent pacemaker implantation compared with patients treated with the edwards sapien 3 prosthesis . Newergeneration tavi devices are characterized by improved device success as compared with earlygeneration devices primarily by a reduction of moderate or severe prosthetic valve regurgitation, which has consistently been associated with increased late mortality.8, 9 documentation of moderate to severe aortic regurgitation has been reported in up to 14% of patients treated with earlygeneration devices,8, 9, 10 and motivated the development of internal skirts and external cuffs to seal the prosthesis to the aortic annulus and reduce paravalvular regurgitation . Complimentary to technical refinements of the devices, dedicated imaging tools have been introduced allowing for precise device positioning within the annular landing zone . In the swiss tavi registry, moderate or severe aortic regurgitation was documented in 0.7% and 1.2% of patients treated with lotus and edwards sapien 3, respectively . Our findings are consistent with the repositionable percutaneous replacement of stenotic aortic valve through implantation of lotus valve system: evaluation of safety and performance (reprise) ii study and the uk lotus registry, reporting moderate or severe aortic regurgitation in 1% and 0.8% of patients, respectively.2, 3 reduction of paravalvular aortic regurgitation results from a combination of both, the full repositionability of the lotus valve allowing for an assessment of the result prior to deployment, and the prosthesis design with an adaptive seal in the distal portion and an outer sleeve.6 a similarly low incidence of moderate to severe aortic regurgitation was documented with the edwards sapien 3 valve that has been refined by an external sealing cuff that mimics a parachute . The incidence of more than mild paravalvular regurgitation decreased from 5.3% to 1.3% (p=0.04) as compared with its predecessor in a previous analysis from the swiss tavi registry including almost 600 patients.11 rates of permanent pacemaker implantation amounted to 34% among patients treated with the lotus valve, and were 2 to 3fold higher compared with patients treated with the edwards sapien 3 prosthesis . Comparable rates of av conductance disturbances and permanent pacemaker implantation have been consistently reported in the reprise ii study (28.6%) and the uk lotus registry (31.8%).2, 3 the effect of permanent pacemaker implantation after tavi on longterm outcomes remains a matter of debate.12, 13 no difference in 1year mortality was documented in patients with a previous permanent pacemaker, a new permanent pacemaker, or no pacemaker in a prospective registry of 353 patients from 2 institutions.12 in contrast, permanent pacemaker implantation after tavi was reported to be an independent predictor of 1year mortality in an analysis of the placement of aortic transcatheter valves (partner i) trial.13 moreover, permanent pacemaker implantation was associated with a longer duration of hospitalization and higher rates of repeat hospitalization at 1 year.14 the degree of pacemaker dependency accompanied by ventricular dyssynchrony may reconcile the differential in clinical findings between studies . Av conductance disturbances along with pacemaker dependency after tavi may be temporary rather than permanent in nature . In a small study of 36 patients with new pacemaker following implantation of a selfexpandable prosthesis, more than half of the patients were pacemaker independent at a median followup of 12 months.15 the rates of the early composite safety end point were comparable between the two devices at 30 days . In line, there were no differences with respect to cardiovascular mortality, myocardial infarction, bleeding, or vascular access site complications . The observed mortality rate (lotus 2.2% versus sapien 3 2.8%) was substantially lower as compared with the sts estimates . The overall incidence of stroke was 4.3% and 3.1% of patients treated with the lotus valve system and the edwards sapien 3 prosthesis, respectively . The incidence of stroke at 30 days was 5.9% in the reprise ii study and 3.9% in the uk lotus registry,2, 3 while large nationwide tavi registries reported stroke rates in the range of 1.5% to 4%.9, 11, 16, 17, 18, 19 although the signal has to be interpreted with caution, several reasons may account for a potential difference in cerebrovascular events between the two devices . A significantly lower rate of prior balloon valvuloplasty among patients treated with the lotus valve as compared with the edwards sapien valve may affect the rates of stroke . In a small study of 87 patients, the volume of new cerebral ischemic lesions as assessed by diffusionweighted magnetic resonance imaging was significantly higher among patients without as compared with patients with prior balloon aortic valvuloplasty.20 in contrast, a recent metaanalysis of 18 studies with 2443 patients demonstrated a trend towards a reduced risk of clinically relevant stroke with direct tavi . However, the findings should be interpreted with caution given the limitations of the nonrandomized studies included in the metaanalysis and the unadjusted nature of the summary measures used.21 the effect of predilatation on clinical outcome is currently being investigated in the transcatheter aortic valve implantation without predilatation (simplify tavi) study (nct 01539746) and the balloon expandable transcatheter aortic valve implantation without predilatation of aortic valve (easeit) study (nct02127580). Moreover, differences in the delivery catheter diameter, flexibility, and steerability may affect the risk of plaque abrasion in the aortic arch . Finally, full repositionability of the lotus valve may increase the inclination of repeated prosthesis placement, which, in turn, has been associated with an increased risk of stroke.22 the present analysis has several limitations . First, there was no random allocation to treatment with the lotus valve or the edwards sapien 3 prosthesis, respectively . Although baseline characteristics between the two treatment arms were comparable, we cannot exclude selection of treatment according to concealed confounders . Second, the number of patients included in the analysis was limited, and the duration of followup did not extend beyond 30 days . However, it constitutes the largest series reported to date and data are consistent with previously reported singlearm registries . Third, differences in balloon valvuloplasty prior to device implantation may have confounded the clinical results . However, our analysis reflects routine clinical practice with the 2 devices by experienced operators . Finally, implantation depth and oversizing have both been associated with an increased rate of conductance disturbances, respectively . First, there was no random allocation to treatment with the lotus valve or the edwards sapien 3 prosthesis, respectively . Although baseline characteristics between the two treatment arms were comparable, we cannot exclude selection of treatment according to concealed confounders . Second, the number of patients included in the analysis was limited, and the duration of followup did not extend beyond 30 days . However, it constitutes the largest series reported to date and data are consistent with previously reported singlearm registries . Third, differences in balloon valvuloplasty prior to device implantation may have confounded the clinical results . However, our analysis reflects routine clinical practice with the 2 devices by experienced operators . Finally, implantation depth and oversizing have both been associated with an increased rate of conductance disturbances, respectively . In a nationwide registry, no statistical difference was found between the repositionable lotus valve system and the balloonexpandable edwards sapien 3 prosthesis with respect to the varc2 early safety outcome for the treatment . The need for new permanent pacemaker implantation was more frequent among patients treated with the lotus valve . The swiss tavi registry is supported by a study grant from the swiss heart foundation and the swiss working group of interventional cardiology, and is sponsored by unrestricted funds from boston scientific, edwards lifesciences, medtronic, st . The study sponsors had no role in the study design, data collection, data analysis, data interpretation, or writing of the report . Jude medical, and received research contracts to the institution from edwards lifesciences and symetis . Dr toggweiler is a proctor for symetis and received speakers' fees from edwards lifesciences and medtronic . Jude medical and has received reimbursement for travel expenses from medtronic, boston scientific, and edwards lifesciences . Dr roffi received institutional research grants from abbott vascular, boston scientific, biotronik, biosensor, and medtronic . Dr windecker has received research contracts to the institution from abbott, boston scientific, biosensors, cordis, medtronic, and st . Dr wenaweser serves as proctor for medtronic, edwards lifesciences, and boston scientific and has received an unrestricted grant from medtronic to the institution (university of bern). Table s1 . Missing baseline characteristics, multiple imputation, and adjusted analyses appendix s1 . Collaborators and swiss transcatheter aortic valve implantation registry investigators.
Leptin is an adipose tissue - derived hormone that has been shown to be related to several metabolic, inflammatory, and hemostatic factors involved in the development of hypertension and cardiovascular disease . Experimental animal studies suggest that higher leptin levels may cause hyperglycemia, elevations in blood pressure (mediated through increased sympathetic activity), and renal dysfunction . In rat models, leptin has been shown to induce natriuresis which may in turn result in an increase in arterial pressure so as to maintain sodium and water balance . Leptin has also been shown to serve as a cofactor of tgf - beta activation, promote renal endothelial cell proliferation, and potentially may play a role in renal glomerulosclerosis [57]. Recent studies have reported that infusion of recombinant leptin into normal rats for 3 weeks fosters the development of focal glomerulos 4.clerosis . However, few human studies have examined the putative association between plasma leptin levels and chronic kidney disease (ckd) in humans . In this context, we examined the independent relation between plasma leptin levels and ckd in a multiethnic sample of us adults, after adjusting for main confounding factors . The current study is based on data from the third national health and nutrition examination survey (nhanes iii). Detailed description of nhanes iii study design and methods are available elsewhere [813]. In brief, nhanes iii included a stratified multistage probability sample representative of the civilian noninstitutionalized us population . Selection was based on counties, blocks, households, and individuals within households and included the oversampling of non - hispanic blacks and mexican americans in order to provide stable estimates of these groups . Subjects were required to sign a consent form before their participation, and approval was obtained from the human subjects committee in the us department of health and human service . The sample included in the current analysis consisted of participants aged greater than 20 years who were randomly assigned to complete an examination in the morning after an overnight fast . We further excluded participants with self - reported cardiovascular disease (n = 434), missing serum creatinine (n = 60) or who were missing data (n = 101) on covariates included in the multivariable model, including systolic or diastolic blood pressure, body mass index (bmi), or cholesterol levels . Serum creatinine was measured using the jaffe kinetic alkaline picrate method performed on a roche hitachi 737 analyzer . Serum creatinine values in nhanes iii were calibrated to the standard creatinine values from the cleveland clinic foundation (ccf) laboratory who used a roche coupled enzymatic assay method that was traceable to an isotope dilution mass spectrometric method using the following deming regression equation: standard creatinine in mg / dl = 0.960 nhanes measured serum creatinine (in mg / dl)0.184 . Glomerular filtration rate (egfr) was estimated from serum creatinine using the 4-variable modification of diet in renal disease (mdrd) study equation as follows: egfr = 175 (serum creatinine in mg / dl) (age in years) (0.742 if female) (1.21 if black). Ckd was defined as an egfr of <60 ml / min/1.73 m, consistent with national kidney foundation kidney disease outcomes quality initiative (kdoqi) stage 3 chronic kidney disease . Age, gender, race / ethnicity, smoking status, alcohol intake (g / day), level of education, history of diabetes and oral hypoglycemic intake or insulin administration, and antihypertensive medication use were assessed using standardized questionnaires . Individuals who had not smoked 100 cigarettes in their lifetimes were considered never smokers; those who had smoked 100 cigarettes in their lifetimes were considered former smokers if they answered negatively to the question do you smoke now? And current smokers if they answered affirmatively . Using height and weight measured during the study examination, body mass index (bmi) rigorous procedures with quality control checks were used in blood collection and details about these procedures are provided in the nhanes laboratory / medical technologists procedures manual [8, 11]. Louis, mo, usa . The assay was a radioimmunoassay (ria) with a polyclonal antibody raised in rabbits against highly purified recombinant human leptin . The minimum detectable concentration of the assay was 0.5 fg / l leptin, and the limit of linearity was 100 recovery of leptin added to serum is 99104% over the linear range of the assay . Within- and between - assay cvs ranged from 3.4% to 8.3% and from 3.6% to 6.2%, respectively [11, 12]. Serum glucose was measured using the modified hexokinase method at the university of missouri diabetes diagnostic laboratory . Diabetes was defined based on the guidelines of the american diabetes association as a serum glucose 126 mg / dl after fasting for a minimum of 8 hours, a serum glucose 200 mg / dl for those who fasted <8 hours before their nhanes visit, a glycosylated hemoglobin value 6.5%, or a self - reported current use of oral hypoglycemic medication or insulin . Seated systolic and diastolic blood pressures were measured using a mercury sphygmomanometer according to the american heart association and seventh joint national committee (jnc7) recommendations . Participants were considered to have hypertension if they reported current blood pressure - reducing medication use and/or had systolic blood pressure 140 mm hg and/or diastolic blood pressure 90 mm hg . Plasma leptin was analyzed both as a continuous variable as well as a categorical variable . For the analysis as a continuous variable, leptin values were log transformed (base e) as a result of their skewed distribution . Using the distribution present in the nhanes iii population, we categorized plasma leptin level into quartiles (4.3 fg / l, 4.48.7 fg / l, 8.816.9 fg / l,> 16.9 fg / l). The multivariable - adjusted odds ratio [(or) (95% confidence interval (ci)] of ckd associated with leptin quartile was calculated with the lowest quartile as the referent, using logistic regression models . Odds ratios were calculated initially after age and sex adjustment and subsequently after additional adjusting for race / ethnicity (non - hispanic whites, non - hispanic blacks, mexican americans, and others), education categories (below high school, high school, above high school), smoking (never smoker, former smoker, current smoker), alcohol intake (continuous), bmi (continuous), diabetes mellitus (absent, present), hypertension (absent, present), and total serum cholesterol (continuous). Trends in the or of ckd across increasing plasma leptin category were determined by modeling median within - quartile leptin level as a continuous variable . To examine the dose - response relationship of the observed association between plasma leptin level and ckd without linearity assumptions, we used flexible nonparametric logistic regression employing the generalized additive modeling approach (r system for statistical computing, available from comprehensive r archive network [http://www.cran.r-project.org/]) to calculate odds ratio of ckds mellitus, adjusting for all covariates in the multivariable model; the predicted odds ratio of ckd was then plotted against increasing leptin levels (on the log scale). Previous studies have shown that serum leptin levels are associated with increased systemic inflammation as measured by c - reactive protein levels, hyperglycemia, high insulin levels, and increased systolic blood pressure, factors that have also been shown to be associated with ckd[17, 21, 22]. Therefore, in a supplementary analysis, to examine if the observed association between plasma leptin and ckd was explained by c - reactive protein levels, fasting insulin, glucose levels, or systolic blood pressure, we adjusted for these variables in the multivariable - adjusted model . Sample weights that account for the unequal probabilities of selection, oversampling, and nonresponse were applied for all analyses using sudaan (version 8.0; research triangle institute, research triangle park, nc) and sas (version 9.2; sas institute, cary, nc) software; standard errors (se) were estimated using the taylor series linearization method . Table 1 presents the characteristics of the study population included in the current analysis . Overall, this study included a broad age range, multiethnic sample of americans with an approximately equal number of men and women . The mean egfr of the study participants was 94 ml / min/1.73 m, and 3.5% had ckd . A positive association between higher leptin quartiles and ckd was present in the age, sex - adjusted model as well as the multivariable model . When analyzed as a continuous variable after log transformation, a positive association was present between leptin and ckd . Table 3 presents the association between plasma leptin levels and ckd within subgroups defined by gender, bmi categories, and diabetes and hypertension . Overall, the association between leptin and ckd was consistently present within these subgroups . Although some of the ors failed to reach conventional levels of statistical significance due to limited sample size and therefore statistical power, tests for interaction were not statistically significant (each p> 0.10 for all stratified analyses). When we employed nonparametric models to graphically examine the dose - response relationship between plasma leptin levels and ckd without linearity assumptions involved in traditional regression models, we observed an overall positive association between plasma leptin and ckd, consistent with the results in tables 1, 2, and 3 . However, there was a steeper association with ckd for plasma leptin levels> 16 fg / l (figure 1). In a supplementary analysis, to examine if the observed association between leptin and ckd was explained by c - reactive protein, a marker of inflammation, or fasting insulin or glucose levels, or systolic blood pressure, we additionally adjusted for these variables to the multivariable - adjusted model . The positive association between leptin and ckd was attenuated, but still present . Compared to quartile 1 of plasma leptin (referent), the multivariable or (95% ci) of ckd was 1.31 (0.70 to 2.44) in quartile 2, 1.22 (0.57 to 2.62) in quartile 3, and 2.72 (1.14 to 6.48) in quartile 4; p - trend = 0.0475 . In a multi - ethnic, population - based sample of us adults, we found that higher plasma leptin levels were positively associated with ckd . This association appeared to be independent of confounders such as age, race - ethnicity, education, bmi, diabetes, and hypertension and appeared to be consistently present in both men and women . Furthermore, the observed association between plasma leptin levels and ckd was present even after adjusting for c - reactive protein and fasting insulin levels, suggesting an association between this adipokine and ckd that is independent of these factors . Several lines of recent evidence suggest that an association between leptin and ckd is plausible . This includes the role of leptin in activating the sympathetic nervous system and causing chronic elevations in blood pressure and renal dysfunction, inducing natriuresis which may result in an increase in arterial pressure so as to maintain sodium and water balance, serving as a cofactor of tgf - beta activation, promoting renal endothelial cell proliferation, and subsequent glomerulosclerosis [57]. It was recently shown that, in rats, infusion of recombinant leptin caused the development of focal glomerulosclerosis . Also, leptin is also reported to be related to insulin resistance and high c - reactive protein levels, both of which have been shown to be related to ckd [21, 25]. A previous study conducted among women with type 1 diabetes reported that plasma leptin levels are independently related to reduced renal function . In a study from south africa conducted among approximately 300 black subjects, okpechi et al . Reported that plasma leptin levels were inversely related to egfr . In another study, common polymorphisms in the lep gene overall, our findings of a positive association between plasma leptin and ckd are in agreement with these previous studies; in addition, we were able to study a large multiethnic sample that includes both men and women and also adjusts for multiple confounders such as bmi, diabetes, hypertension, lipid levels, and c - reactive protein . The main strengths of our study include its population - based nature, inclusion of a representative multiethnic sample, adequate sample size, and the availability of data on confounders for multivariable adjustment . Furthermore, all data were collected following rigorous methodology, including a study protocol with standardized quality control checks . The main limitation of our study is the cross - sectional nature of nhanes, which precludes conclusions regarding the temporal nature of the association between plasma leptin and ckd . Second, defining ckd as egfr <60 ml / min/1.73 m introduces some ascertainment bias . This bias is likely to result in under- or overestimation of odds ratios presented in this report . In summary, in a multiethnic sample of us adults, we found that higher plasma leptin levels are associated with ckd, independent of traditional factors such as age, sex, smoking, alcohol intake, bmi, diabetes, hypertension and serum cholesterol . Our results suggest that leptin may explain part of the reported association between obesity and kidney disease . However, future prospective cohort studies are needed to confirm or refute our findings.
Thyroid hormones failure leads to significant interferences and malfunctions at different physical, mental and social aspects . According to an investigation performed in colorado state of usa at 2000, screening of 25000 people showed that nearly 2.6 million people would be apparently afflicted with hypothyroidism in the next 20 years . A thyroid screening investigation in tehran at 2001 revealed that the prevalence of hypothyroidism in people over 20 years old is 3.5 people per thousand and its subclinical feature prevalence is 2.2 people per thousand; the prevalence of hyperthyroidism is less than one people per thousand and subclinical hyperthyroidism prevalence is 4.2 people per thousand . Mental health is considered as one of the most important indicators of the health and hygiene of a society and is mainly affirmed by the psychologists and other scientists of behavioral and social sciences; this has developed a background for evaluating and assessing mental disorders caused by hypothyroidism . The purpose of mental health is a person s complete ability to perform his / her daily affairs, communicate with his / her family and environment properly, and show no adverse behaviors culturally and socially . Mental health is very important because it is correlated with a person s individual and social performance . In fact, providing mental health can lead to increased efficiency in both personal and social aspects . Many studies have reflected that duration of treatment will be prolonged in patients with lower mental health levels and these patients can be prepared through necessary interventions in order to achieve more adaptation and encounter to the tension of the disease . A study has indicated that psychiatric disorders are usually common in patients with acute hypothyroidism and these disorders are (to some extent) recovered along with the treatment of the causative disease . The effect of hypothyroidism on increase of depression parameters in older people has been proved in another study . Hypothyroidism can also affect the conceptive (perceptive) and temperamental (behavioral) functions of the patient . Mental signs, stress, tension, memory loss (par amnesia), and physical dysfunctions are the factors that cause quality of life deterioration . Quality of life is the state of our good or bad feeling about our own life . Effect of auto - immunity thyrotoxicosis on the quality of life of patients with auto - immune hypothyroidism has been determined . The results of another study have determined that men with hypothyroidism have weaker physical conditions than hypothyroid women . Another study surveying the relationship between the quality of life and hypothyroidism has revealed that the quality of life of most of the patients with hypothyroidism was at an intermediate level . Anyway, psychological factors affect the occurrence of all diseases and the fact that whether their role is related to the beginning, progression, severity or recurrence of the disease or predisposing or reaction to the disease has been a controversial issue and it is variable among different diseases . Generally, the importance of quality of life and mental health in hypothyroid patients is considerable . Changes in mental health condition of these patients have been approved . Also, mental health is one of the most important factors affecting the quality of life . Therefore, in the present study, the mental health condition and the quality of life level of patients with hypothyroidism have been compared with the normal people in order to perform appropriate care programs to increase these patients mental health condition and quality of life . Therefore, the purpose of present study was to compare the quality of life and mental health of hypothyroid patients with normal people referring to motahari clinic, affiliated to shiraz university of medical sciences . This study is a descriptive - analytic investigation performed in motahari clinic of shiraz city . Research samples were 95 patients (more than 20 years old) with hypothyroidism confirmed by specialists; they referred to motahari clinic and were selected through convenience sampling method . Recruitment was performed by attendance of the researcher at the motahari clinic and selection of the patients based on inclusion and exclusion criteria, between october 2014 and august 2015 . A control group of 95 normal people was assigned among the relativesof the other patients and also the personnel of the clinics by convenience sampling method . Then, the sample volume was calculated and determined using med - calc statistical software with 5% error rate and 80% statistical power (attrition of 20%) as 190 cases . N=(z1-2)2(12+22)(2-1)2 =0.05, =0.10, 1=20.1, 2=19.6, sd1=2.95, sd2=2.8, z1-a/2=1.96, z1-b/2=0.85 . Age more than 20 years old and ability to write and read or to participate in the interview were the inclusion criteria . Exclusion criteria were lack of willingness to participate and cognitive disorders or chronic diseases . Eligible patients with hypothyroidism referring to motahari clinic of shiraz university of medical sciences were placed in the test (patients) group . Normal people (control group) were the eligible relative of the other patients (except patients with hypothyroidism) with or with no other diseases except hypothyroidism referring to the motahari clinic without any direct relationship or affinity to patients with hypothyroidism . After approval of the ethics committee of shiraz university of medical sciences (no 93 - 01 - 08 - 8062) and coordination with the clinic, the researcher attended the clinic on all working days of the week and at two work shifts of morning and afternoon and selected the eligible samples . The purpose of the research was explained to the participants, written consent forms were obtained, and they were ensured about the privacy of the data . Then, the individual questionnaire, general health questionnaires (ghq) and the quality of life questionnaire were delivered to the study units . If the participants were not able to fill the questionnaire, the questions were read to them by the researcher and their answers were recorded completely . Data collection tool in this study was a 3 part questionnaire . In the first part, the demographic information was gathered through 5 questions (age, sex, marital status, level of education and occupation) and in the second part, the questions of ghq through 28 questions in 4 domains of physical signs, anxiety, social dysfunction and depression on the basis of a 4- choice likert scale (not at all, normally, more than usual and much more than usual). Reliability coefficient of the whole test were reported as 0.88 and those of the fields were 0.77, 0.81, 0.50 and 0.58, respectively . (2001) evaluated the ghq-28 psychometric properties; the coefficients of test - retest and split half and chorenbach alpha were obtained as 0.70, 0.93 and 0.90, respectively . The simultaneous validity was obtained 0.55 through midlex questionnaire and the construct validity was reported 0.72 to 0.87 . The third part was about the questions of whoqol - bref questionnaire, evaluating the quality of life generally and totally; this part consisted of 26 questions in 4 fields of physical health, mental health, social relations and environmental health (3, 6, 7 and 8 questions for each field respectively) and 2 other miscellaneous questions surveying the health condition and quality of life level in a general manner . Each field was given a score range of 4 - 20, scoring 4 representing the worst and score 20 the best condition in the related field . Evaluated the reliability of whoqol - bref questionnaire in iranian community with test - retest of all domains; physical health, mental health, social relations and environmental health respectively 0.77, 0.77, 0.75, 0.84 . Also, internal consistency of all domains using alpha cronbach between patients and healthy subjects was reported 0.52 - 0.84 . Data were analyzed in spss software version 19 (spss statistics; ibm corporation, chicago, illinois, usa) using descriptive statistical methods and independent t- test, pearson correlation coefficient and anova and p<0.05 was considered statistically significant . Frequencies and percentages were calculated for categorical variables, means and standard deviations for continuous variables . Independent t - test was performed to examine mental health, quality of life and quantitative demographic information . Then, the sample volume was calculated and determined using med - calc statistical software with 5% error rate and 80% statistical power (attrition of 20%) as 190 cases . N=(z1-2)2(12+22)(2-1)2 =0.05, =0.10, 1=20.1, 2=19.6, sd1=2.95, sd2=2.8, z1-a/2=1.96, z1-b/2=0.85 . Age more than 20 years old and ability to write and read or to participate in the interview were the inclusion criteria . Exclusion criteria were lack of willingness to participate and cognitive disorders or chronic diseases . Eligible patients with hypothyroidism referring to motahari clinic of shiraz university of medical sciences were placed in the test (patients) group . Normal people (control group) were the eligible relative of the other patients (except patients with hypothyroidism) with or with no other diseases except hypothyroidism referring to the motahari clinic without any direct relationship or affinity to patients with hypothyroidism . After approval of the ethics committee of shiraz university of medical sciences (no 93 - 01 - 08 - 8062) and coordination with the clinic, the researcher attended the clinic on all working days of the week and at two work shifts of morning and afternoon and selected the eligible samples . The purpose of the research was explained to the participants, written consent forms were obtained, and they were ensured about the privacy of the data . Then, the individual questionnaire, general health questionnaires (ghq) and the quality of life questionnaire were delivered to the study units . If the participants were not able to fill the questionnaire, the questions were read to them by the researcher and their answers were recorded completely . Data collection tool in this study was a 3 part questionnaire . In the first part, the demographic information was gathered through 5 questions (age, sex, marital status, level of education and occupation) and in the second part, the questions of ghq through 28 questions in 4 domains of physical signs, anxiety, social dysfunction and depression on the basis of a 4- choice likert scale (not at all, normally, more than usual and much more than usual). Reliability coefficient of the whole test were reported as 0.88 and those of the fields were 0.77, 0.81, 0.50 and 0.58, respectively . (2001) evaluated the ghq-28 psychometric properties; the coefficients of test - retest and split half and chorenbach alpha were obtained as 0.70, 0.93 and 0.90, respectively . The simultaneous validity was obtained 0.55 through midlex questionnaire and the construct validity was reported 0.72 to 0.87 . The third part was about the questions of whoqol - bref questionnaire, evaluating the quality of life generally and totally; this part consisted of 26 questions in 4 fields of physical health, mental health, social relations and environmental health (3, 6, 7 and 8 questions for each field respectively) and 2 other miscellaneous questions surveying the health condition and quality of life level in a general manner . Each field was given a score range of 4 - 20, scoring 4 representing the worst and score 20 the best condition in the related field . Nejat et al . Evaluated the reliability of whoqol - bref questionnaire in iranian community with test - retest of all domains; physical health, mental health, social relations and environmental health respectively 0.77, 0.77, 0.75, 0.84 . Also, internal consistency of all domains using alpha cronbach between patients and healthy subjects was reported 0.52 - 0.84 . Data were analyzed in spss software version 19 (spss statistics; ibm corporation, chicago, illinois, usa) using descriptive statistical methods and independent t- test, pearson correlation coefficient and anova and p<0.05 was considered statistically significant . Frequencies and percentages were calculated for categorical variables, means and standard deviations for continuous variables . Independent t - test was performed to examine mental health, quality of life and quantitative demographic information . 95 patients (male and female) in the patient group and 95 people (male and female) in the control group were studied . Minimum age in the patient group was 22 years old and maximum age was 70 years . The sample s demographic characteristics different aspects of the quality of life were assessed and compared between the two groups using independent t - test and no significant difference was noted between the test and control groups . In other words, the patients mental health mean score was 59.70, compared with that of the normal people which was 48.68 (p>0.001). In fact, the physical signs, anxiety and depression were higher and more severe in the test (patients) group . In other words, the mental health condition of hypothyroid patients was lower than the normal people . Mean and standard deviation between the quality of life and mental health in hypothyroidism patients and healthy subjects comparison of the mean scores of mental health and quality of life in hypothyroidism patients and normal subjects comparison of demographic data and both of mental health and quality of life showed a significant relationship (p=0.005) just between education and mental health (table 4). Also, evaluation of the relationship between demographic variables and mental health and quality of life revealed that there was a significant difference only between the mental health condition and physical signs (p>0.001) and anxiety (p>0.001) with degree of education . But this significant relationship was not observed in the quality of life and its aspects (p>0.001). Mental health and quality of life based on demographic variables these results also confirm the significant correlation between the mental health and quality of life in hypothyroid patients (p>0.001). The present study was performed to assess and compare the quality of life and mental health between patients with hypothyroidism and normal people . Results showed that hypothyroid patients have significant differences in all aspects of mental health compared with the normal people . It was revealed that these patients have the highest severity of depression, physical signs and anxiety . But there was no significant difference in the quality of life and its aspects between the patients and the normal people . These results regarding the low level of mental health in patients with hypothyroidism compared with the normal people are in agreement with other studies . A study performed to investigate the effect of thyroid disorders on anxiety and depression level of patients showed that anxiety and depression signs in patients with overt hypo- and hyper - thyroidism were more intense than other thyroid disorders . Another investigation has noted that anxiety - but not depression- was higher in patients with differentiated thyroid cancer (dtc) than normal people . Also, another study revealed that neuropsychiatric disorders were very common in hypothyroidism which may be recovered through treatment done regarding the underlying cause (treatment associated with hypothyroidism). Because of the considered aspects of mental health have been evaluated and assessed in the mentioned investigations, it can be said that their results confirm our findings to some extent . On the other hand, because the depression feature has the most severity in patients with hypothyroidism, it can be seen that some investigations confirm it too . For example, the results of a study showed that subclinical hypothyroidism can increase the risk of depression . There was also a compatible study which showed the significant relationship between physical condition and anxiety aspects of mental health and patient s education level . Those results also showed that there was a direct correlation between hypothyroid patient s education level and their social condition; this can be because of lower social and individual adaptation skills in patients with lower education levels to withstand the problems of the disease . Therefore, patients with higher education levels have better physical conditions and lower anxiety levels . Our results are also compatible with other studies results regarding the comparison of the quality of life of patients with hypothyroidism and normal people . The quality of life of patients with different types of thyroid gland cancer undergoing tsh- suppressive thyroxin therapy was assessed in an investigation . These patients were considered as a model of subclinical hyperthyroidism . Finally, the results showed that restoration of euthyroidism (normal function of thyroid gland) had no effect on the quality of life of these patients . But the results of a similar study comparing the mental health condition and the quality of life of patients with hypothyroidism with the normal people were different . It was seen that the quality of life of these patients was lower than the normal people in the mentioned study . Also, another study implied the lower level of quality of life in patients with auto - immune hypothyroidism . This difference can be due to the varieties of the study population, culture and the type of investigation tool . In this regard, the results of a review study on evaluating tools of signs and condition of health and quality of life of hypothyroid patients showed that many of using tools were not appropriate to the purpose of the investigations; hence, they can increase or alter the results . Moreover, there are some compatible studies about the significant correlation between mental health and the quality of life in patients with hypothyroidism . In a study performed to investigate the relationship between mental health and quality of life of patients with coronary arteries disorders, this relationship was reported as significant . Unfortunately, in studies performed to compare the quality of life and mental health of patients with multiple sclerosis (ms) and irritable bowel syndrome (ibs), only the results of the quality of life comparison and mental health comparison between 2 groups were reported, but no results regarding the existence or otherwise of the relationship between the two mentioned variables in one group were reported . In this study, most of the subjects were male with a mean age of 38.93 . In comparison with this result, the other study reported the prevalence of overt and subclinical hypothyroidism more in female, respectively . This result disagrees with that of the current study and it can be due to the selection of subjects from one center . But another study conducted on hypothyroidism patients over 18 years of age, reported that the mean age of the subjects was 44 years old . Therefore, it is recommended that in the future studies in this field, multi - central sampling should be considered . The most important limitation of the present study was the selection of patients from only one specialized clinic (of course, it is the most active clinic in this field in the south of iran). Therefore, generalization of the results of this study to larger fields must be performed with caution and in order to increase the extension capability of these findings, the population and sample size must be larger and multiple . Also, the use of several diagnostic methods for the selection of normal people in future studies is recommended . With regard to the results of the present study indicating lower levels of mental health in patients with hypothyroidism, holding educational programs and protocols about this disease to improve the mental health is suggested . Also, these results indicate the relationship between mental health and quality of life; therefore, it is necessary to pay attention to their importance in such patient s care and treatment programs design.
Genetic disorders caused by mutations in the -globin gene are widely known as the human -hemoglobinopathies, in which -thalassemia and sickle cell disease (scd) are the most prevalent ones, particularly in the mediterranean, africa, and southeast asia, leading to great mortality and morbidity [14]. The high occurrence of the -thalassemia and scd mutations is due to the reason that both cause mild severity of malarial infection in the heterozygous state [57]. However, in the homozygous state, these mutations shorten the lives of affected ones . -thalassemia is caused by the inherited mutations in the -globin gene complex, resulting a total absence or severe decrease in the production of -globin chains [8, 9]. The lack of -globin chain production leads to the accumulations and precipitations of free intracellular -globin chains, which may consequently result in premature hemolysis of red blood cells and apoptosis of erythroid precursor [8, 10, 11]. Therefore, the combining effects of ineffective erythropoiesis, hemolysis, and hypersplenism are the main culprit of severe anemia in -thalassemia patients . It is characterized by the abnormal appearance of the red blood cells which are rigid and sickled . Scd is attributed to a point mutation at the coding sequence of the -globin gene which causes the substitution of glutamate by valine in the glutamic acid at the sixth position of -globin protein, and thus forming a sickle hemoglobin (hbs, 22) when incorporating into a hemoglobin tetramer [13, 14]. Hbs will polymerize inside the red blood cells under hypoxic condition, resulting in the alternation of the shape of red blood cells as well as their function . However, long - term transfusion therapy may cause iron overload in patients from the gradual breakdown of transfused blood which may eventually result in cardiac failure and/or even death . Though the advance in iron chelation can help to remove excess iron in patients, the survival rate is greatly dependent on the iron chelation regimens . Allogeneic hematopoietic stem cell transplantation (hsct) is one of the gene transfer therapies aimed at the underlying molecular causes of scd and -hemoglobinopathies . Several hundred scd and thalassemia patients have successfully experienced hsct with promising results [16, 17]. Nevertheless, there is a great likelihood that hsct will be limited to a small proportion of hemoglobinopathy patients as evidence has shown merely younger patients and those who have not developed significant disease complications have gained the best results in hsct . Also, most successful transplantations have to utilize stem cells from matched sibling donors making hsct a challenging therapy for some patients . Therefore, hsct may not be applicable to many current patients . Transferring of - or -hematopoietic stem cells of patients can be another therapy option for -thalassemia patients . It has taken a long period of time to have the clinical gene transfer protocol being approved since the transduction of human hematopoietic stem cells and gene expression must reach certain efficiency and high level . Despite the fact that this approach has successfully passed its initial human trial, previous studies reported the issues regarding low autoploidy recombination, insertional mutagenesis, and effect of inserted vectors on the expression of nearby genes could possibly limit the application [21, 22]. Apart from gene therapy, fetal hemoglobin reactivation by chemical agents appears promising enough to develop into effective interventions to cure human -hemoglobinopathies . Previous studies have revealed that homozygous -thalassemia patients will not suffer severe anemia until fetal -globin genes are silenced and that patients carrying hereditary persistence of fetal hemoglobin (hpfh), meaning fetal hemoglobin (hbf) is abnormally persisted at high level in adults, will only suffer mild anemia [8, 13, 2327]. More evidences also supported that hpfh can improve the severity of both -thalassemia and scd . Therefore, it have been suggested that increasing the synthesis of fetal hemoglobin (hbf) by reactivating fetal -globin gene can be a potential therapy in patients suffering -thalassemia or scd . It is expected that the pharmacological induction of hbf can correct the globin chain imbalance in -thalassemia patients, while inhibit hbs polymerization in scd patients [2832]. In recent years, much effort has been made to identify the naturally occurring inducers and drug treatments which can increase the synthesis of hbf and promote the expression of fetal -globin gene . Some chemotherapeutic agents, for example, 5-azacytidine and hydroxyurea, (hu) have been reported due to their ability to enhance hbf production [31, 33, 34]. Yet, most of these currently identified hbf - inducing agents exhibit low efficacy and specificity, myelotoxicity, and carcinogenesis as well as modest responses to treatment which greatly limit their usefulness in the clinical practice [5, 35, 36]. Owing to this, (i) discovering novel screening platform for identifying potential inducers with high efficiency and accuracy and (ii) identifying new hbf - inducing agents from the natural world with the combination of efficacy, safety, and ease of use will be high on the agenda . With the aim of determining the therapeutic potency of the novel inducing compounds and studying the underlying regulatory mechanism of the embryonic and fetal human globin genes expression, various in vitro and in vivo screening platforms have been widely utilized . For in vitro models, there are six human cell lines carrying an embryonic - hbf phenotype; they are k562 human chronic myelogenous leukemia cells, m - tat, nsmeg, ocim1, ocmi2, and as - e2, while k562 cell line is one of the most well - known and widely used screening platforms for hbf inducers [13, 30]. Another seven human cell lines which are capable of synthesizing both - and -globin chains are jk-1, kmoe-2, ku812, lama-84, tf-1, tn922, and ap217; yet, ku812 cell line is comparatively unique and useful from the others as it can undergo a spontaneous differentiation which can be observed [30, 3739]. Moreover, human bone marrow cd34 + hematopoietic progenitors drawn from -thalassemia patients and primary erythroid progenitors stem cells (epscs) obtained from peripheral blood are also great in vitro models to study the effect of different hbf - inducing agents [1, 13]. Back to the 1980s, baboons, a nonhuman primate model, have already been used for the study of fetal and adult hemoglobin synthesis during fetal development [40, 41]. There was an influential research conducted by de simone and colleagues that the adult baboon has been shown to respond to erythropoietic stress with the reverse hemoglobin switch during which an increase in the number of hbf - containing erythrocytes (f cells) and an increase in hbf synthesis can be observed in adult baboon [40, 42, 43]. However, this animal model is expensive to purchase, feed, and maintain in conditions appropriate to modern animal husbandry . In order to understand the influence of hbf synthesis and its induction mechanism, till the 90s, researchers have successfully discovered that transgenic mice carrying human a -globin gene, which can act as a new in vivo model for screening novel pharmaceutical compounds for hbf induction in the adult [44, 45]. These inducers can be categorized into several classes based on their mechanisms of action [13, 46] as listed in table 1 . Some of them are classified as chemotherapeutic agents, for example, hydroxyurea (hu), 5-azacytidine (5-aza), decitabine, and citarabine . Hu is also known as a ribonucleotide reductase inhibitor due to its ability of inhibiting dna analysis, while 5-aza, decitabine and citarabine are dna methyltransferase inhibitors who responsible for the hypomethylation of dna [8, 13]. Several short - chain fatty acids (scfas) specifically stimulate transcription in the -globin gene promoter through histone deacetylase hdac inhibition, resulting in global histone hyperacetylation [5, 47]. In contrast, some studies argue that globin histone hyperacetylation induced is not the primary mechanism of scfa; yet, hdac inhibitors are often potent -globin inducers [47, 48]. Rapamycin preferentially induce -globin mrna accumulation, while being only minor for -globin and none for -globin mrnas . As its hbf - inducing effect is not related to cytotoxicity and cell growth inhibition, scientists are very interested in further studying if the enhancement of -globin mrna medicated by rapamycin is associated with xmni polymorphism . There are studies showing that k562 cells treated with dna - binding agents, such as mithramycin, have led to erythroid differentiation and sharp enhancement of -globin mrna level . Through pcr arrested experiments, it is found that these dna - binding drugs were capable of interacting with -globin promoter of human genomic dna . In recent years, researches have been done on immunomodulatory drugs, such as thalidomide, revlimid, and pomalidomide . Their exhibited hbf - inducing activity has been revealed in k562 or primary human erythroid cultures . Further study has demonstrated their activity is associated with the increase in histone acetylation at -globin gene promoter . Erythropoietin (epo) is a cytokine that have been shown to induce hbf production during in vitro differentiation of primary human cells in several trials [47, 5255]. It also stimulates red blood cells production, prolongs the survival of erythroid cells, and decreases the incident of programmed cell death . In recent years, scientists have conducted numerous studies in order to identify the natural remedies that could be possibly applied in treating -hemoglobinopathies, including scd and -thalassemia, summarized in table 2 . The extract of aegle marmelos containing bergatene was found to be responsible for the activation of erythroid differentiation and hbf induction in human leukemic k562 cells [31, 69]. They are powerful inducers of erythroid differentiation, -globin gene expression and hbf synthesis in human erythroid cells . Thus, it is known as a potential therapeutic approach for both -thalassemia and sickle cell anemia . In addition, nicosan (formerly known as niprisan), an ethanol / water extract from nigeria indigenous plants, has successfully demonstrated a significant anti - sickling effects in vitro as well as in vivo [70, 71]. There is evidence demonstrating that angelicin is a powerful inducer of erythroid differentiation, enhancement of the hbf synthesis in erythroid progenitors and -globin mrna accumulation of human leukemia k562 cells [8, 73]. Red wine, especially the skin of grapes, contains resveratrol which mimics the hbf - inducing activity of hu . Since -thalassemia cells exhibit a high level of oxidative stress, which eventually shorten the survival of erythroid cells in -thalassemia patients, resveratrol which exhibits both antioxidant activity and hbf inducing property can become a very promising hbf inducer from the natural world . Rapamycin is isolated from streptomyces hygroscopicus, a bacterial species being found in the soil of easter island . It has the ability to increase hbf production in cultures of erythroid precursors from -thalassemia patients without cytotoxicity or growth - inhibitory effect [8, 13]. It is a dna - binding drug which has the potential to induce -globin mrna accumulation and hbf production in erythroid cells from healthy human subjects as well as -thalassemia patients . Recently, it is reported that an indian almond, called terminalia catappa, has long been used as a traditional herbal treatment for sca in nigeria . Has then demonstrated terminalia catappa distilled water active fraction (tcdwf) from terminalia catappa leaves exhibit a stimulatory effect on the hbf production in primary erythroid prohenitor stem cells (epscs). Terminalia catappa consists of flavonoids, alkaloids, and anthraquinones . Also, through gas chromatography - mass spectrometry (gcms), it is shown that a group of highly related long - chain fatty acids, for example, hexadecanoic acid, 10-octadecenoic acid, and octadecenoic acid, are present in the tcdwf . Yet, further investigation is required in order to confirm the biological activities of these active compounds present in the tcdwf . For the chinese herbal medicine, yisui shengxue granule (yssxg), a complex prescription made up of 11 different kinds of chinese herbal medicines, has shown to be effective in enhancing the hbf expression and inhibiting ineffective hematopoiesis [76, 79]. Current research has further confirmed this complex prescription has the ability to increase -globin gene expression and alter the expression of genes that involved in the survival, proliferation, and terminal differentiation of erythroid cells . Recently, some researchers found that cucurbitacin d (cud) in a chinese medicinal herb, called fructus trichosanthis (ft), exhibits a higher potency in hbf induction compared with hydroxyurea since there is evidence showed cud results in a higher fetal cell percentage and greater hbf content in k562 cells with much lower cytotoxicity . Scientists have started to put effort on appreciating their underlying molecular mechanisms as well as verifying their target molecules . The first piece of evidence regarding the cell signaling of the hbf induction has suggested that, in k562 cells, butyrate induces erythroid differentiation and hemoglobin production through p38 mapk pathway [1, 80]. Several years later, another group of scientists has interestingly revealed that, without drug treatment, the -globin mrna level is increased sufficiently solely by overexpressing mapkk3 and mapkk 6 which are the direct upstream activators of p38 in k562 cells . According to mabaera et al ., p38 mapk signaling pathway is critical for the upregulation of the production of hbf . Different environmental stresses could activate the p38 mapk signal pathway which will subsequently cause apoptosis, cell growth and erythroid differentiation . Various studies have also revealed the effect of numerous hbf - inducing agents, such as butyrate, apicidin, and trichostatin a, are associated with p38 mapk signaling pathway . Therefore, they both indicated p38 mapk pathway plays a vital role in promoting -globin gene expression . During the past few years, researchers come up with different mechanistic models of hbf induction while most of the models are generally based on what are thought to be the primary actions of hbf - inducing agents, for instance, global dna hypomethylation induced by dna methyltransferase inhibitors (dnmt inhibitors) or global histone hyperacetylation induced by histone deacetylase inhibitors (hdac inhibitors), including scfa derivatives . However, when we come across with the recent key experimental findings, including the fact that 5-aza can promote hbf production without dna hypomethylation, that -globin promoter hypomethylation is inadequate to stimulate gene expression, and that the ability of hdac inhibitors to induce hbf is regardless of the potency of hdac inhibitions, some of these proposed mechanistic models are no longer capable to explain those results . Therefore, it is of essence to propose a new model of hbf induction which is valid to most hbf - inducing agents and can adequately account for the recent experimental results ., through integrating recent results of cell - signaling experiments with the stress erythropoiesis model, have proposed a new model called the cell stress signaling model . They suggested that the key effect of most hbf - inducing agents is to activate the cell stress signaling pathways during adult erythropoiesis which subsequently lead to -globin gene expression and hbf production . It is found that certain hbf - inducing agents, such as hu, butyrate (scfa), thalidomide (imid), trichostatin a (hdac inhibitor), and anisomycin, can activate the corresponding cell stress responses, including nitric oxide, oxidative stress (ros), osmotic shock, and protein synthesis inhibition . These cell stress responses will eventually activate the downstream p38 mapk signaling pathway, including downstream kinases and transcription factors, and thus result in -globin gene expression and hbf production . Besides p38 mapk signaling pathway, the potential of camp signaling pathway in hbf production has also been mentioned in the cell stress signaling model . There are findings suggested that in primary erythroid cell cultures, camp response element binding protein (creb) is activated by camp - activated protein kinase a instead of p38 mapk pathway . The phosphorylated creb will then activate the downstream transcription factors and eventually lead to -globin gene expression . Therefore, not only is the cell stress signaling model applicable to most of the hbf - inducing agents but also is able to explain the key findings of some of the previous experiments . In the previous studies, researchers have found out that stimulating erk signaling pathway leads to megakaryocytic differentiation; contrastingly, suppressing erk signaling pathway results in the enhancement of the erythroid phenotype as well as the -globin mrna expression . Moreover, the evidence that erk inhibitor u0126 has the ability to stimulate -globin gene expression and hbf production in human erythroid progenitor cells suggested the inhibition of erk can possibly lead to the promotion of hbf production . The involvement of jnk in erythroid differentiation still remained debatable [76, 8587]. It is reported that pretreatment with hu in k562 cells has led to a significant inhibition of jnk . Moreover, short chain fatty acid derivatives (scfad), such as butyrate and valproate, did not result in jnk phosphorylation; thus, there was not any significant changes on jnk pathway in k562 cells [76, 87]. Nevertheless, in mouse erythroleukemia cells, experimental findings have suggested that activation of jnk is crucial for erythropoietin - induced erythroid differentiation [86, 88]. As a result, whether jnk plays a significant role in hbf induction still remains to be investigated in the future . The potential of reactivating fetal hemoglobin via manipulating the gene transcription of - or -globin has been gained much attention recently . There is escalated number of publications regarding the genetic regulatory mechanism of developmental stage - specific expression of -globin genes . The mechanisms accounting for switching globin - genes expression during the development are highly regulated by cis - acting elements and trans - acting factors which include the locus control region (lcr) [85, 86]. Previous studies have also revealed that lcr has an important of enhancing globin - gene switching potently . Understanding the fetal - to - adult hemoglobin switching is believed to have a clinical relevance of developing novel approaches of reversal of fetal hemoglobin production from adult hemoglobin in patients . Several researchers have applied the knowledge of the regulatory mechanisms of globin - genes expression to design an artificial zinc finger dna - binding domain (zf - dbd) with the aim of manipulating the expression patterns of globin - genes [85, 87]. Therefore, techniques involving small molecule inhibitors or genetic knockdown can be potential applications to reactivate the fetal hemoglobin in the future . Interestingly, besides the discovery of the upstream signaling pathway of activating the -globin gene expression, there are findings supporting the fact that posttranscriptional regulation also involves in regulating the -globin gene expression in response to different stimuli [89, 90]. Weinberg et al . Revealed that instead of acting on the transcription rate of -globin gene in patients, butyrate has the ability to enhance the efficiency of translation of -globin mrna . Moreover, there is evidence proving that the key effect of gtp and doxorubicin derivatives is the elevation of the amount of -globin in patients, at least partially, by enhancing the stability of -globin mrna [91, 92]. Liu et al . Also suggested that, similar to gtp and doxorubicin derivatives, cud can therapeutically induce the production of hbf due to its ability of increasing -globin mrna stability . They have showed even though the change in the half - life of -globin mrna is small, it leads to remarkable changes in the total amount of stable -globin mrna and consequently the amount of functional -globin present in the cell . Therefore, the control of -globin mrna stability can be known as a significant regulatory mechanism of -globin gene expression . In -thalassemia, due to the precipitation of excess -globin chains, rapid cellular apoptosis of early erythroblasts and red blood cell membrane damage are well characterized [9496]. 5-azacytidine, hydroxyurea, myleran, and butyrate had long been applied in clinics for -thalassemia treatment with the aim of stimulating hbf production [97100]. Despite the ability of inducing the production of hbf in -thalassemia, a large portion of identified hbf - inducing agents, such as 5-azacytidine and hydroxyurea, are chemotherapeutic agents which inhibit cell proliferation and cause cell growth arrest . Further, due to their cytotoxic nature, the dose limiting myelotoxicity and potential carcinogenicity have always led to concerns . Therefore, in order to correct the pathophysiology of -thalassemia, it is of essence to improve the underlying erythroid cell survival and proliferation, with the intention that hbf - inducing agents can exert their effect on stimulating the fetal globin expression prior to the activation of irreversible programmed cell death pathway . Owing to this issue, perrine et al . (2002) have conducted a pilot study revealing the combination of butyrate and rhu - erythropoietin (epo), the hematopoietic growth factor that stimulates erythroid proliferation, decreases apoptosis, and prolongs erythroid cell survival and differentiation, has addictive effects in inducing hematologic responses in any -thalassemia patients . This result further suggested that definitive treatment to correct -thalassemia will likely require more than one type of therapeutic regimen; in other words, hematopoietic growth factor, such as exogenous epo will be required for -thalassemia patients in order to respond optimally to any hbf - inducing agent . Hydroxyurea (hu) was first approved by the food and drug administration (fda) for the treatment of sickle cell disease (scd) in 1996; yet, hu only increases hbf production in approximately half of scd patients and is even less effective in enhancing the hbf level for -thalassemia patients [104106]. Additionally, there is a recent clinical trial conducted to assess the therapeutic potential of hqk-1001, an oral butyrate derivative 2,2-dimethylbutyrate sodium salt to treat -thalassemia patients . Although results revealed hqk-1001 exhibits a stimulatory effect on hbf production and -globin gene expression, further study is needed to find out if the surge of hbf production is sufficient to alleviate the complications of -thalassemia including anaemia, chronic haemolysis, and so forth . Nevertheless, up to date, there is no such pharmacological agent(s) has been officially approved by fda for treating patients with -thalassemia . The underlying reason of this is that for pharmacological hbf induction therapy in patients with thalassemia major, it requires the production of -globin chains (plus -globin chains in thalassemia major) reaches 50% of the production of -globin chains in order to result in optimal therapeutic correction of the anemia in patients with -thalassemia; while for that in patients with scd, it only requires the production of hbf reaches 2030% of total circulating hemoglobin for sufficient prevention of sickling effect in scd . Therefore, it is necessary to identify novel pharmacological hbf inducer or alternate therapeutic approaches which can effectively enhance the hbf production to the optimal level and sufficiently reduce the chain imbalance in homozygous -thalassemia . In recent years, (2008) have proposed p38 mapk cell stress signaling pathway and other stress - related pathways may be the keys to understanding hbf induction, owing to the fact that most hbf - inducing agents are, as mentioned before, cytotoxic and many activate the p38 mapk cell stress signaling pathway . This stress signaling model predicts a variety of diverse hbf - inducing compounds and stimuli will activate cell stress signaling pathway which will then activate similar response genes, such as the -globin genes . It is supported by several observation and evidence, for example, the stimulation of -globin gene expression and hbf production in human primary erythroid cells by 5-azacytidine is closely related to p38 mapk phosphorylation and this stimulation is inhibited when treated with p38 mapk inhibitor sb203580 . On the other hand, based on the stress signaling model, mabaera and colleagues (2008) have also made some predictions which require further investigation, for example, the members of stress signaling pathways, ranging from the sensors of cellular stress to the activated transcription factors that bind to the -globin gene promoters, are needed for activating the -globin gene expression as well as hbf production . Nevertheless, concerns have been raised about the possibility of triggering rapid cellular apoptosis in erythroid cells and consequently leading to low blood count when patients, especially those with thalassemia, are receiving chemotherapeutic agents including 5-azacytidine, decitabine, hu and butyrate . Comparing with scd patients, -thalassemia patients are more susceptible to chemotherapeutic agents because treating with chemotherapeutic agents may further encourage rapid cellular apoptosis which has already been well - characterized in -thalassemia patients even they are not exposed to the agents . Also, as mentioned before, accelerated cellular apoptosis of erythroid progenitors in -thalassemia is a significant barrier to definitive therapy because there is a high possibility that the programmed cell death will be established earlier before hbf - inducing agents act its beneficial effect on the globin chain balance in cells . Consequently, chemotherapeutic agents, such as hu, butyrate and 5-azacytidine, may not be the best hbf - inducer for -thalassemia due to the fact that the hbf - inducing property of those agents are largely dependent on the activation of cell stress signaling pathway . In contrast, those chemotherapeutic agents may have beneficial effect on activating -globin gene expression and hbf production in scd; yet, the dose of inducing agents must be strictly monitored in order to ensure it is high enough to activate stress signaling pathway but not too high to trigger cell - cycle arrest or apoptosis in hematopoietic precursor cells that will result in dangerously low blood counts . In light of the lack of promising hbf - inducing agents for -thalassemia treatment, up to now, different research groups have paid lots of effort on identifying the existing or novel natural chinese herbal medicines which have the possibility to effectively induce hbf synthesis without any apparent growth - inhibitory effects . Yisui shenxu granule (yssxg), a complex prescription consisting of 11 chinese herbal medicines, had been used for treating -thalassemia for more than over 20 years [76, 79]. Recently, effort has been put on investigating the efficacy and safety of yssxg by a randomized single - blinded trial . Result has demonstrated it has obvious clinical efficacy, while hepatauxe and splenomegaly were relieved and no adverse reaction was observed . The underlying mechanism for the effect of yssxg is possibly by activating the expression of -globin gene and increase hbf production in order to compensate for the functional deficiency of -globin gene . Zhang and wu have found out that stimulating the gene expression of -globin, epor, spi, and fklf, while hindering the gene expression of ckit, gata1, and gata2, could promote the -globin gene expression and alter the expression of gene which is responsible for the regulation of -globin gene expression and the expression of other genes that are participated in the survival, proliferation, and terminal differentiation of erythroid cells . (2011) have presented the first piece of evidence on the hbf - inducing property of ethanol extract of fructus trichosanthis (ft), one of the most frequently used chinese herbal medicine . Their study has demonstrated ft has the ability to enhance the -globin gene expression as well as hbf synthesis through activating p38 mapk and inhibiting erk signaling pathway . Despite the promising result shown, acute and chronic toxicity test in vivo is strongly required in the future in order to ensure the ethanol extract of ft is safe for clinical use on human . Also, the efficacy of the extract should be examined in clinical evaluation before actual clinical practice . During the same year, another group of researchers have identified cucurbitacin d (cud), a chemical inducing agent that can be found in fructus trichosanthis, is a novel therapeutic agent for treating -thalassemia . There are evidences suggesting that cud could act as a good hbf - inducing agent for -thalassemia patient comparing with hu since cud exhibits a higher amplitude and rapidity in enhancing the hbf production than hu does in k562 cells, and, unlike hu, cud does not show any growth - inhibitory effect even when it is at its optimal activity . Taken all together, natural herbal medicines, which exhibit a higher potency in hbf induction compared with chemotherapeutic agents and have much lower cytotoxicity, will definitely be the novel therapeutic candidates for -thalassemia by targeting the activation of -globin gene expression; yet, some of the candidates are still required further investigation on their safety and efficacy . Chemotherapeutic agents, such as 5-azacytidine, hydroxyurea, myleran, and butyrate, had long been used for -thalassemia treatment by stimulating hbf synthesis; yet, cytotoxicity, growth - inhibitory effect, fear of long - term carcinogenesis, and only modest hbf - inducing activity have limited the clinical usage of these agents in -thalassemia and scd treatment . Also, through understanding the pathology of -thalassemia, it is revealed that most of the identified hbf - inducing agents have limitation on treating -thalassemia . It is because the rapid cellular apoptosis of erythroid progenitors in -thalassemia causes a significant obstacle that overstimulating the cell stress signaling pathway by the hbf inducer may possibly lead to irreversible cellular apoptosis before -globin gene expression and hbf synthesis can be stimulated . With the advancement of biotechnologies, increasing number of studies will be done to explore and optimize new interventions and nature remedies to reactivate hbf synthesis for -thalassemia patients . In the future, it is expected that increasing number of hbf inducing agents could be found from natural remedies and folk medicines all over the world . In this context, further studies are required with the aim of exploring more natural herbal medicines as well as studying the efficacy and safety of from the laboratory to clinical use for the individuals with -hemoglobinopathies.
A girl weighing 3,190 g was delivered by a caesarean section at 38 weeks and five days of gestation . At the time of birth, a systolic murmur was noted during a physical examination, and transthoracic echocardiography revealed a peri - membranous ventricular septal defect (vsd) with a septal aneurysm, a small patent foramen ovale (pfo), and a small right - sided patent ductus arteriosus (pda) from the innominate artery . The vsd was measured as having a diameter of 3.5 mm and a shunt flow less than 2.5 m / sec . Due to the presence of neonatal hyperbilirubinemia, echoencephalography was conducted, and no abnormalities were found . Five months later, the patient was referred to konkuk university medical center for vsd and pda . Her body weight was 6,700 g (25th percentile), and her height was 61.6 cm (10th percentile). Transthoracic echocardiography revealed a vsd approximately 6 mm in size with a minimal aneurysm, a left - sided pda 3.6 mm in diameter from the right aortic arch and an aberrant left subclavian artery . A subsequent computed tomographic scan demonstrated isolation of the left subclavian artery with a right aortic arch, a left pda, and a vsd (fig . The intraoperative findings were a perimembranous vsd, a pfo, a mildly patent right ductus arteriosus, and isolation of the left subclavian artery connected to the left pulmonary artery via a left pda . The left subclavian artery was disconnected from the left pulmonary artery and reimplanted to the left common carotid artery by end - to - side anastomosis with monofilament polypropylene 6 - 0 sutures (fig . Ductus arteriosus is usually located on the left side, between the descending aorta and the junction of the main pulmonary artery and left pulmonary artery . However, ductus arteriosus may also be present on the right side or, very rarely, may occur bilaterally in association with aortic arch anomalies or conotruncal anomalies . In such aortic arch anomalies, isolation of the left subclavian artery with right aortic arch is also uncommon . Here, isolation refers to the fact that the left subclavian artery connects to the pulmonary artery via either the ligamentum arteriosum or a patent ductus arteriosus without any connection to the aorta . Isolation of the left subclavian artery with a right aortic arch is known to be commonly associated with congenital heart disease, but may also occur with normal intracardiac anatomy, although few such cases have been described . Isolation of the left subclavian artery with a right aortic arch may be related to the 22q11 deletion . Bilateral ductus arteriosus and isolation of the left subclavian artery with a right aortic arch can be explained through the hypothetical double aortic arch plan suggested by edward . Regression takes place on two levels in the double aortic arch plan: on one level, regression occurs between the left common carotid artery and the left subclavian artery; and on the other level, regression occurs at the left dorsal aortic root distal to the left ductus arteriosus . And then right ductus arteriosus remains persistent, left ductus arteriosus connects the left subclavian artery to the left pulmonary artery (fig . However, the right ductus arteriosus regressed, and only the left ductus arteriosus remained patent . If the left ductus arteriosus is patent, blood may be supplied to the left subclavian artery via the left ductus arteriosus . If the left ductus arteriosus regresses, the blood supply to the left subclavian artery may involve a mediastinal, thoracic anastomosis, or vertebral pathway . Isolation of the left subclavian artery usually presents with no apparent symptoms in neonates, but it may present with congenital pulmonary steal syndrome, subclavian steal syndrome, or may even present in adults with late symptoms due to sporadic progression . Hayabuchi et al . Reported the case of a three - month - old girl with cerebral atrophy and an underdeveloped left arm . Reported the case of a 15-year - old boy with an underdeveloped left arm . Due to these symptoms and signs, the therapeutic management of isolation of the left subclavian artery remains controversial, especially when it is associated with complicated congenital heart disease . Some authors have suggested that adequate collateral circulation must be ensured, meaning that reconstruction of the isolated subclavian artery is optional, regardless of the symptoms and signs . Successful results have been reported after ligation or device closure of the pda and ligation of the left subclavian artery . However, reconstruction of the left subclavian artery due to pulmonary steal syndrome after right pda closure in bilateral pda has been reported . In one report, ischemic symptoms in the left arm and vertebrobasilar insufficiency occurred years after ligation of the left subclavian artery . Hokari et al . Reported that a man with peutz - jeghers syndrome presented with his first vertigo attacks due to subclavian steal syndrome at 29 years of age . Our patient presented with no symptoms and signs related to subclavian or pulmonary steal syndrome, and had shown normal findings on an echoencephalography study conducted at our medical center due to neonatal hyperbilirubinemia . However, brain computed tomography angiography performed after surgery revealed hypoplasia of the left vertebral artery . We suggest that this hypoplasia would have led to vertebrobasilar insufficiency or underdevelopment of the left arm without surgical reconstruction . Since surgical reconstruction of the isolated left subclavian artery leads to antegrade flow in the left subclavian artery, it can prevent hypoplasia of the left vertebral artery and subclavian / pulmonary steal syndrome . Our case shows that early surgical reconstruction is reasonable, regardless of the symptoms, in cases of isolation of the left subclavian artery.
In 2015, an estimated 477.9/100,000 cases of esophageal cancer (ec) were diagnosed in china, and approximately 375/100,000 people died from this disease [1, 2]. In china, ec occurs in 50.3% (161.3/320.8) of patients aged 60 - 74, and in 19.6% (62.9/320.8) of patients over 75 years of age in [1, 2]. A radiation therapy oncology group study (rtog 8501) demonstrated a survival benefit of the addition of platinum - based chemotherapy to radiation, compared to radiation alone for patients with nonsurgical ec [3, 4]. Rtog 8501 only included about 23.1% (28/121) of elderly patients (70 years). Thus, management of elderly patients with ec remains a therapeutic challenge, and the most relevant treatment modalities are still being debated . Although survival improvement has been observed over the past decade, ec treatment continues to be significantly influenced by age . Moreover, it has also been reported that elderly patients have undergone less surgery, radiotherapy, and chemotherapy than younger patients . To our knowledge, no specific data have been published regarding therapeutic strategies in elderly patients with ec . Despite progress in surgical practice, esophagectomy is associated with significant morbidity and mortality, and 75 years is often considered as the age limit for surgery . External beam radiation therapy (ebrt) was an important treatment strategy for elderly patients . However, a few published results indicate that ebrt combined with brachytherapy in elderly patients with ec . Californium-252 (cf) is a neutron - emitting radionuclide, and cf - based neutron brachytherapy (nbt) has only been implemented in china very recently . Neutron brachytherapy is a form of high linear energy transfer (let) radiotherapy, which has been proven to be effective for treating intracavitary cancers of the cervix when used in combination with ebrt [9, 10]. We performed a retrospective cohort study of 191 patients older than 69 years who were diagnosed with locally advanced esophageal squamous cell cancer (escc), treated with radiation therapy . The main objective was to assess the overall survival and local control rates after ebrt plus neutron brachytherapy for elderly escc patients . We also evaluated the impact of age on treatment tolerance, prognostic factors, and patterns of failure . From january 2001 until november 2012, a total of 191 consecutive patients older than 69 years with localized, advanced escc were referred to our department at the changzhi cancer hospital for radiotherapy and cf nbt . The reasons were as follows: 30 patients were medically inoperable (6 patients were diabetic, 11 had chronic obstructive pulmonary disease, and 13 patients had a prior or concurrent malignancy); 34 patients rejected surgery; 76 patients were too old (75 years or older, 33 of 85 had t4 lesion); and 85 patients had unresectable lesions . Of these, 191 patients were treated with ebrt combined with brachytherapy . Patients with good performance status (at least able to care for himself or herself) and adequate hepatic, renal, and hematologic functions were selected for curative treatment . The patients clinical stage was diagnosed by barium examination, endoscopy, endoscopic ultrasonography, or tumor histology . Megavoltage radiation therapy units were used with a minimum source - to - axis distance of 100 cm . The radiation field extended at least 3 cm superior and inferior to the tumor, with a lateral margin of at least 2 cm . The field included the lesser curvature and bottom of stomach if the tumor invades gastroesophageal junction . Multi - field techniques were used to limit the maximum dose to the spinal cord to 45 gy . The initial anterior - posterior parallel - opposed fields received 30 gy, and the off - cord fields received 20 - 30 gy, for a total dose of 40 - 54 gy in 20 - 27 fractions in 4 - 5.5 weeks . Neutron brachytherapy with a one - balloon applicator (figure 1) was used in conjunction with the cf lzh-1000 remote after - loading system (linden science and technology co., shenzhen, china). The physical characteristics of the cf neutron, the characteristics of the applicator, and the process of nbt were described in detail by liu [12, 13]. The nbt dose was prescribed to the reference point, which was located at 10 mm from the center point of the source capsule in the transverse direction . Figure 1 is an x - ray image taken while the applicator and the simulator source were both inserted into the esophagus of a patient . The source applicator is a custom - made catheter, which not only allows the source wire to travel inside, but also includes a water balloon surrounding the source . The water balloon is 12 cm long, and its diameter can vary depending on the amount of water injected into it . That are eccentric with respect to the axis of the esophagus, the water balloon can be inflated accordingly to keep the source close to the tumor but away from the adjacent normal epithelium . In figure 1, the water balloon can clearly be seen as it is filled with an x - ray contrast agent . The total nbt dose (to the reference point) given to each patient varied between 8 and 25 gy - eq in two to five fractions, with 4 - 5 gy - eq per fraction per week . Images (a - d) showing a 75 years male patient, middle site esophageal squamous cell cancer . The tumor regression conditions before each of the four neutron brachytherapy treatments under an x - ray treatmentplanning simulator from a - d the patients were examined weekly during the ebrt . Weekly blood tests were obtained, and any admission for treatment - related complications was recorded . All adverse events were graded according to the national cancer institute s common terminology criteria for adverse events, version 3.0 . The patients usually underwent follow - up examinations every 3 - 4 months after the completion of treatment . Tumor response and nodal disease were evaluated with repeated computed tomography (ct) scans, barium swallow studies, and endoscopy . The objectives of the study were to evaluate overall acute toxicity and local - regional control rates . Survival was calculated from the date of consultation until death or last follow - up evaluation . The pattern of failure (local and/or regional vs. distant) was defined as the first site of failure . The time to first failure, time to any local failure, and time to any distant metastases were calculated from the date of consultation . Local and regional recurrence included the primary tumor and regional lymph nodes . Overall survival and local - regional control were estimated using the kaplan - meier method . Pearson s test was used to assess measures of association in the frequency data . From january 2001 until november 2012, a total of 191 consecutive patients older than 69 years with localized, advanced escc were referred to our department at the changzhi cancer hospital for radiotherapy and cf nbt . The reasons were as follows: 30 patients were medically inoperable (6 patients were diabetic, 11 had chronic obstructive pulmonary disease, and 13 patients had a prior or concurrent malignancy); 34 patients rejected surgery; 76 patients were too old (75 years or older, 33 of 85 had t4 lesion); and 85 patients had unresectable lesions . Of these, 191 patients were treated with ebrt combined with brachytherapy . Patients with good performance status (at least able to care for himself or herself) and adequate hepatic, renal, and hematologic functions were selected for curative treatment . The patients clinical stage was diagnosed by barium examination, endoscopy, endoscopic ultrasonography, or tumor histology . Megavoltage radiation therapy units were used with a minimum source - to - axis distance of 100 cm . The radiation field extended at least 3 cm superior and inferior to the tumor, with a lateral margin of at least 2 cm . The field included the lesser curvature and bottom of stomach if the tumor invades gastroesophageal junction . Multi - field techniques were used to limit the maximum dose to the spinal cord to 45 gy . The radiation treatments were delivered 5 days / week at 2 gy / fraction . The initial anterior - posterior parallel - opposed fields received 30 gy, and the off - cord fields received 20 - 30 gy, for a total dose of 40 - 54 gy in 20 - 27 fractions in 4 - 5.5 weeks . Neutron brachytherapy with a one - balloon applicator (figure 1) was used in conjunction with the cf lzh-1000 remote after - loading system (linden science and technology co., shenzhen, china). The physical characteristics of the cf neutron, the characteristics of the applicator, and the process of nbt were described in detail by liu [12, 13]. The nbt dose was prescribed to the reference point, which was located at 10 mm from the center point of the source capsule in the transverse direction . Figure 1 is an x - ray image taken while the applicator and the simulator source were both inserted into the esophagus of a patient . The source applicator is a custom - made catheter, which not only allows the source wire to travel inside, but also includes a water balloon surrounding the source . The water balloon is 12 cm long, and its diameter can vary depending on the amount of water injected into it . The water balloon is an essential part of the applicator . For tumors that are eccentric with respect to the axis of the esophagus, the water balloon can be inflated accordingly to keep the source close to the tumor but away from the adjacent normal epithelium . In figure 1, the water balloon can clearly be seen as it is filled with an x - ray contrast agent . The total nbt dose (to the reference point) given to each patient varied between 8 and 25 gy - eq in two to five fractions, with 4 - 5 gy - eq per fraction per week . Images (a - d) showing a 75 years male patient, middle site esophageal squamous cell cancer . The tumor regression conditions before each of the four neutron brachytherapy treatments under an x - ray treatmentplanning simulator from a - d weekly blood tests were obtained, and any admission for treatment - related complications was recorded . All adverse events were graded according to the national cancer institute s common terminology criteria for adverse events, version 3.0 . The patients usually underwent follow - up examinations every 3 - 4 months after the completion of treatment . Tumor response and nodal disease were evaluated with repeated computed tomography (ct) scans, barium swallow studies, and endoscopy . The objectives of the study were to evaluate overall acute toxicity and local - regional control rates . Survival was calculated from the date of consultation until death or last follow - up evaluation . The pattern of failure (local and/or regional vs. distant) was defined as the first site of failure . The time to first failure, time to any local failure, and time to any distant metastases were calculated from the date of consultation . Overall survival and local - regional control were estimated using the kaplan - meier method . Pearson s test was used to assess measures of association in the frequency data . Age of the escc patients who were treated with radiation therapy (nbt and ebrt) ranged from 70 to 84 years (median: 75 years). There were 115 patients aged 70 - 74, and 76 patients aged> 74 years . The cancer stages were categorized according to the 6 edition of the ajcc cancer staging manual, with 72 patients categorized as stage iia, 10 patients categorized as stage iib, and 109 patients were categorized as stage iii . The detailed patient data and log - rank test are provided in table 1 . Patient and tumor characteristics rt radiotherapy alone, os overall survival rate, lcr local control rate the duration of follow - up ranged from 6 to 106 months (median: 30.4 months). The median survival time for the 191 patients was 23.6 months, and the 1-, 2-, 3-, and 5-year rates for overall survival (os) were 68.5%, 48.2%, 40.3%, and 28.7%, respectively . The 1-, 2-, 3-, and 5-year rates for local - regional control (lrc) were 82.2%, 67.0%, 61.8%, and 54.2%, respectively . We used the following nine factors for the univariate analysis of survival rates and local control rate: sex, age, karnofsky score (kps), tumor location, tumor length, tumor t stage, nodal stage, clinical stage, and radiation dose . Among them, three (age, tumor length, and clinical n stage) were found to have relevance to os (p = 0.010, p = 0.016, and p = 0.009, respectively). Age, clinical n stage, and radiation dose were factors that were significantly related to lrc (p = 0.038, p = 0.014, p = 0.014, respectively). In the univariate analysis, the 5-year os (lrc) was 37.3% (58.6%) for patients aged 70 - 74 years, and 14.5% (47.9%) for patients aged> 74 years (p = 0.010 and p = 0.038, respectively, figure 2a and b). In multivariate analysis, age and clinical n stage were associated with os and lrc (p = 0.011 [0.041] and p = 0.005 [0.005]) (table 2). Results of multivariate cox regression analysis of overall survival and local - regional control ci confidence interval, hr hazard ratio, c clinical comparison of the overall survival rate (a), and local control rate (b) between the two treatment groups at the time of the analysis, 80 patients were alive and free of disease, and 5 patients were alive with disease evolution . The main sites of distant metastases were the lung (n = 9), liver (n = 5), brain (n = 2), and bones (n = 8). In 14 patients, metastases developed in more than one organ . Additionally, 15 patients died of mixed causes, including pneumonia, cerebral hemorrhage, and heart infarction . Local - regional recurrence occurred in 59 (59/191, 30.9%) patients, with 9/59 (15.3%) occurrences outside the radiation fields and 50/59 (84.7%) occurrences inside the radiation fields . None of those patients underwent salvage surgery . In terms of acute toxicity, no perforations dysphagia was relieved after the second or third nbt treatment in 87% of the patients, and a temporary feeding tube was not required in most of the patients . Grade 2 esophagitis, expressed by clinical odynophagia, was observed in 64 cases (33.5%), and it was managed with the early introduction of h2 blockers and surface anesthesia at the initiation of the nbt . In total, eight (4.2%) patients had grade 2 irradiation dermatitis . From the time of treatment completion to the development of local - regional recurrence or death, 5 (2.6%) and 15 (7.9%) patients experienced fistula and massive bleeding, respectively . The median time of incidence was 7.0 (3.7 - 55.7) months for fistula and 9.5 (3.2 - 90.9) months for bleeding . As shown in table 3, the incidence of severe, late complications was related to older age (p = 0.027), higher nbt dose / fraction (20 - 25 gy/5f), and higher total dose (> 66 gy). In total, 68.5% of the patients resumed normal swallowing, while 4.2% had some residual dysphagia (non - malignant) requiring intermittent dilatation . Age of the escc patients who were treated with radiation therapy (nbt and ebrt) ranged from 70 to 84 years (median: 75 years). There were 115 patients aged 70 - 74, and 76 patients aged> 74 years . The cancer stages were categorized according to the 6 edition of the ajcc cancer staging manual, with 72 patients categorized as stage iia, 10 patients categorized as stage iib, and 109 patients were categorized as stage iii . The detailed patient data and log - rank test are provided in table 1 . Patient and tumor characteristics rt radiotherapy alone, os overall survival rate, lcr local control rate the duration of follow - up ranged from 6 to 106 months (median: 30.4 months). The median survival time for the 191 patients was 23.6 months, and the 1-, 2-, 3-, and 5-year rates for overall survival (os) were 68.5%, 48.2%, 40.3%, and 28.7%, respectively . The 1-, 2-, 3-, and 5-year rates for local - regional control (lrc) were 82.2%, 67.0%, 61.8%, and 54.2%, respectively . We used the following nine factors for the univariate analysis of survival rates and local control rate: sex, age, karnofsky score (kps), tumor location, tumor length, tumor t stage, nodal stage, clinical stage, and radiation dose . Among them, three (age, tumor length, and clinical n stage) were found to have relevance to os (p = 0.010, p = 0.016, and p = 0.009, respectively). Age, clinical n stage, and radiation dose were factors that were significantly related to lrc (p = 0.038, p = 0.014, p = 0.014, respectively). In the univariate analysis, the 5-year os (lrc) was 37.3% (58.6%) for patients aged 70 - 74 years, and 14.5% (47.9%) for patients aged> 74 years (p = 0.010 and p = 0.038, respectively, figure 2a and b). In multivariate analysis, age and clinical n stage were associated with os and lrc (p = 0.011 [0.041] and p = 0.005 [0.005]) (table 2). Results of multivariate cox regression analysis of overall survival and local - regional control ci confidence interval, hr hazard ratio, c clinical comparison of the overall survival rate (a), and local control rate (b) between the two treatment groups at the time of the analysis, 80 patients were alive and free of disease, and 5 patients were alive with disease evolution . The main sites of distant metastases were the lung (n = 9), liver (n = 5), brain (n = 2), and bones (n = 8). In 14 patients, metastases developed in more than one organ . Additionally, 15 patients died of mixed causes, including pneumonia, cerebral hemorrhage, and heart infarction . Local - regional recurrence occurred in 59 (59/191, 30.9%) patients, with 9/59 (15.3%) occurrences outside the radiation fields and 50/59 (84.7%) occurrences inside the radiation fields . In terms of acute toxicity, no perforations were observed during this treatment period . In total, 88 (46.1%) dysphagia was relieved after the second or third nbt treatment in 87% of the patients, and a temporary feeding tube was not required in most of the patients . Grade 2 esophagitis, expressed by clinical odynophagia, was observed in 64 cases (33.5%), and it was managed with the early introduction of h2 blockers and surface anesthesia at the initiation of the nbt . In total, eight (4.2%) patients had grade 2 irradiation dermatitis . From the time of treatment completion to the development of local - regional recurrence or death, 5 (2.6%) and 15 (7.9%) patients experienced fistula and massive bleeding, respectively . The median time of incidence was 7.0 (3.7 - 55.7) months for fistula and 9.5 (3.2 - 90.9) months for bleeding . As shown in table 3, the incidence of severe, late complications was related to older age (p = 0.027), higher nbt dose / fraction (20 - 25 gy/5f), and higher total dose (> 66 gy). In total, 68.5% of the patients resumed normal swallowing, while 4.2% had some residual dysphagia (non - malignant) requiring intermittent dilatation . To our knowledge, this is the first reported clinical experience of the treatment using nbt and ebrt for elderly patients with escc . We also found that, firstly, nbt + ebrt is safe and beneficial in terms of local control in the radical treatment of elderly patients with escc, and secondly, the os rate was significantly increased, and the late complication rate was significantly decreased in patients aged 70 - 74 years compared to that of patients aged> 74 years . During the treatment period, no severe treatment related complication occurred . Definitive conformal radiotherapy (crt) is considered a feasible nonsurgical treatment in patients with a locally advanced ec, and approximately a 50 - 65% clinical complete response rate, 17 - 26 months of median overall survival, and 30 - 40% 2-year survival rate [15, 16, 17]. In the current study, the os was similar to the results of prior studies, though without chemotherapy [15, 16, 17]. We believe that there are at least two factors that made the cf - based nbt more effective for local tumor than chemotherapy regimens, particularly in the treatment of locally advanced escc . The first factor is related to the high - let nature of fission neutrons, which made them much more effective (compared to the low - let x - ray) in killing the hypoxic tumor cells in the locally advanced cancers . The second factor is related to the fact that water is an effective neutron attenuator that can be conveniently injected into the source applicator during treatment to reduce the neutron dose to the nearby normal tissue . Because there is a significant difference in the elasticities of normal tissue and tumor tissue, the proper injection of water into the source applicator can effectively push away the nearby normal tissue while still keeping the tumor tissue close to the source . Tougeron reported that age> 74 years was associated with worse creatinine clearance (p <0.01) and greater chemotherapy dose reduction at treatment onset due to age (p <0.01), but this had no influence on total crt dose, or os . In the current study, the incidence of late severe complications was significantly related to the factors of higher total dose and nbt dose . In addition to the dose factors, the patients age also significantly increased the incidence of relevant, late complications . While the normally expected side effects (shown in table 4) seem to be quite acceptable, the number of deaths (n = 20 or 10.5%) resulting from fistula, hematemesis, and hemoptysis is high . This may be linked to the late effect of radiation damage, as fatal esophagitis of fistula cases were also observed in the rtog 92 - 07 trial where the ir - based high - dose - rate boost dose of 15 gy in 3 weekly fractions was deemed to be too high . Further ct review is needed to compare the pretreatment tumor length, esophageal tumor wall thickness, and association of tumor with surrounding normal structures with subsequent fistula formation . Comparative toxicity rates, overall survival and local control of selected series ct chemotherapy, bt brachytherapy, erbt external beam radiotherapy, pts patients, y years, ns not stated, os overall survival ld limited disease, lc local control brachytherapy applied between ebrt the major limitation of our study was that the retrospective analysis might have been based on incomplete medical records . Others restrictions were that the study was conducted in a single institution, small sample size, and the lack of predefined factors determining treatment decisions, which were based only on evaluations by the referral doctor and members of a multidisciplinary team . It should nonetheless be recalled that the aim of the study was to retrospectively identify the parameters to be associated with the key therapeutic decision . Our results suggest that elderly patients with escc could benefit from nbt + ebrt without major toxicities . Patient s age, clinical stage n status, and radiation dose could be used to select the appropriate treatment in an elderly patient.
Aggregatibacter actinomycetemcomitans is an inhabitant of the oral cavity and periodontal pathogen . In periodontal disease, the bacterium infects and proliferates within the periodontal pocket, between the gingival tissue and the tooth . The presence of bacteria and their products such as secreted proteins and lps induce an inflammatory response by the host . Inflammation leads to tissue damage and alveolar bone loss that is characteristic of periodontal diseases . A. actinomycetemcomitans has been highly associated with a rapidly progressing form of periodontal disease known as localized aggressive periodontitis (lap) that occurs in adolescents . This bacterium has also been reported to cause non - oral infections such as pneumonia, endocarditis, pericarditis, bacteremia, septicemia, osteomyelitis, synovitis, infectious arthritis, skin infections, urinary tract infections and brain abscesses [46]. A major virulence factor of a. actinomycetemcomitans is the secretion of leukotoxin (ltxa), which induces apoptosis in white blood cells (wbc) from humans and old world primates [710]. Apoptosis induction by ltxa occurs via different pathways such as a mitochondrial signaling pathway that results in collapse of the mitochondrial membrane potential and arrest of oxidative phosphorylation [1113] or by activation of caspase 1 . Furthermore, ltxa has been shown to induce g2/m cell cycle arrest and apoptosis in mouse b - cell hybridoma hs-72 cells . However, the molecular pathway that leads to ltxa induced cellular apoptosis and cell cycle arrest is not well understood . Ltxa is believed to play a crucial role in evasion of the host immune response by the bacterium . Ltxa likely exerts its effects within the periodontal pocket where polymorphonuclear leukocytes and other immune cells infiltrate to control the infection . The receptor for ltxa on wbcs is leukocyte function antigen-1 (lfa-1; cd11a / cd18) [1618]. Lfa-1 is expressed only on wbcs and is normally involved in migration of wbcs to infected and injured tissues . When presented in its activated or exposed state, lfa-1 binds intercellular adhesion molecule-1 (icam-1) on the surface of vascular endothelial cells resulting in adhesion of wbcs to the endothelial lining and subsequent extravasation . Recently, we reported that ltxa preferentially targets immune cells expressing the activated form of lfa-1, resulting in selective depletion of host cells . While studying the interaction between wbcs and vascular endothelial cells, we found that relatively high doses of ltxa irreversibly damaged endothelial cells and caused changes in expression levels of endothelial adhesion molecules . This work provides a novel mechanism for a. actinomycetemcomitans - induced tissue damage during infection . Leukotoxin (ltxa) was purified from culture supernatants of a. actinomycetemcomitans strain nj4500 as previously described . The storage buffer for the purified toxin was 20 mm tris hcl, ph 6.8, 250 mm nacl, and 0.2 mm cacl2 . The typical yield was 0.5 mg/100 ml starting culture . For long - term storage (greater than one month), protein was lyophilized in sterile glass vials and stored at 80 c . Samples were reconstituted in sterile distilled water prior to use and we found that when stored in this manner, ltxa was stable for at least 6 months . All toxin preparations were filtered through a 0.22 m filter prior to use . For experimental setup heat inactivation (65 c for 20 min) has been shown effectively abolishing all toxic effects of ltxa . Human microvascular endothelial cells (immortalized cell line hcmec / d3 were used at a passage number 2832 . Hcmec / d3 were grown in ebm-2 medium (lonza cc-3156), supplemented with 5% fetal bovine serum, 1.4 m hydrocortisone, 5 g / ml ascorbic acid, 1% chemically defined lipid concentrate, 10 mm hepes, and 1 ng / ml human basic fibroblast growth factor . After trypsinization, cells were seeded in 96-well plates pre - coated with 0.3% collagen (5000 cells / well). Medium was supplemented with ltxa at concentrations of 5 g / ml, 500 ng / ml or 50 ng / ml or corresponding to the highest dosage ltxa - buffer alone was added . After 24, 48, 72, 96 and 144 h, proliferation was quantitated using the cck-8 assay, based on the mitochondrial reduction of tetrazolium salt (fluka 96992). Briefly, medium was removed and 100 l of fresh medium and 10 l of cck-8 solution was added . Absorbance was measured after 4 h of incubation with a bmg fluostar optima spectrofluorophotometer ., between 50,000 and 60,000 cells were seeded per well in a collagen coated 12-well plate without treatment or with immediate addition of ltxa - buffer, or ltxa at 5 g / ml, 500 ng / ml, 50 ng / ml or 5 ng / ml . Seventy - two or 96 h after seeding with or without treatment, cells were trypsinized, washed and counted in a malassez haematocytometer in triplicates and results were expressed as cells / cm . Hcmec / d3 cells were grown in pre - coated 12-well plates without treatment, with ltxa - buffer or with ltxa at doses ranging from 5 g / ml to 5 ng / ml for 24, 72 or 96 h. cells were harvested by trypsinization, washed in rpmi containing 10% fcs, then washed in ice - cold pbs and resuspended in 1 ml 80% ethanol . Ethanol was removed after centrifugation and cells were stained in pbs, containing 0.05% triton - x, 0.1 mg / ml rnase a and 15 l propidium iodide for 1 h on ice . After this, cells were resuspended in 3 ml pbs, pelleted by centrifugation and resuspended in 500 l pbs for flow cytometric analysis, performed in duplicates (except 24 h experiment), repeated 34 times . For analysis of apoptosis, cells were grown in 6-well plates without changing of medium for 48 or 72 h without treatment, with ltxa - buffer or with ltxa at doses ranging from 5 g / ml to 5 ng / ml . The pan - caspase inhibitor, z - vad - fmk, at a final concentration of 25 m was added to high concentrations of ltxa (5 g / ml and 500 g / ml) to evaluate apoptosis via caspase activation . Cells were then stained in binding buffer for annexin v - fitc (beckman coulter aposcreen) and 7-aad (bd) for 15 min at room temperature . Another 100 l of binding buffer was added and cells were directly analyzed by flow cytometry (beckman coulter fc-500)., cells were grown on tissue culture dishes with cover glass bottom (fluorodish fd 35 - 100). Cells were either untreated or treated with 5 g / ml ltxa . After 72 h cells were washed with pbs and stained with hoechst 33258 (0.01 mg / ml) for 20 min . Cells were washed again and images were taken directly afterwards at 40 magnification (olympus ix-71). Hcmec / d3 cells were grown in 6-well plates for 48 h without changing of medium and in the presence or not of ltxa 5 ug / ml to 5 ng / ml or ltxa - buffer . After 24 h and 48 h 90 l of supernatant was removed and stained for annexin v (aposcreen beckman coulter) according to combes et al . . Analysis of annexin v positive mp was performed in triplicates, repeated 3 times . For analysis of long - term effects of ltxa, hcmec / d3 cells were seeded in collagen - coated 24-well or 12-well plates with or without ltxa at 5 g / ml, 500 ng / ml, 50 ng / ml, 5 ng / ml or ltxa - buffer . Medium was changed every other day and cells were grown until untreated wells were confluent (three to four days). For short - term ltxa effect evaluation, hcmec / d3 were grown in collagen - coated 24-well or 12-well plates until confluence and then treated for 16 h with ltxa at 5 g / ml, 500 ng / ml, 50 ng / ml, 5 ng / ml, ltxa - buffer or medium alone . Cells were stained for cd54 (mab from beckman coulter im1239u) and cd106 (mab from ebioscience 12 - 1069 - 73) and analyzed by flow cytometry in duplicates, repeated 34 times . Comparison between treatment groups at different time points were performed by two - way anova and bonferroni post test between groups . 1a). To confirm that our preparation contained only ltxa and not other products that could potentially affect cells (eg . Lps, cytolethal distending toxin, endotoxin), ltxa (5 g / ml) was incubated with hl-60 cells and k562 cells . K562 cells are a white blood cell line that does not express lfa-1 and are therefore resistant to ltxa - mediated cytotoxicity . Nearly all the hl-60 cells were annexin v positive, indicating they were undergoing apoptosis (fig . In contrast, k562 cells did not stain with annexin v after ltxa treatment and the buffer- and ltxa - treated curves were superimposable . Thus, cytotoxicity was due to ltxa in our purified preparation . To assess the effect exerted by purified ltxa (fig . 1) on human brain endothelial cells (hcmec / d3) proliferation, cells were treated once with increasing concentrations of ltxa (5 ng / ml5 g / ml) and grown for up to six days . Every day a tetrazolium - salt - based assay (cck-8) was performed as well as cell counts . Proliferation was irreversibly abrogated by a single treatment of high dose ltxa (5 g / ml). At 500 ng / ml a significant decrease in proliferation was observed whereas lower ltxa concentrations or ltxa - buffer had no effect (fig . 2). Whereas untreated cells as well as ltxa - buffer and low dosage ltxa treated cells quintupled after 96 h, 5 g / ml ltxa reduced cell numbers by half, and 500 ng / ml ltxa resulted only in a duplication of cell numbers at 96 h. as shown in fig . 6, hcmec / d3 cells presented with dramatic morphological changes when treated with a single dose of 5 g / ml ltxa . Monolayer formation and even generation of cell cell contacts seemed to be inhibited by ltxa treatment and could not be observed . Hcmec / d3 cells were treated once with increasing concentrations of ltxa (5 ng / ml5 g / ml) and cell cycle analysis was performed at different time points (24, 72, and 96 h). Treatment with ltxa dose - dependently increased the proportion of cells in the g2/m phase but decreased the proportion in the g1 phase (fig . 54% of untreated or ltxa - buffer treated cells were in the g1 phase and 26% in the g2/m phase, compared to 21% of cells in the g1 phase and 70% in the g2/m phase when treated with 5 g / ml ltxa (p <.001, p <.001, respectively). The s phase was reduced from 18% in untreated or ltxa - buffer treated cells to 3% in ltxa treated cells (p <.05). Single dose ltxa treatment (5 g / ml) significantly increased numbers of apoptotic cells (annexin v positive, 7-aad negative) after 48 h and 72 h (fig . 4). After 72 h a mean of 1.9% of hcmec / d3 stained positively for annexin v and were therefore considered apoptotic, compared to a mean of 18% in ltxa 5 g / ml treated cells (p <.001) and 5.9% in ltxa 500 ng / ml treated cells (ns). Addition of the pan - caspase inhibitor z - vad - fmk (25 m) to the 5 g / ml ltxa treatment for 72 h reduced apoptotic cell proportions to 9.8% (p <experiments with z - vad - fmk were only performed for 5 g / ml and 500 ng / ml ltxa treatment or untreated cells for 72 h. after 48 h and 72 h, ltxa treatment (500 ng / ml) significantly increased numbers of annexin v and 7-aad double positive cells (after 72 h 13.7% in 5 g / ml [p <.001] and 5.2% [ns] in 500 ng / ml) were found, compared to untreated (3.2%), ltxa - buffer treated (1.9%) or ltxa in lower concentrations (2.2% in 50 ng / ml, and 2% in 5 ng / ml, respectively). Treatment with z - vad - fmk concomitantly with to ltxa 5 g / ml or 500 ng / ml did not reduce numbers of necrotic cells (16.4% and 5.2%, respectively, untreated 3.2%). Since cellular microparticles positively staining for annexin v are considered markers of early apoptosis we evaluated numbers of endothelial microparticles after 24 h and 48 h incubation with ltxa at increasing concentrations and ltxa - buffer . After 24 h and 48 h increased numbers of annexin v positive microparticles at 5 g / ml ltxa treatment (both p <.001) were found, indicating early signs of apoptosis and/or cellular activation already after 24 h (fig . 5). After 72 h apoptotic endothelial cells, exhibiting the characteristic chromatin condensation were observed by hoechst staining and fluorescence microscopy (fig . 7, hcmec / d3 cells either seeded with or without a single dose of ltxa (long - term) in increasing concentrations (5 ng / ml to 5 g / ml) or treated with ltxa for 16 h after forming a confluent monolayer (short - term), expressed increased levels of both icam-1 (cd54) and vcam-1 (cd106). Percentages of icam-1 positive cells as well as mean fluorescence intensity (mfi) were comparably and dose - dependently upregulated in both long- and short - term treatments . After short - term treatment of a confluent monolayer 87.2% of untreated cells compared to 95.3% of ltxa 500 ng / ml and 99% of ltxa 5 g / ml treated cells expressed icam-1 (long - term icam-1 positive cells untreated 87.2%, ltxa 500 ng / ml 94.2% and ltxa 5 g / ml 96.9%). Mean icam-1 mfi of untreated cells was 28.9 and 119.9 (long - term mfi untreated 30.7 vs. ltxa 5 g / ml 126) after ltxa 5 g / ml treatment . Vcam-1 expression was dose - dependently higher when hcmec / d3 confluent monolayers were treated for a short - term period (16 h). After a 16 h ltxa treatment 6.9% (mfi 9.2) of untreated cells expressed cd106 compared to 26.9% (mfi 11.4) of ltxa 500 ng / ml and 43.7% (mfi 26.9) of ltxa 5 g / ml treated cells . When cells were grown in the presence of ltxa 5.7% (mfi 8.2) of untreated cells and 8.6% (mfi 10.3) of ltxa 500 ng / ml or 18.8% (mfi 9.7) of ltxa 5 g / ml treated cells expressed cd106 (fig . Many pathogenic bacteria produce toxins that amplify or suppress the host immune response by altering cell signaling or transcriptional responses . Evidence to date suggests that a. actinomycetemcomitans ltxa disrupts the host immune response mainly by killing of host immune cells . Our present findings of ltxa - induced apoptosis and activation of microvascular endothelial cells, never previously reported, amplify the spectrum of pathogenetic mechanisms of a. actinomycetemcomitans and provide further explanation for tissue destruction in localized aggressive periodontitis and other diseases associated with this bacterium . As the interface between circulation and site of infection, the vascular endothelium plays a pivotal role in recruitment of leucocytes and launch of immune responses, as well as in the basic function of blood circulation and tissue maintenance . We found that a single dose of purified ltxa administered to human microvascular endothelial cells (hcmec / d3) importantly and irreversibly inhibits cell proliferation by g2/m cell cycle arrest . In addition ltxa induces apoptosis, which is partially caspase - dependent . Both decreased cell viability and apoptotic cell death of endothelial cells due to ltxa impairment of the endothelial barrier function would facilitate tissue invasion and distribution of a. actinomycetemcomitans and ltxa . Indeed, loesche postulated that ltxa is the causative agent of local tissue destruction during lap associated with a. actinomycetemcomitans . In addition, intact blood perfusion of tissue surrounding the sites of inflammation is crucial for an effective host immune response . Damage to the endothelial lining of microvessels by ltxa could severely compromise circulation and impair host defense to infection, further contributing to the pathogenic nature of the bacterium . However, in our study endothelial cells were only affected by ltxa in a relatively high dose rage from 500 ng / ml to 5 g / ml, whereas lfa-1 bearing cells such as macrophages or human pbmcs were already affected at 110 ng / ml and minimal amounts of ltxa have been shown to induce a rapid proinflammatory reaction in human macrophages, already at a ratio of 1 bacterium / macrophage . To our knowledge concentrations of ltxa locally present in patients with lap is not known, however, currently it is not evaluable if higher dosage as used in our experiment do have important clinical relevance . Already after 24 h of ltxa treatment in the highest concentration, hcmec / d3 cells shed significantly increased amounts of annexin v positive microparticles into the cell culture supernatant . Therefore, our finding of increased endothelial microparticle numbers is a clear sign of cell activation and/or apoptosis as an immediate reaction to ltxa treatment . It has been reported that apoptosis of endothelial cells in vitro is associated with the establishment of a pro - inflammatory milieu leading to paracrine induction of icam-1 and vcam-1 and, in turn, increased adhesiveness resulting in adhesion and transmigration of monocytic cells into the vessel wall . In our study, we found that levels of icam-1 and vcam-1 increased on endothelial cells upon treatment with ltxa . Leukocyte recruitment form the circulation to sites of inflammation and infection involves a multistep cascade consisting of leukocyte rolling, firm adhesion, and, ultimately, transmigration . A key step in this process is the interaction of icam-1 with its leukocyte counter receptor, lymphocyte function - associated antigen-1 (lfa-1) [3234]. Whereas under physiological conditions vascular endothelium expresses low levels of icam-1, inflammatory stimuli can significantly increase icam-1 surface expression . In acute and chronic inflammatory diseases, endothelial cells become activated and express increased levels of icam-1, in addition to vcam-1 and e - selectin [3537]. Vcam-1 is implicated in the control of leukocyte rolling in the beginning of leukocyte recruitment process whereas icam-1 accounts for firm arrest . This might be reflected by our results of vcam-1 up - regulation in short - term stimulation whereas icam-1 was upregulated in short- and long - term stimulation . Whether the upregulation of icam-1 and vcam-1 upon ltxa treatment is directly induced by ltxa or caused by paracrine induction due to a pro - apoptotic milieu caused by the toxin however, other pro - inflammatory effects of ltxa such as the activation and secretion of interleukin-1 beta have already been described . It is therefore tempting to speculate that an autocrine stimulation of endothelial cells by their own il-1 could play a role in the observed cam upregulation . Epidemiologic and clinical studies suggest a connection between poor oral health and increased risk of cardiovascular disease (cvd). Perodontopathogens have been found in atherosclerotic plaques and a. actinomycetemcomitans periodontitis has been linked to a higher risk of cardiovascular diseases and atherosclerosis . Recently described elevated icam-1 expression in the aorta of mice which have been systemically challenged with a. actinomycetemcomitans . Endothelial icam-1 upregulation is of special interest since it is one of the basal mechanisms associated with atherosclerotic plaque formation and subsequentially development of cvd . Since endothelial cells do not express lfa-1, a 2 integrin receptor and the natural receptor for ltxa, it is not clear how ltxa may interact with this cell type . However endothelial cells do express 1 and 3 heterodimers on their cell surfaces . While no significant amino acid homology between 1/3 and 2 integrins is known, a certain amount of structural homology does exist . Therefore, even though the toxin does not bind with a high affinity to 1 or 3 as it does to 2 high ltxa dosage might be enough binding stimulus to trigger cell activation and/or cell death . Interestingly, a. actinomycetemcomitans has the ability to invade vascular endothelial cells using platelet - activating factor as its receptor although the bacterial adhesin mediating this interaction is not known . Gangliosides are glycoshingolipids on the surfaces of many cell types and are involved in signaling and membrane protein regulation . We proposed that ltxa may have gained the ability to bind to gangliosides on red blood cells (rbc) because of the similarity of their sugar moieties and structure to the carbohydrate modification of lfa-1 . Similar to our observations on endothelial cells, rbc lysis requires significantly higher doses of ltxa than needed for wbc killing . Hence, it is possible that ltxa interacts with gangliosides on the surface of endothelial cells in a manner similar to that seen in rbcs . In conclusion, we demonstrate that ltxa significantly increased expression levels of icam-1 and vcam-1 in endothelial cells further corroborating pro - inflammatory effects of ltxa . Additionally, ltxa has important anti - proliferative as well as pro - apoptotic effects on microvascular endothelial cells and induces a g2/m phase cell cycle arrest . The presented work underlines not only the important function of ltxa in tissue destruction during a. actinomycetemcomitans infection but also its relevance in cvd.
Osteoporosis is one of the most serious public health problems for elderly people and also a major cause of the bedridden state . This condition / disease has threatened the quality of life in old age and increased medical costs . In particular, the potential for developing osteoporosis increases dramatically after menopause in females, and estrogen deficiency causes rapid bone loss of trabecular regions in hip or lumbar spine . Estrogen deficiency is also associated with decrease in intestinal ca absorption which results in further acceleration of bone loss . Although it is well known that decrease in intestinal ca absorption is attributable to estrogen deficiency, it remains unclear what actions other gonadal hormones have for the association between bone mass and intestinal ca absorption in the estrogen deficiency state . Dehydroepiandrosterone (dhea), secreted mainly from the adrenal gland and ovary, plays a critical physiological role for maintaining steroidogenesis by being used as the available precursor converted to testosterone and estrogens in various peripheral tissues such as bones, liver, brain, and skeletal muscles . Dhea concentration in the blood decreases the following ovariectomy in animals . On the other hand, dhea replacement improves bone mineral density (bmd), especially a trabecular site, in the ovariectomized (ovx) animals [6 - 8]. We previously observed that dhea replacement increased e2 (estradiol) centration in the blood . The presence of estrogen receptors in the intestinal mucosa and estrogen stimulates intestinal calcium absorption via an estrogen receptor . However, to our knowledge, the effect of dhea administration on intestinal ca absorption in estrogen deficiency state has not been studied yet . Accordingly, we hypothesized that dhea administration would increase intestinal ca absorption via increasing e2 concentration in the blood and prevent trabecular bone loss caused by estrogen deficiency . In the present study, we examined bone mass in a trabecular abundant site of lumbar spine and ca balance such as intestinal ca absorption and ca accumulation in ovx rats after 8 weeks of dhea administration . Seventeen female sprague - dawley rats, 6 weeks old, were surgically ovariectomized and randomized into two groups: ovx control rats (oc, n = 8) and ovx rats with dhea treatment (od, n = 9). Briefly, all rats were fed a diet with 1.05% calcium and 1.01% phosphate purchased from clea japan (ce-2, clea japan, inc ., dhea dissolved in sesame oil was administered to the od group intraperitoneally at 20 mg dhea / kg body weight for 8 weeks beginning one week after ovariectomy while the oc group was treated with vehicle only (sesame oil, 0.5 ml). Rats were not treated with dhea or vehicle every fourth day (i.e., they were treated for 3 consecutive days). The rats were kept in individual cages (15 25 19.5 cm) and allowed access to food and distilled water ad libitum . Food consumption and body weight gain the room temperature was maintained at 24 1c, and the humidity at 50 5% . Fluorescent lights were turned on from 8:00 a.m. to 8:00 p.m. animal care and experimental procedures were approved by the animal experimental committee of the university of tsukuba . The lumbar spine, left, and right tibiae of each rat were isolated by dissection, and all the muscle and connective tissue was carefully removed . Thereafter, bmc and bmd value for the l3-l6 lumbar spine were measured by dual - energy x - ray absorptiometry (dxa; aloka dcs-600r instrument). In the present study, we used young ovx rats whose growth of bone is considerably influenced by body mass . Therefore, we normalized body weight for bmc to evaluate the effect of dhea on bone mass . At the each dissection after the adhering connective tissues had been trimmed off, the femoral wet weight was measured . Thereafter, the femoral length, long width, and short width were measured with a caliper . Length was measured from the proximal tip of the femur head to the distal tip of the medial condyle . The mediolateral (long width) and anteroposterior (short width) dimensions were measured at the midpoint of the femur diaphysis . The bone strength of the middle diaphysis of the femur was then tested by measuring the mechanical strength, breaking force with an iio dyn-1255 instruments . The force necessary to produce a break at the center of the femur was measured under the following conditions; the sample space was 1.0 cm, the plunger speed was 100.0 mm / min, the load range was 50.0 kg, and the chart speed was 120.0 cm / min . In this study, two balance studies were carried out to determine the rate of intestinal ca absorption and ca accumulation . Animals were placed in individual metabolic cages (24 20 18 cm). The first phase was carried out on the 4th and 5th day after starting the experimental diets period (metabolic cage phase 1: mc 1). The next phase (mc 2) was carried out at the end of the experimental period . At each phase, urine was collected under acidic conditions by using 2ml 2n hydrochloric acid, thus preventing ca precipitate and putrefaction . All urine was centrifuged at 2,500 rpm for 15 min to eliminate refuse . In the fecal determination, all daily feces were burnt to ash at 550 - 600 for approximately 18 hours, and the resulting ash was dissolved in 1n nitric acid . The level of serum ca was measured by the inductively coupled plasma atomic emission spectroscopy (icap - aes - 575 v nippon jarrell - ash). Intestinal ca absorption and ca accumulation were calculated using the amount of ca intake, the fecal ca excretion and the urinary ca excretion . Statistical significance (p <0.05) was determined using an unpaired student s t - test between groups . Two - way analysis of variance (anova) was used for determining the effects of time and group on intestinal ca absorption and accumulation . Statistical analysis was carried out by one - way anova followed by fisher`s f - test for multiple comparisons among four points in intestinal ca absorption and accumulation . Statistical analysis was performed using spss for windows (version 20.0 j; spss inc ., seventeen female sprague - dawley rats, 6 weeks old, were surgically ovariectomized and randomized into two groups: ovx control rats (oc, n = 8) and ovx rats with dhea treatment (od, n = 9). Briefly, all rats were fed a diet with 1.05% calcium and 1.01% phosphate purchased from clea japan (ce-2, clea japan, inc ., dhea dissolved in sesame oil was administered to the od group intraperitoneally at 20 mg dhea / kg body weight for 8 weeks beginning one week after ovariectomy while the oc group was treated with vehicle only (sesame oil, 0.5 ml). Rats were not treated with dhea or vehicle every fourth day (i.e., they were treated for 3 consecutive days). The rats were kept in individual cages (15 25 19.5 cm) and allowed access to food and distilled water ad libitum . Food consumption and body weight gain the room temperature was maintained at 24 1c, and the humidity at 50 5% . Fluorescent lights were turned on from 8:00 a.m. to 8:00 p.m. animal care and experimental procedures were approved by the animal experimental committee of the university of tsukuba . The lumbar spine, left, and right tibiae of each rat were isolated by dissection, and all the muscle and connective tissue was carefully removed . Thereafter, bmc and bmd value for the l3-l6 lumbar spine were measured by dual - energy x - ray absorptiometry (dxa; aloka dcs-600r instrument). In the present study, we used young ovx rats whose growth of bone is considerably influenced by body mass . Therefore, we normalized body weight for bmc to evaluate the effect of dhea on bone mass . After the adhering connective tissues had been trimmed off, the femoral wet weight was measured . Thereafter, the femoral length, long width, and short width were measured with a caliper . Length was measured from the proximal tip of the femur head to the distal tip of the medial condyle . The mediolateral (long width) and anteroposterior (short width) dimensions were measured at the midpoint of the femur diaphysis . The bone strength of the middle diaphysis of the femur was then tested by measuring the mechanical strength, breaking force with an iio dyn-1255 instruments . The force necessary to produce a break at the center of the femur was measured under the following conditions; the sample space was 1.0 cm, the plunger speed was 100.0 mm / min, the load range was 50.0 kg, and the chart speed was 120.0 cm / min . In this study, two balance studies were carried out to determine the rate of intestinal ca absorption and ca accumulation . Animals were placed in individual metabolic cages (24 20 18 cm). The first phase was carried out on the 4th and 5th day after starting the experimental diets period (metabolic cage phase 1: mc 1). The next phase (mc 2) was carried out at the end of the experimental period . At each phase, urine was collected under acidic conditions by using 2ml 2n hydrochloric acid, thus preventing ca precipitate and putrefaction . All urine was centrifuged at 2,500 rpm for 15 min to eliminate refuse . In the fecal determination, all daily feces were burnt to ash at 550 - 600 for approximately 18 hours, and the resulting ash was dissolved in 1n nitric acid . The level of serum ca was measured by the inductively coupled plasma atomic emission spectroscopy (icap - aes - 575 v nippon jarrell - ash). Intestinal ca absorption and ca accumulation were calculated using the amount of ca intake, the fecal ca excretion and the urinary ca excretion . All the data are expressed as mean se . Statistical significance (p <0.05) was determined using an unpaired student s t - test between groups . Two - way analysis of variance (anova) was used for determining the effects of time and group on intestinal ca absorption and accumulation . Statistical analysis was carried out by one - way anova followed by fisher`s f - test for multiple comparisons among four points in intestinal ca absorption and accumulation . Statistical analysis was performed using spss for windows (version 20.0 j; spss inc ., chicago, il, usa). The body weight gain, food intake, and food efficiency are presented in table 1 . The initial body weight did not differ between the groups . The final body weight and the body weight gain were significantly lower in the od group than in the oc group . The bmc normalized by body weight of the lumbar spine (trabecular - abundant region) in the od group was found to be significantly higher compared to that in the oc group . To eliminate the effect of body weight on growing bone, we normalized body weight for bmc for evaluating the effect of dhea on bone mass . The bmd of the lumbar spine in the od group tended to be higher compared to that in the oc group, but it did reach statistically significant levels . The femoral wet weight normalized by body weight in the od group was found to be significantly higher compared to that in the oc group, but the long and short width and length of femur did not differ between the groups (table 2). The breaking force at femoral diaphysis site (cortical boneabundant) did not differ between the groups (table 2). The intestinal ca absorption, rate of intestinal ca absorption, ca accumulation, and rate of ca accumulation decreased from mc 1 (the 4th and 5th of the experimental diet period) to mc 2 (the end of the experimental period), but the interaction of time and group was not observed . In both mc1 and mc2, all parameters did not differ between the groups . The present study demonstrated that dhea administration increased the bone mass of lumbar spine in ovariectomized rats . On the other hand we found that the bone mass of lumbar spine (trabecular abundant site) was increased after 8 weeks of dhea administration . This result is similar to the result of our previous study and turner et al.s study which showed increase in trabecular bone mass of tibia . Dhea particularly may have a positive effect on trabecular bone mass but not in cortical areas in the diaphysis region . The reason for this site - specific effect remains unclear yet, but our result further supported that dhea administration may be effective for preventing the reduction of trabecular abundant bones at which fracture frequently occurs for postmenopausal women with osteoporosis . In the present study, femoral weight normalized by body weight was higher in rats treated with dhea than in control rats . The reason for the higher femoral weight would be supported by martel et al. Study that the total bmc of femur increased by dhea administration in ovariectomized rats . Also, we measured the femoral strength . To our knowledge, no studies have examined the effect of dhea treatment on the bone strength of diaphysis site . However, we did not observe any effect of dhea on the bone strength at the midpoint of the femur diaphysis . The lack of effect of dhea on the bone strength might be partly influenced by the non - effect of dhea on the bone mass at the diaphysial area . Our previous study showed that e2 concentration in the blood was higher in ovariectomized rats treated with dhea than in ovariectomized control rats . Although evidence of a direct effect of estrogen promoting intestinal ca absorption has been reported, our results revealed that deha administration may have no effect on intestinal ca absorption . As reason for the lack of dhea effect, intestinal ca absorption might have increased by dhea administration in an earlier phase during 8 weeks of the experimental period . Ca accumulation at the end point of the present experiment did not differ between the groups, meaning that the effect of dhea on bone had already reached its ceiling peak level . As another possible reason for the lack of dhea effect, we used 1.05% ca diet which is an enough ca amount for maintaining the bone health of ovariectomized rats . Some studies related to examining intestinal ca absorption used to use 0.3 - 0.5% ca diet condition which can lead to bone loss and induce higher ca absorption . As the result of our previous study, the serum dhea concentration was approximately 9-fold higher in dhea - treated rats than in control rats . Thus, we cautiously assumed that ca absorption would have increased if we treated lower dose of dhea than that in the present study . Therefore, future research that focuses on the dose - dependent effect of dhea administration is necessary . In addition, we observed that dhea administration increased bmc normalized body weight even though it did not increase bmd . During the growing phase, bmd sometimes may not precisely reflect the alteration of bone mass since growth of bone is considerably influenced by body mass . Because bmd is calculated by dividing the bmc with area (cm), the bmd measurement by dxa did not provide the details of bone parameters such as cortical width and periosteal circumferences . Thus, the parameters should be examined in detail by using the methods of pqct, ct, or bone morphometry in future studies, especially when using animals in the growing phase . The present study confirmed the positive effect of dhea on trabecular bone mass of lumbar spine in ovariectomized rats . On the other hand, dhea administration did not affect intestinal ca absorption . We suggest that dhea administration might have limited impact on intestinal ca absorption in estrogen deficiency state.
Chronic hepatitis b virus (hbv) infection is a potentially life - threatening liver disease with serious complications such as cirrhosis and hepatocellular carcinoma . The prevalence of hbv infection varies widely, with rates ranging from 0.1% to 20% worldwide . Iran is an area of intermediate endemicity and the prevalence of chronic hepatitis b infection is reported to be 1.7% in general population . It has been demonstrated that some hbs - ag negative individuals with positive anti - hbc continue to replicate hbv . Thus, the absence of hbs - ag in the blood of apparently healthy individuals may not be sufficient to ensure an hbv - free status . Anti - hbc can be detected throughout the course of both acute and chronic hbv infection . It persists longlife after resolution, therefore the routine blood donor screening for anti - hbc has been implemented in some countries, resulting in a decrease in the risk of post - transfusion hbv infection . In iran, the reported rates range from 5.1% in hamadan and 6.5% in shiraz to as high as 34% in sistan - balouchestan . Nevertheless, the associated figures for isoalted anti - hbc prevalnce in other studies were as low as 0.56% in the united kingdom to as high as 76% in ghana however, most of previous studies reported isolated anti - hbc rates between 2 - 5% . Despite this fact, iranian health policy makers have not introduced anti - hbc screening among blood donors, partly because of the lack of empirical data on the benefit of introducing anti - hbc screening in a donor population with low prevalence of hbv . On the other hand, the indefinite deferral of donors with false - positive anti - hbc is a major disappointing side effect of anti - hbc screening in countries with low endemicity of hbv infection . It is of utmost importance to differentiate whether isolated anti - hbc is due to false positive results or the prior exposure to hbv, because individuals with false - positive anti - hbc can benefit from vaccination and their blood can be safely transfused . To distinguish between the aforementioned conditions, evaluation of response to hb vaccination has been proposed . In the present study, we investigated the anti - hbs seroconversion in 2 groups of blood donors with isolated anti - hbc and their controls after hb vaccination . We also attempted to find the predictors of non - response status among individuals with isolated anti - hbc . Ninety individuals with isolated anti - hbc positive test and 100 healthy persons with negative serological markers of hepatitis b were recruited in the study as case and control groups, respectively . The exclusion criteria were: age> 64 years, previous hbv vaccination, organ transplantation, immunodeficiency disorders, hemodialysis, immunosuppressive therapy, contraindication for hbv vaccination (i.e. Prior history of anaphylactic reaction to vaccine), positive results for hbs - ag, anti - hbs, anti - hcv, or anti - hiv tests and aspartate aminotransferase (ast) or alanine aminotransferase (alt) levels above 3 times normal cutoff values . Also, none of the participants were positive for signs and symptoms of chronic hepatitis and cirrhosis . Prior to intervention, informed consents were obtained from all the participants, in accordance with the guidelines established by the ethics committee of zahedan university of medical sciences . All the participants were tested for hbv - dna by qualitative polymerase chain reaction (pcr) (cinna gen inc ., tehran, iran) that could detect as low as 100 copy / ml of viral genome . The following reagents were added to each tube on ice: 1x pcr mixture 15 ul and taq - dna polymerase 0.4 ul . Then the tubes were shaken and spun thoroughly . The pcr mixture contained primers amplifying 353 bp of the region of the core gene . One drop (20 - 25 ul) of mineral oil was also added to each tube . Then, 10 ul dna (with the use of specified pipette for sampling of dna) was added to the mixture and spun on microfuge for 3 - 5 seconds . The tubes were transferred to preheated thermocycler with the following program: 1 cycle of 60 seconds at 93c, 20 seconds at 61c and 40 seconds at 72c, followed by 35 cycles of 93c, 61c, and 72c for 20, 20, and 40 seconds, respectively . Finally, 10 iu / ml of amplified samples were directly analyzed in a 2% agarose gel without adding loading buffer . Afterwards, all subjects received three doses of 20 g of hepatitis b recombinant vaccine (heberbiovac hbr, havana, cuba) at 0, 1, and 6 months, if applicable (see below). The vaccine was injected intramuscularly into the deltoid muscle . In order to quantify anti - hbs antibodies, individuals with high titer anti - hbs response (anti - hbs> 50 miu / ml) did not receive additional doses of the vaccine . However, others completed the vaccination course, and another blood sample was collected 30 days after the third dose to measure anti - hbs level . Anti - hbs titer 10 miu / ml 30 days after the first vaccination was considered as early primary response . Also, late primary response was defined as anti - hbs titer 10 miu / ml 30 days after the third dose of vaccines . Non - responders were individuals with <10 miu / ml anti - hbs titers after receiving three doses of hb vaccine . Data were analyzed using spss for windows software, version 11.5 (spss inc ., odd s ratios (ors) were chosen to measure the association between dichotomous variables, and the results were adjusted for potential confounders by multivariate logistic modeling . Table 1 presents the demographic and important risk factors of the participants, comparing those with isolated anti - hbc to controls . 1) abnormal level was defined as levels> 1.5 times upper than normal limit 2) ns: not significant 3) linear by linear association (chi - squared for linear trend); exact method participants of the case group were significantly older, and mostly married . The case group had also higher male / female ratio, and more history of transfusion and tattoo (p<0.05). However, history of infection in spouse, familial history of hbv infection, and serum ast and alt levels was not significantly different between cases and controls . Totally, 19 (21.1%) cases and 3 (3%) controls did not seroconvert (non - responder) after three doses of hb vaccine (p<0.0001). However, primary response was observed in 43 (47.8%) isolated anti - hbc positive cases and 92 (92%) controls (p<0.0001), of whom 15 cases and two controls seroconverted after the first dose of hb vaccine (early responder). Furthermore, anti - hbs titer 50 miu / ml 30 days after the first dose of vaccine was significantly higher among cases (31.1% vs. 5%, p<0.0001). Two (2.2%) cases with positive isolated anti - hbc had detectable hbv dna and were therefore, excluded from the analysis . Both of these two hbv dna positive cases did not seroconvert after receiving 3 doses of vaccination . None of the cases and controls showed adverse effects after receiving recombinant hb vaccine . Table 2 compares non - responders (excluding 2 hbv dna positive cases) with primary responders (including early and late responders). According to univariate analysis, serum anti - hbc positivity, age, and marital status were significantly different between the two groups; however, in multivariable analysis, only anti - hbc positivity and age remained independently associated with non - responding status . Indeed, anti - hbc positive subjects were 12.2 times (95% ci: 3.2 - 46.4, p<0.0001) more likely to fail seroconversion . Similarly, age 50 years was 3.6 times more likely to lead in non - responsive state (95% ci: 1.0 - 12.3, p<0.04). Also, anti - hbc positive participants were more likely to develop anti - hbs titer 50 miu / ml 30 days after receiving the first dose of vaccine (table 3). 1) abnormal level was defined as levels> 1.5 times upper than normal limit 2) ns: not significant 3) linear by linear association (chi - squared for linear trend); exact method . * participants with anti - hbs titer 50 miu / ml 30 days after the first dose of vaccine and those with positive hbv dna were excluded as noted earlier, early response (anti - hbs titer 10 miu / ml 30 days after the first vaccination) was found in 17 subjects . We compared early responders to late responders (the table is not included) and found a trend toward a higher seroprotective response rate after the first vaccination in the subjects with abnormal baseline alt levels and those aged>50 years (ors: 6.9 and 8, p<0.02 and p<0.001, respectively). Data were analyzed using spss for windows software, version 11.5 (spss inc ., odd s ratios (ors) were chosen to measure the association between dichotomous variables, and the results were adjusted for potential confounders by multivariate logistic modeling . Table 1 presents the demographic and important risk factors of the participants, comparing those with isolated anti - hbc to controls . 1) abnormal level was defined as levels> 1.5 times upper than normal limit 2) ns: not significant 3) linear by linear association (chi - squared for linear trend); exact method participants of the case group were significantly older, and mostly married . The case group had also higher male / female ratio, and more history of transfusion and tattoo (p<0.05). However, history of infection in spouse, familial history of hbv infection, and serum ast and alt levels was not significantly different between cases and controls . Totally, 19 (21.1%) cases and 3 (3%) controls did not seroconvert (non - responder) after three doses of hb vaccine (p<0.0001). However, primary response was observed in 43 (47.8%) isolated anti - hbc positive cases and 92 (92%) controls (p<0.0001), of whom 15 cases and two controls seroconverted after the first dose of hb vaccine (early responder). Furthermore, anti - hbs titer 50 miu / ml 30 days after the first dose of vaccine was significantly higher among cases (31.1% vs. 5%, p<0.0001). Two (2.2%) cases with positive isolated anti - hbc had detectable hbv dna and were therefore, excluded from the analysis . Both of these two hbv dna positive cases did not seroconvert after receiving 3 doses of vaccination . None of the cases and controls showed adverse effects after receiving recombinant hb vaccine . Table 2 compares non - responders (excluding 2 hbv dna positive cases) with primary responders (including early and late responders). According to univariate analysis, serum anti - hbc positivity, age, and marital status were significantly different between the two groups; however, in multivariable analysis, only anti - hbc positivity and age remained independently associated with non - responding status . Indeed, anti - hbc positive subjects were 12.2 times (95% ci: 3.2 - 46.4, p<0.0001) more likely to fail seroconversion . Similarly, age 50 years was 3.6 times more likely to lead in non - responsive state (95% ci: 1.0 - 12.3, p<0.04). Also, anti - hbc positive participants were more likely to develop anti - hbs titer 50 miu / ml 30 days after receiving the first dose of vaccine (table 3). 1) abnormal level was defined as levels> 1.5 times upper than normal limit 2) ns: not significant 3) linear by linear association (chi - squared for linear trend); exact method . * participants with anti - hbs titer 50 miu / ml 30 days after the first dose of vaccine and those with positive hbv dna were excluded as noted earlier, early response (anti - hbs titer 10 miu / ml 30 days after the first vaccination) was found in 17 subjects . We compared early responders to late responders (the table is not included) and found a trend toward a higher seroprotective response rate after the first vaccination in the subjects with abnormal baseline alt levels and those aged>50 years (ors: 6.9 and 8, p<0.02 and p<0.001, respectively). In our setting, we vaccinated isolated anti - hbc as well as healthy blood donors and monitored their response to hbv vaccination . A total of 19 (21.1%) cases and 3 (3%) controls did not seroconvert after receiving 3 doses of hb vaccine . Prior investigators reported a wide range of non - responding state, sunbul 9.1%, coz yatacho 9.7% . Therefore, at least 20% of isolated anti - hbc positive cases may suffer from occult hepatitis b infection, for whom a diminished ability to mount an anti - hbs response is proposed . Furthermore, in our study, anti - hbs titer 50 miu / ml 30 days after the first dose of vaccine was developed in 28 (31.1%) cases and 5 (5%) controls . Scanty studies have addressed anamnestic rates; however, in a study reported by sunbul and colleagues, out of 33 (42.4%) subjects with isolated anti - hbc showed rapid high seroconversion, while in a report by ural and co - workers, this rate was 41.7% (20 out of 48). Our lower rate could in part be explained by lower prevalence of hbv in our region . In such patients, further vaccination may not be necessary . Finally, 43 (47.8%) subjects with isolated positive anti - hbc and 92 (92%) controls showed primary response after the vaccination protocol . Nevertheless, most of previous researchers assigned their subjects in two groups of responders (either primary or anamnestic) and non - responders . Accordingly, our cumulative responder rate would be 78.9% among subjects with isolated positive anti - hbc, which is in agreement with reports by koh (70.6%), kabir (79.8%), and silva (80%), but lower than what reported by ural (89.6%), pereira (90%), yatacho (90.3%) and sunbul (90.9%)., discrepancies in response rate to hb vaccination among isolated anti - hbc subjects can be explained by serologic kit sensitivity, defining cut off points, and inclusion criteria for borderline results . Therefore, in our region, nearly 80% of isolated anti - hbc subjects can be safely included in the donor pool, simply based on a vaccination protocol . Notably, the isolated anti - hbc subjects with false positive results (47.8% in our setting) are susceptible to hepatitis b infection and should receive a complete course of hb vaccine . However, they may lose hb vaccination because of misinterpretation of anti - hbc results . On the other hand, our results revealed that individuals with isolated positive anti - hbc were significantly older, mostly married, had higher male / female ratio, history of transfusion, tattoo and abnormal serum ast and alt levels when compared with controls . The higher rate of anti - hbc positivity among older subjects mirrors a cohort effect of hbv infections acquired decades ago when hbv was moderately endemic in iran and hbv national vaccination program had not been commenced . Furthermore, only two (2.2%) isolated anti - hbc positive cases had detectable hbv dna . Recent studies suggest that 1 - 2% of anti - hbc reactive units contain low levels of hbv dna . However, in studies conducted on 4930 healthy blood donor volunteers in fars and hamadan provinces, 6.5% and 5.1% were isolated anti - hbc positive cases, among whom 16 (12.3%) and 3 (1.2%) were hbv dna positive, respectively . Having compared non - responders to primary responders (table 2), anti - hbc positivity, age, and marital status showed significant differences in univariable analysis . However, in multivariable analysis, only anti - hbc positivity and age (> 50 years) remained independently associated with non - responding status . On the other hand, when we compared primary responders with those who developed anti - hbs titer 50 miu / ml 30 days after the first dose of vaccine (table 3), positivity for anti - hbc and age> 50 years were the most relevant factors in developing higher anti - hbs titers to hbv vaccine . Therefore, isolated anti - hbc subjects aged> 50 years would more likely either develop anti - hbs titers 50 miu / ml 30 days after receiving the first dose of vaccine or never respond to hb vaccine . As a result, one month after the first dose of hb vaccine in isolated anti - hbc subjects aged> 50 years, we can make a decision regarding whether to advise a strict follow - up (occult hbv infection) or assure the patient that he / she has a resolved hbv infection . Interestingly, when we compared the early and late responders (the table is not included), anti - hbc positivity, abnormal alt level, and age> 50 years were associated with a higher probability of early response . Therefore, anti - hbc positive subjects aged> 50 years with abnormal alt levels should be offered the first dose of hb vaccine and evaluated one month later in order to decide for their future follow - up . Various molecular modalities have been developed to determine if isolated anti - hbc subjects could transmit hbv infection . However, they will further increase the testing costs and require trained personnel and equipment, which may not be possible in many blood banks around the country . Thus, studying the cost effectiveness of monitoring the response to hb vaccination could be worth of considering as a diagnostic tool . We also suggest future studies focusing on evaluating the short - course vaccination protocols such as double - dose / double - time vaccination in order to make sooner decision . In conclusion; using vaccination, we found that half of the subjects with isolated positive anti - hbc were falsely positive for the test and more than 75% of subjects with isolated positive anti - hbc could benefit from vaccination and could be included in donor pool . Moreover, one - fifth of subjects were non - responders and may have occult hbv infection . Because at least 5% of the donor population in iran is anti - hbc positive, blindly rejecting anti - hbc positive donors would cause the exclusion of a consistent number of donors, most of whom could safely be included in the donor pool . Therefore, hb vaccination may be used as a diagnostic tool for clarifying the situation of the subjects with isolated anti - hbc and can help in reducing the blood supply problems . This study was financially supported by zahedan university of medical sciences and iranian blood transfusion organization research center (zahedan branch).
We searched medline (between 1966 and 2007) and the cochrane library central registry of controlled trials (between 1984 and 2007) for relevant publications using the following medical subject heading terms: diabetes and (food or diet). We examined reference lists of those publications to identify additional studies suitable for our purpose . We searched for studies of the effects of two kinds of prescribed diets differing according to proportions of carbohydrate and fat under conditions that the prescribed total energy and protein intake did not differ significantly between groups of patients with type 2 diabetes . We designated one diet as the lfhc diet, which was defined as having a relatively high c / f ratio, and the other as the hflc diet, which had a relatively low c / f ratio . As shown in detail in table 1, in examining these studies, we found that the c / f ratio ranged from 0.60 to 1.56 for the hflc diets and from 1.67 to 7.30 for the lfhc diets . Descriptive statistics of studies included in the meta - analysis c / f / p, proportion of carbohydrate / fat / protein to total energy of the prescribed diet; n / a, not assessed . Among the studies identified, we included only randomized controlled trials with measurements of fasting plasma glucose (fpg) and fasting insulin and intervention periods of 1 week . Studies that included an intervention with a change in the content or quality of carbohydrate such as an increase in fiber and whole grains were excluded because such diets are high in fiber, which in itself ameliorates glycemia and lipemia regardless of changes in the c / f ratio (3,4). Studies of very - low - calorie or enteral (not oral) diets and those in which the dosage of hypoglycemic agents was changed during the intervention period were also excluded . One of three reviewers extracted all studies that met the eligibility criteria, and a second reviewed all extracted data . Extracted data included features of the study design (i.e., crossover or parallel design and presence of a washout period), intervention periods, characteristics of patients (mean age, bmi, percent men, and percent those using hypoglycemia agents). Other extracted data regarded the characteristics of each diet, such as macronutrient composition; a weight - loss diet, which was defined as caloric restriction resulting in weight reduction; a weight - maintenance diet, which was defined by a weight change of 1 kg during the intervention period, and a monounsaturated fat (mufa) diet within the hflc - diet group, which was defined as the addition of mufa to the hflc diet . We also extracted baseline and final means and statistical dispersions of each group for the following metabolic profiles: a1c, fpg, fasting insulin, total cholesterol, fasting triglycerides, ldl cholesterol, hdl cholesterol, and 2-h postprandial levels of glucose and insulin . If vldl cholesterol but not triglyceride data were provided, the triglyceride value was calculated by multiplying vldl cholesterol 5 according to the friedewald formula (5). Also, if hba1 but not a1c data were provided, a1c was estimated by the relation between hba1 and a1c concentrations according to the methodology of kilpatrick et al . (6). If necessary, measures of means and dispersion were approximated from figures in the articles using an image scanner (canoscan lide 500f [resolution 600 dpi]; canon, tokyo, japan). (7), with each included trial evaluated according to randomization, double blinding, withdrawals, and dropouts . The effect on each metabolic profile, which is expressed as the mean difference between lfhc- and hflc - diet groups in individual studies, was calculated by subtracting the change from baseline to final values in the hflc - diet group from that in the lfhc - diet group . The se of the change from baseline values was directly extracted from the reported data or estimated from the ses of the baseline and final values in the lfhc- and hflc - diet groups, assuming a correlation of 0.5 between the baseline and final measures within each group, according to the formula of follmann et al . (8), as follows: we chose the percent change from baseline values because the mean baseline and final values in patients in each study were highly skewed . To estimate percent change, we divided each change from baseline values and its se by the baseline value . When no baseline value was reported, as in some crossover studies, we summarized the intervention effect by the ratio of the difference in final values between lfhc- and hflc - diet groups to the final value in the hflc - diet group and assumed that the baseline se was equal to the final se . This method of estimating percent change has limitations, especially in studies without washout periods . Therefore, we performed a sensitivity analysis to examine the effect of these studies on the results . All percent changes were firstly pooled with a fixed - effects model (9). For each outcome measure, influence analysis was conducted to detect an outlier (i.e., a single estimate with an extreme result), which influenced overall outcome . If heterogeneity was significant, the percent changes were secondarily re - pooled with a random - effects model (9). Publication bias was assessed using two formal methods: begg's test (10) and egger's test (11). The trim - and - fill technique (12) was used to investigate the impact of any suggested bias . We also calculated the weighted mean difference (wmd) in individual trials by multiplying each percent change by the inverse of its se squared . We ecologically examined the mutual association among each metabolic effect of the lfhc diet compared with the hflc diet by spearman's correlation analyses among wmds . To investigate the effect of study characteristics, stratified analyses were performed for the following possible confounders: study design (i.e., whether each trial used a crossover design and, if so, whether the trial had a washout period or data on baseline values), intervention period (<4 vs. 4 weeks), percent the study of female sex (<50 or 50%), mean age (<55 vs. 55 years), bmi (<28 vs. 28 kg / m), percentage using hypoglycemia agents (zero vs. above zero), c / f ratio in the lfhc (> 3 vs. 3) and hflc (> 1 vs. 1) groups, prescription of the mufa diet (yes vs. no), and prescription of a weight - loss or weight - maintenance diet . We additionally conducted linear multivariable regression analyses to determine whether the characteristics of the patients were independent predictors that influenced the effect of the lfhc diet versus that of the hflc diet . In this analysis, age, bmi, and the carbohydrate proportion in the lfhc and hflc diets were entered as continuous variables . All analyses were performed with stata software version 10 (stata corporation, college station, tx). Of 2,203 potentially relevant publications based on search terms and 22 references obtained from manual searches, 19 (1331) met the inclusion criteria . Four articles (19,20,24,31) included two trials in one study, and two articles (27,28) used the same cohort . Studies included in the current analysis had intervention periods ranging from 10 days to 6 weeks and patient numbers ranging from 8 to 42 . Means between - study sds for the mean study characteristics from 22 trials were as follows: age 55 5 years, percent men 63 23, bmi 28 3 kg / m, percent using hypoglycemia agents 52 31, and diabetes duration 6 1 years . Ten studies (15,1821,2326,31) described the number of dropouts, and nine (13,14,16,17,22,2730) did not . None of the 19 articles described methods of randomization, which led to a low quality score for the trial . A crossover design was used in 17 studies (1318,20,21,2331) (with 19 trials), whereas a parallel design was used in two studies (19,22) with three trials . Median carbohydrate / fat proportion of total energy (c / f ratio) in the lfhc and hflc diets was 58%/24% (2.4) and 40%/40% (1.0), respectively . Three studies (19,22,26) with 4 trials prescribed a weight - loss diet, and 11 studies (13,14,1719,21,2325,27,28) with 11 trials provided a mufa diet to the hflc - diet group . There were no significant differences in the reduction in a1c, total cholesterol, and ldl cholesterol between the lfhc and hflc diets . However, the lfhc diet produced significant increases in fasting insulin and triglycerides levels of 8.4% (p = 0.02) and 13.4% (p <0.001), respectively, and a significant reduction in hdl cholesterol compared with that associated with the hflc diet . Two - h glucose and insulin values were higher in the lfhc - diet group than in the hflc - diet group by 10.3% (p <0.001) and 12.8% (p <0.001), respectively . Overall percent changes resulting from lfhc versus hflc diet on metabolic profiles and data on publication bias and their likely effect on the estimates * studies (n) added by the trim - and - fill method . Percent change after adjustment for publication bias by the trim - and - fill method . Begg's, begg's adjusted rank correlation test; egger's, egger's regression asymmetry test . Influence analyses indicated that there were a few outliers for percent change in total (22), hdl (22), and ldl (29) cholesterol (see online appendix tables a1 and a2, available at http://care.diabetesjournals.org/cgi/content/full/dc08-1716/dc1). When these trials were omitted from the analyses, percent change in total cholesterol, hdl cholesterol, and ldl cholesterol significantly changed from 0.0% (95% ci 2.1 to 2.0) to 1.6% (4.5 to 1.3; p = 0.03), from 10.4% (12.2 to 8.6) to 5.6% (2.9 to 8.4; p <0.001), and from 3.0% (6.3 to 0.4) to 0.1% (4.1 to 3.8; p = 0.001), respectively . These outlying trials comprised a large part of study heterogeneity in percent change in total, hdl, and ldl cholesterol (22.2, 59.1, and 53.0%, respectively .) Therefore, they were excluded from the following analyses for the outcome that they affected . After omission of these outliers, there was no evidence of significant study heterogeneity (p> 0.4 for all outcomes). Ecological analyses showed trends indicating that the wmd in fpg was positively associated with that in fasting insulin (r = 0.45; p = 0.04) and triglycerides (r = 0.59; p = 0.004) and that the wmd in fasting insulin and triglycerides was mutually associated (r = 0.43; p = 0.04). These associations remained significant after adjustment for whether a weight - loss diet was prescribed (fpg vs. fasting insulin, r = 0.58 and p = 0.004; fpg vs. triglycerides, r = 0.44 and p = 0.04; and fasting insulin vs. triglycerides, r = 0.44 and p = 0.04). Table 2 also shows data on publication bias and its likely effect on estimates of outcome according to the trim - and - fill method (12). There was a relatively strong suspicion of publication bias for hdl cholesterol (egger's test, p = 0.08 for hdl cholesterol; recommended level of significance, p 0.10 [(32)]). According to results of the compensatory trim - and - fill method, the effect of publication bias would slightly underestimate the adverse effect of the lfhc diet . Results of our stratified analysis to detect characteristics of studies and patients included in our analyses that might have modulated study outcomes are shown in table 3 . Of the 17 studies with a crossover design, 9 with 10 trials (1416,21,2326,29) did not include a washout period, which could lead to an underestimation due to a carryover effect (33). However, the effect of these nine studies on results was not significant for any of the measures . Stratified analysis to examine the effects of characteristics of studies and patients on each metabolic profile * studies having neither a washout period nor baseline data . Parallel study design or cross - sectional design studies that have a washout period and/or baseline data . The elevation in fasting insulin was remarkable (17.1%; p = 0.001) in lfhc diets with a c / f ratio 3 (in this case, an lfhc diet with 60% carbohydrate and 20% fat of total energy) while the c / f ratio in the lfhc diet did not influence triglycerides . There was a greater elevation in triglycerides (21.0%; p<0.001) with the lfhc diet when the lfhc diet and mufa diet were compared; i.e., mufa was replaced with carbohydrate . However, the magnitude of the elevation in fasting insulin did not differ between the mufa diet and non - mufa diet (i.e., regardless of dietary fat quality). Whereas a larger elevation in triglycerides was observed in trials limited to weight - maintenance diets, the lfhc diet did not significantly elevate triglycerides compared with the hflc diet when only trials with weight - loss diets were examined (i.e., diets for weight loss) (p = 0.48). The elevation in fasting insulin was greater in younger and leaner patients in response to the lfhc diet compared with that in response to the hflc diet . Moreover, mean age and bmi were independent predictors of percent change in fasting insulin . Multiple regression analysis indicated that every 1 kg / m of bmi and 1 year of age were independently associated with a greater elevation in fasting insulin by 2.6% (p = 0.002) and 1.7% (p = 0.005), respectively . For patients not taking antihyperglycemic drugs, however, because only a few studies included patients not receiving antihyperglycemic drugs, the results should perhaps be interpreted with caution . Of 2,203 potentially relevant publications based on search terms and 22 references obtained from manual searches, 19 (1331) met the inclusion criteria . Four articles (19,20,24,31) included two trials in one study, and two articles (27,28) used the same cohort . Studies included in the current analysis had intervention periods ranging from 10 days to 6 weeks and patient numbers ranging from 8 to 42 . Means between - study sds for the mean study characteristics from 22 trials were as follows: age 55 5 years, percent men 63 23, bmi 28 3 kg / m, percent using hypoglycemia agents 52 31, and diabetes duration 6 1 years . Ten studies (15,1821,2326,31) described the number of dropouts, and nine (13,14,16,17,22,2730) did not . None of the 19 articles described methods of randomization, which led to a low quality score for the trial . A crossover design was used in 17 studies (1318,20,21,2331) (with 19 trials), whereas a parallel design was used in two studies (19,22) with three trials . Median carbohydrate / fat proportion of total energy (c / f ratio) in the lfhc and hflc diets was 58%/24% (2.4) and 40%/40% (1.0), respectively . Three studies (19,22,26) with 4 trials prescribed a weight - loss diet, and 11 studies (13,14,1719,21,2325,27,28) with 11 trials provided a mufa diet to the hflc - diet group . There were no significant differences in the reduction in a1c, total cholesterol, and ldl cholesterol between the lfhc and hflc diets . However, the lfhc diet produced significant increases in fasting insulin and triglycerides levels of 8.4% (p = 0.02) and 13.4% (p <0.001), respectively, and a significant reduction in hdl cholesterol compared with that associated with the hflc diet . Two - h glucose and insulin values were higher in the lfhc - diet group than in the hflc - diet group by 10.3% (p <0.001) and 12.8% (p <0.001), respectively . Overall percent changes resulting from lfhc versus hflc diet on metabolic profiles and data on publication bias and their likely effect on the estimates * studies (n) added by the trim - and - fill method . Percent change after adjustment for publication bias by the trim - and - fill method . Begg's, begg's adjusted rank correlation test; egger's, egger's regression asymmetry test . Influence analyses indicated that there were a few outliers for percent change in total (22), hdl (22), and ldl (29) cholesterol (see online appendix tables a1 and a2, available at http://care.diabetesjournals.org/cgi/content/full/dc08-1716/dc1). When these trials were omitted from the analyses, percent change in total cholesterol, hdl cholesterol, and ldl cholesterol significantly changed from 0.0% (95% ci 2.1 to 2.0) to 1.6% (4.5 to 1.3; p = 0.03), from 10.4% (12.2 to 8.6) to 5.6% (2.9 to 8.4; p <0.001), and from 3.0% (6.3 to 0.4) to 0.1% (4.1 to 3.8; p = 0.001), respectively . These outlying trials comprised a large part of study heterogeneity in percent change in total, hdl, and ldl cholesterol (22.2, 59.1, and 53.0%, respectively .) Therefore, they were excluded from the following analyses for the outcome that they affected . After omission of these outliers, there was no evidence of significant study heterogeneity (p> 0.4 for all outcomes). Ecological analyses showed trends indicating that the wmd in fpg was positively associated with that in fasting insulin (r = 0.45; p = 0.04) and triglycerides (r = 0.59; p = 0.004) and that the wmd in fasting insulin and triglycerides was mutually associated (r = 0.43; p = 0.04). These associations remained significant after adjustment for whether a weight - loss diet was prescribed (fpg vs. fasting insulin, r = 0.58 and p = 0.004; fpg vs. triglycerides, r = 0.44 and p = 0.04; and fasting insulin vs. triglycerides, r = 0.44 and p = 0.04). Table 2 also shows data on publication bias and its likely effect on estimates of outcome according to the trim - and - fill method (12). There was a relatively strong suspicion of publication bias for hdl cholesterol (egger's test, p = 0.08 for hdl cholesterol; recommended level of significance, p 0.10 [(32)]). According to results of the compensatory trim - and - fill method, the effect of publication bias would slightly underestimate the adverse effect of the lfhc diet . Results of our stratified analysis to detect characteristics of studies and patients included in our analyses that might have modulated study outcomes are shown in table 3 . Of the 17 studies with a crossover design, 9 with 10 trials (1416,21,2326,29) did not include a washout period, which could lead to an underestimation due to a carryover effect (33). Moreover, none of these studies had baseline data . However, the effect of these nine studies on results was not significant for any of the measures . Stratified analysis to examine the effects of characteristics of studies and patients on each metabolic profile * studies having neither a washout period nor baseline data . Parallel study design or cross - sectional design studies that have a washout period and/or baseline data . P <0.05 . The elevation in fasting insulin was remarkable (17.1%; p = 0.001) in lfhc diets with a c / f ratio 3 (in this case, an lfhc diet with 60% carbohydrate and 20% fat of total energy) while the c / f ratio in the lfhc diet did not influence triglycerides . There was a greater elevation in triglycerides (21.0%; p<0.001) with the lfhc diet when the lfhc diet and mufa diet were compared; i.e., mufa was replaced with carbohydrate . However, the magnitude of the elevation in fasting insulin did not differ between the mufa diet and non - mufa diet (i.e., regardless of dietary fat quality). Whereas a larger elevation in triglycerides was observed in trials limited to weight - maintenance diets, the lfhc diet did not significantly elevate triglycerides compared with the hflc diet when only trials with weight - loss diets were examined (i.e., diets for weight loss) (p = 0.48). The elevation in fasting insulin was greater in younger and leaner patients in response to the lfhc diet compared with that in response to the hflc diet . Moreover, mean age and bmi were independent predictors of percent change in fasting insulin . Multiple regression analysis indicated that every 1 kg / m of bmi and 1 year of age were independently associated with a greater elevation in fasting insulin by 2.6% (p = 0.002) and 1.7% (p = 0.005), respectively . For patients not taking antihyperglycemic drugs, the lfhc diet could be more harmful for fasting insulin than the hflc diet . However, because only a few studies included patients not receiving antihyperglycemic drugs, the results should perhaps be interpreted with caution . Although central to mnt, the influences of various dietary c / f ratios on glycemic control and lipid profiles in patients with type 2 diabetes have not been systematically reviewed . Our meta - analysis is the first to quantify the effect of the lfhc diet compared with that of the hflc diet on each metabolic outcome . Our results fundamentally support current dietary guidelines (1) stating that replacing fat with carbohydrate significantly elevates postprandial glucose and insulin levels when total energy intake is consistent . We additionally found that the lfhc diet significantly elevated fasting insulin compared with the hflc diet, with marked elevations noted when the c / f ratio was 3 . Moreover, there were significantly positive relationships among the change in fpg and the magnitude of the elevation in fasting insulin and triglycerides, independent of energy restriction for weight control . Postprandial hyperglycemia with postprandial hyperinsulinemia and failure to maintain glucose homeostasis are often clustered in insulin - resistant individuals, who are representative of those with type 2 diabetes (34). This suggests that an lfhc diet is unfavorable compared with an hflc diet for insulin - resistant patients, at least when energy intake is consistent . However, our findings do not support the benefit of an extremely high - fat diet because the carbohydrate proportion in the hflc diets included in our analyses was 50% . Moreover, we cannot comment on the possible benefit of a high - carbohydrate diet with a high - fiber component because we excluded studies investigating the effect of such a diet . Moreover, there is concern that increased fat intake ad libitum may promote weight gain (35). It is worth repeating that total caloric intake and nutritional content must be appropriate for metabolic control regardless of macronutrient proportions (1). Changes in fpg and a1c did not differ between the two diets despite significant elevations in 2-h and fasting insulin with the lfhc diet . One possible explanation is that the elevation in postprandial glucose level was overcompensated for by increased insulin secretion . However, only three studies concurrently assessed a1c, fasting insulin, and fpg values, with an intervention period of, at most, 6 weeks . Therefore, we could not conclude whether the elevation in postprandial glucose and insulin level achieved by raising the dietary c / f ratio leads to the deterioration of glycemic control represented by elevations in fpg and a1c . A previous meta - analysis suggested that replacing carbohydrate with mufa reduced fasting triglycerides in patients with type 2 diabetes on weight - maintenance diets (36); this was supported by our results . However, it is uncertain whether the effect on triglycerides was caused by the c / f ratio or the ratio of energy from mufa to total energy . Moreover, whether the effect of this replacement was independent of that of a weight - loss diet has not been investigated . According to our stratified analyses, no dose - response relationship between the c / f ratio in the lfhc diet and the elevation in triglycerides was indicated, although replacement of the mufa diet with the lfhc diet induced a greater elevation in triglycerides . Moreover, the lfhc diet did not significantly elevate triglycerides compared with the hflc diet when a weight - loss diet was prescribed . Therefore, controlling total caloric intake and the quality of dietary fat appear to be more important than carbohydrate and fat composition in improving triglycerides levels . In other words, these findings suggest that a high - carbohydrate diet has little harmful effect on triglycerides levels if such a diet provides sufficient energy restriction for weight control . First, although we omitted studies investigating the effect of high - carbohydrate diets that were also high in dietary fiber, it is possible that the additional phytochemicals (including fiber itself), which are inevitably accompanied by a substantial amount of carbohydrate, influence the metabolic effects regardless of the change in c / f ratio . Second, we assumed that energy intake from the two diet groups would be similar if a weight - maintenance diet was equal to an isocaloric diet based on evidence of the meta - analysis by bravata et al . (37) that indicated that weight change was associated with restriction of caloric intake but not reduced carbohydrate content . However, some recent studies showed that low - carbohydrate diets resulted in greater weight loss than low - fat diets despite their similar energy content (38), as is often the case with high - fiber diets (e.g., whole grains) (39). More investigation is needed to determine whether the relationship between change in energy intake and body weight is independent of the proportions of dietary carbohydrate and fat . Third, few studies investigated long - term effects (e.g.,> 2 months) of changing the proportions of carbohydrate and fat on metabolic profiles in patients with type 2 diabetes . Actually, a larger elevation in fasting insulin in association with the lfhc diet was observed for an intervention period of <4 weeks compared with 4 weeks but without statistical significance (p = 0.10). Possibly, a prolonged intervention involving changes in macronutrient composition causes some adaptation of insulin metabolism . Fourth, most studies provided insufficient data about baseline glucose and lipid levels, and few focused on black or asian patients . Therefore, the current meta - analysis provides limited suggestions on identifying patients for whom a low - fat or low - carbohydrate diet is especially effective in terms of their circumstances or metabolic profiles (1). Future studies focused on the following are suggested: 1) providing a possible explanation for the greater adverse effect on the fasting insulin by the lfhc diet than by the hflc diet, especially in younger and leaner individuals; 2) identifying the long - term effect of a low - carbohydrate diet on factors other than metabolic effects (e.g., adaptation in glucose and lipid metabolism, ad libitum energy intake in patients with type 2 diabetes or obesity [(40)]) and the safety of such a diet (e.g., with regard to the digestive system); and 3) addressing whether a subject's medication status and the characteristics of diabetes drugs could influence the effect of changing the dietary c / f ratio in patients with type 2 diabetes . In conclusion, replacement of dietary fat with carbohydrate is not recommended for improvement of insulin resistance in patients with type 2 diabetes under conditions whereby total energy and protein intake and the content of carbohydrate are similar and the proportion of carbohydrate to total energy is 30% . We found that younger and leaner patients had higher fasting insulin responses with the lfhc diet, although the biological mechanism was not fully investigated . The lfhc diet also adversely affects triglycerides and hdl cholesterol compared with the hflc diet . However, energy restriction and dietary fat quality seemed more important for lowering the triglyceride concentration than the proportion of carbohydrate and fat.
Mindfulness refers to an awareness that emerges by paying attention to purpose and to the present moment and nonjudgmentally focusing on the unfolding of one's immediate experience . More recently, mindfulness has been proposed as a cognitive behavior, rather than physiological, paradigm for meditation . Mindfulness aims to develop enhanced awareness of the moment - to - moment experience of perceptible mental processes and forms an important component of meditation practices . Initially, a meditator engages in focused concentration or attention over an object and as one grows in his practice, he leans towards the attentional disengagement or open monitoring . Meditation imbibes an initial phase of mindfulness, making mindfulness a key determinant of meditation practice . During the last decade, several studies have been conducted across the globe to report the development of mindfulness and its effects on health and well - being . One such study conducted on a martial art technique aikido using a mindfulness attention awareness scale (maas) concluded that consistent practice of aikido leads to development of mindfulness . Another study on insomnia in menopausal women reported that postmenopausal women with insomnia are less mindful than women without insomnia, thereby concluding that mindfulness - based interventions, such as meditation, may be beneficial for postmenopausal insomnia . A study assessing the health risk behavior in adolescents concluded that mindfulness possibly shields against decision - making processes that place adolescents at risk for smoking . There are several others studies looking at the effects of mindfulness on neurological and psychiatric diseases and also assessing the levels of mindfulness in normal and diseased individuals . One of the studies reviewing the instruments of measuring mindfulness concluded that the maas was used by most studies (n = 27) and had positive overall quality ratings for most of the psychometric properties reviewed . Given the fact that past studies have looked at the levels of mindfulness in various practices, health and disease conditions, we planned the current study to asses the levels of mindfulness in a moving meditation practice . One of the various forms of mindfulness is the practice of a unique technique called cyclic mediation (cm). It was fundamentally designed for novice practitioners and combines the practice of yoga postures with guided meditation . Cm is known to induce a quiet state of mind, which is compatible with the description of meditation (dhyana or effortless expansion), according to patanjali . Although this moving meditation differs from the classic description of meditation, in which the practitioners remain seated, keeping as still as possible, the mental state in both practices (moving meditation and seated practices) is supposed to be comparable . An essential part of the practice of cm is being aware of sensations arising in the body, which emphasize the mindful component . There have been several studies which have proven the beneficial effects of cm . In one of the studies conducted on middle managers, cm program decreased occupational stress levels and baseline autonomic arousal in 26 asymptomatic, studies conducted to ascertain the effects of cm practice reported a decreased oxygen consumption indicating physiological relaxation as in mindfulness . Few studies looking at the immediate effects of cm concluded that it improves attention, cognition, enhances slow wave sleep, and reduces anxiety . Mindful yoga practices (like cm) may generate the state of mindfulness, which, when evoked recurrently through repeated practice, may accrue into trait or dispositional mindfulness . Despite several studies on cm, none have reported its mindful component . The current study aimed at investigating the level of mindfulness in experienced cyclic meditators . We also report the correlation between the years of meditation experience and the level of mindfulness . One hundred and thirty - three (n = 133) healthy male volunteers (66 meditators and 67 non - meditators) with ages ranging from 25 to 35 years [group mean age standard deviation (s.d . ), 24.6 4.5 for meditators and 24.1 4.7 for non - meditators] participated in the study . Meditators were selected from s - vyasa yoga university, south india and corresponding non - meditators (controls) matched for age, gender, and education were obtained from similar institutes in bangalore, india . Meditators had a minimum 3 years experience of meditation (group mean experience s.d ., 5.12 1.35 years). Non - meditators had no exposure to any yoga practices and were unaware of the aims of the study . Subjects with cognitive deficits ruled out by routine clinical examination were excluded from the study . This study was approved by the institutional ethics committee and a signed informed consent was obtained from all the subjects following explanation of the study . The questionnaire was administered in a classroom setup (for approximately 30 min) and two of the project coordinators were present to supervise the administration and to assist the participants where necessary . All the participants filled out the questionnaire, but for whom more than 10% of the items were missing or whose reports were considered unreliable (i.e., consistently rated the highest or the lowest scores on all items), were excluded from the analyses (n = 06; 4%). The subjects participating in this study had higher educational qualifications with almost 90% of the participants being postgraduates . This was a cross - sectional study, where subjects (meditators) were recruited from s - vyasa yoga university and other universities (non - meditators) by convenience sampling . Maas is a 15-item self - reported single - factor scale that is exclusively focused on attention / awareness component of mindfulness construct . This instrument has been independently used to assess individuals either with or without meditation experience . This scale has been widely used for various studies and has reported positive overall quality ratings for most of the psychometric properties reviewed . Maas is a brief, easy to administer scale, and has therefore been used in wide range of studies related to assessing mindfulness trait . Maas is known to have good reliability ratings and a history of clinical and research use that was developed to assess the core attentional aspect of mindfulness, and the capacity for moment - to - moment attention in particular . The maas consists of 15 items that measure the level of mindfulness (example items are i could be experiencing some emotion and not be conscious of it until some time later, or i find it difficult to stay focused on what's happening in the present). The items are answered on a six - point scale (1 = almost always; 6 = almost never) on which higher scores are an indication of higher trait mindfulness . The maas has been validated in various samples of students (= 0.82) and adults from the general community (= 0.87). The questionnaire was scored by computing a mean of the 15 items in the questionnaire . The data were checked for normality and an independent samples t - test was employed to compare the means of both the groups . We also calculated the partial correlation (r) between the years of meditation experience against the levels of mindfulness . For all the analysis, we present 95% confidence intervals and considered p <0.05 as significant . This was a cross - sectional study, where subjects (meditators) were recruited from s - vyasa yoga university and other universities (non - meditators) by convenience sampling . Maas is a 15-item self - reported single - factor scale that is exclusively focused on attention / awareness component of mindfulness construct . This instrument has been independently used to assess individuals either with or without meditation experience . This scale has been widely used for various studies and has reported positive overall quality ratings for most of the psychometric properties reviewed . Maas is a brief, easy to administer scale, and has therefore been used in wide range of studies related to assessing mindfulness trait . Maas is known to have good reliability ratings and a history of clinical and research use that was developed to assess the core attentional aspect of mindfulness, and the capacity for moment - to - moment attention in particular . The maas consists of 15 items that measure the level of mindfulness (example items are i could be experiencing some emotion and not be conscious of it until some time later, or i find it difficult to stay focused on what's happening in the present). The items are answered on a six - point scale (1 = almost always; 6 = almost never) on which higher scores are an indication of higher trait mindfulness . The maas has been validated in various samples of students (= 0.82) and adults from the general community (= 0.87). The questionnaire was scored by computing a mean of the 15 items in the questionnaire . The data were checked for normality and an independent samples t - test was employed to compare the means of both the groups . We also calculated the partial correlation (r) between the years of meditation experience against the levels of mindfulness . For all the analysis, we present 95% confidence intervals and considered p <0.05 as significant . Maas scores were significantly higher in meditators as compared to with the non - meditators (independent samples t - test, t = 10.391, p <0.001). The 95% confidence interval for the difference in the levels of mindfulness trait between meditators and non - meditators was (1.05, 1.55). We found a positive correlation (r = 0.620) between the years of meditation practice and the levels of trait mindfulness . Mean total scores of meditators and non - meditators on the mindfulness attention awareness scale . In the present study, we studied trait mindfulness and its correlation with duration of meditation practice using a maas . We found that meditators had higher levels of trait mindfulness and were positively correlated with the duration of meditation practice . While there are known differences between buddhist views of mindfulness and modern psychological adaptations, there is broad agreement that a clearly formulated mental training, usually referred to as meditation, the practice of cm involves physical postures (asanas), breath work, physical and mental awareness together leading to a state of meditation . According to patanjali, development of meditation (dhayana) is a process and takes a series of practices, which together are called ashtanga yoga the eightfold path to reach the highest state of consciousness . One reaches the state of mindfulness or meditation or antaranga yoga as a result of continued and consistent practice of the first six limbs of yoga . Our results are very much in accordance with patanajali's concept of the process of development of mindfulness and meditation . Another school of yoga, hatha yoga comprises practices of postures, breath work, and cleansing practices, all aimed at striking a balance between the body and the mind . Consistent practice of these hatha yoga techniques transforms the practitioner and establishes him in the state of mindfulness and meditation . The meditation technique practiced in the current study comprises all these components, which justifies the higher levels of mindfulness in the meditation group . Also, higher levels of trait mindfulness in cm practitioner can be accredited to the years of cm practice, which would have lead to the development of mindful trait in the meditators as signified by the positive correlation between level of mindfulness and the duration of meditation practice . The findings of the present study are in line with earlier studies on trait mindfulness in meditators . Highly experienced zen meditators showed similar trends where levels of mindfulness were found to have strong positive correlation to the years of meditation experience . The results of this study indicate that maas is sensitive to individual differences in levels of mindfulness and suggest that the higher scores among those consciously practicing this skill are due to such training . An 8-week mindfulness - based stress reduction (mbsr) program showed increase in the trait mindfulness of the participants, which mediate the effects of training on clinical outcomes . In a similar study, 8 weeks of yoga training resulted in significant increases in trait mindfulness of practitioners . Another study with cancer patients undergoing 8-weeks of mbsr, showed improved levels of mindfulness and lowered mood disturbances and symptoms of stress . A similar study like ours, comparing two different meditation techniques concluded that meditation improves the levels of mindfulness regardless of the meditation technique . Studies explaining the underlying mechanisms of development of mindfulness have been its stage of infancy . However, if mindfulness is considered to be a component of self - awareness and meditation, one of the studies reports the role of frontal control systems in neuroanatomical models of self - awareness . Several neuroimaging and electroencephalograpy (eeg)/event related potentials (erp) studies have shown changes in activation of prefrontal cortex (pfc) and the anterior cingulate cortex (acc), as well as significant increases in alpha and theta activity during meditation there is substantial evidence of changes in pfc during mindfulness meditation, which is known to be associated with attention, concentration, and emotion regulation . In another study, individuals with higher levels of mindfulness demonstrated less emotional reactivity in the midbrain (amygdala, dorsal acc), which is likely due to an enhanced ability to engage the pfc . Functional magnetic resonance imaging studies comparing experienced mindfulness meditators and novice controls have suggested increased neuronal activity in regions of the brain related to self - awareness (e.g., dorsolateral and medial pfc), particularly momentary self - awareness / self - reference . Majority of the studies on mindfulness meditation and other mindfulness training programs have demonstrated significant changes in the pfc . These findings show promise for the individual's ability to train the mind, changing not only emotional experiences, but also brain structure and functioning; moreover, the ability to do so appears to improve over time as experience with meditation increases . One of the limitations of our study is that the meditators participating in this study lived in a yoga institute and practiced other yoga techniques . Therefore, we are not sure if the development of higher levels of mindfulness is due to the meditation practice or an influence of other yoga practices . Further studies should be conducted on subjects practicing only cm and not adhering to any other yoga practices . Another limitation could be the small sample size . Given the huge number of yoga practitioners in today's date, studies with larger sample sizes are warranted . Consistent practice of moving meditation practices like cm can lead to development of higher levels of mindfulness . This may positively impact mental states and attention, which can in turn help psychological well - being of individuals . This furthers the scope for clinical trials with cm as an intervention in the management of psychological disorders.
The head and neck posture of an individual can influence soft - tissue relationships in the cervical and shoulder region1, 2 . A common concern in the modern workplace is upper extremity disorders arising from overhead work, which is associated with neck and shoulder disorders and pain3 . Long - term overhead working postures result in strain and fatigue of the shoulder muscles because arm elevation is associated with shoulder muscle fatigue4, 5 . Previous studies have focused on risk factor analysis and the development of therapeutic exercises for overhead work - related disorders rather than prevention6, 7 . Some studies have been performed on postural ergonomic interventions including working techniques for overhead work6, 7 . However, we found that few studies have focused on protective ergonomic devices for overhead workers . Therefore, this study investigated a new neck support tying (nst) method that used a thera - band for the prevention of neck and shoulder pain in workers performing overhead work . The new nst method supports the neck during hyperextension and prevents excessive upward rotation of the scapula during overhead work . The purpose of the present study was to investigate the effect of this nst method on cervical rom and shoulder pain after overhead work . The subjects were divided into two groups as follows: a control group consisting of 7 males without nst, and a nst group consisting of 7 males with nst . The initial cervical rom and initial ppts of the ut and mt were not significantly different between the two groups . The initial values of cervical flexion, extension, and right and left lateral flexion in the control group were 63.44.2, 72.86.0, 53.92.9, and 51.35.6 degrees, respectively . The initial values for cervical flexion, extension, and right and left lateral flexion in the nst group were 62.35.1, 72.53.9, 53.33.0, and 52.22.4 degrees, respectively . All participants gave their informed, written consent according to the protocol approved by the human ethics committee of the yonsei university faculty of health science . This study examined a new nst method that uses a thera - band for the prevention of neck and shoulder pain in workers performing overhead work . For the nst method, we used the grey thera - band (60 cm length) which was applied as follows . The midpoint of the thera - band supported the posterior aspect of the neck, and both ends of the thera - band were passed under both axillae, and tied behind the back . The nst provided support for neck hyperextension and prevented excessive upward scapular rotation during overhead work . Cervical flexion, extension, and right and left lateral flexion were measured with a cervical range of motion (crom) instrument (performance attainment associates, st . A dolorimeter pressure algometer (fabrication enterprises, white plains, ny, usa) was used to measure the pressure pain threshold (ppt) of the right side upper trapezius (ut) and the lower trapezius (lt) muscles . A 1-cm rubber plate delivers pressure from the probe to the body, and the pressure is read from a needle gauge . All subjects performed one trial of overhead work with their arms over their heads for 15 min . The overhead work was performed at a height of 25 cm above each subject's head . Differences in cervical rom and ppt between the nst and control groups after the overhead work were tested with the independent t - test using the spss statistical package (version 18.0; spss, chicago, il, usa). The cervical flexion, extension, and lateral flexion angles of the nst group were significantly larger than those of the control group (p <0.05). The cervical flexion, extension, and right and left lateral flexion of control group were 50.48.2, 64.711.3, 41.77.9, 43.29.2 degrees, respectively . The cervical flexion, extension, and right and left lateral flexion of nst group were 61.511.2, 69.46.9, 48.75.6, 49.86.7 degrees, respectively . The ppt of ut of the nst group (7.21.8 lb) was significantly higher than those of the control group (6.32.0 lb) (p <0.05). The ppt of mt of the nst group (5.81.4 lb) was significantly higher than those of the control group (5.01.2 lb) (p <0.05). Repeated and sustained working with elevated arms is known to lead to neck and shoulder pain8 . This study proposed a new neck support tying method using thera - band and investigated its effect on cervical rom and shoulder pain after overhead work . Reductions in rom have implications for the safety and efficiency of functional activities, and lead to a loss of corrective or protective reactions1, 9 . Rom losses can occur from inactivity and structural changes of the tissues in the cervical spine, and result in an increase in connective - tissue density, shortening of collagen tissue, and muscle fibrosis1, 9 . In this study, the cervical flexion, extension, and lateral flexion angles of the nst group shoulder forward flexion with scapular upward rotation requires the activation of the upper trapezius, and overstretches the middle trapezius through scapular protraction3, 5, 7 . The ppts of the ut and mt were significantly lower in the nst group than those of the control group . These results indicate that the nst supported the neck and prevented excessive scapular elevation and upward rotation during overhead work . The thera - band, which provides varied resistance through the range of movement, has been used for rehabilitation in combination with therapeutic exercise10 . It is light and portable, has low resistance, and can be adjusted to accommodate various situations11 . The nst method prevented rom reduction and pain in the cervical and shoulder regions . The nst method can be easily and simply applied using a thera - band and is also inexpensive . We suggest that industrial workers could use the nst method when performing overhead work.
Rates of diabetes mellitus are rising dramatically across the globe, threatening both individual health as well as the stability of national health systems . In the united states, diabetes is the leading cause of adult blindness, kidney failure, and non - traumatic amputations while also playing a central role in the development of cardiovascular disease, the leading killer of those with the disease . With estimates that diabetes currently affects nearly 24 million people in the us and that this number will rise to over 44 million individuals by 2034, the staggering $245 billion spent annually on diabetes - related healthcare costs is sure to rise dramatically . While these costs are unsustainable in the us where the healthcare infrastructure is robust and relatively well - funded, the burden of diabetes in the developing world may be catastrophic . Current projections estimate that 592 million individuals worldwide will have diabetes by 2035, with 77% of those individuals living in middle- or low - income countries with significantly less developed health systems . To prevent this threat from overwhelming health budgets across the globe, identification and elimination of the factors promoting the last decade has witnessed a proliferation of exciting epidemiological and basic science data suggesting that environmental contaminants play a pathogenic role in the development of metabolic disease (reviewed in refs . ). Indeed, while initially implicated in perturbations of sex steroid and thyroid hormone signaling, environmental endocrine disrupting chemicals (edcs) have now been shown to be associated with or to directly alter body weight and glucose homeostasis after either adult or developmental exposure (reviewed in refs . ). Such metabolic disruptors include a diverse array of compounds such as bisphenol a, persistent organic pollutants (pops), phthalates, antifouling agents, and pesticides . Our own recent work has added to this list a novel class of metabolism - perturbing agents, namely phenylsulfamide fungicides, as exemplified by tolylfluanid (tf). In this work, we ve shown that dietary exposure to tf has the capacity to promote weight gain and increase adiposity despite not altering total food intake; rather, these changes appear to occur through altered circadian rhythms of food intake . Moreover, adult mice exposed to tf exhibited glucose intolerance as well as systemic and adipose - specific insulin resistance, phenotypic features commonly seen in the pathogenesis of type 2 diabetes . This new evidence provides further support to the contention that environmental change may play a critical role in the emergence of chronic diseases; however, many questions remain regarding the clinical scenarios in which edcs exert their deleterious metabolic effects . Under most circumstances, homeostatic mechanisms maintain normal energy metabolism and resist the development of cardiometabolic disease; however, several factors operating alone or in concert can lower this resistance to metabolic disease (rmd) and accelerate the progression of obesity, diabetes, hypertension, dyslipidemia, and cardiovascular disease (fig . 1). While accidental or intentional ingestions of a single chemical may be sufficient to cause diabetes in select circumstances (e.g. The rodenticide vacor and the fungicide chlorothalonil), it is unlikely that a single chemical will be sufficient to explain the dramatic explosion in global diabetes rates . However, as there are tens of thousands of unique chemicals to which humans are potentially exposed, coordinate exposure to multiple edcs that additively antagonize critical pathways regulating energy metabolism through complimentary mechanisms may be sufficient to promote cardiometabolic dysfunction, whereas a single signaling disruptor in isolation may be insufficient to drive significant disease . These edc edc interactions may be critical for generating metabolic phenotypes from the chronic, low - dose exposures that characterize human contact with environmental toxicants . Unfortunately, the experimental complexities of analyzing mixtures of metabolic disruptors are immense; however, some recent data demonstrate that such mixtures, at environmentally relevant doses, can disrupt energy handling, suggesting that further studies into common co - exposures are warranted . Alternatively, the potency of a metabolic disruptor might be enhanced by permissive conditions in specific patients that may predispose those individuals to environmentally - mediated metabolic disease . Such factors may include underlying genetics, diet, lifestyle, preexisting diseases, pharmacological treatments, and states of fat redistribution that alter the patient s baseline physiology in such a way as to increase their sensitivity to metabolic disruption . Our recent work demonstrates that dietary exposure to tf increases adiposity, promotes glucose intolerance, and decreases insulin sensitivity both globally and at the level of the adipocyte . Like many metabolic investigations while we had previously shown that ex vivo exposure to tf induced adipocytic insulin resistance in outbred cd1 mice, inbred c57bl/6 mice, 2 strains of rats, and even human adipose tissue, whether the observed effects on global energy homeostasis are influenced (either positively or negatively) by the background genetics of the animal model is not known . In the field of endocrine disruption, this may be particularly relevant as the c57bl/6 strain is known to harbor a polymorphism in the aryl hydrocarbon receptor gene, a molecular target for many putative edcs, including dioxins and dioxin - like polychlorinated biphenyls (pcbs). Intriguingly, since a predominant phenotype of exposure to metabolic disruptors is an increase in adiposity, whether mice with a genetic predilection to accrete adipose tissue exhibit divergent metabolic consequences of edc exposure may suggest that underlying genetics modulate the metabolic risk posed by edcs . Importantly, because many edcs are hydrophobic, ascertaining whether sequestration in fat is potentially protective may shed new light on the mechanisms and metabolic consequences of adipose expansion under edc exposure . Finally, animal models that are resistant to edc - induced metabolic disruption may provide novel insights into detoxification or resistance pathways that may be exploited pharmacologically to treat or prevent edc - induced obesity and diabetes . Built upon and supporting the developmental origins of health and disease hypothesis (dohad), recent evidence demonstrates that exposure to various edcs during critical developmental windows can promote metabolic dysfunction in adulthood . The mechanisms by which remote exposures to edcs disrupt energy homeostasis and how these effects can be inherited in a multigenerational or transgenerational manner are not fully understood . Although epigenetic alterations are implicated, the molecular targets of these epigenetic modifications are imprecisely known . While genome - wide association studies have been generally disappointing with regard to identifying genetic polymorphisms that may explain type 2 diabetes, genes for which the data are strongest, e.g., tcf7l2, should be considered as potential epigenetic targets of edcs that induce metabolic disruption after developmental exposure . More intriguing may be genes implicated in the pathogenesis of neonatal diabetes and maturity onset diabetes of the young (mody). Mutations in genes implicated in these conditions are often inherited in an autosomal dominant fashion and elicit robust metabolic phenotypes, suggesting that edc - induced alterations in these genes or regions that regulate their expression may be sufficient to drive the onset of diabetes . Intriguingly, the link between mody genes and type 2 diabetes in larger cohorts has recently been established . Identifying such potential causes of developmentally - derived diabetes is particularly important since some patients with mody mutations who have historically been treated with insulin can be managed with oral agents (e.g., sulfonylureas), potentially resulting in both better control and reduced morbidity . Determining whether edcs may promote metabolic dysfunction through these pathways is vital as it may provide vital insights into the best approaches to treat patients with environmentally - mediated diabetes . It is clear that the deteriorating quality of our diet plays an important role in promoting the development of obesity and diabetes; moreover, the exportation of the american diet may be a significant contributor to the global plague of metabolic disease . Despite the importance of diet in modulating energy handling, the impact of these global shifts in diet composition on edc action has only recently been interrogated . To date, the interactions between edcs and dietary stressors in the disruption of energy homeostasis has largely been interrogated in the context of high fat feeding . Indeed, potentiation of metabolic disruption by a high fat diet has been demonstrated for bisphenol a, perfluorooctane sulfonate, pops, and chemical mixtures . Interestingly, our metabolic cage analyses suggest that mice exposed to tf have a preference for fat over carbohydrate utilization during the fed state . This suggests that diets rich in carbohydrates, or the simple sugars enriched in a western diet, may be particularly deleterious in the context of exposure to tf and potentially other edcs . This may be significant as the transformation of the american diet over the last 30 years has been one of increased carbohydrate content, particularly the refined grains and simple sugars found in processed foods . As increased fructose intake has been implicated in the explosion in diabetes rates, understanding how edcs interact with secular trends in dietary carbohydrate content and composition will be important for contextualizing the importance of edcs in the current metabolic disease epidemic . Furthermore, as the burden of diabetes spreads to low- and middle - income countries, understanding how edcs interact with specific dietary factors in these countries will be essential for estimating the risk posed by environmental toxicants as drivers of the metabolic disease epidemic in the developing world . An intriguing finding of our work on tf was that adiposity (fat mass as a fraction of total body mass) and weight gain were increased in the presence of this edc despite no change in total food intake . We were, however, able to discern an intriguing change in the circadian pattern of food intake, with tf - exposed mice consuming more food during the normally fasting light - phase . This evidence provides some of the first experimental support for edc - induced disruptions in energy homeostasis arising through perturbations in circadian rhythms . In humans, clinically, the timing of food intake has been shown to contribute to weight gain . Moreover, individuals who consume more calories at night may have a harder time losing weight . Second, do edcs augment the deleterious metabolic effects of disruptions in circadian patterns of food intake that are driven by lifestyle factors that are intentional (e.g., night eating) or forced (e.g., shift work)? And finally, if alterations in circadian rhythms are wholly or partially responsible for edc - induced obesity and diabetes, will restriction of food intake to the normal feeding period be an appropriate treatment approach? Studies examining the lifestyle factors that exacerbate or antagonize the deleterious effects of edcs on energy homeostasis may provide vital insights into both the mechanisms of edc - induced metabolic dysfunction as well as potential avenues to treat toxicant - mediated metabolic disease . Beyond influences of feeding behavior, understanding how edcs modulate central processes such as motivation may help explain difficulties patients have self - regulating food intake and sustaining exercise, as will studies of edc effects on skeletal muscle, which remains an understudied area of metabolic toxicity . Some of the early evidence suggesting edcs have the capacity to alter energy metabolism came from studies examining their ability to promote adipocyte differentiation from preadipocytes or mesenchymal stem cells . In many of these studies the 3t3-l1 cell line is used as a model of adipogenesis, with preadipoctye - to - adipocyte differentiation classically induced by exposure to insulin, a glucocorticoid, and an agent to raise camp levels . The capacity of various edcs to amplify adipose development can be studied by triggering adipogenesis with this differentiation cocktail in the presence or absence of the edc of interest . By this approach, many environmental contaminants have been shown to promote adipocyte differentiation (reviewed in ref . ). As we have recently dissected for the prototypical obesogen tributyltin, most edcs appear to require one or more components of the differentiation cocktail to induce adipogenesis . As such, it is possible that clinical states that modulate those specific pathways could augment the adipogenic capacity of edcs . For example, hyperinsulinemic states, as observed in prediabetes or early type 2 diabetes, may sensitize mesenchymal stem cells and preadipocytes to edc - induced adipocyte differentiation for chemicals requiring co - exposure to insulin to induce adipogenesis . Similarly, clinical states of high adrenergic tone that are predicted to raise intracellular camp levels may potentiate the action of some adipogenic edcs that require pre - activation of camp signaling . One example of this is obstructive sleep apnea (osa), a common disease linked to metabolic dysfunction . Likewise, edcs that require glucocorticoids to promote adipocyte development may exhibit enhanced adipogenic capacity in individuals with hyperactivation of the hypothalamic - pituitary - adrenal (hpa) axis who exhibit high glucocorticoid levels or enhanced glucocorticoid receptor (gr) signaling . Such clinical states include cushing s syndrome, pseudo - cushing s syndrome, osa, or even endogenous obesity . We demonstrated that tf activates adipocytic gr signaling both ex vivo and in vivo, suggesting one mechanism by which this novel endocrine disruptor promotes metabolic dysfunction . It remains to be determined whether clinical states of heightened gr signaling potentiate tf action . It is interesting, however, to speculate that other edcs that inhibit the enzymes responsible for glucocorticoid inactivation, 11-hydroxysteroid dehydrogenases, might exhibit enhanced metabolism - disrupting potency in clinical states of glucocorticoid excess (e.g., dithiocarbamates and organotins). Whether an individual s underlying disease state renders them more susceptible to edc - mediated metabolic disruption has not been studied, but knowledge of the mechanisms by which these environmental toxicants disrupt nutrient handling and the development of metabolic tissues may suggest that, under certain clinical scenarios, some individuals may be primed for environmentally - mediated alterations in energy metabolism . Reciprocal to the concept that underlying diseases might augment an individual s sensitivity to the deleterious metabolic effects of an edc, disease treatments themselves may generate a permissive environment under which edcs induce metabolic dysfunction . As argued for states of endogenous corticosteroid overproduction, pharmacological treatment with glucocorticoids, as employed in the care of individuals with inflammatory diseases, may potentiate the action of edcs working through or in conjunction with gr signaling . Similarly, treatment with adrenergic agonists that raise camp levels may prime preadipocytes for differentiation upon exposure to some edcs . For example, standard treatment approaches to asthma, a highly prevalent lung disease, with 2-adrenergic agonists such as albuterol, may generate a permissive environment for edc - induced adipogenesis . As our knowledge of the mechanisms by which edcs exert deleterious metabolic effects expands, we will be better equipped to predict how various disease states and their treatments may render a subset of patients particularly sensitive to environmental contaminants modulating those pathways . Conversely, knowledge of the mechanisms by which edcs induce metabolic disease may suggest therapeutic avenues to treat environmentally - mediated obesity and diabetes or identify individuals whose exposure to certain edcs should be aggressively limited . Finally, future studies may take advantage of the known mechanisms by which anti - diabetic medications lower blood sugar (e.g., sulfonylurea receptor, peroxisome proliferator - activated receptor-, amp - activated protein kinase, sodium - glucose co - transporter-2, incretin receptors, etc .) To: a.) Unravel the molecular mechanisms of metabolic disruption; b.) Identify potential edc - drug antagonism that limit therapeutic efficacy; and c.) develop molecularly - based approaches to treat edc - mediated diabetes in a new era of precision environmental medicine . A core therapy for myriad metabolic diseases is weight loss . Whether achieved through dietary interventions, exercise, anti - obesity drugs, or surgery, reductions in body weight have multiple salutary effects on energy metabolism and overall health, effectively raising rmd in the disease progression model (fig . 1). However, because adipose tissue serves as a reservoir for lipophilic edcs, mobilization of fat with weight loss is accompanied by a repartitioning of these chemicals from adipose tissue into the circulating plasma compartment . This has been shown in several human studies examining caloric restriction with or without a weight loss drug, as well as for patients undergoing bariatric surgery . The precise impact of this release on global energy homeostasis is poorly characterized, but evidence suggests that the subsequent rise in serum organochlorine levels is associated with changes in muscle metabolism that suggest an impairment in energy handling . A similar study also showed an inverse association between the extent of pollutant liberation and insulin levels . It is interesting to speculate that this reduction in insulin levels could be compensation for edc - induced impairments in hepatic gluconeogenesis as we have shown for dioxin - like pcbs . Importantly, reductions in total fat mass have also been shown to concentrate pollutant levels in adipose tissue, suggesting that adipocyte physiology may also be deleteriously affected by effective increases in edc concentrations in this important metabolic tissue induced through weight loss . Whether this repartitioning of putative metabolic disruptors partially antagonizes further weight loss or its metabolic benefits requires further investigation, as does the hypothesis that adipose expansion itself protects against edc - induced metabolic dysfunction through fat sequestration that limits edc action on non - adipose tissues . Finally, the impact of clinical states of altered adipose development, e.g., congenital and acquired lipodystrophies, on an individual s sensitivity to environmental contaminants necessitates interrogation as individuals with these diseases may be especially vulnerable to edc - induced metabolic dysfunction due to a reduced capacity to safely sequester lipophilic pollutants . An analogous clinical state of edc repartitioning that results from energy shifts and promotes systemic pollutant release is lactation . Many studies from diverse geographical regions have demonstrated the presence of various environmental contaminants in breast milk, including pops such as pcbs and organochlorine pesticides . Importantly, while edc elimination through lactation may be an important mode by which the total body burden of pollutants is reduced in the mother, the subsequent exposure of the developing newborn to these edcs during this critical developmental period may be especially deleterious for the long - term metabolic health of the child as suggested by the dohad hypothesis . Interestingly, while the metabolism - disrupting potency of edcs to which an individual is exposed in adulthood is likely enhanced by that individual s clinical status (e.g. Lifestyle factors, underlying diseases, and medications), it is also possible that early life edc exposures potentiate the adverse metabolic consequences of those same clinical factors later in life . Improved understanding of the impact of exposure to edcs through breast milk on later life energy homeostasis is of critical importance for both predicting risk and developing novel treatment strategies to address pollutant - induced metabolic dysfunction . Our recent work demonstrating that dietary exposure to the phenylsulfamide fungicide tf promotes weight gain, adipose accretion, and glucose intolerance as well as systemic and cellular insulin resistance provides further support to the theory that environmental toxicants likely contribute to the current global epidemic of metabolic disease . While exposure to certain edcs has been shown to be sufficient to initiate the development of diabetes, this occurs rarely . It is more likely that the contribution of edcs to the current epidemic of obesity and diabetes results from coordinate exposures to multiple edcs, each affecting the same or complimentary signaling pathways that regulate energy handling . Alternatively, edc - mediated metabolic dysfunction may be enhanced or accelerated by patient - specific parameters that alter an individual s sensitivity to metabolic disruption, including underlying genetics, diet, lifestyle factors, preexisting diseases, pharmaceuticals, and clinical states of fat redistribution . More comprehensive understanding of the complex interplay between these patient - specific variables and edc action will be essential for determining the contribution of environmental pollutants to the current epidemic of metabolic disease and for devising strategies to address the threat of environmental degradation on human metabolic health.
In this issue of critical care, laporta and coworkers review a multidisciplinary working group's analysis of a case study on the causes of and solutions for staff turnover in an intensive care unit (icu) setting . This issue is of profound significance to health care leaders in western countries because the workforce is shrinking as a result of impending baby boomer retirements and, as the population ages, the demand for intensive care services will grow considerably . These demographic factors are further compounded by the fact that the complexity of care provided in the icu demands professionals who are highly trained and skilled . In this environment, turnover can be costly to the organization because of the significant expenses associated with recruiting and training workers . There are many well documented reasons for staff turnover in the intensive care setting that are highlighted by laporta and coworkers as core reasons . These core reasons include job dissatisfaction due to inflexible scheduling practises, insufficient opportunity for professional development, as well as a lack of collaborative decision making around clinical and practice issues . The authors discuss that data on icu turnover comes from nursing literature and that this research may be applicable to other health care professionals . However, it is important not to assume that reasons for turnover are the same among different groups of health care providers and that staff turnover is something to be avoided at all costs . For example, misra - hebert and coworkers state that one contributor to physician turnover is conflict between the physician's and organization's philosophy and goals . Physician turnover in this case may be beneficial both to the physician and organization if the two parties cannot reconcile their differences and the conflict impacts on the ability of both parties to move forward . There are other important reasons for turnover that should be considered by icu leaders, and these include burnout and generational diversity . Burnout is a prevalent phenomenon in icus, and the nursing literature suggests that issues such as moral distress when engaging in futile care contributes to burnout . In the medical literature causes of physician burnout include volume of work, increased expectations of the public, lack of sleep and the possibility of being sued . The consequence of burnout is that there is a negative impact on quality of care and staff morale, which can ultimately cause turnover . For example, gunderson indicates that physicians who are dissatisfied may engage in inappropriate prescribing patterns . Neuhauser, furthermore, discusses how environments with rigid systems and attitudes among the leadership will decrease staff morale because staff desire flexible policies and autonomy in decision - making . The generational diversity found in the icu environment can also be a source of turnover of staff . It is well documented that generation x (born in 19651980) and the millennial generation (born in 19802000) have a strong desire for more balanced work life than veterans (born in 19251945) and baby boomers (born in 19461964). Research conducted by lorin and coworkers on internal medicine residents of the millenial generation showed that although 41% considered a fellowship in critical care, only 3.4% chose this training because of lack of leisure time and stress levels among faculty and fellows . Clearly, it is important for leaders to be attuned to these generational differences when developing recruitment and retention plans and redesigning the workplace environment . The review from laporta and coworkers also highlights the importance of icu leadership working with frontline staff to create a vision and strategy that addresses the core reasons for turnover . It is essential that this vision be aligned with the vision, mission and values, and strategic plan of the health care organization . Furthermore, the team should assess whether their hospital is highly reputable, has high patient satisfaction, and sufficient resources and equipment to provide care . All of these components are signs of a positive work environment, and leadership can build on these attributes to recruit and retain staff . The other key factor in this process is the use of a team work approach . Team work training in the areas of conflict resolution, learning styles and giving feedback will help the staff to work together to create and achieve an inspiring vision . Although the financial and human resource investments required to engage in this process are considerable, there is substantial evidence in the literature that highly functioning, satisfied teams lead to more efficient patient care and better outcomes . Staff turnover is a critical issue that icu leaders need to understand and address in their unit settings . Attention to this issue with a systematic, evidence - based approach that focuses on team work and collaboration will not only improve retention but will also make the icu a highly competitive and desirable place to work.
Due to the widespread use of imaging modalities, such as ultrasonography, computed tomography (ct), and magnetic resonance imaging, the incidences of incidentally found small cortical renal masses (srms) and renal cell carcinoma (rcc), have increased during the past years . For decades, the standard therapy for patients with clinically suspected rcc consisted of radical nephrectomy, an invasive surgical procedure with high morbidity . However, in a recently published randomized trial of nephron - sparing surgery (nss) in patients with srm yielded comparable oncological outcome with radical nephrectomy . In addition, population - based studies clearly demonstrate an overall survival benefit in patients undergoing nss as a result of preserved renal function [4, 5]. Nephron - preserving procedures, such as partial nephrectomy and image - guided minimally invasive ablative procedures, have therefore increasingly been applied in patients with srm [6, 7]. Initially, image - guided ablative procedures were performed in patients who were not suitable candidates for nss based on significant medical comorbidity, advanced symptomatic disease, or refusal of conventional therapy [6, 8]. Accumulating data on follow - up and oncological safety suggest a broader indication in patients with srm . A particular form of an image - guided ablative procedure is radiofrequency ablation (rfa), which can be performed open or percutaneously . In rfa, an electric current oscillates through an electrode placed centrally in the target tissue . This results in frictional ionic agitation and heat formation in the tissue surrounding the tip of the electrode, causing local protein coagulation and cellular death . Compared with open and laparoscopic (partial) nephrectomy it is a nephron - sparing therapy with low morbidity and mortality, short hospital stay, and acceptable oncologic outcome [8, 10, 12]. Nevertheless, rfa of the kidney can be accompanied by minor and even major complications . Several investigators have postulated the occurrence of bowel perforation as a complication of rfa of renal masses due to the close proximity of bowel [13, 14]. To our current knowledge, only two articles have described such a case [15, 16]. Yet in a large series of 100 percutaneously performed renal rfas, none of the patients had colonic injuries . The reported incidence of bowel perforation complicating renal rfa therefore ranges from 0 to 8.3% [8, 15]. In this article, we describe the first case of appendiceal perforation as a complication of ct - guided percutaneous rfa of an smr . A 60-year - old male patient was referred to our outpatient clinic with an incidental mass in the right kidney, which was recently diagnosed during work - up of his microscopic hematuria . His previous medical history consisted of kidney stone lithotripsy, hypertension treated with a beta - blocker and diuretic, and two episodes of transient ischemic attack . Abdominal ct scan showed a rapidly enhancing, exophytic mass in the lower pole of the right kidney with a maximum diameter of 2.5 cm (fig . The appendix was noticed in a retrocecal position, at a 1.4-cm distance from the renal mass (fig . 1b, c). Based on his mild comorbidity and on the small size of the renal mass, 1a small, exophytic, rapidly enhancing renal mass at the lower pole of the right kidney . B retrocecal position of the appendix on the lateral site of the right kidney in the vicinity of the renal mass (c). * appendix, renal mass a small, exophytic, rapidly enhancing renal mass at the lower pole of the right kidney . B retrocecal position of the appendix on the lateral site of the right kidney in the vicinity of the renal mass (c). * appendix, renal mass our technique of rfa in renal masses has extensively been described in previous articles [17, 18]. In short, after the patient received an antibiotic prophylaxis (1,500 mg cefuroxim) and epidural analgesic before the rfa procedure, he was placed in prone position on the ct table . A planning ct scan was performed to locate the renal mass . Under fluoroscopic ct guidance, a 17 g cool - tip electrode (valleylab, covidien, boulder, co) was placed centrally into the mass . Subsequently a 20 g needle was inserted lateral in the anterior pararenal space for injection of dextrose in water to hydrodissect the renal mass from the surrounding vital tissues, such as the colon and appendix . After the hydrodissection and the positions of the needles were checked with a ct scan of the area of interest, ablation was started . Final temperature after 15 min was> 75c with adequate roll - offs . The rfa procedure was performed by a highly experienced interventional radiologist (w.p .) Who at that time had already performed> 180 percutaneous image - guided ablative procedures (including renal, liver, and lung). A 60-year - old male patient was referred to our outpatient clinic with an incidental mass in the right kidney, which was recently diagnosed during work - up of his microscopic hematuria . His previous medical history consisted of kidney stone lithotripsy, hypertension treated with a beta - blocker and diuretic, and two episodes of transient ischemic attack . Abdominal ct scan showed a rapidly enhancing, exophytic mass in the lower pole of the right kidney with a maximum diameter of 2.5 cm (fig . The appendix was noticed in a retrocecal position, at a 1.4-cm distance from the renal mass (fig . 1b, c). Based on his mild comorbidity and on the small size of the renal mass, 1a small, exophytic, rapidly enhancing renal mass at the lower pole of the right kidney . B retrocecal position of the appendix on the lateral site of the right kidney in the vicinity of the renal mass (c). * appendix, renal mass a small, exophytic, rapidly enhancing renal mass at the lower pole of the right kidney . B retrocecal position of the appendix on the lateral site of the right kidney in the vicinity of the renal mass (c). Our technique of rfa in renal masses has extensively been described in previous articles [17, 18]. In short, after the patient received an antibiotic prophylaxis (1,500 mg cefuroxim) and epidural analgesic before the rfa procedure, he was placed in prone position on the ct table . A planning ct scan was performed to locate the renal mass . Under fluoroscopic ct guidance, a 17 g cool - tip electrode (valleylab, covidien, boulder, co) was placed centrally into the mass . Subsequently a 20 g needle was inserted lateral in the anterior pararenal space for injection of dextrose in water to hydrodissect the renal mass from the surrounding vital tissues, such as the colon and appendix . After the hydrodissection and the positions of the needles were checked with a ct scan of the area of interest, ablation was started . Final temperature after 15 min was> 75c with adequate roll - offs . The rfa procedure was performed by a highly experienced interventional radiologist (w.p .) Who at that time had already performed> 180 percutaneous image - guided ablative procedures (including renal, liver, and lung). On the ct images performed during the procedure, the colon and appendix were considered to be a safe distance (at least 1.0 cm) from the ablative field as a result of the hydrodissection (fig . 2). Adequate ablation of the kidney tumor was achieved without intraprocedural complications . On the first postprocedural day fluid collection with air configuration on the lateral and anterior side of the renal mass as a result of hydrodissection can be seen . Retrocecal appendix () is in the vicinity of the ablative field patient in left lateral decubitus position during rfa procedure . Fluid collection with air configuration on the lateral and anterior side of the renal mass as a result of hydrodissection can be seen . Retrocecal appendix () is in the vicinity of the ablative field five days after the procedure, he presented at our hospital with fever (39.5c) and right lumbar pain . 3) showed a large retroperitoneal fluid collection with air configurations, suggesting retroperitoneal abscess formation, on the lateral side of the right kidney . Moreover, a direct connection was noticed between the cecum and fluid collection, with contrast material in the retroperitoneal abscess, suggesting perforation at the base of the appendix (fig . 3). After ct - guided drainage of the abscess and intravenous antibiotic therapy, the patient remained septic . Therefore, 2 days later laparotomy was performed . Intraoperatively, a retroperitoneally confined abscess was drained . However, due to an extensive local inflammatory reaction affecting the terminal ileum (fig . 4), the approach had to be extended intra - abdominally to allow necessary resection of the ileocecal region followed by primary anastomosis between the ileum and ascending colon and an omental plasty . During this step, histopathologic examination of the resected specimen showed a perforated appendix based on ulcero - phlegmonous and gangrenous inflammation . On day 18 after the rfa 3leakage of contrast material from the perforated cecum / appendiceal base into the right anterior pararenal space suggesting appendiceal perforation . Presence of air configurations in the right anterior pararenal space suggesting abscess formationfig . Oversewn cecal perforation at the base of the necrotic appendix leakage of contrast material from the perforated cecum / appendiceal base into the right anterior pararenal space suggesting appendiceal perforation . Presence of air configurations in the right anterior pararenal space suggesting abscess formation intraoperative view after abscess drainage . Rfa of srm was first applied in 1997 and has proven to be a promising and safe technique since . In a large series of 100 patients with renal tumors treated with rfa, 11 minor and major complications were reported, of which the most common was haemorrhage . . Complications of rfa can generally be divided into two categories: (1) those related to imaging - guided electrode placement and (2) those related to thermal therapy . The latter are more common in kidney rfa compared with other rfa indications, e.g., hepatic rfa, as a result of the proximity of other vital structures, such as the bowel and ureter . Nevertheless, only two articles so far have reported bowel perforation as a complication of renal rfa [15, 16]. The first step in preventing thermal complications is thorough assessment of the tumor location on preprocedural ct scans during the process of patient selection . Percutaneous renal rfa can be performed with the patient prone or in lateral decubitus position . In both positions, vital structures in the vicinity of the target mass will be kept away from the ablative zone by way of gravity . Third, the rfa electrodes can be used to lift the ablated tumor away from vital structures . Examples of invasive methods include hydrodissection with glucose in water or injection of carbon dioxide in between the target tissue and the tissue that needs protection . In our patient, a lateral dissection was performed with glucose in water to dissect the renal mass from the appendix and the colon, which was located caudolateral with respect to the renal mass . Unfortunately, in this way the appendix came even closer to the tract of the needle . Nevertheless, on the ct images performed during the procedure, the appendix was considered to be at sufficient distance from the ablative field . Eventually, this caused the appendiceal perforation . Since this complication, we modified our ablative technique . Currently we start the hydrodissection before placement of the rfa electrode in the target tissue . In addition, instead of injecting 100 cm fluid during hydrodissection, we attach the needle to a continuous drip system . In conclusion, in this article we described a case of appendiceal perforation leading to retroperitoneal abscess formation as a complication of percutaneous rfa of an srm . Although rfa of srm is generally a minimally invasive and safe procedure, one should be aware of the possibility of particular minor and major complications when performing this innovative and promising procedure . If vital structures remain in close vicinity of the ablative field, one should consider treatment options other than rfa.
Have all been reported to affect bond strength,3,4 owing to differences in the degree of conversion, contraction stress and physical properties of the materials selected.57 in addition, the type of bur chosen might affect both the etching effect and the penetration of resin monomer since the roughness of the bur influences smear layer thickness.8 it has also been reported that certain environmental conditions, for example, increased temperature and humidity in the oral cavity, significantly reduces the bond strength.9,10 contamination is also a well - known and important factor affecting bonding performance; in particular, contamination with blood, saliva or gingival crevicular fluid significantly reduces the bond strength1113 due to the inhibition of monomer diffusion, and therefore requires the application of isolation techniques, such as the use of a rubber dam, in the bonding procedure . The routine use of maintenance spray for prolonging the superior performance of dental cutting handpieces is also of importance when considering sources of contamination . Maintenance spray must be used before each autoclaving or chemi - claving, and recently almost all dental offices sterilize the handpieces used with patients by autoclaving or chemi - claving for infection control . Immediately after spraying, the handpiece is briefly operated for several minutes to remove excess spray; however, it has been reported that this usual practice of removing excess spray is ineffective for preventing surface contamination.14 some studies have evaluated the influence of maintenance spray on resin bonding to enamel, and almost of those indicated that contamination of maintenance spray had little effect on bonding.1519 on the other hand, the contamination of maintenance spray to dentin has been some reported to affect the lower bond strength.19 however, the reports have been equivocal,20 and further studies should be needed . The purpose of this study was, therefore, to investigate the influence of contamination with two different types of maintenance sprays on the microtensile bond strength (tbs) of dentin bonded with a 2-step self - etching adhesive system . The null hypothesis tested was that contamination with maintenance spray does not influence the tbs of the bonded dentin . Nine caries - free extracted human molars stored in 0.5% chloramine t solution at 4c was used for tbs study . The teeth were trimmed using a model trimmer (mt-7, j morita tokyo mfg . Tokyo, japan) in order to form a long, flat dentin surface at the mid - crown level . The flat dentin surface was then polished with #600 silicon carbide paper to create a standard smear layer . These specimens were then randomly divided to one of the following three groups, with three teeth in each group: oil - free spray group: dentin surface contaminated with an oil - free maintenance spray for air bearing handpieces (astron cleaner, j. morita mfg . Tokyo, japan) for approximately 1 s at a distance of 23 cm, rinsed with water spray for 30 s, and then air - dried sufficiently . Oil - containing spray group: dentin surface contaminated with an oil - containing maintenance spray for ball bearing handpieces (intra spray, j morita mfg . Corp .) For approximately 1 s at a distance of 23 cm, rinsed with water spray for 30 s, and then air - dried sufficiently . Control group: dentin surface was immediately rinsed with water spray for 30 s and then air - dried sufficiently . All specimens were then treated with a self - etching priming adhesive system (clearfil se bond, kuraray medical, tokyo, japan; also known as clearfil megabond in japan) according to the manufacturer s instructions . The self - etching primer was applied with a three - way syringe to the surfaces for 20 s prior to drying . Bonding agent was then applied to the surface and polymerized by quartz - tungsten - halogen light curing unit for 10 s (new light vl - ii, gc, tokyo, japan). After applying the bonding agent to each specimen, resin composite (clearfil ap - x, shade a2, kuraray medical) was built - up incrementally (in five steps) to a height of 5 mm . Each increment was light - cured for 20 s (new light vl - ii), and the specimens were then stored in distilled water for 24 h at 37c . After storage, each bonded specimen was sectioned into four or five slabs, approximately 0.7-mm thick, perpendicular to the bonded surface using a low - speed diamond saw (isomet, buehler, lake bluff, il, usa) under water cooling . The slabs were trimmed using a superfine - grit diamond bur (sf #114, shofu, kyoto, japan) to an hourglass shape to form a gentle curve along the adhesive interface from both sides, as described by sano et al.21 the width at the narrowest portion was approximately 1.4 mm, and the thickness of the bonded area of each specimen was verified by a digital micrometer (mitutoyo, tokyo, japan). The specimens were then attached to a bencor multi - t testing apparatus (danville engineering co, san ramen, ca, usa) with cyanoacrylate adhesive (model repair ii blue, dentsply - sankin, ohtawara, japan) connected to a universal testing machine (tensilon rtc-1150-tsd, orientec, tokyo, japan). The specimens were then subjected to tbs testing at a crosshead speed of 1 mm / min until failure occurred . The tensile bond strength was calculated as the load at failure (n) divided by the bonded area (mm). Bond strength data were analyzed by one - way anova and the tukey - kramer test . Statistical analysis was performed using a commercially available statistical package (statview 5.0j, sas institute, cary, nc, usa). To determine the mode of failure, both the dentin and composite halves of all fractured specimens were visually inspected under a light microscope (ms-803, moritex, tokyo, japan) at 210 magnification and further observed using a field - emission scanning electron microscope (fe - sem; jsm-6340f, jeol, tokyo, japan) at 15 kv, under the magnifications of 75 to classify the failure mode of each specimen, and 1000 to observe the details of peculiar images . Failure modes were classified as cohesive failure of resin, failure of the adhesive interface (fracture between the dentin or the hybrid layer and the overlying adhesive in the same sample), mixed resin and adhesive (r&a) failure (interfacial and partial cohesive failure of the adhesive only or cohesive failure in the same sample), mixed that included the dentin (failure within the dentin only or mixed failure that included the dentin) or cohesive failure of dentin, wherever relevant . Bonded samples prepared by same procedure as for tbs testing were ground with increasingly finer silicon carbide paper and highly polished with a slurry solution of aluminum polishing suspension (refine tec, co., yokohama, japan) (1 m, 0.3 m, 0.05 m). The samples were then subjected to 32% phosphoric acid (uni - etch, bisco, schaumburg, il, usa) treatment for 30 s and rinsed with tap water for 30 s. the specimens were further treated with 1% sodium hypochlorite solution (wako pure chemical, osaka, japan) for 10 min . All specimens were subsequently dehydrated in ascending grades of ethanol (50%, 70%, 80%, 90%, 95%, 99%, and 99.9%) for 10 min each, and were further desiccated in a box with silica gel for 24 h. the dried specimens were placed on an aluminum stub and sputter - coated with au - pd using a cool sputter coater (sc500a, vg microtech, east sussex, uk). The coated specimens were examined using the fe - sem at 15 kv, under the magnification of 4000. nine caries - free extracted human molars stored in 0.5% chloramine t solution at 4c was used for tbs study . The teeth were trimmed using a model trimmer (mt-7, j morita tokyo mfg . Tokyo, japan) in order to form a long, flat dentin surface at the mid - crown level . The flat dentin surface was then polished with #600 silicon carbide paper to create a standard smear layer . These specimens were then randomly divided to one of the following three groups, with three teeth in each group: oil - free spray group: dentin surface contaminated with an oil - free maintenance spray for air bearing handpieces (astron cleaner, j. morita mfg . Tokyo, japan) for approximately 1 s at a distance of 23 cm, rinsed with water spray for 30 s, and then air - dried sufficiently . Oil - containing spray group: dentin surface contaminated with an oil - containing maintenance spray for ball bearing handpieces (intra spray, j morita mfg . Corp .) For approximately 1 s at a distance of 23 cm, rinsed with water spray for 30 s, and then air - dried sufficiently . Control group: dentin surface was immediately rinsed with water spray for 30 s and then air - dried sufficiently . All specimens were then treated with a self - etching priming adhesive system (clearfil se bond, kuraray medical, tokyo, japan; also known as clearfil megabond in japan) according to the manufacturer s instructions . The self - etching primer was applied with a three - way syringe to the surfaces for 20 s prior to drying . Bonding agent was then applied to the surface and polymerized by quartz - tungsten - halogen light curing unit for 10 s (new light vl - ii, gc, tokyo, japan). After applying the bonding agent to each specimen, resin composite (clearfil ap - x, shade a2, kuraray medical) was built - up incrementally (in five steps) to a height of 5 mm . Each increment was light - cured for 20 s (new light vl - ii), and the specimens were then stored in distilled water for 24 h at 37c . After storage, each bonded specimen was sectioned into four or five slabs, approximately 0.7-mm thick, perpendicular to the bonded surface using a low - speed diamond saw (isomet, buehler, lake bluff, il, usa) under water cooling . The slabs were trimmed using a superfine - grit diamond bur (sf #114, shofu, kyoto, japan) to an hourglass shape to form a gentle curve along the adhesive interface from both sides, as described by sano et al.21 the width at the narrowest portion was approximately 1.4 mm, and the thickness of the bonded area of each specimen was verified by a digital micrometer (mitutoyo, tokyo, japan). The specimens were then attached to a bencor multi - t testing apparatus (danville engineering co, san ramen, ca, usa) with cyanoacrylate adhesive (model repair ii blue, dentsply - sankin, ohtawara, japan) connected to a universal testing machine (tensilon rtc-1150-tsd, orientec, tokyo, japan). The specimens were then subjected to tbs testing at a crosshead speed of 1 mm / min until failure occurred . The tensile bond strength was calculated as the load at failure (n) divided by the bonded area (mm). Bond strength data were analyzed by one - way anova and the tukey - kramer test . Statistical analysis was performed using a commercially available statistical package (statview 5.0j, sas institute, cary, nc, usa). To determine the mode of failure, both the dentin and composite halves of all fractured specimens were visually inspected under a light microscope (ms-803, moritex, tokyo, japan) at 210 magnification and further observed using a field - emission scanning electron microscope (fe - sem; jsm-6340f, jeol, tokyo, japan) at 15 kv, under the magnifications of 75 to classify the failure mode of each specimen, and 1000 to observe the details of peculiar images . Failure modes were classified as cohesive failure of resin, failure of the adhesive interface (fracture between the dentin or the hybrid layer and the overlying adhesive in the same sample), mixed resin and adhesive (r&a) failure (interfacial and partial cohesive failure of the adhesive only or cohesive failure in the same sample), mixed that included the dentin (failure within the dentin only or mixed failure that included the dentin) or cohesive failure of dentin, wherever relevant . To determine the mode of failure, both the dentin and composite halves of all fractured specimens were visually inspected under a light microscope (ms-803, moritex, tokyo, japan) at 210 magnification and further observed using a field - emission scanning electron microscope (fe - sem; jsm-6340f, jeol, tokyo, japan) at 15 kv, under the magnifications of 75 to classify the failure mode of each specimen, and 1000 to observe the details of peculiar images . Failure modes were classified as cohesive failure of resin, failure of the adhesive interface (fracture between the dentin or the hybrid layer and the overlying adhesive in the same sample), mixed resin and adhesive (r&a) failure (interfacial and partial cohesive failure of the adhesive only or cohesive failure in the same sample), mixed that included the dentin (failure within the dentin only or mixed failure that included the dentin) or cohesive failure of dentin, wherever relevant . Three human molars were used . Bonded samples prepared by same procedure as for tbs testing were ground with increasingly finer silicon carbide paper and highly polished with a slurry solution of aluminum polishing suspension (refine tec, co., yokohama, japan) (1 m, 0.3 m, 0.05 m). The samples were then subjected to 32% phosphoric acid (uni - etch, bisco, schaumburg, il, usa) treatment for 30 s and rinsed with tap water for 30 s. the specimens were further treated with 1% sodium hypochlorite solution (wako pure chemical, osaka, japan) for 10 min . All specimens were subsequently dehydrated in ascending grades of ethanol (50%, 70%, 80%, 90%, 95%, 99%, and 99.9%) for 10 min each, and were further desiccated in a box with silica gel for 24 h. the dried specimens were placed on an aluminum stub and sputter - coated with au - pd using a cool sputter coater (sc500a, vg microtech, east sussex, uk). The coated specimens were examined using the fe - sem at 15 kv, under the magnification of 4000. mean and standard deviation (sd) tbs for the specimens of all three tested groups are summarized in table 1 . The non - sprayed control showed significantly higher tbs than the two sprayed groups (p <0.05). There was no significant difference between the two sprayed groups (oil - free spray (n = 14) and oil - containing spray (n = 15)) (p> 0.05). Representative fe - sem micrographs of fractured specimens after the tbs testing are shown in figures 2a, 3a and 4a, and distribution of the failure mode is summarized in figure 5 . Most commonly, a mixture of cohesive failure of the resin and failure of the adhesive interface / hybrid layer (r&a failure) was observed in each group . Failure in the adhesive interface was observed only in the two sprayed groups and not in the control group . The percentage of mixed failure that included the dentin was higher in the control group than in the two sprayed groups . Fe - sem micrographs of the cross - sectioned resin - dentin interfaces in each group are shown in figures 2b, 3b and 4b . Resin tags were evident in all three groups, with no significant difference among the groups . The purpose of this study was to investigate the influence of contamination with two different types of maintenance sprays on the microtensile bond strength (tbs) of dentin bonded with a 2-step self - etching adhesive system, clearfil se bond . Some of the previous studies applied the combined spray of lubricant and water running through the handpiece20 in order to simulate the clinical situation . It has been reported that the spray contents was discharged up to at least 240 min, but the amount of discharge was gradually reduced.14 their results suggested that uniform discharging of spray contents into entire the dentin surface might be difficult . In this study, therefore, the spray was applied directly in order to contaminate the dentin surface, referred to rosa et al and matos et al.18,19 powers et al15 and knight et al17 evaluated the handpiece lubrication on bond strength of enamel using two multi - step etch and rinse adhesive systems (all - bond 2, bisco; opti - bond fl, kerr; and gluma 2000, heraeus kulzer), and they found that the significant difference between the mean bond strengths for the group prepared with a sterilized unlubri - cated handpiece and the group prepared with a lubricated handpiece . However, other studies which evaluate the bondstrengths of oil - contaminated enamel with multi - step etch and rinse adhesives stated that contamination had little effect on bond strength.15,18 rosa et al assumed that etch and rinse adhesive had little effect of oil contamination, because the etchant was efficient in removing much of the oil.18 it has also been some reported about the influence of handpiece lubrication on bond strength, but the results have been equivocal.15,16,19,20 roberts et al investigated using a 2-step etch and rinse adhesive (single bond, 3 m espe), a 2-step self - etch adhesive (clearfil se bond), and a 1-step self - etch adhesive (one - up bond f, tokuyama dental), and resulted that there were no significant differences in dentin bond strength between the non - contaminated control and the spray - contaminated groups regardless of the type of handpiece or use of routine lubrication in each adhesive system.20 on the other hand, matos et al19 reported that the bond strength of clearfil protect bond (kuraray medical), a 2-step self - etching adhesive system which improved on clearfil se bond22 to dentin was lower more than half compared with a non - contaminated group . Our study also revealed that contamination of maintenance spray significantly affected to reduce the tbs of bonded dentin . Differ to etch and rinse adhesive, it is not needed the water spraying before applying self - etch adhesive . Therefore, the adverse effect of maintenance spray on self - etch adhesive might be larger than that on etch and rinse adhesive . In the results of this study, we suggested that the null hypothesis tested in this study that contamination with maintenance sprays does not influence the tbs of dentin bonded with 2-step self - etch adhesive can be rejected . This study also compared two different types of maintenance sprays oil - free spray (astron cleaner) and oil - containing spray (intra spray), but no significant difference was found between the sprays . Intra spray contains iso - paraffin oil for lubrication, and astron cleaner contains ethanol but do not contain any type of oil . In fe - sem micrographs of the fractured surface, the failure within the hybridized dentin area was mainly observed in the oil - containing spray group, and failure at the adhesive interface was rarely observed . Furthermore, the long thick resin tags visible on the fe - sem micrographs of the cross - sectioned resin - dentin interface were the same as those observed in the other groups . These results indicated that the lower tbs in the oil - containing spray group might not be due to the inhibition of resin penetration . Since both spray cans contain liquefied petroleum gas as an aerosol propellant, this might be attributable to decrease in the mechanical properties of the adhesive interfacial area . Further studies are needed to clarify what component was affected on resin bonding . In order to perform ideal bonding, it should be eliminated the all inhibitors on resin bonding in the clinical situation . As already mentioned, contamination of blood or saliva significantly reduces the bond strength12,13 due to the inhibition of resin penetration . In order to prevent cavity surfaces produced by such contaminants, dentists typically use the rubber dam isolation technique, which is useful for creating a suitable environment for resin bonding since it not only isolates the surface from these fluids, but also reduces intraoral humidity . However, the technique is not able to prevent contamination from handpiece maintenance spray since the spray has been reported to discharge for at least 240 minutes;23 thus, the usual practice of removing excess spray by operating the handpiece for just a few minutes is ineffective in preventing the contamination.14 future work should focus on eliminating the contaminants from maintenance sprays in order to improve bonding performance to dentin . Within the limitations of this study, the following conclusions are drawn: contamination from maintenance spray significantly affects the microtensile bond strength to dentin . However, there is no difference between the effects of oil - free and oil - containing maintenance sprays on the reduction in the microtensile bond strength to dentin.
Fungal diseases have markedly increased during the recent years (1); although more increase is observed in opportunistic mycoses . Determining the mycoflora of normal people is important when the role of skin is considered as a reservoir for microorganisms . Infectious diseases, mostly the ones that affect the epidermal or mucous membrane, are serious troubles all over the world because of hygiene and education deficiency . Microbiome of human skin refers to complete collection of microorganisms comprising bacteria, fungi and virus . The kind and quantity of skin microbes are different from one individual to another depending on the location of the body . Fungi are among the most significant groups of these skin pathogens (2). Cutaneous mycoses refer to skin infection and its appendages are involved in yeasts and filamentous fungi . They have the affinity to parasitized tissues with rich keratin and create the skin inflammatory responses and cause severe itching, burning and redness . Moreover, these diseases cause cosmetic outcomes . Candidiasis includes the infections that vary from superficial; for example, oral thrush and vulvovaginitis, to visceral and potentially life - threatening diseases . Superficial infections of dermal inflammation and discomfort are frequent in numerous human societies . Toe webs harbor fungi more than the less occluded parts such as trunk, legs, and arms (2). At present a normal healthy skin in adults is expected to transmit a number of representatives of the genera candida, malassezia and geotrichum as well as few anthropophilic dermatophytes as inhabitants of the normal skin . Direct examination, and culture and molecular analyses are employed to recognize the skin microbial inhabitants . Direct examination is based on finding the microbial elements such as unicellular yeast, mycelium and pseudohyphae . The presence of malassezia spp . In healthy human skin was identified in previous investigation in the nineteenth century . The incidence and density of colonization depended on the activity of sebaceous gland and age . The species most commonly associated with this disease are, malassezia globosa, m. furfur and m. sympodialis (3 - 5). This disorder could be observed in temperate climates, particularly during summer in humid months . The malassezia yeast form generates dicarboxylic acids similar to azelaic acid which prohibit tyrosine kinase that consequences in hypopigmentation of skin (4) diagnosis of this disease is clinically easy and it is confirmed by microscopic examination of skin scraping on potassium hydroxide or using scotch tape . Conversion from yeast form of malassezia to mycelia form was promoted with warm and humid weather . Immunodeficiency, poor hygiene, diabetes and poor nutrition are other factors related to pityriasis versicolor . The seborrheic areas are chest, back, abdomen, neck, and proximal arms . The lesions possess different colors such as yellow, brown, pink, red or hypo pigmented . Keratinophilic fungi are the groups which could infect the skin, hair and nail in human (6, 7). Anthropophilic type of theses fungi can apparently live in healthy human skin which may contribute to transmission of the fungi (8, 9). The current study aimed to investigate the incidence of fungal flora on interdigital spaces of the human foot . Samples were collected from toe webs of 865 girl students who lived in the dormitories of ahvaz jundishapur university of medical sciences in autumn 2008 . The tested web spaces did not show any signs of infection such as erythema, scaling, blistering and itching . The samples were collected into sterilized clean pockets and transferred immediately to the mycology medical laboratory . A part of the samples were digested with 20% koh and screened by a light microscope for fungal elements . Another part of the samples were cultured on sabouraud glucose agar (sga) and sga containing 0.05 mg / ml chloramphenicol and 0.5 mg/ ml cyclohexmide . The filamentous fungi were identified after slide culturing according to their gross and morphological features . Yeasts were identified on the bases of germ tube formation, morphology on the corn meal agar medium, and assay of unease activity . Out of the 865 samples, 616 (71.2%) were positive in direct examination or culture . The culture was positive in 349 (40.3%) specimens; and 22 (% 2.5%) samples were also positive for both direct examination and culture . The most common fungal isolates in direct test were yeast (29.4%), followed by conidia (0 . 92%), melanised hypha (0.35%) and non - septated hypha (0.23%). Skin surface is moderately dry, fairly acidic and dead cells are the prime source of nutrition . Skin has an environment which inhibits the growth of numerous microorganisms, however, some have adapted to living on the skin . Candida and malassezia species are two good examples that inhabit on the skin (3, 4, 10, 11). The current study performed a comprehensive and quantitative analysis of the mycoflora on the surface of interdigital skin of girl students . Overall fungal structure was found on the skin of 30.9% of the students in the direct examination with koh . The rates of the total yeast counts were much higher than those of molds in samples of skin in the direct examination . The species of candida live on skin and cause the frequent skin infection named candidiasis . Candida species were isolated from 1.2% of interdigital spaces of people in the current study . Although skin candidiasis is not life threatening, it can affect the emotional and physical status of the patients . Malassezia species is considered as an opportunistic fungus, since it stays benign until the conditions that convey it to cause infection are provided . In the current study, various fungi which can be considered as possible agents for skin diseases were isolated . In the present study, various fungi were isolated from the skin which could be considered as possible causes of skin diseases . Compared to many fungal diseases, host - fungus association in dermatophytosis was significant because dermatophytes affect immunocompetent persons; however they usually invade just superficial keratinized tissues . Currently, not much has been recognized from the relevant factors which mediate adherence of these fungi to host . The capability of t. rubrum to adhere to epithelial cells is a trait to carbohydrate - specific adhesin on microconidia (12). Morphological observation detected the fibrillar projections in t. mentagrophytes through the adherence stage (13, 14). In the current research various saprophytic fungi actually, many of the fungal spores recovered from the skin can be found in the atmosphere . The present study demonstrated the incidence of fungal flora on interdigital spaces of human foot . The obtained results showed that fungi can survive on the surfaces of skin without showing the sign of infection.
Implant - supported overdenture prostheses can be divided into bar overdentures and single attachment overdentures . Single attachment elements for overdentures include single retentive anchors, single magnet anchors, and individually cast telescopic copings.1 among these, telescopic copings have the benefit of implant splinting found in bar overdentures and the retrievability of single attachment overdentures . However, this method is typically fabricated using gold materials, so it is not an economical treatment option . Also, if an inner crown will be worn, it is difficult to maintain appropriate retentive forces . In this case report, a telescopic implant - supported overdenture prosthesis was made using a new material, polyaryletherketone (paek) based polymer (pekkton ivory, cendres + mtaux sa, biel / bienne, switzerland). It shares benefits of typical telescopic coping, in additional to being highly economical, wear resistant, and light in weight compared to conventional implant overdenture prostheses . 1) presented to the department of prosthodontics at chonnam national university dental hospital . After clinical and radiographic examinations after maxillary teeth extractions and use of provisional maxillary complete denture for six months, six small diameter implant fixtures (3.0 10.0 mm usii, osstem implant co. ltd ., seoul, korea) the definitive prosthesis was planned as a telescopic overdenture using paek based polymer . After a making definitive impression by polyvinylsiloxane (honigum, dmg, hamburg, germany), a polymer telescopic abutment and an outer overdenture frame were fabricated with consideration of the patient's vertical dimension (fig . Telescopic abutment and framework design were laid out by cad software (exocad dental cad, exocad gmbh, darmstadt, germany). The milling machine (s1, vhf camfacture ag, ammerbuch, germany) made the final framework and abutment according to the design . Polymer abutment and titanium link were sandblasted by 110 um grit aluminum oxide, and bonded with primer (sr link, ivoclar vivadent, schaan, liechtenstein) and bonding agent (multilink n, ivoclar vivadent, schaan, leichtenstein). After that, the definitive prosthesis was made by autopolymerized pour - type resin (press lt, retec, rosbach, germany) (fig . 4); the design and weight of the prosthesis were adjusted to achieve acceptable esthetics and phonetics . After 6 months, there were no problems with alveolar bone around the implant fixtures and retention of the overdenture prosthesis . However, no treatment modality meets all criteria for successful treatment, and conventional overdenture material can sometimes be limited by economic, functional, and technical considerations . Now, many new prosthetic materials are available to overcome these limitations, and as in this case, a new polymer can be used to make telescopic crowns and frameworks to obtain satisfactory results . Paek based polymer, pekkton ivory, as used in this case, is a member of the high performance semi - crystalline thermoplastic resin group, recognized for its keto and ether group ratio . Paek has good dimensional stability at high temperature, high chemical and mechanical resistance against wear, and high tensile, fatigue and flexural strength, making it an attractive material with expanded uses in medicine and dentistry.3 however, peek (polyetheretherketone), a conventional paek - based polymer, cannot be used as a permanent material due to its relatively weak physical properties . A new material, pekkton, is mainly composed of pekk (polyetherketoneketone); its molecular structure has an added ketone to the structure of peek with and has a wide range of uses due to its amorphous and crystalline structure . Pekk reveals up to 80% greater compressive strengths than peek, so this polymer may be used in permanent prostheses according to the manufacturer.4 thus, this new polymer can be considered to have greater strength than peek and have greater esthetics titanium, higher resin bond strength than zirconia, and a lighter weight (1.4 g / cm) than metal . Therefore, this material is found to be mechanically suitable for fpd frameworks, milled overdenture bars, clasps, telescopic crowns, and other applications . Despite the reportedly good bond strength, bonding between the titanium link and the telescopic abutment is still very sensitive, with the risk of fracture of the thin abutment wall due to connecting titanium link; hence more studies are needed on such cases . She reported satisfaction with its strength and esthetics, and no negative symptoms . Due to the lack of evidence on the long term retentive capabilities of this material, due to its functional and economic advantages, paek based polymer is a good alternative material to conventional materials and methods in the fabrication of implant overdenture.
The question of structural and mechanical alterations of the arterial wall in the case of renal transplantation has not yet been completely resolved . An insufficiency of endogenous regulators of calcium and phosphate is well known to be a significant factor affecting extraosseous calcifications.1 in dialysis patients and kidney transplant patients, volume overload and disturbances of calcium and phosphate metabolism add to the atherogenic profile, and these serve as independent risk factors for cardiovascular mortality.2 the presence of arterial calcifications was strongly and independently predictive of the outcome in end - stage renal disease (esrd).3 on this basis, estimating the arterial stiffness (as) in esrd is of great interest . Measuring the pulse - wave velocity (pwv) is a reliable means of determining the as.4,5 there are several studies of pwv in patients with renal transplantation.69 notably, 24-hour pwv analysis has not yet been conducted in adults . Meanwhile, some modern devices for ambulatory blood pressure monitoring (abpm) allow the assessment of some as indices, and the approach to the analysis of these indices may be quite similar to that of abpm.10 accordingly, the aim of our study was to assess the feasibility of this approach in the management of patients with renal transplantation . Overall, 41 patients were recruited from the kidney transplant waiting list of the privolzhsky district medical center in nizhniy novgorod, russia . The inclusion criteria were the following: age between 18 years and 55 years, esrd resulting from nondiabetic glomerulopathy (glomerular filtration rate <15 ml / min per 1.73 m), and candidate for a first kidney transplant . Patients with a history of prior kidney transplant or who were candidates for a kidney - pancreas transplant were excluded from the study . The additional exclusion criteria were cardiac rhythm disturbances, body mass index higher than 35 kg / m, severe dyslipidemia, and unstable clinical presentation . Our study group included 27 (65.8%) men and had a mean age of 35.2 years, with a mean systolic blood pressure of 134 mmhg, mean diastolic blood pressure of 86 mmhg, and mean heart rate of 74 beats per minute . All the patients were receiving dialysis at enrollment, including 40 by hemodialysis and one by peritoneal dialysis . Management of patients included the monitoring of calcium and phosphate serum levels and their correction . All measurements were performed before kidney transplantation and at 1 and 20 weeks after transplantation . Approval for the study was obtained from the local research ethics committee, and written informed consent was obtained for each participant . The vasotens technology is an innovative method used for pulse - wave analysis based on oscillometric blood pressure measurements, using the bplab (nizhniy novgorod, russia) device for abpm.11,12 the technology was developed by the petr telegin company in nizhniy novgorod, russia . The recordings are made using a conventional blood pressure cuff for adults . During the blood pressure measurement, the pressure waveforms in the cuff are registered while performing a step - by - step deflation . The separation and timing of the forward and reflected pulse waves are determined by a special mathematical algorithm . The distance for the pwv equation used by the vasotens was measured according to the bplab user s guide requirements . The quality control method consists of a visual assessment of the curves in the vasotens clinical report screen . The pulse time index of norm (ptin) is calculated by the vasotens for the estimation of the 24-hour pwv . The principle of the ptin calculation is where tm is the entire period of monitoring and t1, t2, and tn represent the periods in which the pwv does not exceed the cut - off value of 10 m / second . Essentially, all the data are shown as the mean and standard deviation (sd). We used bpstat software, version 05.00.04 (bplab) to tabulate all the indices of every measured 24-hour waveform automatically . Overall, 41 patients were recruited from the kidney transplant waiting list of the privolzhsky district medical center in nizhniy novgorod, russia . The inclusion criteria were the following: age between 18 years and 55 years, esrd resulting from nondiabetic glomerulopathy (glomerular filtration rate <15 ml / min per 1.73 m), and candidate for a first kidney transplant . Patients with a history of prior kidney transplant or who were candidates for a kidney - pancreas transplant were excluded from the study . The additional exclusion criteria were cardiac rhythm disturbances, body mass index higher than 35 kg / m, severe dyslipidemia, and unstable clinical presentation . Our study group included 27 (65.8%) men and had a mean age of 35.2 years, with a mean systolic blood pressure of 134 mmhg, mean diastolic blood pressure of 86 mmhg, and mean heart rate of 74 beats per minute . All the patients were receiving dialysis at enrollment, including 40 by hemodialysis and one by peritoneal dialysis . Management of patients included the monitoring of calcium and phosphate serum levels and their correction . All measurements were performed before kidney transplantation and at 1 and 20 weeks after transplantation . Approval for the study was obtained from the local research ethics committee, and written informed consent was obtained for each participant . The vasotens technology is an innovative method used for pulse - wave analysis based on oscillometric blood pressure measurements, using the bplab (nizhniy novgorod, russia) device for abpm.11,12 the technology was developed by the petr telegin company in nizhniy novgorod, russia . The recordings are made using a conventional blood pressure cuff for adults . During the blood pressure measurement, the pressure waveforms in the cuff are registered while performing a step - by - step deflation . The separation and timing of the forward and reflected pulse waves are determined by a special mathematical algorithm . The distance for the pwv equation used by the vasotens was measured according to the bplab user s guide requirements . The quality control method consists of a visual assessment of the curves in the vasotens clinical report screen . The pulse time index of norm (ptin) is calculated by the vasotens for the estimation of the 24-hour pwv . The principle of the ptin calculation is where tm is the entire period of monitoring and t1, t2, and tn represent the periods in which the pwv does not exceed the cut - off value of 10 m / second . Essentially, all the data are shown as the mean and standard deviation (sd). We used bpstat software, version 05.00.04 (bplab) to tabulate all the indices of every measured 24-hour waveform automatically . The ptins in different periods before and after renal transplantation are illustrated in figure 1 . As shown in figure 1, before kidney transplantation as our analysis showed, this value did not depend on the duration of the history of the disease or the time of the interdialysis period at which the monitoring was performed . Then, a week after the renal transplantation, we observed a decrease in the ptin in most cases . The mean ptin in the whole group at this period was 27.6 (sd, 11.1). After 20 weeks, the mean ptin in the whole group increased again to 52.0 (sd, 23.6), but the detailed analysis showed that those patients who had a higher value of ptin before transplantation had a higher increase at this time . Using the receiver operating characteristic curve, we determined the cutoff value of ptin that could predict the two ptin states: a state of improvement or a state of decline / without change (figure 2). The cutoff value of ptin at 45% had a sensitivity of 69%, specificity of 76%, and area under the curve of 0.65 to predict these states . For the detailed baseline characteristics of the patient groups separated according to this cut - off point, please see table 1 . As shown in table 1, there was no significant difference between the groups for almost all characteristics except the ptin . The difference in preoperative dialysis period (p = 0.0405) and the tendency toward a difference in age (p = 0.0590) should be noted . The ptin at different periods before and after renal transplantation in the groups with ptin of 45% or higher or less 45% is illustrated in figure 3 . The analysis of variance showed that in the first group, the ptin changed significantly (p <0.05), whereas in the second group, the ptin was not significantly different . The effect of renal transplantation on blood pressure (table 2) was similar to the effect on the ptin . Some authors have noted an improvement in the as after kidney transplantation.68 a number of studies have shown that after kidney transplantation, the disturbances in calcium and phosphate metabolism improve in general, but the media calcification, structural alterations of the arterial wall, and disturbed mechanical vessel wall properties persist.9,1315 in our study, we found that the persistence of these disturbances after kidney transplantation appears to be relatively predictable: there are clear differences in the clinical conditions of patients who develop an excess in the pwv over the cutoff value for either a small or large percentage of the monitoring period . If ptin is 45% or higher before kidney transplantation, there appears to be a good chance that the ptin will improve in the remote period after transplantation . This improvement is particularly important for the management in the initial period after implantation, when increased damage to the arterial wall, resulting in a decreased ptin in most patients, is observed . It is known that the prevalence of postoperative hypertension in patients with renal transplantation is high, and this is one reason for an aggressive therapeutic approach by physicians, reflected by administering more antihypertensive medications.16 the ptin seems to give us an opportunity to optimize this approach . Regarding the influence of hemodialysis on the variability of the pwv, a study in which the measurements were performed immediately before (pre) and 1 hour after (post) the end of each dialysis session should be noted . Cyclic intradialysis changes in the pwv were similar during the three dialysis sessions; as a consequence, all postdialytic pwv values were lower with respect to the predialytic levels.17 as our analysis showed, the ptin did not depend on the time of the interdialysis period at which the monitoring was performed . In our opinion, this mismatching is a result of the smoothing effect of the nocturnal pattern of pwv in ptin, which integrates the pwv values over the course of 24 hours . Thus, the analysis of the 24-hour pulse wave velocity in the management of patients with renal transplantation using ptin is feasible.
Xanthogranulomatous pyelonephritis (xgp) is a rare, distinct and aggressive form of chronic infectious pyelonephritis . It accounts for lesser than 1% of chronic pyelonephritis . Though common in fifth to sixth decade complications can occur in the form of psoas abscess, nephro cutaneous fistula, enterocolonic fistula, paranephric abscess and sepsis . It is essential to suspect and diagnose this condition early to prevent the morbidity and mortality . Owing to its rarity and clinical curiosity a 75-year - old man presented with difficulty in micturition since 15 days, fever, abdominal and flank pain and burning micturition since 7 days . X - ray, ultrasonography (usg) and computed tomography (ct) scan abdomen findings include pyonephrosis and cortical atrophy in the right kidney . Intravenous urography (ivu) revealed non - excretory right kidney . Left kidney showed normal excretion resected specimen was yellowish lobulated renal mass measuring 13 8 6 cm with ureter [figure 1a]. Cut section showed dilated pelvis, calyces and cortical atrophy due to extensive destruction of renal parenchyma, which were covered with thick purulent material [figure 1b]. Histopathology revealed atrophic and dilated renal tubules showing thyroidisation and sclerosed glomeruli and interstitial fibrosis [figure 2]. Many areas showed histiocytes with abundant foamy cytoplasm, lymphoplasmacytic inflammatory cells with foci of polymorph nuclear leukocytes [figure 3 and inset] ziehl nelsen stain was negative for acid fast bacilli . (a) yellowish lobulated renal mass with ureter; (b) dilated pelvis, calyces and cortical atrophy covered with thick purulent material atrophic and dilated renal tubules showing thyroidisation, sclerosed glomeruli and fibrosis . Inset show giant cells and cholesterol clefts (h and e, 400) foamy histiocytes, inflammatory cells . Xgp is a severe chronic renal inflammatory condition leading to focal or diffuse kidney destruction . It may be due to defect in degradation of bacteria in the macrophages especially when associated with infection and obstruction by stones . Three forms of xgp are recognised: diffuse is characterized by diffuse involvement of kidney, segmental by segmental involvement and focal - is located within the cortex . It is often misdiagnosed pre - operatively as pyelonephritis, tuberculosis, perinephric abscess and renal cell carcinoma (rcc). Symptoms include flank or abdominal pain, fever, palpable mass, gross hematuria, pyuria, dysuria and weight loss . In our case, symptoms were attributed to bph . Other organisms include staphylococcus aureus, group b streptococcus, candida, klebsiella and bacteroides . Elevation of serum creatinine and bun in our case can be attributed to impaired renal function . Non - functioning, ct scan is the main stay of diagnostic imaging for xgp . Xgp has been shown to be associated with transitional cell carcinoma of renal pelvis and rcc . They have even been reported in renal allograph . In diffuse or advanced stage xgp, nephrectomy is the treatment option . Focal or segmental xgp if diagnosed early pre - operatively can be treated with antibiotics . Pre- and post - operative broad spectrum antibiotics and symptomatic management are also key factors for successful management and better prognosis . We presented this unusual case of elderly male to stress the importance of through evaluation of renal function who gave a history of recurrent urinary tract obstruction and infection . Chronic renal infection and obstruction are two common etiological factors for xgp . In all patients of prostatic enlargement, renal function must be assessed for the extent of damage . In non - functioning kidney

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